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1
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Culver JN, Vallar M, Burchard E, Kamens S, Lair S, Qi Y, Collum TD, Dardick C, El-Mohtar CA, Rogers EE. Citrus phloem specific transcriptional profiling through the development of a citrus tristeza virus expressed translating ribosome affinity purification system. PLANT METHODS 2025; 21:49. [PMID: 40211356 PMCID: PMC11983876 DOI: 10.1186/s13007-025-01368-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/03/2024] [Accepted: 04/01/2025] [Indexed: 04/14/2025]
Abstract
BACKGROUND The analysis of translationally active mRNAs, or translatome, is a useful approach for monitoring cellular and plant physiological responses. One such method is the translating ribosome affinity purification (TRAP) system, which utilizes tagged ribosomal proteins to isolate ribosome-associated transcripts. This approach enables spatial and temporal gene expression analysis by driving the expression of tagged ribosomal proteins with tissue- or development-specific promoters. In plants, TRAP has enhanced our understanding of physiological responses to various biotic and abiotic factors. However, its utility is hampered by the necessity to generate transgenic plants expressing the tagged ribosomal protein, making this approach particularly challenging in perennial crops such as citrus. RESULTS This study involved the construction of a citrus tristeza virus (CTV) vector to express an immuno-tagged ribosome protein (CTV-hfRPL18). CTV, limited to the phloem, has been used for expressing marker and therapeutic sequences, making it suitable for analyzing citrus vascular tissue responses, including those related to huanglongbing disease. CTV-hfRPL18 successfully expressed a clementine-derived hfRPL18 peptide, and polysome purifications demonstrated enrichment for the hfRPL18 peptide. Subsequent translatome isolations from infected Nicotiana benthamiana and Citrus macrophylla showed enrichment for phloem-associated genes. CONCLUSION The CTV-hfRPL18 vector offers a transgene-free and rapid system for TRAP expression and translatome analysis of phloem tissues within citrus.
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Affiliation(s)
- James N Culver
- Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, MD, USA.
- Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA.
| | - Meinhart Vallar
- Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, MD, USA
| | - Erik Burchard
- USDA, Agricultural Research Service, Appalachian Fruit Research Station, Kearneysville, WV, USA
| | - Sophie Kamens
- Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, MD, USA
| | - Sebastien Lair
- Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, MD, USA
| | - Yiping Qi
- Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA
| | - Tamara D Collum
- USDA, Agricultural Research Service, Appalachian Fruit Research Station, Kearneysville, WV, USA
| | - Christopher Dardick
- USDA, Agricultural Research Service, Appalachian Fruit Research Station, Kearneysville, WV, USA
| | - Choaa A El-Mohtar
- Department of Plant Pathology, Citrus Research and Education Center, University of Florida, Gainesville, FL, USA
| | - Elizabeth E Rogers
- USDA, Agricultural Research Service, Foreign Disease-Weed Science Research Unit, Frederick, MD, USA
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2
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Tezuka T, Nosaka-Takahashi M, Yoshikawa T, Sato Y. RNA Extraction from Rice Immature Embryo Using Laser Capture Microdissection. Methods Mol Biol 2025; 2869:21-27. [PMID: 39499464 DOI: 10.1007/978-1-0716-4204-7_4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2024]
Abstract
Laser capture microdissection (LCM) enables the selective isolation of organs, tissues, and cells from surrounding tissues. Total RNA extracted from small tissue sections can be used for a variety of subsequent analysis such as RNA-seq analysis. Here, we describe a method for isolating embryos from rice ovary sections using LCM and extracting total RNA. We successfully obtained sufficient amount of total RNA from a single rice embryo at the globular embryo stage for RNA-seq analysis.
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Affiliation(s)
- Takumi Tezuka
- National Institute of Genetics, Mishima, Shizuoka, Japan.
| | | | | | - Yutaka Sato
- National Institute of Genetics, Mishima, Shizuoka, Japan
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Luna-García V, de Folter S. Laser-Assisted Microdissection and High-Throughput RNA Sequencing of the Arabidopsis Gynoecium Medial and Lateral Domains. Bio Protoc 2024; 14:e5056. [PMID: 39282231 PMCID: PMC11393044 DOI: 10.21769/bioprotoc.5056] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2024] [Revised: 07/22/2024] [Accepted: 07/23/2024] [Indexed: 09/18/2024] Open
Abstract
For obtaining insights into gene networks during plant reproductive development, having transcriptomes of specific cells from developmental stages as starting points is very useful. During development, there is a balance between cell proliferation and differentiation, and many cell and tissue types are formed. While there is a wealth of transcriptome data available, it is mostly at the organ level and not at specific cell or tissue type level. Therefore, methods to isolate specific cell and tissue types are needed. One method is fluorescent activated cell sorting (FACS), but it has limitations such as requiring marker lines and protoplasting. Recently, single-cell/nuclei isolation methods have been developed; however, a minimum amount of genetic information (marker genes) is needed to annotate/predict the resulting cell clusters in these experiments. Another technique that has been known for some time is laser-assisted microdissection (LAM), where specific cells are microdissected and collected using a laser mounted on a microscope platform. This technique has advantages over the others because no fluorescent marker lines must be made, no marker genes must be known, and no protoplasting must be done. The LAM technique consists in tissue fixation, tissue embedding and sectioning using a microtome, microdissection and collection of the cells of interest on the microscope, and finally RNA extraction, library preparation, and RNA sequencing. In this protocol, we implement the use of normal slides instead of the membrane slides commonly used for LAM. We applied this protocol to obtain the transcriptomes of specific tissues during the development of the gynoecium of Arabidopsis. Key features • Laser-assisted microdissection (LAM) allows the isolation of specific cells or tissues. • Normal slides can be used for LAM. • It allows the identification of the transcriptional profiles of specific tissues of the Arabidopsis gynoecium.
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Affiliation(s)
- Valentín Luna-García
- Unidad de Genómica Avanzada (UGA-Langebio), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (Cinvestav), Irapuato, Mexico
| | - Stefan de Folter
- Unidad de Genómica Avanzada (UGA-Langebio), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (Cinvestav), Irapuato, Mexico
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4
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Mei X, Zhu K, Yan D, Jia H, Luo W, Ye J, Deng X. Developing a simple and rapid method for cell-specific transcriptome analysis through laser microdissection: insights from citrus rind with broader implications. PLANT METHODS 2024; 20:113. [PMID: 39068421 PMCID: PMC11282741 DOI: 10.1186/s13007-024-01242-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/07/2023] [Accepted: 07/18/2024] [Indexed: 07/30/2024]
Abstract
BACKGROUND With the rapid development of single-cell sequencing technology, histological studies are no longer limited to conventional homogenized tissues. Laser microdissection enables the accurate isolation of specific tissues or cells, and when combined with next-generation sequencing, it can reveal important biological processes at the cellular level. However, traditional laser microdissection techniques have often been complicated and time-consuming, and the quality of the RNA extracted from the collected samples has been inconsistent, limiting follow-up studies. Therefore, an improved, simple, and efficient laser microdissection method is urgently needed. RESULTS We omitted the sample fixation and cryoprotectant addition steps. Instead, fresh samples were embedded in Optimal Cutting Temperature medium within 1.5 ml centrifuge tube caps, rapidly frozen with liquid nitrogen, and immediately subjected to cryosectioning. A series of section thicknesses of citrus rind were tested for RNA extraction, which showed that 18 μm thickness yielded the highest quality RNA. By shortening the dehydration time to one minute per ethanol gradient and omitting the tissue clearing step, the resulting efficient dehydration and preserved morphology ensured high-quality RNA extraction. We also propose a set of laser microdissection parameters by adjusting the laser power to optimal values, reducing the aperture size, and lowering the pulse frequency. Both the epidermal and subepidermal cells from the citrus rind were collected, and RNA extraction was completed within nine hours. Using this efficient method, the transcriptome sequencing of the isolated tissues generated high-quality data with average Q30 values and mapping rates exceeding 91%. Moreover, the transcriptome analysis revealed significant differences between the cell layers, further confirming the effectiveness of our isolation approach. CONCLUSIONS We developed a simple and rapid laser microdissection method and demonstrated its effectiveness through a study based on citrus rind, from which we generated high-quality transcriptomic data. This fast and efficient method of cell isolation, combined with transcriptome sequencing not only contributes to precise histological studies at the cellular level in citrus but also provides a promising approach for cell-specific transcriptome analysis in a broader range of other plant tissues.
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Affiliation(s)
- Xuehan Mei
- National Key Lab for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan, 430070, Hubei, China
| | - Kaijie Zhu
- National Key Lab for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan, 430070, Hubei, China
| | - Danni Yan
- National Key Lab for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan, 430070, Hubei, China
| | - Huihui Jia
- National Key Lab for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan, 430070, Hubei, China
| | - Wangyao Luo
- National Key Lab for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan, 430070, Hubei, China
| | - Junli Ye
- National Key Lab for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan, 430070, Hubei, China
| | - Xiuxin Deng
- National Key Lab for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
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Luna-García V, Bernal Gallardo JJ, Rethoret-Pasty M, Pasha A, Provart NJ, de Folter S. A high-resolution gene expression map of the medial and lateral domains of the gynoecium of Arabidopsis. PLANT PHYSIOLOGY 2024; 195:410-429. [PMID: 38088205 DOI: 10.1093/plphys/kiad658] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/25/2023] [Accepted: 11/14/2023] [Indexed: 05/02/2024]
Abstract
Angiosperms are characterized by the formation of flowers, and in their inner floral whorl, one or various gynoecia are produced. These female reproductive structures are responsible for fruit and seed production, thus ensuring the reproductive competence of angiosperms. In Arabidopsis (Arabidopsis thaliana), the gynoecium is composed of two fused carpels with different tissues that need to develop and differentiate to form a mature gynoecium and thus the reproductive competence of Arabidopsis. For these reasons, they have become the object of study for floral and fruit development. However, due to the complexity of the gynoecium, specific spatio-temporal tissue expression patterns are still scarce. In this study, we used precise laser-assisted microdissection and high-throughput RNA sequencing to describe the transcriptional profiles of the medial and lateral domain tissues of the Arabidopsis gynoecium. We provide evidence that the method used is reliable and that, in addition to corroborating gene expression patterns of previously reported regulators of these tissues, we found genes whose expression dynamics point to being involved in cytokinin and auxin homeostasis and in cell cycle progression. Furthermore, based on differential gene expression analyses, we functionally characterized several genes and found that they are involved in gynoecium development. This resource is available via the Arabidopsis eFP browser and will serve the community in future studies on developmental and reproductive biology.
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Affiliation(s)
- Valentín Luna-García
- Unidad de Genómica Avanzada (UGA-LANGEBIO), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Irapuato CP 36824, Guanajuato, México
| | - Judith Jazmin Bernal Gallardo
- Unidad de Genómica Avanzada (UGA-LANGEBIO), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Irapuato CP 36824, Guanajuato, México
| | - Martin Rethoret-Pasty
- Department of Cell & Systems Biology, Centre for the Analysis of Genome Evolution and Function, University of Toronto, 25 Willcocks St., Toronto, ON M5S 3B2, Canada
- Polytech Nice Sophia, Université Côte d'Azur, 930 Rte des Colles, 06410 Biot, France
| | - Asher Pasha
- Department of Cell & Systems Biology, Centre for the Analysis of Genome Evolution and Function, University of Toronto, 25 Willcocks St., Toronto, ON M5S 3B2, Canada
| | - Nicholas J Provart
- Department of Cell & Systems Biology, Centre for the Analysis of Genome Evolution and Function, University of Toronto, 25 Willcocks St., Toronto, ON M5S 3B2, Canada
| | - Stefan de Folter
- Unidad de Genómica Avanzada (UGA-LANGEBIO), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Irapuato CP 36824, Guanajuato, México
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6
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Chavan S, Schnabel E, Saski C, Frugoli J. Fixation and Laser Capture Microdissection of Plant Tissue for RNA Extraction and RNASeq Library Preparation. Curr Protoc 2023; 3:e844. [PMID: 37486164 DOI: 10.1002/cpz1.844] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/25/2023]
Abstract
To study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed by laser capture microdissection (LCM) and processing for RNA recovery. LCM allows the isolation of individual cell types from heterogeneous tissue sections and is particularly suited to plant processing because it does not require the breakdown of cell walls. This approach allows accurate separation of a small volume of cells that can be used to study gene expression profiles in different tissues or cell layers. The technique requires neither separation of cells by enzymatic digestion of any kind nor cell-specific reporter genes, and it allows storage of fixed and embedded tissue for months before capture. The methods for fixation, embedding, sectioning, and capturing of plant cells that we describe yield high-quality RNA suitable for making libraries for RNASeq. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tissue Preparation for Laser Capture Microdissection Basic Protocol 2: Tissue Sectioning Basic Protocol 3: Laser Capture Microdissection of Embedded Tissue Basic Protocol 4: RNA Extraction from Laser Capture Microdissection Samples.
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Affiliation(s)
- Suchitra Chavan
- Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina
| | - Elise Schnabel
- Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina
| | - Christopher Saski
- Department of Plant and Environmental Sciences, Clemson University, Clemson, South Carolina
| | - Julia Frugoli
- Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina
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7
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Pires RC, Ferro A, Capote T, Usié A, Correia B, Pinto G, Menéndez E, Marum L. Laser Microdissection of Woody and Suberized Plant Tissues for RNA-Seq Analysis. Mol Biotechnol 2023; 65:419-432. [PMID: 35976558 DOI: 10.1007/s12033-022-00542-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2021] [Accepted: 07/05/2022] [Indexed: 10/15/2022]
Abstract
An accurate profile of gene expression at a cellular level can contribute to a better understanding of biological processes and complexities involved in regulatory mechanism of woody plants. Laser microdissection is one technique that allows isolation of specific, target cells or tissue from a heterogeneous cell population. This technique entails microscopic visualization of the selected tissue and use a laser beam to separate the desired cells from surrounding tissue. Initial identification of these cells is made based on morphology and/or histological staining. Some works have been made in several tissues and plant models. However, there are few studies of laser microdissection application in woody species, particularly, lignified and suberized cells. Moreover, the presence of high level of suberin in cell walls can be a big challenge for the application of this approach. In our study it was developed a technique for tissue isolation, using laser microdissection of four different plant cell types (phellogen, lenticels, cortex and xylem) from woody tissues of cork oak (Quercus suber), followed by RNA extraction and RNA-Seq. We tested several methodologies regarding laser microdissection, cryostat equipments, fixation treatments, duration of single-cells collection and number of isolated cells by laser microdissection and RNA extraction procedures. A simple and efficient protocol for tissue isolation by laser microdissection and RNA purification was obtained, with a final method validation of RNA-Seq analysis. The optimized methodology combining RNA-Seq for expression analysis will contribute to elucidate the molecular pathways associated with different development processes of the xylem and phellem in oaks, including the lenticular channels formation.
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Affiliation(s)
- Rita Costa Pires
- Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL)/Instituto Politécnico de Beja (IPBeja), 7801-908, Beja, Portugal
| | - Ana Ferro
- Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL)/Instituto Politécnico de Beja (IPBeja), 7801-908, Beja, Portugal.,MED - Mediterranean Institute for Agriculture, Environment and Development, CEBAL - Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo, 7801-908, Beja, Portugal.,Center for Genomics and Systems Biology, New York University Abu Dhabi, NYUAD Campus, 129188, Abu Dhabi, United Arab Emirates
| | - Tiago Capote
- Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL)/Instituto Politécnico de Beja (IPBeja), 7801-908, Beja, Portugal.,MED - Mediterranean Institute for Agriculture, Environment and Development, CEBAL - Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo, 7801-908, Beja, Portugal.,Center for Genomics and Systems Biology, New York University Abu Dhabi, NYUAD Campus, 129188, Abu Dhabi, United Arab Emirates
| | - Ana Usié
- Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL)/Instituto Politécnico de Beja (IPBeja), 7801-908, Beja, Portugal.,MED - Mediterranean Institute for Agriculture, Environment and Development & CHANGE - Global Change and Sustainability Institute, CEBAL - Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo, 7801-908, Beja, Portugal
| | - Bárbara Correia
- Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL)/Instituto Politécnico de Beja (IPBeja), 7801-908, Beja, Portugal.,B-hive Innovations Ltd., Boole Technology Centre, Beevor Street, Lincoln, LN6 7DJ, UK
| | - Glória Pinto
- Department of Biology, Centre for Environmental and Marine Studies (CESAM), University of Aveiro, 3810-193, Aveiro, Portugal
| | - Esther Menéndez
- MED-Mediterranean Institute for Agriculture, Environment and Development & CHANGE - Global Change and Sustainability Institute, Institute for Advanced Studies and Research (IIFA), University of Évora, Polo da Mitra, Ap. 94, 7006-554, Évora, Portugal.,Department of Microbiology and Genetics/CIALE, Universidad de Salamanca, 37007, Salamanca, Spain
| | - Liliana Marum
- Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo (CEBAL)/Instituto Politécnico de Beja (IPBeja), 7801-908, Beja, Portugal. .,MED - Mediterranean Institute for Agriculture, Environment and Development & CHANGE - Global Change and Sustainability Institute, CEBAL - Centro de Biotecnologia Agrícola e Agro-Alimentar do Alentejo, 7801-908, Beja, Portugal.
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8
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Song S, Ma D, Xu C, Guo Z, Li J, Song L, Wei M, Zhang L, Zhong YH, Zhang YC, Liu JW, Chi B, Wang J, Tang H, Zhu X, Zheng HL. In silico analysis of NAC gene family in the mangrove plant Avicennia marina provides clues for adaptation to intertidal habitats. PLANT MOLECULAR BIOLOGY 2023; 111:393-413. [PMID: 36645624 DOI: 10.1007/s11103-023-01333-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/28/2022] [Accepted: 12/29/2022] [Indexed: 06/17/2023]
Abstract
NAC (NAM, ATAF1/2, CUC2) transcription factors (TFs) constitute a plant-specific gene family. It is reported that NAC TFs play important roles in plant growth and developmental processes and in response to biotic/abiotic stresses. Nevertheless, little information is known about the functional and evolutionary characteristics of NAC TFs in mangrove plants, a group of species adapting coastal intertidal habitats. Thus, we conducted a comprehensive investigation for NAC TFs in Avicennia marina, one pioneer species of mangrove plants. We totally identified 142 NAC TFs from the genome of A. marina. Combined with NAC proteins having been functionally characterized in other organisms, we built a phylogenetic tree to infer the function of NAC TFs in A. marina. Gene structure and motif sequence analyses suggest the sequence conservation and transcription regulatory regions-mediated functional diversity. Whole-genome duplication serves as the driver force to the evolution of NAC gene family. Moreover, two pairs of NAC genes were identified as positively selected genes of which AmNAC010/040 may be imposed on less constraint toward neofunctionalization. Quite a few stress/hormone-related responsive elements were found in promoter regions indicating potential response to various external factors. Transcriptome data revealed some NAC TFs were involved in pneumatophore and leaf salt gland development and response to salt, flooding and Cd stresses. Gene co-expression analysis found a few NAC TFs participates in the special biological processes concerned with adaptation to intertidal environment. In summary, this study provides detailed functional and evolutionary information about NAC gene family in mangrove plant A. marina and new perspective for adaptation to intertidal habitats.
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Affiliation(s)
- Shiwei Song
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - Dongna Ma
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - Chaoqun Xu
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - Zejun Guo
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - Jing Li
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - Lingyu Song
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - Mingyue Wei
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - Ludan Zhang
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - You-Hui Zhong
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - Yu-Chen Zhang
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - Jing-Wen Liu
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - Bingjie Chi
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - Jicheng Wang
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - Hanchen Tang
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - Xueyi Zhu
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China
| | - Hai-Lei Zheng
- Key Laboratory of the Ministry of Education for Costal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen, 361102, Fujian, China.
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9
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Tadeo FR, Agustí J, Merelo P, Talón M. Laser Microdissection: A High-Precision Approach to Isolate Specific Cell Types from Any Plant Species for Downstream Molecular Analyses. Methods Mol Biol 2023; 2642:365-373. [PMID: 36944888 DOI: 10.1007/978-1-0716-3044-0_19] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/23/2023]
Abstract
Plants display a great diversity of particular cell types that obviously perform functions and regulations that are essential for successful growth and development, whether under optimal or adverse conditions. The functions performed by each of these particular cell types must be associated with specific transcriptomic, proteomic, and metabolic profiles that cannot be disentangled by analyzing whole plant organs and tissues. Laser microdissection is a technique for the collection of specific cell types in plant organs and tissues comprising heterogeneous cell populations. It has been successfully used for physiological and molecular studies. Laser microdissection can be applied to any plant species as long as it is possible to reliably identify the cell types of interest. Here, we describe step by step, using citrus as a model plant, a fast, simple, easy to perform, and experimentally validated protocol to collect cells from the abscission zone, a specific tissue that is difficult to access and whose activity is important in the response of plants to adverse environmental conditions.
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Affiliation(s)
- Francisco R Tadeo
- Centro de Genómica, Instituto Valenciano de Investigaciones Agrarias, Moncada, Valencia, Spain.
| | - Javier Agustí
- Centro de Genómica, Instituto Valenciano de Investigaciones Agrarias, Moncada, Valencia, Spain
- Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas, Valencia, Spain
| | - Paz Merelo
- Centro de Genómica, Instituto Valenciano de Investigaciones Agrarias, Moncada, Valencia, Spain
- Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas, Valencia, Spain
| | - Manuel Talón
- Centro de Genómica, Instituto Valenciano de Investigaciones Agrarias, Moncada, Valencia, Spain
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Janíček T, Hobza R, Hudzieczek V. Laser Capture Microdissection: From Genomes to Chromosomes, from Complex Tissue to Single-Cell Analysis. Methods Mol Biol 2023; 2672:163-175. [PMID: 37335475 DOI: 10.1007/978-1-0716-3226-0_9] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/21/2023]
Abstract
Laser microdissection (LM) is a powerful tool for various molecular analyses providing pure samples for genomic, transcriptomic, and proteomic studies. Cell subgroups, individual cells, or even chromosomes can be separated via laser beam from complex tissues, visualized under the microscope, and used for subsequent molecular analyses. This technique provides information on nucleic acids and proteins, keeping their spatiotemporal information intact. In short, the slide with tissue is placed under the microscope, imaged by a camera onto a computer screen, where the operator selects cells/chromosomes based on morphology or staining and commands the laser beam to cut the specimen following the selected path. Samples are then collected in a tube and subjected to downstream molecular analysis, such as RT-PCR, next-generation sequencing, or immunoassay.
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Affiliation(s)
- Tomáš Janíček
- Department of Plant Developmental Genetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic.
| | - Roman Hobza
- Department of Plant Developmental Genetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic
| | - Vojtěch Hudzieczek
- Department of Plant Developmental Genetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic
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11
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Regulators of early maize leaf development inferred from transcriptomes of laser capture microdissection (LCM)-isolated embryonic leaf cells. Proc Natl Acad Sci U S A 2022; 119:e2208795119. [PMID: 36001691 PMCID: PMC9436337 DOI: 10.1073/pnas.2208795119] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The superior photosynthetic efficiency of C4 leaves over C3 leaves is owing to their unique Kranz anatomy, in which the vein is surrounded by one layer of bundle sheath (BS) cells and one layer of mesophyll (M) cells. Kranz anatomy development starts from three contiguous ground meristem (GM) cells, but its regulators and underlying molecular mechanism are largely unknown. To identify the regulators, we obtained the transcriptomes of 11 maize embryonic leaf cell types from five stages of pre-Kranz cells starting from median GM cells and six stages of pre-M cells starting from undifferentiated cells. Principal component and clustering analyses of transcriptomic data revealed rapid pre-Kranz cell differentiation in the first two stages but slow differentiation in the last three stages, suggesting early Kranz cell fate determination. In contrast, pre-M cells exhibit a more prolonged transcriptional differentiation process. Differential gene expression and coexpression analyses identified gene coexpression modules, one of which included 3 auxin transporter and 18 transcription factor (TF) genes, including known regulators of Kranz anatomy and/or vascular development. In situ hybridization of 11 TF genes validated their expression in early Kranz development. We determined the binding motifs of 15 TFs, predicted TF target gene relationships among the 18 TF and 3 auxin transporter genes, and validated 67 predictions by electrophoresis mobility shift assay. From these data, we constructed a gene regulatory network for Kranz development. Our study sheds light on the regulation of early maize leaf development and provides candidate leaf development regulators for future study.
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12
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Wu J, Liang J, Lin R, Cai X, Zhang L, Guo X, Wang T, Chen H, Wang X. Investigation of Brassica and its relative genomes in the post-genomics era. HORTICULTURE RESEARCH 2022; 9:uhac182. [PMID: 36338847 PMCID: PMC9627752 DOI: 10.1093/hr/uhac182] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/18/2022] [Accepted: 08/07/2022] [Indexed: 06/16/2023]
Abstract
The Brassicaceae family includes many economically important crop species, as well as cosmopolitan agricultural weed species. In addition, Arabidopsis thaliana, a member of this family, is used as a molecular model plant species. The genus Brassica is mesopolyploid, and the genus comprises comparatively recently originated tetrapolyploid species. With these characteristics, Brassicas have achieved the commonly accepted status of model organisms for genomic studies. This paper reviews the rapid research progress in the Brassicaceae family from diverse omics studies, including genomics, transcriptomics, epigenomics, and three-dimensional (3D) genomics, with a focus on cultivated crops. The morphological plasticity of Brassicaceae crops is largely due to their highly variable genomes. The origin of several important Brassicaceae crops has been established. Genes or loci domesticated or contributing to important traits are summarized. Epigenetic alterations and 3D structures have been found to play roles in subgenome dominance, either in tetraploid Brassica species or their diploid ancestors. Based on this progress, we propose future directions and prospects for the genomic investigation of Brassicaceae crops.
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Affiliation(s)
| | | | | | - Xu Cai
- Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 100081 Beijing, China
| | - Lei Zhang
- Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 100081 Beijing, China
| | - Xinlei Guo
- Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 100081 Beijing, China
| | - Tianpeng Wang
- Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 100081 Beijing, China
| | - Haixu Chen
- Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 100081 Beijing, China
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13
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Qin W, Li Y, Peng B, Liu H, Chen T, Yan X, Zhang Y, Wang C, Yao X, Fu X, Li L, Tang K. A high-efficiency trichome collection system by laser capture microdissection. FRONTIERS IN PLANT SCIENCE 2022; 13:985969. [PMID: 36072328 PMCID: PMC9441851 DOI: 10.3389/fpls.2022.985969] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 07/04/2022] [Accepted: 08/02/2022] [Indexed: 06/15/2023]
Abstract
Trichomes, which are classified as glandular or non-glandular, are hair-like epidermal structures that are present on aerial parts of most plant species. Glandular secretory trichomes (GSTs) have the capacity to secrete and store specialized metabolites, which are widely used as natural pesticides, food additives, fragrance ingredients or pharmaceuticals. Isolating individual trichomes is an essential way for identifying trichome-specific gene functions and discovering novel metabolites. However, the isolation of trichomes is difficult and time-consuming. Here, we report a method to isolate the GSTs from leaf epidermis dispense with fixation using laser capture microdissection (LCM). In this study, 150 GSTs were captured efficiently from Artemisia annua leaves and enriched for artemisinin measurement. UPLC analysis of microdissected samples indicated specific accumulation of secondary metabolites could be detected from a small number of GSTs. In addition, qRT-PCR revealed that the GST-specific structural genes involved in artemisinin biosynthesis pathway were highly expressed in GSTs. Taken together, we developed an efficient method to collect comparatively pure GSTs from unfixed leaved, so that the metabolites were relatively obtained intact. This method can be implemented in metabolomics research of purely specific plant cell populations and has the potential to discover novel secondary metabolites.
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14
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Song JH, Montes-Luz B, Tadra-Sfeir MZ, Cui Y, Su L, Xu D, Stacey G. High-Resolution Translatome Analysis Reveals Cortical Cell Programs During Early Soybean Nodulation. FRONTIERS IN PLANT SCIENCE 2022; 13:820348. [PMID: 35498680 PMCID: PMC9048599 DOI: 10.3389/fpls.2022.820348] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/22/2021] [Accepted: 03/22/2022] [Indexed: 06/14/2023]
Abstract
Nodule organogenesis in legumes is regulated temporally and spatially through gene networks. Genome-wide transcriptome, proteomic, and metabolomic analyses have been used previously to define the functional role of various plant genes in the nodulation process. However, while significant progress has been made, most of these studies have suffered from tissue dilution since only a few cells/root regions respond to rhizobial infection, with much of the root non-responsive. To partially overcome this issue, we adopted translating ribosome affinity purification (TRAP) to specifically monitor the response of the root cortex to rhizobial inoculation using a cortex-specific promoter. While previous studies have largely focused on the plant response within the root epidermis (e.g., root hairs) or within developing nodules, much less is known about the early responses within the root cortex, such as in relation to the development of the nodule primordium or growth of the infection thread. We focused on identifying genes specifically regulated during early nodule organogenesis using roots inoculated with Bradyrhizobium japonicum. A number of novel nodulation gene candidates were discovered, as well as soybean orthologs of nodulation genes previously reported in other legumes. The differential cortex expression of several genes was confirmed using a promoter-GUS analysis, and RNAi was used to investigate gene function. Notably, a number of differentially regulated genes involved in phytohormone signaling, including auxin, cytokinin, and gibberellic acid (GA), were also discovered, providing deep insight into phytohormone signaling during early nodule development.
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Affiliation(s)
- Jae Hyo Song
- Divisions of Plant Sciences and Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, United States
| | - Bruna Montes-Luz
- Divisions of Plant Sciences and Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, United States
| | - Michelle Zibetti Tadra-Sfeir
- Divisions of Plant Sciences and Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, United States
| | - Yaya Cui
- Divisions of Plant Sciences and Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, United States
| | - Lingtao Su
- Department of Electrical Engineering and Computer Science, C.S. Bond Life Science Center, University of Missouri, Columbia, MO, United States
| | - Dong Xu
- Department of Electrical Engineering and Computer Science, C.S. Bond Life Science Center, University of Missouri, Columbia, MO, United States
| | - Gary Stacey
- Divisions of Plant Sciences and Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, United States
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15
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Yamada K, Nakanowatari M, Yumoto E, Satoh S, Asahina M. Spatiotemporal plant hormone analysis from cryosections using laser microdissection-liquid chromatography-mass spectrometry. JOURNAL OF PLANT RESEARCH 2022; 135:377-386. [PMID: 34812978 DOI: 10.1007/s10265-021-01360-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/18/2021] [Accepted: 11/17/2021] [Indexed: 06/13/2023]
Abstract
Laser microdissection (LMD) is used for isolating specific regions or single cells from a wide variety of tissue samples under direct microscopic observation. The LMD method enables the harvest of the cells of interest in a region or specific cells for several analyses, such as DNA/RNA analysis, proteomics, metabolomics, and other molecular analyses. Currently, LMD is used to study various biological events at the tissue or cellular level; it has been used in a wide range of research fields. In this report, we describe techniques for isolating different tissues/specific cells from cryosections of incised Arabidopsis flowering stems by LMD for spatiotemporal quantitative plant hormone analysis. The endogenous indole-3-acetic acid levels in the epidermis/cortex, vascular bundles, and pith of Arabidopsis flowering stems were approximately 19.0 pg mm-3, 33.5 pg mm-3, and 3.32 pg mm-3, respectively, and these endogenous levels were altered spatiotemporally after incision. We also analyzed jasmonic acid from LMD-isolated cells and showed that the endogenous levels increased in the range of approximately 200-3,500 pg mm-3 depending on the tissue and region at 1 h after incision and then decreased to less than 100 pg mm-3 or undetectable levels at 24 h after incision. Quantitative analyses of phytohormones, including jasmonic acid-related molecules, gibberellin, abscisic acid, and cytokinins, could also be performed using the same cell samples. These results showed that spatiotemporal changes in plant hormones could be quantitatively and simultaneously analyzed by LMD-isolated cells from cryosections with positional information. The combination of quantitative analysis by liquid chromatography-mass spectrometry (LC-MS) and sampling by the LMD method provides a comprehensive and quantitative understanding of spatiotemporal changes in plant hormones in a region- and tissue-specific manner. Therefore, LMD-LC-MS methods will contribute to our understanding of the physiological events that control the process of plant growth and development.
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Affiliation(s)
- Kazuki Yamada
- Graduate School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan
| | - Miyuki Nakanowatari
- Graduate School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan
| | - Emi Yumoto
- Advanced Instrumental Analysis Center, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan
| | - Shinobu Satoh
- Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, 305-8572, Japan
| | - Masashi Asahina
- Graduate School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan.
- Advanced Instrumental Analysis Center, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan.
- Department of Biosciences, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan.
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16
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Simeoni F, Skirycz A, Simoni L, Castorina G, de Souza LP, Fernie AR, Alseekh S, Giavalisco P, Conti L, Tonelli C, Galbiati M. The AtMYB60 transcription factor regulates stomatal opening by modulating oxylipin synthesis in guard cells. Sci Rep 2022; 12:533. [PMID: 35017563 PMCID: PMC8752683 DOI: 10.1038/s41598-021-04433-y] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2021] [Accepted: 12/06/2021] [Indexed: 12/04/2022] Open
Abstract
Stomata are epidermal pores formed by pairs of specialized guard cells, which regulate gas exchanges between the plant and the atmosphere. Modulation of transcription has emerged as an important level of regulation of stomatal activity. The AtMYB60 transcription factor was previously identified as a positive regulator of stomatal opening, although the details of its function remain unknown. Here, we propose a role for AtMYB60 as a negative modulator of oxylipins synthesis in stomata. The atmyb60-1 mutant shows reduced stomatal opening and accumulates increased levels of 12-oxo-phytodienoic acid (12-OPDA), jasmonic acid (JA) and jasmonoyl-L-isoleucine (JA-Ile) in guard cells. We provide evidence that 12-OPDA triggers stomatal closure independently of JA and cooperatively with abscisic acid (ABA) in atmyb60-1. Our study highlights the relevance of oxylipins metabolism in stomatal regulation and indicates AtMYB60 as transcriptional integrator of ABA and oxylipins responses in guard cells.
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Affiliation(s)
- Fabio Simeoni
- Dipartimento di Bioscienze, Università degli Studi di Milano, Milan, Italy
| | | | - Laura Simoni
- Dipartimento di Bioscienze, Università degli Studi di Milano, Milan, Italy
| | - Giulia Castorina
- Dipartimento di Scienze Agrarie e Ambientali-Produzione, Territorio, Agroenergia, Università degli Studi di Milano, Milan, Italy
| | | | - Alisdair R Fernie
- Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany
- Center for Plant Systems Biology and Biotechnology, Plovdiv, Bulgaria
| | - Saleh Alseekh
- Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany
| | - Patrick Giavalisco
- Metabolomics Core Facility, Max Planck Institute for Biology of Ageing, Cologne, Germany
| | - Lucio Conti
- Dipartimento di Bioscienze, Università degli Studi di Milano, Milan, Italy
| | - Chiara Tonelli
- Dipartimento di Bioscienze, Università degli Studi di Milano, Milan, Italy
| | - Massimo Galbiati
- Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale Delle Ricerche, Milan, Italy.
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17
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Balestrini R, Sillo F. Plant-Fungal Interactions: Laser Microdissection as a Tool to Study Cell Specificity. Methods Mol Biol 2022; 2536:369-380. [PMID: 35819614 DOI: 10.1007/978-1-0716-2517-0_20] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
In the past 20 years, laser microdissection (LMD) technology has been widely applied to plant tissues, allowing to obtain new information on the role of different cell-type populations during plant development and interactions, including plant-pathogen interactions. The application of a LMD approach allowed verifying the response of plant and pathogen during the progression of the infection in different cell types, focusing both on gene expression in host plants and pathogens. Here, a protocol to apply the LMD approach to study plant and fungal transcript profiles in different cell-type populations is described in detail, from the biological material preparation to RNA extraction and gene expression analyses.
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Affiliation(s)
- Raffaella Balestrini
- National Research Council, Institute for Sustainable Plant Protection (CNR-IPSP), Torino, Italy.
| | - Fabiano Sillo
- National Research Council, Institute for Sustainable Plant Protection (CNR-IPSP), Torino, Italy
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18
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Filipecki M, Żurczak M, Matuszkiewicz M, Święcicka M, Kurek W, Olszewski J, Koter MD, Lamont D, Sobczak M. Profiling the Proteome of Cyst Nematode-Induced Syncytia on Tomato Roots. Int J Mol Sci 2021; 22:ijms222212147. [PMID: 34830029 PMCID: PMC8625192 DOI: 10.3390/ijms222212147] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2021] [Revised: 11/05/2021] [Accepted: 11/07/2021] [Indexed: 02/06/2023] Open
Abstract
Cyst nematodes are important herbivorous pests in agriculture that obtain nutrients through specialized root structures termed syncytia. Syncytium initiation, development, and functioning are a research focus because syncytia are the primary interface for molecular interactions between the host plant and parasite. The small size and complex development (over approximately two weeks) of syncytia hinder precise analyses, therefore most studies have analyzed the transcriptome of infested whole-root systems or syncytia-containing root segments. Here, we describe an effective procedure to microdissect syncytia induced by Globodera rostochiensis from tomato roots and to analyze the syncytial proteome using mass spectrometry. As little as 15 mm2 of 10-µm-thick sections dissected from 30 syncytia enabled the identification of 100–200 proteins in each sample, indicating that mass-spectrometric methods currently in use achieved acceptable sensitivity for proteome profiling of microscopic samples of plant tissues (approximately 100 µg). Among the identified proteins, 48 were specifically detected in syncytia and 7 in uninfected roots. The occurrence of approximately 50% of these proteins in syncytia was not correlated with transcript abundance estimated by quantitative reverse-transcription PCR analysis. The functional categories of these proteins confirmed that protein turnover, stress responses, and intracellular trafficking are important components of the proteome dynamics of developing syncytia.
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Affiliation(s)
- Marcin Filipecki
- Department of Plant Genetics, Breeding and Biotechnology, Institute of Biology, Faculty of Biology and Biotechnology, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland; (M.Ż.); (M.M.); (M.D.K.)
- Correspondence: ; Tel.: +48-22-5932171
| | - Marek Żurczak
- Department of Plant Genetics, Breeding and Biotechnology, Institute of Biology, Faculty of Biology and Biotechnology, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland; (M.Ż.); (M.M.); (M.D.K.)
| | - Mateusz Matuszkiewicz
- Department of Plant Genetics, Breeding and Biotechnology, Institute of Biology, Faculty of Biology and Biotechnology, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland; (M.Ż.); (M.M.); (M.D.K.)
| | - Magdalena Święcicka
- Department of Botany, Institute of Biology, Faculty of Biology and Biotechnology, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland; (M.Ś.); (W.K.); (M.S.)
| | - Wojciech Kurek
- Department of Botany, Institute of Biology, Faculty of Biology and Biotechnology, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland; (M.Ś.); (W.K.); (M.S.)
| | - Jarosław Olszewski
- Veterinary Research Centre, Centre for Biomedicine Research, Centre for Regenerative Medicine, Department of Large Animal Diseases and Clinic, Institute for Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 100, 02-797 Warsaw, Poland;
| | - Marek Daniel Koter
- Department of Plant Genetics, Breeding and Biotechnology, Institute of Biology, Faculty of Biology and Biotechnology, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland; (M.Ż.); (M.M.); (M.D.K.)
| | - Douglas Lamont
- ‘FingerPrints’ Proteomics Facility, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK;
| | - Mirosław Sobczak
- Department of Botany, Institute of Biology, Faculty of Biology and Biotechnology, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland; (M.Ś.); (W.K.); (M.S.)
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19
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Owusu Adjei M, Xiang Y, He Y, Zhou X, Mao M, Liu J, Hu H, Luo J, Zhang H, Feng L, Yang W, Li X, Ma J. Adventitious root primordia formation and development in the stem of Ananas comosus var. bracteatus slip. PLANT SIGNALING & BEHAVIOR 2021; 16:1949147. [PMID: 34288829 PMCID: PMC8525929 DOI: 10.1080/15592324.2021.1949147] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/18/2021] [Revised: 06/24/2021] [Accepted: 06/24/2021] [Indexed: 06/13/2023]
Abstract
There are about 4-6 slips on a fruit, and they are good materials for effective regeneration of Ananas comosus var. bracteatus. Adventitious root (AR) induction is essential for the propagation of Ananas comosus var. bracteatus slips. Growth regulator treatment, and culture medium are imperative factors that affect slip growth and rooting. In order to screen the optimal methods for slips rooting and reveal the anatomic procedure of slip rooting, this study induced slip rooting by different treatment of growth regulator, culture medium, observed the slip stem structure, AR origination and formation procedure through paraffin sections. The results showed that, slip cuttings treated with 100 mg/L of Aminobenzotriazole (ABT) for 6 hrs, cultured in river sand: coconut chaff: garden soil 2:2:1 medium is the optimal method for rooting. The proper supplementary of ABT can enhance the soluble sugar content, soluble protein content, polyphenol oxidase (PPO) activity and peroxidase (POD) enzyme activity, which resulted in the improvement of rooting. The slip stem structure is quite different from other monocots, which consists of epidermis, cortex, and stele with vascular tissues distributed in the cortex and stele. The AR primordia originates from the parenchyma cells located on the borderline between the cortex and stele. The vascular tissues in the AR develop and are connected with vascular tissue of the stem before the AR grew out the stem. The number of primary xylem poles in AR is about 30.
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Affiliation(s)
- Mark Owusu Adjei
- College of Landscape Architecture-Sichuan Agricultural University, Chengdu- Sichuan, China
| | - Yixuan Xiang
- College of Landscape Architecture-Sichuan Agricultural University, Chengdu- Sichuan, China
| | - Yehua He
- Horticultural Biotechnology College of South China Agricultural University, Guangdong, China
| | - Xuzixin Zhou
- College of Landscape Architecture-Sichuan Agricultural University, Chengdu- Sichuan, China
| | - Meiqin Mao
- College of Landscape Architecture-Sichuan Agricultural University, Chengdu- Sichuan, China
| | - Jiawen Liu
- College of Landscape Architecture-Sichuan Agricultural University, Chengdu- Sichuan, China
| | - Hao Hu
- College of Landscape Architecture-Sichuan Agricultural University, Chengdu- Sichuan, China
| | - Jiaheng Luo
- College of Landscape Architecture-Sichuan Agricultural University, Chengdu- Sichuan, China
| | - Huiling Zhang
- College of Landscape Architecture-Sichuan Agricultural University, Chengdu- Sichuan, China
| | - Lijun Feng
- College of Landscape Architecture-Sichuan Agricultural University, Chengdu- Sichuan, China
| | - Wei Yang
- College of Landscape Architecture-Sichuan Agricultural University, Chengdu- Sichuan, China
| | - Xi Li
- College of Landscape Architecture-Sichuan Agricultural University, Chengdu- Sichuan, China
| | - Jun Ma
- College of Landscape Architecture-Sichuan Agricultural University, Chengdu- Sichuan, China
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20
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Gogolev YV, Ahmar S, Akpinar BA, Budak H, Kiryushkin AS, Gorshkov VY, Hensel G, Demchenko KN, Kovalchuk I, Mora-Poblete F, Muslu T, Tsers ID, Yadav NS, Korzun V. OMICs, Epigenetics, and Genome Editing Techniques for Food and Nutritional Security. PLANTS (BASEL, SWITZERLAND) 2021; 10:1423. [PMID: 34371624 PMCID: PMC8309286 DOI: 10.3390/plants10071423] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/13/2021] [Revised: 06/30/2021] [Accepted: 07/07/2021] [Indexed: 12/22/2022]
Abstract
The incredible success of crop breeding and agricultural innovation in the last century greatly contributed to the Green Revolution, which significantly increased yields and ensures food security, despite the population explosion. However, new challenges such as rapid climate change, deteriorating soil, and the accumulation of pollutants require much faster responses and more effective solutions that cannot be achieved through traditional breeding. Further prospects for increasing the efficiency of agriculture are undoubtedly associated with the inclusion in the breeding strategy of new knowledge obtained using high-throughput technologies and new tools in the future to ensure the design of new plant genomes and predict the desired phenotype. This article provides an overview of the current state of research in these areas, as well as the study of soil and plant microbiomes, and the prospective use of their potential in a new field of microbiome engineering. In terms of genomic and phenomic predictions, we also propose an integrated approach that combines high-density genotyping and high-throughput phenotyping techniques, which can improve the prediction accuracy of quantitative traits in crop species.
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Affiliation(s)
- Yuri V. Gogolev
- Federal Research Center Kazan Scientific Center of Russian Academy of Sciences, Kazan Institute of Biochemistry and Biophysics, 420111 Kazan, Russia;
- Federal Research Center Kazan Scientific Center of Russian Academy of Sciences, Laboratory of Plant Infectious Diseases, 420111 Kazan, Russia;
| | - Sunny Ahmar
- Institute of Biological Sciences, University of Talca, 1 Poniente 1141, Talca 3460000, Chile; (S.A.); (F.M.-P.)
| | | | - Hikmet Budak
- Montana BioAg Inc., Missoula, MT 59802, USA; (B.A.A.); (H.B.)
| | - Alexey S. Kiryushkin
- Laboratory of Cellular and Molecular Mechanisms of Plant Development, Komarov Botanical Institute of the Russian Academy of Sciences, 197376 Saint Petersburg, Russia; (A.S.K.); (K.N.D.)
| | - Vladimir Y. Gorshkov
- Federal Research Center Kazan Scientific Center of Russian Academy of Sciences, Kazan Institute of Biochemistry and Biophysics, 420111 Kazan, Russia;
- Federal Research Center Kazan Scientific Center of Russian Academy of Sciences, Laboratory of Plant Infectious Diseases, 420111 Kazan, Russia;
| | - Goetz Hensel
- Centre for Plant Genome Engineering, Institute of Plant Biochemistry, Heinrich-Heine-University, 40225 Dusseldorf, Germany;
- Centre of the Region Haná for Biotechnological and Agricultural Research, Czech Advanced Technology and Research Institute, Palacký University Olomouc, 78371 Olomouc, Czech Republic
| | - Kirill N. Demchenko
- Laboratory of Cellular and Molecular Mechanisms of Plant Development, Komarov Botanical Institute of the Russian Academy of Sciences, 197376 Saint Petersburg, Russia; (A.S.K.); (K.N.D.)
| | - Igor Kovalchuk
- Department of Biological Sciences, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada; (I.K.); (N.S.Y.)
| | - Freddy Mora-Poblete
- Institute of Biological Sciences, University of Talca, 1 Poniente 1141, Talca 3460000, Chile; (S.A.); (F.M.-P.)
| | - Tugdem Muslu
- Faculty of Engineering and Natural Sciences, Sabanci University, 34956 Istanbul, Turkey;
| | - Ivan D. Tsers
- Federal Research Center Kazan Scientific Center of Russian Academy of Sciences, Laboratory of Plant Infectious Diseases, 420111 Kazan, Russia;
| | - Narendra Singh Yadav
- Department of Biological Sciences, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada; (I.K.); (N.S.Y.)
| | - Viktor Korzun
- Federal Research Center Kazan Scientific Center of Russian Academy of Sciences, Laboratory of Plant Infectious Diseases, 420111 Kazan, Russia;
- KWS SAAT SE & Co. KGaA, Grimsehlstr. 31, 37555 Einbeck, Germany
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Callegari Ferrari R, Pires Bittencourt P, Yumi Nagumo P, Silva Oliveira W, Aurineide Rodrigues M, Hartwell J, Freschi L. Developing Portulaca oleracea as a model system for functional genomics analysis of C 4/CAM photosynthesis. FUNCTIONAL PLANT BIOLOGY : FPB 2021; 48:666-682. [PMID: 33256895 DOI: 10.1071/fp20202] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/12/2020] [Accepted: 10/20/2020] [Indexed: 06/12/2023]
Abstract
Previously regarded as an intriguing photosynthetic curiosity, the occurrence of C4 and Crassulacean acid metabolism (CAM) photosynthesis within a single organism has recently emerged as a source of information for future biotechnological use. Among C4/CAM facultative species, Portulaca oleracea L. has been used as a model for biochemical and gene expression analysis of C4/CAM under field and laboratory conditions. In the present work, we focussed on developing molecular tools to facilitate functional genomics studies in this species, from the optimisation of RNA isolation protocols to a method for stable genetic transformation. Eleven variations of RNA extraction procedures were tested and compared for RNA quantity and quality. Also, 7 sample sets comprising total RNA from hormonal and abiotic stress treatments, distinct plant organs, leaf developmental stages, and subspecies were used to select, among 12 reference genes, the most stable reference genes for RT-qPCR analysis of each experimental condition. Furthermore, different explant sources, Agrobacterium tumefaciens strains, and regeneration and antibiotic selection media were tested in various combinations to optimise a protocol for stable genetic transformation of P. oleracea. Altogether, we provide essential tools for functional gene analysis in the context of C4/CAM photosynthesis, including an efficient RNA isolation method, preferred reference genes for RT-qPCR normalisation for a range of experimental conditions, and a protocol to produce P. oleracea stable transformants using A. tumefaciens.
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Affiliation(s)
- Renata Callegari Ferrari
- Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, São Paulo 05508-090, Brasil
| | - Priscila Pires Bittencourt
- Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, São Paulo 05508-090, Brasil
| | - Paula Yumi Nagumo
- Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, São Paulo 05508-090, Brasil
| | - Willian Silva Oliveira
- Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, São Paulo 05508-090, Brasil
| | - Maria Aurineide Rodrigues
- Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, São Paulo 05508-090, Brasil
| | - James Hartwell
- Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK
| | - Luciano Freschi
- Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, São Paulo 05508-090, Brasil; and Corresponding author.
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22
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Ke Y, Podio M, Conner J, Ozias-Akins P. Single-cell transcriptome profiling of buffelgrass (Cenchrus ciliaris) eggs unveils apomictic parthenogenesis signatures. Sci Rep 2021; 11:9880. [PMID: 33972603 PMCID: PMC8110759 DOI: 10.1038/s41598-021-89170-y] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2021] [Accepted: 04/15/2021] [Indexed: 12/04/2022] Open
Abstract
Apomixis, a type of asexual reproduction in angiosperms, results in progenies that are genetically identical to the mother plant. It is a highly desirable trait in agriculture due to its potential to preserve heterosis of F1 hybrids through subsequent generations. However, no major crops are apomictic. Deciphering mechanisms underlying apomixis becomes one of the alternatives to engineer self-reproducing capability into major crops. Parthenogenesis, a major component of apomixis, commonly described as the ability to initiate embryo formation from the egg cell without fertilization, also can be valuable in plant breeding for doubled haploid production. A deeper understanding of transcriptional differences between parthenogenetic and sexual or non-parthenogenetic eggs can assist with pathway engineering. By conducting laser capture microdissection-based RNA-seq on sexual and parthenogenetic egg cells on the day of anthesis, a de novo transcriptome for the Cenchrus ciliaris egg cells was created, transcriptional profiles that distinguish the parthenogenetic egg from its sexual counterpart were identified, and functional roles for a few transcription factors in promoting natural parthenogenesis were suggested. These transcriptome data expand upon previous gene expression studies and will be a resource for future research on the transcriptome of egg cells in parthenogenetic and sexual genotypes.
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Affiliation(s)
- Yuji Ke
- Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Tifton, GA, 31793, USA
| | - Maricel Podio
- Department of Horticulture, University of Georgia, Tifton, GA, 31793, USA
| | - Joann Conner
- Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Tifton, GA, 31793, USA.,Department of Horticulture, University of Georgia, Tifton, GA, 31793, USA
| | - Peggy Ozias-Akins
- Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Tifton, GA, 31793, USA. .,Department of Horticulture, University of Georgia, Tifton, GA, 31793, USA.
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23
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Biological potential of bioactive metabolites derived from fungal endophytes associated with medicinal plants. Mycol Prog 2021. [DOI: 10.1007/s11557-021-01695-8] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
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24
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Velada I, Menéndez E, Teixeira RT, Cardoso H, Peixe A. Laser Microdissection of Specific Stem-Base Tissue Types from Olive Microcuttings for Isolation of High-Quality RNA. BIOLOGY 2021; 10:biology10030209. [PMID: 33801829 PMCID: PMC7999021 DOI: 10.3390/biology10030209] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/17/2021] [Revised: 03/05/2021] [Accepted: 03/08/2021] [Indexed: 01/22/2023]
Abstract
Simple Summary Only a small portion of the stem cells participate in the process of adventitious root formation and the cells/tissues types involved in this process is species-dependent. In olive, it is still unclear which type of cells acquire competence for rooting. Regardless, the entire stem nodal segment (containing a mixture of distinct cell types) continues to be used in studies related to the molecular mechanisms underlying this process. Laser microdissection (LM) technology has been applied to isolate specific tissue and cell types. However, it is difficult to find a standard LM protocol suitable for all plant species and cell types and, thus, LM procedures must be developed and optimized for each particular tissue. In this study, we aimed to evaluate the efficiency of a LM protocol in olive microcuttings stem-base samples. This work presents a simple, rapid and efficient LM procedure for harvesting specific tissue types used for further high-quality RNA isolation. This will encourage future cell type-specific transcriptomic studies, contributing at deciphering rooting-competent cells in olive stems and to better understand the molecular mechanisms underlying the process of adventitious root formation. Abstract Higher plants are composed of different tissue and cell types. Distinct cells host different biochemical and physiological processes which is reflected in differences in gene expression profiles, protein and metabolite levels. When omics are to be carried out, the information provided by a specific cell type can be diluted and/or masked when using a mixture of distinct cells. Thus, studies performed at the cell- and tissue-type level are gaining increasing interest. Laser microdissection (LM) technology has been used to isolate specific tissue and cell types. However, this technology faces some challenges depending on the plant species and tissue type under analysis. Here, we show for the first time a LM protocol that proved to be efficient for harvesting specific tissue types (phloem, cortex and epidermis) from olive stem nodal segments and obtaining RNA of high quality. This is important for future transcriptomic studies to identify rooting-competent cells. Here, nodal segments were flash-frozen in liquid nitrogen-cooled isopentane and cryosectioned. Albeit the lack of any fixatives used to preserve samples’ anatomy, cryosectioned sections showed tissues with high morphological integrity which was comparable with that obtained with the paraffin-embedding method. Cells from the phloem, cortex and epidermis could be easily distinguished and efficiently harvested by LM. Total RNA isolated from these tissues exhibited high quality with RNA Quality Numbers (determined by a Fragment Analyzer System) ranging between 8.1 and 9.9. This work presents a simple, rapid and efficient LM procedure for harvesting specific tissue types of olive stems and obtaining high-quality RNA.
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Affiliation(s)
- Isabel Velada
- MED—Mediterranean Institute for Agriculture, Environment and Development, Institute for Advanced Studies and Research, Universidade de Évora, Pólo da Mitra, Ap. 94, 7006-554 Évora, Portugal; (E.M.); (H.C.)
- Correspondence:
| | - Esther Menéndez
- MED—Mediterranean Institute for Agriculture, Environment and Development, Institute for Advanced Studies and Research, Universidade de Évora, Pólo da Mitra, Ap. 94, 7006-554 Évora, Portugal; (E.M.); (H.C.)
| | - Rita Teresa Teixeira
- BioISI—Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisbon, 1749-016 Lisbon, Portugal;
| | - Hélia Cardoso
- MED—Mediterranean Institute for Agriculture, Environment and Development, Institute for Advanced Studies and Research, Universidade de Évora, Pólo da Mitra, Ap. 94, 7006-554 Évora, Portugal; (E.M.); (H.C.)
| | - Augusto Peixe
- MED—Mediterranean Institute for Agriculture, Environment and Development and Departamento de Fitotecnia, Escola de Ciências e Tecnologia, Universidade de Évora, Pólo da Mitra, Ap. 94, 7006-554 Évora, Portugal;
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25
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Single Cell Type Specific RNA Isolation and Gene Expression Analysis in Rice Using Laser Capture Microdissection (LCM)-Based Method. Methods Mol Biol 2021. [PMID: 33471338 DOI: 10.1007/978-1-0716-1068-8_18] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/05/2024]
Abstract
The success of single cell type-specific gene expression or functional study largely depends on the efficient isolation of high-quality RNA from them. Laser capture microdissection (LCM) is an efficient technique that allows accessing and dissecting out a specific individual cell or cell type from a microscopic heterogeneous tissue in a minimally disruptive way. Here, we describe an efficient and inexpensive LCM-based method for the extraction of RNAs with high yield and integrity from laser-microdissected mesophyll and bundle sheath cells of rice leaf. The integrity of isolated RNA is assessed with bioanalyzer analysis, and the presence of mRNA of a specific gene is validated through RT-PCR. This RNA could further be used for uncovering single cell type-specific gene expression signature using next-generation transcriptome sequence or through regular RT-PCR.
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26
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Shaw R, Tian X, Xu J. Single-Cell Transcriptome Analysis in Plants: Advances and Challenges. MOLECULAR PLANT 2021; 14:115-126. [PMID: 33152518 DOI: 10.1016/j.molp.2020.10.012] [Citation(s) in RCA: 149] [Impact Index Per Article: 37.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/11/2020] [Revised: 09/08/2020] [Accepted: 10/30/2020] [Indexed: 05/22/2023]
Abstract
The rapid and enthusiastic adoption of single-cell RNA sequencing (scRNA-seq) has demonstrated that this technology is far more than just another way to perform transcriptome analysis. It is not an exaggeration to say that the advent of scRNA-seq is revolutionizing the details of whole-transcriptome snapshots from a tissue to a cell. With this disruptive technology, it is now possible to mine heterogeneity between tissue types and within cells like never before. This enables more rapid identification of rare and novel cell types, simultaneous characterization of multiple different cell types and states, more accurate and integrated understanding of their roles in life processes, and more. However, we are only at the beginning of unlocking the full potential of scRNA-seq applications. This is particularly true for plant sciences, where single-cell transcriptome profiling is in its early stage and has many exciting challenges to overcome. In this review, we compare and evaluate recent pioneering studies using the Arabidopsis root model, which has established new paradigms for scRNA-seq studies in plants. We also explore several new and promising single-cell analysis tools that are available to those wishing to study plant development and physiology at unprecedented resolution and scale. In addition, we propose some future directions on the use of scRNA-seq technology to tackle some of the critical challenges in plant research and breeding.
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Affiliation(s)
- Rahul Shaw
- Department of Plant Systems Physiology, Institute for Water and Wetland Research, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands; Department of Biological Sciences and Centre for BioImaging Sciences, National University of Singapore, Singapore 117543, Singapore
| | - Xin Tian
- Department of Plant Systems Physiology, Institute for Water and Wetland Research, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands; Department of Biological Sciences and Centre for BioImaging Sciences, National University of Singapore, Singapore 117543, Singapore
| | - Jian Xu
- Department of Plant Systems Physiology, Institute for Water and Wetland Research, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands; Department of Biological Sciences and Centre for BioImaging Sciences, National University of Singapore, Singapore 117543, Singapore.
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27
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Román Á, Golz JF, Webb AAR, Graham IA, Haydon MJ. Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2020; 102:187-198. [PMID: 31692146 PMCID: PMC7217008 DOI: 10.1111/tpj.14603] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/02/2019] [Accepted: 10/31/2019] [Indexed: 05/28/2023]
Abstract
In multicellular organisms different types of tissues have distinct gene expression profiles associated with specific function or structure of the cell. Quantification of gene expression in whole organs or whole organisms can give misleading information about levels or dynamics of expression in specific cell types. Tissue- or cell-specific analysis of gene expression has potential to enhance our understanding of gene regulation and interactions of cell signalling networks. The Arabidopsis circadian oscillator is a gene network which orchestrates rhythmic expression across the day/night cycle. There is heterogeneity between cell and tissue types of the composition and behaviour of the oscillator. In order to better understand the spatial and temporal patterns of gene expression, flexible tools are required. By combining a Gateway®-compatible split luciferase construct with a GAL4 GFP enhancer trap system, we describe a tissue-specific split luciferase assay for non-invasive detection of spatiotemporal gene expression in Arabidopsis. We demonstrate the utility of this enhancer trap-compatible split luciferase assay (ETSLA) system to investigate tissue-specific dynamics of circadian gene expression. We confirm spatial heterogeneity of circadian gene expression in Arabidopsis leaves and describe the resources available to investigate any gene of interest.
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Affiliation(s)
- Ángela Román
- School of BioSciencesUniversity of MelbourneMelbourneAustralia
- Department of BiologyUniversity of YorkYorkUnited Kingdom
| | - John F. Golz
- School of BioSciencesUniversity of MelbourneMelbourneAustralia
| | - Alex A. R. Webb
- Department of Plant SciencesUniversity of CambridgeCambridgeUnited Kingdom
| | - Ian A. Graham
- Department of BiologyUniversity of YorkYorkUnited Kingdom
| | - Michael J. Haydon
- School of BioSciencesUniversity of MelbourneMelbourneAustralia
- Department of BiologyUniversity of YorkYorkUnited Kingdom
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28
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Gupta S, Chaturvedi P, Kulkarni MG, Van Staden J. A critical review on exploiting the pharmaceutical potential of plant endophytic fungi. Biotechnol Adv 2020; 39:107462. [DOI: 10.1016/j.biotechadv.2019.107462] [Citation(s) in RCA: 171] [Impact Index Per Article: 34.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2019] [Revised: 09/22/2019] [Accepted: 10/22/2019] [Indexed: 02/08/2023]
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29
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Chen M, Bui AQ, Goldberg RB. Using Giant Scarlet Runner Bean (Phaseolus coccineus) Embryos to Dissect the Early Events in Plant Embryogenesis. Methods Mol Biol 2020; 2122:205-222. [PMID: 31975305 DOI: 10.1007/978-1-0716-0342-0_15] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The giant embryo of the scarlet runner bean (Phaseolus coccineus) has been used historically to investigate the molecular and developmental processes that control the early events of plant embryo development. In more recent years, our laboratory has been using scarlet runner bean embryos to uncover the genes and regulatory events that control embryo proper and suspensor region differentiation shortly after fertilization. In this chapter we describe methods that we have developed to isolate scarlet runner bean embryos at the globular stage of development, and capture embryo proper and suspensor regions by either hand dissection or laser capture microdissection (LCM) for use in downstream genomic analysis. These methods are also applicable for use in investigating the early events of common bean (Phaseolus vulgaris) embryo development, a close relative of scarlet runner bean, which also has a giant embryo in addition to a sequenced genome.
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Affiliation(s)
- Min Chen
- Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA, USA
| | - Anhthu Q Bui
- Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA, USA.,Inviata Ltd., Research Triangle Park, Morrisville, NC, USA
| | - Robert B Goldberg
- Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA, USA.
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30
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Laser Microdissection as a Useful Tool to Study Gene Expression in Plant and Fungal Partners in AM Symbiosis. Methods Mol Biol 2020; 2146:171-184. [PMID: 32415603 DOI: 10.1007/978-1-0716-0603-2_13] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Laser microdissection (LMD) technology has been widely applied to plant tissues, offering novel information on the role of different cell-type populations during plant-microbe interactions. In this chapter, protocols to apply the LMD approach to study plant and fungal transcript profiles in different cell-type populations from arbuscular mycorrhizal (AM) roots are described in detail, starting from the biological material preparation to gene expression analyses by RT-PCR and RT-qPCR.
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31
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Giolai M, Verweij W, Lister A, Heavens D, Macaulay I, Clark MD. Spatially resolved transcriptomics reveals plant host responses to pathogens. PLANT METHODS 2019; 15:114. [PMID: 31624491 PMCID: PMC6785889 DOI: 10.1186/s13007-019-0498-5] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/31/2019] [Accepted: 09/27/2019] [Indexed: 05/04/2023]
Abstract
BACKGROUND Thorough understanding of complex model systems requires the characterisation of processes in different cell types of an organism. This can be achieved with high-throughput spatial transcriptomics at a large scale. However, for plant model systems this is still challenging as suitable transcriptomics methods are sparsely available. Here we present GaST-seq (Grid-assisted, Spatial Transcriptome sequencing), an easy to adopt, micro-scale spatial-transcriptomics workflow that allows to study expression profiles across small areas of plant tissue at a fraction of the cost of existing sequencing-based methods. RESULTS We compare the GaST-seq method with widely used library preparation methods (Illumina TruSeq). In spatial experiments we show that the GaST-seq method is sensitive enough to identify expression differences across a plant organ. We further assess the spatial transcriptome response of Arabidopsis thaliana leaves exposed to the bacterial molecule flagellin-22, and show that with eukaryotic (Albugo laibachii) infection both host and pathogen spatial transcriptomes are obtained. CONCLUSION We show that our method can be used to identify known, rapidly flagellin-22 elicited genes, plant immune response pathways to bacterial attack and spatial expression patterns of genes associated with these pathways.
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Affiliation(s)
- Michael Giolai
- John Innes Centre, Norwich Research Park, Norwich, UK
- Earlham Institute, Norwich Research Park, Norwich, UK
| | - Walter Verweij
- Earlham Institute, Norwich Research Park, Norwich, UK
- Enza Zaden, Enkhuizen, NL Netherlands
| | | | | | - Iain Macaulay
- Earlham Institute, Norwich Research Park, Norwich, UK
| | - Matthew D. Clark
- Earlham Institute, Norwich Research Park, Norwich, UK
- Natural History Museum, London, UK
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32
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Schlechter RO, Miebach M, Remus-Emsermann MN. Driving factors of epiphytic bacterial communities: A review. J Adv Res 2019; 19:57-65. [PMID: 31341670 PMCID: PMC6630024 DOI: 10.1016/j.jare.2019.03.003] [Citation(s) in RCA: 102] [Impact Index Per Article: 17.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2018] [Revised: 03/12/2019] [Accepted: 03/13/2019] [Indexed: 12/29/2022] Open
Abstract
Bacteria establish complex, compositionally consistent communities on healthy leaves. Ecological processes such as dispersal, diversification, ecological drift, and selection as well as leaf surface physicochemistry and topology impact community assembly. Since the leaf surface is an oligotrophic environment, species interactions such as competition and cooperation may be major contributors to shape community structure. Furthermore, the plant immune system impacts on microbial community composition, as plant cells respond to bacterial molecules and shape their responses according to the mixture of molecules present. Such tunability of the plant immune network likely enables the plant host to differentiate between pathogenic and non-pathogenic colonisers, avoiding costly immune responses to non-pathogenic colonisers. Plant immune responses are either systemically distributed or locally confined, which in turn affects the colonisation pattern of the associated microbiota. However, how each of these factors impacts the bacterial community is unclear. To better understand this impact, bacterial communities need to be studied at a micrometre resolution, which is the scale that is relevant to the members of the community. Here, current insights into the driving factors influencing the assembly of leaf surface-colonising bacterial communities are discussed, with a special focus on plant host immunity as an emerging factor contributing to bacterial leaf colonisation.
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Affiliation(s)
- Rudolf O. Schlechter
- School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
- Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand
| | - Moritz Miebach
- School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
| | - Mitja N.P. Remus-Emsermann
- School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
- Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand
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33
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Olsen S, Krause K. A rapid preparation procedure for laser microdissection-mediated harvest of plant tissues for gene expression analysis. PLANT METHODS 2019; 15:88. [PMID: 31388345 PMCID: PMC6676614 DOI: 10.1186/s13007-019-0471-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/07/2019] [Accepted: 07/26/2019] [Indexed: 05/31/2023]
Abstract
BACKGROUND Gene expression changes that govern essential biological processes can occur at the cell-specific level. To gain insight into such events, laser microdissection is applied to cut out specific cells or tissues from which RNA for gene expression analysis is isolated. However, the preparation of plant tissue sections for laser microdissection and subsequent RNA isolation usually involves fixation and embedding, processes that are often time-consuming and can lower the yield and quality of isolated RNA. RESULTS Infection sites of the parasitic plant Cuscuta reflexa growing on its compatible host plant Pelargonium zonale were sectioned using a vibratome and dried on glass slides at 4 °C before laser microdissection. High quality RNA (RQI > 7) was isolated from 1 mm2, 3 mm2 and 6 mm2 total surface areas of laser microdissection-harvested C. reflexa tissue, with the yield of RNA correlating to the amount of collected material (on average 7 ng total RNA/mm2). The expression levels of two parasite genes previously found to be highly expressed during host plant infection were shown to differ individually between specific regions of the infection site. By drying plant sections under low pressure to reduce the dehydration time, the induced expression of two wound-related genes during preparation was avoided. CONCLUSIONS Plants can be prepared quickly and easily for laser microdissection by direct sectioning of fresh tissue followed by dehydration on glass slides. We show that RNA isolated from material treated in this manner maintains high quality and enables the investigation of differential gene expression at a high morphological resolution.
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Affiliation(s)
- Stian Olsen
- Department of Arctic and Marine Biology, Faculty of Biosciences, Fisheries and Economics, UiT The Arctic University of Norway, Framstredet 39, 9019 Tromsø, Norway
| | - Kirsten Krause
- Department of Arctic and Marine Biology, Faculty of Biosciences, Fisheries and Economics, UiT The Arctic University of Norway, Framstredet 39, 9019 Tromsø, Norway
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34
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Hua L, Hibberd JM. An optimized protocol for isolation of high-quality RNA through laser capture microdissection of leaf material. PLANT DIRECT 2019; 3:e00156. [PMID: 31468025 PMCID: PMC6710646 DOI: 10.1002/pld3.156] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/23/2019] [Revised: 07/11/2019] [Accepted: 07/17/2019] [Indexed: 05/07/2023]
Abstract
Laser Capture Microdissection is a powerful tool that allows thin slices of specific cell types to be separated from one another. However, the most commonly used protocol, which involves embedding tissue in paraffin wax, results in severely degraded RNA. Yields from low abundance cell types of leaves are particularly compromised. We reasoned that the relatively high temperature used for sample embedding, and aqueous conditions associated with sample preparation prior to microdissection contribute to RNA degradation. Here, we describe an optimized procedure to limit RNA degradation that is based on the use of low-melting-point wax as well as modifications to sample preparation prior to dissection, and isolation of paradermal, rather than transverse sections. Using this approach, high-quality RNA suitable for down-stream applications such as quantitative reverse transcriptase-polymerase chain reactions or RNA-sequencing is recovered from microdissected bundle sheath strands and mesophyll cells of leaf tissue.
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Affiliation(s)
- Lei Hua
- Department of Plant SciencesUniversity of CambridgeCambridgeUK
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35
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Loganathan J, Pandey R, Ambhore NS, Borowicz P, Sathish V. Laser-capture microdissection of murine lung for differential cellular RNA analysis. Cell Tissue Res 2019; 376:425-432. [PMID: 30710174 PMCID: PMC6534428 DOI: 10.1007/s00441-019-02995-y] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2018] [Accepted: 01/15/2019] [Indexed: 12/11/2022]
Abstract
The lung tissue contains a heterogeneous milieu of bronchioles, epithelial, airway smooth muscle (ASM), alveolar, and immune cell types. Healthy bronchiole comprises epithelial cells surrounded by ASM cells and helps in normal respiration. In contrast, airway remodeling, or plasticity, increases surrounding of bronchial epithelium during inflammation, especially in asthmatic condition. Given the profound functional difference between ASM, epithelial, and other cell types in the lung, it is imperative to separate and isolate different cell types of lungs for genomics, proteomics, and molecular analysis, which will improve the diagnostic and therapeutic approach to treat cell-specific lung disorders. Laser capture microdissection (LCM) is the technique generally used for the isolation of specific cell populations under direct visual inspection, which plays a crucial role to evaluate cell-specific effect in clinical and preclinical setup. However, maintenance of tissue RNA quality and integrity in LCM studies are very challenging tasks. It is obvious to believe that the major factor affecting the RNA quality is tissue-fixation method. The prime focus of this study was to address the RNA quality factors within the lung tissue using the different solvent system to fix tissue sample to obtain high-quality RNA. Paraformaldehyde and Carnoy's solutions were used for fixing the lung tissue and compared RNA integrity in LCM captured lung tissue samples. To further confirm the quality of RNA, we measured cellular marker genes in collected lung tissue samples from control and mixed allergen (MA)-induced asthmatic mouse model using qRT-PCR technique. RNA integrity number showed a significantly better quality of RNA in lung tissue samples fixed with Carnoy's solution compared to paraformaldehyde solution. Isolated RNA from MA-induced asthmatic murine lung epithelium, smooth muscle, and granulomatous foci using LCM showed a significant increase in remodeling gene expression compared to control which confirm the quality and integrity of isolated RNA. Overall, the study concludes tissue fixation solvent can alter the quality of RNA in the lung and the outcome of the results.
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Affiliation(s)
- Jagadish Loganathan
- Department of Pharmaceutical Sciences, School of Pharmacy, North Dakota State University, Sudro Hall, Room 203, Fargo, ND, 58108-6050, USA
| | - Roshni Pandey
- Department of Pharmaceutical Sciences, School of Pharmacy, North Dakota State University, Sudro Hall, Room 203, Fargo, ND, 58108-6050, USA
| | - Nilesh Sudhakar Ambhore
- Department of Pharmaceutical Sciences, School of Pharmacy, North Dakota State University, Sudro Hall, Room 203, Fargo, ND, 58108-6050, USA
| | - Pawel Borowicz
- Department of Animal Sciences, North Dakota State University, Fargo, ND, USA
| | - Venkatachalem Sathish
- Department of Pharmaceutical Sciences, School of Pharmacy, North Dakota State University, Sudro Hall, Room 203, Fargo, ND, 58108-6050, USA.
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Verma S, Gautam V, Sarkar AK. Improved laser capture microdissection (LCM)-based method for isolation of RNA, including miRNA and expression analysis in woody apple bud meristem. PLANTA 2019; 249:2015-2020. [PMID: 30976910 DOI: 10.1007/s00425-019-03127-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/29/2018] [Accepted: 03/04/2019] [Indexed: 06/09/2023]
Abstract
Isolation of high-quality RNA, including miRNA, from microscopic woody apple bud meristem using laser capture microdissection-based method. It is often challenging to study the expression of microRNAs (miRNAs) or genes in less accessible inner tissues of tree species rich in polyphenols or polysaccharides. Here, we report a laser capture microdissection (LCM)-based method for efficient and cost-effective isolation and expression analysis of miRNAs and genes in the meristem tissue of woody apple bud. The tissue fixation, processing, infiltration, and sectioning steps were optimized for LCM-based excision and subsequent RNA isolation. Further, we have confirmed that RNA isolated from LCM-derived apple bud meristem contained miRNAs and was of good quantity and quality, sufficient for downstream expression analysis.
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Affiliation(s)
- Swati Verma
- National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, 110067, India
| | - Vibhav Gautam
- National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, 110067, India
- Centre of Experimental Medicine and Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India
| | - Ananda K Sarkar
- National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, 110067, India.
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Brukhin V, Baskar R. A brief note on genes that trigger components of apomixis. J Biosci 2019; 44:45. [PMID: 31180058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/09/2023]
Abstract
Apomixis or asexual reproduction through seeds occurs in about 400 species of flowering plants producing genetically uniform progeny. During apomixis, meiosis is bypassed and embryos develop by parthenogenesis. However, the endosperm could form either autonomously without fertilization or sexually, depending on the plant species. Most probably, a heterochronic expression of sexually expressed genes is one of the reason that causes apomixis. A better understanding of the genetic components regulating apomixis is important for developmental and evolutionary studies and also for engineering apomixis traits into crop plants that may realize a possibility to propagate hybrid vigor in a range of subsequent generations.
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Affiliation(s)
- Vladimir Brukhin
- Dobzhansky Center for Genome Bioinformatics, St. Petersburg State University, 41 Sredniy Prospekt, Vasilievsky Island, Saint Petersburg, Russia 199004
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Rodriguez-Villalon A, Brady SM. Single cell RNA sequencing and its promise in reconstructing plant vascular cell lineages. CURRENT OPINION IN PLANT BIOLOGY 2019; 48:47-56. [PMID: 31071514 DOI: 10.1016/j.pbi.2019.04.002] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/09/2019] [Revised: 04/01/2019] [Accepted: 04/02/2019] [Indexed: 06/09/2023]
Abstract
In the last decade, recent advances in single-cell RNA sequencing coupled with computational algorithms have opened new avenues to study the cell type composition of tissues and organs as well as to infer cell developmental trajectories. These technologies have been used to resolve and map atlases of tissues and organs in many animal species as well as to further order cell developmental trajectories. Despite these advances in animals, many of the current plant cell type expression profiles confound multiple developmental stages preventing an accurate monitoring of cell lineage. In this review, we propose how the application of single-cell sequencing will improve our molecular understanding of cell type differentiation. Using root vascular cells as a model, we highlight the potential of single cell transcriptomics as well as its limitations to monitor the progression of vascular maturation. By comparing cell morphology, functionality and gene expression, we aim to provide a new perspective of plant cell type differentiation.
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Affiliation(s)
| | - Siobhan M Brady
- Department of Plant Biology and the Genome Center, University of California, Davis, CA 95616, USA.
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Gao P, Xiang D, Quilichini TD, Venglat P, Pandey PK, Wang E, Gillmor CS, Datla R. Gene expression atlas of embryo development in Arabidopsis. PLANT REPRODUCTION 2019; 32:93-104. [PMID: 30762127 DOI: 10.1007/s00497-019-00364-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/16/2018] [Accepted: 02/01/2019] [Indexed: 05/24/2023]
Abstract
Embryogenesis represents a critical phase in the life cycle of flowering plants. Here, we characterize transcriptome landscapes associated with key stages of embryogenesis by combining an optimized method for the isolation of developing Arabidopsis embryos with high-throughput RNA-seq. The resulting RNA-seq datasets identify distinct overlapping patterns of gene expression, as well as temporal shifts in gene activity across embryogenesis. Network analysis revealed stage-specific and multi-stage gene expression clusters and biological functions associated with key stages of embryo development. Methylation-related gene expression was associated with early- and middle-stage embryos, initiation of photosynthesis components with the late embryogenesis stage, and storage/energy-related protein activation with late and mature embryos. These results provide a comprehensive understanding of transcriptome programming in Arabidopsis embryogenesis and identify modules of gene expression corresponding to key stages of embryo development. This dataset and analysis are a unique resource to advance functional genetic analysis of embryo development in plants.
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Affiliation(s)
- Peng Gao
- Aquatic and Crop Resource Development, National Research Council Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada
- Global Institute for Food Security, University of Saskatchewan, Saskatoon, SK, S7N 4J8, Canada
| | - Daoquan Xiang
- Aquatic and Crop Resource Development, National Research Council Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada
| | - Teagen D Quilichini
- Aquatic and Crop Resource Development, National Research Council Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada
| | - Prakash Venglat
- Aquatic and Crop Resource Development, National Research Council Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada
- Department of Plant Sciences and Crop Development Centre, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, S7N 5A8, Canada
| | - Prashant K Pandey
- Aquatic and Crop Resource Development, National Research Council Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada
| | - Edwin Wang
- Center for Health Genomics and Informatics, University of Calgary Cumming School of Medicine, Calgary, AB, T2N 4N1, Canada
| | - C Stewart Gillmor
- Laboratorio Nacional de Genómica para la Biodiversidad (Langebio), Unidad de Genómica Avanzada, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Irapuato, Guanajuato, México
| | - Raju Datla
- Aquatic and Crop Resource Development, National Research Council Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada.
- Global Institute for Food Security, University of Saskatchewan, Saskatoon, SK, S7N 4J8, Canada.
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Wang HLV, Chekanova JA. An Overview of Methodologies in Studying lncRNAs in the High-Throughput Era: When Acronyms ATTACK! Methods Mol Biol 2019; 1933:1-30. [PMID: 30945176 PMCID: PMC6684206 DOI: 10.1007/978-1-4939-9045-0_1] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The discovery of pervasive transcription in eukaryotic genomes provided one of many surprising (and perhaps most surprising) findings of the genomic era and led to the uncovering of a large number of previously unstudied transcriptional events. This pervasive transcription leads to the production of large numbers of noncoding RNAs (ncRNAs) and thus opened the window to study these diverse, abundant transcripts of unclear relevance and unknown function. Since that discovery, recent advances in high-throughput sequencing technologies have identified a large collection of ncRNAs, from microRNAs to long noncoding RNAs (lncRNAs). Subsequent discoveries have shown that many lncRNAs play important roles in various eukaryotic processes; these discoveries have profoundly altered our understanding of the regulation of eukaryotic gene expression. Although the identification of ncRNAs has become a standard experimental approach, the functional characterization of these diverse ncRNAs remains a major challenge. In this chapter, we highlight recent progress in the methods to identify lncRNAs and the techniques to study the molecular function of these lncRNAs and the application of these techniques to the study of plant lncRNAs.
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Affiliation(s)
- Hsiao-Lin V Wang
- Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning, Guangxi, China
- Present address: Department of Biology, Emory University, Atlanta, GA, USA
| | - Julia A Chekanova
- Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning, Guangxi, China.
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Kivivirta K, Herbert D, Lange M, Beuerlein K, Altmüller J, Becker A. A protocol for laser microdissection (LMD) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms. PLANT METHODS 2019; 15:151. [PMID: 31889976 PMCID: PMC6913016 DOI: 10.1186/s13007-019-0536-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/21/2019] [Accepted: 12/02/2019] [Indexed: 05/11/2023]
Abstract
BACKGROUND Plant development is controlled by the action of many, often connected gene regulatory networks. Differential gene expression controlled by internal and external cues is a major driver of growth and time specific differentiation in plants. Transcriptome analysis is the state-of-the-art method to detect spatio-temporal changes in gene expression during development. Monitoring changes in gene expression at early stages or in small plant organs and tissues requires an accurate technique of tissue isolation, which subsequently results in RNA of sufficient quality and quantity. Laser-microdissection enables such accurate dissection and collection of desired tissue from sectioned material at a microscopic level for RNA extraction and subsequent downstream analyses, such as transcriptome, proteome, genome or miRNA. RESULTS A protocol for laser-microdissection, RNA extraction and RNA-seq was optimized and verified for three distant angiosperm species: Arabidopsis thaliana (Brassicaceae), Oryza sativa (Poaceae) and Eschscholzia californica (Papaveraceae). Previously published protocols were improved in processing speed by reducing the vacuum intensity and incubation time during tissue fixation and incubation time and cryoprotection and by applying adhesive tape. The sample preparation and sectioning of complex and heterogenous flowers produced adequate histological quality and subsequent RNA extraction from micro-dissected gynoecia reliably generated samples of sufficient quality and quantity on all species for RNA-seq. Expression analysis of growth stage specific A. thaliana and O. sativa transcriptomes showed distinct patterns of expression of chromatin remodelers on different time points of gynoecium morphogenesis from the initiation of development to post-meiotic stages. CONCLUSION Here we describe a protocol for plant tissue preparation, cryoprotection, cryo-sectioning, laser microdissection and RNA sample preparation for Illumina sequencing of complex plant organs from three phyletically distant plant species. We are confident that this approach is widely applicable to other plant species to enable transcriptome analysis with high spatial resolution in non-model plant species. The protocol is rapid, produces high quality sections of complex organs and results in RNA of adequate quality well suited for RNA-seq approaches. We provide detailed description of each stage of sample preparation with the quality and quantity measurements as well as an analysis of generated transcriptomes.
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Affiliation(s)
- Kimmo Kivivirta
- Institute of Botany, Justus-Liebig-University Gießen, Heinrich-Buff-Ring 38, 35392 Gießen, Germany
| | - Denise Herbert
- Institute of Botany, Justus-Liebig-University Gießen, Heinrich-Buff-Ring 38, 35392 Gießen, Germany
| | - Matthias Lange
- Institute of Botany, Justus-Liebig-University Gießen, Heinrich-Buff-Ring 38, 35392 Gießen, Germany
- Present Address: Freelance Trial Monitor and Manager for Non-Interventional Studies, Grolmanstr. 22, 10623 Berlin, Germany
| | - Knut Beuerlein
- Rudolph-Buchheim-Institute of Pharmacology, Justus-Liebig-University Gießen, Schubertstraße 81, 35392 Gießen, Germany
| | - Janine Altmüller
- Cologne Center for Genomics (CCG), University of Cologne, Weyertal 115b, 50931 Köln, Germany
| | - Annette Becker
- Institute of Botany, Justus-Liebig-University Gießen, Heinrich-Buff-Ring 38, 35392 Gießen, Germany
- Rudolph-Buchheim-Institute of Pharmacology, Justus-Liebig-University Gießen, Schubertstraße 81, 35392 Gießen, Germany
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Shen XJ, Wang YY, Zhang YX, Guo W, Jiao YQ, Zhou XA. Overexpression of the Wild Soybean R2R3-MYB Transcription Factor GsMYB15 Enhances Resistance to Salt Stress and Helicoverpa Armigera in Transgenic Arabidopsis. Int J Mol Sci 2018; 19:E3958. [PMID: 30544851 PMCID: PMC6321161 DOI: 10.3390/ijms19123958] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2018] [Revised: 12/04/2018] [Accepted: 12/05/2018] [Indexed: 11/16/2022] Open
Abstract
Plant R2R3-MYB transcription factors (TFs) have been suggested to play crucial roles in the response to diverse abiotic and biotic stress factors but there is little molecular evidence of this role in soybean plants. In this work, we identified and functionally characterized an R2R3-MYB TF, namely, GsMYB15, from the wild soybean ED059. Protein and promoter sequence analysis indicated that GsMYB15 is a typical R2R3-MYB TF and contains multiple stress-related cis-elements in the promoter region. GsMYB15 is located in the nucleus and exhibits transcriptional activation activity. QPCR assays suggested that the expression of GsMYB15 could be induced by NaCl, insect attacks and defense-related hormones (MeJA and SA). Furthermore, GsMYB15 exhibited highest expression in pods compared to other tissues. Functional analysis of GsMYB15 demonstrated that overexpression of GsMYB15 could increase salt tolerance and enhance the resistance to H. armigera larvae in transgenic Arabidopsis plants. Moreover, overexpression of GsMYB15 also affected the expression levels of salt stress- and defense-related genes in the transgenic plants. Feeding with transgenic Arabidopsis plant leaves could significantly suppress the expression levels of immunity-related genes in H. armigera larvae. Overexpression of GsMYB15 also increased mesophyll cell levels in transgenic plants. Taken together, these results provide evidence that GsMYB15 is a positive regulator of salt stress tolerance and insect resistance in transformed Arabidopsis plants.
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Affiliation(s)
- Xin-Jie Shen
- Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China.
| | - Yan-Yan Wang
- Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China.
- Graduate School of the Chinese Academy of Agricultural Science, Beijing 100081, China.
| | - Yong-Xing Zhang
- Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China.
| | - Wei Guo
- Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China.
| | - Yong-Qing Jiao
- Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China.
| | - Xin-An Zhou
- Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China.
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Shunmugam ASK, Bollina V, Dukowic-Schulze S, Bhowmik PK, Ambrose C, Higgins JD, Pozniak C, Sharpe AG, Rozwadowski K, Kagale S. MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat. BMC PLANT BIOLOGY 2018; 18:293. [PMID: 30463507 PMCID: PMC6249822 DOI: 10.1186/s12870-018-1514-z] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/15/2018] [Accepted: 10/31/2018] [Indexed: 05/21/2023]
Abstract
BACKGROUND Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific genes and associated regulatory networks. RESULTS We describe an optimized method termed MeioCapture for simultaneous isolation of uncontaminated male meiocytes from wheat (Triticum spp.), specifically from the pre-meiotic G2 and the five sub-stages of meiotic prophase I. The MeioCapture protocol builds on the traditional anther squash technique and the capillary collection method, and involves extrusion of intact sporogenous archesporial columns (SACs) containing meiocytes. This improved method exploits the natural meiotic synchrony between anthers of the same floret, the correlation between the length of anthers and meiotic stage, and the occurrence of meiocytes in intact SACs largely free of somatic cells. The main advantage of MeioCapture, compared to previous methods, is that it allows simultaneous collection of meiocytes from different sub-stages of prophase I at a very high level of purity, through correlation of stages with anther sizes. A detailed description is provided for all steps, including the collection of tissue, isolation and size sorting of anthers, extrusion of intact SACs, and staging of meiocytes. Precautions for individual steps throughout the procedure are also provided to facilitate efficient isolation of pure meiocytes. The proof-of-concept was successfully established in wheat, and a light microscopic atlas of meiosis, encompassing all stages from pre-meiosis to telophase II, was developed. CONCLUSION The MeioCapture method provides an essential technique to study the molecular basis of chromosome pairing and exchange of genetic information in wheat, leading to strategies for manipulating meiotic recombination frequencies. The method also provides a foundation for similar studies in other crop species.
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Affiliation(s)
| | | | | | | | - Chris Ambrose
- Department of Biology, University of Saskatchewan, Saskatoon, SK Canada
| | - James D. Higgins
- Department of Genetics and Genome Biology, University of Leicester, Leicester, UK
| | - Curtis Pozniak
- Department of Plant Sciences, University of Saskatchewan, Saskatoon, Canada
| | - Andrew G. Sharpe
- National Research Council Canada, Saskatoon, SK Canada
- Global Institute for Food Security, University of Saskatchewan, Saskatoon, Canada
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Rincão MP, de Carvalho MCDCG, Nascimento LC, Lopes-Caitar VS, de Carvalho K, Darben LM, Yokoyama A, Carazzolle MF, Abdelnoor RV, Marcelino-Guimarães FC. New insights into Phakopsora pachyrhizi infection based on transcriptome analysis in planta. Genet Mol Biol 2018; 41:671-691. [PMID: 30235396 PMCID: PMC6136362 DOI: 10.1590/1678-4685-gmb-2017-0161] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2017] [Accepted: 01/02/2018] [Indexed: 11/30/2022] Open
Abstract
Asian soybean rust (ASR) is one of the most destructive diseases affecting soybeans. The causative agent of ASR, the fungus Phakopsora pachyrhizi, presents characteristics that make it difficult to study in vitro, limiting our knowledge of plant-pathogen dynamics. Therefore, this work used leaf lesion laser microdissection associated with deep sequencing to determine the pathogen transcriptome during compatible and incompatible interactions with soybean. The 36,350 generated unisequences provided an overview of the main genes and biological pathways that were active in the fungus during the infection cycle. We also identified the most expressed transcripts, including sequences similar to other fungal virulence and signaling proteins. Enriched P. pachyrhizi transcripts in the resistant (PI561356) soybean genotype were related to extracellular matrix organization and metabolic signaling pathways and, among infection structures, in amino acid metabolism and intracellular transport. Unisequences were further grouped into gene families along predicted sequences from 15 other fungi and oomycetes, including rust fungi, allowing the identification of conserved multigenic families, as well as being specific to P. pachyrhizi. The results revealed important biological processes observed in P. pachyrhizi, contributing with information related to fungal biology and, consequently, a better understanding of ASR.
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Affiliation(s)
- Michelle Pires Rincão
- Programa de Pós-Graduação em Genétiva e Biologia Molecular, Departamento de Biologia Geral, Universidade Estadual de Londrina, Londrina, PR, Brazil
- Laboratory of Plant Biotechnology and Bioinformatics, Embrapa Soja, Londrina, PR, Brazil
| | | | - Leandro Costa Nascimento
- Laboratory of Genomics and Expression (LGE), Instituto de Biologia, Universidade Estadual de Campinas (Unicamp), Campinas, SP, Brazil
| | - Valéria S. Lopes-Caitar
- Programa de Pós-Graduação em Genétiva e Biologia Molecular, Departamento de Biologia Geral, Universidade Estadual de Londrina, Londrina, PR, Brazil
- Laboratory of Plant Biotechnology and Bioinformatics, Embrapa Soja, Londrina, PR, Brazil
| | - Kenia de Carvalho
- Laboratory of Plant Biotechnology and Bioinformatics, Embrapa Soja, Londrina, PR, Brazil
| | - Luana M. Darben
- Laboratory of Plant Biotechnology and Bioinformatics, Embrapa Soja, Londrina, PR, Brazil
| | - Alessandra Yokoyama
- Laboratory of Plant Biotechnology and Bioinformatics, Embrapa Soja, Londrina, PR, Brazil
- Programa de Pós-Graduação em Biotecnologia, Universidade Estadual de Londrina, Londrina, PR, Brazil
| | - Marcelo Falsarella Carazzolle
- Laboratory of Genomics and Expression (LGE), Instituto de Biologia, Universidade Estadual de Campinas (Unicamp), Campinas, SP, Brazil
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Lavarenne J, Guyomarc'h S, Sallaud C, Gantet P, Lucas M. The Spring of Systems Biology-Driven Breeding. TRENDS IN PLANT SCIENCE 2018; 23:706-720. [PMID: 29764727 DOI: 10.1016/j.tplants.2018.04.005] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/06/2017] [Revised: 04/12/2018] [Accepted: 04/16/2018] [Indexed: 05/08/2023]
Abstract
Genetics and molecular biology have contributed to the development of rationalized plant breeding programs. Recent developments in both high-throughput experimental analyses of biological systems and in silico data processing offer the possibility to address the whole gene regulatory network (GRN) controlling a given trait. GRN models can be applied to identify topological features helping to shortlist potential candidate genes for breeding purposes. Time-series data sets can be used to support dynamic modelling of the network. This will enable a deeper comprehension of network behaviour and the identification of the few elements to be genetically rewired to push the system towards a modified phenotype of interest. This paves the way to design more efficient, systems biology-based breeding strategies.
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Affiliation(s)
- Jérémy Lavarenne
- UMR DIADE, Université de Montpellier, IRD, 911 Avenue Agropolis, 34394 Montpellier cedex 5, France; Biogemma, Centre de Recherches de Chappes, Route d'Ennezat, 63720 Chappes, France
| | - Soazig Guyomarc'h
- UMR DIADE, Université de Montpellier, IRD, 911 Avenue Agropolis, 34394 Montpellier cedex 5, France
| | - Christophe Sallaud
- Biogemma, Centre de Recherches de Chappes, Route d'Ennezat, 63720 Chappes, France
| | - Pascal Gantet
- UMR DIADE, Université de Montpellier, IRD, 911 Avenue Agropolis, 34394 Montpellier cedex 5, France.
| | - Mikaël Lucas
- UMR DIADE, Université de Montpellier, IRD, 911 Avenue Agropolis, 34394 Montpellier cedex 5, France
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Chavan S, Schnabel E, Saski C, Frugoli J. Fixation and Laser Capture Microdissection of Plant Tissue for RNA Extraction and RNASeq Library Preparation. ACTA ACUST UNITED AC 2018; 3:14-32. [PMID: 30040248 DOI: 10.1002/cppb.20063] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
In order to study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed by laser capture microdissection (LCM) and processing for RNA recovery. LCM allows the isolation of individual cell types from heterogeneous tissue sections and is particularly suited to plant processing because it does not require the breakdown of cell walls. This approach allows accurate separation of a small volume of cells that can be used to study gene expression profiles in different tissues or cell layers. The technique does not require separation of cells by enzymatic digestion of any kind, does not require cell-specific reporter genes, and allows storage of fixed and embedded tissue for months before capture. The methods for fixation, embedding, sectioning, and capture of plant cells that we describe yield high-quality RNA suitable for making libraries for RNASeq. © 2018 by John Wiley & Sons, Inc.
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Affiliation(s)
- Suchitra Chavan
- Clemson University, Department of Genetics and Biochemistry, Clemson, South Carolina
| | - Elise Schnabel
- Clemson University, Department of Genetics and Biochemistry, Clemson, South Carolina
| | - Christopher Saski
- Clemson University, Clemson University Genomics and Computational Biology Laboratory, Clemson, South Carolina
| | - Julia Frugoli
- Clemson University, Department of Genetics and Biochemistry, Clemson, South Carolina
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Wang Y, Berkowitz O, Selinski J, Xu Y, Hartmann A, Whelan J. Stress responsive mitochondrial proteins in Arabidopsis thaliana. Free Radic Biol Med 2018; 122:28-39. [PMID: 29555593 DOI: 10.1016/j.freeradbiomed.2018.03.031] [Citation(s) in RCA: 47] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/14/2017] [Revised: 03/05/2018] [Accepted: 03/16/2018] [Indexed: 12/27/2022]
Abstract
In the last decade plant mitochondria have emerged as a target, sensor and initiator of signalling cascades to a variety of stress and adverse growth conditions. A combination of various 'omic profiling approaches combined with forward and reverse genetic studies have defined how mitochondria respond to stress and the signalling pathways and regulators of these responses. Reactive oxygen species (ROS)-dependent and -independent pathways, specific metabolites, complex I dysfunction, and the mitochondrial unfolded protein response (UPR) pathway have been proposed to date. These pathways are regulated by kinases (sucrose non-fermenting response like kinase; cyclin dependent protein kinase E 1) and transcription factors from the abscisic acid-related, WRKY and NAC families. A number of independent studies have revealed that these mitochondrial signalling pathways interact with a variety of phytohormone signalling pathways. While this represents significant progress in the last decade there are more pathways to be uncovered. Post-transcriptional/translational regulation is also a likely determinant of the mitochondrial stress response. Unbiased analyses of the expression of genes encoding mitochondrial proteins in a variety of stress conditions reveal a modular network exerting a high degree of anterograde control. As abiotic and biotic stresses have significant impact on the yield of important crops such as rice, wheat and barley we will give an outlook of how knowledge gained in Arabidopsis may help to increase crop production and how emerging technologies may contribute.
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Affiliation(s)
- Yan Wang
- Department of Animal, Plant and Soil Sciences, Australian Research Council Centre of Excellence in Plant Energy Biology, School of Life Sciences, La Trobe University, Bundoora, Victoria, Australia
| | - Oliver Berkowitz
- Department of Animal, Plant and Soil Sciences, Australian Research Council Centre of Excellence in Plant Energy Biology, School of Life Sciences, La Trobe University, Bundoora, Victoria, Australia.
| | - Jennifer Selinski
- Department of Animal, Plant and Soil Sciences, Australian Research Council Centre of Excellence in Plant Energy Biology, School of Life Sciences, La Trobe University, Bundoora, Victoria, Australia
| | - Yue Xu
- Department of Animal, Plant and Soil Sciences, Australian Research Council Centre of Excellence in Plant Energy Biology, School of Life Sciences, La Trobe University, Bundoora, Victoria, Australia
| | - Andreas Hartmann
- Department of Animal, Plant and Soil Sciences, Australian Research Council Centre of Excellence in Plant Energy Biology, School of Life Sciences, La Trobe University, Bundoora, Victoria, Australia
| | - James Whelan
- Department of Animal, Plant and Soil Sciences, Australian Research Council Centre of Excellence in Plant Energy Biology, School of Life Sciences, La Trobe University, Bundoora, Victoria, Australia
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Muñoz-Sanhueza LG, Lee Y, Tillmann M, Cohen JD, Hvoslef-Eide AK. Auxin analysis using laser microdissected plant tissues sections. BMC PLANT BIOLOGY 2018; 18:133. [PMID: 29940865 PMCID: PMC6019200 DOI: 10.1186/s12870-018-1352-z] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/11/2017] [Accepted: 06/15/2018] [Indexed: 05/18/2023]
Abstract
BACKGROUND Quantitative measurement of actual auxin levels in plant tissue is complimentary to molecular methods measuring the expression of auxin related genes. Current analytical methods to quantify auxin have pushed the limit of detection to where auxin can be routinely quantified at the pictogram (pg) level, reducing the amount of tissue needed to perform these kinds of studies to amounts never imagined a few years ago. In parallel, the development of technologies like laser microdissection microscopy (LMD) has allowed specific cells to be harvested from discrete tissues without including adjacent cells. This method has gained popularity in recent years, especially for enabling a higher degree of spatial resolution in transcriptome profiling. As with other quantitative measurements, including hormone quantifications, sampling using traditional LMD is still challenging because sample preparation clearly compromises the preservation of analytes. Thus, we have developed and validated a sample preparation protocol combining cryosectioning, freeze-drying, and capturing with a laser microdissection microscope to provide high-quality and well-preserved plant materials suitable for ultrasensitive, spatially-resolved auxin quantification. RESULTS We developed a new method to provide discrete plant tissues for indole-3-acetic acid (IAA) quantification while preserving the plant tissue in the best possible condition to prevent auxin degradation. The method combines the use of cryosectioning, freeze-drying and LMD. The protocol may also be used for other applications that require small molecule analysis with high tissue-specificity where degradation of biological compounds may be an issue. It was possible to collect the equivalent to 15 mg of very specific tissue in approximately 4 h using LMD. CONCLUSIONS We have shown, by proof of concept, that freeze dried cryosections of plant tissue were suitable for LMD harvest and quantification of the phytohormone auxin using GC-MS/MS. We expect that the ability to resolve auxin levels with both spatial- and temporal resolution with high accuracy will enable experiments on complex processes, which will increase our knowledge of the many roles of auxins (and, in time, other phytohormones) in plant development.
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Affiliation(s)
- Luz G. Muñoz-Sanhueza
- Department of Plant Sciences (IPV), Faculty of Biosciences, Norwegian University of Life Sciences, Norway Campus Ås, Universitetstunet 3, 1430 Ås, Norway
| | - YeonKyeong Lee
- Department of Plant Sciences (IPV), Faculty of Biosciences, Norwegian University of Life Sciences, Norway Campus Ås, Universitetstunet 3, 1430 Ås, Norway
| | - Molly Tillmann
- Department of Horticultural Sciences, Microbial and Plant Genomics Institute, University of Minnesota, 305 Alderman Hall, 1970 Folwell Avenue, Saint Paul, MN 55108 USA
| | - Jerry D. Cohen
- Department of Horticultural Sciences, Microbial and Plant Genomics Institute, University of Minnesota, 305 Alderman Hall, 1970 Folwell Avenue, Saint Paul, MN 55108 USA
| | - Anne Kathrine Hvoslef-Eide
- Department of Plant Sciences (IPV), Faculty of Biosciences, Norwegian University of Life Sciences, Norway Campus Ås, Universitetstunet 3, 1430 Ås, Norway
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Stevens ME, Woeste KE, Pijut PM. Localized gene expression changes during adventitious root formation in black walnut (Juglans nigra L.). TREE PHYSIOLOGY 2018; 38:877-894. [PMID: 29378021 DOI: 10.1093/treephys/tpx175] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/31/2017] [Accepted: 12/20/2017] [Indexed: 05/13/2023]
Abstract
Cutting propagation plays a large role in the forestry and horticulture industries where superior genotypes need to be clonally multiplied. Integral to this process is the ability of cuttings to form adventitious roots. Recalcitrance to adventitious root development is a serious hurdle for many woody plant propagation systems including black walnut (Juglans nigra L.), an economically valuable species. The inability of black walnut to reliably form adventitious roots limits propagation of superior genotypes. Adventitious roots originate from different locations, and root induction is controlled by many environmental and endogenous factors. At the molecular level, however, the regulation of adventitious root formation is still poorly understood. In order to elucidate the transcriptional changes during adventitious root development in black walnut, we used quantitative real-time polymerase chain reaction to measure the expression of nine key genes regulating root formation in other species. Using our previously developed spatially explicit timeline of adventitious root development in black walnut softwood cuttings, we optimized a laser capture microdissection protocol to isolate RNA from cortical, phloem fiber and phloem parenchyma cells throughout adventitious root formation. Laser capture microdissection permitted high-resolution, site-specific analysis of gene expression that differentiated between participatory and non-participatory root progenitor cells. Results indicated mRNA abundance was altered in all nine rooting-related genes in response to auxin treatment in both juvenile and mature cuttings. SCARECROW LIKE-1 (SCL) had the greatest change in expression in juvenile rooting-competent cells at days 16 and 18, with a 24- and 23-fold increase relative to day 0, respectively. Tissues not linked to root organogenesis had little change in SCL expression at similar time points. AUXIN RESPONSE FACTOR (ARF)6 and ARF8 as well as SHORTROOT expression also increased 2- to 4-fold in rooting-competent tissue. The greatest transcript abundance in rooting-competent cuttings was restricted to root progenitor cells, while recalcitrant cuttings had a diffuse mRNA signal among tissue types.
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Affiliation(s)
- Micah E Stevens
- Department of Forestry and Natural Resources, Purdue University, Hardwood Tree Improvement and Regeneration Center (HTIRC), 715 West State Street, West Lafayette, IN 47907, USA
| | - Keith E Woeste
- USDA Forest Service, Northern Research Station, HTIRC, 715 West State Street, West Lafayette, IN 47907, USA
| | - Paula M Pijut
- USDA Forest Service, Northern Research Station, HTIRC, 715 West State Street, West Lafayette, IN 47907, USA
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