Source: VIRGINIA STATE UNIVERSITY submitted to
FUNCTIONAL GENOMICS OF PURSLANE DROUGHT TOLERANCE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0227307
Grant No.
(N/A)
Project No.
VAX-SREN2011
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2011
Project End Date
Sep 30, 2014
Grant Year
(N/A)
Project Director
Ren, S.
Recipient Organization
VIRGINIA STATE UNIVERSITY
(N/A)
PETERSBURG,VA 23803
Performing Department
Agriculture
Non Technical Summary
Drought is the most important abiotic constraint on crop production worldwide, especially under current global climate change conditions. In the US, economic losses due to drought stress are estimated between $6 and $8 billion a year. Due to a lack of unique genetic resources, development of genetically modified drought-tolerant crops has been unsuccessful. Purslane is a potentially useful species that grows in extreme drought conditions, however, little is known about the molecular mechanisms conferring its drought tolerance. The long term goal of this study is to elucidate mechanisms and identify unique genes regulating drought tolerance in purslane, and apply them to genetically engineer drought tolerance for crop species and/or bio-fuel production. The objectives of this proposal are 1) use next generation sequencing technique to identify unique genes regulating drought tolerance in purslane, 2) validate and examine expression patterns for the top 25 genes with enhanced expression by drought stress in both control and drought treatment libraries, and 3) Over-express two unique genes and evaluate their functions in drought tolerance in Arabidopsis thaliana. We propose to use the FLX Titanium sequencing technique to deep sequence both control and drought-treated purslane samples, establish ESTs contigs, and identify novel genes expressed during drought stress. Arabidopsis thaliana will be used as a model to study the functions of isolated genes. The project also serves as a platform for training minority students in plant biotechnology. It will significantly strengthen students' training and research capacity at VSU on biotechnology and its application in agriculture.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2012299104080%
2032420102020%
Goals / Objectives
Objective 1: Establish the expression profile of purslane being challenged by drought stress using FLX Titanium sequencing. Two cDNA libraries will be generated from purslane. Roche 454 GS-FLX Titanium sequencing will be performed. The obtained reads will be assembled and functional annotated using Blast2GO software. Contigs with ESTs expressed mostly in drought treated samples will be established. Objective 2: Validate FLX Titanium sequences and analyze gene expression patterns for the top 25 isolated genes from each cDNA library. Top 25 genes from each cDNA library will be validated using real time RT-PCR. RT-PCR will be used to confirm the sequencing results in drought and control samples and to analyze expression profiles in different tissues and developmental stages. RT-PCR will also be performed on different purslane accessions to determine the gene expression variation among different purslane accessions. Objective 3: Over-express two isolated promising genes and examine their functions on drought tolerance in Arabidopsis thaliana. The full length cDNAs of 2 promising genes will be isolated and the coding FLcDNA for each gene will be cloned behind the CaMV 35S promoter in pBI121 binary vector in replacement of GUS gene, or pCBK05 binary vector. The constructs will be transformed first into Agrobacterium tumefaciens GV3101 and then into Arabidopsis. The cDNA over-expressing lines together with controls will be evaluated for their features in drought stress responses.
Project Methods
Objective 1: Total RNAs will be extracted after drought treatment. Two cDNA libraries will be prepared and subjected to pyrosequencing using the Roche 454 GS-FLX Titanium sequencing platform. The reads will be assembled using Roche GS De Novo Assembler Software and assemblies function annotation will be performed using Blast2GO software. Contigs with ESTs expressed mostly in drought treated samples and those expressed mostly in the control will be established. Objective 2: Total RNAs will be extracted from all needed tissues and accessions. Top 25 genes from each cDNA library will be selected, and primers will be designed for each gene. Real time RT-PCR will be performed for each gene to validate its expression and responding to drought treatment. RT-PCR will also be used to analyze expression profiles in different tissues and developmental stages. In addition, RT-PCR will be performed on different purslane accessions to determine the gene expression variation among different purslane accessions for all selected genes with tubulin or EIF gene serving as a control for RNA quality and quantity. Objective 3: The full length cDNAs of 2 promising genes will be isolated by both 5 prime and 3 prime RACE methods. The coding FLcDNA for each gene will be cloned behind the CaMV 35S promoter in pBI121 binary vector in replacement of GUS gene, or pCBK05 binary vector. The constructs will be transformed first into Agrobacterium tumefaciens GV3101 and then into Arabidopsis. The cDNA over-expressing lines together with controls will be evaluated for their features in drought stress responses.

Progress 10/01/11 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Environmental stresses are key factors affecting agriculture production. Understanding mechanisms and discovering new genes that control plant stress tolerance will provide a new strategy for crop stress improvement. Previously, we discovered that the purslane accession Tokombia, originated from Eritrea, Africa, displays a unique physiological phenotype under drought stress, and thus is extremely heat and drought tolerant. However, the molecular mechanism(s) controlling its stress tolerance are not understood. The current project intends to elucidate how purslane accession tokombia tolerates drought conditions at the genomics level. Tokombia was grown in greenhouse and treated with and without water stress. Total RNAs were isolated from control, early and late drought stress treated samples, and high throughput transcriptome analysis was conducted. For each individual library, a total of 9,728, 9,919, and 8,718 contigs were assembled in the control group, early response group, and the late response group respectively. Using a quick sequence annotation tool (Mercator), analysis against NCBI databases assigned functions to approximately 45% contigs in each group. About 55% of genes in each group are of unknown function. Surprisingly, a similar amount of stress related genes were discovered in each group, with 177 in the control, 209 in the early response, and 177 in the late response. Further analysis of these stress related genes indicated that 67% in the control group, 66% in the early response, and 71% in the late response group were drought or heat related. As a whole. a total of more than 2 million reads with about 1 billion base pairs were generated, and 13,862 genes were identified. Of these, 803 genes were expressed differently among control, early and late drought stress treatments. 37 genes only expressed under drought stress while about 600 genes were completely suppressed by both drought conditions. About 130 genes were either up regulated or down regulated by drought stress. The transcriptome data were validated through qRT-PCR. 5 prime RACE were conducted to identify full length cDNAs for DRM2 and BCL2 genes. The full length cDNA for both were obtained after several rounds of RACE, and confirmed by full sequencing of the cDNAs. FlcDNAs were sub-cloned into binary vector pCBK05 with herbicide resistant marker, and it is ready to transform it into Arabidopsis. In addition, we also examined the genetic variation of arsenic tolerance among purslane accessions and identified two accessions with hypersensitive and two accessions with hyposensitive to arsenic, and an Arabidopsis homolog of ABCC2 was identified to be responsible for this arsenic tolerance in purslane.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Ren S, Weeda S and Mandadi K (2014) Establishment of a genomics tool in purslane to identify genes benefiting agriculture, environments and human health. PAG XXIII, January 2014, San Diego, CA.


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two undergraduate students were recruited and hands-on trained during the reporting period. How have the results been disseminated to communities of interest? Discoveries from this research project were presented at two international professional conferences What do you plan to do during the next reporting period to accomplish the goals? This project was aimed to use next generation sequencing technique to genome-wide identify unique genes regulating drought tolerance in purslane; validate and examine expression patterns for top 25 genes with enhanced expression by drought stress in both control and drought treatment libraries; and over-express three unique genes and evaluate their functions on drought tolerance in Arabidopsis thaliana

Impacts
What was accomplished under these goals? Environmental stresses are key factors affecting agriculture production. In Virginia the combination of heat and drought stresses caused almost 50% of yield loss for corn and peanutand 30% for soybean in 2010. Understanding mechanisms and discovering new genes that control plant stress tolerance will provide a new strategy for crop stress improvement. Previously, we discovered that the purslane accession Tokombia, originated from Eritrea, Africa, displays a unique physiological phenotype under drought stress, and thus is extremely heat and drought tolerant. However, the molecular mechanism(s) controlling its stress tolerance are not understood. The current project intends to elucidate how purslane accession tokombia tolerates drought conditions at the genomics level. In the year one of this project we isolated RNA from control, early and late drought stress treatment samples, performed FLX titanium sequencing. In the second year of the project, we performed detailed bioinformatics analysis for the sequencing data; identified unique genes that were differentially expressed upon drought stress; discovered genetic variation among purslane accessions on arsenic tolerance; and cloned a multidrug resistant gene which is responsible for arsenic tolerance in purslane. In addition, in order to get deep information about purslanetranscriptoms, we further performed large scale RNA-seq analysis using MiSeq. In our first draft of transcriptom sequence, a total of more than 2 million reads with about 1 billion base pairs were generated, and identified unique contigs (genes) of 13,862. Of these, 803 genes were expressed differently among control, early and late drought stress treatments. 37 genes only expressed under drought stress while about 600 genes were completely suppressed by both drought conditions. About 130 genes were either up regulated or down regulated by drought stress. From this pool of genes we identified several candidate genes for our further research objective, including ethylene-responsive transcription factor, DNA methyltransferase (drm2), BTB domain protein, histone h2b, bcl-2 gene and a multidrug export protein. 5 primer RACE will be conducted to identify full length cDNA for some of these candidate genes. In addition, we also examined the genetic variation of arsenic tolerance among purslane accessions and identified two accessions with hypersensitive and two accessions with hyposensitive to arsenic, and an Arabidopsis homolog of ABCC2 was identified to be responsible for this arsenic tolerance.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: American Society of Plant Biologist, July, at Rhode land Plant and Animal Genome Conference, January 2014, at San Diego, CA


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Environmental stresses adversely affect crop productivity of many crop species, causing significant economic losses. In the United States, the direct economic loss due to these environmental stresses is tens of billions of dollars each year. Both basic and applied research is needed to focus on improving crop tolerance to environmental stresses. Previously, we discovered that the purslane accession Tokombia, originating from Eritrea, Africa, displays a unique physiological phenotype under drought stress, and thus is extremely heat and drought tolerant. However, the molecular mechanism(s) controlling its stress tolerance are not understood. The current project intends to elucidate how purslane tolerates drought conditions at the genomic level. Seeds of the Tokombia accession were sown in a growth chamber at VSU's Randolph Farm under natural light conditions. Seedlings were grown under well watered conditions for two weeks, then subjected to drought treatment. Control seedlings were watered regularly so that no drought stress occurred. After two weeks of water-withholding, drought treated seedlings showed initial signs of wilting. We defined this day as Day1. Samples of whole seedlings (including root) were collected, for both the control and drought treatment, at Day 1, 3, 5, 8, 10, 12 and 15. Total RNAs were isolated and equal amounts of RNAs were pooled into early drought response (Day 1 and 3), late drought response (Day 12 and 15), and untreated control (Day 1, 3, 12, and 15). cDNAs were synthesized for all three pooled samples and were subjected to FLX Titanium sequencing. Raw sequences were assembled into respective contigs. As a result, a total of 9,728 contigs were assembled in the control group; 9,919 contigs were in the early response group, and 8,718 were obtained for the late response group. Using a quick sequence annotation tool (Mercator), analysis against NCBI databases assigned functions to approximately 45% of genes (contigs) in each group. About 55% of genes in each group are of unknown function. Surprisingly, a similar amount of stress related genes (contigs) were discovered in each group, with 177 in the control, 209 in the early response, and 177 in the late response. Further analysis of these stress related genes indicated that 67% in the control group, 66% in the early response group, and 71% in the late response group were drought or heat related. Purslane is extremely tolerant to drought and heat stresses, and is considered an extremophyte. The expression of these drought and heat related genes under non-water stressed conditions may explain, at the molecular level, why purslane can tolerate such extreme drought and heat stresses. Constitutive expression of such genes in agriculturally important crops may help to improve abiotic stress tolerance in crops. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Environmental stresses are key factors affecting agriculture production. In Virginia the combination of heat and drought stresses caused almost 50% of yield loss for corn and peanut and 30% for soybean in 2010. Understanding mechanisms and discovering new genes that control plant stress tolerance will provide a new way for crop stress improvement. Purslane is considered an extremophyte that is tolerant to severe drought and heat stresses. However, mechanisms underlying this phenomenon are not clearly understood. Our current research focuses on genomics dissection of this extremophyte under both control and drought stress conditions. This research will advance the body of knowledge on understanding how and why this extremophyte tolerates drought stress, and may provide potential new strategies for improving crop tolerance to environmental stress.

Publications

  • No publications reported this period