Progress 10/01/07 to 06/30/11
Outputs
OUTPUTS: During this reporting period, the project has resulted in two peer-reviewed book chapters (Oliver et al., 2010; Cushman and Oliver, 2011) and one journal article (Oliver et al., 2011). Several additional journal articles are in preparation and will be reported on later. One poster presentation was given by Catherine Espinosa at the 2011 American Society of Plant Biologist (ASPB) on August 6-10, 2011 in Minneapolis, MN. Two additional poster presentations were given by Abou Yobi and Sangho Kang at the Nevada Agricultural Experiment Station Ag Field Day held on September 10, 2011 in Reno, NV. Dr. Cushman delivered two invited lectures to the following audiences: Australian Center for Plant Genomics at the University of Adelaide, Adelaide, AU on July 22, 2011 and at Syngenta, Inc. in Research Triangle Park, NC on September 22, 2011. PARTICIPANTS: This work is a collaborative effort with researchers at the USDA-ARS in Columbia, MO (PI: Melvin Oliver), the division of plant sciences at the University of Missouri, Columbia (Co-PI, Robert Sharp), and USDA-ARS in Lubbock, TX (Co-PI: Paxton Payton). To date, three Ph.D. graduate students (Abou Yobi, Sangho Kang, and Catherine Espinosa) were trained as future independent scientists. A fourth Ph.D. graduate student, Bernard Wone, also assisted with the metabolite analyses in Selaginella and Sporobolus. All students are receiving advising and mentoring from the project director about their various research activities and their career development. One undergraduate student (Lina Castano-Duque) also participated in the project and completed a proteomics project with Selaginella. TARGET AUDIENCES: The project results were published in the scientific literature targeted to the scientific community, specifically to research scientists and undergraduate and graduate students and post-doctoral researcher conducting research on the molecular mechanisms of desiccation tolerance and low water input forage grasses. In addition, outreach by selected oral and poster presentations have targeted selected stakeholder groups. For example, the annual Agriculture Field Day held by the Nevada Agricultural Experiment Station was held on September 11, 2011. PROJECT MODIFICATIONS: None.
Impacts
Three different Sporobolus species, the desiccation tolerant (DT) S. stapfianus Gandoger, and two desiccation sensitive (DS) species, S. pyramidalis (also described as S. indicus var. pyramidalis), and S. fimbriatus (Trin.) Nees. (Poaceae) were evaluated for their potential as low water use forage crops by growing them under three different irrigation regimes. S. stapfianus and S. pyramidalis were not significantly affected by increased water inputs, whereas S. fimbriatus plants were responsive to increasing water. In addition, S. stapfianus had the least biomass production, averaging about three times less than S. pyramidalis and 10 times less than S. fimbriatus. Seed production was also higher in S. fimbriatus, although no clear differences were noted among the three different watering regimes. The forage value of all species contained comparable amounts of protein, tolerable amounts of fiber, and major minerals. Protein and the mineral contents investigated met or exceeded the minimum requirements of beef cattle, with the exception of zinc that appeared low in all the three species. Moreover, all mineral values measured remained below the toxicity level for beef cattle. These results combined with low water consumption (12.33 m3) indicate that these species might be useful as low-water input forage grasses in semi-arid regions of the Western U.S. including Nevada. Unbiased metabolic profiling was used to compare a desiccation sensitive (DS, Selaginella moellendorffii), with a desiccation tolerant (DT, S. lepidophylla) species within the spike or club moss family. The fully hydrated DT species accumulated more sucrose, sugar alcohols, aromatic amino acids, flavonoids, oxidized glutathione as well as a variety of gamma-glutamyl amino acid conjugates and unknown metabolites than did the fully hydrated DS species. Of the total of 302 metabolites identified in this comparative study, 136 (45%) were novel and of these, 82% showed significant differences in abundance between the two species. Within the DT species, a total of 204 metabolites were detected across all five dehydration-rehydration (wet-dry-wet) states and 118 (47%) showed significant changes in abundance in one or more of these states. Remarkably, major sugars (e.g., sucrose, glucose, and trehalose) were found to be very abundant across all stages of the dehydration-rehydration cycle and polyols showed the greatest abundance in the hydrated state. In contrast, nitrogen-rich amino acids and gamma-glutamyl amino acid conjugates accumulated to the greatest concentrations in the dried state. A comparison of protein expression patterns in dehydrated and hydrated S. lepidophylla has now been completed and about 75 different proteins were differentially expressed between the two states. Late embryogenesis abundant (LEA) and heat shock proteins were found to be especially abundant in the dried state. Lastly, functional testing of 10 highly expressed S. lepidophylla genes by ectopic expression in transgenic Arabidopsis is still ongoing. Several LEA proteins have been found to improved osmotic and drought tolerance and are being tested for their ability to confer drought tolerance.
Publications
- Oliver MJ, Cushman JC, Koster KL (2010) Dehydration Tolerance in Plants. Methods in Molecular Biology: Plant Stress Tolerance. 639: 3-24.
- Cushman JC, Oliver MJ, (2011) Understanding vegetative desiccation tolerance using integrated functional genomics approaches within a comparative evolutionary framework. In: Ecological Studies: Desiccation Tolerance in Plants. Eds: Ulrich Luttge, Erwin Beck, and Dorothea Bartels. Springer, Heidelberg. 215: 307-338.
- Oliver MJ, Guo L, Alexander DC, Ryals JA, Wone BWM, Cushman JC. (2011) A sister group metabolomic contrast using untargeted global metabolomic analysis delineates the biochemical regulation underlying desiccation tolerance in Sporobolus stapfianus. The Plant Cell. 23: 1231-1248.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves atmospheric CO2 assimilation in water-limited terrestrial and epiphytic habitats and in CO2-limited aquatic environments. In contrast with C3 and C4 plants, CAM plants take up CO2 from the atmosphere partially or predominantly at night. CAM is taxonomically widespread among vascular plants and is present in many succulent species that occupy semiarid regions, as well as in tropical epiphytes and in some aquatic macrophytes. This water-conserving photosynthetic pathway has evolved multiple times and is found in close to 6% of vascular plant species from at least 35 families. Although many aspects of CAM molecular biology, biochemistry and ecophysiology are well understood, relatively little is known about the evolutionary origins of CAM. Therefore, we recently reviewed five main topic areas that comprise our current understanding of CAM evolution: (1) the permutations and plasticity of CAM, (2) the requirements for CAM evolution, (3) the drivers of CAM evolution, (4) the prevalence and taxonomic distribution of CAM among vascular plants with emphasis on the Orchidaceae and (5) the molecular underpinnings of CAM evolution including circadian clock regulation of gene expression (Silvera et al. 2010a). As a follow up to our original investigation of CAM evolution in the Orchidaceae (Silvera et al. 2009), we published a checklist of orchids of Panama and Costa Rica that included an estimate of their ability to perform CAM based on their d13C values as well as an updated list of all known orchid genera that possess species known to perform CAM (Silvera et al. 2010b). Leaf carbon stable isotopic composition of 1,002 orchid species representing 61% of the total number of orchid species described for Panama and Costa Rica were obtained from herbarium specimens. Carbon isotopic composition of leaf material showed a bimodal distribution with modes at -28 indicating C3 photosynthesis, and at -15, indicating pronounced CAM photosynthesis. Strong CAM was present in 9.5% of species and in 33 of 163 genera studied. Twelve of these genera were not previously known to contain species exhibiting CAM. We also examined the function of CAM in reactive oxygen species (ROS) alleviation using a newly isolated CAM-deficient mutant of a facultative halophyte Mesembryanthemum crystallinum L. Salinity (0.4 M NaCl) induced nocturnal malate synthesis in the leaves of wild-type plant, but not in the mutant. The content of hydrogen peroxide (H2O2) increased over time after salinity treatment, but then declined accompanied by the expression of CAM in the wild-type plants. The CAM-performing wild-type plant grown with 0.4 M NaCl for 12 days showed large diel fluctuation of malate and lower H2O2 content than the mutant. The transcript abundance of a gene encoding Cu/Zn-SOD, a marker of ROS production, was higher in the leaves of mutant plants than in the wild type plants. These results indicated that lower levels of ROS accompanied the performance of CAM, and that CAM might alleviate oxidative stress (Sunagawa et al., 2010). PARTICIPANTS: To date, two graduate students (Katia Silvera, Bahay Gulle) were trained as future independent scientists and are receiving or received advising and mentoring from the project director about their various research activities and their career development. One lab manager (Rebecca Albion, Staff Research Associate II) has been trained on the projects in a wide range of preparative and analytical techniques related to the specific requirement of the projects. TARGET AUDIENCES: The project results and outcomes are being targeted to the scientific community at national and international scientific meetings, specifically to research scientists and undergraduate and graduate students and post-doctoral researchers conducting research on CAM functional genomics and evolution as well as the effects of CAM on reactive oxygen species alleviation. Outreach projects are also being targeted through poster presentations and oral presentations at national and international scientific meetings and poster and display presentations to the lay public. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our investigation into the evolutionary origins of CAM demonstrates several patterns of CAM evolution across the Orchidaceae, as well as several other families that contain a high prevalence of CAM species, has show that multiple independent origins of CAM exist across many taxa, including many reversal events, indicating flexible and parallel evolution of CAM across subfamilies. Ongoing investigations are revealing a complex picture of the molecular underpinnings associated with gene family evolution and recruitment to CAM. We carried out the first comparative studies of wild type and CAM-deficient mutants of M. crystallinum to demonstrate that the operation of CAM contributes to the anti-oxidant strategies of the plant. CAM expression in wild type plants was associated with lower levels of ROS in the leaves compared with those found in the CAM-deficient mutant, indicating that CAM prevents or reduces the production of ROS.
Publications
- Silvera K, Santiago LS, Cushman JC, Winter K. (2009) Crassulacean acid metabolism and epiphytism linked to adaptive radiations in the Orchidaceae. Plant Physiol. 149:1838-1847.
- Silvera K, Neubig KM, Whitten WM, Williams NH, Winter K, Cushman JC. (2010a) Evolution along the Crassulacan acid metabolism continuum. Function Plant Biology. 37: 995-1010 (Cover article).
- Silvera K, Santiago LS, Cushman JC, Winter K. (2010b) Incidence of Crassulacean acid metabolism in the Orchidaceae derived from carbon isotope ratios: a checklist of the flora of Panama and Costa Rica. Botanical Journal of the Linnean Society. 163: 194-222.
- Sunagawa H, Cushman JC, Agarie S. (2010) Crassulacean acid metabolism alleviates reactive oxygen species in the facultative CAM plant, the common ice plant, Mesembryanthemum crystallinum. Plant Production Science. 13: 246-260.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: To better understand the role of CAM and epiphytism in the evolutionary expansion of tropical orchids, we sampled leaf carbon isotopic composition of 1,103 species native to Panama and Costa Rica, performed character state reconstruction and phylogenetic trait analysis of CAM and epiphytism, and related strong CAM, present in 10% of species surveyed, with climatic variables and the evolution of epiphytism in tropical regions. Altitude was the most important predictor of photosynthetic pathway when all environmental variables were taken into account, with CAM being most prevalent at low altitudes. By creating integrated orchid trees to reconstruct ancestral character states, we found that C3 photosynthesis is the ancestral state and that CAM has evolved at least 10 independent times with several reversals. A large CAM radiation event within the Epidendroideae, the most species-rich epiphytic clade of any known plant group, is linked to a Tertiary species radiation that originated 65 million years ago (Silvera et al., 2009). The effects of long-term, seasonal water deficit on berries of Cabernet Sauvignon, a red-wine grape, and Chardonnay, a white-wine grape were analyzed by integrated transcript and metabolite profiling. Over the course of berry development, the steady-state transcript abundance of approximately 6,000 Unigenes differed significantly between the cultivars and the irrigation treatments. Water deficit most affected the phenylpropanoid, ABA, isoprenoid, carotenoid, amino acid and fatty acid metabolic pathways. Targeted metabolites were profiled to confirm putative changes in specific metabolic pathways (Deluc et al., 2009). In order to investigate the unique contribution of individual wine grape (Vitis vinifera) berry tissues and water-deficit to wine quality traits, a survey of tissue-specific differences in protein and selected metabolites was conducted using pericarp (skin and pulp) and seeds of berries from vines grown under well watered and water-deficit stress conditions. Of 1,047 proteins surveyed from pericarp by 2D-PAGE, 90 identified proteins showed differential expression between the skin and pulp. Of 695 proteins surveyed from seed tissue, 163 were identified and revealed that the seed and pericarp proteomes were nearly completely distinct from one another. Water-deficit stress altered the abundance of approximately 7% of pericarp proteins, but had little effect on seed protein expression. Comparison of protein and available mRNA expression patterns showed that 32% pericarp and 69% seed proteins exhibited similar quantitative expression patterns indicating that protein accumulation patterns are strongly influenced by post-transcriptional processes. About half of the 32 metabolites surveyed showed tissue-specific differences in abundance with water-deficit stress affecting the accumulation of seven of these compounds (Grimplet et al., 2009). PARTICIPANTS: To date, four graduate students (Katia Silvera, Bahay Gulle, Matt Wheatley, and Richard Tillett) were trained as future independent scientists and are receiving advising and mentoring from the project director about their various research activities and their career development. Five postdoctoral researchers (Ing Fang Chang, Yuichi Uno, Miguel Rodriguez Milla, Laurent Deluc and Jerome Grimplet) were also trained. One lab manager has been trained on the projects in a wide range of preparative and analytical techniques related to the specific requirement of the projects: Rebecca Albion, Staff Research Associate II. TARGET AUDIENCES: The project results and outcomes are being targeted to the scientific community at national and international scientific meetings, specifically to research scientists and undergraduate and graduate students and post-doctoral researchers conducting research on CAM functional genomics and evolution as well as on molecular mechanisms of water deficits on wine grape fruit and wine quality. Outreach projects are also being targeted through poster presentations and oral presentations at national and international scientific meetings and poster and display presentations to the lay public and local stakeholders at an annual Agriculture Field Day held by the Nevada Agricultural Experiment Station. PROJECT MODIFICATIONS: None.
Impacts
Our investigation into the evolutionary origins of CAM demonstrated several patterns of CAM evolution across the Orchidaceae, including multiple independent origins of CAM, several reversal events indicating the evolutionary flexibility of CAM, and parallel evolution of CAM across subfamilies. Our study shows that parallel evolution of CAM is present among subfamilies of orchids, and correlated divergence between photosynthetic pathways and epiphytism can be explained by the prevalence of CAM in low elevation epiphytes and rapid speciation of high-elevation epiphytes in the Neotropics, contributing to the astounding diversity in the Orchidaceae. Overall, our study reveals biochemical underpinnings and evolutionary interactions between CAM as a water-saving mode of photosynthesis and colonization of epiphytic habitats that have contributed to some of the most substantial plant speciations known to exist (Silvera et al., 2009). Using transcript and metabolite profiling, water deficit was shown to have a significant impact on the metabolism of both red- and white-wine grape berries. The metabolic responses of grapes to water deficit varied with the cultivar and fruit pigmentation. Chardonnay berries, which lack any significant anthocyanin content, exhibited increased photoprotection mechanisms under water deficit conditions. Water deficit increased ABA concentrations in Cabernet Sauvignon, but not Chardonnay berries, consistent with the hypothesis that ABA enhanced proline, sugar and anthocyanin accumulation. This is the first report to show that water deficit had significant effects on transcripts involved in amino acid and fatty acid metabolism in grape berries. The effects of water deficit on metabolism have important impacts on berry constituents that influence flavor and quality characteristics in grapes and wine and might contribute to increased antioxidants and human-health benefits (Deluc et al., 2009). Our proteomics study was the first to report tissue-specific changes in protein expression were surveyed in discrete tissue from the pericarp tissues (skin and pulp) and seed of wine grape by 2D-PAGE under well watered and water deficit stress conditions in addition to performing a limited survey of the tissue-specific expression of selected metabolites. Approximately 7% of the more than 1,000 skin and pulp proteins surveyed showed a two-fold or greater change in abundance in response to water deficit stress indicating that water-deficit stress can have a major impact on protein expression profiles in grape pericarp tissues. From the 695 seed proteins surveyed in the seed, seed protein expression patterns were completely distinct from those in the skin and pulp tissues, mainly due to high concentrations of seed storage proteins. Tissue-specific and water status-dependent differences in metabolite profiles were also evident. Finally, this study was the first to evaluate the relative contribution of mRNA versus protein abundance changes for selected genes in order to evaluate the influence of post-transcriptional regulation of gene expression patterns in wine grape (Grimplet et al., 2009).
Publications
- Silvera K, Santiago LS, Cushman JC, Winter K. (2009) Crassulacean acid metabolism and epiphytism linked to adaptive radiations in the Orchidaceae. Plant Physiol. 149:1838-1847.
- Grimplet J, Ben Jouira H, Wheatley M, Deluc L, Cramer GR, Cushman JC. (2009) Proteomic and selected metabolite analysis of grape berry tissues under well watered and water-deficit stress conditions. Proteomics. 9:2503-2528.
- Deluc LG, Quilici DR, Decendit A, Grimplet J, Wheatley MD, Schlauch KA, Merillon J-M, Cushman JC, Cramer GR. (2009) Water deficit alters differentially metabolic pathways affecting important flavor and quality traits in grape berries of Cabernet Sauvignon and Chardonnay. BMC Genomics. 10:212.
- Uno Y, Rodriguez Milla MA, Maher E, Cushman JC. (2009) Identification of proteins that interact with catalytically active calcium-dependent protein kinases from Arabidopsis. Mol. Gen. Genet. 281:375-390.
- Chang IF, Curran A, Woolsey R, Quilici D, Cushman JC, Mittler R, Harmon A, Harper J. (2009) Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana. Proteomics 9:2967-2985.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The proposed multistate research project brings together outstanding, highly productive photosynthesis investigators from across the country in an integrated effort to broaden our understanding of critically important areas of photosynthesis research. We propose a synergistic, cooperative research program that concentrates on two areas of photosynthetic regulation: 1) Photosynthetic capture and photorespiratory release of CO2. The goal of this research is to determine and modify the biochemical and regulatory factors that impact photosynthetic capture and photorespiratory release of CO2. 2) Developmental and environmental limitations to photosynthesis. The aim of this research is to analyze the limitations and environmental factors that influence photosynthetic productivity at the whole plant and canopy levels. Particular emphasis will be placed on abiotic stresses (temperature, water, and salinity), nitrogen use, and global atmospheric change. This work will integrate understanding to optimize photosynthetic production and yield under current and future environmental conditions. PARTICIPANTS: To date, four graduate students (Katia Silvera, Bahay Gulle, Abou Yobi, and Sangho Kang) are being trained as future independent scientists and are receiving advising and mentoring from the project director about their various research activities and their career development. One lab manager has been trained on the projects in a wide range of preparative and analytical techniques related to the specific requirement of the projects: Rebecca Albion, Staff Research Associate II. TARGET AUDIENCES: The project results and outcomes are being targeted to the scientific community at national and international scientific meetings, specifically to research scientists and undergraduate and graduate students and post-doctoral researchers conducting research on CAM functional genomics and evolution as well as on molecular mechanisms of desiccation tolerance. Outreach projects are also being targeted through poster presentations and oral presentations at national and international scientific meetings and poster and display presentations to the lay public and local stakeholders at an annual Agriculture Field Day held by the Nevada Agricultural Experiment Station. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In Crassulacean (CAM) plants, phosphoenolpyruvate carboxylase (PEPC) catalyzes the initial fixation of atmospheric CO2 into C4-decarboxylic acids. Because this pathway concentrates CO2 in the vicinity of Rubisco, PEPC and its regulation are attractive targets for the genetic engineering of improvements in CO2 fixation. A detailed investigation into the molecular genetic changes present in C3 photosynthesis, weak CAM, and strong CAM plants will help us to define the exact genetic and metabolic requirements for the performance of CAM and to define the mechanisms of CAM evolution. Detail investigation of the molecular mechanisms controlling the unique ability of rare species of resurrection plants to withstand near complete loss of water from vegetative tissues will help us define the exact genetic and metabolic requirements for desiccation tolerance. These reseach efforts will be complemented by the development and enhancement of courses on Plant Breeding, Biotechnology, and Propagation and Plant Environmental Stress Physiology and Ecology, and the development of an integrated research and extension project using Sporobolus as a forage grass. Three major topic areas will be the focus of attention: 1) Integrative functional genomics of CAM: We plan to develop integrated functional genomic tools to identify and functionally test key regulatory components that control the unique metabolic demands of CAM. A deeper knowledge of regulatory and structural requirements of CAM plants might allow CO2 concentrating mechanisms to be genetically improved or transferred to C3 crop plants. 2) CAM evolution: We are in the process of investigating the molecular genetic changes that have occurred during CAM evolution by mapping the occurrence of both weak and strong CAM species. A deeper knowledge of the evolutionary requirements of CAM plants might allow CO2 concentrating mechanisms to be genetically improved or transferred to C3 crop plants. 3) Integrative functional genomics of desiccation tolerance. A focus on functional analysis of drought tolerance genes from a primitive resurrection plant (Selaginella) and a monocot grass resurrection species (Sporobolus) will provide novel targets for future genetic engineering efforts to address drought stress. A deeper knowledge of the regulatory and structural requirements of desiccation tolerance might allow for protection or preservation mechanisms to be genetically engineered in crop plants.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: The resurrection plant Selaginella lepidophylla (Division Lycophyta) belongs to an ancient lineage of vascular plants that can withstand complete desiccation for years and be revived after only a few hours of rehydration. To better understand the molecular basis of dehydration tolerance and to identify candidate genes for functional gene testing, mRNA was collected from tissue sampled over a dehydration-rehydration (wet-dry-wet) regime with samples being collected every 10% loss/gain of fresh weight to capture the full repertoire of expressed genes. In addition to the 3,000 ESTs, sequenced last year, this mRNA will be used to create a new non-normalized cDNA library to be used for 454 pyrosequencing to be completed in the first quarter of 2008. Six candidate genes (five group 2 and 3 late embryogenesis abundant proteins and three cold/salinity/drought stress response proteins) have been cloned into expression vectors and will be transformed into Agrobacterium for
transformation of Arabidopsis plants in 2008. Polar and non-polar metabolites were analyzed from five samples (dry-50% rehydrated-fully hydrated-50% dehydrated-dry) by GC-MS. Although analysis of these samples in not yet completed, many differences in metabolite content were observed, most notably, dry plants accumulated mono- and dissaccharide sugars in high concentrations relative to fully hydrated plants. In addition, a field study of 260 Sporobolus pyrimidalis (DT sensitive) and Sporobolus stapfianus (DT tolerant) (African Inselberg grass) plants grown under three different water input amounts was completed during 2007 in Reno. Plants were watered using a drip irrigation system that delivered only one-tenth the amount of water typically used to irrigate alfalfa. All plants grew well with Sporobolus pyrimidalis exhibiting more robust growth under all three watering regimes tested. Forage analysis of both species is currently in progress. Research results were reported at the
Western Regional Meeting of the American Society of Plant Biologists as both oral and poster presentations.
PARTICIPANTS: Mr. Abou Yobi, a graduate student, was trained on the project during 2007.
Impacts
Understanding the fundamental mechanisms that contribute to the response to drought and salinity stress and the protection or enhancement of yield stability in the face of environmentally variable and stressful conditions have become a critical priority area for agricultural research agencies and corporations. Resurrection plants possess the unique ability to undergo nearly complete water loss in vegetative tissues without loss of viability and thus offer the potential to counteract production losses under stressful conditions. The major outcome expected from this project is the discovery of unique genetic determinants for water-deficit tolerance. To date, gene discovery efforts in vascular resurrection plants have been limited. Therefore, the impact of this project will be to identify genes from the ancient club moss, Selaginella lepidophylla that have been lost during plant evolution. For example, one of the group 3 LEA proteins under investigation is more closely
related to a gene present in DT nematodes, than gene homologs found in higher plants, suggesting that crop plants may lack such genes entirely or lack requisite control of the expression of this gene in order to confer tissue desiccation tolerance. The subject areas of this research project are consistent with the 56.0 Plant Biology (B): Environmental Stress Program priority areas: 1) Water stress, including drought, salt, and flooding stress; 2) Global change stress, including increased carbon dioxide, ozone; and 3) Temperature stress as drought stress often occurs coincidently with high temperatures. Currently, over 35% of the world's land surface is considered to be arid or semi-arid, experiencing precipitation that is inadequate for most agricultural uses. Over 6% of the world's land area and 22% of agricultural land is salt-affected by salinity or sodicity. Agriculture in low lying areas in some developing countries are expected to become increasingly damaged by flooding and
salination as a result of sea level rise and salt water intrusion into groundwater aquifers. In response to these anticipated changes, research into the development of stress-tolerant plants that can help to mitigate the impact of climatic variations is needed.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
The resurrection plant Selaginella lepidophylla (Division Lycophyta) belongs to an ancient lineage of vascular plants that can withstand complete desiccation for years and be revived after only a few hours of rehydration. To better understand the molecular basis of dehydration tolerance, a complementary DNA library was constructed from S. lepidophylla microphyll fronds dehydrated for 2.5 h and used to generate an expressed sequence tag (EST) database (Iturriaga et al., 2006). ESTs were obtained for 1,046 clones representing 874 unique transcripts. Putative functions were assigned to 653 (62.4%) of these clones after comparison with protein databases, whereas 212 (20.2%) sequences having significant similarity to known sequences whose functions are unclear and 181 (17.3%) sequences having no similarity to known sequences. The S. lepidophylla ESTs were compared to the S. moellendorffii EST database using the tBLASTX algorithm. Approximately 36-38% of the S. lepidophylla
ESTs exhibited similarity to the S. moellendorffii ESTs, whereas 62-63% were unique to S. lepidophylla and S. moellendorffii, respectively. For those S. lepidophylla ESTs for which functional assignments could be made, the largest functional categories included photosynthesis (17%), followed by primary metabolism (14%) and disease/defense-related functions (11%). S. lepidophylla had a higher relative percentage of ESTs within the transporter, cell structure, secondary metabolism, molecular chaperones (i.e. heat shock proteins), and abiotic stress-related (i.e. late embryogenesis abundance proteins) functional categories than did S. moellendorffii. Thus, S. lepidophylla may serve as a rich genetic resource for the identification of novel genes associated with environmental stress and dehydration tolerance. A new non-normalized cDNA library was created from tissue sampled over a dehydration-rehydration (wet-dry-wet) regime with samples being collected every 10% loss/gain of fresh weight
to capture the full repertoire of expressed genes. Although our goal is to sequence 25,000 ESTs, funds were only available for attempted sequencing of 3,000 ESTs, which resulted in the production of 2,646 additional ESTs representing 1,963 unique genes. This data has been deposited in GenBank and is publicly available under accession numbers (EH091190- EH093835).
Impacts
Over 35% of the world's land surface is considered to be arid or semi-arid, experiencing precipitation that is inadequate for most agricultural uses. In addition, over 10% of the arable lands are affected by drought and salinity declining average yields for most crops more than 50%. Drought is the leading cause of agricultural productivity losses. Drought is often accompanied by heat, which often leads to further crop losses; in 2000 were estimated to exceed $4 billon. Thus, improving drought tolerance is a critical priority area for agricultural research agencies. Understanding how plant cells tolerate water loss is a vital prerequisite for developing strategies for improving drought tolerance maintaining biomass and seed production under such conditions. Despite their potential utility as a rich source of unique genetic determinants for water-deficit tolerance, gene discovery efforts in vascular resurrection plants have been limited. To effectively identify which
genes play a central adaptive role in dehydration tolerance, we will begin functional testing of genetic determinants that are unique to resurrection plants with a focus on genes derived from S. lepidophylla. This research specifically addresses the USDA-NRI priority of the 22.1 Agricultural Plants and Environmental Stress Program to generate fundamental knowledge of genes and networks involved in water stress responses.
Publications
- Iturriaga, G, Cushman MAF, Cushman JC (2006) An EST catalogue from the resurrection plant Selaginella lepidophylla reveals stress-adaptive genes. Plant Science. 170: 1173-1184.
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