Progress 01/01/11 to 09/30/16
Outputs
Target Audience:Alfalfa breeders and producers; crop scientists and plant biologists who are studying freezing and drought tolerance. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three graduate students, M. Rokebul Anower (PhD, graduated); Praveena Kanchupati (PhD student); Krishna Ghimire (MS student) were trained in this program. MR Anower has graduated and continues research on alfalfa as a postdoc in a top forage research institute in the US. Praveena and Krishna will graduate in 2017.These graduate students had presented their data/findingsat the North American Alfalfa Improvement Conferences, the annual meetings of the American Society of Plant Biologists, and several regional conferences including Northern Plain SunGrant meetings. They were also involved in manuscript preparation.We also trained three undergraduate students (Andrew Nelson, Ping Li, Alex Laugesen) and one visiting graduate student (Yafang Wang from Northwest A&F University, China). How have the results been disseminated to communities of interest?The results have been presented at the North American Alfalfa Improvement Conferences, the annual meetings of the American Society of Plant Biologists, and several regional conferences including Northern Plain SunGrant meetings. Also, the results are published in peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1:Evaluate yellow-flowered alfalfa germplasm that have adapted to diverse growth conditions in South Dakota, especially in the drought and freezing-prone areas in the west, and identify the physiological characteristics associated with stress tolerance in superior alfalfa lines (100% accomplished) Our initial collaborative effort to screen the alfalfa germplasm that had been naturalized to the Grand River National Grassland environment led to the discovery of two germplasms, River side (RS) and Foster ranch (FR), with a superior freezing tolerance at seedling stages when compared with commercial cultivars, Apica (cold tolerant) and CUF-101 (cold susceptible). The better freezing tolerance is reflected by their greater survival rate (74% and 79% for RS and FR, respectively vs 64% and 24% for Apica and CUF-101, respectively based on an average of various tests at different freezing temperatures) and lower membrane leakage (LT50 for cold acclimated plants are -18, -9.6, -8.5, and -5 °C for RS, FR, Apica, and CUF-101, respectively). We also identified a line in the RS population that showed a greater drought tolerance and higher water use efficiency compared with 10 other accessions and varieties. RS showed the smallest reduction in stem elongation (36%), relative growth rate (14%), and shoot dry mass (40%) production under the severest drought (supplying water to only compensate for 25% of transpirational loss) tested in this study relative to the non-drought treatment. While RS showed less biomass production under well-watered conditions, it produced similar or more shoot biomass under drought conditions compared to other accessions. Associated with the drought resistance or less sensitivity to drought, RS showed greater capability to maintain root growth, shoot relative water content, and leaf chlorophyll content compared to other accessions. Different from other accessions, RS showed increasing water use efficiency (WUE) as water deficit became severe, reaching the greatest WUE, while other 10 accessions shower similar but lower WUE. Our physiological and biochemical studies showed that the improved drought tolerance in RS and FR was associated with their abilities to maintain root growth and relative water content in shoots under stress. Objective 2: Discover genes and expression patterns associated with stress tolerance using various tools such as oligonucleotide microarrays, cDNA amplified fragment length polymorphism (cDNA-AFLP), and proteomics.Genes involved in flowering time control will also be studied (35% accomplished) To understand the molecular basis of freezing tolerance in RS and FR, we examined expression of CBF3-like genes in these plants. CBF3 in Arabidopsis plays an important role in cold acclimation and thus freezing tolerance. From 18 CBF3-like genes in alfalfa we identified two potential function homologs of CBF3 in alfalfa based on their induction patterns under cold stress. We also identified an additional CBF3-like gene that showed a different induction pattern from CBF3 but was induced in FR and RS, as well as known freezing tolerant cultivars. FR and RS plants also exhibited distinct patterns of cold induction in a marker gene, CAS15B, suggesting that FR and RS have developed different mechanisms in freezing tolerance. About 65% of the work proposed in Objective 2 was not accomplished due to two main reasons: 1) time constraint and 2) lack of funding. The genome-wide gene discovery proposed in Objective 2 required a large amount of funding support (beyond the Hatch funding support). We submitted a proposal to NIFA twice and were not successful. We won't be able to conduct these experiments until we obtain additional support from NIFA. Objective 3:Determine function and application of a subset of candidate genes in stress tolerance or flowering time control (40% accomplished) We identified a set of candidate genes based on sequence similarity to some key flowering genes in Arabidopsis, such as CO and FCA. CO (CONSTANS) and FCA are two genes involved in flowering time control through the photoperiod pathway and the autonomous pathway, respectively. There are multiple CO-like and FCA-like gene members in alfalfa. Through expression analysis, we narrowed down to three CO-like and two FCA-like genes for function analysis. To confirm the gene function, these genes were cloned from alfalfa and transformed into Arabidopsis to determine their effects on flowering time. Currently we are screening and confirming the Arabidopsis transformants. About 60% of the work proposed in Objective 3 was not accomplished due to two reasons: 1) technical difficulty; 2) time constraint. We initially conducted gene transformation in alfalfa plants and were not successful. We then transformed these genes into Arabidopsis - a common alternative to test the gene function. As mentioned in the summary, we are currently screening and characterizing the Arabidopsis transformants and will continue these experiments and report the results to NIFA in the future.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Anower M.R, Boe A, Auger D, Mott IW, Peel MD, Xu L, Kanchupati P, Wu Y*. (2015) Comparative drought response in eleven diverse alfalfa accessions. Journal of Agronomy and Crop Science. doi:10.1111/jac.12156.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Anower MR, Fennell A, Boe A, Mott I, Peel M, Wu Y*. (2016) Physiological and molecular characterization of alfalfa (Medicago sativa L.) germplasm with improved seedlings freezing tolerance. Crop and Pasture Science (Accepted on 28 January, 2016)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Kanchupati P, Anower MR, Boe A, Wu Y* The CBF-like gene family in alfalfa (Medicago sativa L.): Expression analyses and identification of potential functional homologs of Arabidopsis CBF3. (under revision)
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2015
Citation:
Anower MR, 2015, Abiotic Stress in Alfalfa: Insight into physiological and molecular responses to salinity, drought and freezing 2015 South Dakota State University
- Type:
Other
Status:
Other
Year Published:
2015
Citation:
Genetic Resources
We have identified two alfalfa collections, River side (RS) and Foster ranch (FR), that showed greater drought and freezing tolerance. In addition, RS showed higher water use efficiency. RS and FR can be valuable genetic resources for breeding alfalfa with improved stress tolerance and water use efficiency.
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Alfalfa breeders and producers; crop scientists and plant biologists who are studying freezing and drought tolerance. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three graduate students were trained to conduct the research. One PhD student has successfully defended his dissertation and is now a postdoc at a top forage research institute. How have the results been disseminated to communities of interest?The results were presented at the annual meeting at the American Society of Plant Biologists in Minneapolis. What do you plan to do during the next reporting period to accomplish the goals?We will continue to address physiological processes that are associated with high water use efficiency in River side under drought. We will focus on the stomatal behavior and regulation during drought stress in River side in comparison with those of other alfalfa germplasms. We are currently examining expression of CBF-like genes in River side and Foster ranch in comparison with that of non-cold tolerant alfalfa as well as identifying the cold-inducible candidate genes that are associated with freezing tolerance in River side and Foster ranch.
Impacts
What was accomplished under these goals?
We previously identified an alfalfa germplasm, River side, collected from the Grand River National Grassland in South Dakota that showed a higher water use efficiency under drought compared to several commercial and noncommercial alfalfa germplasms (Anower et al., 2015). Studies were conducted to understand the physiological mechanisms that may contribute to high water use efficiency in River side. In a dehydration assay using detached leaves, River side showed less loss of water during a time course experiment compared to other two alfalfa germplasms used as controls. Examination of leaf morphology revealed that River side had a slightly higher stomatal density compared to the control germplasms. However, River side showed a greater reduction in stomatal conductance under drought especially on the lower surface of the leaf, suggesting a control in stomatal conductance may be the key to reducing water loss under drought. We also noticed that River side had denser leaf hairs compared to the two control germplasms. Currently, we are assaying the sensitivity of stomatal aperture to ABA treatment in these plants. Additional studies were conducted to understand the molecular basis of freezing tolerance in River side and Foster ranch, two alfalfa germplasms. We showed previously that the transcript levels of two CBF-like genes were induced by non-freezing low temperature treatment and were differentially regulated in freezing-tolerant River side and Foster ranch. CBF comprises a multigene family in Medicago and 19 CBF-like members were identified based on protein sequence similarity to Arabidopsis CBFs. Sixteen of the genes were inducible by low temperature treatment in a diploid yellow alfalfa (SD201) and showed different patterns of induction in a time-course experiment. The majority of the cold-inducible CBF-like genes in alfalfa did not show a diurnal expression pattern as shown in CBF3 in Arabidopsis. These genes also show differences in expression in different tissues and at different developmental stages, suggesting these genes may have different roles in low temperature response in alfalfa.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
M. R. Anower, A. Boe, D. Auger, I. W. Mott, M. D. Peel, L. Xu, P. Kanchupati & Y. Wu (2015) Comparative Drought Response in Eleven Diverse Alfalfa Accessions. Journal of Agronomy and Crop Science. doi:10.1111/jac.12156
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Alfalfa breeders and producers, crop scientists and plant biologists who are studying freezing and drought tolerance and flowering time regulation. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two graduate students were trained to conduct the research. How have the results been disseminated to communities of interest? The results were presented at the 14th North American Alfalfa Improvement Conference in Canada. What do you plan to do during the next reporting period to accomplish the goals? We are currently examining expression of other candidate genes in alfalfa to determine how their transcript levels are affected by low temperature treatments and whether they are also associated with cold tolerance. We will continue to identify Arabidopsis transformants (over-expressing genes in wild-type or mutant background) of other flowering candidate genes, and the transformants will be examined for altered flowering time. We will continue to explore the alfalfa transformation methods in order to study gene function directly in alfalfa.
Impacts
What was accomplished under these goals?
We conducted additional studies to characterize physiological and molecular responses in the cold-tolerant alfalfa genotypes that we identified previously. We observed a more rapid and greater induction of several known low-temperature responsive genes in cold-tolerant genotypes compared to a cold-sensitive genotype. We also found that two selected genotypes and the positive control line used in this study differed from each other in which genes were up-regulated and how transcript levels of each gene changed with time. The results suggested that these cold-tolerant alfalfa may have adopted different mechanisms in cold stress response. This notion is supported by our physiological analysis. Measurement of total soluble sugar and relative water content in shoot tissues, two of the physiological parameters that are linked to cold tolerance, showed each genotype had its own pattern of change or regulation. 2. Additional studies were done to examine whether transcript levels of seven candidate genes in flowering time control in alfalfa were regulated by circadian. While all the genes showed clear response to light/darkness (diurnal change) at transcript level, only one gene appeared to have regular circadian pattern of transcript level. The results suggest that the alfalfa gene is potentially an orthologue of the Arabidopsis CONSTANS gene, despite the fact that the gene sequence was less similar to the Arabidopsis gene compared to other alfalfa genes in the same group. We completed transformation of these genes into Arabidopsis for overexpression and obtained transformants for three genes. We will continue to identify transformants for other candidate genes and these transformants will be examined for altered flowering time.
Publications
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Alfalfa breeders and producers, crop scientists and plant biologists who are studying freezing and drought tolerance and flowering time regulation. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two graduate students were trained to conduct the research. In addition, we provided training and research experiences for four undergraduate students. In particular, we hosted and trained an undergraduate researcher from another school who was attending the REU program during the summer on campus. How have the results been disseminated to communities of interest? We presented the results on expression of flowering genes in alfalfa at the NC SunGrant meeting. What do you plan to do during the next reporting period to accomplish the goals? We will study expression of several candidate genes to determine whether some of the genes are associated with improved freezing tolerance in the selected alfalfa lines. We will conduct additional transformation of Arabidopsis plants with flowering candidate genes from alfalfa and characterize the transformants that we have obtained. We will continue to explore the alfalfa transformation methods in order to study gene function directly in alfalfa. A manuscript on alfalfa water use efficiency will be submitted.
Impacts
What was accomplished under these goals?
Last year, we identified two lines of alfalfa that adapted to South Dakota rangeland conditions with greater survival rate under freezing compared to other alfalfa lines and cultivars tested. We conducted additional tests on these alfalfa plants to understand how these plants perform at freezing temperatures. We measured the leaf and stem electrolyte leakage after freezing treatments. Our results showed that the two selections showed less membrane leakage and damage compared to other alfalfa cultivars under various freezing temperatures. As an indication, the freezing temperatures that lead to 50% of cell damage were lower in these selection lines. Pretreatment of alfalfa plants with non-freezing low temperatures (cold acclimation) before freezing significantly reduced membrane leakage in all alfalfa plants. The selected lines again showed greater capability to maintain membrane integrity. The results suggest that these selection lines will potentially show less biomass loss from freezing damage. To understand the molecular basis of freezing tolerance in these plants, we are currently studying expression of some of the genes (~25 genes) that are known to be associated with freezing tolerance in alfalfa and Arabidopsis. Primers were designed and tested. Currently we are conducting qPCR analysis to examine how these genes are expressed in the plants before and after low temperature treatments. We examined expression of seven flowering candidate genes in alfalfa at different developmental stages or photoperiods. Since all the candidate genes come from gene families, the gene expression study helped prioritize the gene list for function analysis. These genes have been cloned and transformed into Arabidopsis for overexpression. Currently, we are transforming these genes into Arabidopsis flowering mutants as part of gene function analysis. We have been trying to transform alfalfa plants with these genes, but it was not successful so far.
Publications
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: 1. Our initial freezing tests identified Foster Ranch and Riverside, two of the South Dakota local collections, among the best in surviving -5 degree (celcius) freezing without cold acclimation. We challenged these plants with lower freezing temperatures. In these tests, we included two positive controls which are freezing tolerant, and a negative control which is susceptible to freezing. We found that non-cold-acclimated Foster Ranch and Riverside showed 74.5% and 69.5% of survival rate at -10 degree, and 73% and 54% of survival rate at -12 degree, respectively. Under both conditions, they showed improved survival rates compared to two positive controls. Negative control showed only 8% survival rate at -12 degree. The freezing test was programmed to reduce the temperature gradually and to hold at the freezing temperate for 2.5 h once the target temperature has reached. We again observed that all the plants showed great freezing tolerance at both -10 and -12 degree after the plants have gone through cold-acclimation for a week. 2. Since differences in plant size and dormancy rate among alfalfa collections complicates drought tolerance screening based on survival rate, we took a different approach to evaluate drought tolerance - measuring biomass production under controlled progressive water deficit conditions. Our goal is to identify alfalfa varieties that can be still productive under drought. Water supply was adjusted based on water use of individual plants grown in a greenhouse in order to maintain similar water deficit levels among different collections. We found one alfalfa population collected from South Dakota grassland showed the least reduction in shoot biomass production under drought. In addition, it used the least amount of water, which resulted in a much higher water use efficiency compared to all other alfalfa tested in the experimental system. We are now conducting additional experiments to understand the uniqueness of this particular alfalfa collection. A manuscript is in preparation. 3. We have cloned 6 genes from alfalfa that are similar to the major flowering genes in Arabidopsis. Expression patterns are now being examined to see how these genes are regulated in alfalfa plants. We have also finished cloning these genes into the vectors for over-expression or gene silencing. We are now transforming these genes into Arabidopsis flowering mutant to see if these alfalfa genes are able to complement the mutation in Arabidopsis. Meanwhile, we are testing different alfalfa transformation protocols so that we can transform these genes into alfalfa for function analysis in the future. PARTICIPANTS: Graduate students: Rokebul Anower and Praveena Kanchupati Undergraduate students: Erik Oines and Alex Lauseng Collaborators: Arvid Boe, Anne Fennell, Lan Xu, Yang Yen, Roger Gates TARGET AUDIENCES: Alfalfa breeders and producers, crop scientists and plant biologists who are studying freezing and drought tolerance and flowering time regulation. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Identification of stress-tolerant alfalfa is critical for breeding alfalfa that can be grown on marginal land and can sustain high yield under stress conditions. Control of flowering time in alfalfa will maximize biomass production as well as improve forage quality.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: 1. We performed freezing tests on 14 alfalfa collections/cultivars, including 9 local rangeland-adapted collections. Young plants were subjected to freezing treatment (-6 celsius) with or without cold acclimation. Without cold acclimation, we found Foster Ranch, one of the local collections, showed the highest survival rate (67%) after freezing. No difference in freezing tolerance was observed among alfalfa collections/cultivars if they experienced cold acclimation, i.e. plants all survived freezing. We are conducting freezing tests at lower freezing temperatures (-10 to -15 Celsius) on these plants to determine their freezing tolerance. 2. We did not see clear difference in drought tolerance among alfalfa collections/cultivars based on survival rate (after drying) due to large data variation. We have redesigned the experiment and will conduct the test again. Drought tolerance will be judged based on the minimal amount of water that is required to keep plants alive and produce biomass. 3. We have identified several gene sequences in the Medicago truncatula genome that show high similarity to flowering genes in Arabidopsis using bioinformatic tools. Some of them have been cloned from alfalfa. We will continue to clone additional genes. Gene expression and molecular genetic analysis will be conducted to confirm their functionality. We presented our initial findings at the annual meeting of the American Society of Plant Biologist and the annual regional SunGrant meeting. PARTICIPANTS: Graduate students: Rokebul Anower and Praveena Kanchupati Undergraduate student: Andrew Nelson Collaborators: Arvid Boe, Anne Fennell, Lan Xu, Yang Yen, Roger Gates TARGET AUDIENCES: Alfalfa breeders and producers, crop scientists and plant biologists who are studying freezing and drought tolerance and flowering time regulation. PROJECT MODIFICATIONS: No
Impacts
Identification of stress-tolerant alfalfa is critical for breeding alfalfa that can be grown on marginal land and can sustain high yield under stress conditions. Control of flowering time in alfalfa will maximize biomass production as well as improve forage quality. We will publish our findings in peer-reviewed research articles.
Publications
- Rokebul Anower, Ivan Mott, Michael Peel, Arvid Boe, Anne Fennell, Yajun Wu. 2011. Physiological Characterization of Abiotic Stress Tolerance in Alfalfa (Medicago Sativa L.). The Annual meeting of the American Society of Plant Biologists. Meeting Abstract (for a poster)
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