Progress 03/01/17 to 02/29/20
Outputs
Target Audience:Scientists working on abiotic stress responses of rice as well as other crop plants Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A graduate student, a postdoctoral fellow and technical professional were not only trained technically (growing rice, stress treatments, metabolite analysis, isolating RNA, qPCR analyses of gene expression and phenotypic assays) but also intellectually in analyzingand interpreting the obtained results from the metabolite analysis, gene expression analysis as well as phenotypic and physiological analysis of mutants under salt and other abiotic stresses. 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?
The serotonin accumulation pattern under salt stress was analyzed in two pairs of contrasting lines differing in their salt sensitivities along with their corresponding salt-tolerant RILs. These include salt-tolerant parent Pokkali, as well as salt tolerant RIL FL478 compared to the salt-sensitive parent IR29. Another pair included salt-tolerant NonaBokra as well as its' tolerant RIL IR63731 and salt-sensitive IR28. In comparison to the salt-sensitive IR29 and IR28, greater accumulation of serotonin was observed both intolerant parents (Pokkali and NonaBokra) and their tolerant RILs, i.e., FL478 and IR63731. These results largely confirmed the potential importance of serotonin accumulation in salt tolerance of rice. We also analyzed serotonin accumulation in rice genotypes with contrasting drought and heat sensitivities to reveal whether serotonin accumulation differs in other abiotic stress-tolerant rice genotypes compared to stress-sensitive genotypes. For this purpose, N-22 (drought and heat tolerant), Dular (drought and heat tolerant), IR64 (drought sensitive), IR36 (drought sensitive), IR52 (heat sensitive) Swarna (drought sensitive) and Moroboreken (heat sensitive) rice genotypes have been utilized.Interestingly, in N-22 (drought and heat tolerant genotype), serotonin levels were decreased both under heat or droughtin roots, whereas the levels were also decreased under heat but not under drought in shoots. In Dular, serotonin levels were almost unaffected in shoots either under drought or heat but decreased in roots under drought but unaltered under heat. On the other hand, both in shoots and roots, the serotonin levels were decreased under PEG-induced drought in IR36 and Swarna, and these levels were almost unaltered in shoots or roots of IR64. Overall, these results argue that serotonin is unlikely to play a role under drought and heat stress responses of rice. In plants, the serotonin biosynthesis involves conversion of tryptophan to tryptamine by tryptophan decarboxylase (TDC) and conversion of tryptamine to serotonin by tryptamine hydroxylase (T5H). Using real-time PCR assays, we have compared analyzed gene expression profiles for TDC and T5Hgenes in salt-tolerant Pokkali, and it's salt tolerant RIL FL478 and salt-sensitive IR29. Similarly, salt-tolerant NonaBokra and salt-sensitive IR28, and it's tolerant RIL IR63731 were also compared. The results indicated thatTDChomologues (Os08g04540andOs08g04560) were highly induced under salt stress in the roots of tolerant genotypes such as the Pokkali, FL478, Nona Bokra, IR63731 compared with the sensitive IR29 and IR28 genotypes. Although both genes were found to be induced by salt stress,Os08g04540shown a much greater induction under salt stress.The genes (Os05g43510andOs10g23900) only displayed mild differences in response to salt stress between Pokkali and IR29 under salt stress, although this was slightly induced in FL478. UnlikeTDCgenes,T5Hgene is represented by a unique (Os12g16720) locus in the rice genome. Using real-time PCR assays, we have analyzed gene expression profiles for T5H in Pokkali, RIL FL478,IR29 as well as in NonaBokra, RIL IR63731, and IR28. The results revealed that T5H expression levels are significantly induced intolerant Pokkali,FL478,NonaBokra, IR63731compared to salt sensitive IR29 and IR28. These results provided additional evidence as to the cause of differential serotonin accumulation in salt-tolerant versus salt-sensitive rice genotypes. Our third objective focused on characterizing the t5h mutant and analyze its phenotypic responses under salt stress as well as other abiotic stresses such as drought, heat, and oxidative stresses. We have obtained three alleles (G650, D2943, and D1137) in the IR64 background that was obtained from the Jane Leach laboratory (Colorado State University) and confirmed the genotype by sequencing the genomic location. To validate the biochemical effects of these mutations on serotonin accumulation, we have analyzed the serotonin abundances in control and salt-treated (150 mM NaCl) seedlings. The serotonin levels in the wild-type IR64 were decreased in shoots under salt stress as was observed for salt-sensitive IR genotypes (IR29 and IR28). The serotonin levels were much higher in the roots of IR64 compared to shoots but were almost unaltered during salt stress. Relative to the wild-type IR64, the serotonin levels were slightly elevated in shoots of mutants under control conditions and these levels were further elevated under salt stress. By contrast, in roots, the serotonin levels were highly decreased (about 6-fold decrease) both under control and salt stress conditions in the mutants. Thus, the overall serotonin analyses between the shots and roots indicated that the mutation has a very strong effect on roots. As explained above, the serotonin biosynthesis in plants involves the conversion of tryptophan to tryptamine and tryptamine to serotonin. Therefore, we also analyzed the tryptophan and tryptamine levels that are upstream compounds of the serotonin biosynthesis in the salt-treated tissues (roots and shoots) of these mutants along with the wild-type IR64. To further investigate the effects of these mutations on the serotonin biosynthetic pathway, we have analyzed the tryptophan and tryptamine abundances in control and salt-treated seedlings. In wild-type as well as in mutants, the tryptophan levels were highly elevated both in shoots and roots although the increase was greater in shoots compared to the roots. The tryptamine levels in the wild-type (IR64) were barely detectedboth under control and salt stress conditions whereas in mutants the levels were remarkably elevatedunder control treatment but slightly decreased under salt stress. These mutants are invaluable for analyzing the role of serotonin in tolerance to salinity as well as other abiotic stresses. Therefore, we conducted salt tolerance assays in hydroponically-grown 3-week-old seedlings as well as soil-grown plants. In both cases, the mutants were highly salt-sensitive implying that serotonin plays an important role in salt tolerance of rice. Morphological and physiological responses further confirmed the salt sensitivity of the mutants compared to the IR64 wild-type plants. In order to test, whether serotonin plays a role under other abiotic stresses, we exposed these mutants (G650, D2943, and D1137) along with the wild-type IR64 to heat, drought as well as oxidative stress (induced by Cu2+) conditions. In all these different stress treatments, the mutant phenotype was similar to the wild type IR64. These results were consistent with the observation that the serotonin accumulation pattern did not differ greatly between drought- or heat-tolerant and -sensitive rice genotypes under drought or heat. Thus, serotonin appears to play a role primarily in response to salt stress in rice. Outcomes/Impacts: Identification of the responsiveness of serotonin and the molecular mechanisms involved in its' accumulation under salt stress has provided a strong basis for future work to determine the physiological role of serotonin in salt tolerance of rice. The results also laid a strong foundation to test whether salt tolerance of rice can be improved by overproducing serotonin accumulation in rice using biotechnological strategies. Collaborations:Vibha Srivastava (University of Arkansas, Fayetteville) and Jan leach (Colorado State University).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Sunkar, R., Ganesan, G., Srivastava, V., Oliver, M., Mort, A. Does Serotonin Play a role in salt tolerance of rice, Phenome, Feb 24-28, 2020, Tucson, AZ
|
Progress 03/01/18 to 02/28/19
Outputs
Target Audience:Scientists working on abiotic stress responses of rice as well as other crop plants Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A postdoctoral fellow has been trained on intellectual and technical (growing rice plants, isolating RNA, performing qPCR analyses, genotypic characterization of mutants) aspects related to the project. How have the results been disseminated to communities of interest?Gave an oral presentation on "Comparative 'omics' analyses for understanding salt tolerance in rice' at the Henan Normal University, Xinxiyang, China, on April 24th, 2018. What do you plan to do during the next reporting period to accomplish the goals?1) Define the serotonin responses to diverse abiotic stresses in rice genotypes with contrasting stress tolerances We have almost completed this objective and we will only verify certain responses for further confirmations. 2) Define TDC and T5H responses to diverse abiotic stresses in rice genotypes with contrasting stress tolerances This objective was also largely completed especially with respect to salt stress responses. We will extend the TDC and T5H gene expression analysis to drought and heat stresses in the contrasting pairs of rice genotypes as proposed. 3) Assess the response of t5h mutant that is defective in serotonin accumulation to diverse abiotic stresses. The three mutant lines (G650, D2943, and D1137) along with wild-type IR64 controls will be evaluated for germination (Yoshida-Agar medium supplemented with NaCl )and seedling development (Yoshida hydroponic medium supplemented with NaCl )under salt stress. Furthermore, the phenotypic and physiological responses under salt, PEG (osmotic), oxidative stresses (Cu2+ and methyl viologen) and heat stress conditions will be undertaken.
Impacts
What was accomplished under these goals?
1) Define the serotonin responses to diverse abiotic stresses in rice genotypes with contrasting stress tolerances The serotonin accumulation pattern was analyzed during the first year in two pairs of contrasting lines differing in their salt sensitivities along with their corresponding salt-tolerant RILs. During the second year of the project, we have measured the tryptophan and tryptamine levels that are upstream compounds of the serotonin biosynthesis in 3-week-old rice seedlings exposed to salt stress in order to gain an insight into the responses of serotonin biosynthetic pathway under salt stress. In shoots, the tryptophan levels were significantly elevated in Pokkali, IR29 as well in their salt tolerant RIL FL478. However, in roots, the tryptophan levels were almost unaffected in all these three rice genotypes. Another contrasting pair and their RIL (NonaBokra, IR28 as well as their tolerant RIL IR63731) exhibited similar responses both in shoots (elevated) and roots (unaffected) under salt stress. Interestingly, the tryptamine levels were elevated in roots of all six genotypes but the in shoots, the levels were elevated in Pokkali, IR29 as well in their RIL FL478 but not in NonaBokra, IR28, and their tolerant RIL IR6373. One of the sub-objectives of this Aim is to analyze serotonin responses in rice genotypes with contrasting drought and heat sensitivities. For this purpose, N-22 (drought and heat tolerant), Dular (drought and heat tolerant), IR64 (drought sensitive), IR36 (drought sensitive), IR52 (heat sensitive) Swarna (drought sensitive) and Moroboreken (heat sensitive) rice genotypes have been utilized. The 3-week-old seedlings grown hydroponically using Yoshida medium were exposed to drought or heat. Drought stress was imposed by supplementing the Yoshida medium with 20%PEG for 72 hrs, whereas the heat stress was imposed by exposing seedlings to 38/30°C (day/night), and the roots and shoots were harvested and the serotonin levels were measured. Interestingly, in N-22 (drought and heat tolerant genotype), the serotonin levels were decreased both under heat or drought stresses in roots, whereas the levels were decreased under heat but not under drought in shoots. In Dular (another drought and heat tolerant genotype), the serotonin levels were almost unaffected in shoots either under drought or heat but decreased in roots under drought but unaltered under heat. On the other hand, both in shoots and roots, the serotonin levels were decreased under PEG-induced drought in IR36 and Swarna, and these levels were almost unaffected in shoots or roots of IR64. These results were inconclusive with respect to the potential role of serotonin under drought and heat stresses in rice and further evaluations are needed. 2) Define TDC and T5H responses to diverse abiotic stresses in rice genotypes with contrasting stress tolerances This objective was largely addressed during the first year. 3) Assess the response of t5h mutant that is defective in serotonin accumulation to diverse abiotic stresses Our third objective is to characterize t5h mutant and analyze phenotypic responses of this mutant under diverse abiotic stresses. Three mutant alleles (G650, D2943, and D1137) in the IR64 background were obtained from Jane leach laboratory (Colorado State University) and molecular characterization of these mutations was confirmed. To validate the effects of these mutations on serotonin accumulation, we have analyzed the serotonin abundances in control and salt-treated (150 mM NaCl) seedlings. The serotonin levels in the wild-type IR64 were decreased in shoots under salt stress as was observed for salt-sensitive IR genotypes (IR29 and IR28). The serotonin levels were much higher in roots of IR64 compared to shoots but were almost unaltered during salt stress. Relative to the wild-type IR64, the serotonin levels were slightly elevated in shoots of mutants under control conditions and these levels were further elevated under salt stress. By contrast, in roots, the serotonin levels were highly decreased (about 6-fold decrease) both under control and salt stress conditions in the mutants. These mutants are invaluable for analyzing the role of serotonin in tolerance to salinity as well as other abiotic stresses. To further investigate the effects of these mutations on serotonin biosynthetic pathway, we have analyzed the tryptophan and tryptamine abundances in control and salt-treated seedlings. In wild-type as well as in mutants, the tryptophan levels were highly elevated both in shoots and roots although the increase was greater in shoots compared to the roots. The tryptamine levels in the wild-type (IR64) were barely detected both in shoots and roots both under control and salt stress conditions whereas in mutants the levels were remarkably elevated in the shoots and roots, both under control as well as salt stress conditions.
Publications
|
Progress 03/01/17 to 02/28/18
Outputs
Target Audience:Scientists working on abiotic stress responses of rice as well as other crop plants. Changes/Problems:We intended to start the project on March 1, 2017 with a graduate student but were unable to recruit a suitable candidate immediately. Recruiting a new graduate student occurred in the middle of August, 2017. Additionally, we could not procure sufficient quantities of seed material for some of the rice genotypes and we have to multiply seeds in our laboratory. Currently, we either recently harvested seeds or in the process of harvesting seeds for different rice genotypes. These hurdles delayed the proposed timelines slightly. What opportunities for training and professional development has the project provided?Ms. Parna Ghosh, a new graduate student is currently being trained in the areas of metabolomics and plant abiotic stress tolerance. Ms. Ghosh is new to the research on plant abiotic stress responses as well as metabolomics analyses. Currently she is being trained in many aspects of the proposal including growing rice, stress treatments, isolating RNA, extracting metabolites for LC/MS, genotyping the mutant and qPCR analyses of gene expression. 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?1) Define the serotonin responses to diverse abiotic stresses in rice genotypes with contrasting stress tolerances Thus far we have analyzed serotonin responses in two pairs of contrasting salt tolerant genotypes (Pokkali and IR29 as well as NonaBokra and IR28) as well as their salt tolerant RILs, FL478 and IR63731. The serotonin responses of the remaining genotypes for salt stress will be analyzed. Similarly, serotonin responses in drought tolerant and drought sensitive lines (N22, Dular, IR64, IR36 and swarna genotypes) as well as in heat tolerant and heat sensitive genotypes (N22, Dular, Moroboreken, and IR52) will be investigated. Since we have received very few seeds for some of these lines we need to multiply seeds, which we have done. The experiments to determine the relevance of serotonin accumulation with respect to drought and heat tolerances will be undertaken. 2) Define TDC and T5H responses to diverse abiotic stresses in rice genotypes with contrasting stress tolerances Thus far our experiments have completed most important analyses for salt stress and analyzing the gene expression profiles in other salt stress-associated rice genotypes is underway. Similarly, the experiments will be undertaken to perform gene expression profiles for TDC genes and T5H gene under drought and heat stresses in the contrasting pairs of rice genotypes as proposed. 3) Assess the response of t5h mutant that is defective in serotonin accumulation to diverse abiotic stresses. Currently we are multiplying the seeds from the mutant lines. Once we obtain sufficient quantities of seeds for each of the three mutant lines, we will evaluate phenotypic and physiological responses under salt, PEG (osmotic), oxidative stresses (Cu2+ and methyl viologen) and heat stresses.
Impacts
What was accomplished under these goals?
1) Define the serotonin responses to diverse abiotic stresses in rice genotypes with contrasting stress tolerances As proposed, we have analyzed the accumulation of serotonin in salt-tolerant parent Pokkali, as well as salt tolerant RIL FL478 compared to the salt-sensitive parent IR29 in response to salt stress. Additionally, we have analyzed serotonin accumulation pattern in another contrasting pair of genotypes, i.e., salt-tolerant NonaBokra as well as it's tolerant RIL IR63731 and in salt-sensitive IR28. In comparison to the salt-sensitive IR29 and IR28 genotypes, a greater accumulation of the serotonin was observed both in tolerant parents (Pokkali and NonaBokra) as well as their tolerant RILs, i.e., FL478 and IR63731. These results largely confirmed the potential importance of serotonin accumulation and salt tolerance in rice. 2) Define TDC and T5H responses to diverse abiotic stresses in rice genotypes with contrasting stress tolerances The serotonin biosynthesis in plants involves the conversion of tryptophan to tryptamine by tryptophan decarboxylase (TDC) and conversion of tryptamine to serotonin by tryptamine hydroxylase (T5H). This objective emphasizes analyzing gene expression profiles for seven putative TDC genes in several rice genotypes that differ in their stress sensitivities. Using real-time PCR assays, we have analyzed gene expression profiles for these genes in salt-tolerant Pokkali, as well as it's salt tolerant RIL FL478 compared to the salt-sensitive IR29 as well as in in another contrasting pair of genotypes, i.e., salt-tolerant NonaBokra and salt-sensitive IR28, as well as it's tolerant RIL IR63731, which are critically important to address the objectives of the proposed study. The results indicated that two of the TDC homologues (Os08g04540 and Os08g04560) were highly induced under salt stress in the roots of tolerant genotypes such as the Pokkali, FL478, Nona Bokra, IR63731 compared with the sensitive IR29 and IR28 genotypes. Although both genes were found to be induced by salt stress, Os08g04540 shown a much greater induction under salt stress. In sensitive (IR29 and IR28) genotypes these genes were only slightly induced (2 fold). The genes (Os05g43510 and Os10g23900) only displayed mild differences in response to salt stress between Pokkali and IR29 under salt stress, although this was slightly induced in FL478. The remaining genes were found to be expressed at extremely low levels, thus it was not possible to determine their stress-responsiveness using real-time PCR assays. Unlike TDC genes, T5H gene is represented by unique (Os12g16720) locus in the rice genome. Using real-time PCR assays, we have analyzed gene expression profiles for T5H in salt-tolerant Pokkali, and it's tolerant RIL FL478 compared to the salt-sensitive IR29 as well as in another contrasting pair, i.e., salt-tolerant NonaBokra and it's tolerant RIL IR63731 as well as in salt-sensitive IR28. The results showed that T5H expression levels are significantly induced in tolerant Pokkali and Fl578 compared to salt sensitive IR29. Similar pattern of expression levels was observed in another contrasting pair, i.e., tolerant NonaBokra and IR63731(greater induction) compared with sensitive IR28. These results provided additional evidences as to the cause of differential serotonin accumulation in salt-tolerant and salt-sensitive rice genotypes 3) Assess the response of t5h mutant that is defective in serotonin accumulation to diverse abiotic stresses Our third objective is to characterize t5h mutant and analyze phenotypic responses of this mutant under diverse abiotic stresses. We have obtained three alleles for this mutant and confirmed the genotype. The mutant lines phenotypic and physiological analyses are underway.
Publications
|
|