Progress 11/15/13 to 11/14/18
Outputs
Target Audience:Target audiences for this research are biotechnology companies, academic scientists, and not-for-profit agricultural research entities such as the International Rice Research Institute (IRRI). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student, one post-doctorate, and one Research Assistant Professor were partially supported and received training on this project. How have the results been disseminated to communities of interest?The results have been disseminated through three publications in 2018-2019. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We finished writing and published our findings on photoprotection in the d1 rice mutant. In collaboration, a field trial was conducted at the International Rice Research Institute (IRRI), which did not show increased yield in d1 under drought. However, we suspect this might have been because of the very mild drought conditions imposed, which did not adequately distinguish between the genotypes. We also published an invited book chapter (indexed in Pub Med) on dwarfing genes in agriculture, of which d1 is one, for a Cold Spring Harbor Perspectives in Biology text. We also finished an in silico study on the relationship between local climate conditions and natural variation among ecotypes, which was subsequently published in Nature Ecology and Evolution. This grant was acknowledged for partial salary support of the first author, Ángel Ferrero-Serrano.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Ferrero-Serrano �, Su Z, Assmann SM. 2018. Illuminating the role of the G? heterotrimeric G protein subunit, RGA1, in regulating photoprotection and photoavoidance in rice. Plant Cell Environ. 41: 451-468. doi: 10.1111/pce.13113 (cover).
- Type:
Book Chapters
Status:
Published
Year Published:
2019
Citation:
Ferrero-Serrano �, Cantos C, Assmann SM. 2019. The Role of Dwarfing Traits in Historical and Modern Agriculture with a Focus on Rice. Cold Spring Harb Perspect Biol. 11: pii: a034645. doi: 10.1101/cshperspect.a034645. PMID: 31358515
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Ferrero-Serrano �, Assmann SM 2019. Phenotypic and genome-wide association of natural variation with the local environment of Arabidopsis. Nature Ecology and Evolution. 3:274-285 doi: 10.1038/s41559-018-0754-5.
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Progress 11/15/16 to 11/14/17
Outputs
Target Audience:Target audiences for this research are biotechnology companies and academic scientists. This research was also presented to graduate students in the Penn State course Plant Biology 513 "Plant Communication and Growth Regulation". Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One postdoctorate was trained on this project in whole plant physiology and RNA-seq analysis. How have the results been disseminated to communities of interest?We have a manuscript on the improved photoprotection in the d1 mutant that we are about to submit to the journal Plant, Cell & Envrionment. What do you plan to do during the next reporting period to accomplish the goals?If we receive the requested second no cost extension for this grant, we will be able to: 1. finish our analysis of potential RLK involvement in the apical meristem and panicle phenotypes and publish with the associated RNA-seq data 2. perform the physiological characterization of our transgenic lines under well-watered vs. drought conditions 3. incorporate results from field trials at IRRI into our manuscript showing improved yield of d1 under drought conditions.
Impacts
What was accomplished under these goals?
What was accomplished 2016-2017 We performed extensive analysis of our leaf RNA-seq data and observed significant regulation of photosynthesis-related genes in the d1 mutant vs. wild type. These results correlated well with the improved photoprotection in d1 that we measured in physiological experiments. The resultant manuscript should be submitted to Plant Cell & Environment within the next two weeks. The abstract draft is provided below: We studied physiological mechanisms of light harvesting and photoprotection of a dwarf rice mutant with erect leaves, d1, in which the RGA1 gene, which encodes the GTP-binding α-subunit of the heterotrimeric G protein, is non-functional. At midday, d1 leaves exhibit a higher Photochemical Reflectance Index (PRI) relative to WT, indicative of reduced de-epoxidation of the xanthophyll cycle and increased photoavoidance capacity, which presumably arises from the erect leaf architecture. RNA-seq analysis of flag leaves revealed the upregulation in d1 of genes encoding proteins associated with photosystem I light harvesting and electron transport, photosystem II assembly, and chlorophyll and carotenoid biosynthesis, consistent with a lower degree of photoinhibitory damage. By contrast HSPs, chlorophyll degradation and ROS scavengers were upregulated in WT. We attribute this dichotomy in transcriptome signatures to the more efficient light harvesting of d1, avoiding excessive light intensities at midday, reducing photoinhibitory damage. When WT and d1 plants were subjected to the same light intensity, d1 plants exhibited a greater capacity to dissipate excess irradiance relative to WT, as determined from increased non-photochemical quenching (NPQ) in d1. The increased capacity for both photoavoidance and photoprotection in d1 plants reduced sustained photoinhibitory damage, as revealed by a higher Fv/Fm in d1. We therefore propose heterotrimeric G proteins as regulators of photoavoidance and photoprotection mechanisms in rice, and highlight the prospect of exploiting modulation of heterotrimeric G protein signaling to increase these characteristics and improve the yield of cereals in the event of abiotic stress. With new personnel in the lab, we resumed our analysis of our transgenic rice lines. A graduate student who recently joined our lab, Megan Sylvia, has been performing immunoblot analysis to quantitatively assess transgene expression, and a new graduate student, Christian Cantos, who was previously employed at the International Rice Research Institute (IRRI), is in the process of performing Southern blots, so we can identify lines with single gene insertions, which will then be subjected to physiological analyses under wild-type and drought conditions. Although we had originally planned to published our RNA-seq data on the d1 vs. wild-type apical meristems and panicles as a simple transcriptome analysis, new results appearing in the literature on other species (maize and Arabidopsis) suggest that the altered morphology of these organs may arise because RLKs can no longer signal appropriately in the absence of the G protein alpha subunit. Therefore, we are planning some tests of protein-protein interaction between rice G protein subunits and rice RLKs implicated from our transcriptome and other analyses. Positive results would significantly strengthen the impact and scope of the manuscript we are preparing. In last year's report, I summarize the absence of conclusive results from our attempted field trials in China on drought tolerance in the rice G alpha mutant, d1 (no funds from USDA/NIFA were used to support those trials). Although we had then planned to just publish our greenhouse results alone, in exciting new developments this month (Sept. 2017), the International Rice Research Institute (IRRI) has agreed to perform field trials on drought resistance of our d1 vs. wild-type lines. We will delay publication of the greenhouse results until we determine the outcome of those field trials. A copy of the correspondence with IRRI is provided below.(Note: photoperiod is not an issue for the variety we are using, Taichung65, and we do have the amount of seed needed for the field trials.) -------- Forwarded Message -------- Subject: Re: query regarding field trials on a drought tolerant rice mutant Date: Thu, 7 Sep 2017 16:47:43 +0800 From: Amelia Henry (IRRI) To: SARAH MARY ASSMANN Hi Sally, nice to hear from you! We would be happy to grow some mutants in the field for you and host your colleague. My main concern is what genetic background the mutants are in, because we have trouble with photoperiod-sensitive/temperate varieties here. Sometimes we plant them but they don't end up flowering within the season. Our dry season is usually planted in Dec-Jan. But we also grow a crop in the wet season (planting in June) when photoperiod sensitive varieties are more likely to flower. (we have rainout shelters to ensure our drought treatments even when it's rainy). The other question is the amount of seed available. For yield trials, we prefer to grow about 3m2 plots that are replicated in both well-watered and drought stress treatments, so it would be good to have >100g per genotype if possible. Kind regards, Amelia -- Amelia Henry Scientist - Drought Physiology International Rice Research Institute (IRRI)
Publications
- Type:
Journal Articles
Status:
Other
Year Published:
2018
Citation:
Ferrero-Serrano, A., Su, Z., Assmann, S.M. The transcriptome signature of the rice Galpha heterotrimeric G protein subunit mutant correlates with improved photoprotection. manuscript in preparation for Plant, Cell & Environment.
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Progress 11/15/15 to 11/14/16
Outputs
Target Audience:Target audiences for this research are biotechnology companies and academic scientists. This research was also presented to graduate students in the Penn State course Plant Biology 513 "Plant Communication and Growth Regulation". Changes/Problems:Our work on our transgenic lines was slowed by lack of appropriate personnel; however we have applied for a no cost extension to facilitate completion of those experiments. What opportunities for training and professional development has the project provided? One postdoctorate was trained on this project in whole plant physiology. How have the results been disseminated to communities of interest?We have published the drought tolerance phenotype of d1 seedlings in a scientific publication in the Journal of Experimental Botany,Vol 67: 3433-3343. What do you plan to do during the next reporting period to accomplish the goals?Revise our manuscript on improved yield in d1 mutants for submission to another journal. Finish the experiments and analyze the results concerning photoprotection in d1 vs. wild-type and determine if the results correlate with gene expression changes seen in our leaf RNA-seq experiments. Finish analysis of transgene expression in our transgenic lines and initiate physiological characterization.
Impacts
What was accomplished under these goals?
We obtained our patent: Methods of improving drought tolerance and seed production in rice US 20160362703 A1 We finished preparation of the manuscript the physiological properties of wild-type vs. d1 seedlings grown under well-watered and drought conditions, including growth and gas exchange parameters, and published it in The Journal of Experimental Botany. We finished analysis of our experiment on flowering and yield of wild-type vs. d1 grown to maturity and showed significantly increased yield of d1 as compared to wild-type under drought conditions. We prepared a manuscript and submitted it to Nature Biotechnology. However, the journal declined the manuscript because it contained only greenhouse and no field data. With coPI Dr. Hong Ma at Fudan University and his colleagues, we attempted field trials (these experiments did not use any USDA-AFRI funding). However, the rainout shelters were non-optimally designed and the fields were located far from Fudan University, so the maintenance of experimental treatments was impaired. Ultimately, no conclusions could be drawn from these experiments. We observed a dark green phenotype of the d1 mutant which led us to hypothesize that the d1 mutant might have an altered capacity to withstand high light stress. We are currently conducting experiments to determine whether d1 has improved photoprotection compared to wild-type.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Ferrero-Serrano, A., and Assmann, S.M. 2016. The alpha subunit of the rice hterotrimeric G protein, RGA1, regulates drought tolerance during the vegetative phase in the dwarf rice mutant, d1. Journal of Experimental Botany 67: 3433-3443
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Progress 11/15/14 to 11/14/15
Outputs
Target Audience:Target audiences for this research are biotechnology companies and academic scientists. This research was presented to Syngenta on December 7, 2014. This research was also presented to graduate students in the Penn State course Plant Biology 513 "Plant Communication and Growth Regulation". Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two post-doctorates were trained on this project, in methods of construct production for rice transformation, production and analysis of RNA-Seq data, yeast two-hybrid screening, and BiFC analysis. One post-doctorate obtained additional experience in gas exchange and physiological measurements. 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?With regard to understanding the mechanistic basis of d1 morphology and yield improvement, we plan to finish analysis of our three RNA-seq datasets and publish them. We plant to finish writing the manuscript and publish our studies on wild-type vs. d1 seedlings. With regard to assessing effects of RGA1 and DEP1-mut over expression on rice morphology and yield, we plan to continue to characterize the transgenic lines for protein expression from the transgene and transgene copy number.
Impacts
What was accomplished under these goals?
RNA-seq data were obtained on 5 different stages of wild-type meristems and 3 different stages of G-protein mutant meristem. This required painstaking and time-consuming hand dissection of meristems, one-by-one, until sufficient material had been collected for the RNA-seq method of transcriptome analysis. We also rescreened the interactors obtained from the first rounds of yeast two-hybrid screening. Unfortunately, only 9 positives were obtained (see table below). We did identify rice THF1 as an interactor of rice Galpha - this protein was previously identified as an interactor of Arabidopsis GPA1. OsTHF1 DEP1.1-RGB1 OsCAX7 DEP1.1 OsFPF1 DEP1.1-RGB1 DEP1.1 Gγs OsXLG1/3 RGG1/2 OsADT6 RGA1 OsKIN2 DEP1.1 OsEPCL1 RGG1/2 OsTLP7 RGG1-RGB1 We finished an experiment measuring the physiological properties of wild-type vs. d1 seedlings grown under well-watered and drought conditions, including growth and gas exchange parameters, and are preparing the manuscript for publication. We also initiated experiments measuring flowering and yield of wild-type vs. d1 grown to maturity, and are currently analyzing the data.
Publications
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Progress 11/15/13 to 11/14/14
Outputs
Target Audience:Target audiences for this research are biotechnology companies and academic scientists. This research has been presented to BASF and will be presented to Syngenta on December 7, 2014. This research was also presented in a 5 minute short talk to USDA-NIFA awardees at the annual PI meeting in May 2014, and has been presented to graduate students in the Penn State course Plant Biology 512 "Plant Resource Acquisition and Utilization". Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three post-doctorates were trained on this project, in methods of construct production for rice transformation, production and analysis of RNA-Seq data, yeast two-hybrid screening, and BiFC analysis. 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?With regard to understanding the mechanistic basis of d1 morphology and yield improvement, we plan to finish analysis of our current RNA-seq datasets and publish them. With regard to elucidating rice heterotrimeric G-protein signaling pathways, we plan to use targeted yeast 2/3 hybrid to retest the positives from the yeast 2/3 hybrid screens (since yeast 2/3 hybrid screening is susceptible to false positives). Those interactors that remain positive after rescreening will be further assessed for interaction in planta using the BiFC method. With regard to assessing effects of RGA1 and DEP1-mut over expression on rice morphology and yield, the T1 generation of our current transgenics will be screened for transgene expression, grown to maturity and seeds collected to obtain homozygous lines for physiological analyses.
Impacts
What was accomplished under these goals?
Constructs were made and transgenic lines were produced (at the Iowa State Tranformation Facility) to overexpress RGA1, the dominant dep1 mutant, and mVenus (vector control). Three libraries were constructed: d1 seedling. WT seedling and panicle. These libraries were screened with 8 different bait combinations for a total of 24 library screenings: a substantial amount of work. The table below summarizes the results of these screenings. BD-RGA1: 3 targets BD-DEP1.1 + HA-RGB1: 22 targets BD-RGB1 + HA-DEP1.1: 7 targets BD-RGB1 alone: 23 targets BG-RGG1 + HA-RGB1: 14 targets BD-GCC2 + HA-RGB1: 17 targets BD-GS3 + HA-RGB1: 34 targets BD-RGG2 + HA-RGB1: 1 target Two sets of RNA-seq data were obtained Two-month-old rice leaf Young panicle at In7 stage undergoing floral organ differentiation with around 2 cm length N =2 for each, for WT and d1 Leaf Panicle Q < 0.05 4703 1487 2 fold 2016 633 up down up down 686 1330 431 202 15 up in both lf and panicle (d1); 21 down in both lf and panicle (d1)
Publications
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