Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to NRP
LIGHT REGULATORY NETWORKS CONNECTING PHYTOCHROMES AND PHOTOPERIOD IN WHEAT DEVELOPMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0224353
Grant No.
2011-67013-30077
Cumulative Award Amt.
$499,607.00
Proposal No.
2010-04200
Multistate No.
(N/A)
Project Start Date
Apr 1, 2011
Project End Date
Mar 31, 2016
Grant Year
2011
Program Code
[A1101]- Plant Health and Production and Plant Products: Biology of Agricultural Plants
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Plant Sciences
Non Technical Summary
Light provides important seasonal information to plants and is one of the major determinants of flowering time. The three phytochrome genes present in grasses (PHYA, PHYB and PHYC) play a central role in the detection of light signals and in monitoring seasonal differences in day-length. We have recently obtained mutants of the wheat PHYC gene using a technique called TILLING and observed significant delays in flowering time under long days. We also observed that the PHYC mutations affected the regulation of other important wheat flowering genes. Our overall goal is to dissect the light signaling network involved in the photoperiodic regulation of flowering in wheat and barley. To achieve this we will generate mutants for the phyA and phyB genes using our wheat TILLING populations and combine them with the available phyC mutants into double and triple mutants. We will also characterize the natural variation of wheat and barley PHYC genes. We will then test the effect of the natural and induced phytochrome mutations on flowering time and on the regulation of other flowering genes, including those involved in vernalization, circadian rhythms, and gibberellic acid biosynthesis and degradation. The analysis of the phytochrome mutants will be complemented with the identification of the proteins that interact with the different phytochromes. Finally, we will duplicate the size of our tetraploid wheat TILLING population to increase the chances of detecting useful mutations in all the wheat genes. We will use this expanded TILLING population to generate a public collection of flowering gene mutants in a common tetraploid wheat background. This proposal will generate basic knowledge about PHYC, which is the least explored of the phytochromes. The PHYC protein interactors responsible for the transduction of the light signals to the photoperiod pathway remain largely unknown. In addition to filling this gap in our basic knowledge, a better understanding of the gene network that integrates light signals is required to precisely manipulate flowering time and optimize wheat and barley adaptation to changing environments. This study will provide barley and wheat breeders well characterized PhyC alleles to design better crosses, to predict the effect of the different alleles in flowering initiation and stem elongation, and to engineer varieties with specific flowering characteristics. A better understanding of the effect of photoperiod on spike development is also important because the number of spikelets per spike is established during this developmental stage. The number of spikelets per spike determines the maximum number of grains per spike, which is an important yield component in wheat. Finally, the expansion of the publicly available tetraploid wheat TILLING population and the generation of a public collection of flowering mutants will be tangible and valuable outcomes of this project.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011549105050%
2011549108050%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1080 - Genetics; 1050 - Developmental biology;
Goals / Objectives
The overall goal of this project is to dissect the phytochrome-mediated light signaling network responsible for the photoperiodic regulation of flowering in wheat. The specific objectives and hypothesis are: 1) Generate phyA and phyB mutants for the A and B genomes of tetraploid wheat by TILLING and combine them with available phyC mutants into double phyCphyA and phyCphyB, to test their effect on flowering time under short and long days. Based on previous rice results, we hypothesize that PHYA may have some redundant role with PHYC in the regulation of photoperiod. However, in contrast with rice, the phy mutants in wheat are expected to delay rather than accelerate flowering time. 2) Analyze the effect of null phyA, phyC, phyCphyA, and co2 mutations on the transcriptome of mature leaves using high-throughput Illumina (R) mRNA-sequencing. Differentially expressed genes detected in the previous experiment and genes with known roles in flowering regulation will also be analyzed by qRT-PCR in different mutant backgrounds and environmental conditions. We hypothesize that phyC mutations will result in the down-regulation of VRN2, FT, and GA20ox1 genes relative to the wild type and that the phyCphyA double mutants will enhance the effects of the phyC mutants. We also hypothesize that the co2 mutants will facilitate the classification of the phy regulated genes into CO2 dependent and independent classes. 3) Identify proteins that interact with PHYC-PHYB dimers in wheat leaves using a yeast-three hybrid system (Y3H pBRIDGE, Clontech) and confirm those interactions in planta. The cDNA library screen will be complemented with targeted Y3H experiments focused on protein-protein interactions between PHYC and wheat bHLH transcription factors (PIFs) homologous to known PIF interactors in other species. During the second part of the grant we will generate mutants of the wheat PIFs that interact with PHYC. We hypothesize that these pif mutants will reduce or eliminate the differences in flowering time between PHYC alleles. 4) Characterize the effect of natural variation of wheat and barley PHYC genes on flowering time. We hypothesize that some of the differences in flowering time observed in the barley and wheat germplasm are associated with allelic differences in PHYC. 5) Double the number of lines in the publicly available tetraploid wheat TILLING population and generate a collection of publicly available flowering gene mutants in a common tetraploid wheat background. We have already produced mutants in this tetraploid background as part of a previous grant. We hypothesize that doubling the TILLING tetraploid collection will greatly increase the chance of detecting truncation mutants in wheat.
Project Methods
The experimental approaches and hypotheses are described below organized by objective. Methods for objective 1. Generation of double phy mutants in wheat: The tetraploid wheat phyC mutants available in our laboratory will be complemented by the screening of phyA and phyB mutants to test putative redundant roles of these phytochromes on the regulation of flowering time by photoperiod. The A and B genome mutants for PhyA and PhyB will be backcrossed twice to Kronos to reduce the load of additional mutations, and then mutations in the A and B genomes will be intercrossed to generate the null mutants. To test the contributions of the phyA, phyB and co2 mutations, we will compare flowering time of the null phyC mutant with that of the phyBphyC, phyAphyC, and phyCco2 double null mutants, under both short and long days. Methods for objective 2. The will study the effect of different mutants on the leaf transcriptome, and the regulation of FT. The transcript levels of FT are closely correlated with those of VRN1 and with flowering time. Whole transcriptome analyses will use new high-throughput sequencing technologies (mRNA-seq) and targeted qRT-PCR experiments. In the mRNA-seq experiments we will compare the wheat leaf transcriptome of null phyA, phyC, phyAphyC, and co2 mutants with non-mutant Kronos plants. Experiments will be performed in controlled environment chambers under long day photoperiod (16 h light / 8 h dark). These experiments will be done using un-vernalized winter Kronos line to keep the plants in the vegetative phase and restrict the differences in transcript levels to the genes affected by the phy mutations upstream of VRN1. Transcript levels of flowering and clock related genes will be measured by qRT-PCR in plants carrying different combinations of phyA, phyB, phyC, vrn-A1, and co2 mutants, tested both under long and short days. Methods for objective 3: To identify proteins that interact with PHYC in wheat we will construct a cDNA library from leaves of diploid winter wheat grown under long day photoperiod. Wheat cDNAs will be fused to the GAL4 activation domain in pGADT7 to generate the library. Since PHYC does not form homodimers and requires hetero-dimerization with the active form of PHYB, we will use the yeast three-hybrid system developed to screen the cDNA for PHYC-PHYB dimer interactors. The PHYC protein will be expressed as a fusion to the GAL4 DNA-BD, while PHYB will be expressed as a third protein using the pBRIDGE system. The most interesting interactions will be validated in planta using a TAP-tagged PHYC transgenic wheat plant. Methods for objective 4: natural variation of wheat and barley PHY genes. We will sequence PHYC from barely and from the A and B genomes of cultivated and wild tetraploid wheats grown at different latitudes to characterize the allelic diversity in this gene. If we identify natural mutations expected to alter the function of PHYC we will develop segregating populations to test their effect. Methods for objective 5: We will use the same TILLING protocols used successfully before to develop our current tetraploid TILLING population to double the number tetraploid wheat TILLING lines.

Progress 04/01/11 to 03/31/16

Outputs
Target Audience:The target audiences of this project include wheat and barley breeders, and cereal geneticists. The knowledge and information generated for the different PHYC, PHYB, VRN1, FT and ELF3 alleles can be used by barley and wheat breeders to design better crosses, and to predict the effect of the different alleles in the different phases of reproductive development. This information is useful for breeders to engineer barley and wheat varieties with specific flowering characteristics. The target audiences of the basic knowledge and reverse genetics resources generated in this study are the different scientific communities working in the understanding of the regulation of flowering in plants. The generation of mutants of multiple flowering genes in a single genetic background provides a useful tool to study epistatic interactions among flowering genes. Wheat researchers and breeders are using these mutants to test the effect of different developmentally regulated processes on their targeted genes and pathways. Finally, all researchers interested in functional studies in wheat have now access to a sequenced tetraploid TILLING population, which provides a tool to rapidly identify mutants in most wheat genes and to characterize gene function. Changes/Problems:There have been no major changes in the proposed approaches and objectives What opportunities for training and professional development has the project provided?This project provided a multidisciplinary environment for training to two postdocs, three PhD students and four undergraduate students. Postdoctoral scholars: Project Scientist Lindsay Shaw (Woman/Australia) has been hired to replace postdoctoral scholar Andrew Chen (he returned to his home country for family reasons). Ph.D. students: Nestor Kippes (Male/Hispanic) is responsible for functional characterization of the PHYB mutants, the vernalization genes, and their connection to the photoperiodic response. Hans Vasquez-Gross initiated his PhD program in genetics and bioinformatics in 2015, with a focus on the development of bioinformatics tools for the sequenced TILLING population. ?Undergraduate students: The Recruitment of the undergraduate through the "UC Davis Academic Program" increased opportunities for minority students. Four undergraduate students (Marielle Palatino, Anna Dominique-Rodriguez, Maisie Borg and Karla Ocampo) and one high school student (David Tan) have received trainings under this project. Graduate Students Receiving degrees: Ph.D. student Rebecca Nitcher (Woman, first generation college student). She has completed her PhD successfully and now is a breeder at Nunhems-Bayer. Ph.D. student Nestor Kippes (Male, Hispanic). He has completed his PhD successfully and is now working as a postdoc in Dr. Dubcovsky's laboratory. How have the results been disseminated to communities of interest?Results have been disseminated in four peer reviewed publications in high impact scientific journals (PNAS, BMC Plant Biology and Journal of Heredity). The PD presented a plenary lecture at the Plant and Animal Genome conferences in San Diego (January 13, 2016) and invited talks in China (Fudan University, Shanghai, August 14, 2015; Shandong Agricultural University, August 17, 2015; Yangling, Shaanxi, August 19, 2015.) and India (Punjab Agricultural University, Ludhiana, India, August 28, 2015) describing the new TILLING resource. Students and postdocs who worked on the project have made five posters and oral presentations in the Plant and Animal Genome XXIV and other scientific meetings. The WheatEXP database has been made public and was announced through Grain Genes. Results have been presented to growers and the wheat industry in field days at UC Davis each yearin May, from 2011 to 2015. 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: We generated loss-of-function mutations for PHYC and PHYB in tetraploid wheat by combining mutations in the A and B genome of each gene (phyCAB and phyBAB). We demonstrated that plants homozygous for these mutations flowered more than 3 month later than wild-type plants under long days but the difference was smaller under short days. These results demonstrated a strong interaction between phytochromes (PHYC and PHYB) and photoperiod. This interaction is further supported by the drastic downregulation of the central photoperiod gene PHOTOPERIOD 1 (PPD1) and its downstream target FLOWERING LOCUS T1 (FT1) in both the phyCAB and phyBAB mutants. These results showed that both genes are required for the promotion of flowering in wheat under long days. Characterization of the elf3 mutant also uncovered a strong epistatic interaction between PPD1 and ELF3, which indicates that the expression of ELF3 is required for the PPD1 acceleration of flowering under long days. We also analyzed the dosage effects of different mutation in the phyBAB and phyCAB and demonstrated that different dosages of phyB and phyC mutations in the different homoeologs can be used to modulate flowering time. For example, the combination of homozygous mutations phyBA phyCB resulted in a 43 days delay in flowering, while homozygous mutations in either gene separately showed no effect on flowering. In the course of this project we also analyzed the effect of mutations in the ELF3, VRN2, CO1, CO2, FT1 and FT2 genes and showed that they can also be used to fine tune wheat flowering time and to improve adaptation of wheat to different environments. Objective 2: We performed replicated RNAseq studies of phyBAB and phyCAB null mutants and their respective wild type isogenic lines. Leaf tissue was harvested from LD-grown four-week-old plants. We sequenced a total of 32 RNA-seq libraries, generating an average of 49.1 million 50 bp single-end reads per sample. We identified 82 genes that were significantly up or down regulated in both the phyBAB and phyCAB null mutants relative to their respective wild-type controls. Among these genes are several well-characterized positive regulators of flowering, including PPD1, FT1 and VRN1, and several circadian clock and clock-output genes, which may also contribute to the observed differences in heading time. Eight-fold more genes were differentially regulated only in the phyBAB null mutant (2,202) than only in the phyCAB null mutant (261). The PHYB-regulated genes were enriched in components of the auxin, gibberellin and brassinosteroid biosynthesis and signaling pathways, and in transcription factors with putative roles in regulating vegetative development and shade-avoidance responses. Several genes involved in abiotic stress tolerance pathways were also found to be regulated by PHYB. In summary, this study showed that PHYB and PHYC are both required for the photoperiodic induction of wheat flowering genes, whereas PHYB alone regulates a large number of additional genes involved in hormone biosynthesis and signaling, shade-avoidance response, and abiotic stress tolerance. This RNAseq study provided a comprehensive overview of the PHYB- and PHYC-mediated transcriptional changes during light signaling, and an initial step towards the dissection of the phytochrome regulatory gene network in wheat. Further dissection of this pathway will be required to explore the individual phytochrome-mediated developmental responses and, in the case of PHYB, to modulate shade avoidance responses in wheat independently of its effect on flowering time. Objective 3: Phytochrome Interacting Factors (PIFs) belong to a small subset of basic helix-loop-helix (bHLH) transcription factors. Upon light-induced nuclear translocation, phytochromes interact with PIFs, subsequently induce rapid phosphorylation and degradation of PIFs to regulate gene expression. In addition to their critical roles in phytochrome signal transduction, PIFs also act to coordinate environmental signals with endogenous clock and hormone signals, such as GA, BRs and Auxin, and function as systems integrators in plant development. We have identified four wheat PIFs that interact with PHYB, PHYC or both in yeast two-hybrid assays. Tilling mutants have been identified for these PIFs and we are currently backcrossing them to wild-type Kronos to reduce background mutations. We are also intercrossing mutations in the A and B genomes to generate loss-of-function mutants for individual PIFs. The functional analysis of PIFs will help us to better understand mechanistically the downstream regulatory networks controlled by wheat PHYB and PHYC. Objective 4: During the course of this project we have completed the evaluation of near isogenic lines carrying the novel FT-B1 allele from the variety "Hope". This rare FT1 allele carries a retrotransposon insertion in its promoter and is expressed at higher levels than the normal allele, particularly under short day photoperiod, resulting in early-flowering under both long and short day photoperiods. We demonstrated that the Hope FT-B1 allele is epistatic to the VERNALIZATION 1 (VRN1) gene. On average, the introgression of Hope FT-B1 into six different genetic backgrounds resulted in an average 2.6 days acceleration of flowering (P<0.0001) and 4.1% increase in spike weight (P=0.0093). These results suggest that the Hope FT-B1 allele will be a useful tool to accelerate floral development with a simultaneous positive effect on grain yield. We have initiated introgression of this allele in several wheat breeding programs. During this project we also completed a study of mutants for the two homologs of FT2 (a paralog of FT1) and demonstrated that in the field, these mutations result in a small but significant delay in flowering and altered spike morphology. Finally, we completed a detailed characterization of genes from the gibberellin (GA) pathway, which play important roles in the regulation of wheat growth, plant height and development. We identified and characterized the genes encoding 2-oxoglutarate-dependent dioxygenases from the GA biosynthetic and inactivation pathways in wheat (bread and durum) and barley. We identified genes encoding the bifunctional enzyme GA 3b,18-dihydroxylase, which produces GA131 from GA9. As both GA54 and GA131 have reduced biological activity compared with GA4 and GA1, these novel wheat and barley genes may play a role in modulating GA signalling in developing grains. ?Objective 5: We took advantage of the redundancy of the polyploid wheat genome to generate sequenced TILLING populations for tetraploid and hexaploid wheat. We developed an exome capture platform including 82,511 genes (286,800 exons) selected from multiple transcriptome analyses and used it to re-sequence the protein coding regions of 1,535 tetraploid and 1,209 hexaploid wheat EMS mutants. Using a stringent threshold, we detected and catalogued 4.26 million induced mutations in tetraploid wheat and 7.32 million in hexaploid wheat. This number of mutations is sufficient to generate truncations or deleterious amino acid changes in most wheat proteins (>38 mutations per kb, per population), facilitating their functional characterization. Since loss-of function mutations in one wheat homoeolog are frequently masked by redundancy in the other homoeologs, wheat breeders and natural selection often act on dominant mutations. This new resource will accelerate the identification of mutations in the duplicated homoeologous genes, which can be combined to generate complete loss-of-function mutations. Since the effect of recessive alleles is usually hidden by homoeolog redundancy in polyploid wheat, these complete loss-of-function mutations will expose phenotypic variation that has not been exploited before. A database searchable by BLAST and a visualization tool have been created. The database is currently used by more than 150 researchers distributed worldwide.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Pearce, S., H. Vazquez-Gross, S.Y. Herin, D. Hane, Y. Wang, Y.Q. Gu, J. Dubcovsky. 2015. WheatExp: an RNA-seq expression database for polyploid wheat. BMC Plant Biology. 15:299. PMC4690421
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Nitcher, R., S. Pearce, G. Tranquilli, X. Zhang, J. Dubcovsky. 2014. Effect of the Hope FT-B1 allele on wheat heading time and yield components. J. Heredity 105:666-675.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Pearce, S.P., K. A.K. Huttly, I.M. Prosser, Y.-D. Li, S.P. Vaughan, B. Gallova, A. Patil, J.A. Coghill, J. Dubcovsky, P. Hedden and A.L. Phillips. 2015 Heterologous expression and transcript analysis of gibberellin biosynthetic genes of grasses reveals novel functionality in the GA3ox family. BMC Plant Biology 15:130
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Kippes N., J.M. Debernardi, H. Vasquez-Gross, B.A. Akpinar, B.H., K. Kato, S. Chao, E. Akhunov and J. Dubcovsky. 2015. Identification of the VERNALIZATION 4 gene reveals the origin of spring growth habit in ancient wheats from South Asia. Proc. Natl. Acad. Sci. U.S.A. 112: E5401E5410. PMC4593092
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Li, C., H. Lin, J. Dubcovsky. 2015. Factorial combinations of protein interactions generate a multiplicity of florigen activation complexes in wheat and barley. The Plant Journal. 84:70-82 (as submitted in previous report)
  • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Pearce, S., N. Kippes, A. Chen, J.M. Debernardi and J. Dubcovsky. 2016. Replicated RNA-seq studies using wheat PHYTOCHROME B and PHYTOCHROME C mutants reveal shared and specific functions in the regulation of flowering and shade-avoidance pathways. Submitted to BMC Plant Biology
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Alvarez, M. A., G. Tranquilli, S. Lewis, N. Kippes and J. Dubcovsky. 2016. Genetic and physical mapping of the earliness per se locus EpsAm1 in Triticum monococcum identifies EARLY FLOWERING 3 (ELF3) as a candidate gene. Plant and Animal Genome XXIV, January 9-13, San Diego. P0843 and W787
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Krasileva K.V., H. Vasquez-Gross, P. Bailey, F. Paraiso, L. Clissold, J. Simmonds, X. Wang, T. Howell, C. Fosker, A. Phillips, S. Ayling, C. Uauy and J. Dubcovsky. 2016. Applying exome-capture technology to uncover 11.5 million mutations in wheat genes. Plant Genetics and Breeding Technologies II: http://viscea.org/index.php/plant-genetics-breeding Vienna, Feb 1-2, 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Vasquez-Gross H., K.V Krasileva, F. Paraiso, X. Wang., T.R. Howell1, P.C. Bailey, S. Ayling, C. Uauy, S. Pearce and J. Dubcovsky. 2016. Using wheat BLAST database to search for mutations and expression. Plant and Animal Genome XXIV, January 9-13, San Diego. C24
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Shaw, L., B. Lv, R. Nichter, C. Li, X. Han, D. Fu, and J. Dubcovsky. 2016. Characterization of Flowering Locus T2 (FT2) in wheat and Brachypodium. Plant and Animal Genome XXIV, January 9-13, San Diego. P0837
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Kippes, N., Chen, A., X. Zhang, A. Lukaszewski, and J. Dubcovsky. 2016. Modulation of the vernalization requirement of hexaploid wheat by non-functional VRN2 alleles. Plant and Animal Genome XXIV, January 9-13, San Diego. P0846


Progress 04/01/14 to 03/31/15

Outputs
Target Audience: The target audience of this project includes wheat and barely breeders and scientists and basic scientists interested in development and gene regulation. The resulting varieties with increased adapataion are targeted to growers and the wheat industry in general. Changes/Problems: Problem Changes:There have been no major changes in the proposed approaches and objectives. The project has generated high impact publications and valuable germplasm and the same trend is projected for the last year. Excellent progress has been made with the sequencing of the 1500 TILLING wheat lines. The sequencing has been completed and half of the data has been analyzed. Since only bioinformatics analyses are pending and the pipeline for mutation detection has been extensively tested, we do not expect major problems to complete this objective. One small problem we detected is that some mutations (<2%) have been masked by the presence of duplicated contigs in the wheat reference genome, since we only use uniquely mapped reads. We are currently developing a separate pipeline to recover these last mutations. We plan to eliminate duplicated contigs from the reference and remap the non-uniquely map reads. This pipeline is under development but has not been tested yet. Even if this fails it will result in the loss of a small proportion of SNPs in recently duplicated genes. What opportunities for training and professional development has the project provided? This project provides a multidisciplinary environment for training one postdoc, two PhD students and one undergraduate student. Project Scientist : Project Scientist Lindsay Shaw (Woman/Australia) has been hired to replace postdoctoral scholar Andrew Chen (he returned to Australia for family reasons). Ph.D. student: Nestor Kippes (Male/Hispanic) is responsible for functional characterization of the vernalization genes and their connection to photoperiod response. Nestor currently supervises one undergraduate student Marielle Palatino (Woman/Hispanic). Undergraduate student: The Recruitment of the undergraduate through the "UC Davis Academic Program" increased opportunities for minority students. Three other undergraduate students (Anna Dominique-Rodriguez, Maisie Borg and Karla Ocampo) and one high school student (David Tan) have also received trainings under this project before. Graduate Students Receiving degrees:Ph.D. student Rebecca Nitcher (Woman, first generation college student) was responsible for the flowering gene FT studies and its connection to photoperiod. She has completed her PhD successfully and now is a breeder at Nunhems-Bayer. How have the results been disseminated to communities of interest? Results have been disseminated in three peer reviewed publications in high impact scientific journals (PNAS, The Plant Journal and PLoS ONE). The PD presented talks at three local and three International Scientific Conferences and Events. Students and postdocs for the project have made three oral presentations in the Plant and Animal Genome XXIII, January 10-14, San Diego. Results have been presented to growers and the wheat industry in Alfalfa & Grains Symposium, Long Beach, CA and improved lines were presented to growers and wheat industry representatives in a field day at UC Davis in May 2014 What do you plan to do during the next reporting period to accomplish the goals? During the last year of this gran we will complete the characterization of the PHYB mutants and the double PHYB-PHYC mutants. We will determine the effect of different dosages of these mutations on heading time. We will also characterize the effect if the double FT1 -FT2 mutants, the quadrupole VRN1-FUL2-FUL3-VRN2 homozygous mutants, and the CO1-CO2-PPD1 mutants on heading time and spike morphology. We will complete the night break experiments in the phyC and phyB mutants and the RNAseq study in the same mutants and the wild type.During 2015 we will submit for publication the final positional cloning of VRN4 and its impact on the discovery of novel allelic variants in the VRN1 gene, which can have a significant impact in winter wheat breeding programs. We will also continue the introgression of the new early flowering FT1 alleles that have a positive effect on the number of grains per spike into the best breeding lines of the UCD wheat breeding program. For the TILLING population we will complete the sequencing and bioinformatics analysis of the 1500 lines and complete the development of a searchable database that will be made openly available in 2015. Currently the partial database is open to wheat researchers by request.

Impacts
What was accomplished under these goals? Impact: Significance of the Project's Findings to Food and Agriculture: This project has demonstrated a unique role of the PHYC on the photoperiodic regulation of wheat flowering time. It has also shown that the wheat PHYC has the ability to form homodimers and to act independently of the other phytochromes, which differentiates wheat PHYC from its rice and Arabidopsis homologs. Our results established the connection between PHYC and the hormone gibberellin, and demonstrated that this hormone plays an important role in the development of wheat spikes. The characterization of induced variation in the phytochromes and downstream flowering genes has generated alleles with different effects on flowering time that can be used to engineer barley and wheat varieties with improved adaptation to changing environments. This project has generated isogenic lines with different flowering alleles, mutants for multiple flowering genes and an increased TILLING population that is publicly available. The sequencing and annotation of the first 750 lines resulted in 1,500,000 mutations in the coding regions of wheat genes. Once completed, the 1500 mutants will provide 3-4 million mutations in the coding regions of wheat genes. This resource is being used to characterize gene functions in wheat and to develop new allelic variation in genes that are valuable for agriculture. Objectives and Accomplishments: Objectives 1 and 2: We generated loss-of-function mutations for PHYC in tetraploid wheat (phyCAB) and showed that plants homozygous for these mutations flowered on average 108 days later than wild-type plants under long days but only 19 days later under short days. These results demonstrated a strong interaction between PHYC and photoperiod. This interaction is further supported by the drastic downregulation of the central photoperiod gene PHOTOPERIOD 1 (PPD1) and its downstream target FLOWERING LOCUS T1 (FT1) in the phyCAB mutant. These two genes are required for the promotion of flowering under long days, suggesting that the PHYC-mediated light activation of PPD1 expression plays a prominent role in the acceleration of wheat flowering under long days. Plants homozygous for the phyCAB mutations also show altered expression profiles of circadian clock and clock-output genes, which may also contribute to the observed differences in heading time. Our results highlight the important differences in the photoperiod pathway between the temperate grasses and the model plant species. These discoveries have been presented in a research paper in PNAS (2014, 111:10037-10044). We have recently found that plants homozygous for loss-of-function mutations in both PHYB homologs (phyBAB) also flower very late under long days. This result suggests that the light-activation of PPD1 transcription is most likely mediated by PHYC-PHYB homodimers. To study the effect of different dosage combinations of mutations in these two genes on wheat flowering we intercrossed the phyBAB and phyCAB. The combination of homozygous mutations phyBA phyCB resulted in a 43 days delay in flowering, while homozygous mutations in either gene separately showed no effect on flowering. These results confirmed that the PHYB-PHYC heterodimers play a critical role in the regulation of flowering time in wheat. Objective 3: We have also demonstrated that wheat PHYC forms signaling active homodimers that translocate into the nucleus in red light to mediate photomorphogenic responses even in the absence of all other phytochromes. This contrasts with results in Arabidopsis and rice, where PHYC is unstable and functionally inactive unless it heterodimerizes with PHYB. The formation of PHYC homodimers was validated by yeast two-hybrid assays and by split-luciferase assays in rice protoplasts. Yeast two-hybrid screens of a cDNA library constructed from 6-8 week-old leaves of diploid wheat T. monococcum (accession G3116) were performed using the C-terminus of the PHYC protein as bait. Three transcription factors were identified in this screen including a PHYTOCHROME INTERACTING FACTOR (PIF), designated as TaPIF4, which also interacts with PHYB. PIFs are well-characterized protein interactors of phytochromes in Arabidopsis, where they play important roles in phytochrome signal transduction as well as in coordination of environmental signals with endogenous clock and hormone signals. Using our tetraploid Kronos TILLING population we have already obtained truncation mutants for both A and B genome copies of TaPIF4. These mutants are currently being backcrossed to wild-type Kronos plants to reduce background mutations and will be then combined by intercrossing to generate a TaPIF4-null mutant. We will use this null mutant to determine the effect of TaPIF4 on PPD1 expression and on wheat flowering time under LD and SD. The additional transcription factors that interact with PHYC identified in the yeast two-hybrid screen are also being backcrossed to wild-type Kronos plants now to reduce background mutations and to generate null mutants to test their effect on flowering. To identify genes transcriptionally regulated by the phytochromes in wheat, we carried out an RNA-seq experiment to compare the transcriptomes of PHYB/phyB-null and PHYC/phyC-null genotypes in a LD photoperiod. We used 4 biological replicates for mutants and wild type and repeated the complete experiment twice. We only retained those genes which exhibit significant differential expression profiles in both experiments to minimize the false discovery rate. We generated >35M sequencing reads for each sample on a HiSeq2500 sequencer. After trimming for quality and adapter contamination, reads were mapped to the latest release of the wheat genome. Genes differentially expressed between genotypes were identified in pairwise comparisons using DESeq. Our preliminary analyses identified 434 high-confidence PHYC-regulated genes and 625 PHYB-regulated genes, with 113 of these genes differentially expressed in both comparisons. We are currently annotating and characterizing these genes. Objective 4: During this year we have completed the characterization of transgenic and mutant lines for FT1 and demonstrated that FT1 is not essential for flowering in wheat. Our study also demonstrated that FT1 plays an important role in the regulation of other FT-like genes (Lv, Nitcher, et al. 2014). We have generated mutants for the two homologs of the FT2 paralog and combined these mutations with the FT1 mutations to study the effect of the double null mutant on flowering time and spike morphology. A study demonstrating a connection between FT1, gibberellins, spike development and photoperiod has also been completed and we published those results in Plant Physiology (Pearce et al. 2013, reported in previous progress reports). Results from this paper provide an entry point to the gene network regulating spike development, a trait that is important for wheat yield potential. Objective 5: We developed an exome capture platform for wheat including 286,800 exons (76% padded with 30 bp of genomic sequence) from 82,511 genes. We then generated a new reference to map these reads including the A and B genomes from the IWGS plus 40,975 contigs (33.8 Mb) generated from the assembling of unmapped reads from 48 Kronos TILLING lines. Using this enriched reference we were able to map 98.2% of the reads (94% as pairs). We used this wheat exome capture to re-sequence 750 EMS mutant lines from out tetraploid (var. Kronos) TILLING population and identified 1,500,000 mutations in the coding regions of wheat genes that are available in a searchable database. Once completed, this resource will provide 3-4 million mutations in the coding regions of wheat genes and the ability to knock out most wheat genes.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Chen, A., C. Li,, W. Hu, M. Lau, H. Lin, N.C. Rockwell, S.S. Martin, J.A. Jernstedt, J.C. Lagarias, and J. Dubcovsky. 2014. PHYTOCHROME C plays a major role in the acceleration of wheat flowering under long days. Proc. Natl. Acad. Sci. U.S.A. 111:10037-10044
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Lv B, Nitcher R., Han X., Wang S., Ni F., Li K., Pearce S., Wu J., Dubcovsky J. and Fu D. (2014) Characterization of FLOWERING LOCUS T1 (FT1) gene in Brachypodium and wheat. Plos One, 9, e94171.
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Li, C., H. Lin, J. Dubcovsky. Factorial combinations of protein interactions generate a multiplicity of florigen activation complexes in wheat and barley. 2015. The Plant Journal. Submitted
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Dubcovsky, J. 2014. Identifying valuable gene variants for wheat improvement. Wolf Awards Conference, May 29, 2014, Jerusalem, Israel.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Dubcovsky, J. 2014. Perceiving, integrating, sending and deciphering environment signals without a nervous system. University of Tel Aviv, June 2, 2014, Tel Aviv, Israel.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Dubcovsky, J. 2014. Integrating temperature and light signals in the regulation of wheat reproductive development. University of Haifa, June 2, 2014, Haifa, Israel.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Dubcovsky, J. 2014. Dissecting the Complex Flowering Pathway in Wheat Using Novel Reverse Genetic Resources. ASA, CSSA, and SSSA Conference, Nov. 2-5, 2014, Long Beach, CA. Keynote speaker at the Ron Phillips Plant Genetics Lectureship.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Dubcovsky, J., 2014. Improving California Wheat for Grain and Forage. Alfalfa & Grains Symposium, Long Beach, CA December 10-12.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Dubcovsky, J. 2015. Exome sequencing of wheat mutant populations opens a new era for wheat functional genetics. IWGSC workshop Plant and Animal Genome XXIII, January 10-14, San Diego.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Vasquez-Gross, H., K. Krasileva, F. Paraiso, X. Wang, T.R. Howell1, P.C. Bailey, S. Ayling, C. Uauy and J. Dubcovsky. 2015. Using the wheat TILLING database to search mutants of interest. Plant and Animal Genome XXIII, January 10-14, San Diego.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Krasileva, K.V., V. Buffalo, S. Ayling, P. Bailey, S. Wang, H. Vasquez-Gross, S. Pearce, E. Akhunov, C. Uauy and J. Dubcovsky. 2014. One plant  multiple genomes: phasing homeologs in polyploid wheat. Plant and Animal Genome XXIII, January 10-14, San Diego
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kippes, N., B.A. Akpinar, H. Vasquez-Gross, S. Chao, A. Eduard, B. Hikmet, K. Kato and J. Dubcovsky. 2015. Positional cloning of wheat vernalization gene VRN-D4 reveals the origin of spring growth habit in ancient hexaploid wheats from India.. Plant and Animal Genome XXIII, January 10-14, San Diego


Progress 04/01/13 to 03/31/14

Outputs
Target Audience: The main target audiences for this project include the wheat and barley breeders. In most environments, there is a very narrow window for optimum flowering time that will maximize wheat and barley grain yield potential. The knowledge and information generated from studying the effect of the different PhyC, VRN1, and FT alleles will be useful for barley and wheat breeders to design better crosses, to predict the effect of the different alleles in flowering initiation and stem elongation, and to engineer varieties with specific flowering characteristics. Another target audience of this project is the scientific community working in the understanding of the regulation of flowering in plants. The generation of flowering mutant collections in a common genetic background will also provide an essential tool to study epistatic interactions among flowering genes. Wheat researchers in different areas will be able to use these mutants to test the effect of different developmentally regulated processes on their targeted genes and pathways. Finally, all researchers interested in functional studies in wheat can benefit from the size increase of our publicly available tetraploid wheat TILLING population, which increases the chances of identifying truncation mutations in any wheat gene. DNAs and seeds of the TILLING populations are publicly available Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project provides a multidisciplinary environment for training a postdoctoral scholar (CoPI Dr. Andrew Chen), two Ph.D. students (Nestor Kippes and Rebecca Nitcher) and several undergraduate students in the integration of genetics, biochemistry and computational tools. The postdoctoral scholar and the Ph.D. students have been mentoring undergraduate students. Recruitment of the undergraduate student through the "UC Davis Academic Program" increases opportunities for minority students. Collaborations in the biochemical aspects of the project have been established with Dr. Clark Lagarias at the Department of Molecular and Cellular Biology, University of California, Davis How have the results been disseminated to communities of interest? Results generated this year have been publicly available through three peer reviewed publications and 10 talks and poster presentations in national and international meetings and symposia by the PI, postdoc and PhD students involved in the project. In three of these talks (2 of them international) the results of these studies were presented as the KEYNOTE lectures. In addition, we developed isogenic lines carrying different alleles of flowering genes that are being distributed for testing in other locations. We have provided TILLING mutants generated in this study for those investigators that requested them and submitted the new germplasm to the National Small Grain Collection. New germplasm was also presented to wheat growers and industry in a well-attended field day at UC Davis. What do you plan to do during the next reporting period to accomplish the goals? We will continue with the planned activities in the grant. We will test the phytochrome C mutants under short day conditions in a photoperiod sensitive background. WE plan to test the recently developed CO1 and CO2 mutants and make the crosses required to test the interactions with the PHYC mutants. We will test the PHYB mutants and make the crosses required to test the interactions with the PHYC and CO1 and CO2 mutants. We will continue with the sequencing by exome capture of the TILLING mutants of our population and with the development of the public database to facilitate the search for mutations in specific genes. We will test the effect of the new flowering alleles in the field to evaluate their effect on yield and other agronomic traits.

Impacts
What was accomplished under these goals? Objective 1: We have generated null mutants for the phyA, phyB, and phyC genes. The phyA mutants show normal flowering, the phyC-null mutants flowered more than 100 days later than the wild type and the phyB mutants failed to flower. This represents a significant change in our previous knowledge of the role of phytochromes in the regulation of flowering time. We have also completed the development of homozygous mutants for CO1 and CO2 and are now combining both genes. These mutations have smaller effects on flowering under long days but larger effects under short days. Objective 2: We completed experiments that demonstrate a strong interaction between PHYC and photoperiod. The phyC-null mutations cause a much larger delay in flowering under LD (114-125 days) than under SD (12-20 days). Transcript analysis revealed that the main photoperiod gene PPD1 and its downstream target FT1, which encodes a mobile florigen protein, were both completely down-regulated in the phyC-null mutants. The introduction of an FT over-expressing allele in the phyC-null mutants accelerated flowering by 20 days under long days confirming that the downregulation of FT in the phyC-null mutants plays an important role in the flowering delay observed in these mutants. Plants homozygous for the phyC-null mutations also show altered profiles of several circadian clock and clock-output genes, which may also contribute to the observed differences in heading time. The transcription studies in the phyC-null mutants have been very useful to clarify previously contradicting results in wheat flowering models and to establish differences and similitudes with the photoperiod pathways in other plant species. Objective 3: In Arabidopsis and rice, PHYC is unstable and functionally inactive unless it heterodimerizes with another phytochrome. We have shown by yeast two-hybrid assays that in contrast to the two model species, the wheat PHYC protein can form homodimers. We have further confirmed this result by split-luciferase assays in rice protoplasts, and demonstrated that PHYC dimers can translocate to the nucleus in the absence of other phytochromes and restore some of the photomorphogenic characteristics of etiolated phy-null Arabidopsis seedlings. We generated a construct expressing a fusion of PhyC and the red fluorescent protein DsRED under the control of 35S promoter. This construct was then transformed into the Arabidopsis phytochrome quintuple mutant (phyABCDE) background, where none of the five Arabidopsis phytochromes is functional. Confocal microscopy analysis of the 35S-PhyC-DsRED/phyABCDE transgenic plants shows a clear nuclear localization pattern for the PhyC protein. This result demonstrated that wheat PhyC protein can accumulate in the nucleus in the absence of other phytochromes. In summary, we demonstrated that when expressed in the Arabidopsis phy-null mutant, wheat PHYC forms signaling active homodimers that translocate into the nucleus under red light and mediate photomorphogenic responses. This represents a significant change in previous knowledge about the roles of PHYC in flowering plants. To identify novel protein interactors of the wheat PhyC, we have constructed cDNA libraries from 6-8 week-old leaves of two diploid wheat accessions. A yeast two-hybrid screening of the cDNA libraries has been conducted using the C-terminus and the complete PhyC protein as baits. We are currently validating and confirming the positive interactions identified in the library screening. Objective 4: We demonstrated that a dominant VRN3 allele for early flowering in barley was associated to an increase in FT copy number and published those results in MGG. We also showed that exogenous gibberellin (GA) application accelerates wheat spike development under short days, but only in wheat lines expressing VRN1. The simultaneous presence of GA and VRN1 results in the upregulation of the floral meristem identity genes SOC1-1 and LFY, whereas inhibition of GA biosynthesis with paclobutrazol precludes the long day induction of these two genes. We demonstrated the upregulation of GA-biosynthetic genes in the apices of plants transferred from short to long days, and in photoperiod insensitive and transgenic wheat plants with increased FT transcription under short days. These results were published in Plant Physiology, and can open the door for studies of early spike development in wheat. Finally, we generated a high density map of the vernalization gene VRN-4 and characterized its epistatic interactions with other flowering genes. These results were published in MGG (see publication list). Objective 5: We duplicated the number of mutants in our tetraploid TILLING population (1368 to 2700) and developed an exon capture platform to start sequencing most of the wheat genes in the mutant lines. We developed a bioinformatics pipeline to identify mutations in the captured sequences from each mutant and to separate those mutations from nucleotide polymorphisms among duplicated genes in the polyploid wheat genome. Analysis of the first 18 mutant sequences demonstrated that we can identify an average of 820 mutations per line with a 95% confidence. The presentation of these preliminary results in scientific meetings has contributed significant changes in the strategies that several of our colleagues are using to identify the function of their target genes. Several scientists are requesting mutants and/or are visiting my lab to screen the TILLING population and obtain their own mutants. The huge number of mutations identified in the wheat TILLING populations is changing the paradigm of what can be done in gene functional analyses in wheat.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: The main target audiences for this project include the wheat and barley breeders. In most environments, there is a very narrow window for optimum flowering time that will maximize wheat and barley grain yield potential. The knowledge and information generated from studying the effect of the different PhyC, VRN1, and FT alleles will be useful for barley and wheat breeders to design better crosses, to predict the effect of the different alleles in flowering initiation and stem elongation, and to engineer varieties with specific flowering characteristics. Another target audience of this project is the scientific community working in the understanding of the regulation of flowering in plants. The generation of flowering mutant collections in a common genetic background will also provide an essential tool to study epistatic interactions among flowering genes. Wheat researchers in different areas will be able to use these mutants to test the effect of different developmentally regulated processes on their targeted genes and pathways. Finally, all researchers interested in functional studies in wheat can benefit from the size increase of our publicly available tetraploid wheat TILLING population, which increases the chances of identifying truncation mutations in any wheat gene. DNAs and seeds of the TILLING populations are publicly available
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Pearce, S., L.S. Vanzetti, J. Dubcovsky. 2013. Exogenous gibberellins induce wheat spike development under short days only in the presence of VERNALIZATION 1. Plant Phys. 163: 14331445
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Kippes N., J. Zhu, A. Chen. L.S. Vanzetti, A. Lukaszewski, H. Nishida, K. Kato, J. Dvorak, J. Dubcovsky (2013) Fine mapping and epistatic interactions of the vernalization gene VRN-D4 in hexaploid wheat. Mol. Genet. Genomics DOI 10.1007/s00438-013-0788-y
  • Type: Other Status: Other Year Published: 2013 Citation: Dubcovsky, J. 2013. Integrating, sending and decoding environmental signals in the wheat flowering response. American Society of Plant Biology. Keynote speaker. April 12-13, 2013, Davis CA
  • Type: Other Status: Other Year Published: 2013 Citation: Dubcovsky, J. 2013. Beyond natural diversity in wheat: generation of new diversity by TILLING. Society of Experimental Botany. Keynote speaker. July 4th, 2013, Valencia, Spain.
  • Type: Other Status: Other Year Published: 2013 Citation: Dubcovsky, J. 2013. Functional genomic tools to understand wheat development. Keynote speaker. 12th International Wheat Genetics Symposium, Okayama Japan, September 8-13, 2013.


Progress 04/01/12 to 03/31/13

Outputs
OUTPUTS: Objective 1: We have generated mutations in the A and B genome copies of PHYA and PHYB genes, and intercrossed them to generate homozygous phyA and phyB null mutants. Under long day (LD) conditions, both phyA and phyB-null mutants flowered at the same time (45 d) as their respective wild-type sister lines whereas the phyC-null mutants flowered more than 100 days later. These results indicate the large effect of PHYC on flowering is not mediated by the PHYA and PHYB genes. We are now combining the phyC-null mutants with the phyA and phyB-null mutants to generate double null mutants and test their effect on flowering under both long and short day (SD) conditions. Objective 2: We completed experiments that demonstrate a strong interaction between PHYC and photoperiod. The phyC-null mutations cause a much larger delay in flowering under LD (114-125 days) than under SD (12-20 days). Transcript analysis revealed that the flowering gene FT, which encodes a mobile florigen protein, was completely down-regulated in the phyC-null mutants. The introduction of an FT over-expressing allele in the phyC-null mutants accelerated flowering by 20 days under long days confirming that the downregulation of FT in the phyC-null mutants plays an important role in the flowering delay observed in these mutants. Objective 3: We have shown by yeast two-hybrid assays that the wheat PhyC protein can form homodimers, a unique characteristic in wheat. In Arabidopsis and rice PHYC only forms heterodimers with PHYB. We have further confirmed this result by split-luciferase assays in rice protoplast, and more recently demonstrated that PHYC dimers can be transferred to the nucleus in the absence of other phytochromes. We generated a construct expressing a fusion of PhyC and the red fluorescent protein DsRED under the control of 35S promoter. This construct was then transformed into the Arabidopsis phytochrome quintuple mutant (phyABCDE) background, where none of the five Arabidopsis phytochromes is functional. Confocal microscopy analysis of the 35S-PhyC-DsRED/phyABCDE transgenic plants shows a clear nuclear localization pattern for the PhyC protein. This result demonstrated that wheat PhyC protein can accumulate in the nucleus in the absence of other phytochromes. To identify novel protein interactors of the wheat PhyC, we have constructed cDNA libraries from 6-8 week-old leaves of two diploid wheat accessions. A yeast two-hybrid screening of the cDNA libraries has been conducted using the C-terminus of the PhyC protein as bait, which is believed to be involved in protein-protein interactions. We are currently validating and confirming the positive interactions identified in the library screening. Objective 4: We demonstrated that a dominant VRN3 allele for early flowering in barley was associated to an increase in FT copy number rather than to SNP differences in the promoter or first intron. The results from this study are partially accepted for publication in Molecular Genetics and Genomics. Objective 5: We have duplicated the number of mutants in our tetraploid tilling population (1368 to 2700) PARTICIPANTS: This project provides a multidisciplinary environment for training a postdoctoral scholar (CoPI Dr. Andrew Chen), two Ph.D. students (Nestor Kippes and Rebecca Nitcher) and several undergraduate students in the integration of genetics, biochemistry and computational tools. The postdoctoral scholar and the Ph.D. students have been mentoring undergraduate students. Recruitment of the undergraduate student through the "UC Davis Academic Program" increases opportunities for minority students. Collaborations in the biochemical aspects of the project have been established with Dr. Clark Lagarias at the Department of Molecular and Cellular Biology, University of California, Davis. TARGET AUDIENCES: Important target audiences for this project include the wheat and barley breeders. In most environments, there is a very narrow window for optimum flowering time that will maximize wheat and barley grain yield potential. The knowledge and information generated from studying the effect of the different PhyC and FT alleles will be useful for barley and wheat breeders to design better crosses, to predict the effect of the different alleles in flowering initiation and stem elongation, and to engineer varieties with specific flowering characteristics. Another target audience of this project is the scientific community working in the understanding of the regulation of flowering in plants. The generation of flowering mutant collections in a common genetic background will also provide an essential tool to study epistatic interactions among flowering genes. Wheat researchers in different areas will be able to use these mutants to test the effect of different developmentally regulated process on their targeted genes and pathways. Finally, all researchers interested in functional studies in wheat can benefit from the increase of the size of our publicly available tetraploid wheat TILLING population, which increases the chances of identifying truncation mutations in any wheat gene. DNAs and seeds of the TILLING populations are publicly available. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Phytochromes have been relatively well studied in model plant species such as Arabidopsis and rice, and T-DNA knockout mutants have been developed for all the phytochrome genes in these species. However, how light signals are transduced from phytochromes to the photoperiodic genes in temperate grasses is still poorly understood. A better understanding of the effect of the different phytochromes on flowering initiation will improve our understanding of flowering regulation in grasses. Generating and characterizing Tilling mutants for all three phytochrome genes in wheat is our first step towards this objective. The >100-day delayed flowering phenotype of the PhyC-null mutants indicates that PHYC plays an essential role in the photoperiodic flowering pathway in temperate grasses. Characterizing the effect of the different PhyC alleles will also be useful for barley and wheat breeders to design better crosses, to predict the effect of the different alleles in flowering initiation and stem elongation, and to engineer varieties with specific flowering characteristics. To unravel the role of PHYC in the photoperiod response in wheat, we are also taking biochemical approaches to determine the proteins that interact with PHYC and transduce the signals to other light regulated genes. Most of the Phytochrome Interacting Factors (PIFs) identified so far are PHYA and PHYB interactors. The PHYC protein interactors responsible for the transduction of the light signals to the photoperiod pathway remain largely unknown. Therefore, the biochemical approaches proposed in this project have the potential to unravel novel interactions and mechanisms. Finally, the expansion of the publicly available tetraploid wheat TILLING population and the generation of a public collection of flowering mutants are valuable outcomes of this project. The expansion of the tetraploid TILLING populations greatly increases the chances of identifying truncation mutations. This is particularly important in a polyploid species, because a large amount of time and resources are devoted to the phenotypic characterization of the mutants, and therefore it is critical to initiate the functional analysis with good mutations. The large amount of work required to combine mutations in the A- and B-genome copies in tetraploid wheat is balanced by the potential to perform gene dosage studies and to test mutations in lethal genes, which cannot be studied in diploid species. The development of wheat TILLING populations with mutation densities high enough to knockout almost every gene makes it possible to generate collections of mutants in wheat similar to the ones available in the model species. The generation of these mutant collections in a common genetic background provides an essential tool to study epistatic interactions among flowering genes. We will make our mutants publicly available by depositing them in the National Small Grain Collection. Wheat researchers in different areas will be able to use these mutants to test the effect of different developmentally regulated process on their targeted genes and pathways.

Publications

  • Peer-reviewed Scientific Journals Chen,A., and J. Dubcovsky. 2012. The wheat gene VERNALIZATION1 is required for the down-regulation of the VRN2 flowering repressor in the leaves and for timely flowering in spring. PLoS Genetics. 8:e1003134. doi: 10.1371/journal.pgen.1003134. Nitcher R., A. Distelfeld, C.T. Tan, L. Yan, J. Dubcovsky. 2013. Increased copy number at the FT-H1 locus is associated with accelerated flowering time in barley. General Genomics and Genetics. Under revision Presentations Kippes, N., J. Zhu, A. Chen, H. Nishida, L. Vanzetti, K. Kato, M. Helguera, J. Dubcovsky. 2012. Fine mapping and epistatic interactions of Vrn-D4 in common wheat (Triticum aestivum L.). Plant and Animal Genome XX, January 14-18, San Diego, CA. P306. Tsai, H., R. Nitcher, T.R. Howell, E. Akhunov, T.H. Tai, J. Dubcovsky, L. Comai. 2012. High-throughput sequencing makes TILLING more fun. Plant and Animal Genome XX, January 14-18, San Diego, CA. W217 Alvarez, M.A., M.E. Faricelli, S. Lewis, G. Tranquilli, M.L. Appendino, J. Dubcovsky. 2012. Eps-Am1, a locus regulating reproductive development in Triticum monococcum, has been delimited to a 50 kb region including 2 genes. Plant and Animal Genome XX, January 14-18, San Diego, CA. P341. Nitcher, R., A. Distelfeld, and J. Dubcovsky. Characterization of barley natural variation at the HvFT1 locus affecting flowering time. ASA, CSSA and SSSA Annual Meeting, Cincinnati, OH- Oct. 21 - Oct. 24, 2012.


Progress 04/01/11 to 03/31/12

Outputs
OUTPUTS: Objective 1 "Generate phyA and phyB mutants for the A and B genomes of tetraploid wheat by TILLING". We have identified mutants from our tetraploid Tilling population for both PhyA-A and PhyA-B copies: mutant #1234 carries a premature stop codon after the PHY domain and mutant #499 has a splicing site mutation in the histidine kinase domain. For PhyB-A, we identified two mutants, one with a splicing site mutation at the end of the PHY domain and the other with a premature stop codon inside the PHY domain. For PhyB-B, we identified a mutant with an amino acid change in the critical GAF domain. All mutants are being backcrossed to Kronos to remove background mutations. Objective 2 "Analyze the effect of null phyC mutations on the transcription of flowering genes". We have generated a null phyC mutant by combining the backcross-two (BC2) phyC-A splice mutant 2327 and phyC-B bilin chromophore binding mutant 807(C323Y). When grown under LD homozygous phyC-A/phyC-B mutants took more than 200 days to flower and were sterile. Transcript analysis revealed that flowering genes VRN1, FT, FT2, ZCCT2, BM3, BM8 and PPD-1 were completely or severely down-regulated in the phyC-A/phyC-B double mutants compared to the wild type. Objective 3 "Identify proteins that interact with PHYC and PHYB in wheat". We split both PHYB and PHYC proteins into two parts: the N terminus, which includes the PAS, GAF and PHY domains and the C terminus including two PAS and one histidine-kinase domains We have generated three yeast baits and three preys for both PHYB and PHYC proteins (N-ter, C-ter and the full-length), and performed auto-activation tests for all of them. For the constructs showing no autoactivation we found that PHYB and PHYC wheat proteins can form homodimers and heterodimers in yeast. We have also generated two constructs to over-express tagged PHYB and PHYC under the control of maize ubiquitin promoter. Both constructs are currently being transformed into wheat and barley, and the generated transgenic plants will be used in the validation of protein interactions in planta. Objective 4 "Characterize the effect of natural variation of wheat and barley PHYC genes on flowering time". We have shown that barley line Amagi Nijo (and other Japanese barley lines) carries an unusual PHYC allele with a mutation in an amino acid that is otherwise conserved in all phytochrome genes from all plants and mosses. To validate the hypothesis that the Amagi Nijo PHYC allele is the one responsible for the differences in flowering time, we have cloned the complete PHYC cDNA from Amagi Nijo including the unique F380S, and we are currently generating a construct to express this PHYC allele under the control of its native promoter in transgenic barley. Objective 5 "Double the number of lines in the publicly available tetraploid wheat TILLING population". We added 768 more lines to our existing tetraploid wheat mutant population to achieve a population of 2304 mutant lines, which is 1.5 times of the previous one. The increased population size will increase our chances of detecting truncation mutants in wheat. PARTICIPANTS: This project provides a multidisciplinary environment for training a postdoctoral scholar (CoPI Dr. Andrew Chen), a Ph.D. student (Nestor Kippes) and several undergraduate students in the integration of genetics, biochemistry and computational tools. The postdoctoral scholar and the Ph.D. student have been mentoring one female undergraduate student each. Recruitment of the undergraduate student through the "UC Davis Academic Program" increases opportunities for minority students. Collaborations in the biochemical aspects of the project have been established with Dr. Clark Lagarias at the Department of Molecular and Cellular Biology, University of California, Davis. A collaboration has been also established with John E. Mullet (Texas A&M University) who is working on the effect of phytochrome C mutations in short day plants. TARGET AUDIENCES: One important target audience for this project is the wheat breeders. In most environments, there is a very narrow window for optimum flowering time that will maximize wheat grain yield potential. The knowledge and information generated from studying the effect of the different PhyC alleles will be useful for barley and wheat breeders to design better crosses, to predict the effect of the different alleles in flowering initiation and stem elongation, and to engineer varieties with specific flowering characteristics. Another target audience of this project is the scientific community working in the understanding of the regulation of flowering in plants. The generation of flowering mutant collections in a common genetic background will also provide an essential tool to study epistatic interactions among flowering genes. We will make our mutants publicly available by depositing them in the National Small Grain Collection. Wheat researchers in different areas will be able to use these mutants to test the effect of different developmentally regulated process on their targeted genes and pathways. Finally, all researchers interested in functional studies in wheat can benefit from the increase of the size of our publicly available tetraploid wheat TILLING population, which increases the chances of identifying truncation mutations in any wheat gene. DNAs and seeds of the TILLING populations are publicly available. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Phytochromes have been relatively well studied in model plant species such as Arabidopsis and rice, and T-DNA knockout mutants have been developed for all the phytochrome genes in these species. However, how light signals are transduced from phytochromes to the photoperiodic genes in temperate grasses is still poorly understood. A better understanding of the effect of the different phytochromes on flowering initiation will improve our understanding of flowering regulation in grasses. Generating and characterizing Tilling mutants for all three phytochrome genes in wheat is our first step towards this objective. The 160-day delayed flowering phenotype of the PhyC knockout mutants indicates that PHYC plays an essential role in the photoperiodic flowering pathway in temperate grasses. Characterizing the effect of the different PhyC alleles will also be useful for barley and wheat breeders to design better crosses, to predict the effect of the different alleles in flowering initiation and stem elongation, and to engineer varieties with specific flowering characteristics. To unravel the role of PHYC in the photoperiod response in wheat, we are also taking biochemical approaches to determine the proteins that interact with PHYC and transduce the signals to other light regulated genes. Most of the Phytochrome Interacting Factors (PIFs) identified so far are PHYA and PHYB interactors. The PHYC protein interactors responsible for the transduction of the light signals to the photoperiod pathway remain largely unknown. Therefore, the biochemical approaches proposed in this project have the potential to unravel novel interactions and mechanisms. Finally, the expansion of the publicly available tetraploid wheat TILLING population and the generation of a public collection of flowering mutants are valuable outcomes of this project. The expansion of the tetraploid TILLING populations greatly increases the chances of identifying truncation mutations. This is particularly important in a polyploid species, because a large amount of time and resources are devoted to the phenotypic characterization of the mutants, and therefore it is critical to initiate the functional analysis with good mutations. The large amount of work required to combine mutations in the A- and B-genome copies in tetraploid wheat is balanced by the potential to perform gene dosage studies and to test mutations in lethal genes, which cannot be studied in diploid species. The development of wheat TILLING populations with mutation densities high enough to knockout almost every gene makes it possible to generate collections of mutants in wheat similar to the ones available in the model species. The generation of these mutant collections in a common genetic background provides an essential tool to study epistatic interactions among flowering genes. We will make our mutants publicly available by depositing them in the National Small Grain Collection. Wheat researchers in different areas will be able to use these mutants to test the effect of different developmentally regulated process on their targeted genes and pathways.

Publications

  • Dubcovsky, J. 2011. Integration of the photoperiod and vernalization pathways in the temperate cereals. NIFA Project Director Meeting Plant and Animal Genome XIX, January 15-19, San Diego, CA. P858.
  • Dubcovsky, J. 2011. Gene networks regulating flowering time in wheat. Key Note speaker. 21st International Triticeae Mapping Initiative (ITMI), Mexico City, September 4 - 9, 2011.
  • Li, C.., A. Distelfeld, A. Comis, and J. Dubcovsky. 2011. Wheat flowering repressor VRN2 and promoter CO2 compete for interactions with NUCLEAR FACTOR-Y complexes. Plant Journal 67:763-773
  • Nitcher, R., A. Distelfeld, J. Dubcovsky. 2011. Effect of HvFT1 promoter haplotypes on barley flowering time. Plant and Animal Genome XIX, January 15-19, San Diego, CA. P327.