Source: UNIVERSITY OF KENTUCKY submitted to NRP
USING RNA INTERFERENCE (RNAI) TO DECONVOLUTE CRYPTOCHROMES' (HVCRYS) INFLUENCES ON GENE EXPRESSION CONTROLLING ABA TITER IMPACTING SEED DO
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0228360
Grant No.
2012-67014-19421
Cumulative Award Amt.
(N/A)
Proposal No.
2011-04375
Multistate No.
(N/A)
Project Start Date
Apr 15, 2012
Project End Date
Apr 14, 2013
Grant Year
2012
Program Code
[A1101]- Plant Health and Production and Plant Products: Biology of Agricultural Plants
Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001
Performing Department
Horticulture
Non Technical Summary
Pre-harvest sprouting (PHS), when high humidity in the field permits imbibition and other phases of germination to occur in the cereal grain prior to harvest, culminates in embryo elongation; the completion of germination on the spike. Such sprouted grains seriously compromise the quality of products made from the flour produced from them, even when mixed with non-sprouted grains, resulting in large economic losses each year. The problem is pervasive and recurrent throughout those regions growing cereals brought about as a response to wet harvest conditions. The occurrence of PHS is one of the major abiotic constraints influencing the production of high quality grain, particularly white wheats. Almost 20 years ago, the economic losses due to PHS were estimated to influence the producer's revenue by an average of -4% (U.S.A), -5% (Canada and the European Union each), and -6% (Australia) per year and these losses have not been mitigated. Additionally, these loss estimates do not reflect the impact PHS has locally because the problem of PHS is widely variable year-to-year depending on the occurrence of weather conditions and can devastate production in a locale. Although there are many morphological, physiological, and environmental factors influencing PHS, a primary cause is poor seed dormancy. The long-term goal of this collaboration is to reduce/eliminate PHS through cellular reprogramming to enhance seed dormancy in cereals.
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
20624101020100%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
2410 - Cross-commodity research--multiple crops;

Field Of Science
1020 - Physiology;
Goals / Objectives
The project goal is to test if lesions in blue light (BL) perception/signal propagation in barley result in a loss of seed dormancy. The rationale behind this project is that reduction of dormancy in barley through alteration of BL sensitivity may be transformative research leading to the enhancement of seed dormancy in wheat through breeding for augmentations in BL sensitivity. The information gathered in barley will legitimize future efforts to enhance the potency of BL dormancy induction in the cereals, particulary wheat, to combat preharvest sprouting (PHS). This will entail characterizing barley RNAi mutants repressed in BL sensing/signaling to determine if barley CRYPTOCHROMES or ELONGATED HYPOCOTYL5 (HY5), a transcription factor known to propagate the blue light signal in Arabidopsis, are involved in enhancing seed dormancy in cereals. Supporting objectives include testing if the coleorhiza is controlling root protrusion from the seed. These results will impact the decision to use superior (for dormancy imposition) HY5 variants in cereals for marker-assisted breeding efforts targeting enhanced seed dormancy, particularly in the white wheats.
Project Methods
The project being pursued involves RNAi-/null-segregant lines targeting barley transcripts HvCRY1a, HvCRY1b, HvCRY2, HvHY5, the transcripts encoding the ABA biosynthetic enzymes HvNCEDs or 1 of 3 ABA catabolic-enzyme isoforms [ABA8' HYDROXYLASE1 (HvABA8'OH1)] (Gubler, Hughes et al. 2008). All of these lines are available or are currently at the T1 stage in the Gubler lab (Frank Gubler pers. com. 2/21/2011). The applicant will participate in the characterization of the degree of expression reduction in the HvCRY1A, HvCRY1B, HvCRY2, and HvHY5 genes in several hvcry1a, hvcry1b, hvcry2, and hvhy5 lines for each RNAi construct through Q-RT-PCR. Once knock-down/-out lines and null-segregants have been identified they will be grown at the same time, in the same area and seeds harvested at the same time points during a seed development time course for a physiological analysis of SD reduction (if any) under blue light compared to darkness. Reductions in BL signaling (Hvcry1, Hvcry2 RNAi lines) or propagation (Hvhy5 RNAi lines) resulting in a repression of SD will be analyzed by examining ABA-content and transcript abundance from ABA- and GA-metabolic enzyme genes. The crux of this project is that if reductions in BL signaling/signal propagation result in a loss of dormancy, then enhancement of this pathway will lead to augmentations in SD.

Progress 04/15/12 to 04/14/13

Outputs
Target Audience: The target audience will eventually be wheat breeders who can utilize the information we have gained through this research to breed pre-harvest sprouting resistant white wheat varieties for use world-wide. Prior to that, the scientific community interested in light-mediated grain dormancy will be targeted in a manuscript describing our efforts (in preparation) and an invited review on light-mediated grain dormancy for publication in Seed Science Research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Seed Hormonal Biology: a) CSIRO Black Mountain: The program leader with whom the fellow worked, Frank Gubler, has recently been elected as a corresponding member to the American Society of Plant Biologists (http://my.aspb.org/resource/resmgr/member_chatter/june_2012.pdf?hhSearchTerms=gubler) for his contributions to our knowledge of hormonal control of aspects of reproduction [11], particularly the hormonal regulation of dormancy and germination in the cereals [12-13]. Although presented by the ASPB, a national organization, it is recognized that the society is international in scope, with a limited number of awards granted to non-US national ASPB members whose research program is of exceptional quality. This history of excellence within the CSIRO Black Mountain’s Seed Biology program has been its hallmark for more than 40 years as Dr. John Jacobsen (retired) had received similar recognition for his work on cereal seed dormancy, work which continues to this day [2, 13-14]. The project I was given upon my arrival was conceptualized through a natural progression of results elucidating cellular components involved in hormone synthesis or sensitivity garnered over the last 10 years using a variety of plant species as models [15-18]. The project can be split into two dove-tailing investigations, both using RNAi technology (a mechanism that was first elucidated at CSIRO, Black Mountain) to down-regulate: 1) aspects of blue light signaling and; 2) the capacity for ABA biosynthesis in barley. This project has delivered some excellent results that are likely to be published in a high impact journal and will build CSIRO’s reputation as a leading research institution in the field of cereal seed biology pre-harvest sprouting. I am also aware of the high profile science that occurs in the Food Futures Flagship under the leadership Matthew Morell. This work aims to improve grain quality and yield. With the delivery of Barley Plus it is expected that there will be more novel cereals delivered to the market. Current work with plant karrikins out of the Smith lab in Western Australia is also of great importance in understanding species strategies for regeneration of fire-ravaged ecosystems [26-30]. 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 major findings are The first demonstration that Cryptochrome 1 (CRY1) is the major blue light receptor that promotes dormancy in barley. This was shown by analyzing the dormancy phenotypes of transgenic barley grains with reduced CRY1. Grains with reduced Cryptochrome 2 showed no altered dormancy phenotype. Neither did lines with reduced CIP8 or HY5. This is a highly novel result that provides the key to unraveling the molecular pathway that directs light regulation of dormancy in cereals. It is expected that this will lead to a publication in a high impact journal. For the first time in cereals reporter lines have been developed to study the location of ABA biosynthesis. These lines will be very useful to study the regulation of ABA biosynthesis in grain development, germination and in abiotic stress responses to drought and cold. This work will lead to a deeper understanding of how this key hormone is regulated and how its production might be manipulated to improve grain dormancy and stress tolerance.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Kushwaha, Rekha, Payne, Christina, Downie, Bruce. 2013. Uses of phage display in Agriculture: A review of food-related protein-protein interactions discovered by biopanning over diverse baits. Computational and Mathematical Methods in Medicine. (Special Issue on Phage Display Informatics) Volume 2013, Article ID 653759, 12 pages. http://dx.doi.org/10.1155/2013/653759 (Published)
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Kushwaha, Rekha, Sch�fermeyer, Kim R., Downie, A. Bruce. 2013. A protocol for phage display and biopanning using recombinant protein baits. Journal of Visualized Experiments. (in press).
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Kushwaha, Rekha, Downie, Bruce, Payne, Christina. 2013. Uses of phage display in Agriculture: Sequence analysis and comparative modeling of Late Embryogenesis Abundant client proteins suggests protein-nucleic acid binding functionality. Computational and Mathematical Methods in Medicine. (Special Issue on Phage Display Informatics; in press)
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Kushwaha, R., Lloyd, T.D., Sch�fermeyer, K.R., Kumar, S., Downie, A.B. 2012. Identification of Late Embryogenesis Abundant (LEA) Protein Putative Interactors Using Phage Display. Int. J. Mol. Sci. 2012, 13.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Nosarzewski, Marta A C, Downie, A. Bruce, Wu, A. Benhong, Archbold, Douglas D. 2012. The role of SORBITOL DEHYDROGENASE in Arabidopsis thaliana. Functional Plant Biology. 39: 462-470. (Published)