The Role of OsWRKY71 and Its Interacting Proteins in Seed Germination and Early Growth of Cereal Grains
Creators: name: Shin, Margaret Ja
Publisher: name: University of Nevada, Las Vegas
Year: 2013

description: During seed germination and early seedling growth, complex molecular and physiological events occur in rice (Oryza sativa) and other cereal grains. As the seed transitions to vegetative tissue, it responds to both favorable and unfavorable environmental conditions and is vulnerable to attack by predation and disease. Although seeds are relatively small and tender in size, extensive and sophisticated molecular networks enables the immobile seed to grow, survive and adapt in its environment. One of the networks I am interested in is in the crosstalk between the gibberellin (GA) and abscisic acid (ABA) signaling pathways. These pathways are interesting because they are largely antagonistic. GA is a hormone that generally promotes germination and growth- related processes while ABA, also a hormone, promotes seed dormancy and represses growth. Although a great deal of research has been dedicated towards understanding these two pathways, the actual mechanism of crosstalk during seed germination is less understood. Any deficiencies in GA and ABA regulation and response may result in altered interpretation of environmental signals and aberrations in seed development and germination, leading to lower grain yields. My research is dedicated towards deciphering the specific role of Oryza sativa WRKY71 (OsWRKY71; amino acid W-R-K-Y) and harpin-induced1-like (HIL) members in the crosstalk between GA and ABA in rice, with the goal that this research will be used to improve cereal grain yield in areas of the world with limited plant productivity. 
The crosstalk between GA and ABA directs the synthesis of ?-amylase, which is an enzyme that breaks down starch in seeds to provide energy for germination. OsWRKY71 was shown to be a transcriptional regulator of ?-amylase and was regulated by both GA and ABA in barley. In this study, I have provided a model of the regulation of OsWRKY71 in seed germination in rice. Although it was previously determined that OsWRKY71 negatively regulated ?-amylase, I show that it positively regulated not only germination but also root growth. To support this, I performed seed germination and root elongation assays using knockout mutants of OsWRKY71. Mutant analysis determined that germination in oswrky71 was delayed for approximately 1 day and was able to recover from the delay. Additionally, after 4 days, oswrky71 seedling roots were nearly 2 cm shorter than wildtype (wt), suggesting that OsWRKY71 may regulate other aspects of plant development. This is further supported by analysis of ?-Glucuronidase (GUS) reporter expression of OsWRKY71p-GUS, which indicated that OsWRKY71 was localized to the third node of rice culms. Thus, the function of OsWRKY71 appears to be more complex and versatile than predicted. 
To further understand the mechanism of OsWRKY71 regulation in rice seed germination, I investigated the role of one of its interacting partners, Oryza sativa harpin-induced1-like 58 (OsHIL58). Using rice aleurone RNA-sequencing data, I found that OsHIL58 was induced upon ABA treatment. Thus, the two proteins may interact during ABA induction. I also annotated the HIL family using in silico methods and identified several other HIL members that were differentially and significantly expressed in the aleurone. One member, OsHIL16, was highly expressed and also coexpressed with an ABA receptor, regulatory component of ABA receptor 9 (RCAR9). Surprisingly, both were repressed by ABA, suggesting that they be involved in the same pathway in the aleurone aside from OsWRKY71 regulation. From this annotation, I also identified and compiled a large family of 104 unique HIL members expressed in various rice tissues. A classification system was designed based on the presence of several conserved amino acid motifs: NPN, RPP, and YQYF. Most HIL members, including OsHIL16 and -58, were Group I members with all three motifs present. These and further analyses suggest that HILs may have multiple roles in plant development, including in seed germination.