School of Agriculture, Food and Ecosystem Sciences - Theses
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ItemMolecular and functional genomic analysis of Phytochrome Interacting Factor 4 during the floral transition of soybean( 2019)Climate change is adversely affecting global food production. Reduced crop yields and increasing food demands are presenting new challenges for developing resilient crop varieties. Legume plant soybean is an important food crop next to cereals. Soybean is a rich source of high-quality vegetable oils, protein, nutraceuticals, and it is widely cultivated across the globe for both human and animal consumption. According to the Australian oilseed federation, soybean cultivation strengthens regional economies and contributes to 2.5 billion dollars of the Australian oilseed industry. To breed better soybean crops, a study of light and temperature perception is important, as these events control the process of flowering which is the first step towards total seed production. On the molecular level, light and temperature signal control is a complex process mediated by a network of photoreceptors and transducers. One such transducer is Phytochrome Interacting Factor 4 (PIF4). It is involved in the perception of external environmental stimuli to integrate the information of daily light and temperature fluctuations with the internal physiology of plants. PIF4 has been studied in the model plant Arabidopsis thaliana, but knowledge about its counterparts in soybean is limited. To understand the function of PIF4 in soybean, an integrated genomics, molecular, and functional characterization approach was employed. The genomic analysis show that there are seven active copies of PIF4 gene in the genome of the cultivated soybean (Glycine max). The seven soybean PIF4 genes have evolved at different time points during the soybean genome duplication events, and phylogeny is suggestive of the existence of a conserved PIF4 clade (PIF4 I), which includes Arabidopsis PIF4 and a clade that groups only the PIF4s of legumes (PIF4II). To assess the gene expression patterns of soybean PIF4s during variable photoperiods and temperature conditions, quantitative RT-PCR was performed. Quantitative RT-PCR results showed that three PIF4s belonging to the PIF4I clade (i.e. GmPIF4a, GmPIF4b, and GmPIF4c) had conserved PIF4 like expression (consistent with Arabidopsis pif4) in inductive photoperiod (short day; SD), whereas two soybean PIF4s belonging to the PIF4II clade (i.e. GmPIF4f and GmPIF4g) exhibited reduced to nil sensitivity to short-days. The mRNA transcript levels of three PIF4s were elevated at 35-degree centigrade as compared to 25-degrees centigrade in short days. In non-inductive photoperiod (long day; LD), all soybean PIF4s were responsive to extended light phase suggesting their functional role in long photoperiods. Further, no significant transcript variation was observed at elevated temperatures in long days, except one PIF4, which expressed at higher levels at 30-degrees centigrade as compared to 25-degrees centigrade. Mining of previously published RNA sequencing data obtained from leaf samples of a short-day soybean cultivar (Bragg) undergoing floral transition showed that one of the PIF4s was differentially regulated with high differences in transcript read values on consecutive short days. This soybean PIF4 was designated as GmPIF4b. GmPIF4b was functionally characterized using ectopic expression in Arabidopsis, mutant complementation in Arabidopsis pif4-101 mutants and gene over-expression in the soybean cultivar, Bragg. Ectopic expression of GmPIF4b in Arabidopsis resulted in elongated hypocotyls and early flowering responses suggesting the function of GmPIF4b in hypocotyl elongation and flowering responses. Arabidopsis pif4-101 mutant contains a mutation in the exon-5 of pif4 gene. This mutant is characterized by shorter hypocotyls and a compact rosette size. Complementation of GmPIF4b in Arabidopsis pif4-101 mutant partially rescued the short hypocotyl and compact rosette phenotypes of pif4-101 mutant. The results were suggestive of both conservation and divergence of GmPIF4b. To gain insights into the function of GmPIF4b in soybean, constitutive overexpression of GmPIF4b in soybean (Bragg) was undertaken. Constitutive overexpression resulted in decreased plant height, reduced leaf surface area, decline in total number of branches per plant, and early flowering in transgenic soybean plants as compared to the wild type. Quantitative RT-PCR was performed to study the transcript levels of soybean florigens; GmFT2a and GmFT5a in the transgenic plants (over expressing GmPIF4b). The transcript levels of GmFT2a and GmFT5a were significantly elevated in transgenic lines compared to the wild type. Late maturing soybean varieties, such as Bragg, can terminate flowering in sub-optimal photoperiods. To study the effect of GmPIF4b over-expression on termination of flowering, transgenic soybean plants (over expressing GmPIF4b) grown in short days were transitioned to non-inductive photoperiod (for 10 long days) at full bloom stage. Interestingly, wild type Bragg plants terminated flowers and produced less pods. Transgenic plants did not terminate their reproductive activity and gave rise to a higher number of pods per plant. To study the dynamics of GmPIF4b protein, immunoblot analysis was undertaken. The results showed that on protein level, GmPIF4b follows a strict diurnal expression pattern under both long and short days, suggesting the involvement of biological clock in regulating GmPIF4b protein expression. Chromatin immunoprecipitation was performed to study the protein-DNA interaction of GmPIF4b protein with the promoters of soybean florigens GmFT2a and GmFT5a. The results suggested that GmPIF4b interacts with the promoters of GmFT2a in short-day treated plants, whereas it interacts with the promoter of GmFT5a in a photoperiod insensitive manner. PIF4 is shown to be involved in nitrate assimilation and phosphate acquisition pathways. Soybeans plants were treated with nitrates or orthophosphates under inductive short days to analyse the GmPIF4b gene expression by RT-PCR. The results showed that the application of nitrates and orthophosphates can affect the levels of GmPIF4b transcript and can modulate the expression pattern of soybean floral identity marker gene, GmAP1. Hence, the outcome of this study can contribute towards developing climate-resilient varieties for the future.