Investigation of genes contributing to whole plant senescence in soybean (glycine max)

Plant senescence is a genetically determined developmental program characterized by systematic degradative processes that involves activation of new gene activity and down-regulation of other genes that ultimately leads to cell, tissue, organ and whole plant death. Elucidating senescence regulatory pathways and participating genes will allow for the development of strategies to improve crop yields and also curtail postharvest losses. Three genes are known to be primary regulators of senescence in soybean; namely, g, D1, and D2. In double and triple mutant combinations these genes confer an evergreen leaf and seed phenotype. The double mutation ggd1d1d2d2 shows an inhibition of degradation of chlorophyll and chlorophyll binding protein, but photosynthesis declines and the leaves still abscise. In the triple mutant GGd1d1d2d2 the leaves maintain the normal photosynthetic capacity, but still abscise. So, while the senescence program is not entirely blocked in the mutant background, it is altered. Studies have shown that the expression patterns of soybean senescence associated genes (SAGs) are regulated differentially by g, D1 and D2. Due to the pivotal regulatory nature of these three genes for senescence, it is important to identify their specific nature. In the first study, an analysis using available soybean genome resources (SoyBase, Phytozome, COGE, etc.) was undertaken. This has resulted in the identification of a gene, Glyma01g41610.2, which encodes a putative transcription factor residing within the marker boundaries of the D1 locus on chromosome 1 which also shares a high level of synteny with a region on chromosome 11 and includes a paralogous gene, Glyma11g03770.2, within the D2 marker boundaries. Similarly, another gene, Glyma01g00510.1, also encodes a putative transcription factor and is located within the marker boundaries of the g locus on chromosome 1. These genes were selected as candidates representing g, D1, and D2 for RT-PCR analysis. None of the initial candidates exhibited a differential expression profile when comparing wildtype and mutant allelic versions in isogenic genetic backgrounds. The D2 and g genes were then selected for Sanger sequencing to determine if sequence differences were responsible for the observed phenotypic variations. No differences in sequence were observed when comparing wild type and mutant allelic forms. However, sequence variations were observed when comparing g in Harosoy versus the reference genome cultivar, Williams 82. A second candidate g gene (Glyma01g00520.4) was selected from the defined marker boundary interval on chromosome 1 but did not demonstrate a differential pattern of expression using RT-PCR. In the second study, additional SAGs up-regulated in various plant species were used to identify candidate soybean ortholog genes that could possibly contribute to whole plant senescence. Some SAGs have been shown to contain a unique senescence response element (SRE) within their promoters that confers a senescencespecific pattern of expression. This is best exemplified by the SAG12 (cysteine protease) gene of Arabidopsis thaliana. The 33 base pair SRE for the SAG12 gene has been shown to harbor a well-conserved 7 base pair sequence that is also found in SAGs from other plant species. To determine if a related SRE could be responsible for regulating soybean SAGs, a genome-wide study of previously identified as SAGs in other plant species was performed using publically available databases to find related genes in soybean. This search has led to the identification of several soybean genes that harbor this SRE. These genes were bioinformatically analyzed using various structural criteria to identify the best potential soybean ortholog for each gene type. Structural criteria included measures of alignment similarity with the Arabidopsis SRE, proximity of the SRE to the transcription start site, gene architecture, polypeptide sequence identity, and phylogenetic and syntenic relationships. Genes meeting the defined structural criteria underwent evaluation for a functional role in soybean senescence through RT-PCR analysis using a suite of isogenic lines exhibiting normal as well as delayed senescence phenotypes. The selected mutants represent different combinations (single, double and triple mutants) of genes g, D1 and D2 that give rise to evergreen leaves and green seed phenotypes. None of the genes selected for functional analysis demonstrated evidence of differential expression among the selected isolines. However, many aditional genes harboring an SRE have yet to be investigated.