Molecular Genetic Analysis of Senescence and the Reversal of Senescence in Soybean Cotyledons [\u3cem\u3eGlycine max\u3c/em\u3e (L.) Bass]

Whole plant senescence is a programmed, endogenously controlled process in which cellular components are degraded and the resultant metabolites sequestered to other plant organs. All cells, with the possible exception of the meristem progenitors, go through cell division, expansion, elaboration of secondary structures to the primary cell wall, quiescence, and finally death. This senescence process is thought to be partially catalyzed by an increase in translation of degradative proteins coupled to a decrease in translation of vital proteins (Skadsen and Cherry, 1983). Inducers of senescence include wounding, fruit ripening, changes in hormone levels (auxin, cytokinins + auxin, Ca2+ + cytokinins, and ethylene), various stresses (anaerobiosis, heat, cold, UV light, Cd2+, and Li+), and pathogen attack (Theologis, 1992). The regulatory mechanisms that facilitate entry of a cell, tissue, or whole plant into this final developmental stage are largely unknown; however, hormonal controls are likely to be involved. A better understanding of the genetic regulation of senescence will be important for studies relating to: 1) normal cellular differentiation, maintenance, and turnover; 2) plant defense mechanisms and the hypersensitive response; and 3) embryogenesis and development. In addition, economic benefits could result from reduction in post-harvest and post-production losses as well as improvement of crop yields. Evidence indicates that soybean cotyledon senescence and induced rejuvenation (i.e.,. the reversal of the senescence process) are associated with changes in gene expression (Marek and Stewart 1992; Skadsen and Cherry 1983). During germination the cotyledons emerge, become green, and develop leaf-like characteristics including photosynthetic and storage mobilizing capability. During normal development, the cotyledons senesce after expansion of the first and second trifoliates. It has been shown (Krul, 1974) that this process can be reversed by the removal of the epicotyl up to the point of no return (PONR), a developmental stage at which up to 90% of nucleic acids and 80% of proteins are lost from the senescing cotyledon. The objectives of this research project were to identify genes that show differential expression during senescence and rejuvenation in the soybean cotyledon system and to isolate clones of genes that are specifically up-regulated in expression during these processes. To accomplish these objectives, Northern blots were used to assay soybean cotyledon mRNA populations during various stages of senescence and rejuvenation using a collection of gene clones known or suspected to be involved in senescence and rejuvenation. These gene clones included: 1) a tomato ethylene forming enzyme (EFE) and several other ethylene up-regulated genes; 2) from carnation, ACC synthase, glutathione stransferase and other unidentified senescence associated genes; 3) from soybean, three lipoxygenases, two vegetative storage proteins, rubisco large and small subunits and an iron superoxide dismutase; 4) from cucumber, malate synthase and isocitrate lyase; and 5) two Arabidopsis genes, Sag2 (cysteine protease) and Sag4 (currently unidentified). In addition, cDNA libraries were constructed from senescent, nonsenescent, and rejuvenated soybean cotyledons. Finally, a cDNA library made from senescing soybean cotyledons was screened for a Sag2 homologue. These studies revealed up-regulation in expression during cotyledon senescence for rubisco large and small subunits, two soybean vegetative storage proteins, and an Arabidopsis cysteine protease. A soybean homologue to the Arabidopsis cysteine protease (Sag2) was cloned from senescing soybean cotyledons and restriction analysis revealed that it contained two Pvull sites