ABSTRACT: The paraveinal mesophyll (PVM) of soybean leaves is a layer of laterally expanded cells sandwiched between the palisade and spongy mesophyll chlorenchyma. The vacuoles of PVM cells contain an abundance of a putative vegetative storage protein, VSP (α, β). VSP is is constitutively produced, but is up-regulated during sink limitation experiments involving flower, fruit, or vegetative bud removal. Soybean vegetative lipoxygenases (Vlx), consisting of 5 isozymes (Vlx, A-D), have been identified as potential storage proteins because they accumulate to high levels with experimental sink limitation and have been co-localized with VSP to the vacuoles of PVM cells. We re-investigated the sub-cellular locations of these enzymes with TEM immuno-cytochemistry. We employed laser micro-dissection to compared RNA expression of PVM cells with mesophyll chlorenchyma cells, and we performed a micro-array analysis of soybean leaf samples representing a time-course, sink-limitation, experiment. We found that none of the Vlx isozymes co-localize with putative storage proteins in PVM vacuoles, and that our sink limitation experiment (typical of those used in the past) induced a strong up-regulation of stress response genes, simultaneous with the up-regulation of the Vlx isozymes. Our findings do not support a storage function for soybean Vlx. The results reported here are from a study designed to evaluate systemic effects of a vegetative bud removal protocol that had been used previously to study accumulation and subsequent mobilization of vegetative storage proteins in soybean. During these experiments plants were grown for 30 days and then all vegetative buds were removed. Bud removal was repeated each day for 15 days (designated the "detip" phase) and then new buds were allowed to regrow (designated the "retip" phase). Samples were collected at 5 day intervals. Total RNA was extracted from selected samples for our microarray experiments. Three replicate time course experiments provided RNA samples for our microarray analysis. Samples were harvested from both bud removal ("detip/retip") plants and untreated controls of the same age that were maintained side-by-side under the same growth conditions. Bud removal plants were designated as "detip" plants during the bud removal phase and "retip" plants during the regrowth phase. Bud removal began at time-course day 30 and lasted for 15 days (designated D0 through D15). Shoot regrowth lasted an additional 15 days (designated R16 through R30). Controls were designate similarly with C0 corresponding to 30 day old plants at the the first day of bud removal and C30 corresponding to day 15 of the regrowth phase. Samples were collected at 5 day intervals. Messenger RNA was extracted from selected samples for our microarray experiments. These were from 30 day old plants ( C0, the last day of growth before detipping), 45 day old plants (C15, D15, at day 15 of bud removal), 50 day old plants (C20, R20, day 5 of shoot regeneration), and 60 old plants (C30, R30, day 15 of regrowth). RNA was extracted from leaf samples using a method developed for tissues containing abundant polysaccharides and polyphenols (Gehrig et al., 2000). In addition, the extracted RNA was further purified using the RNeasy kit (Qiagen, Valencia, CA). Each RNA sample examined (C0, C15, C20, C30, D15, R20, R30; each with a minimum of 3 replicates for a total of 22 samples) was converted to cRNA and hybridized to Affymetrix soybean GeneChips (GEO accession # GPL4592) according to the manufacturer’s instructions (Affymetrix, Inc., Santa Clara, CA).
