Abstract
The soybean vegetative storage protein genes (vspA, and vspB) are regulated in a complex manner developmentally and in response to external stimuli such as wounding and water deficit. The proteins accumulate to almost one-half the amount of soluble leaf protein when soybean plants are continually depodded and have been identified as storage proteins because of their abundance and pattern of expression in plant tissues. We have shown that purified VSP homodimers (VSP alpha and VSP beta) and heterodimers (VSP alpha/beta) possess acid phosphatase activity (alpha = 0.3-0.4 units/mg; beta = 2-4 units/mg; alpha/beta = 7-10 units/mg). Specific activities were determined by monitoring o-carboxyphenyl phosphate (0.7 mM) cleavage at pH 5.5 (VSP alpha) or pH 5.0 (VSP alpha/beta and VSP beta) in 0.15 M sodium acetate buffer at 25 degrees C. These enzymes are active over a broad pH range, maintaining greater than 40% of maximal activity from pH 4.0 to 6.5 and having maximal activity at pH 5.0-5.5. They are inactivated by sodium fluoride, sodium molybdate, and heating at 70 degrees C for 10 min. These phosphatases can liberate Pi from several different substrates, including napthyl acid phosphate, carboxyphenyl phosphate, sugar-phosphates, glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, ATP, ADP, PPi, and short chain polyphosphates. VSP alpha/beta cleaved phosphoenolpyruvate, ATP, ADP, PPi, and polyphosphates most efficiently. Apparent Km and Vmax values at 25 degrees C and pH 5.0 were 42 microM and 2.0 mumol/min/mg, 150 microM and 4.2 mumol/min/mg, and 420 microM and 4.1 mumol/min/mg, for tetrapolyphosphate, pyrophosphate, and phosphoenolpyruvate, respectively.
Highlights
The induction of the genes in wounded tissueappearsto result from wound-induced accumulation of jasmonic acid or its methyl ester, methyl jasmonate. This idea is consistent with the stimulation of usp expression by addition of jasmonic acid or methyl jasmonate to cell cultures or plants(12-14,18, In 1982and 1983,Wittenbach [1,2,3] described proteins that 19).In addition, inhibitorsof lipoxygenase,which should limit accumulated in leaves of soybean when the plants were con- jasmonic acid biosynthesis in wounded tissue, block woundtinually depodded
Ithas been noted that PVM cells are larger than mesophyll cells and have a very large vacuole that probably serves an importantstorage role in these cells
The VSP were so named because they accumulate in PVM vacuoles during periods of high amino acid and carbon flux to andfrom leaf mesophyll cells [1,2,3,4,5,6]
Summary
Sequence Comparison of Acid Phosphatase-I, VSPa, and VSPP-VSPa and VSPP have 45% overall amino acid sequence identity with acid phosphatase-1 [22,23,24]. VSPa andVSPP are glycoproteins, and each contains a potential sitefor asparagine-linked glycosylation (amino acids 129-131 and 130-132; NST and NET (see Ref. 8 for numbering)). This site is not conserved in acid phosphatase-1, but a site nearby (amino acids 142144; NGT) could represent a glycosylation site. The stained gel shown at the topof the elution profiles further illustrates that thedifferent VSP dimersare readily separated by anionexchange chromatography (Mono&).
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