Suppression of lipase activity by Vp and abscisic acid during kernel development

--D.G. Bai, C.W. Magill and J.D. Smith

Reserves, stored as starch, protein and lipid bodies, in endosperm, aleurone and scutellum tissues during seed development, are the main source of nourishment for axis growth during germination. Accumulation of reserves begins at about 13 DAP and continues to maturity. In mature maize kernels, aleurone tissue is characterized by a large number of protein bodies surrounded by lipid bodies. During the early stages of germination the primary source of both phospholipid precursors and the energy required for increased synthetic activity is derived from triacylglycerols in lipid bodies. Lipase, which hydrolyzes storage lipids, is located in the lipid bodies, and changes in lipase activity during germination have been reported.

Electron micrographs of aleurone tissues from wild type, vp/vp and vp5/vp5 kernels were compared at 16, 25 and 35 days after pollination (DAP). The structural integrity of protein and lipid bodies was maintained throughout development in wild type aleurone tissue. The degradation of lipid bodies in vp/vp aleurones was apparent at 16 DAP, and most of the lipid bodies were gone at 25 DAP. Visible degradation of protein bodies was also observed at 25 DAP. At 35 DAP very few lipid bodies were detectable, and most protein bodies were fused together and visibly degraded.

In vp5 aleurone tissue, visible degradation of lipid and protein bodies lagged behind vp. Minimal effects were observed at 16 DAP, and 25 DAP vp5 tissue was similar to that observed at 16 DAP in vp.

Crude homogenate of aleurone and scutellum tissue was assayed for lipase activity. Autolytic activity, degradation of its own lipid bodies, and substrate degradation activity of wild type kernels stayed at very low levels in both tissues throughout the maturation period (<50 nmoles of fatty acid/gFW/min). Aleurone tissue of vp mutants showed high autolytic activity at 16 and 25 DAP, but activity had decreased by 35 DAP. Substrate-degradation activity of vp was highest at 16 DAP and diminished over time. In contrast, vp5 mutants showed continuously increasing activity with or without substrate over time. Autolytic activity in vp5 at 25 DAP was similar to that of vp at 16 DAP.

Table 1. Total fatty acid (nmol FA/gFW/min) synthesized in each tissue homogenate.

High lipase activity occurred earlier in aleurone than scutellum tissue in both mutants, and activity in vp was more pronounced at an earlier developmental stage than in vp5. This is consistent with our observations that axis elongation occurs earlier in vp and that vp expression is less affected by environmental factors than vp5.

The vp mutant is ABA-insensitive, and ABA levels in kernel tissues are comparable to those in normal tissue. Homozygous vp5 kernels are ABA-deficient. They do not synthesize ABA within the kernel, but they contain ABA translocated from the heterozygous maternal plant. ABA levels in these kernels vary with age, but they are usually in the range of 25 to 50% of the ABA found in wild type kernels segregating on the same ear. Both in situ synthesis and translocation of ABA can be inhibited if normal kernel blocks are cultured in vitro on medium containing fluridone. ABA levels in these kernels are about 1% of those found in wild type kernels cultured on standard medium.

Although we did not have fluridone cultured materials at the same ages available, we assayed limited amounts of aleurone and scutellum tissue from 13 DAP kernel blocks cultured on fluridone. Lipase activity in these was similar to that observed in vp tissues at 16 DAP (169 nmFA/gFW/min).

The suppression of lipase during normal kernel development requires both the Vp gene product (presumably a regulatory protein) and the phytohormone abscisic acid. Degree of suppression appears to depend upon the concentration of ABA. Since Vp is always present in normal seed, ABA appears to be the effective regulator. Suppression of lipase may be one of the early control points in a cascade of metabolic events which induce embryo dormancy.


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