University of Florida and USDA/ARS

Mutation of the miniature 1 (mn1) locus is associated with loss of invertase activity

--Michael E. Miller and Prem S. Chourey

We have used histochemical methods of localizing invertase activity on kernel sections as described by Doehlert and Felker (Physiol. Plant. 70:51-57, 1987), to screen large populations of immature kernels for possible mutations showing loss of invertase activity. Among many genotypes tested, including various endosperm starch mutants, only the miniature 1 (mn1) mutant (Lowe and Nelson, Genetics 31:525-533, 1946) was lacking invertase activity (Fig. 1). Further analyses included Mn and mn kernels segregating on the same F2 ear and again the mutants showed no detectable activity.

Spectrophotometric assays for enzyme activity (Tsai et al., Plant Physiol. 46:299-306, 1970) on 12 DAP kernels, harvested from two different crops, confirmed earlier observations based on histochemical assays (Table 1). However, a low level of residual activity was consistently observed in the mutant kernels. Table 1 shows mean values of specific activity derived from 3-5 independent extractions for each genotype. No detectable levels of invertase were found in the upper 2/3 portion of either Mn or mn. In miniature, invertase levels were highly reduced for both cell wall bound and soluble forms indicating that both forms may be due to a single gene. No invertase-inhibitor was exhibited by miniature over normal when predialyzed samples were combined, dialyzed overnight, and assayed. Specific activities of invertase remained as high in the mixed sample as those observed in the mixture of Mn with buffer. Kernels at the 12 DAP stage of development were used as a baseline for this study since invertase levels are known to peak at this stage (Tsai et al., 1970). Spectrophotometric assays on root extracts of Mn and mn showed no detectable differences in invertase activity. Further studies are in progress to determine biochemical, cellular, and molecular basis of loss of invertase activity in the mn genotype.

Table 1. Mean specific activity (µM glucose/mg/min) in 12 DAP kernels (pedical & lower 1/3 endosperm.
  Mn Mn mn mn
  soluble bound soluble bound
Spring 1990 1.73 1.24 .016 .02
Fall 1990 2.10 -- .02 --

Figure 1. Kernels showing histochemical staining for invertase activity. Control = -sucrose, Mn and mn = + sucrose

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