|Shanker, S. - UNIVERSITY OF FLORIDA|
Submitted to: Molecular and General Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 1, 1999
Publication Date: N/A
Interpretive Summary: Gene structure and its relationship to function is a major activity in genetics. Basic knowledge from such studies of economically important genes is also crucial for metabolic engineering and biotechnology so as to obtain crop plants with modified genes for efficient utilization of specific metabolic pathways. ARS scientists in the Crop Genetics & Environmental Research Unit in Gainesville, FL, have previously identified a single gene in maize that controls normal utilization of photosynthesized sucrose in developing seeds. A mutation in this gene that leads to a complete loss of the encoded protein, an invertase enzyme that cleaves sucrose to simple sugars, is the causal basis of highly miniaturized seed showing only 20 to 30 % of the normal seed weight. Another mutation of this gene leads to greatly reduced levels of invertase activity, yet the corresponding RNA levels are not affected. The study reported here is aimed to better understand the molecular basis of this unusual mutation. Our molecular analyses show a change of a single amino acid in a region of the protein that is otherwise well conserved in all invertases and related proteins in plants. Thus, these data have allowed us to identify for the first time a single amino acid, out of a total of ~500, that is essential for the stability of this enzyme. Obviously, all future efforts to engineer this gene in maize and all other crop plants will have to ensure that this amino acid is unchanged so that the protein is stable during seed development.
Technical Abstract: We report here on the molecular nature of an EMS-induced mutant, mn1-89, a leaky semidominant allele of the Miniature1 (Mn1) seed locus that encodes a seed-specific cell wall invertase, INCW2. The mn1-89 locus specifies normal levels of Incw2 transcript but extremely low levels (~6% of normal) of the protein and enzyme activity (Cheng et al., 1996). Sequence analyses of Incw2 clones derived from the parental Mn1 and the mutant genotypes show a C to T transition in the mn1-89 allele, leading to a single amino acid substitution of proline to leucine near the C-terminus of the mutant INCW2 protein. This change is in a motif that is well conserved among all plant invertases and related fructosyltransferase sequences. On the basis of these genetic in planta data, we believe to have identified a thus far unknown proline residue in a GPFG motif as critical for the stability of such proteins.The single base change (C to T) also leads to the elimination of a BglI restriction site in the mutant allele. Indeed, BglI restriction digests of genomic DNAs from Mn1 and mn1-89 genotypes show two and one fragments, respectively. Sequence analyses of RT-PCR-derived Incw clones from mn1-1 predict five amino acid substitutions relative to Mn1. Whether or not these sequences are encoded by the mn1-1 locus or another non-allelic Incw gene in maize genome remains to be elucidated.