A single mutation that increases maize seed weight.

The maize endosperm-specific gene shrunken2 (Sh2) encodes the large subunit of the heterotetrameric starch synthetic enzyme adenosine diphosphoglucose pyrophosphorylase (AGP; EC 2.7.7.27). Here we exploit an in vivo, site-specific mutagenesis system to create short insertion mutations in a region of the gene known to be involved in the allosteric regulation of AGP. The site-specific mutagen is the transposable element dissociation (Ds). Approximately one-third (8 of 23) of the germinal revertants sequenced restored the wild-type sequence, whereas the remaining revertants contained insertions of 3 or 6 bp. All revertants retained the original reading frame 3' to the insertion site and involved the addition of tyrosine and/or serine. Each insertion revertant reduced total AGP activity and the amount of the SH2 protein. The revertant containing additional tyrosine and serine residues increased seed weight 11-18% without increasing or decreasing the percentage of starch. Other insertion revertants lacking an additional serine reduced seed weight. Reduced sensitivity to phosphate, a long-known inhibitor of AGP, was found in the high seed-weight revertant. This alteration is likely universally important since insertion of tyrosine and serine in the potato large subunit of AGP at the comparable position and expression in Escherichia coli also led to a phosphate-insensitive enzyme. These results show that single gene mutations giving rise to increased seed weight, and therefore perhaps yield, are clearly possible in a plant with a long history of intensive and successful breeding efforts.

Enhanced stability of maize endosperm ADP-glucose pyrophosphorylase is gained through mutants that alter subunit interactions

Temperature lability of ADP-glucose pyrophosphorylase (AGP; glucose-1-phosphate adenylyltransferase; ADP: α-d-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), a key starch biosynthetic enzyme, may play a significant role in the heat-induced loss in maize seed weight and yield. Here we report the isolation and characterization of heat-stable variants of maize endosperm AGP. Escherichia coli cells expressing wild type (WT) Shrunken2 (Sh2), and Brittle2 (Bt2) exhibit a reduced capacity to produce glycogen when grown at 42°C. Mutagenesis of Sh2 and coexpression with WT Bt2 led to the isolation of multiple mutants capable of synthesizing copious amounts of glycogen at this temperature. An increase in AGP stability was found in each of four mutants examined. Initial characterization revealed that the BT2 protein was elevated in two of these mutants. Yeast two-hybrid studies were conducted to determine whether the mutant SH2 proteins more efficiently recruit the BT2 subunit into tetramer assembly. These experiments showed that replacement of WT SH2 with the heat-stable SH2HS33 enhanced interaction between the SH2 and BT2 subunits. In agreement, density gradient centrifugation of heated and nonheated extracts from WT and one of the mutants, Sh2hs33, identified a greater propensity for heterotetramer dissociation in WT AGP. Sequencing of Sh2hs33 and several other mutants identified a His-to-Tyr mutation at amino acid position 333. Hence, a single point mutation in Sh2 can increase the stability of maize endosperm AGP through enhanced subunit interactions.

Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene

With the aim of improving the nutritive value of an important grain legume crop, a chimeric gene specifying seed-specific expression of a sulfur-rich, sunflower seed albumin was stably transformed into narrow-leafed lupin (Lupinus angustifolius L.). Sunflower seed albumin accounted for 5% of extractable seed protein in a line containing a single tandem insertion of the transferred DNA. The transgenic seeds contained less sulfate and more total amino acid sulfur than the nontransgenic parent line. This was associated with a 94% increase in methionine content and a 12% reduction in cysteine content. There was no statistically significant change in other amino acids or in total nitrogen or total sulfur contents of the seeds. In feeding trials with rats, the transgenic seeds gave statistically significant increases in live weight gain, true protein digestibility, biological value, and net protein utilization, compared with wild-type seeds. These findings demonstrate the feasibility of using genetic engineering to improve the nutritive value of grain crops.