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Engineering 6-phosphogluconate dehydrogenase improves grain yield in heat-stressed maize
Monday, 2021/01/04 | 08:48:04

Camila Ribeiro, Tracie A. Hennen-Bierwagen, Alan M. Myers, Kenneth Cline, and A. Mark Settles

PNAS December 29, 2020 117 (52) 33177-33185

Significance

Heat stress reduces yield in maize by affecting the number of kernels that develop and the accumulation of seed storage molecules during grain fill. Climate change is expected to increase frequency and duration of high-temperature stress, which will lower grain yields. Here we show that one enzyme in central carbon metabolism is sensitive to high temperatures. By providing a heat-resistant form of the enzyme in the correct subcellular compartment, a larger number of kernels develop per plant during heat stress in the field. This genetic improvement could be included as part of integrated approaches to mitigate yield losses due to climate change.

Abstract

Endosperm starch synthesis is a primary determinant of grain yield and is sensitive to high-temperature stress. The maize chloroplast-localized 6-phosphogluconate dehydrogenase (6PGDH), PGD3, is critical for endosperm starch accumulation. Maize also has two cytosolic isozymes, PGD1 and PGD2, that are not required for kernel development. We found that cytosolic PGD1 and PGD2 isozymes have heat-stable activity, while amyloplast-localized PGD3 activity is labile under heat stress conditions. We targeted heat-stable 6PGDH to endosperm amyloplasts by fusing the Waxy1 chloroplast targeting the peptide coding sequence to the Pgd1 and Pgd2 open reading frames (ORFs). These WPGD1 and WPGD2 fusion proteins import into isolated chloroplasts, demonstrating a functional targeting sequence. Transgenic maize plants expressing WPGD1 and WPGD2 with an endosperm-specific promoter increased 6PGDH activity with enhanced heat stability in vitro. WPGD1 and WPGD2 transgenes complement the pgd3-defective kernel phenotype, indicating the fusion proteins are targeted to the amyloplast. In the field, the WPGD1 and WPGD2 transgenes can mitigate grain yield losses in high–nighttime-temperature conditions by increasing kernel number. These results provide insight into the subcellular distribution of metabolic activities in the endosperm and suggest the amyloplast pentose phosphate pathway is a heat-sensitive step in maize kernel metabolism that contributes to yield loss during heat stress.

 

See https://www.pnas.org/content/117/52/33177

Figure 1: Plastid-localized 6PGDH is required for grain fill and is heat-sensitive. (A) Native PAGE of endosperm protein extracts stained for 6PGDH isozyme activity. B73 and W22 inbred lines show activity of all three isozymes with genetic variation for electrophoretic mobility. The black arrow indicates PGD3 homodimers, and the white arrow indicates homodimers and the heterodimer of PGD1 and PGD2. (B) Homozygous pgd1; pgd2 phenotypes in W22. (BLeft) A mature homozygous double-mutant ear. (BRight) A sagittal kernel section. (C) Ear and sagittal kernel section phenotypes for pgd3 in W22. (CLeftpgd3/+ self-pollination. (CRight) A sectioned pgd3 homozygous kernel. (DN. benthamiana agroinfiltrated leaves expressing PGD1-GFP, PGD2-GFP, and PGD3-GFP fusion proteins. Merged images from spinning disk confocal fluorescence microscopy showing chlorophyll (red) and GFP (green) channels. (E) 6PGDH isozyme activity assay of endosperm extracts heated at 42 °C. Extracts were transferred to ice until all treatments were completed. The black arrow indicates PGD3, and the white arrow points to PGD1 and PGD2 activity. (F) Spectrophotometric assays for total 6PGDH activity of endosperm extracts heated at 42 °C. B73 (diamonds, gray dashed) expresses all isozymes. pgd1; pgd2 double mutants (squares, black solid) only have PGD3 activity, while pgd3 mutants (circles, gray solid) only have PGD1 and PGD2 activity. Error bars indicate SE of three biological replicates.

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