Engineering 6-phosphogluconate dehydrogenase to improve heat tolerance in maize seed development

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 peptide coding sequence to the Pgd1 and Pgd2 open reading frames. 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 on 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.Significance StatementHeat 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.

Overexpression of a Cytosolic 6-Phosphogluconate Dehydrogenase Gene Enhances the Resistance of Rice to Nilaparvata lugens

“…In higher plants, 6PGDH can be divided into cytosolic and plastidic isoforms [16]. We found that Os6PGDH1 has a more closely evolutionary relationship with 6PGDHs of Setaria italica, Sorghum bicolor, and Zea mays, all of which were cytosolic isoforms [18,33,34], confirming that Os6PGDH1 is a cytosolic 6PGDH (Figure 1). Os6PGDH1 was widely expressed in rice tissues but mainly expressed in leaves, outer leaf sheaths, and roots, tissues that are active in anabolic metabolism ( Figure 2); moreover, Os6PGDH1 was induced by mechanical wounding, infestation with gravid BPH females, and JA treatment (Figure 3).…”

Heat Stress-Mediated Constraints in Maize (Zea mays) Production: Challenges and Solutions

“…This discovery of identifying and characterizing HSFs and their role in regulating Hsp genes has provided a fundamental basis for the development of GM maize with the highest heat stress tolerance ( Ahuja et al, 2010 ). Furthermore, the overexpression of chloroplast localized 6-phosphogluconate dehydrogenase (6PGDH) PGD3 displayed an over-accumulation of starch in maize endosperm under heat stress improved grain size and weight, whereas, WPGD1 and WPGD2 transgenes can increase the number of kernels to mitigate losses in high nighttime temperature conditions ( Ribeiro et al, 2020 ). In the metabolic studies of Joshi et al (2021) , a total of 5,136 genes expression were affected in response to heat stress.…”

Maize and heat stress: Physiological, genetic, and molecular insights

“…Gene 6PGDH has two other isoforms localized in the cytoplasm, PGD1 and PGD2, but, only PGD3 is reported as heat liable (Spielbauer et al, 2013). Subsequently, Ribeiro et al (2020) fused chloroplast targeting peptide sequence, Waxy 1 with pgd1 and pgd2, and found that the fusion proteins, WPGD1 and WPGD2, were complementing PGD3 function resulting in higher grain yield and biomass under high night temperature.…”

“…Subsequently, Ribeiro et al. (2020) fused chloroplast targeting peptide sequence, Waxy 1 with pgd1 and pgd2 , and found that the fusion proteins, WPGD1 and WPGD2, were complementing PGD3 function resulting in higher grain yield and biomass under high night temperature.…”