Zhiyong Zhang, Xixi Zheng, Jun Yang, Joachim Messing, and Yongrui Wu

Significance

Nutritional quality and yield are equally important considerations in crop breeding, although they sometimes appear at odds. In this work we made the discovery that these traits are linked through regulation by two transcription factors. Mutations that affect the expression of these transcription factors can improve the nutritional quality of the seed but also can reduce kernel yield and hardness. Therefore future corn-breeding programs should silence zein genes directly, not by blocking transcription factors.

Abstract

The maize endosperm-specific transcription factors opaque2 (O2) and prolamine-box binding factor (PBF) regulate storage protein zein genes. We show that they also control starch synthesis. The starch content in the PbfRNAi and o2 mutants was reduced by ∼5% and 11%, respectively, compared with normal genotypes. In the double-mutant PbfRNAi;o2, starch was decreased by 25%. Transcriptome analysis reveals that >1,000 genes were affected in each of the two mutants and in the double mutant; these genes were mainly enriched in sugar and protein metabolism. Pyruvate orthophosphate dikinase 1 and 2 (PPDKs) and starch synthase III (SSIII) are critical components in the starch biosynthetic enzyme complex. The expression of PPDK1, PPDK2, and SSIII and their protein levels are further reduced in the double mutants as compared with the single mutants. When the promoters of these genes were analyzed, we found a prolamine box and an O2 box that can be additively transactivated by PBF and O2. Starch synthase IIa (SSIIa, encoding another starch synthase for amylopectin) and starch branching enzyme 1 (SBEI, encoding one of the two main starch branching enzymes) are not directly regulated by PBF and O2, but their protein levels are significantly decreased in the o2 mutant and are further decreased in the double mutant, indicating that o2 and PbfRNAi may affect the levels of some other transcription factor(s) or mRNA regulatory factor(s) that in turn would affect the transcript and protein levels of SSIIa and SBEI. These findings show that three important traits—nutritional quality, calories, and yield—are linked through the same transcription factors.

See: http://www.pnas.org/content/113/39/10842.abstract.html?etoc

PNAS September 27 2016; vol.113; no.39: 10842–10847

Fig. 1.

Kernel phenotypes and carbohydrate determination in NG (W64A), PbfRNAi, o2, and PbfRNAi;o2 seeds. (A) Ear (Upper) and kernel (Lower) phenotypes of W64A, PbfRNAi, W64Ao2, and PbfRNAi;o2 seeds. e, embryo; en, endosperm. (Scale bars, 1 mm.) (B) The KW and TW of W64A, PbfRNAi, W64Ao2, and PbfRNAi;o2 seeds. Error bars represent the SD of three independent replicates. (C) SEM images of starch granules in the mature kernels. (Top) Kernel transverse sections. (Middle) SEM images of the kernel peripheral regions indicated by the white-outlined boxes in the top panel; (Lower) SEM images of the kernel central regions indicated by the black-outlined boxes in the top panel. (Scale bars, 10 μm.) (D) Carbohydrate contents in the mature kernels. Error bars represent the SD of five independent replicates. (E) Dosage of O2 in W64A, W64Ao2, and their reciprocal crosses. (F) Dosage effect of O2 on KW and starch content. Data represent the mean ± SD of three independent replicates. In B, D, and F, asterisks indicate a significant difference from W64A (Student’s t test, P < 0.05).