Transcriptional Regulation of Zein Gene Expression in Maize through the Additive and Synergistic Action of opaque2, Prolamine-Box Binding Factor, and O2 Heterodimerizing Proteins

Abstract

Maize (Zea mays) zeins are some of the most abundant cereal seed storage proteins (SSPs). Their abundance influences kernel hardness but compromises its nutritional quality. Transcription factors regulating the expression of zein and other SSP genes in cereals are endosperm-specific and homologs of maize opaque2 (O2) and prolamine-box binding factor (PBF). This study demonstrates that the ubiquitously expressed transcription factors, O2 heterodimerizing proteins (OHPs), specifically regulate 27-kD g-zein gene expression (through binding to an O2-like box in its promoter) and interact with PBF. The zein content of double mutants OhpRNAi;o2 and PbfRNAi;o2 and the triple mutant PbfRNAi;OhpRNAi;o2 is reduced by 83, 89, and 90%, respectively, compared with the wild type. The triple mutant developed the smallest zein protein bodies, which were merely one-tenth the wild type's size. Total protein levels in these mutants were maintained in a relatively constant range through proteome rebalancing. These data show that OHPs, O2, and PBF are master regulators of zein storage protein synthesis, acting in an additive and synergistic mode. The differential expression patterns of OHP and O2 genes may cause the slight differences in the timing of 27-kD g-zein and 22-kD a-zein accumulation during protein body formation.

Maize endosperm-specific transcription factors O2 and PBF network the regulation of protein and starch synthesis

“…On average 21,788 expressed genes were detected for each sample (Fig. S1C), consistent with the previous transcriptome analysis of developing endosperms (9,28). A total of 1,296 up-regulated and 1,513 down-regulated genes were detected on average in the three mutants compared with NG ( Fig.…”

“…However, it is not likely that the reduction of zein proteins contributes significantly to the loss of kernel weight, because in total zein proteins account for only about 6% of the dry seed flour in NG. Moreover, the decrease in zein protein accumulation would result in a compensatory increase in non-zein protein synthesis, which maintains the total protein content in a relatively constant range at maturity (28). In contrast, starch constitutes around 70% of the dry seed mass and therefore is the most preponderant substance in determining the kernel weight.…”

“…PBF and O2 are two endosperm-specific transcription factors that are specifically expressed from 8 to 10 DAP. The two transcription factors control 89% of zein transcription (28). Zein mRNAs then are translated on the polyribosomes of the rough endoplasmic reticulum (RER), and the resulting proteins are translocated into the lumen of the RER, where they are assembled into protein bodies (33).…”

“…The PbfRNAi and o2 mutants and their double mutant PbfRNAi;o2 mutant are all in the W64A background (28). They all exhibit an opaque and soft endosperm phenotype.…”

Breeding for Quality Protein Maize (QPM) Varieties: A Review

“…One of the main focuses of CIMMYT scientists is to develop reliable, easy-to-use markers for endosperm hardness and free amino acid content in the maize endosperm including high throughput, single seed-based DNA extraction, coupled with low-cost, high-density SNP genotyping strategies, and breeder-ready markers for some key adaptive traits in QPM maize to enhance efficiency and cost effectiveness of MAS in QPM breeding programs [58]. Proteomic analysis has been also studied in QPM breeding [28,59,60]. Some case studies conducted on QPM genotypes using molecular approaches are summarized in Table 5.…”