(1) Understanding the molecular mechanisms of the storage-protein gene expression and endosperm filling. 

1)  Construction of the main transcriptional regulatory network of maize storage-protein zein genes 

We revealed that OHP1 and OHP2 specifically regulate 27-kDa γ-zein gene expression through binding to an O2-like box (TTTACGT) in its promoter and interact with PBF1. Through creating the double and triple mutants of o2, PbfRNAi and OhpRNAi, we demonstrated that the three transcription factors regulate 90% of zein gene expression in an additive and synergistic way. So the three transcription factors serve as master regulators of zein gene expression, which determines the hardness and nutritional composition of maize kernels. We further propose a model (Figure 1) for transcriptional regulation of the 22-kDa α- and 27-kDa γ-zein genes, based on the differential expression patterns of the three transcription factors during protein body formation (Zhang et al., 2015; Yang et al., 2016). 

Figure 1. A hypothetical model depicting the transcriptional regulation of the 27-kDa γ- and α-zein genes mediated by O2, PBF1 and OHPs. 

2) Floury3, a PLATZ protein characterized as a new regulator required for maize endosperm development and filling 

RNA polymerase III (RNAPIII) is specialized for transcription of short, abundant nonprotein-coding RNAs, such as tRNAs and 5S rRNA, which have fundamental roles in the protein synthesis. Highly abundant ribosomal proteins, tRNAs and rRNAs are required to achieve high-efficiency functioning of the protein translation machinery in the maize endosperm, which accounts for 90% of the dry seed weight and serves as the main organ of nutrient storage. Transcriptional regulation of protein-coding genes by RNAPII has been extensively studied in almost all model organisms, but regulation of tRNAs and 5S rRNA expression has considerably less understood especially in plants. 

PLATZ (plant AT-rich sequence- and zinc-binding) family proteins are a novel class of plant-specific zinc-dependent DNA-binding proteins and are classified as transcription factors (TFs) in plant TFs databases (Nagano et al., 2001). However, none have yet been genetically characterized to regulate specific genes in plants. We cloned and functionally characterized a maize classic endosperm-specific mutant floury3 (fl3). The fl3 mutation causes severe defects in endosperm development and a dramatic reduction in seed weight. The mutant phenotype only occurs when fl3 is transmitted through the female. fl3 is caused by a dominant mutation and regulated by genomic imprinting, leading to a semi-dominant behavior. Fl3 encodes a PLATZ protein and is determined to be involved in protein-protein interaction with two critical factors, RPC53 and TFC1, in the RNAPIII transcription complex. The fl3 mutation caused significant reduction in levels of many tRNAs and 5S rRNA, and also dramatic alterations in transcriptome (Figure 2). These findings provide new insight into understanding of the endosperm development and storage reserve filling, and also RNAPIII transcription modulation (Li et al., 2017; Wang et al., 2018).

Figure 2. A proposed model for FL3/fl3 controlling the storage filling in the maize endosperm. Floury3 encodes a PLATZ protein and is specifically expressed in starchy endosperm cells. Fl3 interacts with two critical factors, RPC53 and TFC1, in the RNAPIII transcription complex and regulates the endosperm development and storage reserve filling.

3) Maize Oxalyl-CoA Decarboxylase1 Acting Downstream of Opaque7 Degrades Oxalate and Affects the Seed Metabolome and Nutritional Quality 

Oxalate is the simplest dicarboxylic acid, and it is produced in most if not all organisms. In crop plants and many vegetables, it may account for as much as 3–10% of the dry weight and thus plays various functional roles in biological and metabolic processes such as metal tolerance, ion balance, and insect defense (Franceschi and Nakata, 2005). Although oxalate provides many benefits for plant adaption, an excessive accumulation of this acid in vivo is toxic to cell growth (Nakata, 2015) and represents an undesirable trait that affects the nutritional quality of certain vegetables (Weaver et al., 1987; Heaney and Weaver, 1990). Oxalate acts as an anti-nutrient to impair the absorption of Ca and some other minerals. Moreover, ingestion of free oxalate or metal oxalate crystals may cause serious diseases. For example, oxalate can induce breast cancer and Ca oxalate crystals have been shown to be associated with kidney stone composition and urinary metabolic disturbances (Taylor and Curhan, 2007; Lorenz et al., 2013; Kirejczyk et al., 2014; Castellaro et al., 2015).

Figure 3. Maize Oxalyl-CoA Decarboxylase1 Degrades Oxalate. ZmOCD1 catalyzes oxalyl-CoA into formal-CoA and CO2 for oxalate degradation and affects seed storage reserves, metabolome, nutritional quality and endosperm vitreousness.

A putative oxalate degradation pathway and its corresponding enzymes have been proposed in plants. The combined action of four enzymes converts 1 molecule of oxalate into 2 molecules of the final product CO2, with each step catalyzed by oxalyl-CoA synthetase, oxalyl-CoA decarboxylase, formyl-CoA hydrolase and formate dehydrogenase (Foster et al., 2012; Foster and Nakata, 2014; Foster et al., 2016). The gene encoding oxalyl-CoA synthetase has been cloned in maize and Arabidopsis, but the other genes remain unknown (Miclaus et al., 2011; Wang et al., 2011). We cloned a previously undescribed maize opaque endosperm mutant that encodes oxalyl-CoA decarboxylase1 (OCD1). The mutant seeds contain a significantly higher level of oxalate than the wild type, indicating that a defect in oxalate catabolism occurs in ocd1. The maize classic mutant opaque7 (o7) was initially cloned for its high lysine trait, although the gene function was not understood until its homologue in Arabidopsis thaliana was found to encode an oxalyl-CoA synthetase (EC 6.2.1.8), which ligates oxalate and CoA to form oxalyl-CoA. Our enzymatic analysis showed that ZmOCD1 catalyzes oxalyl-CoA, the product of O7, into formyl-CoA and CO2 for degradation. Mutations in ocd1 caused dramatic alterations in metabolome in the endosperm (Figure 3). Our findings demonstrate that ZmOCD1 acts downstream of O7 in oxalate degradation and affects endosperm development, the metabolome and nutritional quality in maize seed (Yang et al., 2018).  

(2) Cloning and functional analysis of a major o2 modifier (qγ27) for endosperm modification in Quality Protein Maize (QPM) 

Quality Protein Maize (QPM) has the potential to benefit millions of people in developing countries who consume maize as their sole protein source. To do this, the scientists have made great efforts to identify the genetic locus or gene responsible for the high amount of lysine in maize endosperm (Mertz et al., 1964; Nelson et al., 1965). More than half a century ago, Oliver Nelson and Edwin Mertz at Purdue University found that the maize opaque2 (o2) mutant produces doubling of the endosperm lysine content, suggesting o2 as a potential target gene for QPM breeding. However, breeding such a QPM hybrid based on o2 takes longer than regular hybrids, primarily because of the complex and unknown components of o2-mediated endosperm modification pathway (Vasal et al., 1980; Bjarnason and Vasal, 1992; Wu and Messing, 2011). 

Previous studies have shown that enhanced expression of 27-kDa γ-zein in QPM is essential for endosperm modification (Lopes and Larkins, 1991; Or et al., 1993; Wu et al., 2010; Holding et al., 2011). Taking advantage of genome-wide association study analysis of a natural population, linkage mapping analysis of a recombinant inbred line population, and map-based cloning, we identified a quantitative trait locus (qγ27) affecting the expression of 27-kDa γ-zein. qγ27 was mapped to the same region as the major o2 modifier (o2 modifier1) on chromosome 7 near the 27-kDa γ-zein locus. qγ27 resulted from 15.26-kb duplication at the 27-kDa γ-zein locus, which increases the level of gene expression (Figure 4). The elevated level of 27-kDa γ-zein is very critical to facilitate the formation of numerous small protein bodies surrounding the starch granules, which is able to partially rebuild the proteinaceous matrix for restoration of kernel vitreousness in QPM. Thus, these findings not only improve our understanding of genetic variation and artificial selection in maize, but also provide a potential genetic resource for QPM breeding (Liu et al., 2016). We developed a molecular marker for this duplication and applied it for molecular breeding of QPM in Yunnan Province.  

Figure 4. Gene duplication at the 27-kDa γ-zein locus. We identified a quantitative trait locus, a gene duplication at the 27-kDa γ-zein locus, which confers enhanced expression of this protein and leads to endosperm modification. This knowledge can effectively be applied in QPM breeding. 

(3) Establishment of the network of the regulation of protein and starch synthesis by maize endosperm-specific transcription factors O2 and PBF1 

Corn is one of the most important food and feed for human beings and livestock, respectively. Nutritional quality and yield are equally important in the corn breeding. In general, corn’s nutritional quality is poor because its main storage protein, zein, is devoid of the essential amino acids lysine and tryptophan. O2 and PBF1 are two endosperm-specific transcription factors for the zein gene expression. The classic mutant opaque2 (o2) is found to improve the seed nutritional value by reducing the synthesis of zein proteins. In practical applications, the o2 mutant has multiple agronomic defects, e.g. soft texture and yield drop. Corn breeders have developed the hard version of o2 mutant by selecting QTLs, generating the quality protein maize (QPM). However, the yield loss has not been overcome, which precludes its wide use. 

We determined that the nutritional quality and yield traits are linked through the same transcription factors. Transcriptome and immunoblotting analyses of mutants showed that both PbfRNAi and o2 affect the expression of several major enzymes of starch synthetic complex in amyloplasts, including PPDKs, SSIII, SSIIa and SBEI. Furthermore, molecular and biochemical experiments prove that the two transcription factors trans-activate the expression of PPDKs and SSIII. We also found that the two transcription factors regulate the expression of the pentose phosphate pathway, which is associated with starch synthesis in corn endosperm. These effects together led to the reduction of starch synthesis and kernel weight in both direct and profound ways (Figure 5). Therefore, future QPM breeding programs should use RNAi specifically against the expression of zein genes instead of the o2 mutant to avoid the yield reduction (Zhang et al., 2016). 

Figure 5. A proposed model for PBF1 and O2 controlling kernel nutritional quality and yield in maize. The maize endosperm-specific transcription factors O2 and PBF1 regulate storage protein zein genes. We show that they also control starch synthesis. Therefore future corn-breeding programs should silence zein genes directly, not by blocking transcription factors.