Research ArticleResearch Article

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

Zhiyong Zhang, Jun Yang and Yongrui Wu
The Plant Cell April 2015, 27 (4) 1162-1172; DOI: https://doi.org/10.1105/tpc.15.00035

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 γ-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 γ-zein and 22-kD α-zein accumulation during protein body formation.

INTRODUCTION

Seed storage proteins are synthesized in great abundance during seed development and serve as a nitrogen sink for the germinating seedling; they are also a staple protein source for humans and livestock. Maize (Zea mays) is one of the most productive crops in the world. Its seeds contain 10% protein, of which >60% are alcohol-soluble proteins called zeins. The abundance of zein proteins is largely attributed to their high level of gene transcription. Zeins are classified into four subfamilies, α-zeins (19 and 22 kD), γ-zeins (50, 27, and 16 kD), β-zeins (15 kD), and δ-zeins (10 and 18 kD), based on their molecular mass and structures (Esen, 1987; Coleman and Larkins, 1999). α-Zeins and γ-zeins are the two major proteins, accounting for 60 to 70% and 20 to 25% of the total zein fraction, respectively, depending on the genetic background (Thompson and Larkins, 1994). Consistent with their protein mass, zein RNA sequences constitute nearly 50% of total endosperm transcripts, of which α- and γ-zeins comprise 30 and 15%, respectively (Hunter et al., 2002). High-throughput sequencing revealed that three-quarters of zein genes (copies) are among the 100 most highly expressed genes in the endosperm, with α- and γ-zein subfamilies contributing the most abundant transcripts (Chen et al., 2014). Although high-level expression of zein genes is critical for the formation of a hard endosperm, which confers strength to withstand mechanical damage during harvesting, transportation, and storage, zein abundance compromises the nutritional value of the grain (Wu and Messing, 2012b), because these proteins are devoid of the essential amino acids lysine and tryptophan (Osborne et al., 1914; Wu et al., 2012).

Opaque2 (O2) is an endosperm-specific transcription factor (TF) belonging to the bZIP family. It has long been known to regulate the 22-kD α-zein and 15-kD β-zein genes by recognizing the O2 box (TCCACGT) in their promoters (Schmidt et al., 1992; Neto et al., 1995). In the o2 mutant, the levels of 22-kD α- and β-zein transcripts and proteins are dramatically reduced. Although the classic O2 box has not been found in the 19-kD α-zein gene promoters, their expression was always observed to be markedly downregulated in the o2 mutant (Schmidt et al., 1992; Or et al., 1993; Wu et al., 2010). Prolamine-box binding factor (PBF), another endosperm-specific DOF (DNA binding one zinc finger) TF, was speculated to play a central role in mediating the synchronized expression of all zein genes by 10 d after pollination (DAP), since most zein genes contain a P box (TGTAAAG) cis-element (Vicente-Carbajosa et al., 1997). However, it was reported recently that silencing Pbf with RNA interference (RNAi) only resulted in decreased expression of 22-kD α-zeins and 27-kD γ-zeins (Wu and Messing, 2012a). Since RNAi is not a knockout mutation, the normal expression of some zein genes with a P box could be explained by the possibilities that either the residue PBF is sufficient for their activation or other DOF TFs play redundant roles with PBF. The combination of o2 and PbfRNAi caused further reductions in 22-kD α-zein levels, confirming that this subfamily is cooperatively regulated by the two TFs through protein-protein interaction (Supplemental Figure 1) (Wu and Messing, 2012a). Sequence comparison identified a cis-element, TTTACGT, in the 27-kD γ-zein promoter, which is similar to the O2 box (TCCACGT) in 22-kD α-zein promoters (Ueda et al., 1992); therefore, it was designated as an O2-like box. O2 does not have a strong binding affinity to the O2-like box, consistent with its constitutive expression in the o2 mutant (Ueda et al., 1992; Hunter et al., 2002). However, when the O2-like box was modified to create an exact O2 binding sequence, the transcription of a reporter gene driven by the 27-kD γ-zein promoter was significantly enhanced when coexpressed with O2 (Ueda et al., 1992). It seems that 22-kD α-zein and 27-kD γ-zein promoters both contain a bifactorial motif that is composed of the closely linked P box and the O2 or O2-like box (Supplemental Figure 1) (Wu and Messing, 2012a), reminiscent of a similar regulatory apparatus in which an unknown bZIP TF might target the O2-like box and cotransactivate the 27-kD γ-zein gene with PBF.

Two maize genes encoding O2 heterodimerizing proteins (OHP1 and OHP2) were identified by screening an endosperm cDNA library with an O2 probe (Pysh et al., 1993). OHP1 and OHP2 were found to be constitutively expressed and could form heterodimers with O2; however, their biological functions are unclear (Pysh et al., 1993), mainly due to the lack of null mutants. Our work here shows that both OHP1 and OHP2 are able to bind the O2-like box and cotransactivate the 27-kD γ-zein promoter through protein-protein interaction with PBF. Using RNAi to knock down the expression of the two Ohp genes, both RNA transcript and protein levels of 27-kD γ-zein were dramatically reduced. We created different combinations of mutants with o2 and PbfRNAi and found that the three TFs regulated the expression of 90% of zein gene family members in an additive and synergistic way. Time-course analysis revealed that Ohp1 and Ohp2 are expressed at significantly higher levels than O2 between 8 and 12 DAP, consistent with the earlier expression of 27-kD γ-zeins than 22-kD α-zeins. Perhaps this temporal difference in expression explains the early appearance of γ-zein in protein bodies.

RESULTS

Temporal and Spatial Expression Patterns of Ohps, O2, and Pbf

OHPs were first thought to encode bZIP TFs that, along with PBF, regulate the expression of the 27-kD γ-zein gene, since they are homologous to O2 (Xu and Messing, 2008). OHP1 and OHP2 are located on chromosomes 1L and 5S, respectively, and are expressed in all tissues tested (Pysh et al., 1993). Our data showed that OHP1 and OHP2 are more highly expressed in cob, root, and leaf than in endosperm, tassel, and stalk (Figure 1). Time-course expression patterns of Ohp1 and Ohp2 in endosperm are similar, but they are totally different from those of Pbf and O2. The expression of Ohp1 and Ohp2 was at maximum at 8 DAP and declined afterward, while that of O2 and Pbf increased steadily, reaching a peak around 25 DAP (Figure 1). Ohps are expressed at much higher levels than O2 and Pbf, especially before 12 DAP; at a late stage of endosperm development (32 DAP), the abundance of their transcripts is lower than that of O2 and Pbf (Figure 1). Ohp1 expression was somewhat higher than Ohp2 expression before 18 DAP but declined afterward (Figure 1).

Figure 1.
Figure 1.

The Expression Patterns of Ohps, O2, and Pbf.

Quantitative RT-PCR analysis of Ohp1, Ohp2, O2, and Pbf in developing endosperms from 8 to 32 DAP and other tissues. All expression levels are normalized to Actin. Four replicates for each sample were made and are illustrated as ±sd.

Endosperm is a highly differentiated organ and is composed of four cell types, aleurone layer, starchy endosperm cells, transfer cell layers, and embryo surrounding cells, each specified to fulfill certain functions during seed development and germination (Olsen and Becraft, 2013). Our results revealed that the mRNAs of O2, Pbf, Ohp1, and Ohp2 have overlapping spatial distributions in starchy endosperm cells, being higher in the outer than the inner area, coincident with the expression patterns of 22-kD α-zein and 27-kD γ-zein genes (Figure 2). The expression of Ohp1 and Ohp2 was also detected in embryo (Figure 2), indicating that they function broadly in seed development.

Figure 2.
Figure 2.

RNA in Situ Hybridization of O2, Pbf, Ohp1, Ohp2, 22-kD α-Zein, and 27-kD γ-Zein Genes in Developing Seeds.

Longitudinal sections of W64A kernels at 12 DAP were hybridized with antisense RNA probes of O2, Pbf, Ohp1, Ohp2, 22-kD α-zein, and 27-kD γ-zein. The positive signals (blue violet) of the six genes were mainly observed in the peripheral area of starchy endosperm. The expression of Ohp1 and Ohp2 was also detected in the embryo, but that of the other four genes was not. No signal was seen in the two sections hybridized with mixed sense probes. e, embryo; se, starchy endosperm; Sense probes-1, mixed sense probes of 22-kD α-zein and 27-kD γ-zein; Sense probes-2, mixed sense probes of O2, Pbf, Ohp1, and Ohp2. Bar = 1 mm.

OHP1 and OHP2 Bind the O2-Like Box in the 27-kD γ-Zein Promoter

To test whether the O2-like box is specifically recognized by OHP1 and OHP2, a 50-bp oligonucleotide (−320 to −271) containing this motif from the 27-kD γ-zein promoter was examined by electrophoretic mobility shift assay (EMSA) (Figure 3). Binding of GST-OHP1 and GST-OHP2 fusion proteins to this DNA sequence could be visualized as retarded bands in the gel. The results revealed that both monomer (band 1) and dimer (band 2) of OHPs are able to bind to this fragment (Figure 3). When two bases of the O2-like box (TTTACGT) were mutated (TTTAAGG), the retarded bands were abolished (Figure 3). The binding specificity of OHPs was verified through the addition of unlabeled intact probes (20 and 100×) in the reaction, which resulted in a gradual loss of all retarded bands, whether recognized by monomer or dimer OHPs. These data indicate that OHP1 and OHP2 can specifically bind the O2-like box in the 27-kD γ-zein promoter.

Figure 3.
Figure 3.

EMSA of OHP1 and OHP2 with the O2-Like Box.

DNA fragments containing the intact O2-like box and one with two nucleotide substitutions were used as probes. The relative amounts of the unlabeled intact probe used for competition are indicated. 1, probe bound by OHP1 or OHP2 monomer; 2, probe bound by OHP1 or OHP2 dimer; Mp, mutated probe.

OHP1 and OHP2 Interact with PBF and Cotransactivate the 27-kD γ-Zein Promoter

The P box and O2-like box are separated by 48 bp in the promoter, suggesting that PBF and OHPs could interact (Ueda et al., 1992; Wu and Messing, 2012a). To test this possibility, pull-down assays were performed. As shown in Figure 4A, GST-tagged OHP1 and OHP2, but not the GST protein, were able to pull down maltose binding protein (MBP)-tagged PBF, indicating that both OHPs can recognize PBF in vitro. This protein-protein interaction was not detected in a previous study (Vicente-Carbajosa et al., 1997), probably due to the sensitivities of the experimental systems employed. To examine these interactions in vivo, we performed a luciferase complementation image (LCI) assay. OHP1, OHP2, and PBF were fused to the C- and N-terminal domains of LUCIFERASE (CLUC and NLUC, respectively). The results showed that cotransfection of PBF-NLUC with either OHP1-CLUC or OHP2-CLUC could produce strong luciferase activity, while individual infiltration of the three vectors with the corresponding empty construct failed to bring about a visible signal (Figure 4B). These results demonstrated that both OHP1 and OHP2 can physically interact with PBF.

Figure 4.
Figure 4.

Interaction of OHPs and PBF and Their Activation of the 27-kD γ-Zein Promoter.

(A) Pull-down experiment showing that OHPs can interact with PBF. Recombinant MBP-PBF was incubated with GST-OHP1 or GST-OHP2, and the boiled supernatants were analyzed with GST and PBF antibodies.

(B) LCI showing that OHPs and PBF interact. Fluorescence signal intensities represent their interaction activities.

(C) Transactivation of the 27-kD γ-zein promoter by OHPs and PBF. A representative image of an N. benthamiana leaf 48 h after infiltration is shown.

(D) Quantitative analysis of the luminescence intensities shown in (C). Three independent determinations were assessed by ImageJ. Error bars represent ±sd.

To investigate the ability of OHPs and PBF to activate the transcription of the 27-kD γ-zein gene, its promoter was fused with the luciferase coding sequence, yielding the reporter vector, P27-LUC, while the coding regions of Ohp1, Ohp2, and Pbf were driven by the cauliflower mosaic virus 35S promoter, giving rise to the effector plasmids. Injection of P27-LUC into a wild tobacco (Nicotiana benthamiana) leaf produced only basal luciferase activity (Figure 4C). Cotransfection of P27-LUC with 35S-PBF increases luciferase activity to a level that was ∼2-fold higher than the control, consistent with a previous study (Marzábal et al., 2008). When the reporter vector was cotransfected with 35S-OHP1 or 35S-OHP2, the luciferase activities were elevated even more than with 35S-PBF, 4- or 5-fold higher than the control, respectively, indicating that OHPs are also transactivators of 27-kD γ-zein expression, comparable to the role of O2 in regulating the 22-kD α-zein genes (Schmidt et al., 1992). Strikingly, transactivation of the 27-kD γ-zein promoter by OHP1 and OHP2 was significantly reinforced by the addition of PBF; both luciferase activities were 6-fold higher than the control, indicating that the interaction of PBF and OHPs had an additive action on the expression of the 27-kD γ-zein gene (Figure 4D).

Silencing of Ohps Results in Dramatically Reduced Expression of the 27-kD γ-Zein Gene

To genetically substantiate the regulatory function of OHPs, null mutants are required to demonstrate their effect on 27-kD γ-zein gene expression. Ohp2 was identified as an allele with a Mu insertion in position −120 relative to the start codon (Supplemental Figures 2A and 2B). However, the Mu insertion did not appear to disrupt Ohp2 transcription (Supplemental Figure 2C); therefore, no discernible change in zein accumulation was observed in homozygous ohp2-Mu1 (Supplemental Figure 2D). Since OHP1 and OHP2 are likely to be functionally redundant (Figure 4), we resorted to RNAi to simultaneously knock down their expression based on the sequence similarity of the two genes (see Methods). Four transgenic events were recovered, and all exhibited silenced expression of Ohp1 and Ohp2 (Figure 5A). Among them, event 6 accumulated 90% less Ohp1 and Ohp2 transcript than the control, while event 3 showed moderate suppression of the two genes. The mRNA levels of 27-kD γ-zein were significantly reduced in all the events, with event 6 being the most affected (Figure 5B). The zein accumulation patterns are exemplified by events 3 and 6, where 27-kD γ-zein transcripts were downregulated to a moderate and a low level, respectively. As shown by SDS-PAGE in Figure 5C, 27-kD γ-zein was barely detected in event 6, while its levels were significantly reduced but still detectable in event 3, consistent with their mRNA levels (Figure 5B). The accumulation of other zeins was not affected in either event, indicating that OHPs specifically regulate 27-kD γ-zein gene expression. Since event 6 had the best silencing effect, it was used in subsequent experiments.

Figure 5.
Figure 5.

Silencing of Ohp1 and Ohp2.

(A) Four transgenic events showing silenced expression of Ohp1 and Ohp2 at 18 DAP. Four biological replicates for each event were made and are illustrated as ±sd. Asterisks indicate significant differences from the wild type (Student’s t test, P < 0.05).

(B) Four transgenic events showing decreased accumulation of 27-kD γ-zein transcripts at 18 DAP. Quantitative RT-PCR was performed as described above.

(C) SDS-PAGE analysis of zein proteins in events 3 and 6. Three kernels each inheriting or not inheriting with the RNAi construct from self-crossed events 3 and 6 were analyzed. Total zein loaded in each lane was equal to 200 μg of maize flour. The size of each band is indicated by numbers.

Effects of o2, PbfRNAi, and OhpRNAi on the Expression of Zein Genes

To compare the synthesis of the main zein components (i.e., 27-kD γ-zein and 22- and 19-kD [19-kD z1A, z1B, and z1D] α-zeins) in the three mutants, which constitute 80 to 90% of total zeins (Thompson and Larkins, 1994), PbfRNAi and OhpRNAi were introgressed into W64A and W64Ao2 for several generations (see Methods). The gene expression and protein levels of 22-kD α-zeins and 27-kD γ-zein were dramatically reduced in o2 and OhpRNAi, respectively, and they were both strongly downregulated in PbfRNAi (Figures 6A and 6B) (Wu and Messing, 2012a). However, these mutations had variable effects on the expression of other zein genes. Although 19-kD α-zein genes had not been found to contain the O2 box or other elements recognized by O2 (Schmidt et al., 1992), their expression was markedly affected in o2 (Or et al., 1993; Wu et al., 2010). Consistent with the transcript levels, the accumulation of 19-kD α-zeins was perceptibly decreased, although not as much as the 22-kD α-zeins, consistent with prior observations (Or et al., 1993; Wu et al., 2010). Neither the mRNA nor the protein levels of 27-kD γ-zein in o2 were significantly altered compared with the wild-type levels; in OhpRNAi, the 22- and 19-kD α-zein genes were observed to produce somewhat lower levels of mRNAs (P < 0.05) but normal amounts of proteins (Figures 5C and 6), indicating that this amount of decrease of their transcripts is still not sufficient to cause translational reduction. PbfRNAi had no significant effect on overall mRNA levels of 19-kD α-zein genes, except for a decrease in z1D transcript levels (P < 0.05). Again, the accumulation of 19-kD α-zein proteins was not affected (Figures 6A and 6B).

Figure 6.
Figure 6.

Expression Analysis of Zeins in Different Combinations of OhpRNAi, PbfRNAi, and o2.

(A) Quantitative RT-PCR measurement of the expression levels of α-zein (22-kD z1C, 19-kD z1A, 19-kD z1B, and 19-kD z1D) and γ-zein genes in all mutant combinations at 18 DAP. The data shown are from two to four biological replicates per sample and are illustrated as ±sd. Asterisks indicate significant differences from W64A (Student’s t test, P < 0.05).

(B) SDS-PAGE analysis of zein proteins in all mutant combinations. Total zein loaded in each lane was from 200 μg of maize flour. Genotypes corresponding to each lane are indicated in (A). The size of each band is indicated by the number beside it. M, protein markers from top to bottom correspond to 37, 25, 20, 15, and 10 kD.

To study the effects of the genetic interaction of O2, Pbf, and Ohps on zein gene expression, three double mutants (PbfRNAi;o2, OhpRNAi;o2, and PbfRNAi;OhpRNAi) and one triple mutant (PbfRNAi;OhpRNAi;o2) were created. The most striking double mutant combinations affecting zein gene expression were PbfRNAi;o2 and OhpRNAi;o2. As expected of the two double mutants, 27-kD γ-zein mRNA and protein accumulated at very low levels but did not exhibit further reduction compared with PbfRNAi and OhpRNAi single mutants, indicating that the impacts of PbfRNAi and OhpRNAi on 27-kD γ-zein expression were epistatic to the effect of o2. In contrast, the mRNA and protein levels of 22- and 19-kD α-zeins were dramatically further reduced compared with those in the single mutants (Figures 6A and 6B). Some 22-kD α-zeins could be detected in o2 by SDS-PAGE, but they were completely missing in the double mutants PbfRNAi;o2 and OhpRNAi;o2 (Figure 6B); the 19-kD α-zein transcripts and proteins were also reduced to barely detectable levels, compared with the sizable amounts that remained in o2, indicating that either PbfRNAi or OhpRNAi has additive and synergistic effects on α-zein gene expression in o2. The combination of PbfRNAi and OhpRNAi was seen to have discernible additive effects on the expression of the 22-kD and 19-kD z1D α-zeins but far less than in the other two double mutants (Figure 6A); as a consequence, α-zeins detected by SDS-PAGE hardly showed further reduction compared with the single mutants (Figure 6B). In the triple mutant, transcript and protein levels of 19-kD α-zeins were further reduced, although the extent was much less (Figure 6B).

To determine global effects on protein synthesis, quantitative measurements of zein and nonzein proteins were performed for these mutants. In the single mutants, o2 had the most effect on the accumulation of zeins, followed by PbfRNAi and OhpRNAi. In W64A, seed flour contained 6.74% zeins based on seed dry weight (zeins/seed flour × 100%), while o2 had 2.92%, 57% lower than the wild type. PbfRNAi and OhpRNAi accumulated 4.90 and 5.70% zeins, 27 and 15% lower, respectively, than the wild type (Table 1). In the double mutants, zein contents dropped drastically in PbfRNAi;o2 and OhpRNAi;o2, to 0.76 and 1.15%, respectively, which is 89 and 83% less than that in the wild type; in PbfRNAi;OhpRNAi, the zein level was only slightly reduced compared with PbfRNAi, consistent with the observation from SDS-PAGE (Figure 6B, Table 1). Among all the mutants, the triple mutant accumulated the least amount of zeins, merely 0.70% of seed flour, which was 90% less than in the wild type (Table 1). For all mutants, the levels of nonzein proteins were proportionately increased to compensate for zein reduction, maintaining total protein levels in a relatively fixed window apparently through proteome rebalancing (Figure 7, Table 1) (Holding and Larkins, 2009; Schmidt et al., 2011; Wu and Messing, 2012b, 2014; Wu et al., 2012).

View this table:
Table 1. Protein Contents and PB Diameters of Different Mutant Combinations
Figure 7.
Figure 7.

Proteome Rebalancing in Different Mutant Combinations.

As zein levels decreased, the levels of nonzeins increased to compensate. Values shown are mg of protein per 100 mg of dry seed flour. Total protein content is the sum of zein and nonzein contents. Error bars represent ±sd with three replicates.

Development of Protein Bodies and Effects on Kernel Texture

Zeins are synthesized on the polyribosomes of the rough endoplasmic reticulum and deposited into its lumen to form the protein bodies (PBs) (Larkins and Davies, 1975; Burr and Burr, 1976; Larkins and Hurkman, 1978; Lending and Larkins, 1989). If zein protein accumulation is reduced, the most affected feature is the size of the PBs (Wolf et al., 1967; Wu and Messing, 2010a). Indeed, the average diameter of PBs in each mutant was positively correlated with the amount of zeins (Figure 8; Table 1). Strikingly, the size of PBs in the triple mutant was only about one-tenth that in the wild type, consistent with the lowest level of zeins (Figure 8; Table 1).

Figure 8.
Figure 8.

Transmission Electron Microscopy of PBs in Different Mutant Combinations.

The fourth starchy endosperm cell layer at 18 DAP was analyzed for each genotype. Each genotype is indicated above the corresponding panel. CW, cell wall; RER, rough endoplasmic reticulum; SG, starch granule. Bars in full images = 1 μm; bars in enlarged inset images = 200 nm.

OhpRNAi had a vitreous phenotype, while all the other mutants were opaque, indicating that the sole loss of 27-kD γ-zein was insufficient to cause opacity (Supplemental Figure 3). This result is consistent with previous observations in which simultaneous silencing of 27- and 16-kD γ-zeins failed to create an opaque kernel phenotype (Wu and Messing, 2010a, 2010b).

DISCUSSION

Endosperm-Specific and Nonspecific TFs Regulate Seed Storage Protein Gene Expression

We previously speculated the existence of a novel bZIP TF, other than O2, that regulates 27-kD γ-zein gene expression through its interaction with PBF, based on the fact that 22-kD α-zein and 27-kD γ-zein genes contain similar components of cis-elements in their promoters (Ueda et al., 1992; Wu and Messing, 2012a). Since OHPs are related to O2 in the maize bZIP family, they were obviously candidates for investigation (Pysh et al., 1993; Xu and Messing, 2008). It turned out that the regulatory model hypothesized for 22-kD α-zein genes can be appropriated for 27-kD γ-zein as well (Supplemental Figure 1). OHP1 and OHP2 can both bind the O2-like box and transactivate the 27-kD γ-zein gene through interaction with PBF (Figures 3 and 4). In the absence of null mutations in Ohp1 and Ohp2, RNAi was an alternative approach to study multicopy gene function (Segal et al., 2003; Wu and Messing, 2010a, 2012b). Indeed, based on SDS-PAGE, the levels of 27-kD γ-zein were dramatically reduced in the most highly RNAi-suppressed event (Figure 5C).

All zein genes are specifically expressed in endosperm from 10 to 12 DAP. It is believed that their temporal and spatial specificities are conferred by endosperm-specific TFs (Vicente-Carbajosa et al., 1997). The discovery of genes homologous to O2 and PBF in other cereal species supports this empirical hypothesis (Albani et al., 1997; Conlan et al., 1999; Oñate et al., 1999; Onodera et al., 2001; Mena et al., 2002; Yamamoto et al., 2006; Kawakatsu et al., 2009). However, in dicots, the seed specificity of storage protein gene expression is not necessarily regulated by seed-specific TFs. The main storage proteins in Arabidopsis thaliana seed are 2S albumins and 12S cruciferins (Bäumlein et al., 1994). These proteins are specifically expressed in the seed and transcriptionally regulated by two classes of TFs, including B3s such as ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEAFY COTYLEDON2 (LEC2) (Giraudat et al., 1992; Bäumlein et al., 1994; Parcy et al., 1994; Stone et al., 2001) and bZIPs (bZIP10 and bZIP25) (Lara et al., 2003). The B3 TFs ABI3, FUS3, and LEC2 are all seed-specific, but bZIP10 and bZIP25, which are O2-related TFs, are generally expressed in all tissues examined. In fact, their transcripts accumulate in roots, shoots, and leaves, where their levels are much higher than in the developing seed (Lara et al., 2003). In our work, Ohp1 and Ohp2 were found to be expressed about 1 order of magnitude higher in cobs, roots, and leaves than in 18-DAP endosperm (Figure 1). Since the protein-protein interactions of B3 and bZIP10/25 in Arabidopsis and PBF and OHPs in maize are mandatory for the transactivation of their targeted genes, it appears that only one class of TFs is seed-specific, and their cotransactivators need not be seed-specific to ensure the seed fidelity of storage protein synthesis. However, the 16-kD γ-zein gene, a homeologous copy of the 27-kD γ-zein gene arising from maize allotetraploidization, has a deletion in the promoter, which eliminates the P box and O2-like box, but it is still specifically expressed in endosperm, indicating that other regulators, possibly including epigenetic factors, can determinate the endosperm specificity of zein gene expression (Wu and Messing, 2012a).

Differential Temporal Patterns of O2, Pbf, and Ohp Expression in Maize Endosperm

The deposition of zeins in PBs is temporally and spatially organized (Lending and Larkins, 1989, 1992). γ-Zeins and β-zeins appear first and are thought to initiate PB formation. Immunotransmission electron microscopy revealed that γ- and β-zeins are localized in the peripheral region of PBs, while α- and δ-zeins, synthesized subsequently, accumulate in the central area and enlarge the PBs (Lending and Larkins, 1989). Although the spatial expression patterns of O2, Pbf, and Ohps in starchy endosperm cells are similar (Figure 2), their temporal patterns are different (Figure 1). In addition, since the o2 mutant had much less effect on the transcription of the 27-kD γ-zein and did not cause a perceivable decrease in the protein level compared with OhpRNAi (Figures 5 and 6), O2 appears to be only a minor regulator of the 27-kD γ-zein. From 8 to 12 DAP, Ohp1 and Ohp2 are expressed at significantly higher levels than O2, and one could envision that, with the onset of Pbf expression, activation of the 27-kD γ-zein should be more rapid than that of the 22-kD α-zeins. To test this, we performed RT-PCR to measure their expression. As shown in Supplemental Figure 4A, expression of the 27-kD γ-zein gene occurred by 10 DAP, while that of 22-kD α-zeins did not occur until 12 DAP. In the zein accumulation pattern observed by SDS-PAGE at 12 DAP, the predominant band is the 27-kD γ-zein, and 22-kD α-zeins do not yet appear (Supplemental Figure 4B). This difference in 27-kD γ-zein and 22-kD α-zein accumulation is consistent with the differential temporal expression patterns of O2 and Ohps and perhaps is related to the mechanism of PB formation (Figure 9).

Figure 9.
Figure 9.

Hypothetical Model Depicting the Transcriptional Regulation of the 27-kD γ- and α-Zein Genes Mediated by O2, PBF, and OHPs.

Due to the significantly higher expression of OHPs than O2 before 12 DAP, the 27-kD γ-zein gene is expressed earlier than the α-zein genes; therefore, the 27-kD γ-zein protein is more predominant than α-zein proteins in the early-forming PBs. Afterward, the amount of α-zein proteins increases rapidly, enlarging the PBs, when the level of O2 is increased. For the regulation of 22-kD (and 19-kD) α-zein genes, O2 is the main transactivator, while PBF and OHP have additive and synergistic actions on their expression.

Regulation of Seed Storage Protein Gene Expression in Maize

One of the striking features of the zein gene family is its expression level, with γ- and α-zeins being the most highly expressed subfamilies (Hunter et al., 2002; Chen et al., 2014). In our work here, we found that the synthesis of most zeins can be massively affected at the transcriptional level by the combination of o2 with either PbfRNAi or OhpRNAi. The triple mutant synthesized 90% less zeins than the wild type (Figure 6, Table 1). However, due to proteome rebalancing, the total seed protein content approached a level that did not deviate much from the wild type by compensatory synthesis of nonzein proteins (Figure 7, Table 1) (Holding and Larkins, 2009; Schmidt et al., 2011; Wu and Messing, 2012b, 2014; Wu et al., 2012). Despite this, the triple mutant accumulated the lowest level of total protein, indicating that the combination of the three mutations can have pleiotropic effects on protein synthesis and proteome rebalancing. As PBs are specialized organelles for the storage of zein proteins, it is not surprising that their sizes correlate well with the remaining amounts of zeins.

As shown in Figure 6A, all the single mutants had variable negative effects on transcript levels of 19-kD α-zeins, but only o2 accumulated discernibly reduced amounts of these proteins. However, the combination of o2 with either PbfRNAi or OhpRNAi caused a sharp decline in the levels of all α-zein transcripts and proteins (Figures 6A and 6B), indicating that O2 has the greatest impact on the expression of all α-zein genes and PBF and OHPs have additive and synergistic effects on their expression (Figure 9). However, a regulatory cis-motif in 19-kD α-zein promoters that is specifically targeted by O2 has not been identified. Thus, the regulatory machinery involved in DNA-protein recognition between O2, PBF, OHPs, and perhaps other unknown factors and cis-elements remains to be identified for the 19-kD α-zeins (Figure 9).

METHODS

Genetic Materials

Coding sequences of Ohp1 (L00623) and Ohp2 (L06478) from maize (Zea mays) were used to search against the UniformMu Transposon Resource (http://www.maizegdb.org/documentation/uniformmu/index.php), and Mu04042 and Mu06960 were found to contain a Mu insertion in Ohp2. Through PCR amplification and sequencing, the two insertions were found to be in the same position, at −120 bp relative to the start codon of Ohp2. Therefore, the two mutants were designated as Ohp2-Mu1.

Because coding sequences of Ohp1 and Ohp2 share 89% identity, a 664-bp Ohp1 cDNA fragment was amplified for OhpRNAi construction. The method for making PbfRNAi was described elsewhere (Wu and Messing, 2012a). The OhpRNAi cassette was coupled with the visible green fluorescent protein (GFP) marker under the control of the 10-kD δ-zein promoter to facilitate scoring transgenic-positive progeny seeds (Supplemental Figure 5) (Wu et al., 2013). Four positive events were recovered following the standard transformation protocol (Frame et al., 2002). All primers used for plasmid construction are listed in Supplemental Table 1. PbfRNAi was introgressed into W64A and W64Ao2 for three generations and self-crossed for two generations, yielding the homozygous single and double mutants W64APbfRNAi and W64APbfRNAi;o2. The newly generated OhpRNAi in the Hi-II genetic background was backcrossed into W64A and W64Ao2 for two generations, yielding the single and double mutants W64AOhpRNAi/+ and W64AOhpRNAi/+;o2. The positive OhpRNAi progeny were screened by viewing GFP fluorescence in kernels with Dark Reader visualization glasses under an SL9S Spot lamp (Clare Chemical Research). The selected progeny (W64AOhpRNAi/+ and W64AOhpRNAi/+;o2) were crossed with W64APbfRNAi and W64APbfRNAi;o2, yielding the double and triple mutants W64APbfRNAi/+;OhpRNAi/+ and W64APbfRNAi/+;OhpRNAi/+;o2. Since RNAi is dominant, the eight genetic materials used in this study are designated as W64A, W64Ao2, PbfRNAi, OhpRNAi, PbfRNAi;o2, OhpRNAi;o2, PbfRNAi;OhpRNAi, and PbfRNAi;OhpRNAi;o2, regardless of the homozygosity or heterozygosity of RNAi.

Quantitative and Standard RT-PCR

Total RNA was extracted from developing endosperms and other tissues using TRIzol reagent (Invitrogen) and purified with the RNeasy Mini Kit after DNase 1 digestion (Qiagen). The concentration and purity of the RNA were determined using a NanoDrop 2000 spectrophotometer. The SuperScript III First Strand Kit (Invitrogen) was used for reverse transcription. Standard RT-PCR was performed with REDTaq Reaction Mix (Sigma-Aldrich) and quantitative RT-PCR with SYBR Green I. The comparative CT method (ΔΔCT method) was employed for the relative quantification of gene expression, in which the maize Actin gene was used as the reference. Statistically significant differences of target gene expression from different mutants were calculated by Student’s t test. All primers are listed in Supplemental Table 1.

Expression and Purification of Recombinant Proteins

The expression constructs for GST-OHP1 and GST-OHP2 were generated by cloning the corresponding open reading frames (ORFs) into the BamHI and EcoRI sites of pGEX-4T-1 (Amersham). The ORF of Pbf was fused to the MBP tag of the expression vector pMAL-C2X (New England Biolabs). All recombinant proteins were affinity-purified following the manufacturer’s manual.

EMSA

The two oligonucleotide probes of the 27-kD γ-zein promoter described in Figure 3, containing the intact and mutated O2-like box, were synthesized and labeled with biotin at the 3′ end (Thermo). The probes were mixed with the purified protein at 25°C for 20 min in a reaction buffer containing 1× binding buffer, 2.5% glycerol, 5 mM MgCl2, 50 ng/μL poly(dI∙dC), and 0.05% Nonidet P-40. The mixture was separated by 6% native PAGE in 0.5× Tris/borate/EDTA buffer. Subsequent procedures were performed according to the instructions of the LightShift Chemiluminescent EMSA Kit (Thermo). The luminescence was visualized on the Tanon-5200 Chemiluminescent Imaging System (Tanon Science and Technology).

GST Pull-Down Assay

GST or GST-OHP fused proteins were mixed with MBP-tagged PBF in 1 mL of GST binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.3), and the mixture was kept at 4°C for 4 h with rotating. After washing with GST binding buffer, Glutathione Sepharose 4B (Amersham) was added to each mixture and then kept rotating for another 2 h at 4°C. After washing three times with GST binding buffer, the GST resin was boiled in SDS loading buffer, and the supernatants were analyzed by immunoblotting. For protein gel blot analysis, the eluted proteins were separated by 15 or 12% SDS-PAGE and transferred onto a nitrocellulose membrane (Whatman). The anti-GST or anti-PBF protein and the secondary antibody, anti-rabbit-IgG conjugated to horseradish peroxidase, was used at a 1:5000 dilution. Protein bands were visualized using the Tanon-5200 system.

LCI Assay and Transactivation of 27-kD γ-Zein Promoter

ORFs of Pbf, Ohp1, and Ohp2 were cloned into JW771 (NLUC) and JW772 (CLUC), respectively, yielding PBF-NLUC and OHP1/OHP2-CLUC constructs for the LCI assay, following the protocol described elsewhere (Gou et al., 2011).

The reporter P27-LUC was generated by insertion of the 27-kD γ-zein promoter (1052 bp) into the HindIII and BamHI sites of the pLL00R vector. Effectors 35S-OHP1, 35S-OHP2, and 35S-PBF were created by cloning their corresponding coding sequences into pRI101 vector (Takara). The empty vector was used as a negative control.

Agrobacterium tumefaciens GV3101 harboring the above constructs was infiltrated into 5-week-old Nicotiana benthamiana leaves using a needleless syringe for LCI and transactivation analyses. After growing for 48 h under the condition of 16 h of light and 8 h of dark, leaves were injected with 0.94 mM luciferin, and the resulting luciferase signals were captured using the Tanon-5200 image system. These experiments were repeated at least three times with similar results. Quantitative analysis was performed using ImageJ software (http://rsb.info.nih.gov/ij/).

RNA in Situ Hybridization

W64A kernels at 12 DAP were used for RNA in situ hybridization. cDNA fragments of target genes were inserted into the pBluescript SK+ vector for RNA probe synthesis. The primers for amplifying these fragments are listed in Supplemental Table 1. The antisense and sense RNA probes were synthesized by in vitro transcription using T7 and T3 RNA polymerase, with DIG RNA Labeling Mixture (Roche). Tissue processing and in situ hybridization experiments using 10-mm sections were performed according to the methods described previously (Cox and Goldberg, 1988; Langdale, 1994).

The sections were observed and imaged with a light stereomicroscope (Leica M165 FC).

Protein Quantification

At least 10 mature kernels for each sample were ground into fine powder with a coffee grinder, and 100 mg of flour was weighed for the extraction of zein and nonzein proteins. The patterns of zein accumulation were analyzed by SDS-PAGE. Protein quantification was performed with the Compat-Able Protein Assay Preparation Reagent Kit and the BCA Protein Assay Kit (Pierce). All measurements were replicated at least three times.

Transmission Electron Microscopy

Kernels at 18 DAP of W64A and different mutants were sliced, fixed, dehydrated, and embedded as described previously (Wu and Messing, 2010a). Ultrathin sections of the samples were cut with a diamond knife on a Leica EMUC6-FC6 ultramicrotome and imaged at 80 kV with a Hitachi H-7650 transmission electron microscope.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers NM_001111951 for O2, NM_001111930 for Pbf, L00623 for Ohp1, and L06478 for Ohp2.

Supplemental Data

Acknowledgments

We thank the reviewers for their critical review of this article and their invaluable suggestions on the editing of the language. The PbfRNAi used in the work was originally generated and obtained from Joachim Messing’s laboratory at the Waksman Institute of Microbiology, Rutgers University. This research was supported by the National Natural Science Foundation of China (Grants 31371630, 91335109, and 31422040 to Y.W.) and a Chinese Thousand Talents Program grant (to Y.W.).

AUTHOR CONTRIBUTIONS

Z.Z., J.Y., and Y.W. designed and performed the research. Z.Z., J.Y., and Y.W. analyzed the data. Z.Z., J.Y., and Y.W. wrote the article.

Footnotes

  • www.plantcell.org/cgi/doi/10.1105/tpc.15.00035

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Yongrui Wu (yrwu{at}sibs.ac.cn).

  • 1 These authors contributed equally to this work.

Glossary

TF
transcription factor
DAP
days after pollination
RNAi
RNA interference
EMSA
electrophoretic mobility shift assay
LCI
luciferase complementation image
PB
protein body
ORF
open reading frame
  • Received January 14, 2015.
  • Revised April 6, 2015.
  • Accepted April 9, 2015.
  • Published April 21, 2015.

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