Article

Opaque-2 Is a Transcriptional Activator That Recognizes a Specific Target Site in 22-kD Zein Genes

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Abstract

opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family.

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... An analysis of the expression level of DEGs detected by previous studies but not by us 164 showed that nearly all of them exhibited low fold-changes (|log 2 FC| < 1) in our dataset 165 (Supplemental Figure 2B). 166 To determine the genome-wide direct target genes of O2, we performed a ChIP-Seq assay on two 167 biological replicates of B73 endosperm at 15 DAP using a previously described anti-O2 antibody 168 (Schmidt et al., 1992). As a negative control, we performed the same assay in duplicates using 169 endosperm from B73o2. ...
... By exclusion, the 189 unbound modulated genes (here referred to as the indirect targets of O2, which are likely to 190 include genes whose transcription is regulated by TFs downstream to O2) included 877 O2-191 7 activated genes and 800 O2-repressed genes, while the group that was detected as bound but 192 unmodulated by O2 included 3,096 genes ( Figure 1C). 193 To evaluate the accuracy of our identification of direct O2 targets, we examined the differential 194 expression and peak association of several canonical O2 targets including O2 itself,cyPPDK1,195 azs22-4, LKR/SDH, b-32, and the 15-kD β-zein gene (Lohmer et al., 1991;Schmidt et al., 1992;196 Cord Neto et al., 1995;Gallusci et al., 1996;Maddaloni et al., 1996;Kemper et al., 1999). The 197 results showed that nearly all of these genes are identified as direct O2 targets by our data Supplemental Data Set 1) but was not associated with an O2 peak. ...
... Profiling the read distribution over the same annotated gene 244 models using a similar approach confirmed this pattern (Supplemental Figure 7). These results 245 were consistent with the reported localization of several known O2-binding sites, which were 246 detected within the 300-bp region upstream of the translation start sites (Schmidt et al., 1992; 247 Wu and Messing, 2012). We used two independent approaches to verify binding of O2 to the O2 248 peaks. ...
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Development of the cereal endosperm involves cell differentiation processes that enable nutrient uptake from the maternal plant, accumulation of storage products and their utilization during germination. However, little is known about the regulatory mechanisms that link cell differentiation processes with those controlling storage product synthesis and deposition, including the activation of zein genes by the maize (Zea mays) bZIP transcription factor Opaque-2 (O2). Here, we mapped in vivo binding sites of O2 in B73 endosperm, and compared the results with genes differentially expressed in B73 and B73o2. We identified 186 putative direct O2 targets and 1,677 indirect targets, encoding a broad set of gene functionalities. Examination of the temporal expression patterns of O2 targets revealed at least two distinct modes of O2-mediated gene activation. Two O2-activated genes, bZIP17 and NAKED ENDOSPERM2 (NKD2), encode transcription factors, which can in turn co-activate other O2-network genes with O2. NKD2 (with its paralog NKD1) was previously shown to be involved in regulation of aleurone development. Collectively, our results provide insights into the complexity of the O2-regulated network and its role in regulation of endosperm cell differentiation and function.
... bZIP factors are the first identified transcription factors to regulate starch biosynthesis [21,22], and they appear to be one of the main regulators of this process. bZIP regulators have been well characterized in several crops, including Oryza sativa L. [23,24], Zea mays L. [10], Hordeum vulgare L. [25], and Triticum aestivum L. [26,27]. bZIP factors play multiple roles during starch synthesis, but their precise roles vary in different species. ...
... For example, one bZIP transcription factor, OsbZIP58, regulates six genes during starch metabolism in rice [10,28,29], while the identified candidate TabZIPs are only involved in the regulation of granule-bound starch synthase I (GBSSI)/Waxy (Wx) and branching enzyme II (BEII) during starch biosynthesis in wheat [26]. Furthermore, the binding motif of bZIPs seems to be considerably the same in rice [11], barley [25], and maize [23], but it is still not clear whether a similar phenomenon exists in other cereal species. bZIPs show broad affinities with different motifs in the process of regulating starch biosynthesis. ...
... Aside from direct regulation of starch synthetic enzymes, some bZIPs are also involved in the regulation of starch synthesis via indirect ways ( Table 2). For example, O2 can regulate the gene expression of α-and β-zein [23], b-32 [21], and pyruvate orthophosphate dikinase 1 (PPDK1) [10,22] via recognition of the O2 box, GA/TGAPyPuTGPu, and TCCACGTAGA sequences in their promoters, respectively. Moreover, two heterodimerizing proteins of O2, OHP1 and OHP2, can regulate the synthesis of 27-kDa γ-zein and 22-kDa α-zein [86,87] through the recognition of the O2-like motif in the promoters of the target genes. ...
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Starch is the main food source for human beings and livestock all over the world, and it is also the raw material for production of industrial alcohol and biofuel. A considerable part of the world’s annual starch production comes from crops and their seeds. With the increasing demand for starch from food and non-food industries and the growing loss of arable land due to urbanization, understanding starch biosynthesis and its regulators is essential to produce the desirable traits as well as more and better polymers via biotechnological approaches in cereal crops. Because of the complexity and flexibility of carbon allocation in the formation of endosperm starch, cereal crops require a broad range of enzymes and one matching network of regulators to control the providential functioning of these starch biosynthetic enzymes. Here, we comprehensively summarize the current knowledge about regulatory factors of starch biosynthesis in cereal crops, with an emphasis on the transcription factors that directly regulate starch biosynthesis. This review will provide new insights for the manipulation of bioengineering and starch biosynthesis to improve starch yields or qualities in our diets and in industry.
... Though Mertz, Bates, and Nelson identified opaque-2 as the sole cause for zein reduction when the mutant was first described, its ability to cause the resultant proteomic rebalancing remained undetermined until 1990 . No doubt in part due to advances in genetic technologies, such as cDNA cloning and sequencing, southern blots, and fusion protein production, Schmidt et al. first hypothesized opaque-2's function as a regulatory protein directly interacting with zein transcription in 1990, and he would later prove himself correct in 1992 (Schmidt et al., 1990;Schmidt et al., 1992). The 1992 paper clearly identified opaque-2 as a leucine zipper transcriptional activator for, specifically, the 22-kD α-zein genes. ...
... Concurrent to Schmidt's work in California identifying O2 as a transcription factor for 22kD α-zein, researchers in Italy were conducting almost identical work with O2 and its effect on the 'b-32' gene, a 32-kD albumin (a water-soluble, globular type protein) (Lohmer, 1991). Lohmer et al. postulated that O2 was the transcriptional activator for the b-32 gene and this theory was widely accepted despite Schmidt's skepticism detailed in his 1992 paper (Lohmer et al., 1991;Schmidt et al., 1992). Taken together, these two papers trailblazed the way for other research groups to further investigate O2 and its transcriptional targets. ...
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In 2017, twelve Quality Protein Popcorn (QPP) inbred lines were developed and selected as premier dent by popcorn crosses fit for hybridization and testing. These QPP inbred lines were derived from specific Quality Protein dent Maize (QPM) by ConAgra Brands® popcorn line crosses to produce high lysine, vitreous popcorn lines capable of near-equal popping characteristics compared to the original popcorn parents. The QPP hybridization project commenced in the summer of 2018 utilizing these 12 inbred QPP lines and crossing them in a full diallel. Since then, the production of QPP hybrids has employed a diverse set of selection factors evaluating agronomic, popping quality, protein quality, and sensory traits. In 2021, six QPP hybrids were selected for continued evaluation based on agronomic, protein, and popping characteristics, and two QPP hybrids were ultimately selected based on the results from a sensory study. Advisor: David R. Holding
... Opaque2 (O2) is an endosperm-specific TF belonging to the bZIP family that regulates the expression of most zein genes (Schmidt et al., 1990;Schmidt et al., 1992;Li et al., 2015;Zhan et al., 2018). In the o2 mutant, the synthesis of 22-kD α-and β-zein proteins is strikingly reduced (Kodrzycki et al., 1989;Schmidt et al., 1992;Cord Neto et al., 1995;Zhang et al., 2015). ...
... Opaque2 (O2) is an endosperm-specific TF belonging to the bZIP family that regulates the expression of most zein genes (Schmidt et al., 1990;Schmidt et al., 1992;Li et al., 2015;Zhan et al., 2018). In the o2 mutant, the synthesis of 22-kD α-and β-zein proteins is strikingly reduced (Kodrzycki et al., 1989;Schmidt et al., 1992;Cord Neto et al., 1995;Zhang et al., 2015). Due to proteome rebalancing, the levels of nonzein proteins are increased and compensate for the zein reduction, maintaining total protein levels in o2 endosperm that do not deviate much from those of WT in a genetic-dependent manner (Zhang et al., 2015). ...
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One-sentence summary: VP1 directly regulates embryo scutellum development and nutrient assimilation, which is required for protein reallocation from the endosperm to embryo. ABSTRACT During maize (Zea mays) seed development, the endosperm functions as the major organ for storage of photoassimilate, serving to nourish the embryo. α-Zeins and globulins (GLBs) predominantly accumulate in the maize endosperm and embryo, respectively. Here, we show that suppression of α-zeins by RNA interference (αRNAi) in the endosperm results in more GLB1 being synthesized in the embryo, thereby markedly increasing the size and number of protein storage vacuoles. Glb genes are strongly expressed in the middle-to-upper section of the scutellum, cells of which are significantly enlarged by αRNAi induction. Elimination of GLBs caused an apparent reduction in embryo protein level, regardless of whether α-zeins were expressed or suppressed in the endosperm, indicating that GLBs represent the dominant capacity for storage of amino acids allocated from the endosperm. It appears that protein reallocation is mostly regulated at the transcriptional level. Genes differentially expressed between wild-type and αRNAi kernels are mainly involved in sulfur assimilation and nutrient metabolism, and many are transactivated by VIVIPAROUS-1 (VP1). In vp1 embryos, misshapen scutellum cells contain notably less cellular content and are unable to respond to αRNAi induction. Our results demonstrate that VP1 is essential for scutellum development and protein reallocation from the endosperm to embryo.
... The Opaque2 gene (O2) encodes an endosperm-specific transcriptional 13 activator which is specifically expressed in the subaleurone layers of maize endosperm during seed development. (Schmidt et al., 1992). ...
... Fourteen proteins affected by mutant allele (o2) in maize were identified across two different backgrounds (wild type v.s. a dent type W64Ao2 and wild type vs a flint type F2o2) via 2-D PAGE (Damerval and De Vienne, 1993). The protein was shown to activate the transcription of the 22-kDa α-zein (Schmidt et al., 1992), and 14 kD β-zein genes (Neto et al., 1995), together with the β-32, a ribosome inactivating protein (RIP) (Lohmer et al., 1991) and cyPPDK1 genes (Maddaloni et al., 1996). The PBF (prolamin-box binding factor) was reported to bind the prolamin box in zein gene promoters and interact with O2 (Vicente-Carbajosa et al., 1997). ...
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I have been working on a Quality Protein Popcorn breeding project where QPM conversion is carried out simultaneously for several elite popcorn germplasms. During my study in the graduate program, I led the following aspects of the Quality Protein Popcorn Breeding Project: 1. Identified suitable QPMs as opaque-2 allele donors. 2.Examined the feasibility of quick introgression of the opaque-2 allele into popcorn lines via marker-assisted selection. 3. Monitored modification by SDS-PAGE zein profiling and light box phenotypic selection to make sure multiple modifier loci for opaque-2 were incorporated each time generation advancement was carried out. 4. Carried out high throughput DNA extraction using the BioSprint Workstation and performed genotyping to guide field selection. 5. Principle Component Analysis (PCA) of amino acid profiles from UPLC (Ultra Performance Liquid Chromatography) of candidate QPP lines along with corresponding parental lines (QPM and popcorn). (in collaboration with Dr. Ruthie Angelovici from University of Missouri Columbia, MO) Advisor: David R. Holding
... We find that the overexpression of OsDLK specifically modulates the expression of NtCf9, the tobacco homologue of Cold-Box Factor 4 ( Figure 4B), while the responses of other cold-related transcripts did not show a significant difference compared to non-transformed tobacco cells ( Figure 4A). Recombinantly expressed OsDLK bound to a specific DNA motif, 11 bp in length ( Figure 5B-D), which has been known to be a target for transcription factor OPAQUE 2, a transcriptional key regulator that activates the accumulation of seed storage proteins by binding to this motif by virtue of its leucin zipper [24,25]. Interestingly, there exist several functional analogies between OPAQUE 2 and OsDLK. ...
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... The mutant opaque2 (o2) located on chromosome-7 encodes less active leucine zipper transcriptional factor leading to enhanced lysine and tryptophan in maize endosperm (Schmidt et al. 1992). opaque16 (o16), another recessive mutant of Robertson's Mutator (Mu) stock, located on chromosome-8 was found to be associated with enhanced lysine and tryptophan in maize endosperm (Yang et al. 2005;Hossain et al. 2008aHossain et al. , b, 2017Sarika et al. 2017). ...
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Maize has emerged as an important crop for food, feed and various applications. Utilization of hybrid technology has resulted in a quantum jump in grain production worldwide. However, ever-increasing population pressure coupled with climate change warrant many fold increase in productivity in a shorter time frame. Emergence of newer diseases and insect-pests further pose a great challenge to even sustain the production. Malnutrition has become a major health issue, thereby causing severe socioeconomic losses. However, discovery of new genes and quantitative trait loci (QTLs) for higher grain yield, plant architecture, resistance/tolerance to various biotic and abiotic stresses, nutritional quality and specialty traits, and also availability of suitable donors provide great opportunity to breed improved hybrids with higher productivity, better resilience to biotic and abiotic stresses, and higher nutritional quality. Genomics-assisted breeding, doubled haploid and gene editing technology provide great impetus to further accelerate the breeding cycle. Here, we discussed the present status, opportunities and challenges in maize breeding.
... Zeins account for >60% endosperm proteins and are extremely deficient in the essential amino acid lysine, thereby resulting in poor nutritional value of the maize grain 11,12 . Opaque2 (O2) is a bZIP transcription factor that mainly regulates the expression of α-zein and β-zein genes [13][14][15] . In the o2 mutant endosperm, the amount of zein proteins is dramatically reduced, but the total protein level remains relatively constant by a complementary increase of non-zein proteins. ...
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Copy number variation (CNV) is a major source of genetic variation and often contributes to phenotypic variation in maize. The duplication at the 27-kDa γ-zein locus (qγ27) is essential to convert soft endosperm into hard endosperm in quality protein maize (QPM). This duplication is unstable and generally produces CNV at this locus. We conducted genetic experiments designed to directly measure DNA rearrangement frequencies occurring in males and females of different genetic backgrounds. The average frequency with which the duplication rearranges to single copies is 1.27 × 10 −3 and varies among different lines. A triplication of γ27 gene was screened and showed a better potential than the duplication for the future QPM breeding. Our results highlight a novel approach to directly determine the frequency of DNA rearrangements, in this case resulting in CNV at the qγ27 locus. Furthermore, this provides a highly effective way to test suitable parents in QPM breeding.
... during drought (Almeida et al. 2014, Lu et al. 2010, Semagn et al. 2013, Vargas et al. 2006, Zhao et al. 2017. QTLs/ genes have been identified for morphological traits and yield components in maize using map-based cloning strategies, such as Brachytic 2 (Br2) (Xing et al. 2015), gln1-4 (Martin et al. 2006), Opaque-2 (o2) (Schmidt et al. 1992), unbranched2 (ub2) (Chuck et al. 2014), and ZmCLA4 (Zhang et al. 2014), which can be useful targets for marker assisted selection (MAS) in modern maize breeding programs. In addition, there is growing evidence suggesting that ear height-to-plant height ratio (EHPH), grain weight per ear (GW), and kernel ratio (KR) are closely associated with drought tolerance and yield, EHPH (phenotypic correlation coefficient (r p ) = 0.318~0.578), ...
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... To date, numerous genes associated with maize grainrelated traits have been identified, including Opaque-2 (O2; Schmidt et al. 1987Schmidt et al. , 1992, Sugary-1 (Su1; Pan and Nelson 1984), Shrunken-2 (Sh2), and Brittle-2 (Bt2; Dickinson and Preiss 1969). Several major QTLs, such as qGW4.05 ...
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Heterosis has been extensively applied for many traits during maize breeding, but there has been relatively little attention paid to the heterosis for kernel size. In this study, we evaluated a population of 301 recombinant inbred lines derived from a cross between 08-641 and YE478, as well as 298 hybrids from an immortalized F2 (IF2) population to detect quantitative trait loci (QTLs) for six kernel-related traits and the mid-parent heterosis (MPH) for these traits. A total of 100 QTLs, six pairs of loci with epistatic interactions, and five significant QTL × environment interactions were identified in both mapping populations. Seven QTLs accounted for over 10% of the phenotypic variation. Only four QTLs affected both the trait means and the MPH, suggesting the genetic mechanisms for kernel-related traits and the heterosis for kernel size are not completely independent. Moreover, more than half of the QTLs for each trait in the IF2 population exhibited dominance, implying that dominance is more important than other genetic effects for the heterosis for kernel-related traits. Additionally, 20 QTL clusters comprising 46 QTLs were detected across ten chromosomes. Specific chromosomal regions (bins 2.03, 6.04–6.05, and 9.01–9.02) exhibited pleiotropy and congruency across diverse heterotic patterns in previous studies. These results may provide additional insights into the genetic basis for the MPH for kernel-related traits.
... 关键词 Opaque2, 转录因子, DAP-Seq, 转录因子结合位点, 调控网络 Opaque2(O2)是玉米胚乳特异表达的转录因子 (transcription factor, TF), O2蛋白含有碱性氨基酸基序/ 亮氨酸拉链, 属于bZIP家族 [1,2] . O2通过结合"ACGT"基 序来激活玉米储藏蛋白如α-醇溶蛋白的编码基因在胚 乳中特异表达, 当O2突变后导致玉米籽粒的醇溶蛋白 大幅降低同时提高籽粒中赖氨酸的含量 [3,4] . 另外, O2 突变还造成胚乳呈现软质不透明的特性, 从而增加玉 米的对病虫害及机械损伤的易感性, 说明O2参与玉米 的抗性过程 [4] . ...
... The corresponding TFs have also been successively identified. O2, an endosperm-specific bZIP TF, binds to the O2-box to transactivate the expression of 22-kDa α-zein and 15-kDa β-zein genes (6)(7)(8). Recently, a genome-wide strategy of ChIP-Seq combined with differential expression analysis also identified other O2 binding motifs, like TGACGTGG (9, 10). O2 can regulate the expression of most zeins except for 16-kDa γ-zein, illustrating a central role for O2-regulating zein expression. ...
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Grain starch and protein are synthesized during endosperm development, prompting the question of what regulatory mechanism underlies the synchronization of the accumulation of secondary and primary gene products. We found that two endosperm-specific NAC transcription factors, ZmNAC128 and ZmNAC130, have such a regulatory function. Knockdown of expression of ZmNAC128 and ZmNAC130 with RNA interference (RNAi) caused a shrunken kernel phenotype with significant reduction of starch and protein. We could show that ZmNAC128 and ZmNAC130 regulate the transcription of Bt2 and then reduce its protein level, a rate-limiting step in starch synthesis of maize endosperm. Lack of ZmNAC128 and ZmNAC130 also reduced accumulation of zeins and nonzeins by 18% and 24% compared with nontransgenic siblings, respectively. Although ZmNAC128 and ZmNAC130 affected expression of zein genes in general, they specifically activated transcription of the 16-kDa γ-zein gene. The two transcription factors did not dimerize with each other but exemplified redundancy, whereas individual discovery of their function was not amenable to conventional genetics but illustrated the power of RNAi. Given that both the Bt2 and the 16-kDa γ-zein genes were activated by ZmNAC128 or ZmNAC130, we could identify a core binding site ACGCAA contained within their target promoter regions by combining Dual-Luciferase Reporter and Electrophoretic Mobility Shift assays. Consistent with these properties, transcriptomic profiling uncovered that lack of ZmNAC128 and ZmNAC130 had a pleiotropic effect on the utilization of carbohydrates and amino acids.
... In this work it was found that a large part of the increase in the content of transcripts that encode for structured proteins can be explained by the incredible increase in the expression of the genes of the zein family. The transcriptional control of genes encoding 22kD zein proteins is mediated by opaque-2 (o2), a well-known TF that is a key transcriptional regulator during the middle phase [65]. Interestingly, Opaque-2 (Zm00001d018971) was identified as a regulator of several zeins in our networks analysis which help to support our predictions. ...
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Abstract: Intrinsic protein disorder is a physicochemical attribute of some proteins lacking tridimensional structure and is collectively known as intrinsically disordered proteins (IDPs). Interestingly, several IDPs have been associated with protective functions in plants and with their response to external stimuli. To correlate the modulation of the IDPs content with the developmental progression in seed, we describe the expression of transcripts according to the disorder content of the proteins that they codify during seed development, from the early embryogenesis to the beginning of the desiccation tolerance acquisition stage. We found that the total expression profile of transcripts encoding for structured proteins is highly increased during middle phase. However, the relative content of protein disorder is increased as seed development progresses. We identified several intrinsically disordered transcription factors that seem to play important roles throughout seed development. On the other hand, we detected a gene cluster encoding for IDPs at the end of the late phase, which coincides with the beginning of the acquisition of desiccation tolerance. In conclusion, the expression pattern of IDPs is highly dependent on the developmental stage, and there is a general reduction in the expression of transcripts encoding for structured proteins as seed development progresses. We proposed maize seeds as a model to study the regulation of protein disorder in plant development and its involvement in the acquisition of desiccation tolerance in plants.
... Grain starch and protein synthesis, which is regulated by a few common TFs (Zhang et al., 2019;Zhang et al., 2016), are coordinated during endosperm development. O2 was initially identified as a transcriptional regulator of 22-kD α-zein genes (Schmidt et al., 1992), but was later found to regulate a large number of genes by experimental (Gallusci et al., 1996;Kemper et al., 1999;Lohmer et al., 1991;Maddaloni et al., 1996) and genomic(Hunter et al., 2002;Li et al., 2015;Zhan et al., 2018) studies. The nutritional quality and yield traits, in terms of the synthesis of zein proteins and starch, are coordinately regulated by O2, which directly transactivates the expression of PPDKs and SSIII with another endosperm-specific TF, PBF1 (Zhang et al., 2016). ...
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The endosperm‐specific transcription factor Opaque2 (O2) acts as a central regulator for endosperm filling, but its functions have not been fully defined. Regular o2 mutants exhibit a non‐vitreous phenotype, so we used its vitreous variety Quality Protein Maize to create EMS‐mutagenesis mutants for screening o2 enhancers (oen). A mutant (oen1) restored non‐vitreousness and produced a large cavity in the seed due to severely depleted endosperm filling. When oen1 was introgressed into inbred W64A with a normal O2 gene, the seeds appeared vitreous but had a shrunken crown. oen1 was determined to encode Shrunken1 (Sh1), a sucrose synthase (SUS, EC 2.4.1.13). Maize contains three SUS‐encoding genes (Sh1, Sus1, and Sus2) with Sh1 contributing predominantly to the endosperm. We determined SUS activity and found a major and minor reduction in oen1 and o2, respectively. In o2;oen1‐1, SUS activity was further decreased. We found all Sus gene promoters contain at least one O2 binding element that can be specifically recognized and be transactivated by O2. Sus1 and Sus2 promoters had a much stronger O2 transactivation than Sh1, consistent with their transcript reduction in o2 endosperm. Although sus1 and sus2 alone or in combination had no perceptible phenotype, either of them could dramatically enhance seed opacity and cavity in sh1, indicating that transactivation of Sus1 and Sus2 by O2 supplements SUS‐mediated endosperm filling in maize. Our findings demonstrate that O2 transcriptionally regulates the metabolic source entry for protein and starch synthesis during endosperm filling.
... These are mainly grouped into four classes according to their solubility in alcohol solutions: a (19-and 22-kD zeins), b (15-kD zein), g (16-, 27-and 50-kD zeins) and d (10-and 18-kD zeins) (Wallace et al. 1990). It has been reported that 22-kD azein is directly bound by the transcription factor O2 (Lending and Larkins 1989;Schmidt et al. 1992;Muth et al. 1996). Moreover, other zeins, such as the 10-, 15and 19-kD zeins, show decreased accumulation of mRNA transcripts (Cord et al. 1995;Hunter et al. 2002;. ...
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Uncovering the genetic basis of seed development will provide useful tools for improving both crop yield and nutritional value. However, the genetic regulatory networks of maize (Zea mays) seed development remain largely unknown. The maize opaque endosperm and small germ 1 (os1) mutant has opaque endosperm and a small embryo. Here, we cloned OS1 and show that it encodes a putative transcription factor containing an RWP‐RK domain. Transcriptional analysis indicated that OS1 expression is elevated in early endosperm development, especially in the basal endosperm transfer layer (BETL), conducting zone (CZ), and central starch endosperm (CSE) cells. RNA sequencing (RNA‐Seq) analysis of the os1 mutant revealed sharp downregulation of certain genes in specific cell types, including ZmMRP‐1 and Meg1 in BETL cells and a majority of zein‐ and starch‐related genes in CSE cells. Using a haploid induction system, we show that wild‐type endosperm could rescue the smaller size of os1 embryo, which suggests that nutrients are allocated by the wild‐type endosperm. Therefore, our data imply that the network regulated by OS1 accomplishes a key step in nutrient allocation between endosperm and embryo within maize seeds. Identification of this network will help uncover the mechanisms regulating the nutritional balance between endosperm and embryo.
... Just like other SSP genes, the spatiotemporal expression pattern of Glu-1 is primarily controlled at the transcriptional level involving a series of cis-acting motifs and trans-acting factors [11,12]. In term of trans-acting factors, four families of transcription factors (TFs), bZIP, DOF, MYB, and B3, were reported to be involved in SSP regulation [13][14][15][16][17][18]. Numerous cis motifs have also been identified in the promoters of SSP genes and several of them have been characterized functionally in model plants. ...
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Wheat high-molecular-weight glutenin subunits (HMW-GS) determine dough elasticity and play an essential role in processing quality. HMW-GS are encoded by Glu-1 genes and controlled primarily at transcriptional level, implemented through the interactions between cis-acting elements and trans-acting factors. However, transcriptional mechanism of Glu-1 genes remains elusive. Here we made a comprehensive analysis of cis-regulatory elements within 1-kb upstream of the Glu-1 start codon (−1000 to −1) and identified 30 conserved motifs. Based on motif distribution pattern, three conserved cis-regulatory modules (CCRMs), CCRM1 (−300 to −101), CCRM2 (−650 to −400), and CCRM3 (−950 to −750), were defined, and their functions were characterized in wheat stable transgenic lines transformed with progressive 5′ deletion promoter::GUS fusion constructs. GUS staining, qPCR and enzyme activity assays indicated that CCRM2 and CCRM3 could enhance the expression level of Glu-1, whereas the 300-bp promoter (−300 to −1), spanning CCRM1 and core region (−100 to −1), was enough to ensure accurate Glu-1 initiation at 7 days after flowering (DAF) and shape its spatiotemporal expression pattern during seed development. Further transgenic assays demonstrated that CCRM1-2 (−300 to −209) containing Complete HMW Enhancer (−246 to −209) was important for expression level but had no effect on expression specificity in the endosperm. In contrast, CCRM1-1 (−208 to −101) was critical for both expression specificity and level of Glu-1. Our findings not only provide new insights to uncover Glu-1 transcription regulatory machinery but also lay foundations for modifying Glu-1 expression.
... O2 is specifically expressed in the endosperm as early as 6 days after pollination and encodes a bZIP-family TF ( Fig. 1) (Li et al. 2014;Schmidt et al. 1990). Previous studies showed that O2 directly regulates many target genes associated with storage functions, including zeins, through binding to a number of conserved cis-motifs collectively known as the O2 box (Cord Neto et al. 1995;Frizzi et al. 2010;Hartings et al. 2011;Hunter et al. 2002;Jia et al. 2007Jia et al. , 2013Li et al. 2015;Muth et al. 1996;Schmidt et al. 1987Schmidt et al. , 1990Schmidt et al. , 1992Zhang et al. 2015Zhang et al. , 2016 (Bhat et al. 2004;Hwang et al. 2004;Jin et al. 2014;Pysh et al. 1993;Pysh and Schmidt 1996;Qiao et al. 2016;Vicente-Carbajosa et al. 1997;Wang et al. 1998;Yilmaz et al. 2009;Zhang et al. 2012). Except for a few genes that are primarily expressed in the endosperm (e.g., PBF and O2 itself), most of these proteins are encoded by genes that are ubiquitously expressed throughout the plant life cycle (Fig. 1). ...
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The endosperm of angiosperms is a zygotic seed organ that stores nutrient reserves to support embryogenesis and seed germination. Cereal endosperm is also a major source of human calories and an industrial feedstock. Maize opaque endosperm mutants commonly exhibit opaque, floury kernels, along with other abnormal seed and/or non-seed phenotypes. The opaque endosperm phenotype is sometimes accompanied by a soft kernel texture and increased nutritional quality, including a higher lysine content, which are valuable agronomic traits that have drawn attention of maize breeders. Recently, an increasing number of genes that underlie opaque mutants have been cloned, and their characterization has begun to shed light on the molecular basis of the opaque endosperm phenotype. These mutants are categorized by disruption of genes encoding zein or non-zein proteins localized to protein bodies, enzymes involved in endosperm metabolic processes, or transcriptional regulatory proteins associated with endosperm storage programs.
... Only a handful of these 153 loci are known or well characterized. For instance, opaque-2 (O2), which encodes a basic leucine zipper protein transcription factor (Schmidt et al., 1992;Deng et al., 2017), was significantly associated with the level of amino acids leucine, ornithine, phenylalanine, glycine, tyrosine, valine and histidine in the mature kernel in this study (Table 1, Data S2). ...
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Primary metabolism plays pivotal roles in normal plant growth, development, and reproduction. As maize is a major crop worldwide, the primary metabolites produced by maize plants are of immense importance from calorific and nutritional perspectives. Here a genome-wide association study (GWAS) of 61 primary metabolites using a maize association panel containing 513 inbred lines identified 153 significant loci associated with the level of these metabolites in four independent tissues. The genome-wide expression level of 760 genes were also linked with metabolite levels within the same tissue. On average, the genetic variants at each locus or transcriptional variance of each gene identified here were estimated to have a minor effect (4.4%-7.8%) on the primary metabolic variation. Thirty-six loci or genes were prioritized as being worthy of future investigation, either with regard to functional characterization or their utility for genetic improvement. This target list includes the well-known opaque 2 (O2) and lkr/sdh genes as well as many less well-characterized genes. During our investigation of these 36 loci, we analyzed the genetic components and variations underlying the trehalose, aspartate and aromatic amino acid pathways thereby functionally characterizing four genes involved in maize primary metabolism.
... The higher accumulation of lysine and tryptophan might be due to regulation of genes operating in amino acid biosynthesis pathway, or other unknown mechanisms. O2 located on chromosome 7 codes for a DNA binding protein belonging to basic leucine zipper class of transcriptional factors, and acts as transcriptional activator of 19-and 22-kDa α-zein genes [35,36]. The mutant o2-based proteininduces an overall reduction of 50-70% in zein protein which increases non-zein proteins proportionally, resulting inan increase of lysine content twice than that in normal maize [37]. ...
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The enhancement of lysine and tryptophan in maize is so far basedon opaque2(o2) mutant, that along with the endosperm-modifiersled to development of Quality Protein Maize[QPM]. Though many mutants improving the endospermic protein quality were discovered, they could not be successfully deployed. Recently discovered opaque16 (o16)mutant enhances the lysine and tryptophan content in maize endosperm. In the present study, the influence of o16 on the endosperm modification was analyzed in four F2 populations, two each segregating for o16 allele alone and in combination with o2. The recessive o16o16 seed endosperm was found to be vitreousphenotypically similar to wild-O16O16. The mutant did not influence the degree of kernel opaqueness in o2o2 genetic background as opaqueness in o2o2/O16O16 and o2o2/o16o16 was similar. Grain hardness of o16o16 was comparable with the normal and QPM maize. The pattern of microscopic organization of proteinaceous matrix and starch granules, and zein profiling of the storage protein in o16o16 were found to be similar with normal maize endosperm, but distinct from the o2o2-soft genotype. The pattern in o2o2/o16o16 was unique and different from o2o2 and o16o16 as well. Here we demonstrated the effects of o16 on physico-biochemical characteristics of endosperm and report of o16 possessing negligible influence on kernel modification and hardness, which holds a great significance in maize quality breeding programme.
... Opaque1 encodes a myosin XI protein which plays an important role in endoplasmic reticulum motility and protein body formation in the endosperm [57]. A homolog of Opaque2 (O2) gene [58], Sobic.001G056700 was~12 kb from SNP 1:4291408, significantly associated with Leu/Pyruvate (P = 1.07E-06). ...
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Background: In sorghum (Sorghum bicolor), one paramount breeding objective is to increase grain quality. The nutritional quality and end use value of sorghum grains are primarily influenced by the proportions of tannins, starch and proteins, but the genetic basis of these grain quality traits remains largely unknown. This study aimed to dissect the natural variation of sorghum grain quality traits and identify the underpinning genetic loci by genome-wide association study. Results: Levels of starch, tannins and 17 amino acids were quantified in 196 diverse sorghum inbred lines, and 44 traits based on known metabolic pathways and biochemical interactions amongst the 17 amino acids calculated. A Genome-wide association study (GWAS) with 3,512,517 SNPs from re-sequencing data identified 14, 15 and 711 significant SNPs which represented 14, 14, 492 genetic loci associated with levels of tannins, starch and amino acids in sorghum grains, respectively. Amongst these significant SNPs, two SNPs were associated with tannin content on chromosome 4 and colocalized with three previously identified loci for Tannin1, and orthologs of Zm1 and TT16 genes. One SNP associated with starch content colocalized with sucrose phosphate synthase gene. Furthermore, homologues of opaque1 and opaque2 genes associated with amino acid content were identified. Using the KEGG pathway database, six and three candidate genes of tannins and starch were mapped into 12 and 3 metabolism pathways, respectively. Thirty-four candidate genes were mapped into 16 biosynthetic and catabolic pathways of amino acids. We finally reconstructed the biosynthetic pathways for aspartate and branched-chain amino acids based on 15 candidate genes identified in this study. Conclusion: Promising candidate genes associated with grain quality traits have been identified in the present study. Some of them colocalized with previously identified genetic regions, but novel candidate genes involved in various metabolic pathways which influence grain quality traits have been dissected. Our study acts as an entry point for further validation studies to elucidate the complex mechanisms controlling grain quality traits such as tannins, starch and amino acids in sorghum.
... Like the regulation of SSRGs, the expression of SSP genes is also mainly regulated by TFs at the transcription level (Verdier & Thompson, 2008;Xi & Zheng, 2011). Up to the present, several TFs belonging to bZIP, Dof, and MYB families have been shown to regulate SSP gene expression, such as SPA (bZIP), WPBF (Dof), GAMYB-D (MYB), and TaGAMyb (MYB) in wheat (Albani et al., 1997;Dong et al., 2007;Plessis et al., 2013;Guo et al., 2015) and O2 (bZIP), ZmbZIP22 (bZIP), and PBF (Dof) in maize (Lohmer et al., 1991;Schmidt et al., 1992, Holdsworth et al., 1995Vicente Carbajosa et al., 1997;Marzabal et al., 2008). Attractively, some TFs were even found to regulate the expression of both SSP genes and SSRGs. ...
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Starch in wheat grain provides humans with carbohydrates and influences the quality of wheaten food. However, no transcriptional regulator of starch synthesis has been identified first in common wheat (Triticum aestivum) due to the complex genome. Here, a novel bZIP family transcription factor TubZIP28 was found to be preferentially expressed in the endosperm throughout grain‐filling stages in Triticum urartu, the A genome donor of common wheat. When TubZIP28 was over‐expressed in common wheat, the total starch content increased by c. 4%, which contributed to c. 5% raise in the thousand kernel weight (TKW). The grain weight per plant of over‐expression wheat was also elevated by c. 9%. Both in vitro and in vivo assays showed that TubZIP28 bound to the promoter of cytosolic AGPase and enhanced both the transcription and activity of the latter. Knock‐out of the homolog TabZIP28 in common wheat resulted in declines of both the transcription and activity of cytosolic AGPase in developing endosperms and c. 4% reduction of the total starch in mature grains. To the best of our knowledge, TubZIP28 and TabZIP28 are transcriptional activators of starch synthesis first identified in wheat, and they could be superior targets to improve the starch content and yield potential of wheat.
... Then, after further cell division, cell expansion and endoreduplication, the embryo and endosperm enlarge significantly. Genetic studies have identified a large number of genes involved in key steps of the regulation of embryogenesis and the biosynthesis of endosperm storage compounds, such as opaque2 (o2) [18], Shrunken2 [19], and knotted1 (kn1) [20]. However, little is known about the developmental pattern of uncompleted double-fertilized maize ovules, which are known as unpollinated kernels (KUP). ...
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Background Maize kernel filling, which is closely related to the process of double fertilization and is sensitive to a variety of environmental conditions, is an important component of maize yield determination. Silk is an important tissue of maize ears that can discriminate pollen and conduct pollination. Therefore, investigating the molecular mechanisms of kernel development and silk senescence will provide important information for improving the pollination rate to obtain high maize yields. Results In this study, transcript profiles were determined in an elite maize inbred line (KA105) to investigate the molecular mechanisms functioning in self-pollinated and unpollinated maize kernels and silks. A total of 5285 and 3225 differentially expressed transcripts (DETs) were identified between self-pollinated and unpollinated maize in a kernel group and a silk group, respectively. We found that a large number of genes involved in key steps in the biosynthesis of endosperm storage compounds were upregulated after pollination in kernels, and that abnormal development and senescence appeared in unpollinated kernels (KUP). We also identified several genes with functions in the maintenance of silk structure that were highly expressed in silk. Further investigation suggested that the expression of autophagy-related genes and senescence-related genes is prevalent in maize kernels and silks. In addition, pollination significantly altered the expression levels of senescence-related and autophagy-related genes in maize kernels and silks. Notably, we identified some specific genes and transcription factors (TFs) that are highly expressed in single tissues. Conclusions Our results provide novel insights into the potential regulatory mechanisms of self-pollinated and unpollinated maize kernels and silks.
... Among TFs that regulate SE development, OPAQUE-2 (O2) is a basic leucine zipper (bZIP) TF that has long been recognized as key to regulating the biosynthesis and accumulation of nutrient materials in SE. First shown to activate the expression of the 22-kD α-zein gene (Schmidt et al., 1990(Schmidt et al., , 1992, a genome-wide transcriptional regulatory network study showed that O2 also regulates several additional zein storage protein genes, genes for carbon fixation (PPDK1 and PPDK2), and additional downstream transcription factors (GBF and Myblike TFs) . Further, proteomic studies showed that protein levels of Granule-Bound Starch Synthase I (GBSSI), Starch Synthase IIa (SSIIa), and Starch Branching Enzyme I (SBEI) were reduced in o2 mutants, indicating that O2 may also play an important role in starch biosynthesis, albeit indirectly (Jia et al., 2013;Zhang et al., 2016). ...
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Maize endosperm plays important roles in human diet, animal feed and industrial applications. Knowing the mechanisms that regulate maize endosperm development could facilitate the improvement of grain quality. This review provides a detailed account of maize endosperm development at the cellular and histological levels. It features the stages of early development as well as developmental patterns of the various individual tissues and cell types. It then covers molecular genetics, gene expression networks, and current understanding of key regulators as they affect the development of each tissue. The article then briefly considers key changes that have occurred in endosperm development during maize domestication. Finally, it considers prospects for how knowledge of the regulation of endosperm development could be utilized to enhance maize grain quality to improve agronomic performance, nutrition and economic value.
... AtZIP10, AtZIP25 and AtZIP53 in Arabidopsis could regulate the expression level of storage protein genes by binding to their promoter region or interacting with other transcription factors, which were also reported in crops including maize, barley, rice, sorghum and wheat (Schmidt et al., 1992;Pirovano et al., 1994;Wu et al., 1998;Onate et al., 1999;Onodera et al., 2001;Lara et al., 2003;Alonso et al., 2009). A candidate gene encoding a bZIP transcription factor (TraesCS6A02G096300) specifically expressed in grain was found in q6A-1 (Supplementary Table S7). ...
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In wheat breeding, improved quality traits, including grain quality and dough rheological properties, have long been a critical goal. To understand the genetic basis of key quality traits of wheat, two single-locus and five multi-locus GWAS models were performed for six grain quality traits and three dough rheological properties based on 19, 254 SNPs in 267 bread wheat accessions. As a result, 299 quantitative trait nucleotides (QTNs) within 105 regions were identified to be associated with these quality traits in four environments. Of which, 40 core QTN regions were stably detected in at least three environments, 19 of which were novel. Compared with the previous studies, these novel QTN regions explained smaller phenotypic variation, which verified the advantages of the multi-locus GWAS models in detecting important small effect QTNs associated with complex traits. After characterization of the function and expression in-depth, 67 core candidate genes involved in protein/sugar synthesis, histone modification and the regulation of transcription factor were observed to be associated with the formation of grain quality, which showed that multi-level regulations influenced wheat grain quality. Finally, a preliminary network of gene regulation that may affect wheat quality formation was inferred. This study verified the power and reliability of multi-locus GWAS methods in wheat quality trait research, and increased the understanding of wheat quality formation mechanisms. The detected QTN regions and candidate genes in this study could be further used for gene cloning and marker-assisted selection in high-quality breeding of bread wheat.
... Unnamed__8 CRE (also known as Unnamed__10, Unnamed__12 and Unnamed__14) was found in OsTPP3 and OsTPP5 promoters. Unnamed__8 CRE acts as an Opaque-2 (O2) target site in maize, where O2 regulates gene expression in developing endosperm (Schmidt et al. 1992;Izawa et al. 1993;Neto et al., 1995). Plant_AP-2-like CRE was found in OsTPP8, which was also found in promoters of starch biosynthesis core genes of maize along with ABA-responsive CREs (Huang et al. 2016;Qu et al. 2019). ...
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Trehalose-6-phosphate phosphatase (TPP) plays a key role in trehalose metabolism in plants. Here, we performed comprehensive in silico analyses and identified 12 OsTPPs (Oryza sativa TPPs) utilizing various bioinformatics tools. Phylogenetic tree, accomplished with OsTPPs and TPPs from 11 monocot and dicot species, was divided mainly into two clades, each clade containing six OsTPPs. Exon–intron distribution was related to phylogenetic clades. All OsTPPs are distributed within nine chromosomes (chr.), except Chr. 1, Chr. 5 and Chr. 11. OsTPPs were found to be stable in nature according to the 3-D structure prediction. Cis-regulatory elements (CREs) were also analyzed using 2 kb upstream of start codon for each gene to predict their biological functions. We categorized all CREs in five distinct groups based on core elements, stress response, cellular development, hormonal regulation, and unknown function, distributed in a range of 3–14 CREs in each group. Interestingly, our expression analysis showed that OsTPPs were more upregulated in response to drought and cold stresses compared to salt stress. Abundance of stress-related CREs found signifies TPPs’ possible role in stress response, which may facilitate to find related transcription factors and unveil complex molecular mechanisms during stress response.
... TFs were involved in transcriptional regulation of gluten genes through binding their cis-elements in promoters (Albani et al., 1997;Xi and Zheng, 2011). For example, the bZIP TFs could bind GCN4 motifs of gliadin and glutenin genes in maize (O2) (Schmidt et al., 1992;Vicente-Carbajosa et al., 1998). The Dof TFs (PBF) could activate α-gliadin and Glu-1 genes in wheat (Zhu et al., 2018) by binding P-box elements. ...
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Two advanced wheat lines BAd7-209 and BAd23-1 without the functional gene GPC-B1 were obtained from a cross between common wheat cultivar Chuannong 16 (CN16) and wild emmer wheat accession D97 (D97). BAd7-209 showed superior quality parameters than those of BAd23-1 and CN16. We found that the components of glutenins and gliadins in BAd7-209 and BAd23-1 were similar, whereas BAd7-209 had higher amount of glutenins and gliadins than those of BAd23-1. RNA sequencing analysis on developing grains of BAd7-209 and BAd23-1 as well as their parents revealed 382 differentially expressed genes (DEGs) between the high-grain protein content (GPC) (D97 + BAd7-209) and the low-GPC (CN16 + BAd23-1) groups. DEGs were mainly associated with transcriptional regulation of the storage protein genes, protein processing in endoplasmic reticulum, and protein export pathways. The upregulated gluten genes and transcription factors (e.g., NAC, MYB, and bZIP) may contribute to the high GPC in BAd7-209. Our results provide insights into the potential regulation pathways underlying wheat grain protein accumulation and contribute to make use of wild emmer for wheat quality improvement.
... TFs play critical roles as key regulators for gene expression during maize kernel development. The first characterized TF in maize is a bZIP family protein OPAQUE2 (O2) (Schmidt et al. 1987), regulating genes in nearly all zein families during endosperm development (Schmidt et al. 1990(Schmidt et al. , 1992Muth et al. 1996). The o2 mutation results in the opaque kernel phenotype with reduced levels of 22-kD α-zeins and increased lysine content (Mertz et al. 1964;Schmidt et al. 1987). ...
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Maize (Zea mays) is a leading cereal crop in the world. The maize kernel is the storage organ and the harvest portion of this crop and is closely related to its yield and quality. The development of maize kernel is initiated by the double fertilization event, leading to the formation of a diploid embryo and a triploid endosperm. The embryo and endosperm are then undergone independent developmental programs, resulting in a mature maize kernel which is comprised of a persistent endo-sperm, a large embryo, and a maternal pericarp. Due to the well-characterized morphogenesis and powerful genetics , maize kernel has long been an excellent model for the study of cereal kernel development. In recent years, with the release of the maize reference genome and the development of new genomic technologies, there has been an explosive expansion of new knowledge for maize kernel development. In this review, we overviewed recent progress in the study of maize kernel development, with an emphasis on genetic mapping of kernel traits, transcriptome analysis during kernel development , functional gene cloning of kernel mutants, and genetic engineering of kernel traits.
... Exploring the regulatory network of zein genes is important for the nutritional improvement of maize grains. Several factors have been identified that directly regulate zein gene expression, including O2, Pbf1, Ohp1, Ohp2, ZmbZIP22, ZmMADS47, NAC128, and NAC130 (Schmidt et al., 1992;Pysh and Schmidt, 1996;Vicente-Carbajosa et al., 1997;Wu and Messing, 2012;Zhang et al., 2015Zhang et al., , 2016Zhang et al., , 2019bQiao et al., 2016;Yang et al., 2016;Li et al., 2018). In general, these genes additively and cooperatively regulate the expression of zein genes. ...
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Grain filling in maize (Zea mays) is regulated by a group of spatiotemporally synchronized transcription factors (TFs), but the factors that coordinate their expression remain unknown. We used the promoter of the grain filling-specific TF gene Opaque2 (O2) to screen upstream regulatory factors and identified a B3 domain TF, ZmABI19, that directly binds to the O2 promoter for transactivation. zmabi19 mutants displayed developmental defects in the endosperm and embryo, and mature kernels were opaque and reduced in size. The accumulation of zeins, starch and lipids dramatically decreased in zmabi19 mutants. RNA sequencing revealed an alteration of the nutrient reservoir activity and starch and sucrose metabolism in zmabi19 endosperms, and plant phytohormone signal transduction and lipid metabolism in zmabi19 embryos. Chromatin immunoprecipitation followed by sequencing coupled with differential expression analysis identified 106 high-confidence direct ZmABI19 targets. ZmABI19 directly regulates multiple key grain filling TFs including O2, Prolamine-box binding factor 1, ZmbZIP22, NAC130, and Opaque11 in the endosperm and Viviparous1 in the embryo. A number of phytohormone-related genes were also bound and regulated by ZmABI19. Our results demonstrate that ZmABI19 functions as a grain filling initiation regulator. ZmABI19 roles in coupling early endosperm and embryo development are also discussed.
... Some of the classical opaque mutants were identified as mutations in transcription factors, such as Opaque2 (O2). O2 is a DNA-binding protein belonging to the bZIP transcription factor that recognizes specific target sites and activates downstream target genes, including 14 kDa β-zein, 10 kD a-zein, 19 kD a-zein, and 22 kDa α-zein [19][20][21][22][23][24]. O2 kernels contain over 70% higher lysine content than wild-type kernels [25]. ...
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Maize kernels are the harvested portion of the plant and are related to the yield and quality of maize. The endosperm of maize is a large storage organ that constitutes 80–90% of the dry weight of mature kernels. Maize kernels have long been the study of cereal grain development to increase yield. In this study, a natural mutation that causes abnormal kernel development, and displays a shrunken kernel phenotype, was identified and named “shrunken 2008 (sh2008)”. The starch grains in sh2008 are loose and have a less proteinaceous matrix surrounding them. The total storage protein and the major storage protein zeins are ~70% of that in the wild-type control (WT); in particular, the 19 kDa and 22 kDa α-zeins. Map-based cloning revealed that sh2008 encodes a GT-2 trihelix transcription factor, ZmThx20. Using CRISPR/Cas9, two other alleles with mutated ZmThx20 were found to have the same abnormal kernel. Shrunken kernels can be rescued by overexpressing normal ZmThx20. Comparative transcriptome analysis of the kernels from sh2008 and WT showed that the GO terms of translation, ribosome, and nutrient reservoir activity were enriched in the down-regulated genes (sh2008/WT). In short, these changes can lead to defects in endosperm development and storage reserve filling in seeds.
... Bzip , , , [13][14][15] opaque floury fl3 , RNA III 5S rRNA tRNA [16] (basal endosperm transfer layer, BETL) ...
... PBF can activate the γ-zein gene (γZ) promoter, and the accumulation of γ-zein protein occurs in the peripheral cells of starchy endosperm induced by PBF, better than expressed (Marzábal et al. 2008). Another important player in the regulation of SSPs in maize is the bZIP family member O2, which regulates 22-kD α-zein and 15-kD β-zein genes through the O2 box (TCCACGT;Neto et al. 1995;Schmidt et al. 1992). In addition, studies have found that O2 heterodimerizing proteins specifically regulate the expression of the 27-kD γ-zein gene and interact with PBF . ...
Article
Plant seeds, which are unique reproductive organs of gymnosperms and angiosperms, are used for edible, medicinal and industrial purposes. Transcription factors (TFs) are master regulators of plant growth, development, and stress responses. This review describes in detail the functions of TFs in regulating seed development. Different TFs, or even different TF families, may have similar functions in seed development. For example, WUSCHEL-related homeobox (WOX), LEC2/FUS3/ABI3 (AFL) and HEME ACTIVATOR PROTEIN3 (HAP3) families can control plant seed embryonic initiation and development. In contrast, some members of the same TF family may have completely opposite roles. For instance, AtMYB76 and AtMYB89 inhibit the accumulation of seed oil, whereas AtMYB96 promotes seed fatty acid accumulation in Arabidopsis thaliana. Compared with the number of studies that have addressed regulation by single TFs, only a few have focused on multiple-TF regulatory networks. This review should be useful as a reference for future studies on regulatory networks of TF complexes. This article is protected by copyright. All rights reserved.
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Cytosolic malate dehydrogenase (MDH) is a key enzyme that regulates the interconversion between malate and oxaloacetate (OAA). However, its role in modulating storage compound accumulation in maize endosperm is largely unknown. Here, we characterized a novel naturally occurring maize mdh4‐1 mutant, which produces small, opaque kernels and exhibits reduced starch but enhanced lysine content. Map‐based cloning, functional complementation, and allelism analyses identified ZmMdh4 as the causal gene. Enzymatic assays demonstrated that ZmMDH4 predominantly catalyzes the conversion from OAA to malate. In comparison, the activity of the mutant enzyme, which lacks one glutamic acid (Glu), was completed abolished, demonstrating that the Glu residue was essential for ZmMDH4 function. Knocking down ZmMdh4 in vivo led to a substantial metabolic shift towards glycolysis and a dramatic disruption in the activity of the mitochondrial complex I, which was correlated with transcriptomic alterations. Taken together, these results demonstrate that ZmMdh4 regulates the balance between mitochondrial respiration and glycolysis, ATP production, and endosperm development, through a yet unknown feedback regulatory mechanism in mitochondria.
Chapter
Seed storage proteins are synthesised in the endoplasmic reticulum and accumulate in protein bodies in the cotyledon and endosperm storage tissues during seed development. Their synthesis relies on remobilisation of nitrogen from vegetative tissues. They are adapted for storing rapidly and in an inert form a high concentration of remobilisable nitrogen. Upon germination, they provide the seedling with amino acids during the heterotrophic growth phase. They also represent a major protein source for humans and livestock. Although having limiting quantities of essential amino acids, notably lysine and methionine, a combination of cereal and legume seed proteins, for example constitutes a good dietary amino acid balance for monogastric animals. Seed proteins have been classified by their differing solubilities. With the benefit of sequence data, we now know that within a solubility class different sequence types may exist. Interestingly, despite their contrasted primary sequences, the conservation of short sequence motifs shows that different storage protein classes are derived from a common evolutionary ancestor. Storage proteins embrace a large range of valuable technological properties, which find varied uses in the food industry. Key Concepts • Seed storage proteins have evolved to store nitrogen efficiently as a source for the germinating seedling. • Most monocotyledons store mainly prolamin storage proteins, whereas dicotyledons store mainly globulins. • Prolamins consist mainly of short tandem repeats of noncharged amino acids, which renders them insoluble in water. • Globulins are assembled into multimers that reduce their solubility. • Seeds contain antinutritional compounds that reduce digestibility of the storage proteins by humans or livestock unless eliminated by breeding or processing. • The secondary/tertiary structure of cereal glutelins confers the property of viscoelasticity, of fundamental importance to breadmaking. • Storage proteins are encoded by multigene families, and mutations having a major effect on their accumulation are mostly in trans, at loci regulating their synthesis or deposition in protein bodies. • The seed's structure and its development are highly adapted to permit a rapid accumulation of storage products during the limited period of seed maturation. • Storage protein deposition relies on remobilisation of nitrogen from vegetative tissue. • Storage protein remobilisation during germination involves activation of preexisting proteases and induction of de novo synthesis.
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Half-size ATP binding cassette G (ABCG) transporters participate in the growth and development of plants by transporting substrates. The VviABCG20 gene is highly expressed in seed and plays an important role in seed development/abortion. However, little is known about the function of the VviABCG20 promoter (pVviABCG20) and its regulatory factors. In our study, we obtained pVviABCG20s from 15 seeded and seedless grape varieties and there were two types of ‘a’ and ‘b’ with 41 bp non-deletion or deletion, respectively. The pVviABCG20 activity was higher in seeds, siliques, flowers and roots of pVviABCG20-GUS Arabidopsis. The GUS activity analysis revealed that the activities of P4 (-586 bp) to P7 (-155 bp) were becoming increasingly weaker, and the P7 activity almost disappears compared with the pVviABCG20 (P0, -1604). Yeast one-hybrid and GUS activity analysis indicated that VviDof14 binds to the AAAG element in the P7′ (-586 bp) fragment of the pVviABCG20 and regulated the activity negatively. The quantitative real-time PCR analysis suggested that the expression of VviDof14 in Thompson seedless seeds was higher than that in Pinot noir. Our study laid the foundation for further analysis of the functions of the pVviABCG20 and its regulator VviDof14 in grape seed development/abortion.
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The organic acid oxalate occurs in microbes, animals and plants; however, excessive oxalate accumulation in vivo is toxic to cell growth and decreases the nutritional quality of certain vegetables. However, the enzymes and functions required for oxalate degradation in plants remain largely unknown. Here, we report the cloning of a maize (Zea mays) opaque endosperm mutant that encodes oxalyl-CoA decarboxylase1 (EC4.1.1.8; OCD1). Ocd1 is generally expressed and is specifically induced by oxalate. The ocd1 mutant seeds contain a significantly higher level of oxalate than the wild type, indicating that the ocd1 mutants have a defect in oxalate catabolism. 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 the metabolome in the endosperm. Our findings demonstrate that ZmOCD1 acts downstream of O7 in oxalate degradation and affects endosperm development, the metabolome, and nutritional quality in maize seeds.
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Zeins are the most abundant storage proteins in maize (Zea mays) kernels, thereby affecting the nutritional quality and texture of this crop. 27-kD γ-zein is highly expressed and plays a crucial role in protein body formation. Several transcription factors (TFs) (O2, PBF1, OHP1 and OHP2) regulate the expression of the 27-kD γ-zein gene, but the complexity of its transcriptional regulation is not fully understood. Here, using probe affinity purification and mass spectrometry analysis, we identified ZmbZIP22, a TF that binds to the 27-kD γ-zein promoter. ZmbZIP22 is a bZIP type TF that is specifically expressed in endosperm. ZmbZIP22 bound directly to the ACAGCTCA box in the 27-kD γ-zein promoter and activated its expression in wild tobacco (Nicotiana benthamiana) cells. 27-kD γ-zein gene expression was significantly reduced in CRISPR/Cas9-generated zmbzip22 mutants. ChIP-Seq (chromatin immunoprecipitation coupled to high-throughput sequencing) confirmed that ZmbZIP22 binds to the 27-kD γ-zein promoter in vivo and identified additional direct targets of ZmbZIP22. ZmbZIP22 can interact with PBF1, OHP1 and OHP2 but not O2. Transactivation assays using various combinations of these TFs revealed multiple interaction modes for the transcriptional activity of the 27-kD γ-zein promoter. Therefore, ZmbZIP22 regulates 27-kD γ-zein gene expression together with other known TFs.
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By utilizing a homologous transient-expression system, we have shown that a 58-bp sequence from the gamma-class 27-kDa zein promoter, spanning from -307 to -250 relative to the transcription start site, confers a high level of transcriptional activity on a truncated plant viral promoter. The transcriptional activity mediated by the 58-bp sequence is orientation independent, and it is further enhanced as a result of its multimerization. A similarly high level of transcriptional activity was also observed in protoplasts isolated from leaf tissue-derived maize suspension cells. In vitro binding and DNase I footprinting assays with nuclear protein prepared from cultured endosperm cells revealed the sequence-specific binding of a nuclear factor(s) to a 16-nucleotide sequence present in the 58-bp region. The nuclear factor binding sequence includes the -300 element, a cis-acting element highly conserved among different zein genes and many other cereal storage protein genes. A 23-bp oligonucleotide sequence containing the nuclear factor binding site is sufficient for binding the nuclear factor in vitro. It also confers a high level of transcriptional activity in vivo, but in an orientation-dependent manner. Four nucleotide substitutions in the -300 element drastically reduced binding and transcriptional activation by the nuclear factor. The same nuclear factor is abundant in the developing kernel endosperm and binds to the -300 element region of the 27-kDa or the alpha-class zein promoter. These results suggest that the highly conserved -300 element is involved in the common regulatory mechanisms mediating the coordinated expression of the zein genes.
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Highly homogenous α zein protein was isolated from maize kernels in an environment‐friendly process using 95% ethanol as solvent. Due to the polyploidy and genetic polymorphism of the plant source the application of high resolution separation methods in conjunction with precise analytical methods, such as MALDI‐ToF‐MS, is required to accurately estimate homogeneity of products that contain natural zein protein. The α zein protein product revealed two main bands in SDS PAGE analysis, one at 25 kDa and one at 20 kDa apparent molecular mass. Yet, high resolution 2D gel electrophoresis revealed approx. five protein spot groups in each row, the first at ca. 25 kDa and the second at ca. 20 kDa. Peptide mass fingerprinting data of the proteins in the two dominant SDS PAGE bands matched to 30 amino acid sequence entries out of 102 non‐redundant data base entries. MALDI‐ToF‐MS peptide mapping of the proteins from all spots indicated the presence of only α zein proteins. The most prominent ion signals in the MALDI mass spectra of the protein mixture of the 25 kDa SDS gel band after in‐gel digestion were found at m/z 1272.6 and m/z 2009.1, and the most prominent ion signals of the protein mixture of the 20 kDa band after in‐gel digestion were recorded at m/z 1083.5 and m/z 1691.8. These ion signals have been found typical for α zein proteins and may serve as marker ion signals which upon chymotryptic digestion reliably indicate the presence of α zein protein in two hybrid corn products. This article is protected by copyright. All rights reserved
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The DOF (DNA binding with one finger) family of plant-specific transcription factors (TF) was first identified in maize in 1995. Since then, DOF proteins have been shown to be present in the whole plant kingdom, including the unicellular alga Chlamydomonas reinhardtii. The DOF TF family is characterised by a highly conserved DNA binding domain (DOF domain), consisting of a CX2C-X21-CX2C motif, which is able to form a zinc finger structure. Early in the study of DOF proteins, their relevance for seed biology became clear. Indeed, the PROLAMIN BINDING FACTOR (PBF), one of the first DOF proteins characterised, controls the endosperm-specific expression of the zein genes in maize. Subsequently, several DOF proteins from both monocots and dicots have been shown to be primarily involved in seed development, dormancy and germination, as well as in seedling development and other light-mediated processes. In the last two decades, the molecular network underlying these processes have been outlined, and the main molecular players and their interactions have been identified. In this review, we will focus on the DOF TFs involved in these molecular networks, and on their interaction with other proteins.
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In seeds, the endosperm is a crucial organ that plays vital roles in supporting embryo development and determining seed weight and quality. Starch is the predominant storage carbohydrate of the endosperm and accounts for ∼70% of the mature maize kernel weight. Nonetheless, because starch biosynthesis is a complex process that is orchestrated by multiple enzymes, the gene regulatory networks of starch biosynthesis, particularly amylose and amylopectin biosynthesis, have not been fully elucidated. Here, through high-throughput RNA sequencing, we developed a temporal transcriptome atlas of the endosperms of high-amylose maize and common maize at 5-, 10-, 15- and 20-day after pollination and found that 21,986 genes are involved in the programming of the high-amylose and common maize endosperm. A coexpression analysis identified multiple sequentially expressed gene sets that are closely correlated with cellular and metabolic programmes and provided valuable insight into the dynamic reprogramming of the transcriptome in common and high-amylose maize. In addition, a number of genes and transcription factors were found to be strongly linked to starch synthesis, which might help elucidate the key mechanisms and regulatory networks underlying amylose and amylopectin biosynthesis. This study will aid the understanding of the spatiotemporal patterns and genetic regulation of endosperm development in different types of maize and provide valuable genetic information for the breeding of starch varieties with different contents.
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Recent breakthroughs in transcriptome analysis and gene characterization have provided valuable resources and information about the maize endosperm developmental program. The high temporal‐resolution transcriptome analysis has yielded unprecedented access to information about the genetic control of seed development. Detailed spatial transcriptome analysis using laser‐capture microdissection has revealed the expression patterns of specific populations of genes in the four major endosperm compartments: the basal endosperm transfer layer (BETL), aleurone layer (AL), starchy endosperm (SE), and embryo‐surrounding region (ESR). Although the overall picture of the transcriptional regulatory network of endosperm development remains fragmentary, there have been some exciting advances, such as the identification of OPAQUE11 (O11) as a central hub of the maize endosperm regulatory network connecting endosperm development, nutrient metabolism, and stress responses, and the discovery that the endosperm adjacent to scutellum (EAS) serves as a dynamic interface for endosperm‐embryo crosstalk. In addition, several genes that function in BETL development, AL differentiation, and the endosperm cell cycle have been identified, such as ZmSWEET4c, Thk1, and Dek15, respectively. Here, we focus on current advances in understanding the molecular factors involved in BETL, AL, SE, ESR, and EAS development, including the specific transcriptional regulatory networks that function in each compartment during endosperm development. This article is protected by copyright. All rights reserved.
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Background and objectives Quality protein maize (QPM) nutritional quality involves high total protein content but reduced zein accumulation in maize endosperm, leading to increased non‐zein proteins, hence higher levels of essential amino acids such as lysine and tryptophan. The aim of this study was to assess the effect of low nitrogen (N) conditions in one location, and optimum conditions in three locations, on zein proteins in 10 QPM hybrids developed by the International Maize and Wheat Improvement Centre (CIMMYT) compared to two non‐QPM checks. Findings With a few exceptions, total protein content, and γ‐ and α‐zein peak areas were significantly reduced under low N compared to optimal conditions, while β‐zein increased. Non‐QPM hybrids had higher protein content than QPM hybrids, and QPM hybrids had higher γ‐zein than non‐QPM in all environments. Higher α‐zein values than other zein types were observed in non‐QPM than in QPM genotypes in all environments. Significance and novelty Low N significantly reduced protein content and all the zein fractions except for β‐zeins, which were increased. The advantages of a decrease in zein fractions in QPM under low N conditions were offset by a large decrease in grain protein content.
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The synthesis of seed storage protein (SSP) is mainly regulated at the transcriptional level. However, few transcriptional regulators of SSP synthesis have been characterized in common wheat (Triticum aestivum) owing to the complex genome. As the A genome donor of common wheat, Triticum urartu could be an elite model in wheat research considering its simple genome. Here, a novel NAC family transcription factor TuSPR from T. urartu was found preferentially expressed in developing endosperm during grain‐filling stages. In common wheat transgenically overexpressing TuSPR, the content of total SSPs was reduced by c. 15.97% attributed to the transcription declines of SSP genes. Both in vitro and in vivo assays showed that TuSPR bound to the cis‐element 5’‐CANNTG‐3’ distributed in SSP gene promoters and suppressed the transcription. The homolog in common wheat TaSPR shared a conserved function with TuSPR on SSP synthesis suppression. The knock‐down of TaSPR in common wheat resulted in 7.07%‐20.34% increases in the total SSPs. Both TuSPR and TaSPR could be superior targets in genetic engineering to manipulate SSP content in wheat, and this work undoubtedly expands our knowledge of SSP gene regulation.
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Maize (Zea mays) is a cereal crop of global food importance. However, the deficiency of essential amino acids, more importantly lysine, methionine and tryptophan, in the major seed storage zein proteins makes corn nutritionally of low value for human consumption. The idea of improving maize nutritional value prompted the search for maize natural mutants harboring low zein contents and higher amount of lysine. These studies resulted in the identification of more than dozens of maize opaque mutants in the previous few decades, o2 mutant being the most extensively studied one. However, the high lysine contents but soft kernel texture and chalky endosperm halted the widespread application and commercial success of maize opaque mutants, which ultimately paved the way for the development of Quality Protein Maize (QPM) by modifying the soft endosperm of o2 mutant into lysine-rich hard endosperm. The previous few decades have witnessed a marked progress in maize zein research. It includes elucidation of molecular mechanism underlying the role of different zein genes in seed endosperm development by cloning different components of zein family, exploring the general organization, function and evolution of zein family members within maize species and among other cereals, and elucidating the cis-and trans-regulatory elements modulating the regulation of different molecular players of maize seed endosperm development. The current advances in high quality reference genomes of maize lines B73 and Mo17 plus the completion of ongoing pan genome sequencing projects of more maize lines with NGS technologies are expected to revolutionize maize zein gene research in near future. This review highlights the recent advances in QPM development and its practical application in the post genomic era, genomic and physical composition and evolution of zein family, and expression, regulation and downstream role of zein genes in endosperm development. Moreover, recent genomic tools and methods developed for functional validation of maize zein genes are also discussed.
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Key message: We review the current knowledge regarding the regulation of zein storage proteins biosynthesis and protein body formation, which are crucial processes for the successful accumulation of nutrients in maize kernels. Storage proteins in the seeds of crops in the grass family (Poaceae) are a major source of dietary protein for humans. In maize (Zea mays), proteins are the second largest nutrient component in the kernels, accounting for ~ 10% of the kernel weight. Over half of the storage proteins in maize kernels are zeins, which lack two essential amino acids, lysine and tryptophan. This deficiency limits the use of maize proteins in the food and feed industries. Zeins are encoded by a large super-gene family. During endosperm development, zeins accumulate in protein bodies, which are derived from the rough endoplasmic reticulum. In recent years, our knowledge of the pathways of zein biosynthesis and their deposition within the endosperm has been greatly expanded. In this review, we summarize the current understanding of zeins, including the genes encoding these proteins, their expression patterns and transcriptional regulation, the process of protein body formation, and other biological processes affecting zein accumulation.
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Zeins are the predominant storage proteins in maize (Zea mays) seeds, while Opaque2 (O2) is a master transcription factor for zein-encoding genes. How the activity of O2 is regulated to respond to external signals is yet largely unknown. Here, we show an E3 ubiquitin ligase ZmRFWD3 interacts with O2 and positively regulates its activity by enhancing its nuclear localization. Ubiquitination of O2 enhances its interaction with ZmIMP1, the α-subunit of Importin-1 in maize, thus enhances its nuclear localization ability. We further show ZmRFWD3 can be phosphorylated by a sucrose-responsive protein kinase, ZmSnRK1, which leads to its degradation. We demonstrated that O2's activity is in response to sucrose levels through the ZmSnRK1-ZmRFWD3-O2 signaling axis. Intriguingly, we found sucrose levels, as well as the ZmRFWD3 levels and the cytonuclear distribution of O2 exhibit diurnal patterns in developing endosperm, leading to the diurnal transcription of O2-regulated zein genes. Loss-of-function of ZmRFWD3 disrupts the diurnal patterns of O2 cytonuclear distribution and zein biosynthesis, and consequently changes the C/N ratio in mature seeds. Thereby, we identify a SnRK1-ZmRFWD3-O2 signaling axis that transduces the source-to-sink signals and coordinates C and N assimilation in developing maize seeds.
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Food shortages arise more frequently owing to unpredictable crop yield losses caused by biotic and abiotic stresses. With advances in molecular biology and marker technology, a new era of molecular breeding has emerged that has greatly accelerated the pace of plant breeding. High-throughput genotyping technology and phenotyping platforms have enabled large-scale marker-trait association analysis, such as genome-wide association studies, to precisely dissect the genetic architecture of plant traits. Large-scale mapping of agronomically important quantitative trait loci, gene cloning and characterization, mining of elite alleles/haplotypes, exploitation of natural variations, and genomic selection have paved the way towards genomics-assisted breeding (GAB). With the availability of more and more informative genomic datasets, GAB would become a promising technique to expedite the breeding cycle for crop improvement.
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A comparison of the DNA and protein sequences of a group of zein cDNA clones reveals that they share extensive sequence homology and probably originated from a common ancestral gene. A comparison of clones corresponding to Mr 22,000 polypeptides shows they are 92% homologous, while five clones corresponding to the Mr 19,000 zeins vary in homology from 75 to 95%. The clones corresponding to the Mr 22,000 proteins are 60-65% homologous to clones encoding the Mr 19,000 zein proteins. A clone corresponding to the Mr 15,000 zein has little homology to either the Mr 22,000 or 19,000 zeins. Clones corresponding to both the Mr 22,000 and 19,000 zeins have two putative polyadenylation signals. S1 nuclease mapping indicates that the first polyadenylation signal following the stop codon is utilized by the Mr 22,000 sequences, while primarily the second polyadenylation signal is utilized by the Mr 19,000 sequences.
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The deposition of zein protein in maize endosperm is under the control of several regulatory loci. The isolation of DNA sequences corresponding to Opaque-2 (O2), one of such loci, is described in this paper. The mutable allele, o2-m5 was first induced moving the Ac transposable element present at the wx-m7 allele to the O2 locus. Genetic data suggest that a functional Ac element is responsible for the observed somatic mutability of o2-m5. The isolation of genomic clones containing flanking sequences corresponding to the O2 gene was possible by screening an o2-m5 genomic libary with a probe corresponding to internal Ac sequences usually absent in the defective element Ds. Out of 27 clones isolated with homology to the central part of Ac element, only clones 6IP and 21IP generated a 2.5 kb internal fragment size of an active Ac element when digested with PvuII restriction enzyme. A sequence representing a XhoI fragment of 0.9 kb lying, in the 6IP clone, adjacent to the Ac elements, was subcloned and utilized to prove that it corresponded to a part of the O2 gene. To obtain this information we made use of: (1) DNAs from several reversions originating from the unstable (o2mk-(r) allele, which, when digested with SstI, showed a correct 3.4 kb fragment typical of non-inserted alleles of the O2 locus; and (2) recessive alleles of the O2 locus which were devoid of a 2.0 kb mRNA, present on the contrary in the wild type and in other zein regulating mutants different from O2.
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The G-box is a cis-acting element found within the promoters of many plant genes where it mediates expression in response to a variety of different stimuli. This palindromic DNA motif (CCACGTGG) is composed of two identical half sites, the base pairs of which we have numbered -4 to +4 (numbering from 5' to 3'). Both half sites are involved in the binding of the bZIP protein GBF1, a member of the GBF family of Arabidopsis thaliana. Here we demonstrate using the random binding site selection method that GBF1 interacts with, in addition to the palindromic G-box, other DNA motifs that fall into seven distinct groups. All groups share the ACGT core sequence, common to most DNA motifs bound by plant bZIP proteins so far characterized. Our studies demonstrate that a high affinity GBF1 binding site is further defined by the following two parameters: first, all sites contain a G residue at position +3 (as in ACGTG) and secondly, only certain base pair combinations are allowed at positions -4, -3 and +4. Two of the identified groups (TGACGTGG and TGACGTGT) contain the base pairs TG at positions -4 and -3 and hence resemble the binding sites of another class of plant bZIP proteins (TGACGT/C binding proteins). However, GBF1 only interacts with the TGACGT sequence if the two 3' distal nucleotides (positions +3 and +4) are occupied by GG or GT. These data define the differences between a G-box binding protein and TGACGT/C binding proteins. The N-terminal domain of GBF1 is defined by a high proline content. Such regions were also identified in proteins related to GBF1. We demonstrate that this N-terminal proline-rich domain of GBF1, when fused to a heterologous DNA binding domain, stimulates transcription in both plant protoplasts and mammalian cells. These extensive DNA binding studies and the characterization of the GBF1 activation domain will facilitate both the identification of regulatory elements and the in vivo function of GBF1.
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Tissue-specific expression of the maize anthocyanin Bronze-1 (Bz1) gene is controlled by the products of several regulatory genes. These include C1 or Pl and R or B that share homology to the myb proto-oncogenes and myc-like genes, respectively. Bz1 expression in embryo tissues is dependent on C1 and an R-sc allele of R. Transient expression from mutated and deleted versions of the Bz1 promoter fused to a luciferase reporter gene was measured in C1, Rscm2 embryos after gene transfer by microprojectiles. This analysis revealed that the sequences between -76 base pairs (bp) and -45 bp and a 9-bp AT-rich block between -88 bp and -80 bp were critical for Bz1 expression. The -76 bp to -45 bp region includes two short sequences that are homologous to the consensus binding sites of the myb- and myc-like proteins. Site-specific mutations of these "myb" and "myc" sequences reduced Bz1 expression to 10% and 1% of normal, respectively. Additionally, a trimer of a 38-bp oligonucleotide containing these myb and myc sites increased the expression of a cauliflower mosaic virus 35S minimal promoter by 26-fold. This enhancement was dependent on both C1 and R. Because the sites critical for Bz1 expression are homologous to the myb and myc consensus binding sequences and the C1 and R proteins share homology with the myb and myc products, respectively, we propose that C1 and R interact with the Bz1 promoter at these sites.
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Protein targeting to the nucleus has been studied extensively in animal and yeast systems; however, nothing is known about nuclear targeting in plants. The Opaque-2 (O2) gene produces a regulatory protein that is responsible for inducing transcription of the alpha-zein class of storage proteins in maize kernels. The cloned O2 gene encodes a protein that contains a leucine zipper DNA binding domain that can interact with zein gene promoters. We have used immunolocalization to show that the O2 protein is present in nuclei in the maize endosperm tissues known to produce alpha-zeins. In addition, neither embryo tissue from wild-type kernels nor endosperm from kernels harboring a null o2 allele contain the O2 protein. Analysis of a transposable, element-induced o2 allele, o2-m20, revealed that sectors of endosperm cells contained the nuclear-localized O2 protein, indicating excision of the transposable element. To study further the nuclear transport of the O2 protein, we have transformed this gene, under the control of a constitutive promoter, into tobacco. Plants were shown to have detectable levels of steady-state O2 mRNA and O2 protein. Immunolocalization of O2 protein in transformed tobacco plants indicated that the O2 protein was transported into tobacco nuclei. Therefore, we have developed a system to study nuclear targeting in plants and have established that the nuclear transport machinery is similar in monocots and dicots.
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The maize locus, Opaque-2, controls the expression in developing endosperm of structural genes encoding a family of storage proteins, the 22 kd zeins, and an abundant albumin, termed b-32. It is shown that the promoter of the b-32 gene is activated in vivo in the presence of the O2 gene product and that the information necessary for this activation resides in a 440 bp DNA fragment containing five O2 binding sites (GATGAPyPuTGPu). Two of these sites are embedded in copies of the 'endosperm box', a motif thought to be involved in endosperm-specific expression, which is also represented in 22 kd zein promoters. The O2 protein is also shown to be capable of binding in vitro and activating in vivo, its own promoter.
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Wheat transcription factors HBP-1a and HBP-1b bind to the hexamer motif, ACGTCA, of wheat histone gene promoters. HBP-1b also binds to the hexamer motif in the promoter of the 35S RNA gene of cauliflower mosaic virus, whereas HBP-1a does not. A cDNA clone encoding HBP-1b was isolated on the basis of its binding specificity to the hexamer motif. The deduced amino acid sequence indicates that HBP-1b, like HBP-1a, belongs to a leucine zipper class of transcription factors. Mutational analyses of the HBP-1a and -1b encoded cDNAs revealed that truncated polypeptides containing the leucine zipper and basic regions are sufficient for DNA binding. HBP-1a and -1b form homodimers, as expected from earlier studies on this class of transcription factors, but did not form heterodimers. Although the hexamer motif or its homologs exist in several plant genes, HBP-1a and -1b exhibited the highest binding affinity to the hexamer motif in the histone promoters, suggesting that both DNA binding proteins are involved in transcriptional regulation of wheat histone genes.
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Four cis-acting elements, designated as Boxes I, II, III and IV, have previously been identified as functionally relevant components of the light-responsive chalcone synthase (CHS) promoter in parsley (Petroselinum crispum). This paper describes the isolation of three cDNAs encoding proteins which bind specifically to Box II, one of two cis-acting elements found within a 52 bp CHS promoter region shown here to be sufficient for light responsiveness in parsley. The deduced amino acid sequences of all three proteins reveal conserved basic and leucine zipper domains characteristic of transcription factors of the bZIP class. Nucleotide sequences recognized by these factors contain an ACGT motif common to many cis-acting elements. Therefore, we have termed the proteins CPRF-1, -2 and -3 (Common Plant Regulatory Factor). The characteristics of CPRF-1 binding to Box II and the timing of transient CPRF-1 mRNA accumulation during light exposure of previously dark-grown parsley cells are consistent with the hypothesis that this factor participates in the light-mediated activation of the CHS gene in parsley.
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The mechanism by which phytohormones, like abscisic acid (ABA), regulate gene expression is unknown. An activity in nuclear extracts that interacts with the ABA response element (ABRE) from the 5' regulatory region of the wheat Em gene was identified. A complementary DNA clone was isolated whose product is a DNA binding protein (EmBP-1) that interacts specifically with an 8-base pair (bp) sequence (CACGTGGC) in the ABRE. A 2-bp mutation in this sequence prevented binding of EmBP-1. The same mutation reduced the ability of the ABRE to confer ABA responsiveness on a viral promoter in a transient assay. The 8-bp EmBP-1 target sequence was found to be conserved in several other ABA-responsive promoters and in promoters from plants that respond to signals other than ABA. Similar sequences are found in promoters from mammals, yeast, and in the major late promoter of adenovirus. The deduced amino acid sequence of EmBP-1 contains conserved basic and leucine zipper domains found in transcription factors in plants, yeast, and mammals. EmBP-1 may be a member of a highly conserved family of proteins that recognize a core sequence found in the regulatory regions of various genes that are integrated into a number of different response pathways.
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The ocs-elements comprise a family of related 20-base pair DNA sequences with dyad symmetry that are functional components of the promoters of several genes introduced into the plant nucleus by Agrobacterium transformation or infection by DNA viruses. We describe the isolation and characterization of a maize cDNA that encodes a protein, OCSBF-1, that binds specifically to ocs-element sequences. The 21-kilodalton OCSBF-1 protein was encoded by a single copy, intron-less gene. The gene was differentially expressed in maize plants. Developing leaves had a gradient of OCSBF-1 mRNA with the basal portion of the leaves, which contain dividing and differentiating cells, having 40-fold to 50-fold higher levels of OCSBF-1 transcripts than the apical portion of the leaves, where the cells are fully differentiated. Roots and shoots of young plants had levels of OCSBF-1 mRNA similar to the basal portions of developing leaves. OCSBF-1 contained a small basic amino acid region and a potential leucine zipper motif homologous to the DNA-binding domains of the basic region-leucine zipper family of transcription factors such as Jun and GCN4. A truncated protein with the amino-terminal 76 amino acids of OCSBF-1, encompassing the basic domain and leucine zipper motif, still bound to ocs-element sequences in vitro. OCSBF-1 was able to bind to a site within each half of the ocs-element as well as to animal AP-1 and CREB sites.
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The C1, B and R genes regulating the maize anthocyanin biosynthetic pathway encode tissue-specific regulatory proteins with similarities to transcriptional activators. The C1 and R regulatory genes are usually responsible for pigmentation of seed tissues, and the B-Peru allele of B, but not the B-I allele, can substitute for R function in the seed. In this study, members of the B family of regulatory genes were delivered to intact maize tissues by high velocity microprojectiles. In colorless r aleurones or embryos, the introduction of the B-Peru genomic clone or the expressed cDNAs of B-Peru or B-I resulted in anthocyanin-producing cells. Luciferase produced from the Bronze1 anthocyanin structural gene promoter was induced 100-fold when co-introduced with the expressed B-Peru or B-I cDNAs. This quantitative transactivation assay demonstrates that the proteins encoded by these two B alleles are equally able to transactivate the Bronze1 promoter. Analogous results were obtained using embryogenic callus cells. These observations suggest that one major contribution towards tissue-specific anthocyanin synthesis controlled by the various alleles of the B and R genes is the differential expression of functionally similar proteins.
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The structure of the zein regulatory gene Opaque 2 of Zea mays has been determined by sequence analysis of genomic and cDNA clones. The size of O2 mRNA is 1751 bp [poly(A) tail not included] containing a major open reading frame (ORF) of 1380 bp preceded by three short ORFs of 3, 21 and 20 amino acid residues. The main ORF comprises 1362 bp and is composed of six exons ranging in size from 465 to 61 bp and five introns of 678 bp to 83 bp. A putative protein 454 amino acids long was derived by the theoretical translation of the genomic sequences corresponding to exons. The opaque 2 protein contains a domain similar to the leucine zipper motif identified in DNA binding proteins of animal protooncogenes such as fos, jun and myc, and in the transcriptional activators GCN4 and C/EBP. The region of 30 amino acid residues next to the leucine repeats towards the N terminus is rich in basic amino acids and is also homologous to a domain present in fos, jun and GCN4. Moreover, in the carboxy terminal region an amino acid motif closely resembling a metal binding domain is present.
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Zeins, the storage proteins of maize seed, are encoded by a large multigene family that is regulated developmentally and expressed in a tissue-specific manner during endosperm development. The synthesis of these proteins is affected by mutations, such as opaque-2, that cause a reduction in the accumulation of zein proteins and mRNAs. We used nuclear run-on transcription assays to analyze the expression of zein genes in developing normal and opaque-2 endosperms and to map the origin of these transcripts with respect to the coding and noncoding regions of the genes. These analyses demonstrate that zein gene expression is regulated transcriptionally and posttranscriptionally in developing endosperm. Transcription of genes encoding alpha-zeins is inhibited significantly in opaque-2 mutants, with expression of those encoding the M(r) 22,000 proteins being almost totally blocked. Other gene subfamilies were affected but to a lesser extent.
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A comparison of the DNA and protein sequences of a group of zein cDNA clones reveals that they share extensive sequence homology and probably originated from a common ancestral gene. A comparison of clones corresponding to Mr 22,000 polypeptides shows they are 92% homologous, while five clones corresponding to the Mr 19,000 zeins vary in homology from 75 to 95%. The clones corresponding to the Mr 22,000 proteins are 60-65% homologous to clones encoding the Mr 19,000 zein proteins. A clone corresponding to the Mr 15,000 zein has little homology to either the Mr 22,000 or 19,000 zeins. Clones corresponding to both the Mr 22,000 and 19,000 zeins have two putative polyadenylation signals. S1 nuclease mapping indicates that the first polyadenylation signal following the stop codon is utilized by the Mr 22,000 sequences, while primarily the second polyadenylation signal is utilized by the Mr 19,000 sequences.
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A 30-amino-acid segment of C/EBP, a newly discovered enhancer binding protein, shares notable sequence similarity with a segment of the cellular Myc transforming protein. Display of these respective amino acid sequences on an idealized alpha helix revealed a periodic repetition of leucine residues at every seventh position over a distance covering eight helical turns. The periodic array of at least four leucines was also noted in the sequences of the Fos and Jun transforming proteins, as well as that of the yeast gene regulatory protein, GCN4. The polypeptide segments containing these periodic arrays of leucine residues are proposed to exist in an alpha-helical conformation, and the leucine side chains extending from one alpha helix interdigitate with those displayed from a similar alpha helix of a second polypeptide, facilitating dimerization. This hypothetical structure is referred to as the "leucine zipper," and it may represent a characteristic property of a new category of DNA binding proteins.
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The genomic organization of the zein structural genes and of regulatory loci influencing their expression suggests that control of zein gene expression will involve interactions between cis elements in the flanking DNA sequences and products from trans-acting genes. The interaction between fragments from the 5' flanking region of a zein gene and specific, double-stranded oligonucleotides with crude nuclear extracts from maize endosperm have been studied by nitrocellulose filter binding, gel retention and DNase I footprinting assays. Specific binding of a nuclear factor was observed and the exact position of the protein binding site was determined. The 22-nt binding site included 14 bp of a 15-bp sequence conserved in all zein genes.
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Heavy and light chain zein genes from maize, affected by different regulatory loci, are related in sequence and structure. The two kinds of genes code for a signal peptide, a head region of 67 amino acids, six and a half to eight repetitive blocks of 20 amino acids each and a short tail piece. This block structure would allow inter or intragenic recombination giving rise to heterogeneous zein genes. Length variation in zein polypeptides is also due to the occurrence of termination mutations within some genes. Homology between heavy and light chain zein genes extends to the flanking sequences where a short region at the 5' end of the transcript can base pair with the 3' end and may have regulatory implications.
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Approximately 10% of the maize kernel's dry weight consists of protein, of which the storage proteins of the endosperm are a major portion. Zein proteins represent the major component of the storage proteins, which provide nitrogen and amino acids for the early development of the seedling. The developing maize kernel synthesizes large amounts of storage proteins between 10 and 50 days after pollination. The maximum rate of synthesis occurs between days 15 and 25. Zeins have an exceptionally high content of the amino acids glutamine, leucine, alanine, and proline and are low in lysine and tryptophan. They were originally defined as the protein fraction (prolamin) of maize meal that could be extracted by aqueous alcohol solutions. The zein multigene family is complex. About 100 zein genes are divided into at least four subfamilies. The protein structure of zeins from four of these subfamilies (SF1-SF4) is similar. The regulation of zein protein biosynthesis may be related to this subfamily organization. The ZAPs have unique features and may play a special role in the organization of the protein bodies.
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During the development of maize (Zea mays L.) ears it was found possible to remove kernels at two-day intervals without disturbing the development of the remaining kernels. Using this method it was possible to examine, by RNA hybridization experiments, the zein specific mRNAs during the development of wild-type and opaque-2 kernels. The amounts of zein proteins synthesized at various developmental stages in both genotypes was shown to be strictly correlated to the amounts of zein mRNA present in the endosperm. The opaque-2 mutation resulted in reduced accumulation of the zein mRNAs, particularly those coding for the 21,000 proteins, and did not affect translation, as previously suggested. Furthermore, hybridizations of restriction endonuclease digested and electrophoretically fractionated maize DNA with zein cDNA probes showed no differences between wild-type and opaque-2 plants in the genome organization of the zein genes. It is suggested that the opaque-2 mutation acts directly upon zein gene transcription.
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This paper reports that the opaque-6 (o6) mutation of maize, which causes seedling lethality and interferes in the endosperm with the synthesis of zeins and b-32 protein, is a proline requiring mutant functionally allelic to proline-1 (pro-1). Furthermore, immunological studies on the b-32 content of ten independently originated o6 and pro-1 alleles demonstrated that four alleles contain an apparently normal b-32 protein while the others are either devoid of it or contain trace amounts of cross-reacting proteins of lower molecular weight.
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Maize endosperms accumulate during development a large amount of storage proteins (zeins). The rate of zein accumulation is under the control of several regulatory genes. Two of these, the opaque-2 and opaque-6 mutants, lower the zein level, thus improving the nutritional quality of maize meals. An endosperm protein of Mr 32 000 (b-32) appears to be correlated with the zein level. The b-32 protein is encoded by the opaque-6 gene which, in turn, is activated by opaque-2. We report the purification, amino-acid composition and peptide map of b-32 protein. Furthermore we demonstrate that the protein exists as a monomer likely located in the soluble cytoplasm. As a step towards the isolation of a complementary-DNA clone for b-32 protein, the purification of its corresponding mRNA is described.
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DNAs were synthesized by using zein mRNAs from normal and opaque-2 versions of the maize inbred W64A. The hybridization of zein mRNAs and cDNAs was more complex than that of ovalbumin mRNA and cDNA which was used as a standard. The hybridization of zein mRNAs and cDNAs occurred over 2.5 decades of R0t and was best described by two kinetic components based on computer analysis using a least-squares procedure to analyze the data. Compared to the ovalbumin standard, the two components of the reaction involving mRNAs from the normal inbred had complexities of 200 and 3950 nucleotides. The complexity of these components was 338 and 3330 nucleotides for the reaction involving mRNAs from the opaque-2 mutant. When the hybrid formation of normal mRNA and cDNA was determined by hydroxylapatite chromatography rather than by S1 nuclease digestion, only one kinetic component was observed with a complexity of 270 nucleotides. By hybrid-arrested translation of normal mRNA with cDNA prepared from opaque-2 mRNA it was demonstrated that a portion of the reduced sequence complexity in the mutant was due to a reduction in the mRNAs for the 22 000 molecular weight prezein proteins. By hybridizing the cDNAs to maize DNA we determined that the zein genes were present in 1-5 copies/haploid genome.
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The nucleotide sequence of the zein genomic clone (W22)Z7 and its flanking sequences from the W22 inbred line of maize is reported. The sequence is 1587 bp long and contains 444 bp of 5' noncoding sequence and 342 bp of 3 ' noncoding sequence. The Z7 sequence belongs to a large complex multigene family and is a member of the B49 subfamily. It is 86% homologous to other known sequences from the same subfamily, but contains four in-frame termination codons caused by single base changes. Its flanking regions contain the usual eukaryotic transcriptional signals.
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A nomenclature system for maize (Zea mays L.) prolamins, zeins, is proposed. According to this system, the protein that is rich in proline, glutamine, leucine and/or alanine, and is extracted with aqueous alcohol solutions containing a reducing agent, is considered to be zein. Zein is separated into three distinct classes (α, β and γ) based on differences in solubility, amino acid composition, electrophoretic, chromatographic, and immunological properties, N-terminal amino sequences and complete amino acid sequences deduced from nucleic acid sequences. α-Zein constitutes 75–85% of the total zein and is made up of Mr-21,000–25,000 polypeptides plus a Mr-10,000 minor polypeptide. β-Zein includes Mrr-17,000–18,000 methionine-rich polypeptides and constitutes 10–15% of the total zein. γ-Zein is made up of one size class, Mr-27,000 proline-rich polypeptide, that constitutes 5–10% of the total zein.
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We have examined the fragments of maize nuclear DNA that are homologous to three cloned cDNAs prepared from zein mRNA. Southern blots of high molecular weight ( > 40 kb) maize nuclear DNA cleaved with BamHI, HindIII or EcoRI were hybridized to three zein cDNA plasmid probes. Multiple restriction fragments in a wide range of size classes were observed to hybridize with all three probes. Our results indicate the occurrence of families of genes in the maize genome that are related by their sequences to a given zein mRNA sequence.
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Zein messenger RNAs from maize endosperm were purified by successive oligo(dT)-cellulose chromatography and sucrose gradient centrifugation. Polyacrylamide gel electrophoresis under denaturing conditions revealed the presence of two size classes of zein messenger RNAs of Mr 3.5 x 10(5) and 4.10 x 10(5). The mRNA was shown to synthesize the major zein polypeptides, to have a base composition characteristic of a poly(A)-containing RNA and to be transcribed by reverse transcriptase into complementary DNA. The r0t1/2 of the hybridization curve of cDNA hybridized to an excess of mRNA was shown to be 7 x 10(-2) M . s indicating that about 15 non-cross-hybridizing sequences are present in the zein mRNA preparations. The kinetics of cDNA annealing with an excess of maize DNA from 2 n cells suggest a ten-times reiteration of each mRNA sequence. This result is confirmed from saturation experiments, where in cDNA excess to DNA, the number of zein genes per haploid maize genome was estimated as about 120 copies. Similar experiments carried out on DNA from normal and mutant endosperms (3n cells) indicate the absence of large amplifications or deletions of zein genes in the tissue devoted to zein synthesis.
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Sodium dodecylsulfate-polyacrylamide gel electrophoresis reveals that zein prepared from normal maize inbred (Zea mays L.) contains six separable components. Z1 and Z2 are the predominant species, with molecular weights of 21,800 and 19,000 daltons. Amino acid analysis of these two components shows that both are rich in glutamic acid, leucine, and proline, but low in lysine. Of the four minor bands, Z3, Z4, Z5, and Z6, the latter two exist only in trace amounts. A mutation at the opaque-2 locus severely suppresses the synthesis of Z1. The nonallelic mutant, opaque-7, strongly suppresses the synthesis of Z3 and Z4, while slightly reducing Z2. On the other hand, the floury-2 mutant appears to reduce the synthesis of these six proteins in the same relative proportion. In the double mutant combinations, opaque-2 apparently is epistatic to opaque-7 and floury-2 in the synthesis of zein components. The glutelin fraction shows a more complex banding pattern; however, qualitative differences are not apparent among the mutant lines examined.