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The kernel length is a crucial determinant of maize (Zea mays L.) yield; however, only a limited number of genes regulating kernel length have been validated, thus leaving our understanding of the mechanisms governing kernel length incomplete. We previously identified a maize kernel mutant, defective kernel219 (dek219), which encodes the DICER-LIKE1 protein that is essential for miRNA biogenesis. The present study revealed that dek219 consistently exhibits a stable phenotype characterized by a reduced kernel length. Further analysis indicated that dek219 may reduce the kernel length by inhibiting the expression of genes involved in regulating kernel length. By employing miRNA-target gene prediction, expression analysis, and correlation analysis, we successfully identified nine transcription factors that potentially participate in the regulation of kernel length under the control of DEK219. Among them, the upregulation fold change of HEAT SHOCK TRANSCRIPTION FACTOR17 (HSF17) expression was the highest, and the difference was most significant. The results of transient expression analysis and electrophoretic mobility shift assay (EMSA) indicated that HSF17 can inhibit the expression of DEFECTIVE ENDOSPERM18 (DE18), a gene involved in regulating kernel length. Furthermore, the hsf17 mutant exhibited a significant increase in kernel length, suggesting that HSF17 functions as a negative regulator of kernel length. The results of this study provide crucial evidence for further elucidating the molecular regulatory mechanism underlying maize kernel length and also offer valuable genetic resources for breeding high-yielding maize varieties.

Deep sowing is an efficient strategy for maize to ensure the seedling emergence rate under adverse conditions such as drought or low temperatures. However, the genetic basis of deep-sowing tolerance-related traits in maize remains largely unknown. In this study, we performed a genome-wide association study on traits related to deep-sowing tolerance, including mesocotyl length (ML), coleoptile length (CL), plumule length (PL), shoot length (SL), and primary root length (PRL), using 255 maize inbred lines grown in three different environments. We identified 23, 6, 4, and 4 quantitative trait loci (QTLs) associated with ML, CL, PL, and SL, respectively. By analyzing candidate genes within these QTLs, we found a γ-tubulin-containing complex protein, ZmGCP2, which was significantly associated with ML, PL, and SL. Loss of function of ZmGCP2 resulted in decreased PL, possibly by affecting the cell elongation, thus affecting SL. Additionally, we identified superior haplotypes and allelic variations of ZmGCP2 with a longer PL and SL, which may be useful for breeding varieties with deep-sowing tolerance to improve maize cultivation.

Banded leaf and sheath blight (BLSB) disease, incited by Rhizoctonia solani, is destructive, difficult to manage and gravely threatens maize (Zea mays L.) production across continents. Plant transcription factors (TFs) act as functional nodes that integrate defense signals to activate relevant immune outlets via large-scale transcriptional reprogramming of the expression of massive sets of defense-related genes (DRGs). Recent studies revealed complex changes in the maize transcriptome during BLSB infection. More than 30 TFs belonging to the WRKY, NAC, TCP, bHLH, and bZIP families, among others, have been putatively identified as core genes inducible in maize by the virulence factors of R. solani. Sadly, no progress has been made in characterizing these TFs in maize resistance to BLSB. Having reviewed the progress made so far, we propose future studies to prioritize functional characterization of the potential TFs and their manipulation through genome editing technology as well as the use of synthetic TFs to improve maize resistance to BLSB.

The MOR (Morphogenesis-related NDR kinase) signaling network, initially identified in yeast, exhibits evolutionary conservation across eukaryotes and plays indispensable roles in the normal growth and development of these organisms. However, the functional role of this network and its associated genes in maize (Zea mays) has remained elusive until now. In this study, we identified a total of 19 maize MOR signaling network genes, and subsequent co-expression analysis revealed that 12 of these genes exhibited stronger associations with each other, suggesting their potential collective regulation of maize growth and development. Further analysis revealed significant co-expression between genes involved in the MOR signaling network and several genes related to cold tolerance. All MOR signaling network genes exhibited significant co-expression with COLD1 (Chilling tolerance divergence1), a pivotal gene involved in the perception of cold stimuli, suggesting that COLD1 may directly transmit cold stress signals to MOR signaling network genes subsequent to the detection of a cold stimulus. The findings indicated that the MOR signaling network may play a crucial role in modulating cold tolerance in maize by establishing an intricate relationship with key cold tolerance genes, such as COLD1. Under low-temperature stress, the expression levels of certain MOR signaling network genes were influenced, with a significant up-regulation observed in Zm00001d010720 and a notable down-regulation observed in Zm00001d049496, indicating that cold stress regulated the MOR signaling network. We identified and analyzed a mutant of Zm00001d010720, which showed a higher sensitivity to cold stress, thereby implicating its involvement in the regulation of cold stress in maize. These findings suggested that the relevant components of the MOR signaling network are also conserved in maize and this signaling network plays a vital role in modulating the cold tolerance of maize. This study offered valuable genetic resources for enhancing the cold tolerance of maize.

ADP-Glc pyrophosphorylase (AGPase), which catalyzes the transformation of ATP and glucose-1-phosphate (Glc-1-P) into adenosine diphosphate glucose (ADP-Glc), acts as a rate-limiting enzyme in crop starch biosynthesis. Prior research has hinted at the regulation of AGPase by phosphorylation in maize. However, the identification and functional implications of these sites remain to be elucidated. In this study, we identified the phosphorylation site (serine at the 31st position of the linear amino acid sequence) of the AGPase large subunit (Sh2) using iTRAQTM. Subsequently, to ascertain the impact of Sh2 phosphorylation on AGPase, we carried out site-directed mutations creating Sh2-S31A (serine residue replaced with alanine) to mimic dephosphorylation and Sh2-S31D (serine residue replaced with aspartic acid) or Sh2-S31E (serine residue replaced with glutamic acid) to mimic phosphorylation. Preliminary investigations were performed to determine Sh2 subcellular localization, its interaction with Bt2, and the resultant AGPase enzymatic activity. Our findings indicate that phosphorylation exerts no impact on the stability or localization of Sh2. Furthermore, none of these mutations at the S31 site of Sh2 seem to affect its interaction with Bt2 (smaller subunit). Intriguingly, all S31 mutations in Sh2 appear to enhance AGPase activity when co-transfected with Bt2, with Sh2-S31E demonstrating a substantial five-fold increase in AGPase activity compared to Sh2. These novel insights lay a foundational groundwork for targeted improvements in AGPase activity, thus potentially accelerating the production of ADP-Glc (the primary substrate for starch synthesis), promising implications for improved starch biosynthesis, and holding the potential to significantly impact agricultural practices.

ADP-Glc pyrophosphorylase (AGPase) is a pivotal enzyme catalyzing the conversion of ATP and glucose-1-phosphate (Glc-1-P) to adenosine diphosphate glucose (ADP-Glc), thereby serving as a rate-limiting factor in starch biosynthesis in crops. Although previous investigations have suggested phosphorylation-based regulation of AGPase in maize, the explicit modulation mechanisms have yet to be elucidated. This research evaluated the effect of point mutations at phosphorylation sites (identified using iTRAQTM AB SCIEX, Framingham, MA, USA) on the subcellular localization and activity of the AGPase small subunit Bt2, and its interaction with the large subunit Sh2, in maize. Despite the induction of point mutations, subcellular localization of the Bt2 subunit remained unaltered, primarily within the cytoplasm and nucleus. The interaction between Bt2 and Sh2 subunits continued, mainly in the chloroplast. Notably, an increase in AGPase activity was observed in the case of simulated phosphorylation point mutations, whereas dephosphorylation activity significantly diminished relative to the wild type. These findings demonstrate that point mutations do not affect the subcellular localization of the Bt2 subunit or its interaction with the Sh2 subunit, but substantially modulate AGPase activity. This study provides critical insights into the role of point mutations in enhancing AGPase activity, thus potentially accelerating the production of ADP-Glc, the primary substrate for starch synthesis, promising implications for improved starch biosynthesis in maize.

In order to investigate the crucial role of ZmBT1 in starch accumulation during maize grain development and analyze the expression and distribution of ZmBT1 in various maize tissues, we prepared a polyclonal antibody. Specifically, we successfully expressed the recombinant plasmid pGEX-6p-ZmBT1-C (382-437aa) and purified Gst-ZmBT1-C as the antigen for antibody preparation. Our results confirmed that the ZmBT1 protein in maize tissues can be specifically recognized by the ZmBT1 antibody. Through Western blotting, we observed that the expression protein of ZmBT1 varied by tissues, with the highest content in the grain and endosperm. Furthermore, we employed a combination of Western blotting and quantitative real-time PCR to show that the expression level of ZmBT1 can be influenced by plant hormones. This finding suggests that ZmBT1 plays a critical role in the accumulation of starch and opens up new avenues for functional studies of this protein.

Abstract Background Phytohormone abscisic acid (ABA) is involved in the regulation of a wide range of biological processes. In Arabidopsis, it has been well-known that SnRK2s are the central components of the ABA signaling pathway that control the balance between plant growth and stress response, but the functions of ZmSnRK2 in maize are rarely reported. Therefore, the study of ZmSnRK2 is of great importance to understand the ABA signaling pathways in maize. Results In this study, 14 ZmSnRK2 genes were identified in the latest version of maize genome database. Phylogenetic analysis revealed that ZmSnRK2s are divided into three subclasses based on their diversity of C-terminal domains. The exon-intron structures, phylogenetic, synteny and collinearity analysis indicated that SnRK2s, especially the subclass III of SnRK2, are evolutionally conserved in maize, rice and Arabidopsis. Subcellular localization showed that ZmSnRK2 proteins are localized in the nucleus and cytoplasm. The RNA-Seq datasets and qRT-PCR analysis showed that ZmSnRK2 genes exhibit spatial and temporal expression patterns during the growth and development of different maize tissues, and the transcript levels of some ZmSnRK2 genes in kernel are significantly induced by ABA and sucrose treatment. In addition, we found that ZmSnRK2.10, which belongs to subclass III, is highly expressed in kernel and activated by ABA. Overexpression of ZmSnRK2.10 partially rescued the ABA-insensitive phenotype of snrk2.2/2.3 double and snrk2.2/2.3/2.6 triple mutants and led to delaying plant flowering in Arabidopsis. Conclusion The SnRK2 gene family exhibits a high evolutionary conservation and has expanded with whole-genome duplication events in plants. The ZmSnRK2s expanded in maize with whole-genome and segmental duplication, not tandem duplication. The expression pattern analysis of ZmSnRK2s in maize offers important information to study their functions. Study of the functions of ZmSnRK.10 in Arabidopsis suggests that the ABA-dependent members of SnRK2s are evolutionarily conserved in plants. Our study elucidated the structure and evolution of SnRK2 genes in plants and provided a basis for the functional study of ZmSnRK2s protein in maize.

Starch, the major component of cereal grains, affects crop yield and quality and is widely used in food and industrial applications. The biosynthesis of maize starch is a complex process involving a series of functional enzymes. However, the sophisticated regulatory mechanisms of starch biosynthetic genes have not been fully elaborated. The basic/helix-loop-helix (bHLH) transcription factors are widely distributed in eukaryotes and participate in many physiological processes. In this study, 202 bHLH encoding genes were identified in the maize genome by Blast method. ZmICE1 gene, which belongs to the ICE subfamily of the bHLH family, was obtained and expressed mainly in maize filling endosperm and co-expressed with 14 starch biosynthesis genes. Based on the comparative analyses across different plant species, we revealed that the gene structures and protein domains of the ICE subfamily were conserved between monocots and dicots, suggesting their functional conservation feature. Yeast activation and subcellular localization assays suggested that ZmICE1 had transcriptional activation activity and localized in the nucleus. Yeast one-hybrid assays confirmed that ZmICE1 could directly bind to the promoters of ZmSSIIa and ZmGBSSI. Transient gene expression analysis in maize endosperm revealed that ZmICE1 positively regulated the expression of ZmSSIIa, but inhibited the expression of ZmGBSSI. Our results indicated that ZmICE1 could function as a regulator of maize starch biosynthesis.

The process of starch biosynthesis is a major developmental event that affects the final grain yield and quality in maize (Zea mays L.), and transcriptional regulation plays a key role in modulating the expression of the main players in the pathway. ZmBt2, which encodes the small subunits of AGPase, is a rate-controlling gene of the pathway; however, much remains unknown about its transcriptional regulation. Our earlier study identifies a short functional fragment of ZmBt2 promoter (394-bp), and further shows it contains multiple putative cis-acting regulatory elements, demonstrating that several transcription factors may govern ZmBt2 expression. Here, we identified a novel TCP transcription factor (TF), ZmTCP7, that interacted with the functional fragment of the ZmBt2 promoter in a yeast one hybrid screening system. We further showed that ZmTCP7 is a non-autonomous TF targeted to the nucleus and predominantly expressed in maize endosperm. Using promoter deletion analyzes by transient expression in maize endosperm protoplasts combined with electrophoretic mobility shift assays, we found that ZmTCP7 bound to GAACCCCAC elements on the ZmBt2 promoter to suppress its expression. Transgenic overexpression of ZmTCP7 in maize caused a significant repression of ZmBt2 transcription by ~77.58%, resulting in a 21.51% decrease in AGPase activity and a 9.58% reduction in the endosperm starch content of transgenic maize. Moreover, the expressions of ZmBt1, ZmSSI, ZmSSIIa, and ZmSSIIIa were increased, while those of ZmSh2 and ZmSSIV reduced significantly in the endosperm of the transgenic maize. Overall, this study shows that ZmTCP7 functions as a transcriptional repressor of ZmBt2 and a negative regulator of endosperm starch accumulation, providing new insights into the regulatory networks that govern ZmBt2 expression and starch biosynthesis pathway in maize.

Maize (Zea mays L.) is one of the most important food crops in the world, and starch is the main component of its endosperm. Transcriptional regulation plays a vital role in starch biosynthesis. However, it is not well understood in maize. We report the identification of the transcription factor ZmNAC126 and its role in regulation of starch synthesis in maize. Transcriptional expression of ZmNAC126 was higher in maize endosperm and kernels than in roots or stems. ZmNAC126 shared a similar expression pattern with starch synthesis genes during seed development, and its expression pattern was also consistent with the accumulation of starch. ZmNAC126 is a typical transcription factor with a transactivation domain between positions 201 and 227 of the amino acid sequence, is located in the nucleus, and binds to CACG repeats in vitro. Yeast one-hybrid assay revealed that ZmNAC126 bound the promoters of ZmGBSSI, ZmSSIIa, ZmSSIV, ZmISA1, and ZmISA2. Transient overexpression of ZmNAC126 in maize endosperm increased the activities of promoters pZmSh2, pZmBt2, pZmGBSSI, pZmSSIIIa, and pZmBT1 but inhibited the activities of pZmISA1 and pZmISA2. ZmNAC126 thus acts in starch synthesis by transcriptionally regulating targeted starch synthesis-related genes in maize kernels.

Maize is a main food and feed crop with great production potential and high economic benefits. Improving its photosynthesis efficiency is crucial for increasing yield. Maize photosynthesis occurs mainly through the C4 pathway, and NADP-ME (NADP-malic enzyme) is a key enzyme in the photosynthetic carbon assimilation pathway of C4 plants. ZmC4-NADP-ME catalyzes the release of CO2 from oxaloacetate into the Calvin cycle in the maize bundle sheath. Brassinosteroid (BL) can improve photosynthesis; however, its molecular mechanism of action remains unclear. In this study, transcriptome sequencing of maize seedlings treated with epi-brassinolide (EBL) showed that differentially expressed genes (DEGs) were significantly enriched in photosynthetic antenna proteins, porphyrin and chlorophyll metabolism, and photosynthesis pathways. The DEGs of C4-NADP-ME and pyruvate phosphate dikinase in the C4 pathway were significantly enriched in EBL treatment. Co-expression analysis showed that the transcription level of ZmNF-YC2 and ZmbHLH157 transcription factors was increased under EBL treatment and moderately positively correlated with ZmC4-NADP-ME. Transient overexpression of protoplasts revealed that ZmNF-YC2 and ZmbHLH157 activate C4-NADP-ME promoters. Further experiments showed ZmNF-YC2 and ZmbHLH157 transcription factor binding sites on the −1616 bp and −1118 bp ZmC4 NADP-ME promoter. ZmNF-YC2 and ZmbHLH157 were screened as candidate transcription factors mediating brassinosteroid hormone regulation of the ZmC4 NADP-ME gene. The results provide a theoretical basis for improving maize yield using BR hormones.

Abstract Background Endosperm-trait related genes are associated with grain yield or quality in maize. There are vast numbers of these genes whose functions and regulations are still unknown. The biolistic system, which is often used for transient gene expression, is expensive and involves complex protocol. Besides, it cannot be used for simultaneous analysis of multiple genes. Moreover, the biolistic system has little physiological relevance when compared to cell-specific based system. Plant protoplasts are efficient cell-based systems which allow quick and simultaneous transient analysis of multiple genes. Typically, PEG-calcium mediated transfection of protoplast is simple and cost-effective. Notably, starch granules in cereal endosperm may diminish protoplast yield and integrity, if the isolation and transfection conditions are not accurately measured. Prior to this study, no PEG-calcium mediated endosperm protoplast system has been reported for cereal crop, perhaps, because endosperm cells accumulate starch grains. Results Here, we showed the uniqueness of maize endosperm-protoplast system (EPS) in conducting endosperm cell-based experiments. By using response surface designs, we established optimized conditions for the isolation and PEG-calcium mediated transfection of maize endosperm protoplasts. The optimized conditions of 1% cellulase, 0.75% macerozyme and 0.4 M mannitol enzymolysis solution for 6 h showed that more than 80% protoplasts remained viable after re-suspension in 1 ml MMG. The EPS was used to express GFP protein, analyze the subcellular location of ZmBT1, characterize the interaction of O2 and PBF1 by bimolecular fluorescent complementation (BiFC), and simultaneously analyze the regulation of ZmBt1 expression by ZmMYB14. Conclusions The described optimized conditions proved efficient for reasonable yield of viable protoplasts from maize endosperm, and utility of the protoplast in rapid analysis of endosperm-trait related genes. The development of the optimized protoplast isolation and transfection conditions, allow the exploitation of the functional advantages of protoplast system over biolistic system in conducting endosperm-based studies (particularly, in transient analysis of genes and gene regulation networks, associated with the accumulation of endosperm storage products). Such analyses will be invaluable in characterizing endosperm-trait related genes whose functions have not been identified. Thus, the EPS will benefit the research of cereal grain yield and quality improvement.

Abstract Background Short internodes contribute to plant dwarfism, which is exceedingly beneficial for crop production. However, the underlying mechanisms of internode elongation are complicated and have been not fully understood. Results Here, we report a maize dwarf mutant, dwarf2014 (d2014), which displays shortened lower internodes. Map-based cloning revealed that the d2014 gene is a novel br2 allele with a splicing variation, resulting in a higher expression of BR2-T02 instead of normal BR2-T01. Then, we found that the internode elongation in d2014/br2 exhibited a pattern of inhibition-normality-inhibition (transient for the ear-internode), correspondingly, at the 6-leaf, 12-leaf and 14-leaf stages. Indeed, BR2 encodes a P-glycoprotein1 (PGP1) protein that functions in auxin efflux, and our in situ hybridization assay showed that BR2 was mainly expressed in vascular bundles of the node and internode. Furthermore, significantly higher auxin concentration was detected in the stem apex of d2014 at the 6-leaf stage and strictly in the node region for the ear-internode at the 14-leaf stage. In such context, we propose that BR2/PGP1 transports auxin from node to internode through the vascular bundles, and excessive auxin accumulation in the node (immediately next to the intercalary meristem) region suppresses internode elongation of d2014. Conclusions These findings suggest that low auxin levels mediated by BR2/PGP1 in the intercalary meristem region are crucial for internode elongation.

Glycogen synthase kinase-3 (GSK-3) is a nonreceptor serine/threonine protein kinase that is involved in diverse processes, including cell development, photomorphogenesis, biotic and abiotic stress responses, and hormone signaling. In contrast with the deeply researched GSK family in Arabidopsis and rice, maize GSKs’ common bioinformatic features and protein functions are poorly understood. In this study, we identified 11 GSK genes in the maize (Zea mays L.) genome via homologous alignment, which we named Zeama;GSKs (ZmGSKs). The results of ZmGSK protein sequences, conserved motifs, and gene structures showed high similarities with each other. The phylogenetic analyses showed that a total of 11 genes from maize were divided into four clades. Furthermore, semi-quantitative RT-PCR analysis of the GSKs genes showed that ZmGSK1, ZmGSK2, ZmGSK4, ZmGSK5, ZmGSK8, ZmGSK9, ZmGSK10, and ZmGSK11 were expressed in all tissues; ZmGSK3, ZmGSK6, and ZmGSK7 were expressed in a specific organization. In addition, GSK expression profiles under hormone treatments demonstrated that the ZmGSK genes were induced under BR conditions, except for ZmGSK2 and ZmGSK5. ZmGSK genes were regulated under ABA conditions, except for ZmGSK1 and ZmGSK8. Finally, using the yeast two-hybrid and BiFC assay, we determined that clads II (ZmGSK1, ZmGSK4, ZmGSK7, ZmGSK8, and ZmGSK11) could interact with ZmBZR1. The results suggest that clade II of ZmGSKs is important for BR signaling and that ZmGSK1 may play a dominant role in BR signaling as the counterpart to BIN2. This study provides a foundation for the further study of GSK3 functions and could be helpful in devising strategies for improving maize.

Abstract Background Utilization of heterosis in maize could be critical in maize breeding for boosting grain yield. However, the genetic architecture of heterosis is not fully understood. To dissect the genetic basis of yield-related traits and heterosis in maize, 301 recombinant inbred lines derived from 08 to 641 × YE478 and 298 hybrids from the immortalized F2 (IF2) population were used to map quantitative trait loci (QTLs) for nine yield-related traits and mid-parent heterosis. Results We observed 156 QTLs, 28 pairs of loci with epistatic interaction, and 10 significant QTL × environment interactions in the inbred and hybrid mapping populations. The high heterosis in F1 and IF2 populations for kernel weight per ear (KWPE), ear weight per ear (EWPE), and kernel number per row (KNPR) matched the high percentages of QTLs (over 50%) for those traits exhibiting overdominance, whereas a notable predominance of loci with dominance effects (more than 70%) was observed for traits that show low heterosis such as cob weight per ear (CWPE), rate of kernel production (RKP), ear length (EL), ear diameter (ED), cob diameter, and row number (RN). The environmentally stable QTL qRKP3–2 was identified across two mapping populations, while qKWPE9, affecting the trait mean and the mid-parent heterosis (MPH) level, explained over 18% of phenotypic variations. Nine QTLs, qEWPE9–1, qEWPE10–1, qCWPE6, qEL8, qED2–2, qRN10–1, qKWPE9, qKWPE10–1, and qRKP4–3, accounted for over 10% of phenotypic variation. In addition, QTL mapping identified 95 QTLs that were gathered together and integrated into 33 QTL clusters on 10 chromosomes. Conclusions The results revealed that (1) the inheritance of yield-related traits and MPH in the heterotic pattern improved Reid (PA) × Tem-tropic I (PB) is trait-dependent; (2) a large proportion of loci showed dominance effects, whereas overdominance also contributed to MPH for KNPR, EWPE, and KWPE; (3) marker-assisted selection for markers at genomic regions 1.09–1.11, 2.04, 3.08–3.09, and 10.04–10.05 contributed to hybrid performance per se and heterosis and were repeatedly reported in previous studies using different heterotic patterns is recommended.

Abstract Background Starch biosynthesis in endosperm is a key process influencing grain yield and quality in maize. Although a number of starch biosynthetic genes have been well characterized, the mechanisms by which the expression of these genes is regulated, especially in regard to microRNAs (miRNAs), remain largely unclear. Results Sequence data for small RNAs, degradome, and transcriptome of maize endosperm at 15 and 25 d after pollination (DAP) from inbred lines Mo17 and Ji419, which exhibit distinct starch content and starch granule structure, revealed the mediation of starch biosynthetic pathways by miRNAs. Transcriptome analysis of these two lines indicated that 33 of 40 starch biosynthetic genes were differentially expressed, of which 12 were up-regulated in Ji419 at 15 DAP, one was up-regulated in Ji419 at 25 DAP, 14 were up-regulated in Ji419 at both 15 and 25 DAP, one was down-regulated in Ji419 at 15 DAP, two were down-regulated in Ji419 at 25 DAP, and three were up-regulated in Ji419 at 15 DAP and down-regulated in Ji419 at 25 DAP, compared with Mo17. Through combined analyses of small RNA and degradome sequences, 22 differentially expressed miRNAs were identified, including 14 known and eight previously unknown miRNAs that could target 35 genes. Furthermore, a complex co-expression regulatory network was constructed, in which 19 miRNAs could modulate starch biosynthesis in endosperm by tuning the expression of 19 target genes. Moreover, the potential operation of four miRNA-mediated pathways involving transcription factors, miR169a-NF-YA1-GBSSI/SSIIIa and miR169o-GATA9-SSIIIa/SBEIIb, was validated via analyses of expression pattern, transient transformation assays, and transactivation assays. Conclusion Our results suggest that miRNAs play a critical role in starch biosynthesis in endosperm, and that miRNA-mediated networks could modulate starch biosynthesis in this tissue. These results have provided important insights into the molecular mechanism of starch biosynthesis in developing maize endosperm.

Starch biosynthesis is a complex and highly controlled process that requires coordinated activities among multiple enzymes. Starch synthase III (SSIII) is the largest protein in the starch synthase complex and its function is to lengthen long-chain amylopectin in starch synthesis. It potentially affects the activity of other key enzymes in starch synthesis through protein–protein interactions; therefore, its function and regulation play a predominant role in starch synthesis. In this review, we summarized the main research of SSIII including its biochemical characteristics, structural features, expression atlas, and regulation means. Structural features and expressional analysis indicated that SSIIIa is the main functional protein in maize endosperm rather than SSIIIb-a and SSIIIb-b, even though they are similar in the tertiary structures. The regulation investigation of SSIIIa showed that there are 13 transcription factors that control the transcription of SSIIIa. Interaction network analysis showed that SSIIIa could be involved with ten other key enzymes in starch synthesis. In conclusion, this review considerably extends our understanding of SSIII and provides the theoretical basis for improving starch synthesis by SSIII in maize.

Starch phosphorylase (PHO) is a multimeric enzyme with two distinct isoforms: plastidial starch phosphorylase (PHO1) and cytosolic starch phosphorylase (PHO2). PHO1 specifically resides in the plastid, while PHO2 is found in the cytosol. Both play a critical role in the synthesis and degradation of starch. This study aimed to report the detailed structure, function, and evolution of genes encoding PHO1 and PHO2 and their protein ligand-binding sites in eight monocots and four dicots. “True” orthologs of PHO1 and PHO2 of Oryza sativa were identified, and the structure of the enzyme at the protein level was studied. The genes controlling PHO2 were found to be more conserved than those controlling PHO1; the variations were mainly due to the variable sequence and length of introns. Cis-regulatory elements in the promoter region of both genes were identified, and the expression pattern was analyzed. The real-time quantitative polymerase chain reaction indicated that PHO2 was expressed in all tissues with a uniform pattern of transcripts, and the expression pattern of PHO1 indicates that it probably contributes to the starch biosynthesis during seed development in Zea mays. Under abscisic acid (ABA) treatment, PHO1 was found to be downregulated in Arabidopsis and Hordeum vulgare. However, we found that ABA could up-regulate the expression of both PHO1 and PHO2 within 12 h in Zea mays. In all monocots and dicots, the 3D structures were highly similar, and the ligand-binding sites were common yet fluctuating in the position of aa residues.

Proper development of the maize kernel is of great significance for high and stable maize yield to ensure national food security. Gibberellin (GA), one of the hormones regulating plant growth, is involved in modulating the development of maize kernels. Cellulose, one of the main components of plant cells, is also regulated by gibberellin. The mechanism of hormone regulation during maize grain development is highly complicated, and reports on GA-mediated modulation of cellulose synthesis during maize grain development are rare. Our study revealed that during grain growth and development, the grain length and bulk density of GA-treated corn kernels improved significantly, and the cellulose content of grains increased, while seed coat thickness decreased. The transcription factor basic region/leucine zipper motif 53 (bZIP53), which is strongly correlated with cellulose synthase gene 1 (CesA1) expression, was screened by transcriptome sequencing and the expression of the cellulose synthase gene in maize grain development after GA treatment was determined. It was found that bZIP53 expression significantly promoted the expression of CesA1. Further, analysis of the transcription factor bZIP53 determined that the gene-encoded protein was localized in the cell and nuclear membranes, but the transcription factor bZIP53 itself showed no transcriptional activation. Further studies are required to explore the interaction of bZIP53 with CesA1.

Starch phosphorylase is a member of the GT35-glycogen-phosphorylase superfamily. Glycogen phosphorylases have been researched in animals thoroughly when compared to plants. Genetic evidence signifies the integral role of plastidial starch phosphorylase (PHO1) in starch biosynthesis in model plants. The counterpart of PHO1 is PHO2, which specifically resides in cytosol and is reported to lack L80 peptide in the middle region of proteins as seen in animal and maltodextrin forms of phosphorylases. The function of this extra peptide varies among species and ranges from the substrate of proteasomes to modulate the degradation of PHO1 in Solanum tuberosum to a non-significant effect on biochemical activity in Oryza sativa and Hordeum vulgare. Various regulatory functions, e.g., phosphorylation, protein–protein interactions, and redox modulation, have been reported to affect the starch phosphorylase functions in higher plants. This review outlines the current findings on the regulation of starch phosphorylase genes and proteins with their possible role in the starch biosynthesis pathway. We highlight the gaps in present studies and elaborate on the molecular mechanisms of phosphorylase in starch metabolism. Moreover, we explore the possible role of PHO1 in crop improvement.

AGPase catalyzes a key rate-limiting step that converts ATP and Glc-1-p into ADP-glucose and diphosphate in maize starch biosynthesis. Previous studies suggest that AGPase is modulated by redox, thermal and allosteric regulation. However, the phosphorylation of AGPase is unclear in the kernel starch biosynthesis process. Phos-tagTM technology is a novel method using phos-tagTM agarose beads for separation, purification, and detection of phosphorylated proteins. Here we identified phos-tagTM agarose binding proteins from maize endosperm. Results showed a total of 1733 proteins identified from 10,678 distinct peptides. Interestingly, a total of 21 unique peptides for AGPase sub-unit Brittle-2 (Bt2) were identified. Bt2 was demonstrated by immunoblot when enriched maize endosperm protein with phos-tagTM agarose was in different pollination stages. In contrast, Bt2 would lose binding to phos-tagTM when samples were treated with alkaline phosphatase (ALP). Furthermore, Bt2 could be detected by Pro-Q diamond staining specifically for phosphorylated protein. We further identified the phosphorylation sites of Bt2 at Ser10, Thr451, and Thr462 by iTRAQ. In addition, dephosphorylation of Bt2 decreased the activity of AGPase in the native gel assay through ALP treatment. Taking together, these results strongly suggest that the phosphorylation of AGPase may be a new model to regulate AGPase activity in the starch biosynthesis process.

The development of maize foundation parents is an important part of genetics and breeding research, and applying new genetic information to produce foundation parents has been challenging. In this study, we focused on quantitative trait loci (QTLs) and general combining ability (GCA) of Ye478, a widely used foundation parent in China. We developed three sets of populations for QTL mapping and to analyze the GCA for some agronomic traits. The assessment of 15 traits resulted in the detection of 251 QTLs in six tested environments, with 119 QTLs identified through a joint analysis across all environments. Further analyses revealed that most favorable alleles for plant type-related traits were from Ye478, and more than half of the favorable alleles for yield-related traits were from R08, another foundation parent used in southwestern China, suggesting that different types of foundation parents carried different favorable alleles. We observed that the GCA for most traits (e.g., plant height and 100-kernel weight) was maintained in the inbred lines descended from the foundation parents. Additionally, the continuous improvement in the GCA of the descendants of the foundation parents was consistent with the main trend in maize breeding programs. We identified three significant genomic regions that were highly conserved in three Ye478 descendants, including the stable QTL for plant height. The GCA for the traits in the F7 generation revealed that the QTLs for the given traits per se were affected by additive effects in the same way in different populations.

The process of starch biosynthesis is a major developmental event that affects the final grain yield and quality in maize (Zea mays L.), and transcriptional regulation plays a key role in modulating the expression of the main players in the pathway. ZmBt2, which encodes the small subunits of AGPase, is a rate-controlling gene of the pathway; however, much remains unknown about its transcriptional regulation. Our earlier study identifies a short functional fragment of ZmBt2 promoter (394-bp), and further shows it contains multiple putative cis-acting regulatory elements, demonstrating that several transcription factors may govern ZmBt2 expression. Here, we identified a novel TCP transcription factor (TF), ZmTCP7, that interacted with the functional fragment of the ZmBt2 promoter in a yeast one hybrid screening system. We further showed that ZmTCP7 is a non-autonomous TF targeted to the nucleus and predominantly expressed in maize endosperm. Using promoter deletion analyzes by transient expression in maize endosperm protoplasts combined with electrophoretic mobility shift assays, we found that ZmTCP7 bound to GAACCCCAC elements on the ZmBt2 promoter to suppress its expression. Transgenic overexpression of ZmTCP7 in maize caused a significant repression of ZmBt2 transcription by ~77.58%, resulting in a 21.51% decrease in AGPase activity and a 9.58% reduction in the endosperm starch content of transgenic maize. Moreover, the expressions of ZmBt1, ZmSSI, ZmSSIIa, and ZmSSIIIa were increased, while those of ZmSh2 and ZmSSIV reduced significantly in the endosperm of the transgenic maize. Overall, this study shows that ZmTCP7 functions as a transcriptional repressor of ZmBt2 and a negative regulator of endosperm starch accumulation, providing new insights into the regulatory networks that govern ZmBt2 expression and starch biosynthesis pathway in maize. [ABSTRACT FROM AUTHOR]

The process of starch biosynthesis is a major developmental event that affects the final grain yield and quality in maize (Zea mays L.), and transcriptional regulation plays a key role in modulating the expression of the main players in the pathway. ZmBt2, which encodes the small subunits of AGPase, is a rate-controlling gene of the pathway; however, much remains unknown about its transcriptional regulation. Our earlier study identifies a short functional fragment of ZmBt2 promoter (394- bp), and further shows it contains multiple putative cis-acting regulatory elements, demonstrating that several transcription factors may govern ZmBt2 expression. Here, we identified a novel TCP transcription factor (TF), ZmTCP7, that interacted with the functional fragment of the ZmBt2 promoter in a yeast one hybrid screening system. We further showed that ZmTCP7 is a non-autonomous TF targeted to the nucleus and predominantly expressed in maize endosperm. Using promoter deletion analyzes by transient expression in maize endosperm protoplasts combined with electrophoretic mobility shift assays, we found that ZmTCP7 bound to GAACCCCAC elements on the ZmBt2 promoter to suppress its expression. Transgenic overexpression of ZmTCP7 in maize caused a significant repression of ZmBt2 transcription by 77.58%, resulting in a 21.51% decrease in AGPase activity and a 9.58% reduction in the endosperm starch content of transgenic maize. Moreover, the expressions of ZmBt1, ZmSSI, ZmSSIIa, and ZmSSIIIa were increased, while those of ZmSh2 and ZmSSIV reduced significantly in the endosperm of the transgenic maize. Overall, this study shows that ZmTCP7 functions as a transcriptional repressor of ZmBt2 and a negative regulator of endosperm starch accumulation, providing new insights into the regulatory networks that govern ZmBt2 expression and starch biosynthesis pathway in maize. [ABSTRACT FROM AUTHOR]