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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.

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.

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.

Seed development is a crucial biological process affecting crop yield. Brassionsteroids (BRs) plays essential role in seed development. Although part of genes participated in BR regulate seed development have been cloned in maize, their genetic and mechanisms are still unclear. In the previous experiment, we found that exogenous spray BL promote seed development. Here, RNA-sequence analysis of BL treated seeds revealed that 12 candidate function genes and 62 candidate transcription factors among the differentially expressed genes (DEGs) were regulated by BL. Combining with previous QTL and GWAS research results in maize and homologous gene analysis, ZmSLG1 was screened out from 12 candidate function genes for further study. Overexpression of ZmSLG1 displayed round seed and delayed embryo development in maize. Moreover, overexpression of ZmSLG1 decreased BR content and increased expression of BR synthesis related genes in seed. To understand the mechanism of BL regulating ZmSLG1 expression, ZmWRKY82 was identified from 62 candidate transcription factors and examined its function. Transient expression, EMSA and ChIP analysis showed ZmWRKY82 can directly bind to the ZmSLG1 promoter to regulate ZmSLG1 expression. Collectively, exogenous BL regulate the ZmSLG1 gene expression through ZmWRKY82 transcription factor, and ZmSLG1 negatively regulates the endogenous BR contents in seed.HighlightWe conclude that ZmWRKY82-ZmSLG1 is required for BR homeostasis and that modification of ZmSLG1 expression to an appropriate level may provide a way to increase yield.

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.

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.

The process of starch biosynthesis is a major developmental event that affects the final grain yield and quality in maize (

Summary Rice sheath blight (RSB) is an economically significant disease affecting rice yield worldwide. Genetic resistance to RSB is associated with multiple minor genes, with each providing a minor phenotypic effect, but the underlying dominant resistance genes remain unknown. A genome‐wide association study (GWAS) of 259 diverse rice varieties, with genotypes based on a single nucleotide polymorphism (SNP) and haplotype, was conducted to assess their sheath blight reactions at three developmental stages (seedlings, tillering and booting). A total of 653 genes were correlated with sheath blight resistance, of which the disease resistance protein RPM1 (OsRSR1) and protein kinase domain‐containing protein (OsRLCK5) were validated by overexpression and knockdown assays. We further found that the coiled‐coil (CC) domain of OsRSR1 (OsRSR1‐CC) and full‐length OsRLCK5 interacted with serine hydroxymethyltransferase 1 (OsSHM1) and glutaredoxin (OsGRX20), respectively. It was found that OsSHM1, which has a role in the reactive oxygen species (ROS) burst, and OsGRX20 enhanced the antioxidation ability of plants. A regulation model of the new RSB resistance though the glutathione (GSH)‐ascorbic acid (AsA) antioxidant system was therefore revealed. These results enhance our understanding of RSB resistance mechanisms and provide better gene resources for the breeding of disease resistance in rice.

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 one of the three major crops worldwide based on its yield and quality. Starch is crucial to both the yield and quality of maize as it accounts more than 60% of the seed weight, and its structure influences the quality of the crop. Starch synthase I (SSI) contributes to the majority of the starch synthase activity in the maize endosperm. An in-depth understanding of the starch synthesis regulatory mechanism would provide opportunities for improving the yield and quality of maize. In this study, ZmPLATZ2, a plant AT-rich sequence and zinc-binding protein (PLATZ) transcription factor related to starch synthesis, was selected based on co-expression analysis. The semiquantitative RT-PCR and qRT-PCR assays revealed that ZmPLATZ2 had a high expression in the endosperm, and reached the peak at 12 days after pollination (DAP). Different treatments demonstrated that ZmPLATZ2 was downregulated by the presence of sucrose. Subsequent transactivation and subcellular localization analyses showed that ZmPLATZ2 was localized in the nuclei without transactivation. Yeast one-hybrid and transient expression in maize endosperm indicated that ZmPLATZ2 could bind to the promoters of ZmSSI, ZmISA1, and ZmISA2 and increase their gene expression. After ZmPLATZ2 overexpression in rice, four starch synthesis genes were significantly upregulated in the transgenic plant, including the OsSSI gene. In vitro DAP-seq data showed that ZmPLATZ2 could bind to the CAAAAAAA element. In conclusion, our data support that ZmPLATZ2 binds to the CAAAAAAA element in the ZmSSI promoter and mediates the Glu signal pathway.

Endosperm of cereal crops is the main component of its grain. Improvement of endosperm traits will bolster grain yield and quality. Functional analysis of endosperm trait-related genes often requires the use of an endosperm cell system. Here, we describe a protocol for the isolation and transfection of maize endosperm cell protoplast. The endosperm protoplast system can be used for several molecular studies including protein subcellular localization, protein-protein interaction by bimolecular fluorescence complementation (BiFC), protein immunoblotting, transient gene expression, and regulatory analyses by qRT-PCR.

Cell cycle is precisely controlled by numerous regulators. Although extensive studies have identified the cell cycle regulators in rice and Arabidopsis and classified into different gene families, relatively few cell cycle regulators were identified in maize. In this study, 110 putative core cell cycle–related genes in the maize genome were identified through sequence alignment analysis. Among them, 54 members of cyclin family were divided into the A-, B-, C-, D-, F-, L-, T-, and SDS-type subfamilies; 16 members of CDK family were partitioned into the A-, B-, C-, E-, F-, and G-type subfamilies; 19 members of E2F-DP family were separated into the E2F, PAP, DPB, CK, and DEL subfamilies; the remaining genes were nine ICK/KRPs, three EL2/SIMs, three CKSs, five Rbs, and one Wee1 kinase. Maize cell cycle–related genes unequally distributed on all chromosomes, and thirty-six pairs of them were identified as paralogs. The prediction of protein interaction found that 91 proteins were able to interact with each other forming 939 pairs of interactions with a probability more than 0.7. The expression analyses revealed that most of the cell cycle–related genes were expressed in all tissues. The transcription level of some genes in young tissues was higher than that in mature tissues of maize. Meanwhile, some genes from different families shared the similar expression pattern. Overall, the sequence characteristics and expression patterns of maize cell cycle–related genes vary with different gene families. Our results provide basic information for elucidating the functions of these genes in maize.

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.

The peduncle vascular system of maize is critical for the transport of photosynthetic products, nutrients, and water from the roots and leaves to the ear. Accordingly, it positively affects the grain yield. However, the genetic basis of peduncle vascular bundle (PVB)-related traits in maize remains unknown. Thus, 15 PVB-related traits of 386 maize inbred lines were investigated at three locations (Yongcheng, 17YC; Kaifeng, 20KF; and Yuanyang, 20YY). The repeatability for the 15 traits ranged from 35.53% to 92.13%. A genome-wide association study was performed and 69 non-redundant quantitative trait loci (QTL) were detected, including 9, 41, and 27 QTL identified at 17YC, 20KF, and 20YY, respectively. These QTL jointly explained 4.72% (SLL) to 37.30% (NSVB) of the phenotypic variation. Eight QTL were associated with the same trait at two locations. Furthermore, four pleiotropic QTL were identified. Moreover, one QTL (qPVB44), associated with NSVB_20KF, was co-localized with a previously reported locus related to kernel width, implying qPVB44 may affect the kernel width by modulating the number of small vascular bundles. Examinations of the 69 QTL identified 348 candidate genes that were classified in five groups. Additionally, 26 known VB-related homologous genes (e.g. VLN2, KNOX1, and UGT72B3) were detected in 20 of the 69 QTL. A comparison of the NSVB between a Zmvln2 EMS mutant and its wild type elucidated the function of the candidate gene ZmVLN2. These results are important for clarifying the genetic basis of PVB-related traits and may be useful for breeding new high-yielding maize cultivars.

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.

BackgroundUtilization 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.ResultsWe 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 F1and IF2populations 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 QTLqRKP3–2was identified across two mapping populations, whileqKWPE9, 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, andqRKP4–3, 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.ConclusionsThe 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.

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. Electronic supplementary material The online version of this article (10.1186/s12864-019-5945-1) contains supplementary material, which is available to authorized users.

Plant shoot and root growth is regulated by multiple factors, including the hormone auxin. A maize brachytic2 (br2) mutant displays a shortened shoot and root with high auxin concentration in their apexes, where BR2 encodes an auxin transporter P-glycoprotein1 (PGP1). We employed comparative transcriptome sequencing to explore the BR2/PGP1-mediated patterns for shoot and root growth among br2, wild type (WT), and hybrid F1 (br2 × WT). The results show 352 genes in the br2 shoot and 234 genes in the br2 root are differentially expressed compared to WT and F1. Zm00001d045203 and Zm00001d031562 were identified as two auxin-responsive genes in both the shoot and root. The Zm00001d045203 gene is down-regulated in br2 and encodes an Aux/IAA inhibitor involved in the SCFTIR1/AFB-mediated auxin signaling pathway. The up-regulated Zm00001d031562 gene encodes a small GTPase belonging to the Rab/Ypt family, which takes part in auxin-mediated responses in Arabidopsis. Cytology analysis reveals that reduced cell elongation affects the shoot and root growth of br2. The Zm00001d043477 gene is down-regulated and encodes a member of the cellulose synthase protein family, possibly reducing cell elongation in the br2 shoot and root. These results reveal the specific regulation of auxin actions and cell responses by BR2/PGP1.

MicroRNA164 (miR164) plays a key role in leaf and flower development, lateral root initiation, and stress responses. However, little is known about the regulatory roles of miR164 during seed development, particularly in maize. The aim of this study was to discover the developmental function of miR164 in maize seed. Small RNA sequencing (sRNA-seq) was performed at two key stages. The results indicated that miR164 was down-regulated during maize seed development. In addition, degradome library sequencing and transient expression assays identified the target genes for miR164. Two microRNA (miRNA) pairs, miR164-NAM, ATAF, and CUC32 (NAC32) and miR164-NAC40, were isolated. The developmental function of miR164 was determined by analyzing the differentially expressed genes (DEGs) between the wild-type and miR164 transgenic lines using RNA sequencing (RNA-seq) and by screening the DEGs related to NAC32 and NAC40 via co-expression and transient expression analysis. These results identified two beta-expansin genes, EXPB14 and EXPB15, which were located downstream of the NAC32 and NAC40 genes. This study revealed, for the first time, a miR164-dependent regulatory pathway, miR164-NAC32/NAC40-EXPB14/EXPB15, which participates in maize seed expansion. These findings highlight the significance of miR164 in maize seed development, and can be used to explore the role of miRNA in seed development.

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.

Starch synthesis is an essential feature of crop filling, but knowledge of the molecular mechanisms regulating the expression of starch synthesis genes (SSGs) is currently limited to transcription factors (TFs). Here, we obtained transcriptome, small RNAome, and DNA methylome data from maize (Zea mays) endosperms during multiple developmental stages and established a regulatory network atlas of starch synthesis. Transcriptome analysis showed a sharp transition at 9-10 days after pollination, when genes involved in starch and sucrose metabolism are upregulated and starch accumulates rapidly. Expression pattern analysis established a comprehensive network between SSGs and TFs. During maize endosperm development, the miRNAs with preferential repression of the expression of TFs, particularly the TFs regulating SSG expression, were extensively downregulated. Specifically, ZmMYB138 and ZmMYB115 affected the transcriptional activities of Du1/Wx and Ae1/Bt2 genes at their respective promoter regions. Remarkably, the two TFs were negatively regulated by the copious expression of Zma-miR159k-3p at the post-transcriptional level. This suggests that miRNAs are important regulators of starch synthesis. Moreover, with the exclusion of the TFs, the expression of both SSGs and miRNAs was globally regulated by DNA methylation. Altogether, the present results (i) establish the regulatory functions of miRNAs and DNA methylation in starch synthesis and (ii) indicate that DNA methylation functions as a master switch.

Palmitic acid, the most common saturated free fatty acid, can lead to lipotoxicity and apoptosis when overloaded in non-fat cells. Palmitic acid accumulation can induce pancreatic &beta, cell dysfunction and cardiac myocyte apoptosis. Under various cellular stresses, the activation of p53 signaling can lead to cell cycle arrest, DNA repair, senescence, or apoptosis, depending on the severity/type of stress. Nonetheless, the precise role of p53 in lipotoxicity induced by palmitic acid is not clear. Here, our results show that palmitic acid induces p53 activation in a dose- and time-dependent manner. Furthermore, loss of p53 makes cells sensitive to palmitic acid-induced apoptosis. These results were demonstrated in human colon carcinoma cells (HCT116) and primary mouse embryo fibroblasts (MEF) through analysis of DNA fragmentation, flow cytometry, colony formation, and Western blots. In the HCT116 p53&minus, /&minus, cell line, palmitic acid induced greater reactive oxygen species formation compared to the p53+/+ cell line. The reactive oxygen species (ROS) scavengers N-acetyl cysteine (NAC) and reduced glutathione (GSH) partially attenuated apoptosis in the HCT116 p53&minus, cell line but had no obvious effect on the p53+/+ cell line. Furthermore, p53 induced the expression of its downstream target genes, p21 and Sesn2, in response to ROS induced by palmitic acid. Loss of p21 also leads to more palmitic acid-induced cell apoptosis in the HCT116 cell line compared with HCT116 p53+/+ and HCT116 p53&minus, In a mouse model of obesity, glucose tolerance test assays showed higher glucose levels in p53&minus, mice that received a high fat diet compared to wild type mice that received the same diet. There were no obvious differences between p53&minus, and p53+/+ mice that received a regular diet. We conclude that p53 may provide some protection against palmitic acid- induced apoptosis in cells by targeting its downstream genes in response to this stress.

BackgroundShort 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.ResultsHere, we report a maize dwarf mutant,dwarf2014(d2014), which displays shortened lower internodes. Map-based cloning revealed that thed2014gene is a novelbr2allele with a splicing variation, resulting in a higher expression ofBR2-T02instead of normalBR2-T01. Then, we found that the internode elongation ind2014/br2exhibited a pattern of inhibition-normality-inhibition (transient for the ear-internode), correspondingly, at the 6-leaf, 12-leaf and 14-leaf stages. Indeed,BR2encodes a P-glycoprotein1 (PGP1) protein that functions in auxin efflux, and our in situ hybridization assay showed thatBR2was mainly expressed in vascular bundles of the node and internode. Furthermore, significantly higher auxin concentration was detected in the stem apex ofd2014at 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 ofd2014.ConclusionsThese findings suggest that low auxin levels mediated by BR2/PGP1 in the intercalary meristem region are crucial for internode elongation.

Tassel architecture is an important trait in maize breeding and hybrid seed production. In this study, we investigated total tassel length (TTL) and tassel branch number (TBN) in 266 F2:3 families across six environments and in 301 recombinant inbred lines (RILs) across three environments, where all the plants were derived from a cross between 08-641 and Ye478. We compared the genetic architecture of the two traits across two generations through combined analysis. In total, 27 quantitative trait loci (QTLs) (15 in F2:3; 16 in RIL), two QTL × environment interactions (both in F2:3), 11 pairs of epistatic interactions (seven in F2:3; four in RIL) and four stable QTLs in both the F2:3 and RILs were detected. The RIL population had higher detection power than the F2:3 population. Nevertheless, QTL × environment interactions and epistatic interactions could be more easily detected in the F2:3 population than in the RILs. Overall, the QTL mapping results in the F2:3 and RILs were greatly influenced by genetic generations and environments. Finally, fine mapping for a novel and major QTL, qTTL-2-3 (bin 2.07), which accounted for over 8.49% of the phenotypic variation across different environments and generations, could be useful in marker-assisted breeding.

Maize is an important food crop and industrial material owing to its high starch content. However, the mechanism of starch synthesis is not fully elucidated, especially with regard to the expression and regulation of starch synthetic genes. The APETALA2/Ethylene Responsive Factor (AP2/ERF) family plays a crucial role in various biological processes via regulating gene expression. In this study, the ZmEREB94 gene was identified through co-expression analysis. Bioinformatics analysis confirmed that ZmEREB94 belongs to the AP2/ERF family. Expression pattern analysis showed that this protein is strongly expressed in the maize endosperm. A ZmEREB94-GFP fusion protein was localized in the nuclei of onion epidermal cells, and ZmEREB94 showed strong transcriptional activation activity, which indicated that this protein is a transcription factor. In addition, yeast-one hybrid assays and transient expression in maize endosperm showed that ZmEREB94 could directly bind to the ZmSSI promoter and indirectly regulate ZmSh2 and ZmGBSSI expression. Our results revealed that ZmEREB94 might act as a key regulator of starch synthesis in maize.

Sucrose acts as a signaling molecule for genes critical to starch biosynthesis in maize endosperm. Previously, we showed that sucrose could regulate starch biosynthesis in maize via transcription factors. To better understand the complex regulation of starch biosynthesis, the 10days after pollination endosperm from Zea mays L. B73 inbred line was collected and treated with sucrose for small RNA sequencing. The sequencing results revealed that 24 known miRNAs and 190 novel miRNAs were significantly differentially expressed in response to sucrose. In addition, most of target mRNAs were characterized as transcription factors, mainly including, MYB, ARF, NAC, AP2/ERF, WRKY, and GRAS, which play important roles in starch biosynthesis and seed development in maize endosperm. The expression profiles of miR398a/b and miR159b/j/k followed opposite expression trends to their target genes when analyzed by qPCR. In conclusion, these results show that sucrose regulates the expression of starch synthetic genes through miRNAs.

The erectophile plant architecture in maize is responsible for high plant density tolerance, yet the genetic basis for this relationship remains elusive, especially for how canopy architecture and plant height related traits at different positions respond to plant density. In this study, nine canopy traits and six plant height (PH) traits were evaluated across four environments under low plant density (57,000 plants/ha, LD) and high plant density (114,000 plants/ha, HD), using a set of 301 recombinant inbred lines originating from two foundation parents in China, the inbred lines YE478 and 08-641. In total, 176 quantitative trait loci (QTLs) for plant architecture related traits (94 only in LD, 44 only in HD and 38 under both densities) and 36 QTL clusters were detected via combined analysis. We identified 21 sharing QTL regions associated with plant height, leaf width and leaf angle at different positions. These results suggest that plant architecture-related traits were greatly influenced by density-specific and environment-specific alleles, and epistasis, QTL × environment interaction and QTL pleiotropy also play essential roles for plant architecture via complex interactions. Though PH-related traits, leaf widths and leaf angles at different positions could be partially affected by several common QTLs, there are still different genetic mechanisms of plant architecture response to plant density. Furthermore, elite line YE478 provided most of the favorable plant architecture alleles for high-density tolerance. Five QTL clusters containing six major QTLs, were useful for further studies of plant architecture and will provide helpful information for ideal plant type, high-density tolerance and marker-assisted selection.

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.

Additional file 7: Figure S5. The morphological characteristics of the stems of d2014 and WT.

Table S8. QTL × environment interactions for yield-related traits detected in the RILs under four environments and the IF2 population under three environments. a RIL, recombinant inbred lines; IF2, the immortalized F2. b AE indicates the additive by designed environment interaction effect. E1, E2, E3, and E4, represent 2014JH, 2015JH, 2016CZ, and 2016JH, respectively. c h2(AE, %) is the contribution rate of additive by environment interaction effect. d DE indicates the dominance by designed environment interaction effect. E1, E2, E3, and E4, represent 2014JH, 2015JH, 2016CZ, and 2016JH, respectively. e h2(DE, %) is the contribution rate of dominance by environment interaction effect. *, **, *** indicate significance at p

Table S5. Pearson’s correlation coefficients between phenotypic performance per se in IF2 population and mid-parent heterosis for those traits. *** Significant at p

Table S3. Pearson’s correlation coefficients among yield-related traits in the RILsa across four environments, the IF2c populations across three environments, and the mid-parent heterosis (MPH) dataset. * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. The absolute values of Pearson’s correlation coefficients (r2) with bold are equal to or larger than 0.5. EWPE, ear weight per ear; CWPE, cob weight per ear; ED, ear diameter; CD, cob diameter; EL, ear length; RN, row number; KNPR, kernel number per row; KWPE, kernel weight per row; RKP, rate of kernel production. a Pearson’s correlation coefficients (r2) in the first row for each trait are for the RILs. b Pearson’s correlation coefficients (r2) in the second row for each trait are for the IF2 population. c Pearson’s correlation coefficients (r2) in the third row for each trait are for mid-parent heterosis. (DOCX 1433 kb)

Figure S2. Phenotypic variance explained (PVE) by QTL and epistasis interaction (EPI, digenic interaction) effects for nine yield-related traits in the RILs, IF2 population and the MPH dataset. The total PVE for QTL and epistasis was estimated on ICIM-ADD and ICIM-EPI, respectively. ICIM, inclusive composite interval mapping. RIL, recombinant inbred line. IF2, immortalized F2. MPH, mid-parent heterosis. EWPE, ear weight per ear; CWPE, cob weight per ear; EL, ear length; ED, ear diameter; CD, cob diameter; RN, row number; KNPR, kernel number per ear; KWPE, kernel weight per ear; RKP, rate of kernel production. (PPTX 37 kb)

Table S1. List of abbreviations and definitions for the yield-related traits recorded at harvest in a sample of ten ears per plot. (DOCX 16 kb)

Figure S2. Phenotypic variance explained (PVE) by QTL and epistasis interaction (EPI, digenic interaction) effects for nine yield-related traits in the RILs, IF2 population and the MPH dataset. The total PVE for QTL and epistasis was estimated on ICIM-ADD and ICIM-EPI, respectively. ICIM, inclusive composite interval mapping. RIL, recombinant inbred line. IF2, immortalized F2. MPH, mid-parent heterosis. EWPE, ear weight per ear; CWPE, cob weight per ear; EL, ear length; ED, ear diameter; CD, cob diameter; RN, row number; KNPR, kernel number per ear; KWPE, kernel weight per ear; RKP, rate of kernel production. (PPTX 37 kb)

Table S5. Pearson’s correlation coefficients between phenotypic performance per se in IF2 population and mid-parent heterosis for those traits. *** Significant at p

Table S2. The performance of yield-related traits in the RILs, the IF2 population, and the MPH dataset across all environments. EWPE, ear weight per ear; CWPE, cob weight per ear; ED, ear diameter; CD, cob diameter; EL, ear length; RN, row number; KNPR, kernel number per row; KWPE, kernel weight per row; RKP, rate of kernel production. a F1, The cross of 08–641 × YE478. RIL, the recombinant inbred lines. b IF2 and MPH indicate the immortalized F2 population and mid-parent heterosis, respectively. c Mean ± SE represents mean across all environments ± standard error. d MPH (%) = (F1-(P1 + P2)/2)/((P1 + P2)/2)*100%. (DOCX 18 kb)

Additional file 3: Figure S1. The br2 allelism test for d2014.

Additional file 4: Figure S2. Comparison of certain internode length between d2014 and WT at the 12-leaf, 14-leaf, and 20-leaf stages.

Table S3. Pearson’s correlation coefficients among yield-related traits in the RILsa across four environments, the IF2c populations across three environments, and the mid-parent heterosis (MPH) dataset. * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. The absolute values of Pearson’s correlation coefficients (r2) with bold are equal to or larger than 0.5. EWPE, ear weight per ear; CWPE, cob weight per ear; ED, ear diameter; CD, cob diameter; EL, ear length; RN, row number; KNPR, kernel number per row; KWPE, kernel weight per row; RKP, rate of kernel production. a Pearson’s correlation coefficients (r2) in the first row for each trait are for the RILs. b Pearson’s correlation coefficients (r2) in the second row for each trait are for the IF2 population. c Pearson’s correlation coefficients (r2) in the third row for each trait are for mid-parent heterosis. (DOCX 1433 kb)

Additional file 2: Table S2. Plant height separation performance in F2 and BC1 populations.

Additional file 6: Figure S4. The analysis of protein domains of PGP1-T01 and PGP1-T02.

Table S2. The performance of yield-related traits in the RILs, the IF2 population, and the MPH dataset across all environments. EWPE, ear weight per ear; CWPE, cob weight per ear; ED, ear diameter; CD, cob diameter; EL, ear length; RN, row number; KNPR, kernel number per row; KWPE, kernel weight per row; RKP, rate of kernel production. a F1, The cross of 08–641 × YE478. RIL, the recombinant inbred lines. b IF2 and MPH indicate the immortalized F2 population and mid-parent heterosis, respectively. c Mean ± SE represents mean across all environments ± standard error. d MPH (%) = (F1-(P1 + P2)/2)/((P1 + P2)/2)*100%. (DOCX 18 kb)

Table S6. Pearson’s correlation coefficients between phenotypic variation and genome marker heterozygosity. a Correlation between performance per se in IF2 population and percentage of heterozygous loci. b Correlation between mid-parent heterosis and the percentage of heterozygous loci. ***, significant at p

Table S1. List of abbreviations and definitions for the yield-related traits recorded at harvest in a sample of ten ears per plot. (DOCX 16 kb)