Genome-Wide Association Study Identifies Quantitative Trait Loci and Candidate Genes Involved in Deep-Sowing Tolerance in Maize (Zea mays L.).

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.

DEK219 and HSF17 Collaboratively Regulate the Kernel Length in Maize.

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.

Serine 31 Phosphorylation-Driven Regulation of AGPase Activity: Potential Implications for Enhanced Starch Yields in Crops.

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.

Identification of Morphogenesis-Related NDR Kinase Signaling Network and Its Regulation on Cold Tolerance in Maize.

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.

Epi-Brassinolide Regulates ZmC4 NADP-ME Expression through the Transcription Factors ZmbHLH157 and ZmNF-YC2.

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.

Pleiotropic ZmICE1 Is an Important Transcriptional Regulator of Maize Endosperm Starch Biosynthesis.

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 novel ZmTCP7 transcription factor targets AGPase-encoding gene ZmBt2 to regulate storage starch accumulation in maize.

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.

Genome-Wide Characterization and Phylogenetic Analysis of GSK Genes in Maize and Elucidation of Their General Role in Interaction with BZR1.

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.

Comparative Study of Starch Phosphorylase Genes and Encoded Proteins in Various Monocots and Dicots with Emphasis on 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.

Isolation and Transfection of Maize Endosperm Protoplasts.

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.

Dissection of the genetic architecture of peduncle vascular bundle-related traits in maize by a genome-wide association study.

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.

Molecular Functions and Pathways of Plastidial Starch Phosphorylase (PHO1) in Starch Metabolism: Current and Future Perspectives.

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.

Genome-wide identification of ZmSnRK2 genes and functional analysis of ZmSnRK2.10 in ABA signaling pathway in maize (Zea mays L).

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.

Gibberellin induced transcription factor bZIP53 regulates CesA1 expression in maize kernels.

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.

Identification of rice (Oryza sativa L.) genes involved in sheath blight resistance via a genome-wide association study.

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.

Coordinated regulation of starch synthesis in maize endosperm by microRNAs and DNA methylation.

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.

Optimization of isolation and transfection conditions of maize endosperm protoplasts.

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.

Identification of quantitative trait loci for kernel-related traits and the heterosis for these traits in maize (Zea mays L.).

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.

Loss of p53 Sensitizes Cells to Palmitic Acid-Induced Apoptosis by Reactive Oxygen Species Accumulation.

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 ß-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-/- 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-/- 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-/-. In a mouse model of obesity, glucose tolerance test assays showed higher glucose levels in p53-/- mice that received a high fat diet compared to wild type mice that received the same diet. There were no obvious differences between p53-/- 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.

Maize brachytic2 (br2) suppresses the elongation of lower internodes for excessive auxin accumulation in the intercalary meristem region.

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.