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Maize, a monoecious crop, is a model plant for studying the regulation of floral organ development. In a previous study, we found that ZmMYB150 is significantly downregulated in sterile anthers by analyzing transcriptome data of the cytoplasmic male sterility (CMS) line C48-2 and its maintainer line 48-2. To elucidate the functional mechanism of ZmMYB150 in maize reproductive development, we cloned ZmMYB150, and sequence analysis showed it is a typical R2R3-MYB transcription factor (TF). Tissue-specific analysis revealed ZmMYB150 to be specifically highly in anthers. The subcellular localization in tobacco leaves proved that ZmMYB150 is located in the nucleus. Co-expression analysis identified 876 genes that may be coexpressed with ZmMYB150, and sequence alignment analysis showed that the coexpressed genes ZmMYB116 and ZmPIP5K6 are homologous to Arabidopsis male fertility-related genes. Furthermore, a yeast two-hybrid (Y2H) assay revealed that ZmMYB150 interacts with ZmPIP5K6. qRT-PCR analysis showed that ZmMYB150 is specifically highly expressed at the late stage of normal anther development, but no expression during the whole stages of sterile anther development. Tobacco transient expression system was used to confirm that ZmMYB150 can be regulated by miR159e-3p at the translational level. Our results lay a foundation for further understanding of the regulatory mechanism of MYB TFs in maize reproductive development.

Male sterility is widely utilized for hybrid seed production. In this study, two new found male sterile mutants SauS4 and SauS5 were obtained from space flighted seeds of maize inbred line RP125. Then, genetic analysis, molecular markers identification, and cytological observation were conducted to confirm their male sterile types. For genetic analysis, the above two male sterile mutants were continuously backcrossed with two maize inbred line 18Hong and RP125, and four stable male sterile lines SauS4(18Hong), SauS5(18Hong), SauS4(RP125), SauS5(RP125) were generated by six-generation backcross. Restoring and maintaining relationship analysis showed that both Hui313 and Zifeng1 didn’t rescue the male sterility SauS4(18Hong) and SauS5(18Hong). Using CMS mitochondria-specific primers for PCR detection suggested that only a 440 bp band unique to CMS-T type was amplified in SauS4(18Hong), SauS5(18Hong), SauS4(RP125), and SauS5(RP125). Sequencing results showed that these bands sequences were identical in DNA level which compared with T-urf13. Cytological observations showed that the main abortion stages of SauS4 and SauS5 were at the middle stage of uninucleate microspores under the two nuclear backgrounds of 18Hong and RP125, exhibiting the characteristics of sporophyte sterility. All the above results pointed out the two male sterile mutants SauS4 and SauS5 belonged to the CMS-T type. Interestingly, some mitochondrial genome difference between SauS4(RP125) and SauS5(RP125) were revealed by AFLP analysis.

C-type cytoplasmic male sterility (CMS-C), one of the three major CMS types in maize, has a promising application prospect in hybrid seed production. However, the complex genetic mechanism underlying the fertility restoration of CMS-C remains poorly understood. The maize inbred line A619 is one of the rare strong restorer lines carrying the restorer gene Rf4, but different fertility segregation ratios are found in several F2 populations derived from crosses between isocytoplasmic allonucleus CMS-C lines and A619. In the present study, the segregation ratios of fertile to sterile plants in the (CHuangzaosi × A619) F2 and BC1F1 populations (36.77:1 and 2.36:1, respectively) did not follow a typical monogenic model of inheritance, which suggested that some F2 and BC1F1 plants displayed restored fertility even without Rf4. To determine the hidden locus affecting fertility restoration, next-generation sequencing-based QTL-seq was performed with two specific extreme bulks consisting of 30 fertile and 30 sterile rf4rf4 individuals from the F2 population. A major QTL related to fertility restoration, designated qRf8-1, was detected on the long arm of chromosome 8 in A619. Subsequently, qRf8-1 was further validated and narrowed down to a 17.93-Mb genomic interval by insertion and deletion (InDel) and simple sequence repeat (SSR) marker-based traditional QTL mapping, explaining 12.59% (LOD = 25.06) of the phenotypic variation. Thus, using genetic analyses and molecular markers, we revealed another fertility restoration system acting in parallel with Rf4 in A619 that could rescue the male sterility of CHuangzaosi. This study not only expands the original fertility restoration system but also provides valuable insights into the complex genetic mechanisms underlying the fertility restoration of CMS-C.

Retrotransposon insertion in Brachytic2 generated a new incomplete recessive dwarf allele after spaceflight can moderately reduce plant height in heterozygous and potentially improve maize yield. Plant height and ear height are two important agronomic traits in maize breeding. In this study, two dwarf mutants short internode length1 (sil1) and short internode length2 (sil2) were obtained from two of 398 spaceflighted seeds of inbred line 18-599. The decrease in longitudinal cell number and cell length led to the shortened internodes of sil1 and sil2. A Ty1-copia LTR-retrotransposon, termed ZmRE-1, inserted in the fifth exon of Brachytic2 (Br2) was identified in sil1 and sil2 at exactly the same site, which indicated the transposition of ZmRE-1 probably correlated with the spaceflight. This new dwarf mutant allele was named as br2-sil in this study. The insertion of ZmRE-1 not only led to the loss of normal transcript of Br2 allele, but also reduced the transcript expression of br2-sil allele. Chop-qPCR displayed that the promoter region DNA methylation level of br2-sil allele in sil1 was higher than that of Br2 allele in WT-sil1. We speculated that the increased methylation level might downregulate the br2-sil expression. There was no difference in the seed-setting rate between sil1 and WT-sil1. Meanwhile, br2-sil could reduce plant and ear height effectively in Br2/br2-sil genotype without negative effects on grain yield. Therefore, the application of br2-sil in breeding has the potential to improve the grain yield per unit area through increasing the planting density.

Background The tricarboxylic acid (TCA) cycle is crucial for cellular energy metabolism and carbon skeleton supply. However, the detailed functions of the maize TCA cycle genes remain unclear. Results In this study, 91 TCA genes were identified in maize by a homology search, and they were distributed on 10 chromosomes and 1 contig. Phylogenetic results showed that almost all maize TCA genes could be classified into eight major clades according to their enzyme families. Sequence alignment revealed that several genes in the same subunit shared high protein sequence similarity. The results of cis-acting element analysis suggested that several TCA genes might be involved in signal transduction and plant growth. Expression profile analysis showed that many maize TCA cycle genes were expressed in specific tissues, and replicate genes always shared similar expression patterns. Moreover, qPCR analysis revealed that some TCA genes were highly expressed in the anthers at the microspore meiosis phase. In addition, we predicted the potential interaction networks among the maize TCA genes. Next, we cloned five TCA genes located on different TCA enzyme complexes, Zm00001d008244 (isocitrate dehydrogenase, IDH), Zm00001d017258 (succinyl-CoA synthetase, SCoAL), Zm00001d025258 (α-ketoglutarate dehydrogenase, αKGDH), Zm00001d027558 (aconitase, ACO) and Zm00001d044042 (malate dehydrogenase, MDH). Confocal observation showed that their protein products were mainly localized to the mitochondria; however, Zm00001d025258 and Zm00001d027558 were also distributed in the nucleus, and Zm00001d017258 and Zm00001d044042 were also located in other unknown positions in the cytoplasm. Through the bimolecular fluorescent complimentary (BiFC) method, it was determined that Zm00001d027558 and Zm00001d044042 could form homologous dimers, and both homologous dimers were mainly distributed in the mitochondria. However, no heterodimers were detected between these five genes. Finally, Arabidopsis lines overexpressing the above five genes were constructed, and those transgenic lines exhibited altered primary root length, salt tolerance, and fertility. Conclusion Sequence compositions, duplication patterns, phylogenetic relationships, cis-elements, expression patterns, and interaction networks were investigated for all maize TCA cycle genes. Five maize TCA genes were overexpressed in Arabidopsis, and they could alter primary root length, salt tolerance, and fertility. In conclusion, our findings may help to reveal the molecular function of the TCA genes in maize.

Maize is one of the most important crops worldwide, but it suffers from salt stress when grown in saline-alkaline soil. There is therefore an urgent need to improve maize salt tolerance and crop yield. In this study, the SsNHX1 gene of Suaeda salsa, which encodes a vacuolar membrane Na+/H+ antiporter, was transformed into the maize inbred line 18-599 by Agrobacterium-mediated transformation. Transgenic maize plants overexpressing the SsNHX1 gene showed less growth retardation when treated with an increasing NaCl gradient of up to 1%, indicating enhanced salt tolerance. The improved salt tolerance of transgenic plants was also demonstrated by a significantly elevated seed germination rate (79%) and a reduction in seminal root length inhibition. Moreover, transgenic plants under salt stress exhibited less physiological damage. SsNHX1-overexpressing transgenic maize accumulated more Na+ and K+ than wild-type (WT) plants particularly in the leaves, resulting in a higher ratio of K+/Na+ in the leaves under salt stress. This result revealed that the improved salt tolerance of SsNHX1-overexpressing transgenic maize plants was likely attributed to SsNHX1-mediated localization of Na+ to vacuoles and subsequent maintenance of the cytosolic ionic balance. In addition, SsNHX1 overexpression also improved the drought tolerance of the transgenic maize plants, as rehydrated transgenic plants were restored to normal growth while WT plants did not grow normally after dehydration treatment. Therefore, based on our engineering approach, SsNHX1 represents a promising candidate gene for improving the salt and drought tolerance of maize and other crops.

Maize male sterile mutant 40 (ms40) was obtained from the progeny of ethyl methanesulfonate (EMS) treated inbred line RP125. Genetic analysis showed that it was controlled by a single recessive nuclear gene. Cytological observation of anthers revealed that abnormal cuticles and disappearing of Ubisch bodies presented in ms40. Moreover, its tapetum exhibited delayed degradation and blocked the formation of abnormal microspore. Using map-based cloning, ms40 locus was located in a 282-kb interval on chromosome 4, five annotated genes were predicted within this region. PCR-based sequencing detected a single nonsynonymous SNP (G>A) which changed glycine (G) to arginine (A) in the seventh exon of Zm00001d053895, while no difference was found for the other four genes between ms40 and RP125. Zm00001d053895 encodes the bHLH transcription factor bHLH51 which protein was located at nuclear. Phylogenetic analysis presented that bHLH51 had the highest homology with Sb04g001650, a tapetum degeneration retardation (TDR) bHLH transcription factor in Sorghum bicolor. Co-expression analysis exposed a total of 1192 genes coexpressed with Zm00001d053895 in maize, 647 out of 1192 were anther-specific genes. In summary, these findings are conducive to the marker-assisted selection of ms40 in hybrid breeding and laid a foundation for further studies on the mechanisms of male fertility.

A novel genic male-sterile mutant ms40 was obtained from EMS treated RP125. The key candidate gene ZmbHLH51 located on chromosome 4 was identified by map-based cloning. This study further enriched the male sterile gene resources for both production applications and theoretical studies of abortion mechanisms. Maize male-sterile mutant 40 (ms40) was obtained from the progeny of the ethyl methanesulfonate (EMS) treated inbred line RP125. Genetic analysis indicated that the sterility was controlled by a single recessive nuclear gene. Cytological observation of anthers revealed that the cuticles of ms40 anthers were abnormal, and no Ubisch bodies were observed on the inner surface of ms40 anthers through scanning electron microscopy(SEM). Moreover, its tapetum exhibited delayed degradation and then blocked the formation of normal microspores. Using map-based cloning strategy, the ms40 locus was found to locate in a 282-kb interval on chromosome 4, and five annotated genes were predicted within this region. PCR-based sequencing detected a single non-synonymous SNP (G > A) that changed glycine (G) to arginine (A) in the seventh exon of Zm00001d053895, while no sequence difference between ms40 and RP125 was found for the other four genes. Zm00001d053895 encodes the bHLH transcription factor ZmbHLH51 which is localized in the nucleus. Phylogenetic analysis showed that ZmbHLH51 had the highest homology with Sb04g001650, a tapetum degeneration retardation (TDR) bHLH transcription factor in Sorghum bicolor. Co-expression analysis revealed a total of 1192 genes co-expressed with ZmbHLH51 in maize, 647 of which were anther-specific genes. qRT-PCR results suggested the expression levels of some known genes related to anther development were affected in ms40. In summary, these findings revealed the abortion characteristics of ms40 anthers and lay a foundation for further studies on the mechanisms of male fertility.

The callose synthase enzyme genes (Cals) generally plays an important role in resisting to environmental stresses as well as in regulating the microspore development of higher plant. However till now, few researches about ZmCals genes have been reported in maize. In this study, ten ZmCals genes were identified, and they are distributed on four chromosomes in maize. All ZmCals proteins contain Glucan-synthase-domain and Fks1-domain. RNA-seq data from public databases were analyzed and the result suggested that ZmCals involved in the development of various tissues, and a strong expression presented especially in young tissue. qRT-PCR analysis shown that most of ZmCals are highly expressed in root, stem and leaf at jointing stage (V6 stage) with maize inbred line B73. Seven out of 10 ZmCals genes display higher expression during maize anther development especially from stage 6 to stage 8b, the dynamic accumulation process of callose is also observed during these period with aniline blue staining. Above results indicated multiple ZmCals may participate in the deposition of callose in maize anther. Therefore, ZmCals are necessary not only for reproductive organ but also for nutritive organ during maize growth and development. This study lays certain foundation for further investigating the roles of the callose synthase enzymes genes in maize.

Aluminum (Al) toxicity is the primary limiting factor that affects crop yields in acid soil. However, the genes that contribute to the Al tolerance process in maize are still poorly understood. Previous studies have predicted that ZmAT6 is a novel protein which could be upregulated under Al stress condition. Here, we found that ZmAT6 is expressed in many tissues and organs and can be dramatically induced by Al in both the roots and shoots but particularly in the shoots. The overexpression of ZmAT6 in maize and Arabidopsis plants increased their root growth and reduced the accumulation of Al, suggesting the contribution of ZmAT6 to Al tolerance. Moreover, the ZmAT6 transgenic maize plants had lower contents of malondialdehyde and reactive oxygen species (ROS), but much higher proline content and even lower Evans blue absorption in the roots compared with the wild type. Furthermore, the activity of several enzymes of the antioxidant system, such as peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), increased in ZmAT6 transgenic maize plants, particularly SOD. Consistently, the expression of ZmSOD in transgenic maize was predominant upregulated by Al stress. Taken together, these findings revealed that ZmAT6 could at least partially confer enhanced tolerance to Al toxicity by scavenging ROS in maize.

Aluminum (Al) toxicity is the primary limiting factor that affects crop yields in acid soil. However, the genes that contribute to the Al tolerance process in maize are still poorly understood. Previous studies have predicted that ZmAT6 is a novel protein which could be upregulated under Al stress condition. Here, we found that

Background C-type cytoplasmic male sterility (CMS-C) is one of the three major types of cytoplasmic male sterility (CMS) in maize. Rf4 is a dominant restorer gene for CMS-C and has great value in hybrid maize breeding, but little information concerning its functional mechanism is known. Results To reveal the functional mechanism of Rf4, we developed a pair of maize near-isogenic lines (NILs) for the Rf4 locus, which included a NIL_rf4 male-sterile line and a NIL_Rf4 male fertility-restored line. Genetic analysis and molecular marker detection indicated that the male fertility of NIL_Rf4 was controlled by Rf4. Whole-genome sequencing demonstrated genomic differences between the two NILs was clustered in the Rf4 mapping region. Unmapped reads of NILs were further assembled to uncover Rf4 candidates. RNA-Seq was then performed for the developing anthers of the NILs to identify critical genes and pathways associated with fertility restoration. A total of 7125 differentially expressed genes (DEGs) were identified. These DEGs were significantly enriched in 242 Gene Ontology (GO) categories, wherein 100 DEGs were involved in pollen tube development, pollen tube growth, pollen development, and gametophyte development. Homology analysis revealed 198 male fertility-related DEGs, and pathway enrichment analysis revealed that 58 DEGs were enriched in cell energy metabolism processes involved in glycolysis, the pentose phosphate pathway, and pyruvate metabolism. By querying the Plant Reactome Pathway database, we found that 14 of the DEGs were involved in the mitochondrial tricarboxylic acid (TCA) cycle and that most of them belonged to the isocitrate dehydrogenase (IDH) and oxoglutarate dehydrogenase (OGDH) enzyme complexes. Transcriptome sequencing and real-time quantitative PCR (qPCR) showed that all the above TCA cycle-related genes were up-regulated in NIL_Rf4. The results of our subsequent enzyme-linked immunosorbent assay (ELISA) experiments pointed out that the contents of both the IDH and OGDH enzymes accumulated more in the spikelets of NIL_Rf4 than in those of NIL_rf4. Conclusion The present research provides valuable genomic resources for deep insight into the molecular mechanism underlying CMS-C male fertility restoration. Importantly, our results indicated that genes involved in energy metabolism, especially some mitochondrial TCA cycle-related genes, were associated with maize CMS-C male fertility restoration. Electronic supplementary material The online version of this article (10.1186/s12870-018-1409-z) contains supplementary material, which is available to authorized users.

Magnesium (Mg) is an essential mineral element for normal plant growth and development, and the CorA/MRS2/MGT-type Mg transporters play a significant role in maintaining Mg homeostasis in plants. In total, 12 maize CorA-like Mg2+ transporters have been identified, but none of them had been functionally characterized. Accordingly, we cloned and functionally characterized ZmMGT12 from the maize CorA-like gene family. ZmMGT12 exhibited the structure typical of Mg2+ transporters, i.e., two conserved TM domains and a GMN tripeptide motif. ZmMGT12, Arabidopsis AtMGT6, and rice OsMRS2-6 shared high protein sequence identity and thus clustered in the same phylogenetic branch, suggesting that they could be homologs. A functional complementation assay in the Salmonella typhimurium MM281 mutant indicated that ZmMGT12 possessed Mg2+ transport ability. ZmMGT12 was expressed in roots, stems, and leaves, with the highest expression in leaves. Moreover, ZmMGT12 expression was induced by light and exhibited a circadian expression pattern. In addition, the expression level of ZmMGT12 in leaf tissue was related to chlorophyll synthesis. Overexpression of ZmMGT12 in Arabidopsis caused no phenotypic change in transgenic plants, including in fresh shoot weight, chlorophyll content, shoot Mg2+ content, and chloroplast Mg2+ content. Together, these results suggest that ZmMGT12 is a Mg2+ transporter and may play a role in Mg transport into chloroplasts.

OsEAT1 (DTD), a stamen-specific bHLH transcription factor, is a master regulator in rice male reproduction. To elucidate its functional mechanism in maize male reproduction, the maize orthologue ZmbHLH122 of OsEAT1 was isolated in this study. Sequence analysis revealed that the ZmbHLH122 coding sequence consists of 1422 bp which encode a protein of 473 amino acids. Bioinformatic analysis confirmed that ZmbHLH122 belongs to the bHLH TF family. The subcellular localization of ZmbHLH122-eGFP in rice protoplasts showed that ZmbHLH122 is a nuclear-localized protein. Yeast one-hybrid assays demonstrated that ZmbHLH122 exhibits weak transactivation ability. Genome-wide coexpression analysis identified 751 potential collaborators of ZmbHLH122, most of which are highly expressed in maize anthers and tassels, and sequence alignment analysis revealed that 43 coexpressed genes are homologous to Arabidopsis male fertility-related genes. Expression pattern analysis confirmed that ZmbHLH122 and its potential collaborators Ms23 (ZmbHLH16), ZmbHLH51, ZmbHLH66 (Ms32) and ZmMYB74 are preferentially expressed in the maize male reproductive organs, especially those at the uninucleate and early binucleate stages. Yeast two-hybrid assays revealed that ZmMYB74 directly interacts with two maize stamen development regulators, Ms23 (ZmbHLH16) and ZmbHLH51, and indirectly collaborates with ZmbHLH122. Our study lays the foundation for further understanding of the ZmbHLH122 molecular function in maize male reproduction.

Aluminum (Al) toxicity is one of the primary factors limiting crop production in acid soils worldwide. The cell wall is the major target of Al toxicity owing to the presence of many Al binding sites. Previous studies have found that XTH, encoding xyloglucan endohydrolase (XEH) and xyloglucan endotransglucosylase (XET), could participate in cell wall extension and affect the binding ability of the cell wall to Al by impeding the activities of these two enzymes. In this study, we found that ZmXTH, an XTH gene in maize, was involved in Al detoxification. The Al-induced up-regulation of ZmXTH occurred in the roots, prominently in the root tips. Additionally, the expression of ZmXTH was specifically induced by Al3+ but no other divalent or trivalent cations. Compared with the wild-type Arabidopsis, ZmXTH overexpressing plants grew more healthy and had decreased Al content in their root and root cell wall after Al stress. Overall, the results suggest that ZmXTH could confer the Al tolerance of transgenic Arabidopsis plants by reducing the Al accumulation in their roots and cell walls.

Chalcone synthases (CHS, EC 2.3.1.74) are key enzymes that catalyze the first committed step in flavonoid biosynthesis. In this study, we isolated a chalcone synthase, named NtCHS6, from Nicotiana tabacum. This synthase shared high homology with the NSCHSL (Y14507) gene and contained most of the conserved active sites that are in CHS proteins. The phylogenetic analysis suggested that NtCHS6 protein shared a large genetic distance with other Solanaceae CHS proteins and was the most closely-related to an uncharacterized CHS from Solanum lycopersicum. The expression analysis indicated that NtCHS6 was abundantly expressed in leaves, especially in mature leaves. By scrutinizing its upstream promoter sequences, multiple cis-regulatory elements involved in light and drought responsive were detected. Furthermore, NtCHS6 expression decreased significantly under dark treatment and increased significantly under drought stress. Our results suggested that NtCHS6 expression exhibited both light responsiveness and drought responsiveness, and might play important roles in ultraviolet protection and drought tolerance.

Natural antisense transcripts (NATs) are a prominent and complex class of regulatory RNAs. Using strand-specific RNA sequencing, we identified 1769 sense and antisense transcript pairs (NAT pairs) in two maize inbreds with different sensitivity to drought, as well as in two derivative recombination inbred lines (RILs). A significantly higher proportion of NATs relative to non-NATs are specifically expressed under water stress (WS). Surprisingly, expression of sense and antisense transcripts produced by NAT pairs is significantly correlated, particularly under WS. We found an unexpected large proportion of NATs with protein coding potential, as estimated by ribosome release scores. Small RNAs significantly accumulate within NAT pairs, with 21 nt smRNA particularly enriched in overlapping regions of these pairs of genes. The abundance of these smRNAs is significantly altered in the leafbladeless1 mutant, suggesting that these genes may be regulated by the tasiRNA pathway. Further, NATs are significantly hypomethylated and include fewer transposable element sequences relative to non-NAT genes. NAT gene regions also exhibit higher levels of H3K36me3, H3K9ac, and H3K4me3, but lower levels of H3K27me3, indicating that NAT gene pairs generally exhibit an open chromatin configuration. Finally, NAT pairs in 368 diverse maize inbreds and 19 segregating populations were specifically enriched for polymorphisms associated with drought tolerance. Taken together, the data highlight the potential impact of that small RNAs and histone modifications have in regulation of NAT expression, and the significance of NATs in response to WS.

Gro/Tup1 proteins act as negative transcriptional regulators and play crucial roles in many growth and developmental processes in a wide range of organisms. However, our understanding of Gro/Tup1 protein functions in plants is confined to the model plant Arabidopsis. Here, 11 Gro/Tup1 genes, which were characterized by the typical LisH and WD40 repeat domains, were identified in maize through a genome-wide survey. A phylogenetic analysis revealed that maize Gro/Tup1 proteins could be divided into three subfamilies, in which members shared similar protein and gene structures. The predicted maize Gro/Tup1 genes were distributed on seven chromosomes and segmental duplication contributed to their expansion. Many predicted cis-elements associated with hormones, biotic- or abioticstress responses, meristem and seed development, and circadian rhythms, were found in their putative promoter regions. A potential associated protein analysis identified a large number of candidates, including transcription factors, chromatin-modifying enzymes, protein kinases, and ubiquitinconjugating enzymes. An expression profile derived from the RNA-seq data indicated that Gro/Tup1 genes in maize were widely expressed in various organs and tissues. Quantitative real-time PCR revealed that these genes responded to at least one hormone or abiotic stress, either in roots or in shoots. Our study provides useful information on the Gro/Tup1 genes in maize and will facilitate the further functional validation of these genes in growth and development, hormone responses, and biotic- or abiotic-stress resistance.

A novel male-sterile maize mutant male sterility 39 (ms39) was obtained from offspring of the commercial hybrid Chuandan No. 9 that had been carried into outer space. A previous study demonstrated that ms39 is controlled by a single recessive nuclear gene, located on the long arm of chromosome 3. Here, we used 1073 mutant individuals derived from the (ms39 × Mo17) F2 population and sequentially developed new primers to identify markers supporting the fine mapping of ms39. A 365-kb region on chromosome 3 flanked by markers L8 and M30 at a genetic distance of 0.18 and 0.47 cM, respectively, was identified. According to the reference sequence of ZmB73_Ref-Gen_v4, 12 candidate genes were identified within the 365-kb mapping region. Based on cloning and sequence BLAST analysis of the 12 candidate genes, a four-base-pair deletion was found within the exon of Zm00001d043909, which encoded callose synthase12. This four-base-pair deletion resulted in a frameshift mutation in ms39, leading to the earlier termination of the coding protein, and ultimately caused abnormal performance of the callose synthase. Additionally, cytological observation was conducted on a sister cross population (ms39/ms39 × ms39/Ms39). These observations showed that the tapetum cells of the ms39 mutant appeared abnormal from the dyad stage, and aborted microspores were observed during pollen development. These results lay the foundation for the cloning of ms39 and exploration of the molecular mechanism underlying aborted pollen development in ms39 maize.

C-type cytoplasmic male sterile (CMS-C) line plays an important role in hybrid seed production in maize. However, mechanisms of pollen abortion and fertility restoration remain unclear. This study aimed to investigate the mechanisms of CMS-C pollen abortion and fertility restoration, particularly based on epigenetics, and to understand the relationship between male fertility performance and DNA methylation status. Methylation-sensitive amplification polymorphism (MSAP) technique was conducted to analyze DNA methylation levels and patterns of tassels at pollen-mother cell, tetrad, mononuclear, and binuclear stages among four half-sib hybrids and their parents with different fertility. Results showed that DNA methylation levels in fertility restored hybrids were higher than those in sterility maintained hybrids. Nine CCGG sites, which displayed methylation polymorphism between male-fertile and male-sterile hybrids, were screened. To validate MSAP results, we performed methylation-sensitive PCR (MS-PCR) and found consistent observations in cloned methylation sites. Interestingly, a specific site named 16–1 was discovered in the region of Rf5 gene, which is one of the restorer genes of CMS-C. Thus, DNA methylation may participate in regulating the expression of fertility restorer genes of maize CMS-C.

Although the root system is indispensable for absorption of nutrients and water, it is poorly studied in maize owing to the difficulties of direct measurement of roots. Here, 103 maize lines were used to compare root architectures under well-watered and water-stressed conditions. Significant genetic variation, with medium to high heritability and significant correlations, was observed for root traits. Total root length (TRL) and total root surface area (TSA) had high phenotypical diversity, and TRL was positively correlated with TSA, root volume, and root forks. The first two principal components explained 94.01% and 91.15% of total root variation in well-watered and water-stressed conditions, respectively. Thus, TRL and TSA, major contributors to root variation, can be used as favorable selection criteria at the seedling stage. We found that stiff stalk and non-stiff stalk groups (temperate backgrounds) showed relatively higher mean values for root morphological diversity than the TST group (tropical/subtropical background). Of the tested lines, 7, 42, 45, and 9 were classified as drought sensitive, moderately sensitive, moderately drought tolerant, and highly drought tolerant, respectively. Seven of the 9 extremely drought tolerant lines were from the TST group, suggesting that TST germplasms harbor valuable genetic resources for drought tolerance that could be used in breeding to improve abiotic stress tolerance in maize.

Abstract Basic helix-loop-helix (bHLH) transcription factors play key roles in plant male reproduction. More than 14 bHLH proteins related to pollen development have been cloned from rice andArabidopsis. However, little is known about the role of the bHLH family in maize microspore development. In this study, the bHLH transcription factor ZmbHLH16 was cloned. ZmbHLH16 shares high similarity with the OsTIP2 (OsbHLH142) protein, a master regulator of the developmental coordination of male reproduction in rice. Expression characterization analysis showed that ZmbHLH16 is preferentially expressed in male reproductive organs and is located in the nucleus. Through nucleotide variation analysis, 36 polymorphic sites in ZmbHLH16, including 23 SNPs and 13 InDels, were detected among 78 maize inbred lines. Neutrality tests and linkage disequilibrium analysis showed that ZmbHLH16 experienced no significant evolutionary pressure. A yeast one-hybrid experiment showed that the first 80 residues in the N-terminus of ZmbHLH16 had transactivation activity, whereas the full length did not. To identify potential ZmbHLH16 interactors, 395 genes that shared similar expression patterns in a genome-wide search were obtained through coexpression analysis. Among these genes, the transcription factor ZmbHLH51 had an expression pattern and subcellular localization similar to those of ZmbHLH16. The interaction between ZmbHLH51 and ZmbHLH16 was verified in yeast cells. In addition to the typical bHLH domain, other regions of ZmbHLH16 were necessary and adequate for its heterodimerization with ZmbHLH51. Our results contribute to a solid foundation for further understanding the functions and mechanisms of ZmbHLH16.

Although C-type cytoplasmic male sterility (CMS-C) is one of the most attractive tools for maize hybrid seed production, the detailed regulation network of the male sterility remains unclear. In order to identify the CMS-C sterility associated genes and/or pathways, the comparison of the transcriptomes between the CMS-C line C48-2 and its isonuclear-alloplasmic maintainer line N48-2 at pollen mother cell stage (PS), an early development stage of microspore, and mononuclear stage (MS), an abortive stage of microspore, were analyzed. 2,069 differentially expressed genes (DEGs) between the two stages were detected and thought to be essential for the spikelet development of N48-2. 453 of the 2,069 DEGs were differentially expressed at MS stage between the two lines and thought to be participated in the process or the causes of microspore abortion. Among the 453 DEGs, 385 (84.99%) genes were down-regulated and only 68 (15.01%) genes were up-regulated in C48-2 at MS stage. The dramatic decreased expression of the four DEGs encoding MYB transcription factors and the DEGs involved in “polyamine metabolic process”, “Cutin, suberine and wax biosynthesis”, “Fatty acid elongation”, “Biosynthesis of unsaturated fatty acids” and “Proline metabolism” might play an important role in the sterility of C48-2. This study will point out some directions for detailed molecular analysis and better understanding of sterility of CMS-C in maize.

ZmMGT10 was specifically expressed in maize roots and induced by a deficiency of magnesium. Overexpression of ZmMGT10 restored growth deficiency of the Salmonella typhimurium MM281 strain and enhanced the tolerance in Arabidopsis to stress induced by low magnesium levels by increasing uptake of Mg2+ via roots. CorA/MRS2/MGT-type Mg2+ transporters play a significant role in maintaining magnesium (Mg) homeostasis in plants. Although the maize CorA/MRS2/MGT family comprises of 12 members, currently no member has been functionally characterized. Here, we report the isolation and functional characterization of ZmMGT10 from the maize MRS2/MGT gene family. ZmMGT10 has a typical structure feature which includes two conserved TMs near the C-terminal end and an altered AMN tripeptide motif. The high sequence similarity and close phylogenetic relationship indicates that ZmMGT10 is probably the counterpart of Arabidopsis AtMGT6. The complementation of the Salmonella typhimurium mutated MM281 strain indicates that ZmMGT10 possesses the ability to transport Mg2+. ZmMGT10 was specifically expressed in the plant roots and it can be stimulated by a deficiency of Mg. Transgenic Arabidopsis plants which overexpressed ZmMGT10 grew more vigorously than wild-type plants under low Mg conditions, exhibited by longer root length, higher plant fresh weight and chlorophyll content, suggesting ZmMGT10 was essential for plant growth and development under low Mg conditions. Further investigations found that high accumulation of Mg2+ occurred in transgenic plants attributed to improved Mg2+ uptake and thereby enhanced tolerance to Mg deficiency. Results from this investigation illustrate that ZmMGT10 is a Mg transporter of maize which can enhance the tolerance to Mg deficient conditions by improving Mg2+ uptake in the transgenic plants of Arabidopsis.

The transcription factor ZmbHLH16, the maize ortholog of OsTIP2 (OsbHLH142), was isolated in the present study. Tissue expression analysis showed that ZmbHLH16 is preferentially expressed in male reproductive organs. Subcellular location analysis of ZmbHLH16 via rice protoplast indicated that it is located in the nucleus. Through nucleotide variation analysis, 36 polymorphic sites in ZmbHLH16, including 23 single nucleotide polymorphisms and 13 InDels, were detected among 78 maize inbred lines. Neutrality tests and linkage disequilibrium analysis showed that ZmbHLH16 experienced no significant evolutionary pressure. Yeast one-hybrid experiment showed that the first 80 residues in the N-terminus of ZmbHLH16 had transactivation activity, whereas the full length did not. Genome-wide coexpression analysis showed that 395 genes were coexpressed with ZmbHLH16. Among these genes, the transcription factor ZmbHLH51 had similar expression pattern and identical subcellular localization to those of ZmbHLH16. Subsequently, the interaction between ZmbHLH51 and ZmbHLH16 was verified by yeast two-hybrid experiment. Through yeast two-hybrid analysis of series truncated ZmbHLH16 fragments, we found not only the typical bHLH domain [175-221 amino acids (a.a.)], but also that the 81-160 a.a. and 241-365 a.a. of ZmbHLH16 could interact with ZmbHLH51. All these results lay the foundation for further understanding the functions of ZmbHLH16., Summary: ZmbHLH16, the maize ortholog of OsTIP2 (OsbHLH142), its subcellular location, molecular evolution, transcriptional activity domains and heterodimerization domains with ZmbHLH51 are analyzed.

Phosphorus (P) deficiency is one of the major limiting factors in maize production in many developing countries. This experiment was conducted to evaluate multiple low-P tolerance criteria and identify the suitable maize germplasm for our future low-P tolerance breeding. A total of 456 diverse maize inbreds were evaluated for low-P tolerance at seedling stage using four shoot-related traits and six root-related traits measured under applied phosphorus (AP) and non-applied phosphorus (NAP) conditions. Analysis of variance revealed significant genetic variation among genotypes for all tested traits. Medium-to-high heritability estimates were obtained for most traits. Total dry weight (DW) was highly inheritable while the widely used root/shoot ratio had only an intermediate level of heritability. Based on the synthetic index (SI), the tested inbreds were classified into three groups representing low, moderate and high tolerance to low-P stress. Regression model built based on selection criteria for low-P tolerance explained 67.8 and 76.8 % of variation for DW under NAP and AP conditions, respectively. Using low-P tolerance index for DW and SI as selection criteria, 23 and 109 maize inbreds were identified as germplasm resources that were extremely tolerant and sensitive to low-P stress, respectively, which could be further used for genetic improvement of low-P tolerance.

In the present study, a mutated chimericorf364, namedorf366-c, was identified in a maize C type cytoplasmic male sterile line (CMS-C) by PCR and RT-PCR. Four base pair adjacent base substitution mutations (CAAA to TTTT) and eight bp insertion mutations were found inorf366-ccompared withorf364. ORF366-C was predicted to contain one membrane-spanning domain using TMHMM online software. Real-time quantitative PCR analysis showed thatorf366-cwas upregulated in the CMS-C line in comparison to its maintainer line at the uninucleate stage. The protokaryotic protein expression oforf366-cinEscherichia colishowed that ORF366-C could be a cytotoxic protein. All the results indicated thatorf366-cmay be associated with maize CMS-C.

Vitamin A deficiency has become a worldwide problem. Biofortified foods can potentially be an inexpensive, locally adaptable, and long-term solution to dietary-nutrient deficiency. In order to improve the β-carotene content in maize grain by breeding and minimize vitamin A deficiency, a complete diallel cross was designed with eight inbred lines of maize, and 64 combinations were obtained in this study. The experimental combinations were planted in Yunnan and Sichuan provinces, respectively, with a random complete block design. The β-carotene contents in the grains of the experimental materials were analyzed by high-performance liquid chromatography. Among the tested materials, the effect difference of general combining ability of the β-carotene content was significant; however, the effect difference of the special combining ability and the reciprocal effect were not significant. The β-carotene content of maize grain was not influenced significantly by the cross and the reciprocal cross. There was a significant correlation about the β-carotene content in the maize grains between the F1 and their parents. The combinations with high β-carotene content were obviously influenced by the environment, and the mean value of β-carotene content for the experimental materials planted in Ya'an of Sichuan was higher than that planted in Yuanjiang of Yunnan, with the results being significant at the 0.01 level.

High-temperature (HT) stress is a major environmental stress that limits cotton growth, metabolism, and yield worldwide. The identification and characterization of thermotolerance is restricted by the plant growth environment and growth stage. In this study, four genotypes of upland cotton (Gossypium hirsutum L.) with known field thermotolerance were evaluated under normal and HTs at the seedlings stage in a growth cabinet with 11 physiological, biochemical, and phenotypic assays. Consistent with previous field observations, the thermotolerance could be identified by genotype differences at the seedling stage under HT in a growth cabinet. Comparative transcriptome analysis was performed on seedlings of two contrasting cotton genotypes after 4 and 8 h of HT exposure. Gene ontology analysis combined with BLAST annotations revealed a large number of HT-induced differentially expressed genes (4,698) that either exhibited higher expression levels in the heat-tolerant genotype (Nan Dan Ba Di Da Hua) compared with the heat-sensitive genotype (Earlistaple 7), or were differentially expressed only in Nan Dan Ba Di Da Hua. These genes encoded mainly protein kinases, transcription factors, and heat-shock proteins, which were considered to play key roles in thermotolerance in upland cotton. Two heat-shock transcription factor genes (homologs of AtHsfA3, AtHsfC1) and AP2/EREBP family genes (homologs of AtERF20, AtERF026, AtERF053, and AtERF113) were identified as possible key regulators of thermotolerance in cotton. Some of the differentially expressed genes were validated by quantitative real-time PCR analysis. Our findings provide candidate genes that could be used to improve thermotolerance in cotton cultivars.

The control of flowering is not only important for reproduction, but also plays a key role in the processes of domestication and adaptation. To reveal the genetic architecture for flowering time and photoperiod sensitivity, a comprehensive evaluation of the relevant literature was performed and followed by meta analysis. A total of 25 synthetic consensus quantitative trait loci (QTL) and four hot-spot genomic regions were identified for photoperiod sensitivity including 11 genes related to photoperiod response or flower morphogenesis and development. Besides, a comparative analysis of the QTL for flowering time and photoperiod sensitivity highlighted the regions containing shared and unique QTL for the two traits. Candidate genes associated with maize flowering were identified through integrated analysis of the homologous genes for flowering time in plants and the consensus QTL regions for photoperiod sensitivity in maize (Zea mays L.). Our results suggest that the combination of literature review, meta-analysis and homologous blast is an efficient approach to identify new candidate genes and create a global view of the genetic architecture for maize photoperiodic flowering. Sequences of candidate genes can be used to develop molecular markers for various models of marker-assisted selection, such as marker-assisted recurrent selection and genomic selection that can contribute significantly to crop environmental adaptation.

DNA methylation plays an important role in gene expression regulation during biological development in plants. This study adopted methylation sensitive amplification polymorphism (MSAP) to compare the levels and patterns of cytosine methylation at CCGG sites in maize genome. The tissues assayed included seedlings and tassels of C-type cytoplasmic male sterility (C Huang Zao Si, C 48-2) and its maintainer lines. For each tissue, both C Huang Zao Si and C 48-2 were more methylated than their corresponding maintainers not only on MSAP ratios, but also on the full methylation levels. In different nuclear backgrounds, the two tissues were more methylated in Huang Zao Si than in 48-2, although the two lines shared the same cytoplasm. Full methylation of internal cytosine was the dominant type in the maize genome. In addition, four different classes of methylation patterns were identified in tassels between C-CMS lines and their maintainer lines; these were specific-methylation, demethylation, hypo-methylation, and hyper-methylation. The results obtained demonstrated the power of the MSAP technique for large-scale DNA methylation detection in the maize genome, and suggested the possible association between DNA methylation polymorphism and C-type cytoplasmic male sterility.

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants and has been applied in the commercial production of hybrid seeds. Two CMS lines A1 and A2 of maize were obtained previously by a transgenic experiment. In this study, we conducted cytological observation of developmental microspores with CMS line A1, A2 and their maintainer line (18 red) using paraffin section technology. We also analyzed DNA methylation levels at different developmental stages using high performance liquid chromatography (HPLC). Our results showed that the pollen abortion of A1 and A2 mainly happened from the tetrad stage to the middle of mononuclear stage. Another abortive phenomenon found in CMS line A2 occurred at the pollen mother cell stage. The DNA methylation level of leaf increased rapidly from the seedling stage to the shooting stage in 18 red, while it remained constant in A1 and A2. For the tassel, the DNA methylation levels in 18 red increased gradually during the anther development, while a peak of DNA methylation level occurred in A1 and A2 at the tetrad stage, corresponding to the abortion period of microspore. This result suggested that the level of DNA methylation in the tassels is associated with the pollen abortion characteristics in CMS lines. In summary, our results implied a connection between pollen abortion and epigenetic regulation in maize CMS.

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful and widely used technique for the measurement of gene expression. Reference genes, which serve as endogenous controls ensure that the results are accurate and reproducible, are vital for data normalization. To bolster the literature on reference gene selection in maize, ten candidate reference genes, including eight traditionally used internal control genes and two potential candidate genes from our microarray datasets, were evaluated for expression level in maize across abiotic stresses (cold, heat, salinity, and PEG), phytohormone treatments (abscisic acid, salicylic acid, jasmonic acid, ethylene, and gibberellins), and different tissue types. Three analytical software packages, geNorm, NormFinder, and Bestkeeper, were used to assess the stability of reference gene expression. The results revealed that elongation factor 1 alpha (EF1α), tubulin beta (β-TUB), cyclophilin (CYP), and eukaryotic initiation factor 4A (EIF4A) were the most reliable reference genes for overall gene expression normalization in maize, while GRP (Glycine-rich RNA-binding protein), GLU1(beta-glucosidase), and UBQ9 (ubiquitin 9) were the least stable and most unsuitable genes. In addition, the suitability of EF1α, β-TUB, and their combination as reference genes was confirmed by validating the expression of WRKY50 in various samples. The current study indicates the appropriate reference genes for the urgent requirement of gene expression normalization in maize across certain abiotic stresses, hormones, and tissue types.

Simple sequence repeats (SSRs) have been widely used in maize genetics and breeding, because they are co-dominant, easy to score, and highly abundant. In this study, we used whole-genome sequences from 16 maize inbreds and 1 wild relative to determine SSR abundance and to develop a set of high-density polymorphic SSR markers. A total of 264 658 SSRs were identified across the 17 genomes, with an average of 135 693 SSRs per genome. Marker density was one SSR every of 15.48 kb. (C/G)n, (AT)n, (CAG/CTG)n, and (AAAT/ATTT)n were the most frequent motifs for mono, di-, tri-, and tetra-nucleotide SSRs, respectively. SSRs were most abundant in intergenic region and least frequent in untranslated regions, as revealed by comparing SSR distributions of three representative resequenced genomes. Comparing SSR sequences and e-polymerase chain reaction analysis among the 17 tested genomes created a new database, including 111 887 SSRs, that could be develop as polymorphic markers in silico. Among these markers, 58.00, 26.09, 7.20, 3.00, 3.93, and 1.78% of them had mono, di-, tri-, tetra-, penta-, and hexa-nucleotide motifs, respectively. Polymorphic information content for 35 573 polymorphic SSRs out of 111 887 loci varied from 0.05 to 0.83, with an average of 0.31 in the 17 tested genomes. Experimental validation of polymorphic SSR markers showed that over 70% of the primer pairs could generate the target bands with length polymorphism, and these markers would be very powerful when they are used for genetic populations derived from various types of maize germplasms that were sampled for this study.