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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.

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.

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.

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.

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.

The charging and etching effects of hydride and fluoride gases in plasma immersion ion implantation (PIII) doping experiments have been investigated. Both phenomena can affect implant profile and dose, alter device structure, and degrade device reliability. In addition, charge accumulation significantly reduces implant energy. Helium is a good choice as a diluting gas for PIII doping processes due to its low ionization rate, inertness, and minimal etching and charging effects.

A general relation between the implanted dose and the processing time for plasma immersion ion implantation (PHI) can be established through the dynamic sheath model. In practice, etching and charging effects have to be taken into account in PIII dose estimation.Plasma immersion ion implantation (PII) has been tested in fabrication of semiconductor devices with shallow junctions and in hydrogénation of poly-Si thin film transistors (TFT). PIII doping is more suitable than conventional implantation for such applications because of its high dose rate at lower energy. Since the dose rate in PIII does not depend on the area being treated, the effective current will be higher if a larger implanted area is involved. However, the relation between dose and time is not always straightforward. During PIII processing possible etching and charging will affect the total accumulated doses. This paper presents a model for each which allows a proper compensation to be performed.

The charging effects of plasma immersion ion implantation (PIII) doping experiments have been investigated using a dynamic sheath model and PDP1 plasma simulation code. When the target has a dielectric film, charge accumulation during PIII can have a profound impact on doping results. Under certain process conditions, it can significantly reduce implant energy and dose and thereby alter the implant profile. In addition, it may degrade device reliability, especially for ultralarge‐scale integrated circuit devices. In order to minimize charging effects, shorter pulse widths along with moderate values of plasma density and pulse potential should be used.