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Starch is an important nutrient component of maize kernels, and starch granule size largely determines kernel waxiness, viscosity, and other physiochemical and processing properties. To explore the genetic basis of maize starch granule size, 266 tropical, subtropical, and temperate inbred lines were subjected to genome-wide association analyses with an array of 56,110 random single nucleotide polymorphisms (SNPs). In the present panel, the kernel starch granule size ranged from 7–15.8 µm long and 6.8–14.3 µm wide. Fourteen significant SNPs were identified as being associated with the length of starch granules and 9 with their width. One linkage disequilibrium block flanking both sides of a significant SNP was defined as a quantitative trait locus (QTL) interval, and seven QTLs were mapped for both granule length and width. A total of 79 and 88 candidate genes associated with starch length and width, respectively, were identified as being distributed on QTL genomic regions. Among these candidate genes, six with high scores were predicted to be associated with maize starch granule size. A candidate gene association analysis identified significant SNPs within genes GRMZM2G419655 and GRMZM2G511067, which could be used as functional markers in screening starch granule size for different commercial uses.

Inhibitors of cyclin-dependent kinases (ICKs) are key regulators of cyclin-dependent kinase activities and cell division. Herein, we identified eight ICKs in maize, which we named Zeama;ICKs (ZmICKs). Primary sequencing and phylogenetic analyses were used to divide the ZmICK family into two classes: group B and group C. Subcellular localization analysis of ZmICK:enhanced green fluorescent protein (eGFP) fusion constructs in tobacco leaf cells indicated that ZmICKs are principally nuclear. Co-localization analysis of the ZmICKs and maize A-type cyclin-dependent kinase (ZmCDKA) was also performed using enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) fusion constructs. The ZmICKs and ZmCDKA co-localized in the nucleus. Semi-quantitative RT-PCR analysis of the ZmICKs showed that they were expressed at different levels in all tissues examined and shared similar expression patterns with cell cycle-related genes. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that ZmICK1, ZmICK2, ZmICK3, and ZmICK4 interact with ZmCDKA1 and ZmCDKA3. Interestingly, ZmICK7 interacts with D-type cyclins. Transformed and expressed ZmCDKA1 and ZmICKs together in fission yeast revealed that ZmICK1, ZmICK3, and ZmICK4 can affect ZmCDKA1 function. Moreover, the C-group of ZmICKs could interact with ZmCDKA1 directly and affect ZmCDKA1 function, suggesting that C-group ZmICKs are important for cell division regulation.

Sucrose is not only the carbon source for starch synthesis, but also a signal molecule. Alone or in coordination with ABA, it can regulate the expression of genes involved in starch synthesis. To investigate the molecular mechanisms underlying this effect, maize endosperms were collected from Zea mays L. B73 inbred line 10 d after pollination and treated with sucrose, ABA, or sucrose plus ABA at 28 °C in the dark for 24 h. RNA-sequence analysis of the maize endosperm transcriptome revealed 47 candidate transcription factors among the differentially expressed genes. We therefore speculate that starch synthetic gene expression is regulated by transcription factors induced by the combination of sucrose and ABA. ZmEREB156, a candidate transcription factor, is induced by sucrose plus ABA and is involved in starch biosynthesis. The ZmEREB156-GFP-fused protein was localized in the nuclei of onion epidermal cells and ZmEREB156 protein possessed strong transcriptional activation activity. Promoter activity of the starch-related genes Zmsh2 and ZmSSIIIa increased after overexpression of ZmEREB156 in maize endosperm. ZmEREB156 could bind to the ZmSSIIIa promoter but not the Zmsh2 promoter in a yeast one-hybrid system. Thus, ZmEREB156 positively modulates starch biosynthetic gene ZmSSIIIa via the synergistic effect of sucrose and ABA.

R2R3-MYB proteins (2R-MYBs) are one of the main transcription factor families in higher plants. Since the evolutionary history of this gene family across the eukaryotic kingdom remains unknown, we performed a comparative analysis of 2R-MYBs from 50 major eukaryotic lineages, with particular emphasis on land plants. A total of 1548 candidates were identified among diverse taxonomic groups, which allowed for an updated classification of 73 highly conserved subfamilies, including many newly identified subfamilies. Our results revealed that the protein architectures, intron patterns and sequence characteristics were remarkably conserved in each subfamily. At least four subfamilies were derived from early land plants, 10 evolved from spermatophytes and 19 from angiosperms, demonstrating the diversity and preferential expansion of this gene family in land plants. Moreover, we determined that their remarkable expansion was mainly attributed to whole genome and segmental duplication, where duplicates were preferentially retained within certain subfamilies that shared three homologous intron patterns (a, b and c) even though up to 12 types of patterns existed. Through our integrated distributions, sequence characteristics and phylogenetic tree analyses, we confirm that 2R-MYBs are old and postulate that 3R-MYBs may be evolutionarily derived from 2R-MYBs via intragenic domain duplication.