Heat shock transcription factors (HSFs) play important roles in plant growth, development, and stress responses. However, the function of these transcription factors in abiotic stress responses in maize (Zea mays) remains largely unknown. In this study, we characterized a novel HSF transcription factor gene, ZmHsf08, from maize. ZmHsf08 was highly homologous to SbHsfB1, BdHsfB1, and OsHsfB1, and has no transcriptional activation activity. The expression profiles demonstrated that ZmHsf08 was differentially expressed in various organs of maize and was induced by salt, drought, and abscisic acid (ABA) treatment. Moreover, the overexpression of ZmHsf08 in maize resulted in enhanced sensitivity to salt and drought stresses, displaying lower survival rates, higher reactive oxygen species (ROS) levels, and increased malondialdehyde (MDA) contents compared with wild-type (WT) plants. Furthermore, RT-qPCR analyses revealed that ZmHsf08 negatively regulates a number of stress/ABA-responsive genes under salt and drought stress conditions. Collectively, these results indicate that ZmHsf08 plays a negative role in response to salt and drought stresses in maize.

TLC (TRAM/LAG/CRN8) proteins play important roles in ceramide metabolism and mycotoxin resistance. Herein a comparative genomics analysis of TLCs was performed in 31 plant and 3 species from other kingdoms, with an emphasis mainly on maize. TLCs were conserved across kingdoms and expanded in angiosperms, largely due to whole-genome/segmental duplication (WGD/SD) under purifying selection. Phylogeny reconstruction by maximum-likelihood method uncovered five TLC clades, subsequently named as TRAM/LAG, CLN8, PS-TLC, TM136 and TLCD clades. Each clade of TLCs shared specific transmembrane regions and motif composition. Divisions of conserved motifs to subunits may have occurred in TM136-type TLCs. Focusing on maize, five WGD and two DNA-mediated transposed duplication (TD) pairs were discovered, accounting for 61.11% ZmTLCs. Combined with further expression analysis, significant divergence was found in expression patterns between most maize WGD pairs, indicating subfunctionalization or/and neofunctionalization. Moreover, ZmTLC5, a deduced parental copy in a TD pair, was highly induced under FB₁ and fungus pathogen injection and exhibited potential capacity to respond to environmental stimuli. Additionally, population genetics analysis showed that ZmTLC10 in the CLN8-clade may have experienced significant positive selection and differentiated between wild and inbred maize populations. Overall, our results help to decipher the evolutionary history of TLCs in maize and plants, facilitating further functional analysis of them.

Stress-associated proteins (SAPs), a class of A20/AN1 zinc finger proteins, play vital roles in plant stress response. However, investigation of SAPs in maize has been very limited. Herein, to better trace the evolutionary history of SAPs in maize and plants, 415 SAPs were identified in 33 plant species and four species of other kingdoms. Moreover, gene duplication mode exploration showed whole genome duplication contributed largely to SAP gene expansion in angiosperms. Phylogeny reconstruction was performed with all identified SAPs by the maximum likelihood (ML) method and the SAPs were divided into five clades. SAPs within the same clades showed conserved domain composition. Focusing on maize, nine ZmSAPs were identified. Further promoter cis-elements and stress-induced expression pattern analysis of ZmSAPs indicated that ZmSAP8 was a promising candidate in response to drought stress, which was the only AN1-AN1-C2H2-C2H2 type SAP in maize and belonged to clade I. Additionally, ZmSAP8 was located in the nucleus and had no transactivation activity in yeast. Overexpressing ZmSAP8 enhanced the tolerance to drought stress in Arabidopsis thaliana, with higher seed germination and longer root length. Our results should benefit the further functional characterization of ZmSAPs.

Drought is a key factor affecting plant growth and development. Heat shock transcription factors (Hsfs) have been reported to respond to diverse abiotic stresses, including drought stress. In the present study, functional characterization of maize heat shock transcription factor 05 (ZmHsf05) gene was conducted. Homologous analysis showed that ZmHsf05 belongs to Class A2 Hsfs. The mRNA expression level of ZmHsf05 can be affected by drought, high temperature, salt, and abscisic acid (ABA) treatment. Ectopic overexpression of ZmHsf05 in rice (Oryza sativa) could significantly enhance the drought tolerance. Faced with drought stress, transgenic rice exhibited better phenotypic performance, higher survival rate, higher proline content, and lower leaf water loss rate, compared with wild-type plant Zhonghua11. Additionally, we assessed the agronomic traits of seven transgenic rice lines overexpressing ZmHsf05 and found that ZmHsf05 altered agronomical traits in the field trials. Moreover, rice overexpressing ZmHsf05 was more sensitive to ABA and had either a lower germination rate or shorter shoot length under ABA treatment. The transcription level of key genes in the ABA synthesis and drought-related pathway were significantly improved in transgenic rice after drought stress. Collectively, our results showed that ZmHsf05 could improve drought tolerance in rice, likely in an ABA-dependent manner.

As major component in cereals grains, starch has been one of the most important carbohydrate consumed by a majority of world’s population. However, the molecular mechanism for regulation of biosynthesis of starch remains elusive. In the present study, ZmES22, encoding a MADS-type transcription factor, was modestly characterized from maize inbred line B73. ZmES22 exhibited high expression level in endosperm at 10 days after pollination (DAP) and peaked in endosperm at 20 DAP, indicating that ZmES22 was preferentially expressed in maize endosperm during active starch synthesis. Transient expression of ZmES22 in tobacco leaf revealed that ZmES22 protein located in nucleus. No transactivation activity could be detected for ZmES22 protein via yeast one-hybrid assay. Transformation of overexpressing plasmid 35S::ZmES22 into rice remarkedly reduced 1000-grain weight as well as the total starch content, while the soluble sugar was significantly higher in transgenic rice lines. Moreover, overexpressing ZmES22 reduced fractions of long branched starch. Scanning electron microscopy images of transverse sections of rice grains revealed that altered expression of ZmES22 also changed the morphology of starch granule from densely packed, polyhedral starch granules into loosely packed, spherical granules with larger spaces. Furthermore, RNA-seq results indicated that overexpressing ZmES22 could significantly influence mRNA expression levels of numerous key regulatory genes in starch synthesis pathway. Y1H assay illustrated that ZmES22 protein could bind to the promoter region of OsGIF1 and downregulate its mRNA expression during rice grain filling stages. These findings suggest that ZmES22 was a novel regulator during starch synthesis process in rice endosperm.