Beijiu Cheng

Background: Plant respiratory burst oxidase homolog (Rboh) gene family produces reactive oxygen species (ROS), and it plays key roles in plant–microbe interaction. Most Rboh gene family-related studies mainly focused on dicotyledonous plants; however, little is known about the roles of Rboh genes in gramineae. Results: A total of 106 Rboh genes were identified in seven gramineae species, including Zea mays, Sorghum bicolor, Brachypodium distachyon, Oryza sativa, Setaria italica, Hordeum vulgare, and Triticum aestivum. The Rboh protein sequences showed high similarities, suggesting that they may have conserved functions across different species. Duplication mode analysis detected whole-genome/segmental duplication (WGD)/(SD) and dispersed in the seven species. Interestingly, two local duplication (LD, including tandem and proximal duplication) modes were found in Z. mays, S. italica and H. vulgare, while four LD were detected in T. aestivum, indicating that these genes may have similar functions. Collinearity analysis indicated that Rboh genes are at a stable evolution state in all the seven species. Besides, Rboh genes from Z. mays were closely related to those from S. bicolor, consistent with the current understanding of plant evolutionary history. Phylogenetic analysis showed that the genes in the subgroups I and II may participate in plant-AM fungus symbiosis. Cis-element analysis showed that different numbers of elements are related to fungal induction in the promoter region. Expression profiles of Rboh genes in Z. mays suggested that Rboh genes had distinct spatial expression patterns. By inoculation with AM fungi, our transcriptome analysis showed that the expression of Rboh genes varies upon AM fungal inoculation. In particularly, ZmRbohF was significantly upregulated after inoculation with AM fungi. pZmRbohF::GUS expression analyses indicated that ZmRbohF was induced by arbuscular mycorrhizal fungi in maize. By comparing WT and ZmRbohF mutant, we found ZmRbohF had limited impact on the establishment of maize-AM fungi symbiosis, but play critical roles in regulating the proper development of arbuscules. Conclusions: This study provides a comprehensive analysis of the evolution relationship of Rboh genes in seven gramineae species. Results showed that several Rboh genes regulate maize-AM fungal symbiosis process. This study provides valuable information for further studies of Rboh genes in gramineae.

High salinity seriously affects crop growth and yield. Abscisic acid-, stress-, and ripening-induced (ASR) proteins play an important role in plant responses to multiple abiotic stresses. In this study, we identified a new salt-induced ASR gene in rice (OsASR6) and functionally characterized its role in mediating salt tolerance. Transcript levels of OsASR6 were upregulated under salinity stress, H₂O₂ and abscisic acid (ABA) treatments. Nuclear and cytoplasmic localization of the OsASR6 protein were confirmed. Meanwhile, a transactivation activity assay in yeast demonstrated no self-activation ability. Furthermore, transgenic rice plants overexpressing OsASR6 showed enhanced salt and oxidative stress tolerance as a result of reductions in H₂O₂, malondialdehyde (MDA), Na/K and relative electrolyte leakage. In contrast, OsASR6 RNAi transgenic lines showed opposite results. A higher ABA content was also measured in the OsASR6 overexpressing lines compared with the control. Moreover, OsNCED1, a key enzyme of ABA biosynthesis, was found to interact with OsASR6. Collectively, these results suggest that OsASR6 serves primarily as a functional protein, enhancing tolerance to salt stress, representing a candidate gene for genetic manipulation of new salinity-resistant lines in rice.

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.

Starch synthesis is a complex process that influences crop yield and grain quality in maize. Many key enzymes have been identified in starch biosynthesis; however, the regulatory mechanisms have not been fully elucidated. In this study, we identified a DOF family gene, ZmDOF36, through transcriptome sequencing analysis. Real-time PCR indicated that ZmDOF36 was highly expressed in maize endosperm, with lower expression in leaves and tassels. ZmDOF36 is a typical DOF transcription factor (TF) that is localized to the nucleus and possesses transcriptional activation activity, and its transactivation domain is located in the C-terminus (amino acids 227–351). Overexpression of ZmDOF36 can increase starch content and decrease the contents of soluble sugars and reducing sugars. In addition, abnormal starch structure in transgenic maize was also observed by scanning electron microscopy (SEM). Furthermore, the expression levels of starch synthesis-related genes were up-regulated in ZmDOF36-expressing transgenic maize. ZmDOF36 was also shown to bind directly to the promoters of six starch biosynthesis genes, ZmAGPS1a, ZmAGPL1, ZmGBSSI, ZmSSIIa, ZmISA1, and ZmISA3 in yeast one-hybrid assays. Transient expression assays showed that ZmDOF36 can activate the expression of ZmGBSSI and ZmISA1 in tobacco leaves. Collectively, the results presented here suggest that ZmDOF36 acts as an important regulatory factor in starch synthesis, and could be helpful in devising strategies for modulating starch production in maize endosperm.

Multidrug and toxic compound extrusion (MATE) proteins are a group of secondary active transporters, which widely exist in all living organisms and play important role in the detoxication of endogenous secondary metabolites and exogenous agents. However, to date, no systematic and comprehensive study of this family is reported in maize. Here, a total of 49 MATE genes (ZmMATE) were identified and divided into seven groups by phylogenetic analysis. Conserved intro–exon structures and motif compositions were investigated in these genes. Results by gene locations indicated that these genes were unevenly distributed among all 10 chromosomes. Tandem and segmental duplications appeared to contribute to the expansion and evolution of this gene family. The K _a/ K _s ratios suggested that the ZmMATE has undergone large-scale purifying selection on the maize genome. Interspecies microsynteny analysis revealed that there were independent gene duplication events of 10 ZmMATE. In addition, most maize MATE genes exhibited different expression profiles in diverse tissues and developmental stages. Sixteen MATE genes were chosen for further quantitative real-time polymerase chain reaction analysis showed differential expression patterns in response to aluminum treatment. These results provide a useful clue for future studies on the identification of MATE genes and functional analysis of MATE proteins in maize.