Cloning and Bioinformatics Analysis of ZmZHD9 Gene
ZmZHD9 gene was cloned from Y882 and sequencing analysis showed that the open reading frame was 303 bp encoding a putative protein of 100 amino acids residues of 10.37 kDa with an isoelectric point of 8.93. Phylogenetic relationship analysis of ZmZHD9 with 17 Arabidopsis ZF-HD TFs and 14 Oryza sativa L. ZF-HD TFs showed that ZmZHD9 belongs subfamily of MIF (Mini Zinc finger). ZmZHD9 was clustered into group with OsMIF1, OsMIF3, AtMIF2 and AtMIF2 (Fig. 1A). Multiple alignments showed that ZmZHD9 proteins contained a Zinc finger domain at its N terminus (Fig. 1B), which suggested that ZmZHD9 was a member of subgroup MIF in maize.
Subcellular Localization and Transcriptional Activation Activity Analysis of ZmZHD9 protein
To detect the subcellular localization of the ZmZHD9 protein, the ORF of ZmZHD9 gene without the termination codon was cloned and the expression vector for pMDC83-ZmZHD9-GFP fusion protein was constructed. The recombinant vector pMDC83-ZmZHD9-GFP and pMDC83-GFP were introduced into Nicotiana benthamiana leaves and observed under a laser scanning confocal microscopy. As shown in Fig. 2, the pMDC83-ZmZHD9-GFP fusion protein was exclusively detected in the nucleus, whereas Green fluorescent protein (GFP) control distributed evenly in the nucleus and the cytoplasm, indicating that ZmZHD9 gene encoded a nuclear localization protein.
To detect the transcriptional function of the ZmZHD9 as a transcription factor in maize, we performed the yeast two-hybrid procedure to evaluate its transactivation activity. The complete ORF region of ZmZHD9 was fused to the GAL4 DNA-binding domain in the pGBKT7 vector and transformed into Yeast strain AH109. As shown in Fig. 3, all transgenic yeasts grew well on SD/-Trp medium. The yeast cells harboring the pGBKT7-ZmZHD9 plasmid grew as well as the positive control and exhibited positive for α-galactosidase activity on SD/-Trp/-His/-Ade/X-α-gal medium, suggesting that the ZmZHD9 protein had transactivation activity to act as a transcriptional activator.
Stress-Related Regulatory Elements analysis in the Promoter of ZmZHD9
Cis-elements in promoter regions of genes always play crucial roles in stress responses. Cis-elements analysis showed that there were numerous stress-related regulatory elements such as TATA-box, ARE, low-temperature responsive (LTR), dehydration and salicylic acid (SA) and W box in the promoter region of 2000 bp upstream of the ATG start codon, indicating that ZmZHD9 was involved in plant hormone-related signal transduction and response to abiotic stress (Table 1).
Table 1
Cis-element analysis of the ZmZHD9 in promotor sequence
Cis-Element
|
Target Sequences
|
Number
|
Function
|
ABRE
|
ACGTG
|
2
|
ABA-responsive
|
ARE
|
AAACCA
|
4
|
antioxidant, drought and salt responsive
|
DRE core
|
GCCGAC
|
1
|
dehydration responsive
|
LTR
|
CCGAAA
|
1
|
low-temperature and salt responsive
|
TCA-element
|
CCATCTTTTT
|
2
|
salicylic acid responsive
|
W box
|
TTGACC
|
1
|
drought and salt responsive
|
TATA-box
|
TATATA
|
3
|
drought, cold and salt responsive
|
O2-site
|
GATGACATGG
|
1
|
zein metabolism regulation
|
P-box
|
CCTTTTG
|
1
|
gibberellin responsive
|
Expression Profiling Analysis of ZmZHD9 in Different Tissues and Response to abiotic stress
Tissue-specific expression was performed by qRT-PCR and the result indicated that ZmZHD9 showed high expression in leaf, root and ear, with weak expression detected in stem, and marginally expression observed in tassel (Fig. 4A). In addition, the expression patterns of ZmZHD9 of leaves under various treatments, including drought, salt, high temperature and ABA were also investigated. Under drought treatment, the ZmZHD9 transcript level was pronouncedly induced until reaching the highest level at 4 h, which greater than 2.6-fold of the initial level and followed by decreased at the last time point (Fig. 4B). ZmZHD9 was also induced by salt treatment and reached the highest level by 1.8-fold at 24 h after treatment (Fig. 4C). After treatment with high temperature, the expression of ZmZHD9 gradually decreased and reached the lowest level at 48 h, then sharply increased by 9-fold at 60 h than that 48 h (Fig. 4D). The expression of ZmZHD9 was also increased by exogenous ABA treatment and reached the highest level at 12 h (Fig. 4E). These results indicate that ZmZHD9 is highly expressed in leaves and that its expression is induced by abiotic stress, such as drought, high salinity and ABA.
Overexpression of ZmZHD9 Enhances Drought Tolerance in Transgenic Maize Plants
To investigate the function of ZmZHD9, we performed Agrobacterium-mediated transformation and obtained single-copy homozygous T3 transgenic lines. Finally, three independent ZmZHD9-OE transgenic lines (OE8, OE13, and OE17) were selected for subsequent experiments. qRT-PCR analysis showed that the expression levels of ZmZHD9 were higher in three transgenic plants than that in control plants (WT), respectively, indicating that the transgenic plants were successfully generated as designed (Fig. 5A). Under normal conditions, there was no visible morphological differences in phenotype between 3 transgenic lines (OE8, OE13, OE17) and control (WT), while after natural drought stress for 10 days, the leaves of WT plants appeared severe dehydration and wilting symptoms, whereas the leaves of ZmZHD9-OE transgenic plants had significantly delayed leaf rolling (Fig. 5B). the leaf relatively water content (RWC) and proline of three transgenic lines exhibited higher levels than that of WT plants (Fig. 5C and 5D), respectively. Moreover, the relative electrolyte leakage levels (REL) and malondialdehyde (MDA) contents of ZmZHD9-OE plants were lower than WT plants (Fig. 5E and 5F). Compared to the WT plants, antioxidant reductases activity of SOD and POD were significantly higher in ZmZHD9-OE plants (Fig. 5G and 5H). Taken together, the results indicated that the overexpression of ZmZHD9 can regulate physiological processes that increase tolerance of transgenic maize plants to drought stress.
Expression Patterns Analysis of ZmZHD9 in Transgenic Plants under Different Abiotic Stress
The expression patterns of ZmZHD9 gene under abiotic stress in transgenic plants were performed by qRT-PCR. Under drought and salt stress, the expression of ZmZHD9 was upregulated in transgenic lines that was increased 2.2-fold and 3.3-fold as compared to those in the wild-type plants, respectively (Fig. 6A, 6B). In contrast, ZmZHD9 was inhibited with much lower expression level under high temperature stress (Fig. 6C). Under ABA treatment, ZmZHD9 was induced, with 2.7-fold increased expression level than that in wild-type (Fig. 6D). These results are consistent with the expression of ZmZHD9 gene in non-transgenic plants, indicated that ZmZHD9 play important role in response to abiotic stress.
Altered Expression of Drought Stress Responsive Genes in Transgenic Plants
To further investigate the pathway regulated by ZmZHD9 under drought stress, transcript levels of six stress-responsive genes were verified in WT and transgenic maize plants under normal and drought stress. The transcript levels of ZmPC5S1, ZmABI2, ZmED22, ZmSNC1, ZmDRE2A, and ZmLEA3 had no significant difference between WT and OE plants under normal conditions. Under drought stress, the expression level of ZmDREB2A were upregulated in ZmZHD9 transgenic lines that was increased over 3-fold as compared in the wild-type plants (Fig. 7E). ZmP5CS1, ZmSAC1, and ZmLEA3 were induced by ZmZHD9 in transgenic plants with about 2.5-fold increased expression level in comparison to that of the WT plants (Fig. 7A, 7D, and 7F). Among all the detected genes, ZmABI2 and ZmRD22 were upregulated in ZmZHD9 transgenic lines by less than 2-fold as compared to those in wild-type plants (Fig. 7B, 7C). All these results suggest that ZmZHD9 may enhance maize resistance to drought through activate the expression of stress-responsive genes.