| Maize is one of the most important crops in the world. Environmental stresses,such as drought, salinity and low temperature, have adverse effects on maize growthand yield. Adversity-resistance breeding has been the important breeding objectives.To gain stress tolerance-related functional genes and cultivate new stress-tolerantcrops using these genes is one of the most effective measures to respond abiotic stressand increase maize yield. Stuides showed that transcription factors have importantfunctions on improving crop tolerance to various stresses. Overexpression of aspecific transcription factor related to stress response can regulate the expression ofmultiple downstream stress-responsive genes at the same time and consequentlyimprove comprehensive resistances of crops. At present, this method has become aresearch hotspot in the genetic improvement of plant resistance breeding.Homeodomain-leucine zipper (HD-Zip) proteins are unique to plants and have diversefunctions in regulating plant growth and stress response. Thus, isolating HD-Zipgenes related to stress response has important significance for maizeadversity-resistance breeding. In this study, we performed genome-wide survey ofHD-Zip genes in maize, and characterized their structure features, phylogeneticrelationships and drought-induced expression patterns. We subsequently study thefunctions of Zmhdz10gene in abiotic stress response by overexpressing this gene inplants. The main results are as follows:1. By Blast homologous search, a total of55non-redundant HD-Zip genes wereidentified in the maize genome and characterized for their structure and evolution, etc.Results showed that most of the genes have highly conserved sequences, and therewas no significant difference in their structure among different plant species. The55HD-Zip genes were further divided into four classes (I-IV) based on phylogeny.Chromosomal location of these genes revealed that they are distributed unevenlyacross all the10chromosomes. Analysis of gene duplication events showed thatsegmental duplicated genes accounted for62%of the maize HD-Zip genes,suggesting that segmental duplication play an important role in the expansion ofmaize HD-ZIP gene family.2. Three types of stress-related cis-elements, including ABRE, LTRE and DREwere identified in the2kb promoter regions upstream of the transcription start site ofthe17HD-Zip I genes. Results showed that all the HD-Zip I genes except for Zmhdz16contained one or more stress-related cis-elements in their promoter regions.By comparing the distribution of the three regulatory elements in the promoter regions,the duplicated genes were found to exhibit significant differences in their promotersequences. Expression levels of the HD-Zip I genes under drought treatment wereinvestigated by quantitative real-time PCR. Of the17HD-Zip I genes, the expressionsof12genes were obviously up-regulated, whereas those of the other5genes weredown-regulated. Expression profiles of the duplicated genes were also compared, andthe results revealed that the duplicated genes exhibited the similar expression patternsunder drought treatment.3. According to phylogenetic relationships and drought-induced expressionpatterns, a drought strongly indued gene, Zmhdz10, was cloned from maize inbredline B73. Sequence analysis showed that the full-length cDNA of Zmhdz10is825-bp,which encodes a protein of274amino acids. The cDNA sequence of Zmhdz10shares100%sequence identity to the predicted sequence in the maize B73annotateddatabase. Further expression analysis showed that Zmhdz10was also induced by saltstress and ABA, and tissue-specific expression indicated that Zmhdz10wasconstitutively expressed in roots, stems, leaves, tassels, ears, silks and seedlings, butthe highest expression was detected in leaves. Transient expression showed that theGFP signal was detected only in the nucleus of the onion cell for Zmhdz10-GFPfusion protein using bombarding transformation. Yeast hybrid assays showed thatZmhdz10can activate the expression of the reporter genes, and specifically bind tothe pseudopalindromic sequence CAAT(A/T)ATTG.4. Zmhdz10overexpression transgenic plants have no significant differences inthe morphology compared with control plants. After drought and salt treatments,Zmhdz10overexpression transgenic plants showed significantly enhanced tolerance todrought and salt stresses. Relative electrolyte leakage, malondialdehyde and prolinewere tested in WT and transgenic plants. Results showed that relative electrolyteleakage and malondialdehyde content in Zmhdz10overexpression plants weresignificantly lower than that in WT plants under stressed conditions, but prolinecontent was higher than that in WT plants. In addition, ABA sensitivity assayindicated that Zmhdz10overexpression transgenic plants exhibit significantlyincreased sensitivity to ABA.5. Zmhdz10overexpression Arabidopsis plants also showed significantlyimproved tolerance to drought and salt stresses. After drought treatment, expression levels of the tested stress/ABA-responsive marker genes, including P5CS1, RD22,RD29B and ABI1, were significantly higher than detected in WT plants, suggestingthat expression of these genes were actived in transgenic plants. No significantdifference in the expression of ERD1was found between WT and transgenic plantsafter drought treatment, which was proved to involve in the ABA-independentpathways as described previously.In conclusion, a total of55HD-Zip genes were identified in the maize genomeby Blast homologous search. According to the analysis of gene structure, phylogeneticrelationships and drought-induced expression patterns, Zmhdz10gene was cloned, andits molecular biological characteristics were also analyzed in this study.Overexpression of Zmhdz10could significantly increase tolerance to drought and saltstresses, which provides an excellent gene resource for improving crop resistancethrough genetic engineering methods. Moreover, the increased sensitivity to ABA andup-regulated expression of stress/ABA-responsive functional genes of transgenicplants indicate that Zmhdz10can regulate the expression of stress tolerance-relatedfunctional genes in response to abiotic stress through the ABA signal transductionpathway. |
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