Os01g0128300
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Annotated Information
Function
The MHZ4 gene encodes a protein of 228 amino acids that harbors an N-terminal signal peptide and four transmembrane domains as predicted by the SMART program (http://smart.embl-heidelberg.de/) (Figure 2B). The MHZ4 sequence shared 47% identity and 64% similarity with Arabidopsis ABA4 (AT1G67080), which is required for neoxanthin formation in the ABA biosynthesis pathway through an unknown mechanism [32].
Expression
MHZ4 accumulation was examined by semiquantitative RT-PCR. The transcripts were detected in all organs from vegetative to reproductive stages and found to be more abundant in young leaves (Figure 3A). Transgenic rice plants harboring MHZ4 promoter::β-glucuronidase (GUS) construct were also generated and GUS staining assay was performed to evaluate the promoter activity. In etiolated seedlings, MHZ4 expression was detected in both roots and coleoptiles, and the signals were also present in the vascular tissues of roots (Figure 3B, a to e). In root apexes, GUS signals were observed in the putative quiescent center (QC) and root caps (Figure 3B, f). In field-grown plants, MHZ4 was expressed in leaf blades, young stem nodes, the base of axillary buds and adventitious roots derived from the nodes (Figure 3B, g to j). In reproductive organs, the expression of MHZ4 was detected in the anthers and pistil of young flowers, lemma of mature flowers, and parts of developing grains (Figure 3B, k to n).
Evolution
Phylogenetic analysis revealed that MHZ4 protein is conserved from cyanobacteria to higher plants and is more closely related to homologues from monocotyledonous plants (Figure 2E).
Ethylene Response Phenotypes
The mhz4 is identified previously in our screen for ethylene-response mutants in rice [45]. For dark-grown wild type (WT) seedlings, ethylene inhibited root growth but promoted coleoptile elongation in a dose-dependent manner (Figure 1A–C). The roots of etiolated mhz4 seedlings were about 10% shorter than that of WT seedlings under normal conditions. Upon ethylene treatment, mhz4 roots were insensitive to ethylene inhibition at lower concentrations (≤1 ppm) but displayed mild growth inhibition at higher concentrations (10 to 100 ppm), e.g. about 20% inhibition in mhz4 compared with about 70% inhibition in the WT at 10 ppm ethylene (Figure 1A and 1B). This indicates that the roots of mhz4 are less sensitive to ethylene. On the other hand, the coleoptiles of mhz4 were slightly but significantly (P<0.004) longer than that of WT seedlings in the absence of ethylene and were much longer than that of WT under all concentrations of ethylene treatment, indicating that the coleoptiles of mhz4 are hypersensitive to ethylene (Figure 1A and 1C). These results indicate that mhz4 mutation oppositely affects ethylene responses in roots and coleoptiles of etiolated rice seedlings.
To further confirm the ethylene responsiveness of mhz4 mutant, we examined expressions of ethylene-inducible genes originally identified from a chip analysis (GSE51153; [45]) and a RNA-seq analysis (SRP041468). Two genes including disease resistance response gene and peptidylprolyl isomerase gene were found to be induced by ethylene in both shoots and roots of WT seedlings (Figure 1D). Four genes (OsERF002, OsRRA5, OsRAP2.8 and OsIAA20) were found to be induced mainly in roots and four genes (SHR5, OsERF063, OsERF073 and photosystem II 10 kDa polypeptide gene) mainly in shoots of WT seedlings (Figure 1E). In mhz4 shoots, the ethylene inducible genes were constitutively expressed at a level higher than that in ethylene-treated WT. Ethylene treatment of mhz4 did not further increase the transcript levels probably due to their expression levels were already very high. It should be noted that we used shoots instead of coleoptiles for the gene expression analyses because similar ethylene responses were found for coleoptiles and shoots [46]. In mhz4 roots, the transcripts of ethylene inducible genes remained at a similar or lower level when compared to those in WT in the absence of ethylene, and these genes showed no or only slight inductions compared to their ethylene inductions in WT roots (Figure 1D, E). These results indicate enhanced and reduced ethylene response in mhz4 shoots/coleoptiles and roots, respectively, at gene expression level
Labs working on this gene
1.Biao Ma, Cui-Cui Yin, Si-Jie He, Xiang Lu, Wan-Ke Zhang, Shou-Yi Chen, Jin-Song Zhang State Key Lab of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China 2.Tie-Gang Lu Biotechnology Research Institute/National Key Facility for Genetic Resources and Gene Improvement, Chinese Academy of Agricultural Sciences, Beijing, China
