Os04g0473900

DWA1 encodes an enzymatic megaprotein conserved in vascular plants，and DWA1 controls drought resistance by regulating drought-induced cuticular wax deposition in rice.

Annotated Information

Function

DWA1 encodes an enzymatic megaprotein (2,391 aa in length) conserved in vascular plants, including an oxidoreductase-like domain, a prokaryotic nonribosomal peptide synthetase-like module, an AMP-binding domain, and an allene oxide synthase-like domain. The AMP-binding domain exhibits in vitro enzymatic activity in activating long-chain fatty acids to form acyl-CoA. Expression pattern analysis shows that DWA1 is preferentially expressed in vascular tissues and epidermal layers and strongly induced by drought stress. The dwa1 mutant was highly sensitive to drought stress compared to the WT and DWA1-functional complementation （DWA1-FC）plants restored drought resistance. Further dissections uncovers that the dwa1 mutant was impaired in cuticular wax accumulation and significantly suppressed many wax-related genes expression under drought conditions and reduced levels of very-long-chain fatty acids. Conversely, plants overexpressing DWA1 were significantly up-regulated many wax-related genes expression under drought conditions and elevated levels of very-long-chain fatty acids. The results suggest that DWA1 controls drought resistance by regulating drought-induced cuticular wax deposition in rice.

Mutation

Fig.1 Identification of the drought-hypersensitive dwa1 mutant in rice, in which functional complementation of dwa1 restore the drought resistance(from reference)

(1)There is an insertion of T-DNA in the sixth exon of DWA1 (Fig.1A).
(2)RT-qPCR analysis of DWA1 in WT and mutant tissues(leaf and panicle) indicate that dwa1 is a loss of-function mutant because of no transcript found in the mutant(Fig.1B).
(3)During the course of drought treatment, the mutant wilted and exhibited leaf rolling earlier than the WT.After severe drought treatment followed by rewatering, more than 90% of the WT plants survive, whereas the dwa1 mutant plants are almost dead(Fig.1C and D).
(4)Water loss rate of mutant is faster than WT(Fig.1E).
(5)DWA1-functional complementation(DWA1-FC)lines which is inserted the full-length genomic fragment of DWA1 into the dwa1 mutant restore drought resistance(Fig.1F and G).
(6)Plant height at the reproductive stage are also restored in the DWA1-FC lines(Fig.1H).

Expression

Fig.2 Expression profiles of DWA1 under normal and stress conditions.(from reference)

Fig.3 Drought-induced expression of DWA1.(from reference)

(1)qRCR results show that DWA1 expression level is very low and undetectable at the vegetative stages but is relatively high at the reproductive stages(Fig.2A).
(2)Using a GUS reporter gene driven by a DWA1 promoter detects that DWA1 expression is mainly in the mature organs, such as in mature leaves (Fig.2B 1-2), axillary bud (Fig.2B3), young panicle (Fig.2B4),spikelet (Fig.2B5), stem (Fig.2B6), young panicle (Fig.2B7), pistil (Fig.2B8), stamen (Fig.2B9), young embryo (Fig.2B10), SEM picture of rice leaf cuticle (Fig.2B11), cross-sections of stem (Fig.2B12) and leaf (Fig.2B13),and semithin sections of leaf (Fig.2B14-15).In addition,SEM observation shows that the GUS staining in leaves is mainly distributed along the silica-cork cell lines in the cuticle.Cross-sections and semithin sections indicate that DWA1 is expressed mainly in vascular tissues and epidermal cell layers.
(3)What's more,after various phytohormones treatment,DWA1 expresses higher,such as abscisic acid, jasmonic acid, indole-3-acetic acid, and gibberellic acid (Fig.2C).
(4)Under drought treatment,GUS staining shows thatDWA1 is induced.For example,in Fig.3B,GUS staining in leaf before (1 and 3) and after (2 and 4) drought stress is shown.

Evolution

Fig.4 Unrooted phylogenetic tree and domain architecture of DWA1 protein orthologs in vascular plants and microbial relatives(from reference)

At present,DWA1,likely to a megaenzyme,has five domains at least. The N terminus of DWA1 is an oxidoreductase-like domain,and closely followed by the AMP-binding domain(A domain)Then,two repeats of a phosphopantetheine-binding subdomain follow the A domain and feature a thiolation domain (T domain).An allene oxide synthase (AOS)-like domain is located between the second and third repeats of the left-handed β-helix (LbH) domain at the C-terminal region(Fig.4).What's more,homologous sequences with DWA1are all from microorganisms.Nonribosomal peptide synthetase(NRPS),a megaenzyme needed for nonribosomal peptides synthesis in bacteria and filamentous fungi,is a major branch of the AMP-binding enzyme superfamily.More importantly,both these homologous sequences and DWA1 have an NRPS module, including adenylation domain, followed by a thiolation domain catalyzing the activation and thiolation reaction of an amino acid instead of a carboxyl substrate for acyl-CoA synthetase.These features suggest that DWA1 and its plant homologs may be derived from a prokaryotic NRPS rather than the acyl-CoA synthetase family, which possesses only the A domain.

Labs working on this gene

• National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan,China