Functional Analysis Of SIZ1in Dendrobium

Orchidaceae is one of the largest families in the flowering plants with high ornamentaland commercial value. Dendrobium is the largest genera of Orchidaceae and mainlydistributed in tropical and subtropical regions. Sumoylation is a post-translational regulatoryprocess in diverse cellular processes in eukaryotes, involving in reversible conjugation ofsmall ubiquitin-like modifier(SUMO) proteins to target substrate proteins in order to modifytheir functions. SIZs have been reported to play a pivotal role in controlling sumoylationpathway as SUMO E3ligases. Although the characteristic features and sumoylationmechanisms of SIZ1protein have been well understood in Arabidopsis, the involvement ofSUMO E3ligases in sumoylation in monocot crops is poorly understood. Currently, there isno information regarding the roles of SIZ1in Dendrobium.Recently, we have reported the isolation of Dendrobium SIZ1(DnSIZ1), Dendrobiumhomologs of Arabidopsis SIZ1. In this paper, we functionally characterized the putativeSUMO E3ligase based on the analysis of bioinformatics and expression patterns, we alsofurther investigated the ectopic expression of DnSIZ1in Arabidopsis, focusing on its roles inthe phenotypes of transgenic plants, regulation of floral transition, and accumulation ofSUMO conjugates. The main results were as follows:1、Bioinformatics analysis indicated that DnSIZ1possessed predicted domains, such asSAP, zf-MIZ (MIZ/SP-RING zinc finger) and plant-specific PHD domain, which were highlyconserved in SIZ/PIAS-type proteins, respectively. Based on amino acid sequence alignmentand their phylogenetic relationship among DnSIZ1and proteins of other species, DnSIZ1andSIZ/PIAS proteins of other species shared high sequence identity with respect to theirconserved structure, especially similar to Arabidopsis and rice. The predicted analysis ofsubcellular localization established that DnSIZ1may localize in the nucleus.2、The temporal and spatial expression patterns of DnSIZ1in Dendrobium weredetected by semi-quantitive RT-PCR. The results suggested that DnSIZ1constitutivelyexpressed in almost all the tissues/organs including root, stem, leaf, flower and flower bud,with the higher expression level in old leaf, young leaf and young stem, but to a lesser extentin root and flower. 3、The transcription level of DnSIZ1gene was analyzed by RT-PCR under different stressconditions. Our results showed that expression of DnSIZ1gene could be affected by a broadrange of abiotic stress factors, such as high and low temperature, drought, abscisic acid (ABA)and wounding treatment. The transcription level of DnSIZ1were strongly up-regulated whentreated with45℃heat shock,4℃cold and wounding, but induced only weakly by ABA anddrought treatments in short term, indicating that DnSIZ1possibly functioned as a mediator ofmultiple environmental stress signaling processes.4、To determine the subcellular localization of DnSIZ1protein, we constructed a fusionplasmid of pBEGFP-DnSIZ1expressed under the regulation of cauliflower mosaic virus(CaMV)35S promoter. When the construct was permanently expressed in wild-type (Col-0)Arabidopsis, its subcellular localization was monitored by confocal laser scanning analysis.The results demonstrated that DnSIZ1protein was localized in the nucleus.5、To further understand the function of DnSIZ1, we also generated pCAMBIA1300-DnSIZ1recombination plasmid, which was transformed into Arabidopsis siz1-2and wild-typeby Agrobacterium tumefactions using the floral dip method. Two overexpressing (OE1, OE3)and complementary (HB4, HB17) transgenic plants were obtained by hygromcin screeningand PCR analysis.6、Phenotype analysis showed that, no detectable differences were observed in themorphological characters of overexpressing lines compared with wild-type plants undercontrol conditions, and the siz1-2phenotypes of dwarfism with leaf curl failed to recover incomplementary lines. Investigation of ABA hypersensitive phenotype for different transgenicplants showed that the germination rates in overexpressing lines were slightly higher thanwild-type at48h after stratification, while the germination rates were strongly enhanced incomplementary lines relative to siz1-2, although they were lower than wild-type. Furthermore,analysis of germination for seeds grown on MS medium containing0.6μM ABA revealed thatthe germination rates were comparable between overexpressing lines and wild-type, whereascomplementary lines were intermediate between wild-type and siz1-2. Therefore, DnSIZ1partially functionally complemented an ABA-mediated inhibition of seed germination insiz1-2, which may involve in response to ABA signaling pathways.7、Under normal growth conditions, rosette leaf numbers at flowering in overexpressing lines and wild-type plants were observed to an equivalent extent, rosette leaf numbers atflowering in complementary lines relative to siz1-2were slightly higher, but were obviouslylower than that in wild-type. Under vernalization treatment at4℃for three weeks, floweringtime of overexpressing lines was similar to that of wild-type plants, whereas complementarylines could substantially restore to wild-type levels. Thus, the early flowering phenotype ofsiz1-2was suppressed by expressing DnSIZ1gene under vernalization treatment, indicatingthat DnSIZ1could represses the flowering mainly through vernalization-induced floraltransition. In contrast to AtSIZ1, which negatively regulated flowering mainly through anSA-dependent pathway, we may make a conclusion that DnSIZ1was a negative regulator offlowering transition depending on vernalization pathway.8、DnSIZ1protein also participated in response to heat shock-induced accumulation ofSUMO conjugates. Under control conditions, SUMO conjugates in wild-type, siz1-2,complementary or overexpressing transgenic plants were accumulated to a lesser extent.However, when exposed to42℃heat shock for30min, overexpressing lines showed slightlyenhanced levels of SUMO conjugates when compared to wild-type, while complementarylines significantly produced more SUMO conjugates than Arabidopsis siz1-2, suggesting thatDendrobium SIZ1could functionally complement Arabidopsis SIZ1in the SUMOconjugation pathway. Take together, these results suggested that DnSIZ1protein exhibitedSUMO E3ligase activity that facilitated the SUMO modification pathway.