*4.12. Statistical Analysis*

All data were plotted in Excel 2013 (Microsoft Inc., Redmond, WA, USA) and Sigmaplot 12.0 (Systat Software Inc., San Jose, CA, USA). Data were analyzed by one-way analysis of variance (ANOVA) and means separated by Duncan's multiple range test (DMRT) (*p* < 0.05, *p* < 0.01) in SPSS version 22.0 software (IBM Corp., Armonk, NY, USA).

#### **5. Conclusions**

Three *DoRWA* genes, named *DoRWA1*, *DoRWA2* and *DoRWA3*, were cloned from the medicinal orchid, *D. o*ffi*cinale*. Phylogenetic analysis revealed that DoRWA3 was clustered with the identified acetyltransferase genes (i.e., AtRWA2, PtRWA-C, PtRWA-D) into one branch. Interestingly, the *cis*-elements of the three *DoRWA* genes had the ABA-responsive element and their expression patterns were sensitive to ABA treatment. The results of subcellular localization showed that the three DoRWA proteins were localized in the ER, and not in the GA. The *O*-acetyl groups shared a similar trend as WSPs in different organs. qRT-PCR and RNA-seq results showed that *DoRWA3* was mainly expressed in the organs where the *O*-acetyl groups accumulated, displaying significantly higher expression than *DoRWA1* and *DoRWA2* in different organs, except for roots. *DoRWA3* was co-expressed with key genes related to the synthesis of WSPs, so it is regarded as a candidate gene that codes for an acetyltransferase. The acetylation level of polysaccharides in seeds, leaves and stems of the three *A. thaliana* OE transgenic lines was significantly higher than in WT, indicating that *DoRWA3* has a similar function as *AtRWA2*.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/17/6250/ s1. Figure S1: Alignment of amino acid sequences of RWA proteins in *Dendrobium o*ffi*cinale*, *Arabidopsis thaliana* and *Populus trichocarpa*; Figure S2: Gene structure of three *DoRWA* genes. Figure S3: Predictions of subcellular localization of DoRWA1 (A), DoRWA2 (B), DoRWA3 (C) and subcellular localization of DoRWA3-YFP with Golgi apparatus localization marker G-rk (D); Figure S4: Comparison of germination rate and root length between wild type (WT) and overexpression (OE) transgenic plants; Table S1: Primers designed for PCR.

**Author Contributions:** Conceptualization, J.D., C.S. and J.A.T.d.S.; methodology, C.S.; software, C.S., C.H., Z.Y.; validation, C.Z., H.W. and M.Z.; formal analysis, C.S.; investigation, C.S.; resources, J.D.; data curation, J.D.; writing—original draft preparation, C.S. and J.A.T.d.S.; writing—review and editing, C.S. and J.A.T.d.S.; visualization, C.S. and J.A.T.d.S.; supervision, J.D.; project administration, J.D.; funding acquisition, J.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the project "Cultivation of new varieties of *Dendrobium o*ffi*cinale* in Guangdong Province", grant number Y334041001.

**Acknowledgments:** The authors are grateful to Rufang Deng for assistance with subcellular localization analysis. We also thank the editor and reviewers for providing suggestions that improved the quality of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**



#### **References**


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