Ectopic Expression of Hawthorn SND1 Gene in Tobacco

Secondary cell walls are largely composed of three main polymers: cellulose, hemicellulose, and lignin. These polymers are normally required to enable vascular plants not only to build strong xylem conduits for the transport of water and minerals but also to attain strong mechanical support for the plant body (Raven et al., 1999). The formation of the secondary cell wall is mainly regulated at the transcriptional level, and most of what is known about the regulation comes from the model herbaceous plant Arabidopsis thaliana. NAC (NAM-ATAF1, 2-CUC2) and MYB act as the key master switches that control secondary cell wall deposition (Taylor-Teeples et al., 2015).

NAC proteins are plant-specific transcription factors (TFs) and have been shown to function in plant development processes and abiotic and/or biotic stress responses. In Arabidopsis, there are two types NAC TFs acting as on-off switches that take part in regulating secondary wall formation in vascular cells and fibre cells. The first type of NAC TFs (VASCULAR- RELATED NAC-DOMAIN) VND6 and VND7 contribute to both secondary wall biosynthesis and programmed cell death of the vessels in both root and shoot tissues (Kubo et al., 2005; Yamaguchi et al., 2008). The second type of NAC TFs consists of NST3 (NAC secondary wall thickening promoting factor 3)/SND1, NST1 and NST2, which participate in thickening the secondary wall both in vascular fibre cells and secondary xylem fibre cells (Zhong and Ye, 2014). Arabidopsis NST3/SND1 is specifically expressed in vascular fibres and xylem fibres, and expressing SND1 under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter can cause ectopic secondary wall deposition in non-sclerenchyma cells.

Protein binding assays have demonstrated that SND1 can bind to a DNA section with the sequence (T/A)NN(C/T)(T/C/G)TNNNNNNNA(A/C)GN(A/C/T)(A/T), named SNBE (secondary wall NAC binding element) (Zhong et al., 2008). Among the SND1-regulated transcription factors, MYB46 and MYB83 have been shown to be direct targets (Zhong et al., 2007). In particular, MYB46 and MYB86, functioning as another level of molecular switches, redundantly turn on the entire secondary wall biosynthetic programme (Zhong and Ye, 2015).

From early transcriptome data of soft-endocarp and hard-endocarp hawthorns (Dai et al., 2013), we found four NAC family transcription factors that were strongly down-regulated in the fruits of soft-endocarp hawthorn compared to the fruits of hard- endocarp hawthorn. We suspected that they might participate in the biosynthesis of lignin or secondary cell walls. In this study, transgenic tobacco plants with overexpressing CpSND1 driven by the CaMV35S promoter presented growth inhibition, upward-curling leaves, which are similar to the phenotypes observed when the SND1 gene of Arabidopsis was overexpressed. Our results indicate that the SND1 function is conserved in different plants.