JIA-2018-09

2008 CHEN Ke-qin et al. Journal of Integrative Agriculture 2018, 17(9): 2007–2014 Secondary wall formation has been intensively studied during xylem cell differentiation. Seven genes from VND1 to VND7 , which form a subclade, are preferentially expressed in developing vascular cells and are induced during xylem cell differentiation (Kubo et al . 2005; Yamaguchi et al . 2008; Ohashi-Ito et al . 2010; Zhou et al . 2014). The expression of VND7 can restore defects in secondary cell wall formation in the fibre cells of inflorescence stems of nst1 / nst3 double mutants (Yamaguchi et al . 2011). The second type of NAC TFs consists of NST3 (NAC secondary wall thickening promoting factor3)/SND1 (secondary wall- associated NAC domain protein1), 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 SND1 expressing under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter can cause ectopic secondary wall deposition in non-sclerenchyma cells. Both the dominant suppression SND1 mutant and the nst1/snd1 double mutant show a significant reduction in secondary wall thickness between the vascular and xylem fibres; and in the nst1/ nst2/snd1 triple mutant, the secondary cell wall in fibres is completely absent in Arabidopsis (Zhong 2006; Mitsuda et al . 2007; Zhong and Ye 2015b). In addition, the process of anther dehiscence to release pollen requires a thick secondary wall in the interior cortex of the anther, and it has been found that anthers in nst1/nst2 double mutants cannot crack, while the single mutant is normal; these observations indicate that NST1 and NST2 are functionally redundant during the secondary wall formation in anthers (Mitsuda et al . 2005). 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, MYB83, SND3, MYB103 and KNAT7 have been shown to be direct targets of NAC TFs, including SND1, NST1, NST2, VND6 and VND7 (Zhong et al . 2007; McCarthy et al . 2009; Ko et al . 2014; Zhong and Ye 2014). These NAC genes were shown to be expressed in fibres and vessels specifically, and dominant repression of their expression levels led to a reduction in secondary wall thickening, indicating that they play important roles involved in the regulation of secondary wall biosynthesis (Zhong and Ye 2015a). In particular, MYB46 and MYB86, functioning as another level of molecular switches, redundantly turn on the entire secondary wall biosynthetic programme (Zhong et al . 2007; Ko et al . 2014; Zhong and Ye 2015a). In addition to targeting the downstream TFs, SND1 can also activate the synthesis enzymes of the secondary wall directly, such as CESA4, CESA7 and CESA8 (Zhong et al . 2010b). In this study, transgenic Arabidopsis plants with overexpressed CpSND1 (a SND1 gene isolated from Crataegus pinnatifida ) driven by the CaMV35S promoter presented growth inhibition, upward-curling leaves, sepal dysplasia and sterile phenotypes, which are similar to the phenotypes observed when AtSND1 is overexpressed. In addition, in transgenic Arabidopsis plants, the genes involved in lignin, cellulose and xylan biosynthesis were significantly induced. 2. Materials and methods 2.1. Plant materials and growth conditions Trees of Crataegus pinnatifida accession H8 (hard-endocarp hawthorn) were maintained in the National Hawthorn Germplasm Repository at Shenyang, China. The cDNA of C . pinnatifida accession H8 was used as the template for CpSND1 cloning. Arabidopsis Columbia (Col-0) was used as the transgenic plants. The rice protoplast for cellular localization was obtained from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Arabidopsis plants were maintained in a growth chamber under short-day conditions (8 h light/16 h dark) at 23°C for 2 wk and then in long-day conditions (16 h light/8 h dark) at 23°C. 2.2. Arabidopsis thaliana transformation To overexpress the CpSND1 gene in Arabidopsis , coding sequences of CpSND1 were inserted into the plant expression vector pRI101-AN, containing a CaMV35S promoter and a nopaline synthase (NOS) terminator. The primers used for vector construction are listed in Appendix A. The reconstructed plasmid pRI101- CpSND1 was introduced into A . thaliana (Col-0) by Agrobacterium tumefaciens (strain GV3101)-mediated transformation using the floral dip method. Arabidopsis flowers were dipped into A . tumefaciens GV3101 suspended in a 1/2 MS liquid containing 10% sucrose and 0.02% Silwet, and then the plants were covered with a black bag and incubated in a growth chamber at 23°C for 1 d and finally allowed to grow in a growth chamber as usual. The collected seeds were screened in 1/2 MS medium supplemented with 30 mg L -1 kanamycin, and transgenic seedlings were identified by PCR. Total RNAwas extracted from the seedlings of the T 1 generation. In addition, the T 1 transgenic lines were used for further analyses. 2.3. Phylogenetic tree graphics and bioinformatics The predicted amino acid sequences of NACs were aligned