Identification of the GhD14 in G. hirsutum
The AtD14 protein sequence was used as the query sequence to against the G. hirsutum genome (Hu et al. 2019) to obtain the D14 protein in G. hirsutum. The sequences GH_A02G1790.1 and GH_D03G0270.1 in the cotton genome were the most similar to AtD14 and were chosen as the candidate orthologs of AtD14 and named GhD14A and GhD14D, respectively. The GhD14A and GhD14D have one intron, contain 816 base pairs (bp) (Fig. S1A), 271 amino acids (Fig. 1A) and have a molecular weight of 19.9 kDa and 30.0 kDa, respectively.
Conserved domain and promoter analysis of GhD14s
The AtD14 protein structure in A. thaliana has been reported as an α/β hydrolase (Li et al. 2020). We compared the protein sequences of GhD14A, GhD14D and AtD14, and identified eight α-helixes, five η-helixes and seven β-strands in D14 protein (Fig. 1B, Fig. S1B, C). There are three catalytic residues, S95, D217 and H246 in GhD14A and GhD14D protein sequences (Fig. 1B). The sequence similarities of AtD14 with GhD14A and GhD14D were 52.7% and 53.5%, respectively. As GhD14A and GhD14D have 97.4% similarity in coding sequences, we cloned GhD14D from G. hirsutum cultivar Xuzhou 142 and used it as the representative GhD14 gene for cis-elements distribution, subcellular location, GUS and overexpression analysis.
The cis-element distribution of 1,715 bp GhD14D promoter sequence and promoter driven GUS expression were investigated to study the potential function of GhD14D. The GhD14D promoter has high A and T content, and typical cis-elements TATA-box and CAAT-box (Fig. S2). The auxin responsiveness cis-element AUXRR-core and jasmonic acid methyl ester (MeJA) responsiveness regulatory element CGTCA-motif were also present in the GhD14D promoter region (Fig. S2), indicating that auxin and MeJA might function in regulating GhD14D gene expression via combine the corresponding cis-elements present in the promoter of GhD14D.
Phylogenetic analysis of GhD14s
To explore the evolutionary relationships among GhD14D and other fifteen D14s from typical higher plant species, we performed phylogenetic analysis of D14 proteins from G. hirsutum and other fifteen higher plant species using MEGA 7.0 software with Neighboring-Joining (NJ) method and 1000 bootstrap replications. The phylogenetic tree showed that the D14s in sixteen higher plants have the same evolutionary origin. The D14s was resolved into two evolutionary branches in phylogenetic tree and both of which contain D14 proteins from monocotyledons and dicotyledons. Meanwhile, GhD14s was more closely related to eudicots, and was most closely related to T. cacao, with moderate bootstrap support of 79% (Fig. S3).
GhD14D is localized to the cytoplasm and nucleus
The pTF486-GhD14D:GFP vector was constructed to investigate the subcellular location of GhD14D protein and DAPI staining was used to test whether the GhD14D protein was localized in the nucleus. The pTF486-GhD14D:GFP was transiently expressed in A. thaliana protoplasts and the cells were stained with DAPI, and then examined using confocal microscopy. The results showed that GhD14D-GFP fusion protein was accumulated throughout the nucleus and cytoplasm and was also co-localized with DAPI (Fig. 2A). The pCHF3-GhD14D:GFP was bombarded into tobacco epidermal cells by agroinfiltration to further confirm the subcellular localization of GhD14D protein. We found the green fluorescence of GhD14D-GFP distributed in the cytoplasm and nucleus and the fluorescence of GhD14D-GFP coincided with the DAPI fluorescence, confirming that GhD14D protein is co-localized in the cytoplasm and nucleus (Fig. 2B). Both GhD14D-GFP fusion protein and DAPI methods in A. thaliana protoplast and tobacco epidermal cells clearly showed strong nucleus and cytoplasm localization, indicating that the hydrolase GhD14D functions in the cytoplasm and nucleus.
Expression patterns of GhD14A/D
Expression patterns of GhD14A/D in fibers at 0, 3, 5, 10, 15 and 20 days post-anthesis (DPA) and root, stem, leaf, and flower were investigated to explore the function of GhD14 using quantitative real-time PCR (qRT-PCR). Since GhD14A and GhD14D have 97.43% coding sequence similarity and they can’t separate with specific primer, the expression level of GhD14A/D was investigated. As shown in Fig. 2, GhD14A/D gene transcripts were increased during the fiber development with peak value at 20 DPA (Fig. 3A), and abundantly expressed in flower and stem (Fig. 3B), indicating that GhD14A/D may function in fiber, flower and stem development.
In order to test the tissue specific expression of GhD14D, the GhD14D promoter driving GUS gene expression in A. thaliana was visualized by histochemical staining of transgenic A. thaliana. The color of transgenic A. thaliana seedlings represents the promoter driven GUS gene expression. As shown in Fig. 4, the GhD14D promoter-driven GUS gene washighly expressed in buds, stems and flowers (Fig. 4). These observations demonstrated that GhD14D may play important role in buds, stems and flowers.
GhD14A/D gene silencing in cotton increased branch angles and reduced fiber length
Virus-induced gene silencing (VIGS) is an efficient and rapid method to reduce gene transcripts and investigate gene functions in plants (Gu et al. 2014). To further uncover potential functions of GhD14 in cotton, the VIGS strategy was used to reduce GhD14A/D transcript levels in G. hirsutum. Positive control of GhPDS gene silence shows in Fig. S5. Expression levels of the GhD14A/D gene were detected using qRT-PCR strategy and the result showed that GhD14A/D gene expression was significantly reduced in GhD14A/D-silenced plants compared with that in the control plants (CLCrVA) (Fig. 5A). The phenotype of branch angles of control and VIGS plants (GhD14A/D-V1, GhD14A/D-V2 and GhD14A/D-V3) showed that the monopodial branch angles were significantly increased in GhD14A/D-silenced plants compared with the negative control plants (CK). The branch angles of GhD14A/D-silenced plants were increased about two times compared with the CK (Fig. 5B, C, Fig. S4). In order to explore the potential functions of GhD14A/D in cotton fiber development. The fiber length was observed after GhD14A/D silencing, and the result showed that the cotton fiber length was significantly reduced in GhD14A/D-silenced plants (Fig. 5D, E), indicating the GhD14A/D gene may play a critical role in cotton fiber elongation. In general, reducing GhD14A/D gene expression led to wider branch angles and decreased fiber length, suggesting that GhD14A/D functions in cotton architecture and fiber development.
GhD14A/D gene silencing in cotton reduce the transcripts of secondary cell wall biosynthesis genes
The cotton fiber is produced by the specific elongation of cell in ovule epidermal and cell wall biosynthesis is required during the fiber elongation process. To further investigate the mechanism of GhD14A/D in regulating fiber development, the relative expression level of secondary cell wall biosynthesis genes (Sun et al. 2017) was investigated in GhD14A/D-silenced plants. As shown in Fig. 6, The six genes (GhLBD30, GhCesA7, GhMYB46, GhXCP1, GhIRX8 and GhXCP2) related secondary cell wall biosynthesis were down regulated in GhD14A/D-silenced plant, while other three genes (GhIRX10, GhCesA8 and GhCesA4) related secondary cell wall biosynthesis have no significant difference in expression levels compare with the negative control (CLCrVA) (Fig. 6). This result demonstrates that downregulating of GhD14 gene expression reduced the transcripts of genes involved in secondary cell wall biosynthesis.
Overexpression of Ghd14d rescued the d14 mutant phenotype in Arabidopsis
Overexpression of GhD14D in the d14 Arabidopsis mutant, a heterologous complementation approach, was performed to further explore the function of GhD14D in stem development and shoot branching, since AtD14 is the receptor of SLs and has essential functions in the SL signal transduction process (Chevalier et al. 2014). The pCAMBIA1305-35S-GhD14D was constructed and transformed into A. thaliana mutant d14-1. The seven transgenic lines were obtained after PCR detection (Fig. S6). The third generation of six transgenic lines with stable and highly expressed GhD14A/D was used for further analysis. As shown in Fig. 6, mutant d14-1 plants had smaller length-width ratio of leaf (Fig. 7A, B) and more shoot branches as well as shorter plant height (Fig. 6C, D, E) compared with wild-type A. thaliana plants. Overexpression of GhD14D in A. thaliana d14-1 mutant restored the leaf phenotypes and the number and length of branches to the phenotype of wild-type plants (Fig. 7). The phenotype of the d14-1 mutant with more shoot branches and short stature could be rescued by GhD14D and there was no significant phenotype difference between wild-type A. thaliana lines and d14-1/35S:GhD14D transgenic lines (Fig. 7). These results suggested that GhD14D and AtD14 may have similar functions in regulating branching number, plant height, petiole length, and length-width ratio of leaf.