In China, modern agricultural cultivation of cotton requires moderately short and compact varieties to adapt to mechanical operations (Su et al. 2018). The short and compact characteristics are mainly controlled by plant architecture, which is regulated by plant hormones, such as auxins and strigolactones (SLs) (Beveridge et al. 2003; McSteen et al. 2005; Ongaro et al. 2008; Domagalska et al. 2011). SLs have been recognized as a new class of hormones that participate in regulating plant architectures in terms of shoot branching, lateral roots, shoot gravitropism and stem secondary thickening (Wang et al. 2020). Branching is a highly plastic determinant of plant shape to allow plants to respond to environmental stresses (Evers et al. 2011). Shoot branching is a ubiquitous phenomenon in higher plants and a basic characteristic of plant growth, that is essential in determining plant architecture.
Cotton is an important source of protein and oil and cotton fiber is commonly used as natural fiber in the textile industry, hence, regulating growth and controlling branching are essential for cotton cultivation. Several key genes involved in the SL biosynthesis signaling pathway and participating in plant architecture development, especially shoot-branching have been identified (Beveridge et al. 2010). In this study, a homolog of AtD14 was cloned from upland cotton cultivar Xuzhou 142 and named GhD14s. GhD14s showed high identity with AtD14 (53.5%). The D14 functional domains were conserved in cotton and the model plants rice (monocotyledon) and Arabidopsis (dicotyledon) (Fig. 1). Phylogenetic analysis showed that the D14s in higher plants have a close relationship with D14s from other higher plants (Supplementary Figure S3). The conservation of D14 among different species indicates it play critical role in higher plants.
Subcellular localization results showed that GhD14D is localized to the nucleus and cytoplasm, which is consistent with the localization results of the D14 genes in Arabidopsis and rice (Chevalier et al. 2014; Yao et al. 2018). The subcellular localization results provided evidence that GhD14D functions in both the nucleus and cytoplasm. GUS staining showed that D14 in rice was mainly expressed in parenchyma cells surrounding the xylem in leaves, stems and axillary buds (Arite et al. 2009). In this study, the GhD14D pro::GUS gene expression was mainly observed in the buds, stems and flowers (Fig. 4). The transcripts of GhD14D in cotton were also significantly accumulated in stem, leaf, flower and 20 DPA fibers (Fig. 3). These results demonstrate that GhD14D functions in stem, leaf, flower and fiber development. Previous studies in other plants found that D14 was involved in the development of stem, leaf and flower: The d14 mutant had a larger stomatal aperture in leaf, slower abscisic acid (ABA)-mediated stomatal closure, lower anthocyanin and reduced plant senescence under drought stress (Li et al. 2020). OsMADS57 interacts with TEOSINTE BRANCHED1 (OsTB1) and targets OsD14 to control the outgrowth of axillary buds in rice (Guo et al. 2013).
The OsD14 is the receptor of SL protein, which acts as a new component of the SL-dependent branching inhibition pathway and inhibits rice tillering (Arite et al. 2009). AtD14 hydrolyzes SLs into a D-ring-derived intermediate CLIM and irreversibly binds CLIM to trigger SL signal transduction, thus regulating shoot branching in Arabidopsis (Yao et al. 2016). Furthermore, overexpression of GhD14D in the d14 mutant, was performed to further explore the function of GhD14D in stem development and shoot branching. As a result, overexpression of GhD14D in AtD14 mutant (d14-1) reduced the mean number of branches and restored the plant height (Fig. 7). Meanwhile, the branching and leaf phenotypes were restored to wild-type plants phenotype and there was no significant difference between wild-type plants and d14-1/35S::GhD14D transgenic lines. The phenotype of the d14-1 mutant with fewer shoot branches and higher stature could be rescued via GhD14D overexpression in d14 mutant, suggesting that GhD14D and AtD14 may have similar functions in regulating branching number, plant height, petiole length, and length-width ratio of leaf. Consistently, the OsD14 gene also restored the phenotype of d14 to the wild type both in plant height and tiller development in rice (Yao et al. 2018).
The GhD14A/D had high expression levels in flower, stem, leaf, and 20 DPA fibers, but was weakly expressed in cotton root (Fig. 3). In rice, transcripts of D14 were highly accumulated in leaves and the first leaf buds, but not in root tip (Arite et al. 2009). In petunia, high expression levels of DAD2 were observed in axillary bud and leaf, but not in root (Hamiaux et al. 2012). In chrysanthemum, DgD14 had the highest expression level in stem, followed by node, and was only weakly expressed in root (Wen et al. 2015). These results reflect that the D14 gene may not be involved in root structural development. The GhD14A/D showed high expression level at 20 DPA fibers (Fig. 3), and this stage is the secondary cell wall thickening stage (Zhang et al. 2015). Meanwhile, silencing the GhD14A/D gene expression reduced the fiber length (Fig. 5) and the expression levels of the secondary cell wall biosynthesis related genes (Fig. 6). These results indicating that GhD14 might play a role in fiber development through impact secondary cell wall biosynthesis of cotton fiber. In the future, molecular mechanisms and regulatory relationships between GhD14 and secondary cell wall biosynthesis needs to be further investigated to deep our understanding of the strigolactone and secondary cell wall biosynthesis.
The GhD14A/D gene silencing enhanced fruit branch angles and reduced fiber length in cotton (Fig. 5), indicating that the GhD14A/D transcripts accumulation level affects cotton plant architecture and fiber length. The plant architecture is also regulated by auxin, cytokinin, and gibberellic acid (GA). D14 is the receptor of SL, which involved in plant stature and inhibition of plant shoot branching (Arite et al. 2009). The strigolactone signal can regulate the auxin polar transport and the cytokinin content in the stem (Ferguson et al. 2009). In the future, molecular mechanisms and regulatory relationships between GhD14 and auxin or cytokinin should be examined to deepen our understanding of strigolactone signaling in cotton architecture. Although we did not investigate this, we speculate that GhD14 might regulate branch number and angles through auxin and GA in cotton. In the future, molecular mechanisms and regulatory relationships between GhD14 and auxin or GA should be examined to deepen our understanding of SL signaling in cotton architecture.