| Abstract: | The trans-Golgi network (TGN) plays a central role in cellular secretion and has been implicated in sorting cargo destined for the plasma membrane. Previously, the Arabidopsis (Arabidopsis thaliana) echidna (ech) mutant was shown to exhibit a dwarf phenotype due to impaired cell expansion. However, ech also has a previously uncharacterized phenotype of reduced male fertility. This semisterility is due to decreased anther size and reduced amounts of pollen but also to decreased pollen viability, impaired anther opening, and pollen tube growth. An ECH translational fusion (ECHPro:ECH-YELLOW FLUORESCENT PROTEIN) revealed developmentally regulated tissue-specific expression, with expression in the tapetum during early anther development and microspore release and subsequent expression in the pollen, pollen tube, and stylar tissues. Pollen viability and production, along with germination and pollen tube growth, were all impaired. The ech anther endothecium secondary wall thickening also appeared reduced and disorganized, resulting in incomplete anther opening. This did not appear to be due to anther secondary thickening regulatory genes but perhaps to altered secretion of wall materials through the TGN as a consequence of the absence of the ECH protein. ECH expression is critical for a variety of aspects of male reproduction, including the production of functional pollen grains, their effective release, germination, and tube formation. These stages of pollen development are fundamentally influenced by TGN trafficking of hormones and wall components. Overall, this suggests that the fertility defect is multifaceted, with the TGN trafficking playing a significant role in the process of both pollen formation and subsequent fertilization.Pollen production and release is a critical stage in plant development that typically involves gene expression from over half of the genome. The extent of genomic involvement in pollen development is illustrated by the high frequency of mutations that result in a failure of male fertility; these can be a consequence of the failure of pollen development or pollen release, dehiscence. Detailed analysis of male-sterile mutants in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) has improved the basic understanding of pollen and anther development (Scott et al., 2004; Ma, 2005; Wilson and Zhang, 2009; Cui et al., 2012); however, there are multiple aspects of pollen formation that are still unclear, and many defects result in uncharacterized effects of reduced fertility or complete sterility.The ECHIDNA (ECH) gene was initially identified from expression profiling of the vascular cambium in poplar (Populus spp.) and associated with secondary xylem formation (Hertzberg et al., 2001). The Arabidopsis ech mutant was shown to have a bushy stature with defects in root and hypocotyl elongation, which was linked to defective cell expansion and elongation (Gendre et al., 2011). Analysis of roots in the ech mutant and complementation analyses in yeast showed that the ECH protein impacts on cell expansion by mediating trans-Golgi network (TGN) secretory trafficking but does not affect endocytosis (Gendre et al., 2011). However, in addition to the defects associated with plant stature, the ech mutant also displays a previously unreported phenotype of reduced fertility.Pollen development occurs in a specialized organ, the stamen, which comprises anthers that hold the developing pollen supported by a filament containing the vasculature connections. Stamen primordia arise from divisions in the L1, L2, and L3 layers in the floral meristem. Divisions in the L2 layer result in four clusters of archesporial cells that subsequently form the central sporogenous cells, which are surrounded by four maternal cell layers: the tapetum, middle cell layer, endothecium, and outer epidermis (Scott et al., 2004). The structure of the maternal anther cell layers has been shown to be critical for the production and release of functional pollen, as demonstrated in a number of male-sterile mutants, which have defects in cell division and early stages of differentiation of the tapetum and sporogenous cells. For example, mutants of the Leu-rich repeat receptor kinase EXTRA SPOROGENOUS CELLS (EXS)/EXCESS MICROSPOROCYTES1 (Canales et al., 2002; Zhao et al., 2002) and its ligand TAPETAL DETERMINANT1 (Jia et al., 2008) result in sterility due to the formation of additional male sporocytes and a lack of tapetal cells.The tapetum has been shown to be critical for functional pollen formation, with many of the characterized male-sterile mutants exhibiting abnormal tapetal development, including DYSFUNCTIONAL TAPETUM1 (DYT1; Zhang et al., 2006; Zhu et al., 2008), TAPETAL DEVELOPMENT AND FUNCTION1 (TDF1; Zhu et al., 2008), ABORTED MICROSPORES (AMS; Sorensen et al., 2003; Xu et al., 2010), and MALE STERILITY1 (MS1; Wilson et al., 2001; Ito and Shinozaki, 2002). After differentiation, the tapetum layer becomes metabolically highly active and plays an essential role in the biosynthesis and secretion of sporopollenin and other wall materials for the developing pollen, prior to breakdown via programmed cell death (Ariizumi and Toriyama, 2011). A frequently observed phenotype in male-sterile mutants is enlarged tapetal cells that show defects in secretion and subsequent alterations in programmed cell death breakdown (Wilson and Zhang, 2009). This indicates the important role that the tapetum plays in the regulation of pollen development and, in particular, the passage of materials to the central locule for viable pollen production.Male-sterile phenotypes have also been identified due to a failure of pollen release, dehiscence. Secondary thickening occurs specifically in the endothecium layer of the anther; this layer and the presence of selective thickening within it are critical to generate the differential forces that are required for anther dehiscence and pollen release (Wilson et al., 2011; Nelson et al., 2012). The importance of this secondary thickening is demonstrated in the myb26 mutant (Yang et al., 2007) and in the double NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 NAC SECONDARY WALL THICKENING PROMOTING FACTOR2 (nst1 nst2) mutant (Mitsuda et al., 2005), which lack endothecium thickening and, as a result, fail to dehisce (Nelson et al., 2012).Previous investigations of the ech mutation indicated that it is impaired in TGN secretion, resulting in dwarf plants with defects in root and hypocotyl cell elongation. The ech mutant also has an uncharacterized phenotype of impaired male fertility; therefore, a detailed analysis of reproduction in the ech mutant was conducted. ECH expression was seen in the anther tapetum during the early stages of tapetal development and microspore release but was subsequently detected in the pollen, pollen tube, and stylar tissues. The reduced fertility was linked to decreased anther size and pollen production but also to reductions in pollen viability, anther opening, and pollen tube growth. The anther wall thickening was reduced and disorganized in ech, possibly as a consequence of altered secretion of wall materials through the TGN. The male-sterile myb26 mutant has defects in anther endothecium wall thickening resulting in a failure of dehiscence; the ech myb26 double mutant exhibits the phenotypes of both mutants and fails to produce secondary thickening, indicating that the ECH-mediated pathway is acting independently of or upstream through MYB26, possibly by providing the components required for secondary cell wall thickening. The reduction in male fertility, therefore, is likely to be a consequence of multiple effects due to altered secretion in the anther because of impaired TGN transport in the ech mutant; the resulting defects are associated with tapetum and pollen wall development but also anther dehiscence and pollen tube formation. |
