KNS4/UPEX1: A Type II Arabinogalactan β-(1,3)-Galactosyltransferase Required for Pollen Exine Development

Abstract

Pollen exine is essential for protection from the environment of the male gametes of seed-producing plants, but its assembly and composition remain poorly understood. We previously characterized Arabidopsis (Arabidopsis thaliana) mutants with abnormal pollen exine structure and morphology that we named kaonashi (kns). Here we describe the identification of the causal gene of kns4 that was found to be a member of the CAZy glycosyltransferase 31 gene family, identical to UNEVEN PATTERN OF EXINE1, and the biochemical characterization of the encoded protein. The characteristic exine phenotype in the kns4 mutant is related to an abnormality of the primexine matrix laid on the surface of developing microspores. Using light microscopy with a combination of type II arabinogalactan (AG) antibodies and staining with the arabinogalactan-protein (AGP)-specific b-Glc Yariv reagent, we show that the levels of AGPs in the kns4 microspore primexine are considerably diminished, and their location differs from that of wild type, as does the distribution of pectin labeling. Furthermore, kns4 mutants exhibit reduced fertility as indicated by shorter fruit lengths and lower seed set compared to the wild type, confirming that KNS4 is critical for pollen viability and development. KNS4 was heterologously expressed in Nicotiana benthamiana, and was shown to possess b-(1,3)-galactosyltransferase activity responsible for the synthesis of AG glycans that are present on both AGPs and/or the pectic polysaccharide rhamnogalacturonan I. These data demonstrate that defects in AGP/pectic glycans, caused by disruption of KNS4 function, impact pollen development and viability in Arabidopsis.Pollen, the male gametophyte of all seed plants, is crucial for reproductive success. Due to the harsh environmental conditions that pollen must survive in, it has developed an elaborate and specialized cell wall. However, despite its importance our knowledge of the fine structure and assembly of the pollen grain wall is poorly understood (Newbigin et al., 2009;Ariizumi and Toriyama, 2011;Quilichini et al., 2015;Shi et al., 2015). Arabidopsis (Arabidopsis thaliana) provides an ideal genetic and cell biological model to dissect the assembly of the components of the pollen grain wall. Arabidopsis pollen grains have a typical surface structure that consists of an inner intine layer, which is usually made up of cellulose and pectin, and the outer exine, which is largely composed of sporopollenin (Li et al., 1995). The exine is further subdivided into inner nexine and outer sexine. The sexine has a three-dimensional (3D) structure composed of many columns called baculae and a roof called tectum. These two structures give the Arabidopsis pollen surface a reticulate appearance with a uniform mesh size. The nexine is composed of outer nexine I (foot layer), which contains sporopollenin, and inner nexine II (endexine; Ariizumi and Toriyama, 2011;Jiang et al., 2013). A recent study suggests that arabinogalactan proteins (AGPs) are key constituents of nexine with express...

PRX9 and PRX40 Are Extensin Peroxidases Essential for Maintaining Tapetum and Microspore Cell Wall Integrity during Arabidopsis Anther Development

“…In the model plant Arabidopsis, tapetum hypertrophy has not been seen as a stress phenotype but instead was reported as a developmental phenotype associated with a wide collection of mutants, including roxy1, roxy2, aborted microspores, male ste-rility1, defective in tapetal development and function1, dysfunctional tapetum1, myb33, myb65, bhlh010, bhlh089, bhlh091, kaonashi4/uneven pattern of exine1, gus negative1, gus nega-tive4, and fat tapetum (Sanders et al, 1999;Wilson et al, 2001;Sorensen et al, 2002Sorensen et al, , 2003Millar and Gubler, 2005;Zhang et al, 2006;Xing and Zachgo, 2008;Zhu et al, 2008Zhu et al, , 2015Phan et al, 2011;Suzuki et al, 2017). Besides KAONASHI4/UNEVEN PATTERN OF EXINE1, which is a glycosyl transferase, these genes encode regulatory genes and transcription factors or are unmapped mutant alleles.…”

PRX9andPRX40are extensin peroxidases essential for maintaining tapetum and microspore cell wall integrity duringArabidopsisanther development

“…In the model plant Arabidopsis thaliana, tapetum hypertrophy has been seen as a developmental phenotype associated with a wide collection of mutants, including roxy1, roxy2, ams, ms1, tdf1, dyt1, myb33, myb65, bhlh010, bhlh089, bhlh091, kns4/upex1, gne1, gne4, and fat tapetum (Sorensen et al, 2003;Wilson et al, 2001;Zhu et al, 2008;Zhang et al, 2006;Sanders et al, 1999;Sorensen et al, 2002;Millar and Gubler, 2005;Suzuki et al, 2017;Phan et al, 2011;Xing and Zachgo, 2008;Zhu et al, 2015).…”

Golgi-localized exo-β1,3-galactosidases involved in AGP modification and root cell expansion in Arabidopsis

“…AGP glycosylation is initiated by proline hydroxylation in the ER and is then advanced in the Golgi apparatus by specialized glycosyl transferases (GTs) (Rose and Lee, 2010; Hijazi et al, 2014). The glycosyl transferase family 31 (GT31) is responsible for the synthesis of the hydroxyproline galactose and the β-1,3-galactan backbone of AGPs (Basu et al, 2015; Basu et al, 2015; Ogawa-Ohnishi and Matsubayashi, 2015; Suzuki et al, 2017). Defects in β-1,3-galactan biosynthesis are associated with various developmental phenotypes including defects in cell expansion (Basu et al, 2015; Ogawa-Ohnishi and Matsubayashi, 2015; Suzuki et al, 2017).…”

“…The glycosyl transferase family 31 (GT31) is responsible for the synthesis of the hydroxyproline galactose and the β-1,3-galactan backbone of AGPs (Basu et al, 2015; Basu et al, 2015; Ogawa-Ohnishi and Matsubayashi, 2015; Suzuki et al, 2017). Defects in β-1,3-galactan biosynthesis are associated with various developmental phenotypes including defects in cell expansion (Basu et al, 2015; Ogawa-Ohnishi and Matsubayashi, 2015; Suzuki et al, 2017). The side chains, which branch off from the β-1,3-galactan back bone via β-1,6 linkages, are synthesized by GALT29A and/or GT31 family enzymes (Geshi et al, 2013; Dilokpimol et al, 2014).…”