Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Dr. Héctor Torres (INGEBI-CONICET), Vuelta de Obligado 2490, Buenos Aires, C1428ADN, Argentina.
Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Biodiversidad y Biología Experimental, Intendente Güiraldes 2160, Ciudad Universitaria, Pabellón II, C1428EGA, Buenos Aires, Argentina.
J Exp Bot. 2024 Jul 23;75(14):4415-4427. doi: 10.1093/jxb/erae269.
Major constituents of the plant cell walls are structural proteins that belong to the hydroxyproline-rich glycoprotein (HRGP) family. Leucine-rich repeat extensin (LRX) proteins contain a leucine-rich domain and a C-terminal domain with repetitive Ser-Pro3-5 motifs that are potentially to be O-glycosylated. It has been demonstrated that pollen-specific LRX8-LRX11 from Arabidopsis thaliana are necessary to maintain the integrity of the pollen tube cell wall during polarized growth. In HRGPs, including classical extensins (EXTs), and probably in LRXs, proline residues are converted to hydroxyproline by prolyl-4-hydroxylases (P4Hs), thus defining novel O-glycosylation sites. In this context, we aimed to determine whether hydroxylation and subsequent O-glycosylation of Arabidopsis pollen LRXs are necessary for their proper function and cell wall localization in pollen tubes. We hypothesized that pollen-expressed P4H4 and P4H6 catalyze the hydroxylation of the proline units present in Ser-Pro3-5 motifs of LRX8-LRX11. Here, we show that the p4h4-1 p4h6-1 double mutant exhibits a reduction in pollen germination rates and a slight reduction in pollen tube length. Pollen germination is also inhibited by P4H inhibitors, suggesting that prolyl hydroxylation is required for pollen tube development. Plants expressing pLRX11::LRX11-GFP in the p4h4-1 p4h6-1 background show partial re-localization of LRX11-green fluorescent protein (GFP) from the pollen tube tip apoplast to the cytoplasm. Finally, immunoprecipitation-tandem mass spectrometry analysis revealed a decrease in oxidized prolines (hydroxyprolines) in LRX11-GFP in the p4h4-1 p4h6-1 background compared with lrx11 plants expressing pLRX11::LRX11-GFP. Taken together, these results suggest that P4H4 and P4H6 are required for pollen germination and for proper hydroxylation of LRX11 necessary for its localization in the cell wall of pollen tubes.
植物细胞壁的主要成分是结构蛋白，属于羟脯氨酸丰富糖蛋白 (HRGP) 家族。富含亮氨酸的重复伸展蛋白 (LRX) 蛋白含有亮氨酸丰富的结构域和 C 端结构域，其中含有重复的 Ser-Pro3-5 基序，这些基序可能被 O-糖基化。已经证明，拟南芥花粉特异性 LRX8-LRX11 对于维持花粉管细胞壁在极化生长过程中的完整性是必要的。在 HRGPs 中，包括经典伸展蛋白（EXTs），并且可能在 LRXs 中，脯氨酸残基被脯氨酰-4-羟化酶（P4Hs）转化为羟脯氨酸，从而定义了新的 O-糖基化位点。在这种情况下，我们旨在确定拟南芥花粉 LRXs 的羟化和随后的 O-糖基化是否对其在花粉管中的正确功能和细胞壁定位是必要的。我们假设花粉表达的 P4H4 和 P4H6 催化 LRX8-LRX11 中 Ser-Pro3-5 基序中脯氨酸单位的羟化。在这里，我们表明 p4h4-1 p4h6-1 双突变体表现出花粉萌发率降低和花粉管长度略有减少。P4H 抑制剂也抑制花粉萌发，表明脯氨酰羟化是花粉管发育所必需的。在 p4h4-1 p4h6-1 背景下表达 pLRX11::LRX11-GFP 的植物显示 LRX11-绿色荧光蛋白（GFP）从花粉管尖端质外体部分重新定位到细胞质。最后，免疫沉淀-串联质谱分析显示，与表达 pLRX11::LRX11-GFP 的 lrx11 植物相比，p4h4-1 p4h6-1 背景下的 LRX11-GFP 中的氧化脯氨酸（羟脯氨酸）减少。综上所述，这些结果表明 P4H4 和 P4H6 对于花粉萌发以及 LRX11 的正确羟化是必需的，这对于其在花粉管细胞壁中的定位是必需的。
