Calabuig-Serna Antonio, Sancho-Oviedo Daniel, Rivas-Sendra Alba, Mata-Nicolás Estefanía, Arjona-Mudarra Paloma, Mir Ricardo, Seguí-Simarro Jose María
Cell Biology Group, COMAV Institute, Universitat Politècnica de València, 46022 Valencia, Spain.
Ann Bot. 2025 Sep 2;136(1):99-110. doi: 10.1093/aob/mcaf054.
Microspore embryogenesis is a convenient inducible system to study the changes associated with the developmental reprogramming of cells. In this work, Brassica napus microspore cultures were used to study the role in the embryogenic switch of callose and pectin cell wall composition, which depends on Ca2+ levels.
We used different chemicals to modulate Ca2+, callose and pectin methyl-esterification, including Ca(NO3)2, inositol 1,4,5-trisphosphate, 2-deoxy-d-glucose, benzyl alcohol, chitosan, epigallocatechin gallate and pectin methyl-esterase. Ca2+ distribution, callose and cellulose were imaged with FluoForte, Aniline Blue and Pontamine Fast Scarlet stainings, respectively, and observed with confocal microscopy.
Inhibition of callose synthesis with 2-deoxy-d-glucose demonstrated that callose is essential for induction of microspore embryogenesis. A moderate increase of Ca2+ levels with Ca(NO3)2 or inositol 1,4,5-trisphosphate promoted increased callose synthesis and deposition in the cell wall. However, the use of benzyl alcohol and chitosan to permeabilize the plasma membrane and allow for Ca2+ influx was not positive, because this prevented embryo development by inducing callus formation. Benzyl alcohol did not affect callose and cellulose deposition, but chitosan induced the formation of callose plugs, similar to those formed in response to pathogen attack. Inhibition of pectin methyl-esterase activity with epigallocatechin gallate during the first 3 days of culture produced ~70 % more embryos, but prolonged exposures were negative. Instead, increased pectin methyl-esterase activity during the first 3 days was not positive, but when applied for 7 days, embryos increased by ~60 %.
Together, these results confirm the relevant role of calcium and callose during the first stages of microspore induction and suggest that the levels of pectin methyl-esterification in the cell wall are dynamic and that different cell wall compositions are required during the different stages of microspore embryogenesis.
小孢子胚胎发生是一种方便的诱导系统，用于研究与细胞发育重编程相关的变化。在本研究中，利用甘蓝型油菜小孢子培养物来研究胼胝质和果胶细胞壁成分在胚胎发生转变中的作用，这取决于钙离子水平。
我们使用不同的化学物质来调节钙离子、胼胝质和果胶甲基化，包括硝酸钙、肌醇1,4,5 -三磷酸、2 -脱氧 - d -葡萄糖、苄醇、壳聚糖、表没食子儿茶素没食子酸酯和果胶甲酯酶。分别用FluoForte、苯胺蓝和滂胺固红染色对钙离子分布、胼胝质和纤维素进行成像，并通过共聚焦显微镜观察。
用2 -脱氧 - d -葡萄糖抑制胼胝质合成表明，胼胝质对于诱导小孢子胚胎发生至关重要。用硝酸钙或肌醇1,4,5 -三磷酸适度提高钙离子水平可促进细胞壁中胼胝质合成和沉积增加。然而，使用苄醇和壳聚糖使质膜通透以允许钙离子内流并不理想，因为这会通过诱导愈伤组织形成而阻碍胚胎发育。苄醇不影响胼胝质和纤维素沉积，但壳聚糖会诱导形成胼胝质栓，类似于对病原体攻击的响应所形成的栓。在培养的前3天用表没食子儿茶素没食子酸酯抑制果胶甲酯酶活性可使胚胎产量增加约70%，但延长处理时间则产生负面效果。相反，在培养的前3天增加果胶甲酯酶活性并不理想，但处理7天时，胚胎数量增加约60%。
总之，这些结果证实了钙和胼胝质在小孢子诱导初期的相关作用，并表明细胞壁中果胶甲基化水平是动态的，且在小孢子胚胎发生的不同阶段需要不同的细胞壁组成。
