Laboratório de Biotecnologia, Centro de Biociências e Biotecnologia (CBB), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Avenida Alberto Lamego 2000, Campos dos Goytacazes, Rio de Janeiro 28013-602, Brazil; Unidade de Biologia Integrativa, Setor de Genômica e Proteômica, CBB, UENF, Brazil.
Laboratório de Melhoramento Genético Vegetal, LMGV, UENF, Brazil.
J Proteomics. 2022 Feb 10;252:104434. doi: 10.1016/j.jprot.2021.104434. Epub 2021 Nov 21.
Understanding the mechanisms that endow a somatic cell with the ability to differentiate into a somatic embryo, which could result in numerous biotechnological applications, is still a challenge. The objective of this work was to identify some of the molecular and physiological mechanisms responsible for the acquisition of embryogenic competence during somatic embryogenesis in Carica papaya L. We performed a broad characterization of embryogenic (EC) and nonembryogenic calli (NEC) of using global and mitochondrial proteomic approaches, histomorphology, histochemistry, respiratory activity, and endogenous hormonal and hydrogen peroxide (HO) contents. EC and NEC presented remarkable differences in anatomical and histochemical characteristics, with EC showing a higher reactivity for the presence of proteins and neutral polysaccharides. Our results demonstrate that mitochondrial metabolism affects the embryogenic competence of C. papaya callus. The EC presented higher participation of alternative oxidase (AOX) enzymes, higher total cell respiration and presented a stronger accumulation of mitochondrial stress response proteins. Differential accumulation of auxin-responsive Gretchen Hagen 3 (GH3) family proteins in EC was related to a decrease in the content of free 2,4-dichlorophenoxyacetic acid (2,4-D). EC also showed higher endogenous HO contents. HO is a promising molecule for further investigation in differentiation protocols for C. papaya somatic embryos. SIGNIFICANCE: To further advance the understanding of somatic embryogenesis, we performed a broad characterization of embryogenic and nonembryogenic callus, through global and mitochondrial proteomic approaches, histomorphology, histochemistry, respiratory activity, and endogenous hormonal and hydrogen peroxide contents. Based on these results, we propose a working model for the competence of papaya callus. This model suggests that GH3 proteins play an important role in the regulation of auxins. In addition, embryogenic callus showed a greater abundance of stress response proteins and folding proteins. Embryogenic callus respiration occurs predominantly via AOX, and the inhibition of its activity is capable of inhibiting callus differentiation. Although the embryogenic callus presented greater total respiration and a greater abundance of oxidative phosphorylation proteins, they had less COX participation and less coupling efficiency, indicating less ATP production.
理解赋予体细胞分化为体细胞胚的能力的机制，这可能导致许多生物技术应用，仍然是一个挑战。本工作的目的是鉴定一些分子和生理机制，这些机制负责在 Carica papaya L. 的体细胞胚胎发生过程中获得胚胎发生能力。我们使用全局和线粒体蛋白质组学方法、组织形态学、组织化学、呼吸活性以及内源激素和过氧化氢 (HO) 含量对胚胎发生 (EC) 和非胚胎发生 (NEC) 愈伤组织进行了广泛的表征。EC 和 NEC 在解剖学和组织化学特征上表现出显著差异，EC 对蛋白质和中性多糖的存在表现出更高的反应性。我们的结果表明，线粒体代谢影响 C. papaya 愈伤组织的胚胎发生能力。EC 表现出更高的替代氧化酶 (AOX) 酶参与度、更高的总细胞呼吸和更强的线粒体应激反应蛋白积累。EC 中生长素应答 Gretchen Hagen 3 (GH3) 家族蛋白的差异积累与游离 2,4-二氯苯氧乙酸 (2,4-D) 含量的降低有关。EC 还表现出更高的内源 HO 含量。HO 是进一步研究 C. papaya 体细胞胚胎分化方案的有前途的分子。意义：为了进一步深入了解体细胞胚胎发生，我们通过全局和线粒体蛋白质组学方法、组织形态学、组织化学、呼吸活性以及内源激素和过氧化氢含量对胚胎发生和非胚胎发生愈伤组织进行了广泛的表征。基于这些结果，我们提出了一个木瓜愈伤组织能力的工作模型。该模型表明，GH3 蛋白在生长素的调节中起着重要作用。此外，胚胎发生愈伤组织表现出更多的应激反应蛋白和折叠蛋白。胚胎发生愈伤组织呼吸主要通过 AOX 发生，其活性的抑制能够抑制愈伤组织分化。尽管胚胎发生愈伤组织表现出更高的总呼吸和更多的氧化磷酸化蛋白，但它们的 COX 参与度较低，偶联效率较低，表明 ATP 产量较低。
