Cotton roots are the major source of gossypol biosynthesis and accumulation

Zhao et al. BMC Plant Biology (2020) 20:88 https://doi.org/10.1186/s12870-020-2294-9 RESEARCH ARTICLE Open Access Cotton roots are the major source of gossypol biosynthesis and accumulation Tianlun Zhao1, Qianwen Xie1, Cong Li1, Cheng Li1, Lei Mei1, John Z. Yu2, Jinhong Chen1 and Shuijin Zhu1* Abstract Background: Gossypol is a specific secondary metabolite in Gossypium species. It not only plays a critical role in development and self-protection of cotton plants, but also can be used as important anti-cancer and male contraceptive compound. However, due to the toxicity of gossypol for human beings and monogastric animals, the consumption of cottonseeds was limited. To date, little is known about the gossypol metabolism in cotton plants. Results: In this study, we found that cotyledon was the primary source of gossypol at the seed germination stage. But thereafter, it was mainly originated from developing roots. Grafting between glanded and glandless cotton as well as sunflower rootstocks and cotton scion revealed that gossypol was mainly synthesized in the root systems of cotton plants. And both glanded and glandless cotton roots had the ability of gossypol biosynthesis. But the pigment glands, the main storage of gossypol, had indirect effects on gossypol biosynthesis. In vitro culture of root and rootless seedling confirmed the strong gossypol biosynthesis ability in root system and the relatively weak gossypol biosynthesis ability in other organs of the seedling. Expression profiling of the key genes involved in the gossypol biosynthetic pathway also supported the root as the major organ of gossypol biosynthesis. Conclusions: Our study provide evidence that the cotton root system is the major source of gossypol in both glanded and glandless cottons, while other organs have a relatively weak ability to synthesize gossypol. Gossypol biosynthesis is not directed related to the expression of pigment glands, but the presence of pigment glands is essential for gossypol accumulation. These findings can not only clarify the complex regulation network of gossypol metabolism, but it could also accelerate the crop breeding process with enhanced commercial values. Keywords: Cotton, Gossypol biosynthesis, Plant grafting, Organ culture, Gene expression profiling Background Cotton (Gossypium spp.) is one of the most important economic crops in the world. Cotton not only produces natural fiber for textile industry, but it also provides a large quantity of cottonseeds which contain high-quality protein and oil [1]. It is estimated that every kilogram fiber yield is coupled with 1.65 kg cottonseeds, which contain approximately 21% oil and 23% protein [2]. However, cottonseeds cannot be used directly due to the presence of gossypol, a toxic substance to human beings and monogastric animals [3]. On the other hand, gossypol plays an important role in self-protection of cotton plants [4–6]. * Correspondence: 1 Department of Agronomy, Zhejiang University, Hangzhou 310058, Zhejiang, China Full list of author information is available at the end of the article Gossypol was first characterized by Adams et al. in 1938 through a series of classic studies [7]. It is a polyphenolic aldehyde which constitutes 20–40% of the pigment glands weight and accounts for 0.4–1.7% of the whole cottonseed kernel. As a phytoalexin, gossypol provides constitutive and inducible resistance against pests and pathogens [8–13]. Besides, gossypol can be used as anti-cancer [14–16], anti-bacterial [17, 18] and male contraceptive reagent [19–21]. There are two different enantiomers of gossypol, (+)-gossypol and (−)-gossypol. Based on previous studies, the biological activity of (−)-gossypol is stronger than that of (+)-gossypol [22– 24]. Additionally, the difference in ratio of enantiomers in cottonseeds might affect the poultry production when they were used as poultry feed [25]. Several key genes involved in the pathway of the gossypol biosynthesis have been identified and characterized, such as terpene synthase genes, GhTPS1 and © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhao et al. BMC Plant Biology (2020) 20:88 GhTPS2 [26], and hmg1 and hmg2 that encode the limiting enzymes of isoprenoid biosynthesis pathway [27]. Other key genes, which usually exist as gene families, of the gossypol biosynthesis pathway, include CAD1-A, CAD1-C2 [28] and cdn1-C4 [29]. In addition, GaCYP706B1 was isolated as the gene encoding cadinene-8-hydroxylase, the key enzyme in the hemigossypol biosynthesis [30]. GaWRKY1, a transcription factor involved in regulation of sesquiterpene biosynthesis which affects the expression of CAD-1, has also been identified [31]. A recent study characterized four key genes in the downstream of gossypol biosynthetic pathway [32]. With tremendous progress in molecular biology, gene transformation, virus-induced gene silencing (VIGS) and CRISPR/Cas9 system have been applied to study cotton traits [2, 33, 34]. These molecular tools together with other genetic and physiological approaches provide a good opportunity to dissect the complex mechanism underlying gossypol metabolism. To make an apparent relationship between pigment gland and gossypol content in cotton plants is very important. Punit et al. found that different genotypes had an impact on the distribution of pigment glands [35]. And the gossypol content in cotton was closely related to the genotypes of pigment glands. Singh and Weaver proposed that the gossypol content was highly correlated with the number of pigment glands [36], except in Gossypium somalense Huntch, which had almost no gossypol in seed although the presence of normal pigment glands [37]. Silencing CYP706B1, a key gene in the gossypol biosynthesis pathway, Ma et al. found that the gossypol content significantly decreased but the pigment glands still formed as normal plant, and eliminating the pigment glands through VIGS led to decline greatly in gossypol content [33]. Therefore, the relationship between gossypol and pigment glands was related but remains unclear, which need more research to clarify. Understanding the metabolism of gossypol is essential for developing cotton cultivars with low gossypol seeds that have a wider utilization potential. However, only few studies have been carried out to elucidate the biosynthesis and transportation of gossypol. Smith proposed that gossypol was synthesized in cotton root b (...truncated)