Coordinated Accumulation of (+)-δ-Cadinene Synthase mRNAs and Gossypol in Developing Seeds of Gossypium hirsutum and a New Member of the cad1 Family from G. arboreum

Abstract

A new member of the (+)-δ-cadinene synthase (CAD1) family was isolated from a Gossypium arboreum cDNA library. This cDNA encodes a protein that showed 97.3%, 96.9%, and 79.2% sequence identities with the proteins encoded by previously isolated cDNAs of cad1-C1, cad1-C14, and cad1-A, respectively. It may be grouped into the cad1-C subfamily as cad1-C2. Seeds of a glanded cotton cultivar, G. hirsutum cv. Sumian-6, were collected at different intervals during maturation, and the cad1 mRNA levels were analyzed by quantitative RT-PCR. The transcripts could be detected in seeds of 27 DPA (days postanthesis) and increased dramatically along with the seed maturation, which coordinated with an increase in sesquiterpene cyclase activities and subsequently the accumulation of gossypol. The transcription level detected with primers specific to cad1-C (including at least C1, C14, and cdn1) was higher than that detected with primers specific to cad1-A, and mRNA was detected also with cad1-C2-specific primers. This investigation indicates that, in developing seeds of the glanded cotton cultivar, genes of both the CAD1-C and CAD1-A subfamilies are expressed and there is an active biosynthesis of cadinene-type sesquiterpenes.Plant defense mechanisms involve the induced expression of a complex assortment of genes, including those coding for enzymes catalyzing the formation of antimicrobial secondary metabolites (phytoalexins). In cotton [Gossypium hirsutum L. and Gossypium arboreum L. (Malvaceae)], the production of cadinene-type sesquiterpenes was induced clearly in plants inoculated with the phytopathogenic bacterium Xanthomonas campestris 1,2 and in cell suspension cultures treated with elicitors prepared from the wilt disease-causing fungus Verticillium dahliae. 3 However, cotton sesquiterpenes, including the phenolic aldehyde gossypol, also are constitutive components present in subepidermal pigment glands. 4,5 The occurrence of pigment glands is a distinctive character in the tribe Gossypieae of the family Malvaceae. 4 The glands are found throughout the plant, and they are particularly dense in cotyledons of mature seeds of most cotton cultivars, and high levels of sesquiterpenes (mainly gossypol) are accumulated and deposited in seed glands. 6,7 Sesquiterpene cyclase catalyzes the cyclization of linear farnesyl diphosphate (FPP), the first step committed to the formation of various cyclic sesquiterpenes. [8][9][10] Previous investigations have characterized three cDNAs of the cotton sesquiterpene cyclase, (+)-δ-cadinene synthase (CAD1), from G. arboreum. On the basis of sequence similarities, they were grouped into two subfamilies, cad1-C (including C1 and C14) and cad1-A. 11,12 A cDNA from G. hirsutum, cdn1, which is over 95% identical to G. arboreum cad1-C1 and cad1-C14, has been isolated recently. 13 In addition, the enzyme was purified from bacterial-inoculated G. hirsutum folia tissues. 14 In G. arboreum cell suspension cultures, mRNA levels of both cad1-C and cad1-A subfamilies were increased by 10 times at 6-10 h p...

Gossypol: phytoalexin of cotton

“…During ovule (embryo) development, biosynthesis of gossypol is tightly regulated: before 15 DPA (day post-anthesis), gossypol is undetectable; from 15-20 DPA, gossypol begin to be synthesized and then accumulated rapidly in later stages, and after 30 DPA the pigment glands containing mostly gossypol can be viewed clearly. Consistent with this, transcripts of CDN-C and CYP706B1 are also undetectable before 15 DPA, then start to accumulate at about 20 DPA and peak at about 40 DPA (Meng et al, 1999).…”

“…After multiple hydroxylation and oxidation steps, it is converted to hemigossypol and then coupled into gossypol. Several enzymes involved in this process have been isolated and characterized, including 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) (Joost et al, 1995), farnesyl diphosphate synthase (FPS) (Liu et al, 1999a), (+)--cadinene synthase (CDN) Chen et al, 1995;Meng et al, 1999;Tan et al, 2000), a P450 monooxygenase (CYP706B1) (Luo et al, 2001;Wang et al, 2003), P450 reductase (Yang et al, 2010), as well as a methyltransferase (Liu et al, 1999b). Recently, evidences suggest that the CYP82D109 (P450) is potentially a new gene involved in this pathway (Wagner et al, 2015) (Figure 1 B).…”

“…Moreover, the total proteins isolated from cotton cotyledons treated with pathogen elicitors was able to catalyze the conversion of FPP into (+)--cadinene at a high rate, indicating that (+)--cadinene is a critical precursor or intermediate for phytoalexin biosynthesis Davila-Huerta et al, 1995;Davis and Essenberg, 1995). Later characterization of multiple (+)--cadinene synthase (CDN) proteins with different sequences revealed that it is encoded by a family includes at least CDN-A, CDN-B, CDN-C and CDN-D subfamilies Benedict et al, 2001;Chen et al, 1995;Davis et al, 1996;Davis and Essenberg, 1995;Meng et al, 1999), and each has variable expression pattern in different tissues (Benedict et al, 2004;Meng et al, 1999;Tan et al, 2000) and can be induced by pathogens (Davila-Huerta et al, 1995;Davis et al, 1996;Liu et al, 1999a) (Figure 1B).…”

Characterization of GaWRKY1, a Cotton Transcription Factor That Regulates the Sesquiterpene Synthase Gene (+)-δ-Cadinene Synthase-A

“…However, this does not exclude the possibility that GaWRKY1 may also regulate CAD1-C genes in other organs. In developing seeds, for example, genes of CAD1-A and CAD1-C showed similar temporal patterns of expression (Meng et al, 1999). Alternatively, GaWRKY1 may function as a repressor of CAD1-C genes in flower.…”

“…Formation of gossypol and related sesquiterpene aldehydes is also developmentally regulated, and these secondary metabolites accumulate in pigmented glands of aerial tissues and in epidermal and subepidermal cells of roots (Bell and Stipanovic, 1977;Halloin and Bell, 1979;Meng et al, 1999). All cotton sesquiterpene phytoalexins identified so far have a cadinene-type skeleton, and biochemical and molecular evidence has suggested that (1)-d-cadinene is the biosynthetic precursor (Chen et al, 1995;DavilaHuerta et al, 1995;Luo et al, 2001).…”