Jinggao Liu

Stereoselective coupling of hemigossypol to form (+)-gossypol in moco cotton is mediated by a dirigent protein

The terpenoid gossypol, a secondary metabolite found in the cotton plant, is synthesized by a free radical dimerization of hemigossypol. Gossypol exists as an atropisomeric mixture because of restricted rotation around the central binaphthyl bond. The dimerization of hemigossypol is regiospecific in cotton. In the case of some moco cotton, the dimerization also exhibits a high level of stereoselectivity. The mechanism that controls this stereoselective dimerization is poorly understood. In this paper, we demonstrate that a dirigent protein controls this stereoselective dimerization process. A partially purified protein preparation from cotton flower petals, which by itself is unable to convert hemigossypol to gossypol, converts hemigossypol with a 30% atropisomeric excess into (+)-gossypol when combined with an exogenous laccase, which by itself produces racemic gossypol.

The enzymatic formation of δ-cadinene from farnesyl diphosphate in extracts of cotton

Abstract Incubation of extracts of cotton stele tissue which had been infected with Verticillium dahliae led to the enzymatic incorporation of [1-3H]-farnesyl diphosphate into hydrocarbons soluble in hexane-ethyl acetate. The mixture of [3H]-hydrocarbons was separated into components by HPLC. The hydrocarbon with a retention time of 17.5–18.0 min, biosynthetically prepared from (1RS)-[1- 2 H ]-(E,E)- farnesyl diphosphate, was analysed by GC-MS. The data not only agree with the reported mass spectrum of δ-cadinene but also support the proposed hydride shift in the biosynthesis of δ-cadinene from farnesyl diphosphate.

The enzymatic cyclization of nerolidyl diphosphate by δ-cadinene synthase from cotton stele tissue infected with verticillium dahliae

Abstract Soluble preparations of cotton stele tissue infected with Verticillium dahliae containing δ-cadinene synthase convert (1- RS )-[1- 2 H]- E,E -farnesyl diphosphate to [5- 2 H]- and [11- 2 H]-δ-cadinene and convert [4,4,13,13,13- 2 H 5 ]-nerolidyl diphosphate to [8,8,15,15,15- 2 H 5 ]-δ-cadinene. These data imply that nerolidyl diphosphate is an intermediate in the enzymatic cyclization of the natural substrate E,E -farnesyl diphosphate to δ-cadinene by δ-cadinene synthase and involves the conversion of E,E -farnesyl diphosphate to nerolidyl diphosphate followed by cyclization to cis -germacradienyl cation, a 1,3-hydride shift, a second cyclization to a cadinanyl cation and deprotonation to δ-cadinene. Kinetic analyses of induced δ-cadinene synthase mRNA, δ-cadinene synthase activity and formation of sesquiterpenoid phytoalexins in cotton stele tissue infected with Verticillium dahliae show that 12 hr after fungal inoculation the δ-cadinene synthase mRNA was at a maximum level. The tissue injected with H 2 O in place of fungal inoculation showed no detectable δ-cadinene synthase mRNA or δ-cadinene synthase activity after 12 to 96 hr. After 12 hr, 54% of the δ-cadinene synthase activity had developed, but no phytoalexins were detected, the midpoint in the formation of the phytoalexins was 48 hr. These data, together with the enzyme analyses, support the conclusion that Verticillium dahliae initiates a signal in the stele tissue that results in an increased steady-state level of δ-cadinene synthase mRNA and an increased activity of δ-cadinene synthase which functions in the conversion of E,E -farnesyl diphosphate → nerolidyl diphosphate → δ-cadinene that is metabolically converted to desoxyhemigossypol, desoxyhemigossypol-6-methyl ether, hemigossypol and hemigossypol-6-methyl ether.

Reduced levels of cadinane sesquiterpenoids in cotton plants expressing antisense (+)-δ-cadinene synthase

Cotton plants were transformed with an antisense construct of cdn1-Cl, a member of a complex gene family of delta-(+)cadinene (CDN) synthase. This synthase catalyzes the cyclization of (E,E)-farnesyl diphosphate to form CDN, and in cotton, it occupies the committed step in the biosynthesis of cadinane sesquiterpenoids and heliocides (sesterterpenoids). Southern analyses of the digestion of leaf DNA from R(o), T(o), and T(1) plants with Hind III, Pst I and Kpn I restriction enzymes show the integration of antisense cdn1-C1 cDNA driven by the CaMV 35S promoter into the cotton genome. Northern blots demonstrate the appearance of cdn synthase mRNA preceding CDN synthase activity and the formation of gossypol in developing cottonseed. T(2) cottonseed show a reduced CDN synthase activity and up to a 70% reduction in gossypol. In T(1) leaves the accumulated amounts of gossypol, hemigossypolone and heliocides are reduced 92.4, 83.3 and 68.4%, respectively. These data demonstrate that the integration of antisense cdn1-C1 cDNA into the cotton genome leads to a reduction of CDN synthase activity and negatively impacts on the biosynthesis of cadinane sesquiterpenoids and heliocides in cotton plants.

Glandless seed and glanded plant research in cotton. A review

Recently the world has been entangled by insufficient food such as the lack of rice which threatens the safety of world food and affect sustainable development of the world economy, resulting in rising of food price. To address this issue, cotton appears as a possible source of both fiber and food. The research in recent years indeed showed bright prospects for this expectation. However, gossypol stored in the glands of cotton is toxic to nonruminant animals and humans, which wastes large amounts of cottonseed protein that could potentially provide the annual protein requirements for half a billion people. Gossypium species are characterized by their lysigenous glands containing terpenoid aldehydes, important secondary phytoalexins consisting mainly of gossypol, which constitute one of the important plant’s defense system against pests and diseases. The best approach to address this issue is to create glandless seed and glanded plant cotton. A breakthrough in this field would realise the fulfilment of making cotton both a fiber and a food crop, which would be a feat of great magnitude for sustainable development of agriculture. Research on the relationship between glands and their secondary inclusions at the molecular level would be one approach for genetic engineering to control the glands and gossypol content. In this article, we review recent progress on glands and gossypol content for diverse gland types in Gossypium species, inheritance of glands and gossypol content, traditional breeding of glandless seeds and glanded plant cotton, the terpenoid aldehyde biosynthesis pathway, molecular cloning of the related genes, the strategy for genetic engineering, and future prospects.

Phytotoxicity of fusaric acid and analogs to cotton

We developed a cotton cotyledonary leaf bioassay to test the phytotoxicity of fusaric acid (5-butylpicolinic acid), picolinic acid and related analogs. The compounds were dissolved in aqueous Tween 80, and 20 μL of the test solution was placed at three positions on the leaf, and a needle was used to puncture the leaf through each drop; the results were evaluated after 48 h. In contrast to previous studies, we found the carboxylic acid group is essential for phytotoxicity. Nicotinic acid was considerably less phytotoxic than picolinic acid and conversion of picolinic acid to the amide or N-oxide decreased phytotoxicity. Increasing the alkyl chain length at the 5-position on picolinic acid from two up to five carbons atoms increased phytotoxicity. Fusaric acid methyl ester, the most phytotoxic compound tested, is a naturally occurring compound; as such it has potential as a herbicide in organic farming.

Total and Percent Atropisomers of Gossypol and Gossypol-6-methyl Ether in Seeds from Pima Cottons and Accessions of Gossypium barbadense L

Gossypol occurs naturally in the seed, foliage, and roots of the cotton plant ( Gossypium ) as atropisomers due to restricted rotation around the binaphthyl bond. The atropisomers differ in their biological activities. (-)-(R)-Gossypol is more toxic and exhibits significantly greater anticancer activity than the (+)-(S)-atropisomer. Most commercial Upland ( Gossypium hirsutum ) cottonseeds have an (R)- to (S)-gossypol ratio of approximately 2:3, but some Pima ( Gossypium barbadense ) seeds have an excess of (R)-gossypol. There is no known source of cottonseed with an (R)- to (S)-gossypol ratio of greater than approximately 70:30. Cottonseed with a high percentage of (R)-gossypol would be of value to the pharmaceutical industry. It was theorized that G. barbadense cotton might be a source of this desirable high (R)-gossypol seed trait. There are 671 different accessions of G. barbadense in the U.S. Cotton Germplasm Collection, few of which had been characterized with respect to their (R)- to (S)-gossypol ratio. This work completed that analysis and found considerable variation in the atropisomer ratio. Approximately half of the accessions have an excess of (R)-gossypol, and 52 accessions have essentially a 1:1 ratio. The highest percentage of (R)-gossypol was found in accessions GB26 (68.2%) and GB283 (67.3%). Surprisingly, five accessions had 5% or less of (R)-gossypol: GB516 (5.0%), GB761 (4.5%), GB577 (4.3%), GB719 (3.7%), and GB476 (2.3%). These accessions may be useful in a breeding program to reduce (R)-gossypol in Pima seed, which is a concern to the dairy industry because of the toxicity and male antifertility activity of this atropisomer. Also, GB710 was devoid of gossypol.

Phylogeny and pathogenicity of Fusarium oxysporum isolates from cottonseed imported from Australia into California for dairy cattle feed

A unique biotype of the Fusarium wilt pathogen, Fusarium oxysporum Schlecht. f.sp. vasinfectum (Atk) Sny. & Hans., found in Australia in 1993 is favored by neutral or alkaline heavy soils and does not require plant parasitic nematodes to cause disease. This makes it a threat to 4-6 million acres of USA Upland cotton ( Gossypium hirsutum L.) that is grown on heavy alkaline soil and currently is not affected by Fusarium wilt. In 2001-2002, several shiploads of live cottonseed were imported into California for dairy cattle feed. Thirteen F. oxysporum f.sp. vasinfectum isolates and four isolates of a Fusarium spp. that resembled F. oxysporum were isolated from the imported cottonseed. The isolates, designated by an AuSeed prefix, formed four vegetative compatibility groups (VCG) all of which were incompatible with tester isolates for 18 VCGs found in the USA. Isolate AuSeed14 was vegetatively compatible with the four reference isolates of Australian biotype VCG01111. Phylogenetic analyses based on EF-1α, PHO, BT, Mat1-1, and Mat1-2 gene sequences separated the 17 seed isolates into three lineages (race A, race 3, and Fusarium spp.) with AuSeed14 clustering into race 3 lineage or race A lineage depending on the genes analyzed. Indel analysis of the EF-1α gene sequences revealed a close evolutionary relationship among AuSeed14, Australian biotype reference isolates, and the four Fusarium spp. isolates. The Australian seed isolates and the four Australian biotype reference isolates caused disease with root-dip inoculation, but not with stem-puncture inoculation. Thus, they were a vascular incompetent pathotype. In contrast, USA race A lineage isolates readily colonized vascular tissue and formed a vascular competent pathotype when introduced directly into xylem vessels. The AuSeed14 isolate was as pathogenic as the Australian biotype, and it or related isolates could cause a severe Fusarium wilt problem in USA cotton fields if they become established.

Glandless Seed and Glanded Plant Research in Cotton

Recently the world has been entangled by insufficient food such as the lack of rice which threatens the safety of world food and affect sustainable development of the world economy, resulting in rising of food price. To address this issue, cotton appears as a possible source of both fiber and food. The research in recent years indeed showed bright prospects for this expectation. However, gossypol stored in the glands of cotton is toxic to nonruminant animals and humans, which wastes large amounts of cottonseed protein that could potentially provide the annual protein requirements for half a billion people. Gossypium species are characterized by their lysigenous glands containing terpenoid aldehydes, important secondary phytoalexins consisting mainly of gossypol, which constitute one of the important plant’s defense system against pests and diseases. The best approach to address this issue is to create glandless seed and glanded plant cotton. A breakthrough in this field would realise the fulfilment of making cotton both a fiber and a food crop, which would be a feat of great magnitude for sustainable development of agriculture. Research on the relationship between glands and their secondary inclusions at the molecular level would be one approach for genetic engineering to control the glands and gossypol content. In this article, we review recent progress on glands and gossypol content for diverse gland types in Gossypium species, inheritance of glands and gossypol content, traditional breeding of glandless seeds and glanded plant cotton, the terpenoid aldehyde biosynthesis pathway, molecular cloning of the related genes, the strategy for genetic engineering, and future prospects.

Nuclear magnetic resonance (NMR) studies on the biosynthesis of fusaric acid from Fusarium oxysporum f. sp. vasinfectum.

Fusarium oxysporum is a fungal pathogen that attacks many important plants. Uniquely pathogenic strains of F. oxysporum f. sp. vasinfectum were inadvertently imported into the United States on live cottonseed for dairy cattle feed. These strains produce exceptionally high concentrations of the phytotoxin fusaric acid. Thus, fusaric acid may be a critical component in the pathogenicity of these biotypes. This study investigated the biosynthesis of fusaric acid using (13)C-labeled substrates including [1,2-(13)C(2)]acetate as well as (13)C- and (15)N-labeled aspartate and [(15)N]glutamine. The incorporation of labeled substrates is consistent with the biosynthesis of fusaric acid from three acetate units at C5-C6, C7-C8, and C9-C10, with the remaining carbons being derived from aspartate via oxaloacetate and the TCA cycle; the oxaloacetate originates in part by transamination of aspartate, but most of the oxaloacetate is derived by deamination of aspartate to fumarate by aspartase. The nitrogen from glutamine is more readily incorporated into fusaric acid than that from aspartate.