Glandless Seeds Research Articles

Single-cell transcriptome atlas identified novel regulators for pigment gland morphogenesis in cotton.

Single-cell transcriptome atlas identified novel regulators for pigment gland morphogenesis in cotton.

Single-cell transcriptomic analysis reveals the developmental trajectory and transcriptional regulatory networks of pigment glands in Gossypium bickii

Comprehensive utilization of cottonseeds is limited by the presence of pigment glands and its inclusion gossypol. The ideal cotton has glandless seeds but a glanded plant, a trait found in only a few Australian wild cotton species, including Gossypium bickii. Introgression of this trait into cultivated species has proved to be difficult. Understanding the biological processes toward pigment gland morphogenesis and the associated underlying molecular mechanisms will facilitate breeding of cultivated cotton varieties with the trait of glandless seeds and glanded plant. In this study, single-cell RNA sequencing (scRNA-seq) was performed on 12 222 protoplasts isolated from cotyledons of germinating G. bickii seeds 48 h after imbibition. Clustered into 14 distinct clusters unsupervisedly, these cells could be grouped into eight cell populations with the assistance of known cell marker genes. The pigment gland cells were well separated from others and could be separated into pigment gland parenchyma cells, secretory cells, and apoptotic cells. By integrating the pigment gland cell developmental trajectory, transcription factor regulatory networks, and core transcription factor functional validation, we established a model for pigment gland formation. In this model, light and gibberellin were verified to promote the formation of pigment glands. In addition, three novel genes, GbiERF114 (ETHYLENE RESPONSE FACTOR 114), GbiZAT11 (ZINC FINGER OF ARABIDOPSIS THALIANA 11), and GbiNTL9 (NAC TRANSCRIPTION FACTOR-LIKE 9), were found to affect pigment gland formation. Collectively, these findings provide new insights into pigment gland morphogenesis and lay the cornerstone for future cotton scRNA-seq investigations.

Comparative Transcriptomic Analysis to Identify the Genes Related to Delayed Gland Morphogenesis in Gossypium bickii.

Cotton is one of the major industrial crops that supply natural fibers and oil for industries. This study was conducted to understand the mechanism of delayed gland morphogenesis in seeds of Gossypium bickii. In this study, we compared glandless seeds of G. bickii with glanded seeds of Gossypium arboreum. High-throughput sequencing technology was used to explore and classify the expression patterns of gland-related genes in seeds and seedlings of cotton plants. Approximately 131.33 Gigabases of raw data from 12 RNA sequencing samples with three biological replicates were generated. A total of 7196 differentially-expressed genes (DEGs) were identified in all transcriptome data. Among them, 3396 genes were found up-regulated and 3480 genes were down-regulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were performed to identify different functions between genes unique to glandless imbibed seeds and glanded seedlings. Co-expression network analysis revealed four modules that were identified as highly associated with the development of glandless seeds. Here the hub genes in each module were identified by weighted gene co-expression network analysis (WGCNA). In total, we have selected 13 genes involved in transcription factors, protein and MYB-related functions, that were differentially expressed in transcriptomic data and validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). These selected genes may play an important role for delayed gland morphogenesis. Our study provides comprehensive insight into the key genes related to glandless traits of seeds and plants, and can be further exploited by functional and molecular studies.

Isolation of Cottonseed Extracts That Affect Human Cancer Cell Growth

Cottonseeds are classified as glanded or glandless seeds depending on the presence or absence of gossypol glands. Glanded cottonseed has anticancer property and glandless cottonseed was reported to cause cancer in one animal study. It is important to investigate the effect of bioactive components from cottonseeds. Our objectives were to isolate ethanol extracts from cottonseeds and investigate their effects on human cancer cells. A protocol was developed for isolating bioactive extracts from seed coat and kernel of glanded and glandless cottonseeds. HPLC-MS analyzed the four ethanol extracts but only quercetin was identified in the glandless seed coat extract. Residual gossypol was detected in the glanded and glandless seed kernel extracts and but only in the glanded seed coat extract. Ethanol extracts were used to treat human cancer cells derived from breast and pancreas followed by MTT assay for cell viability. Ethanol extracts from glanded and glandless cottonseed kernels and gossypol significantly decreased breast cancer cell mitochondrial activity. Ethanol extract from glanded cottonseed kernel and gossypol also significantly decreased pancreas cancer cell mitochondrial activity. These results suggest that ethanol extracts from cottonseeds, like gossypol, contain anticancer activities.

Cottonseed Extracts and Gossypol Regulate Diacylglycerol Acyltransferase Gene Expression in Mouse Macrophages.

Plant bioactive polyphenols have been used for the prevention and treatment of various diseases since ancient history. Cotton ( Gossypium hirsutum L.) seeds are classified as glanded or glandless depending on the presence or absence of pigment glands, which contain polyphenolic gossypol. Diacylglycerol acyltransferases (DGATs) are integral membrane proteins that catalyze the last step of triacylglycerol biosynthesis in eukaryotes. Understanding the regulation of DGATs will provide information for therapeutic intervention for obesity and related diseases. However, little was known if DGAT gene expression was regulated by natural products. The objective of this study was to investigate the effects of cottonseed extracts and gossypol on DGAT gene expression in mouse RAW264.7 macrophages. Mouse cells were treated with different concentrations of cottonseed extracts, gossypol, and lipopolysaccharides (LPS) for various times. Quantitative polymerase chain reaction assay showed that coat extract of glanded seeds had a modest effect on DGAT1 and minimal effect on DGAT2 mRNA levels. Kernel extract of glanded seeds had a minimal effect on DGAT1 but increased DGAT2 mRNA levels more than 20-fold. Coat extract of glandless seeds and LPS had minimal effects on DGAT mRNA levels. Kernel extract of glandless seeds did not have much effect on DGAT1 and slightly increased DGAT2 mRNA levels. Gossypol increased DGAT1 and DGAT2 mRNA levels by up to three-fold and more than 80-fold, respectively. The coefficient correlations ( R2) between DGAT2 mRNA levels and glanded kernel extract and gossypol concentrations were 0.82-0.99. This study suggests that Dgat2 is an inducible gene rapidly responding to stimulators such as polyphenols whose protein product DGAT2 plays an important role in fat biosynthesis. We conclude that gossypol and ethanol extract from glanded cottonseed kernel are strong stimulators of DGAT2 gene expression and that they may be novel agents for intervention of lipid-related dysfunction via increasing DGAT2 gene expression in target tissues.

Gene expression profiling during gland morphogenesis of a mutant and a glandless upland cotton

To identify genes involved in pigment gland morphogenesis in cotton, gene expression was profiled using genechip (Affymetrix) during pigment gland morphogenesis in cotton variety Xiangmian-18, which has glandless seeds but glanded plants, and a glandless line, N5. The results showed that 303 genes were differentially expressed by a factor greater than two during gland morphogenesis; 59% (180) of these genes shared similarity with known genes in GenBank. These genes play roles in defense response, response to oxidative stress, peroxidase activity, and other metabolic pathways. KOBAS (KEGG Orthology-Based Annotation System) indicate that these genes are involved in 68 biochemical pathways. These findings suggest that the related defense response, gossypol biosynthesis pathway and other complex regulation may be associated with pigment gland morphogenesis in cotton. The results may provide a basis for further study and serve as a guide for related research.

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.

Introgression of a gene for delayed pigment gland morphogenesis from Gossypium bickii into upland cotton

The presence of gossypol and its derivatives above the WHO/FAO standards (0.02–0.04%) in cotton seed oil and meal limits its usage as food and feed. To the contrary, the presence of pigment glands filled with gossypol and its derivatives helps to protect cotton plants from phytophageous pests. Thus a desirable cultivar would have glandless seeds on a glanded plant. This paper describes results on the successful introgression of this trait from Gossypium bickii into cultivated upland cotton. Five different tri-specific hybrids (ABH1, ABH2, ABH3, ABH4 and ABH5) were obtained by crossing the amphidiploid F1 (G. arboreum × G. bickii) with different gland genotypes of G. hirsutum as male parent. The hybrids were highly sterile, and their chromosome configuration at meiosis metaphase 1 (M1) in pollen mother cell (PMC) was 2n = 52 = 41.04 I + 4.54 II + 0.57 III + 0.04 IV. All five hybrids were similar in morphological characters, except for the gland expression and gossypol contents. The hybrid (ABH3) derived from genotype Gl2Gl2gl3gl3 of upland cotton (a single gene dominant line) had completely introgressed the target trait of G. bickii. While ABH1 and ABH2, which derived from recessive (gl2gl2gl3gl3) or dominant (GlGl) glandless upland cotton genotypes, had glandless seeds too, but the density and size of the glands on the plant were reduced significantly.

Inheritance of the delayed gland morphogenesis trait in Australian wild species ofGossypium

Five Australian wild cotton species with the delayed gland morphogenesis trait, as well asG arboreum, G davidsonii and four different gland genotypes ofG hirsutum, Gl2Gl2Gl3Gl2, Gl2Gl2gl3gl3, gl2gl2Gl3G3, and gl2gl2gl3gl3, were used in this experiment and 10 interspecific hybrids were obtained by the crossing among them. According to the gland expression on the seeds and plants of the interspecific hybrids, the inheritance of the delayed gland morphogenesis trait of Australian wild cotton species was opened out as follows: ( i ) the inheritance of the delayed gland morphogenesis trait was almost the same among the 5 Australian wild cotton species, and the gene or genes which controlled this trait may be located in the same loci. (ii) The glandless seed trait of the Australian wild cotton species was dominant over the glanded seed trait ofG arboreum, a genome A species, and the seeds of interspecific hybrid F1 between them were glandless. However, it was recessive over the glanded character of genome D species,G davidsonii, and their F1 was a typical glanded one. (iii) The glandless seed trait of the Australian wild cotton species was recessive or incomplete dominant over the glanded cotton but dominant over the glandless cotton ofG hirsutum, and the glandless genes (gl2gl2gl3gl3 of upland cotton had great weakening effect on the glanded plant trait of the Australian wild cotton species on the other hand. For the two main glanded genes of upland cotton, the delayed gland morphogenesis trait of the Australian wild cotton species was dominant epistatic over glandless genes, gl2gl2gl3gl3, and one of the glanded genes, Gl2Gl2, but was recessive epistatic over the other glanded gene, Gl3Gl3. Therefore, it is much convenient to use Gl2Gl2gl3gl3 as the upland cotton parent in the interspecific hybridization and backcrossing afterward, in order to produce the upland cotton germplasm with glandless seeds and glanded plant trait.

Development of high-gossypol cotton plants with low-gossypol seeds using trispecies bridge crosses and in vitro culture of seed embryos

The objective of this work is to develop an upland cotton, Gossypium hirsutum L. [2n = 4x =52, 2(AD)h], having a reduced level of gossypol in the seeds for food and feed uses, and a high level of gossypol in the remaining organs to limit pest incidence. Using G. sturtianum Willis (2n = 2x = 26, 2C1) as donor and G. thurberi Torado (2n = 2x = 26, 2D1) or G. raimondii Ulbrich (2n = 2x = 26, 2D5) as bridge species, two trispecies hybrids G. thurberi– G. sturtianum– G. hirsutum and G. hirsutum– G. raimondii– G. sturtianum were synthesized. Both trispecies hybrids were male sterile. Recurrent backcrossing to G. hirsutum as pollinator and selfing of the second backcross (BC) progenies resulted in seeds which were rescued by in vitro culture. In total, 1208 flowers of the trispecies hybrids and their BC progenies yielded 192 seed embryos from which 62 plants were obtained. Cytogenetic analyses indicated a relatively high frequency of chromosome pairing and chiasmata. The gland levels in backcross seeds ranged from glandless seeds to normally glanded seeds. All vegetative parts of those hybrids were glanded, but a wide range of variability for gland density was observed on leaf, stem, bract and calyx. Plants derived from seeds having a reduced level of gossypol constitute very interesting germplasm to develop a cultivated glanded cotton with low-gossypol seeds.

Effects of processing on the oligosaccharides of oilseed and legume protein meals

The effects of (alkanol)-hexane, (alkanol/water)-hexane and (alkanol/ammonia/water)-hexane extraction systems on the removal of low-molecular-weight sugars from soybean, cottonseed, field pea and mung bean were studied. The total content of soluble sugars of hexane-extracted meals varied between 4.79 and 12.12%, depending on the type of seed and solvent extraction system used. The predominant sugar of cottonseed was raffinose. Sucrose and stachyose were present in smaller quantities. However, glanded cottonseed contained about four times more stachyose than glandless seeds. Seeds were extracted with methanol, ethanol and isopropanol, with or without 10% (w/w) ammonia, possibly containing 50% (v/v) water. The above extraction systems removed up to 81% of the total flatulence-causing sugars. Methanol was the most effective alkanol used, while isopropanol exhibited the poorest extraction efficiency. (Methanol/ammonia)-treated meals contained from 0.38 to 2.86% raffinose; however, all extraction systems were less effective in removing stachyose and verbascose. Results of this study imply that the removal of flatulence-causing sugars depends on their molecular weight, the seed's structural parameters, interaction within the seed components and polarity of the solvent systems used.

Effect of refeeding diets containing cottonseed flour with traces of gossypol on rat liver and testis

The aim of the present work was to show that cottonseed flour introduced into rehabilitation diets is not nutritionally harmful. The cottonseed flour obtained from glandless seeds contains traces of gossypol. As gossypol is known to have pathological hepatic and testicular actions, the effects on hepatic variables and on the histological appearance of liver and testis of diets containing cottonseed flour were compared with those without cottonseed flour. Seventy growing male Wistar rats were divided into two groups. The control group (C) received a balanced diet (200 g casein + 3 g methionine/kg) for 78 d and the experimental group was fed on a low-protein diet (20 g casein + 3 g methionine/kg) for 29 d. After the period of protein deprivation, the depleted rats were divided into four groups and each group was refed with a different diet, for 49 d: a balanced diet (200 g casein + 3 g methionine/kg; rC), maize-soya-bean flour-milk (60:30:5, by wt; MSM), maize-cottonseed flour-milk (60:30:5, by wt; MCM) or rice-cottonseed flour-milk (60:30:5, by wt; RCM). Each diet was supplemented with a vitamin mixture (10 g/kg) and a salt mixture (40 g/kg). In the liver, protein, total lipid, fatty acid composition and nucleic acid contents were determined after 29 d of protein deprivation and after 2, 14, 29, 49 d of refeeding. The refeeding of rats with either diet MSM or RCM promoted a higher growth than that with diet MCM or diet rC. In all groups, a progressive decrease in relative liver weights with age was observed. The highest values were obtained with diets rC and MSM. After 49 d of refeeding, the lowest values for lipid, DNA and protein:RNA and the highest values for RNA and RNA:DNA were obtained with the cottonseed diets (MCM and RCM). Rats fed on the cottonseed-flour diets for a 49 d period after severe protein malnutrition showed no change in size or shape of their hepatocytes compared with those obtained with the control casein diet, and all stages of spermatogenesis occurred normally and spermatozoa were accurately formed. Therefore, our findings show that a low level of gossypol administrated during 49 d, even to an organism weakened by protein malnutrition, does not affect hepatic variables and the histological appearance of liver and testis.

Development of a Cotton Plant with Glandless Seeds, and Glanded Foliage and Fruiting Forms1

Pigment glands in cotton (Gossypium spp.) contain gossypol and related terpenoid aldehyde compounds and these glands are found throughout most aboveground parts, including seeds. These glands protect the plant from insect pests, but gossypol in the embryo of the seed is undesirable because it is toxic to nonruminant animals. The objectives of the present study were to identify a hexaploid cotton plant that possessed glands in most of the aboveground parts, except the seed, and initiate the transfer of this trait into a tetraploid cotton. A fertile hexaploid (2N= 78) plant from an interspecific cross of tetraploid (2N=52) Gossypium hirsutum L. ✕ diploid (2N=26) G. sturtianum Willis showed a phenotype having glandless seed, and glanded foliage and fruiting forms. This phenotype had flowerbud and seed gossypol percentages of 0.29 and 0.02, respectively. Fertile pentaploid F1 plants from crossing the hexaploid and the tetraploid G. hirsutum Texas marker‐1 (TM‐1) stock appeared to produce as much seed cotton as the tetraploid Texas marker‐1 at Brownsville, TX. However, the F1 plant produced 0.31% flowerbud and only 0.02% seed gossypol. The flowerbud and seed gossypol percentages of TM‐1 were 0.58 and 1.29, respectively. These results show that fertile hexaploid germplasm in cotton has been developed that possesses the unique characteristic of storing gossypol in glands in the vegetative foliar and fruiting plant parts but not in the embryo of the seed.

Action of the Genes Controlling the Character Glandless Seed in Cotton1

Action of the Genes Controlling the Character Glandless Seed in Cotton¹

The Use of Glandless Seed to Determine the Amount of Natural Crossing in Gossypium Hirsutum1

The Use of Glandless Seed to Determine the Amount of Natural Crossing in <i>Gossypium</i> Hirsutum¹