Parental legacy versus regulatory innovation in salt stress responsiveness of allopolyploid cotton (Gossypium) species.

Polyploidy provides an opportunity for evolutionary innovation and species diversification, especially under stressful conditions. In allopolyploids, the conditional dynamics of homoeologous gene expression can be either inherited from ancestral states pre-existing in the parental diploids or novel upon polyploidization, the latter potentially permitting a wider range of phenotypic responses to stresses. To gain insight into regulatory mechanisms underlying the diversity of salt resistance in Gossypium species, we compared global transcriptomic responses to modest salinity stress in two allotetraploid (AD-genome) cotton species, Gossypium hirsutum and G. mustelinum, relative to their model diploid progenitors (A-genome and D-genome). Multivariate and pairwise analyses of salt-responsive changes revealed a profound alteration of gene expression for about one third of the transcriptome. Transcriptional responses and associated functional implications of salt acclimation varied across species, as did species-specific coexpression modules among species and ploidy levels. Salt responsiveness in both allopolyploids was strongly biased toward the D-genome progenitor. A much lower level of transgressive downregulation was observed in the more salt-tolerant G. mustelinum than in the less tolerant G. hirsutum. By disentangling inherited effects from evolved responses, we show that expression biases that are not conditional upon salt stress approximately equally reflect parental legacy and regulatory novelty upon allopolyploidization, whereas stress-responsive biases are predominantly novel, or evolved, in allopolyploids. Overall, our work suggests that allopolyploid cottons acquired a wide range of stress response flexibility relative to their diploid ancestors, most likely mediated by complex suites of duplicated genes and regulatory factors.

Cotton roots are the major source of gossypol biosynthesis and accumulation.

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.

Salt-tolerance diversity in diploid and polyploid cotton (Gossypium) species.

The development of salt-tolerant genotypes is pivotal for the effective utilization of salinized land and to increase global crop productivity. Several cotton species comprise the most important source of textile fibers globally, and these are increasingly grown on marginal or increasingly saline agroecosystems. The allopolyploid cotton species also provide a model system for polyploid research, of relevance here because polyploidy was suggested to be associated with increased adaptation to stress. To evaluate the genetic variation of salt tolerance among cotton species, 17 diverse accessions of allopolyploid (AD-genome) and diploid (A- and D-genome) Gossypium were evaluated for a total of 29 morphological and physiological traits associated with salt tolerance. For most morphological and physiological traits, cotton accessions showed highly variable responses to 2 weeks of exposure to moderate (50 mm NaCl) and high (100 mm NaCl) hydroponic salinity treatments. Our results showed that the most salt-tolerant species were the allopolyploid Gossypium mustelinum from north-east Brazil, the D-genome diploid Gossypium klotzschianum from the Galapagos Islands, followed by the A-genome diploids of Africa and Asia. Generally, A-genome accessions outperformed D-genome cottons under salinity conditions. Allopolyploid accessions from either diploid genomic group did not show significant differences in salt tolerance, but they were more similar to one of the two progenitor lineages. Our findings demonstrate that allopolyploidy in itself need not be associated with increased salinity stress tolerance and provide information for using the secondary Gossypium gene pool to breed for improved salt tolerance.

Gossypium barbadense and Gossypium hirsutum genomes provide insights into the origin and evolution of allotetraploid cotton.

Allotetraploid cotton is an economically important natural-fiber-producing crop worldwide. After polyploidization, Gossypium hirsutum L. evolved to produce a higher fiber yield and to better survive harsh environments than Gossypium barbadense, which produces superior-quality fibers. The global genetic and molecular bases for these interspecies divergences were unknown. Here we report high-quality de novo-assembled genomes for these two cultivated allotetraploid species with pronounced improvement in repetitive-DNA-enriched centromeric regions. Whole-genome comparative analyses revealed that species-specific alterations in gene expression, structural variations and expanded gene families were responsible for speciation and the evolutionary history of these species. These findings help to elucidate the evolution of cotton genomes and their domestication history. The information generated not only should enable breeders to improve fiber quality and resilience to ever-changing environmental conditions but also can be translated to other crops for better understanding of their domestication history and use in improvement.

Genotypic differences in leaf secondary metabolism, plant hormones and yield under alone and combined stress of drought and salinity in cotton genotypes.

Drought and salinity stress highly affect the plant growth and production around the world. Secondary metabolites play a main role in adaptation to the environment and in overcoming stress conditions. In order to investigate the effect of drought and salinity, alone or in combination, on secondary metabolism-related enzyme activities, plant hormones and yield parameters, a greenhouse pot experiment was conducted using two cotton genotypes Zhongmian 23 (salt tolerant) and Zhongmian 41 (salt sensitive). Results showed that single and combined drought and salinity stresses caused remarkable decrease in plant height, bolls and lint yield in the order as follows: D + S > salinity > drought, and Zhongmian 41 > Zhongmian 23. Lower H2 O2 and superoxide but higher proline content and secondary metabolism-related enzyme activities were observed in Zhongmian 23 under drought and salinity, both alone and combined, compared with control in Zhongmian 41. Our findings suggest that controlling reactive oxygen species (ROS) levels and increasing activities of secondary metabolism-related enzymes in Zhongmian 23 might be an effective mechanism to reduce the negative effects of drought and salinity stress. However, cinnamyl alcohol dehydrogenase (CAD), and shikimate dehydrogenase (SKDH) activities were markedly decreased in Zhongmian 41 under salinity stress alone as compared with control. Meanwhile, Zhongmian 23 had higher expression levels of genes related to secondary metabolism (c.f. phenylalanine ammonia-lyase, PAL; polyphenol oxidase, PPO and CAD) under the three stresses compared to Zhongmian 41. The content of flavonoids and phenols were significantly enhanced under drought and D + S, with higher accumulation in Zhongmian 23. Phenols content in Zhongmian 23 remained unchanged under salinity as relative to control, but were significantly reduced in Zhongmian 41. In addition, callose content, chitinase activities and abscisic acid (ABA) and Indole-3-acetic acid (IAA) were more induced in Zhongmian 23 under drought, salinity and D + S, than in Zhongmian 41. Our results suggest that high tolerance to D + S stress in Zhongmian 23 is closely related to elevated callose, chitinase, flavonoids and phenols contents and higher secondary metabolism-related enzyme activities and their transcript levels.

Genome-Wide Identification of R2R3-MYB Genes and Expression Analyses During Abiotic Stress in Gossypium raimondii.

The R2R3-MYB is one of the largest families of transcription factors, which have been implicated in multiple biological processes. There is great diversity in the number of R2R3-MYB genes in different plants. However, there is no report on genome-wide characterization of this gene family in cotton. In the present study, a total of 205 putative R2R3-MYB genes were identified in cotton D genome (Gossypium raimondii), that are much larger than that found in other cash crops with fully sequenced genomes. These GrMYBs were classified into 13 groups with the R2R3-MYB genes from Arabidopsis and rice. The amino acid motifs and phylogenetic tree were predicted and analyzed. The sequences of GrMYBs were distributed across 13 chromosomes at various densities. The results showed that the expansion of the G. Raimondii R2R3-MYB family was mainly attributable to whole genome duplication and segmental duplication. Moreover, the expression pattern of 52 selected GrMYBs and 46 GaMYBs were tested in roots and leaves under different abiotic stress conditions. The results revealed that the MYB genes in cotton were differentially expressed under salt and drought stress treatment. Our results will be useful for determining the precise role of the MYB genes during stress responses with crop improvement.

Genome sequence of cultivated Upland cotton (Gossypium hirsutum TM-1) provides insights into genome evolution.

Gossypium hirsutum has proven difficult to sequence owing to its complex allotetraploid (AtDt) genome. Here we produce a draft genome using 181-fold paired-end sequences assisted by fivefold BAC-to-BAC sequences and a high-resolution genetic map. In our assembly 88.5% of the 2,173-Mb scaffolds, which cover 89.6%∼96.7% of the AtDt genome, are anchored and oriented to 26 pseudochromosomes. Comparison of this G. hirsutum AtDt genome with the already sequenced diploid Gossypium arboreum (AA) and Gossypium raimondii (DD) genomes revealed conserved gene order. Repeated sequences account for 67.2% of the AtDt genome, and transposable elements (TEs) originating from Dt seem more active than from At. Reduction in the AtDt genome size occurred after allopolyploidization. The A or At genome may have undergone positive selection for fiber traits. Concerted evolution of different regulatory mechanisms for Cellulose synthase (CesA) and 1-Aminocyclopropane-1-carboxylic acid oxidase1 and 3 (ACO1,3) may be important for enhanced fiber production in G. hirsutum.

The novel approach to enhance seed security: dual anti-counterfeiting methods applied on tobacco pelleted seeds.

Seed security is of prime importance for agriculture. To protect true seeds from being faked, more secure dual anti-counterfeiting technologies for tobacco (Nicotiana tabacum L.) pelleted seed were developed in this paper. Fluorescein (FR), rhodamine B (RB), and magnetic powder (MP) were used as anti-counterfeiting labels. According to their different properties and the special seed pelleting process, four dual-labeling treatments were conducted for two tobacco varieties, MS Yunyan85 (MSYY85) and Honghua Dajinyuan (HHDJY). Then the seed germination and seedling growth status were investigated, and the fluorescence in cracked pellets and developing seedlings was observed under different excitation lights. The results showed that FR, RB, and MP had no negative effects on the germination, seedling growth, and MDA content of the pelleted seeds, and even some treatments significantly enhanced seedling dry weight, vigor index, and shoot height in MS YY85, and increased SOD activity and chlorophyll content in HHDJY as compared to the control. In addition, the cotyledon tip of seedlings treated with FR and MP together represented bright green fluorescence under illumination of blue light (478 nm). And the seedling cotyledon vein treated with RB and MP together showed red fluorescence under green light (546 nm). All seeds pelleted with magnetic powder of proper concentration could be attracted by a magnet. Thus, it indicated that those new dual-labeling methods that fluorescent compound and magnetic powder simultaneously applied in the same seed pellets definitely improved anti-counterfeiting technology and enhanced the seed security. This technology will ensure that high quality seed will be used in the crop production.

[Determination of amino acid contents in cottonseeds using near infrared reflectance spectroscopy].

A total of 445 samples with great variability in amino acid contents were harvested for different seasons in different regions for developing calibration equations of amino acid content in cottonseeds. The spectral data of cotton kernel powder was processed using the first derivative mathematical treatment combined with SNV and de-trend, as well as modified partial least squares (MPLS) regression method. The chemometric models for 17 amino acids present in cottonseed were developed, and 12 of them were excellent for the determination of related amino acids, namely asparagic acid, threonine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, and arginine, with RPDc of 3.735-7.132 and determination coefficient (r2) of 0.910-0.979 in external validation. For those 12 amino acids, their values predicted by NIRS are comparable to those obtained by the chemical method with good accuracy. The RPDc of serine, methionine, tyrosine and proline were 2.205 -2.814, and their determination coefficient (r2) were 0.800-0.830 in external validation. For those 4 amino acids, the values from NIRS are not so accurate as chemical analysis, but could be used in sample screening in cotton breeding program. While the equation for cystine was useless as its RPDc was only 1.358, which was not suitable for estimating its content in cottonseeds.

Cadmium-induced ultramorphological and physiological changes in leaves of two transgenic cotton cultivars and their wild relative.

The present study describes cadmium-induced alterations in the leaves as well as at the whole plant level in two transgenic cotton cultivars (BR001 and GK30) and their wild relative (Coker 312) using both ultramorphological and physiological indices. With elevated levels of Cd (i.e. 10, 100, 1000 microM), the mean lengths of root, stem and leaf and leaf width as well as their fresh and dry biomasses linearly decreased over their respective controls. Moreover, root, stem and leaf water absorption capacities progressively stimulated, which were high in leaves followed by roots and stems. BR001 accumulated more cadmium followed by GK30 and Coker 312. Root and shoot cadmium uptakes were significantly and directly correlated with each other as well as with leaf, stem and root water absorption capacities. The ultrastructural modifications in leaf mesophyll cells were triggered with increase in Cd stress regime. They were more obvious in BR001 followed by GK30 and Coker 312. Changes in morphology of chloroplast, increase in number and size of starch grains as well as increase in number of plastoglobuli were the noticed qualitative effects of Cd on photosynthetic organ. Cd in the form of electron dense granules could be seen inside the vacuoles and attached to the cell walls in all these cultivars. From the present experiment, it can be well established that both apoplastic and symplastic bindings are involved in Cd detoxification in these cultivars. Absence of tonoplast invagination reveals that Cd toxic levels did not cause water stress in any cultivars. Additionally, these cultivars possess differential capabilities towards Cd accumulation and its sequestration.

Cadmium-induced functional and ultrastructural alterations in roots of two transgenic cotton cultivars.

The toxic effect of cadmium (Cd) at increasing concentrations was studied with special attention being given to the root morphological and ultrastructural changes in two transgenic cotton cultivars viz. BR001 and GK30 and their wild relative viz. Coker 312. In comparison to their respective controls, low concentration (10 and 100microM) of Cd greatly stimulated seed germination, while it was inhibited by highest concentration of Cd (1000microM) in case of two transgenic cultivars. However, in Coker 312 the seed germination percentage progressively decreased over the control at all Cd levels. Various physiological and morphological parameters of the root and whole plant in both transgenic cotton cultivars and their relative wild cotton genotype respond differently towards the Cd toxicity. Bioavailability of Cd was concentration-dependent where seedling root captured more Cd as compared to shoot. BR001 accumulated more Cd followed by GK30, while Coker 312 was less Cd accumulator. The ultrastructural modifications in the root tip cells of both the transgenic cotton cultivars and their wild relative were also dose-dependent. With the increase in Cd levels, the fine structures of their root cells also invariably changed. Increase in plasmolysis of the plasma membrane, greater number of nucleoli and vacuoles and enlarged vacuoles could be observed in both transgenic cotton cultivars. In comparison to them, Coker 312 showed relatively well developed ultrastructures of the root tips except enlarged vacuoles and greater number of mitochondria. Moreover, the accumulation of Cd in the form of electron dense granules and crystals both in vacuoles and attached to cell walls were visible in both transgenic cotton cultivars and their wild relative. These results suggest that both transgenic cotton cultivars and their wild relative cotton genotype responded positively towards Cd stress at seedling stage, the internal Cd-detoxification might be through apoplastic and symplastic binding. Moreover, as a whole BR001 proved to be sensitive whereas; GK30 and Coker 312 were found as tolerant.

[Studies on the cytological characters of the interspecific hybrid F1 among the cultivated species in Gossypium and their genetic relationship].

Interspescific hybridization among four cultivated species in Gossypium (G. herbaceum, G. arboreum, G. hirsutum and G. barbadense) were carried out to produce dispecific hybrids F1, (G. arboreum x G. herbaceum) F1 and (G. hirsutum x G. barbadense) F1, and quadrispecific hybrid F1, which was produced by crossed the chromosome doubled (G. arboreum x G. herbaceum) F1 with (G. hirsutum x G. barbadense) F1. In order to study the evolution relationship among the four cultivated species in Gossypium, the characteristic of chromosome behavior during the meiosis and pollen viability in those interspecific hybrids F1 were studied in this paper. The results showed that the diploid interspecific hybrid, (G. arboreum x G. herbaceum) F1, had a four-chromosome-ring, the chromosome configuration was 2n = 26 = 11 II + 1 IV. And the normal pollen percent was 50.71%, which showed the character of typical gamete semi-sterility, and approved that there was a chromosome translocation between the two diploid cotton species, G. arboreum and G. herbaceum. For the allotetraploid species interspecific hybrid F1, (G. hirsutum x G. barbadense) F1, most of the chromosomes at metaphase I could be paired into bivalents, with a few number of univalents, trivalents, and quardrivalents. The chromosome configuration was 2n = 52 = 0.78 I +22.24 II +0.94 III +0.98 IV, with a normal pollen rate of 54. 84%. The experiment showed that there were a few chromosome translocation or chromosome inversion between the two allotetraploid cotton species, G. hirsutum and G. barbadenses. The meiosis of the quardrispecific hybrid F1 was abnormal, and the loss of chromosomes was common. Most of the chromosomes could not synapse at metaphase I, which led to many univalents and some multivalents. The chromosome configuration of the quardrispecific hybrid F1 was 2n = 52 = 5.45 I +14.41 II +2.44 III +1.59 IV +0.63 V +0.15 VI, and the normal pollen rate was 6.87%, which showed that the relationship of four cultivated cotton species was relatively closed. It is possible to produce a new germplasm with the good characters of the four cultivated species through genetic recombination.