Anthocyanin gene enrichment in the distal region of cotton chromosome A07: mechanisms of reproductive organ coloration.

Introduction: The biosynthesis of secondary metabolites like anthocyanins is often governed by metabolic gene clusters (MGCs) in the plant ancestral genome. However, the existence of gene clusters specifically regulating anthocyanin accumulation in certain organs is not well understood. Methods and results: In this study, we identify MGCs linked to the coloration of cotton reproductive organs, such as petals, spots, and fibers. Through genetic analysis and map-based cloning, we pinpointed key genes on chromosome A07, such as PCC/GhTT19, which is involved in anthocyanin transport, and GbBM and GhTT2-3A, which are associated with the regulation of anthocyanin and proanthocyanidin biosynthesis. Our results demonstrate the coordinated control of anthocyanin and proanthocyanidin pathways, highlighting the evolutionary significance of MGCs in plant adaptation. The conservation of these clusters in cotton chromosome A07 across species underscores their importance in reproductive development and color variation. Our study sheds light on the complex biosynthesis and transport mechanisms for plant pigments, emphasizing the role of transcription factors and transport proteins in pigment accumulation. Discussion: This research offers insights into the genetic basis of color variation in cotton reproductive organs and the potential of MGCs to enhance our comprehension of plant secondary metabolism.

Correction: Co-expression of GR79 EPSPS and GAT generates high glyphosate-resistant alfalfa with low glyphosate residues.

[This corrects the article DOI: 10.1007/s42994-023-00119-3.].

GhTPPA_2 enhancement of tobacco sugar accumulation and drought tolerance.

Trehalose metabolism plays an important role in plant growth and response to abiotic stress. Trehalose-6-phosphate (Tre6P) can help regulate sugar homeostasis and act as an indication signal for intracellular sugar levels. Crop productivity can be greatly increased by altering the metabolic level of endogenous trehalose in plants, which can optimize the source-sink connection. In this study, the upland cotton GhTPP protein family was first homologously compared and 24 GhTPP genes were found. Transcriptome analysis revealed that GhTPP members had obvious tissue expression specificity. Among them, GhTPPA_2 (Gh_A12G223300.1) was predominantly expressed in leaves and bolls. The results of subcellular localization showed that GhTPPA_2 is localized in the chloroplast. Via PlantCare, we analyzed the promoters and found that the expression of GhTPPA_2 may be induced by light, abiotic stress, and hormones such as abscisic acid, ethylene, salicylic acid and jasmonic acid. In addition, GhTPPA_2 was overexpressed (TPPAoe) in tobacco, and we found that the TPPase activity of TPPAoe tobacco increased by 66 %. Soluble sugar content increased by 39 % and starch content increased by 27 %. Whereas, the transgenic tobacco had obvious growth advantages under 100 mM mannitol stress. Transcriptome sequencing results showed that the differential genes between TPPAoe and control were considerably enriched in functions related to photosynthesis, phosphate group metabolism, and carbohydrate metabolism. This study shows that GhTPPA_2 is involved in regulating sugar metabolism, improving soluble sugar accumulation and drought stress tolerance of tobacco, which provides theoretical basis for research on high yield and drought tolerance of crops.

Co-expression of GR79 EPSPS and GAT generates high glyphosate-resistant alfalfa with low glyphosate residues.

Weed competition seriously threatens the yield of alfalfa, the most important forage legume worldwide, thus generating herbicide-resistant alfalfa varieties is becoming a necessary cost-effective strategy to assist farmers for weed control. Here, we report the co-expression of plant codon-optimized forms of GR79 EPSPS (pGR79 EPSPS) and N-acetyltransferase (pGAT) genes, in alfalfa, via Agrobacterium-mediated transformation. We established that the pGR79 EPSPS-pGAT co-expression alfalfa lines were able to tolerate up to tenfold higher commercial usage of glyphosate and produced approximately ten times lower glyphosate residues than the conventional cultivar. Our findings generate an elite herbicide-resistant germplasm for alfalfa breeding and provide a promising strategy for developing high-glyphosate-resistant and low-glyphosate-residue forages. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00119-3.

Natural variation in Beauty Mark is associated with UV-based geographical adaptation in Gossypium species.

BACKGROUND: Anthocyanins, a class of specialized metabolites that are ubiquitous among plant species, have attracted a great deal of attention from plant biologists due to their chemical diversity. They confer purple, pink, and blue colors that attract pollinators, protect plants from ultraviolet (UV) radiation, and scavenge reactive oxygen species (ROS) to facilitate plant survival during abiotic stress. In a previous study, we identified Beauty Mark (BM) in Gossypium barbadense as an activator of the anthocyanin biosynthesis pathway; this gene also directly led to the formation of a pollinator-attracting purple spot. RESULTS: Here, we found that a single nucleotide polymorphism (SNP) (C/T) within the BM coding sequence was responsible for variations in this trait. Transient expression assays of BM from G. barbadense and G. hirsutum in Nicotiana benthamiana using luciferase reporter gene also suggested that SNPs in the coding sequence could be responsible for the absent beauty mark phenotype observed in G. hirsutum. We next demonstrated that the beauty mark and UV floral patterns are associated phenotypes and that UV exposure resulted in increased ROS generation in floral tissues; BM thus contributed to ROS scavenging in G. barbadense and wild cotton plants with flowers containing the beauty mark. Furthermore, a nucleotide diversity analysis and Tajima's D Test suggested that there have been strong selective sweeps in the GhBM locus during G. hirsutum domestication. CONCLUSIONS: Taken together, these results suggest that cotton species differ in their approaches to absorbing or reflecting UV light and thus exhibit variations in floral anthocyanin biosynthesis to scavenge reactive ROS; furthermore, these traits are related to the geographic distribution of cotton species.

Genome-wide identification and expression analysis of the cotton patatin-related phospholipase A genes and response to stress tolerance.

MAIN CONCLUSION: Patatin-related phospholipase A genes were involved in the response of Gossypium hirsutum to drought and salt tolerance. pPLA (patatin-related phospholipase A) is a key enzyme that catalyzes the initial step of lipid hydrolysis, which is involved in biological processes, such as drought, salt stress, and freezing injury. However, a comprehensive analysis of the pPLA gene family in cotton, especially the role of pPLA in the response to drought and salt tolerance, has not been reported so far. A total of 33 pPLA genes were identified in this study using a genome-wide search approach, and phylogenetic analysis classified these genes into three groups. These genes are unevenly distributed on the 26 chromosomes of cotton, and most of them contain a few introns. The results of the collinear analysis showed that G. hirsutum contained 1-5 copies of each pPLA gene found in G. arboreum and G. raimondii. The subcellular localization analysis of Gh_D08G061200 showed that the protein was localized in the nucleus. In addition, analysis of published upland cotton transcriptome data revealed that six GhPLA genes were expressed in various tissues and organs. Two genes (Gh_A04G142100.1 and Gh_D04G181000.1) were highly expressed in all tissues under normal conditions, showing the expression characteristics of housekeeping genes. Under different drought and salt tolerance stresses, we detected four genes with different expression levels. This study helps to clarify the role of pPLA in the response to drought and salt tolerance.

Genetic and transcriptome analysis of a cotton leaf variegation mutant.

In eukaryotic photosynthetic organisms, chloroplast is not only a site for photosynthesis, but it also have a vital role in signal transduction mechanisms. Plants exhibit various colors in nature with various mutants induced by EMS, whose traits are regulated by developmental and environmental factors, making them ideal for studying the regulation of chloroplast development. In this study, the cotton leaf variegated mutant (VAR) induced by EMS was used for this experiment. Genetic analysis revealed that VAR phenotype was a dominant mutation and by performing freehand section inspection, it was noticed that the vascular bundles of VAR were smaller. Chloroplast ultrastructure showed that the stacking of grana thylakoid was thinner and the starch granules were increased significantly in VAR comparedto wild type (WT). Transcriptome analysis found that the KEGG was enriched in photosynthesis pathway, and GO was abundant in zinc ion transmembrane transport, electron transporter and cation binding terms. In addition, GhFTSH5 expression in VAR was significantly higher than WT and the promoter sequence of GhFTSH5 had differences. The results showed that the VAR plant had altered GhFTSH5 expression and disrupted chloroplast structure, which in turn affects plant photosynthesis. More importantly, this study lays a foundation for further analyzing molecular mechanism of cotton variegated phenotypes.

Metagenomic analysis of the soil microbial composition and salt tolerance mechanism in Yuncheng Salt Lake, Shanxi Province.

The soil in Yuncheng Salt Lake has serious salinization and the biogeographic environment affects the composition and distribution of special halophilic and salt-tolerant microbial communities in this area. Therefore, this study collected soils at distances of 15, 30, and 45 m from the Salt Lake and used non-saline soil (60 m) as a control to explore the microbial composition and salt tolerance mechanisms using metagenomics technology. The results showed that the dominant species and abundance of salt-tolerant microorganisms changed gradually with distance from Salt Lake. The salt-tolerant microorganisms can increase the expression of the Na+/H+ antiporter by upregulating the Na+/H+ antiporter subunit mnhA-G to respond to salt stress, simultaneously upregulating the genes in the betaine/proline transport system to promote the conversion of choline into betaine, while also upregulating the trehalose/maltose transport system encode genes to promote the synthesis of trehalose to resist a high salt environment.

Integrating advancements in root phenotyping and genome-wide association studies to open the root genetics gateway.

Plant adaptation to challenging environmental conditions around the world has made root growth and development an important research area for plant breeders and scientists. Targeted manipulation of root system architecture (RSA) to increase water and nutrient use efficiency can minimize the adverse effects of climate change on crop production. However, phenotyping of RSA is a major bottleneck since the roots are hidden in the soil. Recently the development of 2- and 3D root imaging techniques combined with the genome-wide association studies (GWASs) have opened up new research tools to identify the genetic basis of RSA. These approaches provide a comprehensive understanding of the RSA, by accelerating the identification and characterization of genes involved in root growth and development. This review summarizes the latest developments in phenotyping techniques and GWAS for RSA, which are used to map important genes regulating various aspects of RSA under varying environmental conditions. Furthermore, we discussed about the state-of-the-art image analysis tools integrated with various phenotyping platforms for investigating and quantifying root traits with the highest phenotypic plasticity in both artificial and natural environments which were used for large scale association mapping studies, leading to the identification of RSA phenotypes and their underlying genetics with the greatest potential for RSA improvement. In addition, challenges in root phenotyping and GWAS are also highlighted, along with future research directions employing machine learning and pan-genomics approaches.

DgCspC gene overexpression improves cotton yield and tolerance to drought and salt stress comparison with wild-type plants.

Drought and high salinity are key limiting factors for cotton quality and yield. Therefore, research is increasingly focused on mining effective genes to improve the stress resistance of cotton. Few studies have demonstrated that bacterial Cold shock proteins (Csps) overexpression can enhance plants stress tolerance. Here, we first identified and cloned a gene DgCspC encoding 88 amino acids (aa) with an open reading frame (ORF) of 264 base pairs (bp) from a Deinococcus gobiensis I-0 with high resistance to strong radiation, drought, and high temperature. In this study, heterologous expression of DgCspC promoted cotton growth, as exhibited by larger leaf size and higher plant height than the wild-type plants. Moreover, transgenic cotton lines showed higher tolerance to drought and salts stresses than wild-type plants, as revealed by susceptibility phenotype and physiological indexes. Furthermore, the enhanced stresses tolerance was attributed to high capacity of cellular osmotic regulation and ROS scavenging resulted from DgCspC expression modulating relative genes upregulated to cause proline and betaine accumulation. Meanwhile, photosynthetic efficiency and yield were significantly higher in the transgenic cotton than in the wild-type control under field conditions. This study provides a newly effective gene resource to cultivate new cotton varieties with high stresses resistance and yield.

Insights Into MicroRNA-Mediated Regulation of Flowering Time in Cotton Through Small RNA Sequencing.

The timing of flowering is a key determinant for plant reproductive. It has been demonstrated that microRNAs (miRNAs) play an important role in transition from the vegetative to reproductive stage in cotton; however, knowledge remains limited about the regulatory role of miRNAs involved in flowering time regulation in cotton. To elucidate the molecular basis of miRNAs in response to flowering time in cotton, we performed high-throughput small RNA sequencing at the fifth true leaf stage. We identified 56 and 43 miRNAs that were significantly up- and downregulated in two elite early flowering cultivars (EFC) compared with two late flowering cultivars (LFC), respectively. The miRNA targets by RNA sequencing analysis showed that GhSPL4 in SBP transcription factor family targeted by GhmiR156 was significantly upregulated in EFCs. Co-expression regulatory network analysis (WGCNA) revealed that GhSOC1, GhAP1, GhFD, GhCOL3, and GhAGL16 act as node genes in the auxin- and gibberellin-mediated flowering time regulatory networks in cotton. Therefore, elucidation of miRNA-mediated flowering time regulatory network will contribute to our understanding of molecular mechanisms underlying flowering time in cotton.

Multi-Dimensional Molecular Regulation of Trichome Development in Arabidopsis and Cotton.

Plant trichomes are specialized epidermal cells that are widely distributed on plant aerial tissues. The initiation and progression of trichomes are controlled in a coordinated sequence of multiple molecular events. During the past decade, major breakthroughs in the molecular understanding of trichome development were achieved through the characterization of various trichomes defective mutants and trichome-associated genes, which revealed a highly complex molecular regulatory network underlying plant trichome development. This review focuses on the recent millstone in plant trichomes research obtained using genetic and molecular studies, as well as 'omics' analyses in model plant Arabidopsis and fiber crop cotton. In particular, we discuss the latest understanding and insights into the underlying molecular mechanisms of trichomes formation at multiple dimensions, including at the chromatin, transcriptional, post-transcriptional, and post-translational levels. We summarize that the integration of multi-dimensional trichome-associated genes will enable us to systematically understand the molecular regulation network that landscapes the development of the plant trichomes. These advances will enable us to address the unresolved questions regarding the molecular crosstalk that coordinate concurrent and ordered the changes in cotton fiber initiation and progression, together with their possible implications for genetic improvement of cotton fiber.

Glyphosate-induced GhAG2 is involved in resistance to salt stress in cotton.

KEY MESSAGE: The transcription of GhAG2 was strongly enhanced by glyphosate treatment. Overexpression of GhAG2 could improve plant tolerance to salt and salicylic acid stress. Although glyphosate has been widely used as an herbicide over the past decade owing to its high efficacy on weed controls and worldwide commercialization of glyphosate-resistant crops, little is known about the glyphosate-induced responses and transcriptional changes in cotton plants. Here, we report the identification of 26 differentially expressed genes after glyphosate treatment, among which, six highly up-regulated sequences share homology to cotton expressed sequence tags (ESTs) responsive to abiotic stresses. In addition, we cloned GhAG2, a gene whose transcription was strongly enhanced by glyphosate treatment and other abiotic stresses. Transgenic GhAG2 plants showed improved tolerance to salt, and salicylic acid (SA) stress. The results could open the door to exploring the function of the GhAG2 proteins, the glyphosate-induced transcriptional profiles, and the physiological biochemical responses in cotton and other crops. GhAG2 could also be used to improve salt stress tolerance through breeding and biotechnology in crops. Furthermore, these results could provide guidelines to develop a glyphosate-inducible system for controlled expression of targeted genes in plants.

Overexpression of GhKTI12 Enhances Seed Yield and Biomass Production in Nicotiana Tabacum.

Crop molecular breeding primarily focuses on increasing the trait of plant yield. An elongator-associated protein, KTI12, is closely associated with plant biomass and yield. KTI12 is involved in developmental processes of most organs, including the leaf, root, flower, and seed, through regulating cell division and differentiation. Previous work has shown that in upland cotton (Gossypium hirsutum), GhKTI12 regulates plant height, flowering, and tolerance to salt and drought stress. However, little is known about the molecular regulation mechanism of GhKTI12 in plant developmental processes. In this study, we identified the main GhKTI12 (Gh_D02G144400) gene and transformed it into tobacco (Nicotonia tabacum cv NC89). From seven transgenic lines, we obtained three (OE5, OE6 and OE8) with high expression of GhKTI12; compared with wild type plants, these three lines exhibited larger plant size, later flowering, and higher seed yield. Microscopic observation revealed that the number of leaf epidermal cells and stem parenchyma cells was increased by ~55%. Biochemical analysis showed that chlorophyll content and starch accumulation were significantly increased in younger leaves at the top canopy of transgenic plants, which may contribute to improved photosynthetic rate and, in turn, increased seed yield. To understand the molecular mechanism of GhKTI12 in transgenic plants development, two lines (OE6 and OE8) with higher expression levels of GhKTI12 were used as representative plants to conduct RNA-seq analysis. Through transcriptome analysis of the plant's shoot apical meristematic tissue of these two lines, we identified 518 upregulated genes and 406 downregulated genes common to both overexpression lines. A large number of cellular component genes associated with cell division and differentiation, such as RD21, TET8, KTN80, AOX1, AOX2, CP1, and KIC, were found to be upregulated, and genes showing the most downregulation included MADS-box genes related to flowering time, such as MADS6, AP1, AP3, AGL8, AGL6, SEP1, and SEP2. Downregulation of these genes caused delayed flowering time and longer vegetative stage during development. Combined with the upregulation of the yield-related gene RD21, the GhKTI12 transgenic plants could produce a higher seed yield. We here show that the overexpression of GhKTI12 could positively improve key agronomic traits in tobacco by regulating cell proliferation, photosynthesis, and organ development, and suggest that homologs of GhKTI12 may also be important in the genetic improvement of other crop plants.

Increasing floral visitation and hybrid seed production mediated by beauty mark in Gossypium hirsutum.

Hybrid crop varieties have been repeatedly demonstrated to produce significantly higher yields than their parental lines; however, the low efficiency and high cost of hybrid seed production has limited the broad exploitation of heterosis for cotton production. One option for increasing the yield of hybrid seed is to improve pollination efficiency by insect pollinators. Here, we report the molecular cloning and characterization of a semidominant gene, Beauty Mark (BM), which controls purple spot formation at the base of flower petals in the cultivated tetraploid cotton species Gossypium barbadense. BM encodes an R2R3 MYB113 transcription factor, and we demonstrate that GbBM directly targets the promoter of four flavonoid biosynthesis genes to positively regulate petal spot development. Introgression of a GbBM allele into G. hirsutum by marker-assisted selection restored petal spot formation, which significantly increased the frequency of honeybee visits in G. hirsutum. Moreover, field tests confirmed that cotton seed yield was significantly improved in a three-line hybrid production system that incorporated the GbBM allele. Our study thus provides a basis for the potentially broad application of this gene in improving the long-standing problem of low seed production in elite cotton hybrid lines.

Correction: Syed, T., et al. Current Insights on Vegetative Insecticidal Proteins (Vip) as Next Generation Pest Killers. Toxins 2020, 12, 522.

The authors wish to make the following corrections to their paper [...].

Construction of Gossypium barbadense Mutant Library Provides Genetic Resources for Cotton Germplasm Improvement.

Allotetraploid cotton (Gossypium hirsutum and Gossypium barbadense) are cultivated worldwide for its white fiber. For centuries, conventional breeding approaches increase cotton yield at the cost of extensive erosion of natural genetic variability. Sea Island cotton (G. barbadense) is known for its superior fiber quality, but show poor adaptability as compared to Upland cotton. Here, in this study, we use ethylmethanesulfonate (EMS) as a mutagenic agent to induce genome-wide point mutations to improve the current germplasm resources of Sea Island cotton and develop diverse breeding lines with improved adaptability and excellent economic traits. We determined the optimal EMS experimental procedure suitable for construction of cotton mutant library. At M6 generation, mutant library comprised of lines with distinguished phenotypes of the plant architecture, leaf, flower, boll, and fiber. Genome-wide analysis of SNP distribution and density in yellow leaf mutant reflected the better quality of mutant library. Reduced photosynthetic efficiency and transmission electron microscopy of yellow leaf mutants revealed the effect of induced mutations at physiological and cellular level. Our mutant collection will serve as the valuable resource for basic research on cotton functional genomics, as well as cotton breeding.

Current Insights on Vegetative Insecticidal Proteins (Vip) as Next Generation Pest Killers.

Bacillus thuringiensis (Bt) is a Gram negative soil bacterium. This bacterium secretes various proteins during different growth phases with an insecticidal potential against many economically important crop pests. One of the important families of Bt proteins is vegetative insecticidal proteins (Vip), which are secreted into the growth medium during vegetative growth. There are three subfamilies of Vip proteins. Vip1 and Vip2 heterodimer toxins have an insecticidal activity against many Coleopteran and Hemipteran pests. Vip3, the most extensively studied family of Vip toxins, is effective against Lepidopteron. Vip proteins do not share homology in sequence and binding sites with Cry proteins, but share similarities at some points in their mechanism of action. Vip3 proteins are expressed as pyramids alongside Cry proteins in crops like maize and cotton, so as to control resistant pests and delay the evolution of resistance. Biotechnological- and in silico-based analyses are promising for the generation of mutant Vip proteins with an enhanced insecticidal activity and broader spectrum of target insects.

Genome-Wide Characterization and Expression Analysis of NHX Gene Family under Salinity Stress in Gossypium barbadense and Its Comparison with Gossypium hirsutum.

Cotton is an important economic crop affected by different abiotic stresses at different developmental stages. Salinity limits the growth and productivity of crops worldwide. Na+/H+ antiporters play a key role during the plant development and in its tolerance to salt stress. The aim of the present study was a genome-wide characterization and expression pattern analysis under the salinity stress of the sodium-proton antiporter (NHX) of Gossypium barbadense in comparison with Gossypium hirsutum. In G. barbadense, 25 NHX genes were identified on the basis of the Na+_H+ exchanger domain. All except one of the G. barbadenseNHX transporters have an Amiloride motif that is a known inhibitor of Na+ ions in plants. A phylogenetic analysis inferred three classes of GbNHX genes-viz., Vac (GbNHX1, 2 and 4), Endo (GbNHX6), and PM (GbNHX7). A high number of the stress-related cis-acting elements observed in promoters show their role in tolerance against abiotic stresses. The Ka/Ks values show that the majority of GbNHX genes are subjected to strong purifying selection under the course of evolution. To study the functional divergence of G. barbadenseNHX transporters, the real-time gene expression was analyzed under salt stress in the root, stem, and leaf tissues. In G. barbadense, the expression was higher in the stem, while in G. hirsutum the leaf and root showed a high expression. Moreover, our results revealed that NHX2 homologues in both species have a high expression under salinity stress at higher time intervals, followed by NHX7. The protein-protein prediction study revealed that GbNHX7 is involved in the CBL-CIPK protein interaction pathway. Our study also provided valuable information explaining the molecular mechanism of Na+ transport for the further functional study of Gossypium NHX genes.

GFF3sort: a novel tool to sort GFF3 files for tabix indexing.

BACKGROUND: The traditional method of visualizing gene annotation data in JBrowse is converting GFF3 files to JSON format, which is time-consuming. The latest version of JBrowse supports rendering sorted GFF3 files indexed by tabix, a novel strategy that is more convenient than the original conversion process. However, current tools available for GFF3 file sorting have some limitations and their sorting results would lead to erroneous rendering in JBrowse. RESULTS: We developed GFF3sort, a script to sort GFF3 files for tabix indexing. Specifically designed for JBrowse rendering, GFF3sort can properly deal with the order of features that have the same chromosome and start position, either by remembering their original orders or by conducting parent-child topology sorting. Based on our test datasets from seven species, GFF3sort produced accurate sorting results with acceptable efficiency compared with currently available tools. CONCLUSIONS: GFF3sort is a novel tool to sort GFF3 files for tabix indexing. We anticipate that GFF3sort will be useful to help with genome annotation data processing and visualization.