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IntroductionVerticillium wilt (VW) caused by Verticillium dahliae is a soil-borne vascular fungal disease that severely affects cotton yield and fiber quality. Sugar metabolism plays an important role in the growth and pathogenicity of V. dahliae. However, limited information is known about the sugar transporter genes and their roles in the growth and pathogenicity of V. dahliae.MethodIn this study, genome-wide identification of sugar transporter genes in V. dahliae was conducted and the expression profiles of these genes in response to root exudates from cotton varieties susceptible or resistant to V. dahliae were investigated based on RNA-seq data. Tobacco Rattle Virus-based host-induced gene silencing (TRV-based HIGS) and artificial small interfering RNAs (asiRNAs) were applied to investigate the function of candidate genes involved in the growth and pathogenic process of V. dahliae.ResultsA total of 65 putative sugar transporter genes were identified and clustered into 8 Clades. Of the 65 sugar transporter genes, 9 were found to be induced only by root exudates from the susceptible variety, including VdST3 and VdST12 that were selected for further functional study. Silencing of VdST3 or VdST12 in host plants by TRV-based HIGS reduced fungal biomass and enhanced cotton resistance against V. dahliae. Additionally, silencing of VdST12 and VdST3 by feeding asiRNAs targeting VdST12 (asiR815 or asiR1436) and VdST3 (asiR201 or asiR1238) inhibited fungal growth, exhibiting significant reduction in hyphae and colony diameter, with a more significant effect observed for the asiRNAs targeting VdST12. The inhibitory effect of asiRNAs on the growth of V. dahliae was enhanced with the increasing concentration of asiRNAs. Silencing of VdST12 by feeding asiR815+asiR1436 significantly decreased the pathogenicity of V. dahliae.DiscussionThe results suggest that VdST3 and VdST12 are sugar transporter genes required for growth and pathogenicity of V. dahliae and that asiRNA is a valuable tool for functional characterization of V. dahliae genes.

The Commonwealth Scientific and Industrial Research Organisation (CSIRO) cotton breeding program is the sole breeding effort for cotton in Australia, developing high performing cultivars for the local industry which is worth∼AU$3 billion per annum. The program is supported by Cotton Breeding Australia, a Joint Venture between CSIRO and the program’s commercial partner, Cotton Seed Distributors Ltd. (CSD). While the Australian industry is the focus, CSIRO cultivars have global impact in North America, South America, and Europe. The program is unique compared with many other public and commercial breeding programs because it focuses on diverse and integrated research with commercial outcomes. It represents the full research pipeline, supporting extensive long-term fundamental molecular research; native and genetically modified (GM) trait development; germplasm enhancement focused on yield and fiber quality improvements; integration of third-party GM traits; all culminating in the release of new commercial cultivars. This review presents evidence of past breeding successes and outlines current breeding efforts, in the areas of yield and fiber quality improvement, as well as the development of germplasm that is resistant to pests, diseases and abiotic stressors. The success of the program is based on the development of superior germplasm largely through field phenotyping, together with strong commercial partnerships with CSD and Bayer CropScience. These relationships assist in having a shared focus and ensuring commercial impact is maintained, while also providing access to markets, traits, and technology. The historical successes, current foci and future requirements of the CSIRO cotton breeding program have been used to develop a framework designed to augment our breeding system for the future. This will focus on utilizing emerging technologies from the genome to phenome, as well as a panomics approach with data management and integration to develop, test and incorporate new technologies into a breeding program. In addition to streamlining the breeding pipeline for increased genetic gain, this technology will increase the speed of trait and marker identification for use in genome editing, genomic selection and molecular assisted breeding, ultimately producing novel germplasm that will meet the coming challenges of the 21st Century.

Verticillium wilt, caused by the soil-borne fungus Verticillium dahliae, is one of the most devastating diseases in cotton (Gossypium spp.). Lignin in the cell wall forms a physical barrier to inhibit pathogen invasion, and defense-induced lignification reinforces secondary cell wall to prevent pathogens from further spreading. Cinnamyl alcohol dehydrogenases (CADs) catalyze the production of three main monolignols, p-coumaryl- (H), coniferyl- (G), and sinapyl-alcohols (S), which are the fundamental blocks of lignin. Here, we identified CAD genes in G. hirsutum, analyzed their expression profiles in cotton leaf, stem, and root from different developmental stages, and selected GhCAD35, GhCAD45, and GhCAD43, which were consistently induced by V. dahliae inoculation in G. hirsutum cultivars resistant or susceptible to V. dahliae. On the basis of confirmation of the in vitro enzymatic activity of the three proteins in generation of the three monolignols, we used virus-induced gene silencing (VIGS) to investigate the effects of silencing of GhCAD35, GhCAD45, or GhCAD43 on resistance to V. dahliae as well as on deposition and the composition of lignin. Silencing each of the three CADs impaired the defense-induced lignification and salicylic acid biosynthesis in stem, and compromised resistance to V. dahliae. Moreover, our study showed that silencing the three GhCADs severely affected the biosynthesis of S-lignin, leading to a decrease of the syringyl/guaiacyl (S/G) ratio. Heterogeneous overexpression of GhCAD35, GhCAD45, or GhCAD43 in Arabidopsis enhanced disease resistance. Taken together, our study demonstrates a role of the three GhCADs in defense-induced lignin biosynthesis and resistance to V. dahliae in G. hirsutum.

Many factors, including illumination, affect anthocyanin biosynthesis and accumulation in plants. light quality is the key factor affecting the process of photoinduced anthocyanin biosynthesis and accumulation. We observed that the red color of the Upland cotton accession Huiyuan with the R1 mutation turned to normal green color under light-emitting diodes (LEDs), which inspired us to investigate the effect of red and blue lights on the biosynthesis and accumulation of anthocyanins. We found that both red and blue lights elevated accumulation of anthocyanins. Comparative transcriptomic analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and GSEA, revealed that genes differentially expressed under different light conditions were enriched with the pathways of circadian rhythm, phenylpropanoid biosynthesis, anthocyanin biosynthesis, and flavone and flavonol biosynthesis. Not surprisingly, all the major structural genes related to biosynthesis of anthocyanins, including the key regulatory MYB transcription factor (GhPAP1D) and anthocyanin transporter (GhGSTF12), were induced by red or blue light treatment. However, LARs and MATEs related to biosynthesis of proanthocyanidins were more significantly up-regulated by red light radiation than by blue light radiation. Vice versa, the accumulation of anthocyanins under red light was not as high as that under blue light. In addition, we demonstrated a potential role of GhHY5, a key regulator in plant circadian rhythms, in regulation of anthocyanin accumulation, which could be achieved via interaction with GhPAP1D. Together, these results indicate different effect of red and blue lights on biosynthesis and accumulation of anthocyanins and a potential module including GhHY5 and GhPAP1D in regulation of anthocyanin accumulation in cotton. These results also suggest that the substrates responsible the synthesis of anthocyanins under blue light is diverted to biosynthesis of proanthocyanidin under red light.

Verticillium wilt, caused by Verticillium dahliae, is one of the most damaging and widespread soil-borne cotton diseases. The molecular mechanisms underlying the cotton defense against V. dahliae remain largely elusive. Here, we compared the transcriptional differences between Upland cotton cultivars: one highly resistant (HR; Shidalukang 1) and one highly susceptible (HS; Junmian 1). This was done at multiple time points after V. dahliae inoculation, which identified 2010 and 1275 differentially expressed genes (DEGs) in HR and HS, respectively. Plant hormone signal transduction-related genes were enriched in HR, whereas genes related to lignin biosynthesis were enriched in both HR and HS. Weighted gene co-expression network analysis (WGCNA) using the 2868 non-redundant genes differentially expressed between the V. dahliae infected and uninfected samples in HR or HS identified 10 different gene network modules and 22 hub genes with a potential role in regulating cotton defense against V. dahliae infection. GhGDH2, encoding glutamate dehydrogenase (GDH), was selected for functional characterization. Suppressing the expression level of GhGDH2 by virus-induced gene silencing (VIGS) in HS led to inhibition of the salicylic acid (SA) biosynthesis/signaling pathways and activation of the jasmonic acid (JA) biosynthesis/signaling pathways, which resulted in an increase of 42.1% JA content and a reduction of 78.9% SA content in cotton roots, and consequently enhanced V. dahliae resistance. Our finding provides new insights on the molecular mechanisms of cotton resistance to V. dahliae infection and candidate genes for breeding V. dahliae resistance cotton cultivars by genetic modification.

Silicon (Si) plays an important role in improving soil nutrient availability and plant carbon (C) accumulation and may therefore impact the biogeochemical cycles of C, nitrogen (N), and phosphorus (P) in terrestrial ecosystems profoundly. However, research on this process in grassland ecosystems is scarce, despite the fact that these ecosystems are one of the most significant accumulators of biogenic Si (BSi). In this study, we collected the aboveground parts of four widespread grasses and soil profile samples in northern China and assessed the correlations between Si concentrations and stoichiometry and accumulation of C, N, and P in grasses at the landscape scale. Our results showed that Si concentrations in plants were significantly negatively correlated (p < 0.01) with associated C concentrations. There was no significant correlation between Si and N concentrations. It is worth noting that since the Si concentration increased, the P concentration increased from less than 0.10% to more than 0.20% and therefore C:P and N:P ratios decreased concomitantly. Besides, the soil noncrystalline Si played more important role in C, N, and P accumulation than other environmental factors (e.g., MAT, MAP, and altitude). These findings indicate that Si may facilitate grasses in adjusting the utilization of nutrients (C, N, and P) and may particularly alleviate P deficiency in grasslands. We conclude that Si positively alters the concentrations and accumulation of C, N, and P likely resulting in the variation of ecological stoichiometry in both vegetation and litter decomposition in soils. This study further suggests that the physiological function of Si is an important but overlooked factor in influencing biogeochemical cycles of C and P in grassland ecosystems.

Verticillium wilt caused by Verticillium dahliae is a destructive cotton disease causing severe yield and quality losses worldwide. WRKY transcription factors play important roles in plant defense against pathogen infection. However, little has been reported on the functions of WRKYs in cotton's resistance to V. dahliae. Here, we identified 5, 5, and 10 WRKY70 genes in Gossypium arboreum, Gossypium raimondii, and Gossypium hirsutum, respectively, and investigated the expression profiles of all GhWRKY70 genes in various cotton tissues and in response to hormone treatment or V. dahliae infection. Reverse transcription-quantitative PCR analysis showed that GhWRKY70D13 was expressed higher in roots and stems than in other tissues, and up-regulated after V. dahliae inoculation. Knock-down of GhWRKY70D13 improved resistance to V. dahliae in both resistant and susceptible cotton cultivars. Comparative analysis of transcriptomes generated from wild-type and stable RNAi (RNA interference) plant with down-regulated GhWRKY70D13 showed that genes involved in ethylene (ET) and jasmonic acid (JA) biosynthesis and signaling were significantly upregulated in the GhWRKY70D13 RNAi plants. Consistently, the contents of 1-aminocyclopropane-1-carboxylic (ACC), JA, and JA-isoleucine levels were significantly higher in the GhWRKY70D13 RNAi plants than in wild-type. Following V. dahliae infection, the levels of ACC and JA decreased in the GhWRKY70D13 RNAi plants but still significantly higher (for ACC) than that in wild-type or at the same level (for JA) as in non-infected wild-type plants. Collectively, our results suggested that GhWRKY70D13 negatively regulates cotton's resistance to V. dahliae mainly through its effect on ET and JA biosynthesis and signaling pathways.

Phytolith-occluded carbon (PhytOC) can be preserved in soils or sediments for thousands of years and might be a promising potential mechanism for long-term terrestrial carbon (C) sequestration. As the principal pathway for the return of organic matters to soils, the forest litter layers make a considerable contribution to terrestrial C sequestration. Although previous studies have estimated the phytolith production fluxes in the above-ground vegetations of various terrestrial ecosystems, the storages of phytoliths and PhytOC in litter layers have not been thoroughly investigated, especially in forest ecosystems. Using analytical data of silica, phytoliths, return fluxes and storages of forest litter, this study estimated the phytolith and PhytOC storages in litter layers in different forest types in southern China. The results indicated that the total phytolith storage in forest litter layers in southern China was 24.34 ± 8.72 Tg. Among the different forest types, the phytolith storage in bamboo forest litter layers (15.40 ± 3.40 Tg) was much higher than that in other forests. At the same time, the total PhytOC storage reached up to 2.68 ± 0.96 Tg CO2 in forest litter layers in southern China, of which approximately 60% was contributed by bamboo forest litter layers. Based on the current litter turnover time of different forest types in southern China, a total of 1.01 ± 0.32 Tg of PhytOC per year would be released into soil profiles as a stable C pool during litter decomposition, which would make an important contribution to the global terrestrial long-term biogeochemical C sink. Therefore, the important role of PhytOC storage in forest litter layers should be taken into account in evaluating long-term forest C budgets.

Cytoplasmic linker-associated proteins (CLASPs) are microtubule-associated proteins (MAPs) involved in regulation of dynamics of microtubules (MTs) that play an important role in plant growth and development. In this study, we identified cotton CLASP genes and investigated the function of GhCLASP2. GhCLASP2 was mainly expressed in stem and developing fibers, especially in fibers of the secondary cell wall deposition stage. Ectopic expression of GhCLASP2 in Arabidopsis increased the branching number of leaf trichomes and rescued the defective phenotypes of clasp-1. In cotton, overexpression of GhCLASP2 increased fiber strength, probably related to enhanced expression levels of tubulin, cellulose synthase, and expansin genes. Suppression of GhCLASP2 caused shorter internodes and semi-dwarfism, abnormal flower stigma, aborted anthers without pollen grains, and sterility. These changed phenotypes were similar to those observed in the Arabidopsis clasp-1 mutant. GhCLASP2 was co-localized with MTs according to transient experiment. These results suggest that GhCLASP2 functions similarly as AtCLASP, acting as a MAP and controlling cotton growth and development by regulating MTs.

The objective of this study was to increase understanding about the mechanism by which polyamines (PAs) promote the conversion of embryogenic calli (EC) into somatic embryos in cotton (Gossypium hirsutum L.). We measured the levels of endogenous PAs and H2O2, quantified the expression levels of genes involved in the PAs pathway at various stages of cotton somatic embryogenesis (SE), and investigated the effects of exogenous PAs and H2O2 on differentiation and development of embryogenic calli. Putrescine (Put), spermidine (Spd) and spermine (Spm) significantly increased from the EC stage to the early phase of embryo differentiation. The levels of Put then decreased until the somatic embryo stage whereas Spd and Spm remained nearly the same. The expression profiles of GhADC genes were consistent with changes in Put during cotton SE. The H2O2 concentrations began to increase significantly at the EC stage, during which time both GhPAO1 and GhPAO4 expressions were highest and PAO activity was significantly increased. Exogenous Put, Spd, Spm and H2O2 not only enhanced embryogenic callus growth and embryo formation, but also alleviated the effects of D-arginine and 1, 8-diamino-octane, which are inhibitors of polyamine synthesis and PAO activity. Overall, the results suggest that both PAs and their metabolic product H2O2 are essential for the conversion of EC into somatic embryos in cotton.

A wide range of RNA species interacting with microRNAs (miRNAs) form a complex gene regulation network and play vital roles in diverse biological processes. In this study, we performed a genome-wide identification of endogenous target mimics (eTMs) for miRNAs and phased-siRNA-producing loci (PHAS) in soybean with a focus on those involved in lipid metabolism. The results showed that a large number of eTMs and PHAS genes could be found in soybean. Additionally, we found that lipid metabolism related genes were potentially regulated by 28 miRNAs, and nine of them were potentially further regulated by a number of eTMs with expression evidence. Thirty-three miRNAs were found to trigger production of phasiRNAs from 49 PHAS genes, which were able to target lipid metabolism related genes. Degradome data supported miRNA- and/or phasiRNA-mediated cleavage of genes involved in lipid metabolism. Most eTMs for miRNAs involved in lipid metabolism and phasiRNAs targeting lipid metabolism related genes showed a tissue-specific expression pattern. Our bioinformatical evidences suggested that lipid metabolism in soybean is potentially regulated by a complex non-coding network, including miRNAs, eTMs and phasiRNAs, and the results extended our knowledge on functions of non-coding RNAs.

Cytoplasmic linker-associated proteins (CLASPs) are microtubule-associated proteins (MAPs) involved in regulation of dynamics of microtubules (MTs) that play an important role in plant growth and development. In this study, we identified cotton CLASP genes and investigated the function of GhCLASP2. GhCLASP2 was mainly expressed in stem and developing fibers, especially in fibers of the secondary cell wall deposition stage. Ectopic expression of GhCLASP2 in Arabidopsis increased the branching number of leaf trichomes and rescued the defective phenotypes of clasp-1. In cotton, overexpression of GhCLASP2 increased fiber strength, probably related to enhanced expression levels of tubulin, cellulose synthase, and expansin genes. Suppression of GhCLASP2 caused shorter internodes and semi-dwarfism, abnormal flower stigma, aborted anthers without pollen grains, and sterility. These changed phenotypes were similar to those observed in the Arabidopsis clasp-1 mutant. GhCLASP2 was co-localized with MTs according to transient experiment. These results suggest that GhCLASP2 functions similarly as AtCLASP, acting as a MAP and controlling cotton growth and development by regulating MTs. [ABSTRACT FROM AUTHOR]

The objective of this study was to increase understanding about the mechanism by which polyamines (PAs) promote the conversion of embryogenic calli (EC) into somatic embryos in cotton (Gossypium hirsutum L.). We measured the levels of endogenous PAs and H2O2, quantified the expression levels of genes involved in the PAs pathway at various stages of cotton somatic embryogenesis (SE), and investigated the effects of exogenous PAs and H2O2 on differentiation and development of EC. Putrescine (Put), spermidine (Spd), and spermine (Spm) significantly increased from the EC stage to the early phase of embryo differentiation. The levels of Put then decreased until the somatic embryo stage whereas Spd and Spm remained nearly the same. The expression profiles of GhADC genes were consistent with changes in Put during cotton SE. The H2O2 concentrations began to increase significantly at the EC stage, during which time both GhPAO1 and GhPAO4 expressions were highest and PAO activity was significantly increased. Exogenous Put, Spd, Spm, and H2O2 not only enhanced embryogenic callus growth and embryo formation, but also alleviated the effects of D-arginine and 1, 8-diamino-octane, which are inhibitors of PA synthesis and PAO activity. Overall, the results suggest that both PAs and their metabolic product H2O2 are essential for the conversion of EC into somatic embryos in cotton. [ABSTRACT FROM AUTHOR]

A wide range of RNA species interacting with microRNAs (miRNAs) form a complex gene regulation network and play vital roles in diverse biological processes. In this study, we performed a genome-wide identification of endogenous target mimics (eTMs) for miRNAs and phased-siRNA-producing loci (PHAS) in soybean with a focus on those involved in lipid metabolism. The results showed that a large number of eTMs and PHAS genes could be found in soybean. Additionally, we found that lipid metabolism related genes were potentially regulated by 28 miRNAs, and nine of them were potentially further regulated by a number of eTMs with expression evidence. Thirty-three miRNAs were found to trigger production of phasiRNAs from 49 PHAS genes, which were able to target lipid metabolism related genes. Degradome data supported miRNA- and/or phasiRNA-mediated cleavage of genes involved in lipid metabolism. Most eTMs for miRNAs involved in lipid metabolism and phasiRNAs targeting lipid metabolism related genes showed a tissue-specific expression pattern. Our bioinformatical evidences suggested that lipid metabolism in soybean is potentially regulated by a complex non-coding network, including miRNAs, eTMs, and phasiRNAs, and the results extended our knowledge on functions of non-coding RNAs. [ABSTRACT FROM AUTHOR]

Vegetable oils high in oleic acid are considered to be advantageous because of their better nutritional value and potential industrial applications. The oleic acid content in the classic safflower oil is normally 10-15% while a natural mutant (ol) accumulates elevated oleic acid up to 70% in seed oil. As a part of our investigation into the molecular features of the high oleic (HO) trait in safflower we have profiled the microRNA (miRNA) populations in developing safflower seeds expressing the ol allele in comparison to the wild type high linoleic (HL) safflower using deep sequencing technology. The small RNA populations of the mid-maturity developing embryos of homozygous ol HO and wild type HL safflower had a very similar size distribution pattern, however, only ∼16.5% of the unique small RNAs were overlapping in these two genotypes. From these two small RNA populations we have found 55 known miRNAs and identified two candidate novel miRNA families to be likely unique to the developing safflower seeds. Target genes with conserved as well as novel functions were predicted for the conserved miRNAs. We have also identified 13 miRNAs differentially expressed between the HO and HL safflower genotypes. The results may lay a foundation for unraveling the miRNA-mediated molecular processes that regulate oleic acid accumulation in the HO safflower mutant and developmental processes in safflower embryos in general. [ABSTRACT FROM AUTHOR]

Gossypium hirsutum, Verticillium dahliae, GhWRKY70D13, ribonucleic acid interference, jasmonic acid, ethylene. [Extracted from the article]