Single-cell landscape of long and short glandular trichomes in Nicotiana tabacum leaves.

Glandular trichomes (GTs) play a crucial role in plant defenses and the synthesis of secondary metabolites. Understanding the developmental trajectory of GTs is essential for unraveling their functional significance and potential applications. Here we established a comprehensive single-cell atlas of Nicotiana tabacum leaves, a model plant for GT studies. The atlas included a total of 40,433 cells and successfully captured both long GTs (LGTs) and short GTs (SGTs) from Nicotiana leaves. The developmental trajectories of these trichomes were delineated, revealing potential disparities in epidermal development. Comparative analysis of Arabidopsis and Nicotiana trichome development indicated limited similarity between Arabidopsis epidermal non-glandular trichomes and Nicotiana LGTs and SGTs, implying the essentiality of studying the genes directly involved in the development of Nicotiana GTs for a proper and comprehensive understanding of GT biology. Overall, our results provide profound insights into the developmental intricacies of the specialized GTs.

A reference genome of Commelinales provides insights into the commelinids evolution and global spread of water hyacinth (Pontederia crassipes).

Commelinales belongs to the commelinids clade, which also comprises Poales that includes the most important monocot species, such as rice, wheat, and maize. No reference genome of Commelinales is currently available. Water hyacinth (Pontederia crassipes or Eichhornia crassipes), a member of Commelinales, is one of the devastating aquatic weeds, although it is also grown as an ornamental and medical plant. Here, we present a chromosome-scale reference genome of the tetraploid water hyacinth with a total length of 1.22 Gb (over 95% of the estimated size) across 8 pseudochromosome pairs. With the representative genomes, we reconstructed a phylogeny of the commelinids, which supported Zingiberales and Commelinales being sister lineages of Arecales and shed lights on the controversial relationship of the orders. We also reconstructed ancestral karyotypes of the commelinids clade and confirmed the ancient commelinids genome having 8 chromosomes but not 5 as previously reported. Gene family analysis revealed contraction of disease-resistance genes during polyploidization of water hyacinth, likely a result of fitness requirement for its role as a weed. Genetic diversity analysis using 9 water hyacinth lines from 3 continents (South America, Asia, and Europe) revealed very closely related nuclear genomes and almost identical chloroplast genomes of the materials, as well as provided clues about the global dispersal of water hyacinth. The genomic resources of P. crassipes reported here contribute a crucial missing link of the commelinids species and offer novel insights into their phylogeny.

Identification of sugar transporter genes and their roles in the pathogenicity of Verticillium dahliae on cotton.

Introduction: Verticillium 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. Method: In 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. Results: A 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. Discussion: The 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.

Genomic insights into the evolution of Echinochloa species as weed and orphan crop.

As one of the great survivors of the plant kingdom, barnyard grasses (Echinochloa spp.) are the most noxious and common weeds in paddy ecosystems. Meanwhile, at least two Echinochloa species have been domesticated and cultivated as millets. In order to better understand the genomic forces driving the evolution of Echinochloa species toward weed and crop characteristics, we assemble genomes of three Echinochloa species (allohexaploid E. crus-galli and E. colona, and allotetraploid E. oryzicola) and re-sequence 737 accessions of barnyard grasses and millets from 16 rice-producing countries. Phylogenomic and comparative genomic analyses reveal the complex and reticulate evolution in the speciation of Echinochloa polyploids and provide evidence of constrained disease-related gene copy numbers in Echinochloa. A population-level investigation uncovers deep population differentiation for local adaptation, multiple target-site herbicide resistance mutations of barnyard grasses, and limited domestication of barnyard millets. Our results provide genomic insights into the dual roles of Echinochloa species as weeds and crops as well as essential resources for studying plant polyploidization, adaptation, precision weed control and millet improvements.

Corrigendum: GhWRKY70D13 Regulates Resistance to Verticillium dahliae in Cotton Through the Ethylene and Jasmonic Acid Signaling Pathways.

[This corrects the article DOI: 10.3389/fpls.2020.00069.].

GhWRKY70D13 Regulates Resistance to Verticillium dahliae in Cotton Through the Ethylene and Jasmonic Acid Signaling Pathways.

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.

A transcriptomic profile of topping responsive non-coding RNAs in tobacco roots (Nicotiana tabacum).

BACKGROUND: Non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long ncRNAs (lncRNAs) and circular RNAs (circRNAs), accomplish remarkable variety of biological functions. However, the composition of ncRNAs and their interactions with coding RNAs in modulating and controlling of cellular process in plants is largely unknown. Using a diverse group of high-throughput sequencing strategies, the mRNA, miRNA, lncRNA and circRNA compositions of tobacco (Nicotiana tabacum) roots determined and their alteration and potential biological functions in response to topping treatment analyzed. RESULTS: A total of 688 miRNAs, 7423 non-redundant lncRNAs and 12,414 circRNAs were identified, among which, some selected differentially expressed RNAs were verified by quantitative real-time PCR. Using the differentially expressed RNAs, a co-expression network was established that included all four types of RNAs. The number of circRNAs identified were higher than that of miRNAs and lncRNAs, but only two circRNAs were present in the co-expression network. LncRNAs appear to be the most active ncRNAs based on their numbers presented in the co-expression network, but none of them seems to be an eTM (endogenous Target Mimicry) of miRNAs. Integrated with analyses of sequence interaction, several mRNA-circRNA-miRNA interaction networks with a potential role in the regulation of nicotine biosynthesis were uncovered, including a QS-circQS-miR6024 interaction network. In this network miR6024 was significantly down-regulated, while the expression levels of its two targets, circQS and its host gene QS, were sharply increased following the topping treatment. CONCLUSIONS: These results illustrated the transcriptomic profiles of tobacco roots, the organ responsible for nicotine biosynthesis. mRNAs always play the most important roles, while ncRNAs are also expressed extensively for topping treatment response, especially circRNAs are the most activated in the ncRNA pool. These studies also provided insights on the coordinated regulation module of coding and non-coding RNAs in a single plant biological sample. The findings reported here indicate that ncRNAs appear to form interaction complex for the regulation of stress response forming regulation networks with transcripts involved in nicotine biosynthesis in tobacco.

Characterization and evolution of gene clusters for terpenoid phytoalexin biosynthesis in tobacco.

MAIN CONCLUSION: The study performed genome-wide identification, characterization and evolution analysis of gene clusters for phytoalexin terpenoid biosynthesis in tobacco, and specifically illustrated ones for capsidiol, an efficient defensive specialized metabolite. Terpenoid phytoalexins play an important role in plant self-defense against pest and pathogen attack. Terpenoid biosynthesis involves terpene synthase and cytochrome P450, which always locate and function as cluster(s). In this study, we performed genome-wide investigation of metabolic gene clusters involved in terpenoid production in tobacco (Nicotiana tabacum). Due to the complexity of the tobacco genome, we modified a published prediction pipeline to reduce the influence of the large number of repeats and to improve the annotation of tobacco genes with respect to their metabolic functions. We identified 1181 metabolic gene clusters with 34 of them potentially being involved in terpenoid biosynthesis. Through integration with transcriptome and metabolic pathway annotation analyses, 3 of the 34 terpenoid biosynthesis-related gene clusters were determined to be high-confidence ones, with 2 involved in biosynthesis of capsidiol, a terpenoid recognized as 1 of the effective resistance compounds in the Nicotiana species. The capsidiol-related gene cluster was conserved in N. sylvestris, N. tomentosiformis and N. attenuate. Our findings demonstrate that phytoalexins in tobacco can arise from operon-like gene clusters, a genomic pattern characterized as being beneficial for rapid stress response, gene co-regulation, co-function and co-heredity.

Genome-wide identification of oil biosynthesis-related long non-coding RNAs in allopolyploid Brassica napus.

BACKGROUND: Long noncoding RNAs (lncRNAs) are transcripts longer than 200 bp that do not encode proteins but nonetheless have been shown to play important roles in various biological processes in plants. Brassica napus is an important seed oil crop worldwide and the target of many genetic improvement activities. To understand better the function of lncRNAs in regulating plant metabolic activities, we carried out a genome-wide lncRNA identification of lncRNAs in Brassica napus with a focus on lncRNAs involved in lipid metabolism. Twenty ribosomal RNA depleted strand specific RNA-seq (ssRNA-seq) datasets were generatred using RNAs isolated from B. napus seeds at four developmental stages. For comparison we also included 30 publically available RNA-seq datasets generated from poly(A) enriched mRNAs isolated from from various Brassica napus tissues in our analysis. RESULTS: A total of 8905 lncRNA loci were identified, including 7100 long intergenic noncoding RNA (lincRNA) loci and 1805 loci generating long noncoding natural antisense transcript (lncNAT). Many lncRNAs were identified only in the ssRNA-seq and poly(A) RNA-seq dataset, suggesting that B. napus has a large lncRNA repertoire and it is necessary to use libraries prepared from different tissues and developmental stages as well as different library preparation approaches to capture the whole spectrum of lncRNAs. Analysis of coexpression networks revealed that among the regulatory modules are networks containing lncRNAs and protein-coding genes related to oil biosynthesis indicating a possible role of lncRNAs in the control of lipid metabolism. One such example is that several lncRNAs are potential regulators of BnaC08g11970D that encodes oleosin1, a protein found in oil bodies and involved in seed lipid accumulation. We also observed that the expression levels of B. napus lncRNAs is positively correlated with their conservation levels. CONCLUSIONS: We demonstrated that the B. napus genome has a large number of lncRNA and that these lncRNAs are expressed broadly across many developmental times and in different tissue types. We also provide evidence indicating that specific lncRNAs appear to be important regulators of lipid biosynthesis forming regulatory networks with transcripts involved in lipid biosynthesis. We also provide evidence that these lncRNAs are conserved in other species of the Brassicaceae family.

Simultaneous silencing of GhFAD2-1 and GhFATB enhances the quality of cottonseed oil with high oleic acid.

Cottonseed oil has become an important source of edible oil due to its significant cost advantage. However, there is a growing concern over its fatty acid composition and nutritional value. In Gossypium hirsutum, GhFAD2-1 and GhFATB encoding the microsomal oleate desaturase and palmitoyl-acyl carrier protein thioesterase, respectively, play critical roles in regulating the proportions of saturated and polyunsaturated fatty acids in cottonseed lipids. In this study, RNAi technology was used to simultaneously inhibit the expression levels of GhFAD2-1 and GhFATB to improve the quality of cottonseed oil by increasing oleic acid content. Transgenic cotton plants with reduced levels of both target genes were successfully generated. In mature seed kernels of transgenic plants, the content of oleic acid was 38.25%, accordingly increasing by 156.96%, while the content of palmitic acid and linoleic acid was 19.15% and 36.68%, decreasing by 21.28% and 33.92%, respectively, compared with that of the control. The total oil content in transgenic and control kernels was 22.48% and 29.83%, respectively. The reduced oil level in transgenic seeds was accompanied by a reduction in seed index, thereby causing disadvantageous effects on seed germination potentiality and seed vigor, particularly under cool stress conditions. Our results demonstrated the feasibility of simultaneous manipulation of multiple genes using RNAi technology and showed the important role of oil content in seed development and vigor. Our findings provide insight into the physiological significance of the fatty acid composition in cottonseeds.

Regulation of Nicotine Biosynthesis by an Endogenous Target Mimicry of MicroRNA in Tobacco.

The interaction between noncoding endogenous target mimicry (eTM) and its corresponding microRNA (miRNA) is a newly discovered regulatory mechanism and plays pivotal roles in various biological processes in plants. Tobacco (Nicotiana tabacum) is a model plant for studying secondary metabolite alkaloids, of which nicotine accounts for approximately 90%. In this work, we identified four unique tobacco-specific miRNAs that were predicted to target key genes of the nicotine biosynthesis and catabolism pathways and an eTM, novel tobacco miRNA (nta)-eTMX27, for nta-miRX27 that targets QUINOLINATE PHOSPHORIBOSYLTRANSFERASE2 (QPT2) encoding a quinolinate phosphoribosyltransferase. The expression level of nta-miRX27 was significantly down-regulated, while that of QPT2 and nta-eTMX27 was significantly up-regulated after topping, and consequently, nicotine content increased in the topping-treated plants. The topping-induced down-regulation of nta-miRX27 and up-regulation of QPT2 were only observed in plants with a functional nta-eTMX27 but not in transgenic plants containing an RNA interference construct targeting nta-eTMX27. Our results demonstrated that enhanced nicotine biosynthesis in the topping-treated tobacco plants is achieved by nta-eTMX27-mediated inhibition of the expression and functions of nta-miRX27. To our knowledge, this is the first report about regulation of secondary metabolite biosynthesis by an miRNA-eTM regulatory module in plants.

Development of a 63K SNP Array for Cotton and High-Density Mapping of Intraspecific and Interspecific Populations of Gossypium spp.

High-throughput genotyping arrays provide a standardized resource for plant breeding communities that are useful for a breadth of applications including high-density genetic mapping, genome-wide association studies (GWAS), genomic selection (GS), complex trait dissection, and studying patterns of genomic diversity among cultivars and wild accessions. We have developed the CottonSNP63K, an Illumina Infinium array containing assays for 45,104 putative intraspecific single nucleotide polymorphism (SNP) markers for use within the cultivated cotton species Gossypium hirsutum L. and 17,954 putative interspecific SNP markers for use with crosses of other cotton species with G. hirsutum. The SNPs on the array were developed from 13 different discovery sets that represent a diverse range of G. hirsutum germplasm and five other species: G. barbadense L., G. tomentosum Nuttal × Seemann, G. mustelinum Miers × Watt, G. armourianum Kearny, and G. longicalyx J.B. Hutchinson and Lee. The array was validated with 1,156 samples to generate cluster positions to facilitate automated analysis of 38,822 polymorphic markers. Two high-density genetic maps containing a total of 22,829 SNPs were generated for two F2 mapping populations, one intraspecific and one interspecific, and 3,533 SNP markers were co-occurring in both maps. The produced intraspecific genetic map is the first saturated map that associates into 26 linkage groups corresponding to the number of cotton chromosomes for a cross between two G. hirsutum lines. The linkage maps were shown to have high levels of collinearity to the JGI G. raimondii Ulbrich reference genome sequence. The CottonSNP63K array, cluster file and associated marker sequences constitute a major new resource for the global cotton research community.

Transcriptome and complexity-reduced, DNA-based identification of intraspecies single-nucleotide polymorphisms in the polyploid Gossypium hirsutum L.

Varietal single nucleotide polymorphisms (SNPs) are the differences within one of the two subgenomes between different tetraploid cotton varieties and have not been practically used in cotton genetics and breeding because they are difficult to identify due to low genetic diversity and very high sequence identity between homeologous genes in cotton. We have used transcriptome and restriction site-associated DNA sequencing to identify varietal SNPs among 18 G. hirsutum varieties based on the rationale that varietal SNPs can be more confidently called when flanked by subgenome-specific SNPs. Using transcriptome data, we successfully identified 37,413 varietal SNPs and, of these, 22,121 did not have an additional varietal SNP within their 20-bp flanking regions so can be used in most SNP genotyping assays. From restriction site-associated DNA sequencing data, we identified an additional 3090 varietal SNPs between two of the varieties. Of the 1583 successful SNP assays achieved using different genotyping platforms, 1363 were verified. Many of the SNPs behaved as dominant markers because of coamplification from homeologous loci, but the number of SNPs acting as codominant markers increased when one or more subgenome-specific SNP(s) were incorporated in their assay primers, giving them greater utility for breeding applications. A G. hirsutum genetic map with 1244 SNP markers was constructed covering 5557.42 centiMorgan and used to map qualitative and quantitative traits. This collection of G. hirsutum varietal SNPs complements existing intra-specific SNPs and provides the cotton community with a valuable marker resource applicable to genetic analyses and breeding programs.

Identification of wounding and topping responsive small RNAs in tobacco (Nicotiana tabacum).

BACKGROUND: MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are two major classes of small RNAs. They play important regulatory roles in plants and animals by regulating transcription, stability and/or translation of target genes in a sequence-complementary dependent manner. Over 4,000 miRNAs and several classes of siRNAs have been identified in plants, but in tobacco only computational prediction has been performed and no tobacco-specific miRNA has been experimentally identified. Wounding is believed to induce defensive response in tobacco, but the mechanism responsible for this response is yet to be uncovered. RESULTS: To get insight into the role of small RNAs in damage-induced responses, we sequenced and analysed small RNA populations in roots and leaves from wounding or topping treated tobacco plants. In addition to confirmation of expression of 27 known miRNA families, we identified 59 novel tobacco-specific miRNA members of 38 families and a large number of loci generating phased 21- or 24-nt small RNAs (including ta-siRNAs). A number of miRNAs and phased small RNAs were found to be responsive to wounding or topping treatment. Targets of small RNAs were further surveyed by degradome sequencing. CONCLUSIONS: The expression changes of miRNAs and phased small RNAs responsive to wounding or topping and identification of defense related targets for these small RNAs suggest that the inducible defense response in tobacco might be controlled by pathways involving small RNAs.

The ANTHER INDEHISCENCE1 gene encoding a single MYB domain protein is involved in anther development in rice.

Using a two-element iAc/Ds transposon-tagging system, we identified a rice (Oryza sativa L. cv Nipponbare) recessive mutant, anther indehiscence1 (aid1), showing partial to complete spikelet sterility. Spikelets of the aid1 mutant could be classified into three types based on the viability of pollen grains and the extent of anther dehiscence. Type 1 spikelets (approximately 25%) were sterile due to a failure in accumulation of starch in pollen grains. Type 2 spikelets (approximately 55%) had viable pollen grains, but anthers failed to dehisce and/or synchronize with anthesis due to failure in septum degradation and stomium breakage, resulting in sterility. Type 3 spikelets (approximately 20%) had normal fertility. In addition, aid1 mutant plants had fewer tillers and flowered 10 to 15 d later than the wild type. The Ds insertion responsible for the aid1 mutation was mapped within the coding region of the AID1 gene on chromosome 6, which is predicted to encode a novel protein of 426 amino acids with a single MYB domain. The MYB domain of AID1 is closely related to that of the telomere-binding proteins of human, mouse, and Arabidopsis, and of single MYB domain transcriptional regulators in plants such as PcMYB1 and ZmIBP1. AID1 was expressed in both the leaves and panicles of wild-type plants, but not in mutant plants.