Chromosome-scale assembly and gene editing of Solanum americanum genome reveals the basis for thermotolerance and fruit anthocyanin composition.

Solanum americanum serves as a promising source of resistance genes against potato late blight and is considered as a leafy vegetable for complementary food and nutrition. The limited availability of high-quality genome assemblies and gene annotations has hindered the exploration and exploitation of stress-resistance genes in S. americanum. Here, we present a chromosome-level genome assembly of a thermotolerant S. americanum ecotype and identify a crucial heat-inducible transcription factor gene, SaHSF17, essential for heat tolerance. The CRISPR/Cas9 system-mediated knockout of SaHSF17 results in remarkably reduced thermotolerance in S. americanum, exhibiting a significant suppression of multiple HSP gene expressions under heat treatment. Furthermore, our transcriptome analysis and anthocyanin component investigation of fruits indicated that delphinidins are the major anthocyanins accumulated in the mature dark-purple fruits. The accumulation of delphinidins and other pigment components during fruit ripening in S. americanum coincides with the transcriptional regulation of key genes, particularly the F3'5'H and F3'H genes, in the anthocyanin biosynthesis pathway. By integrating existing knowledge, the development of this high-quality reference genome for S. americanum will facilitate the identification and utilization of novel abiotic and biotic stress-resistance genes for improvement of Solanaceae and other crops.

Genome-wide identification and characterization of tomato 14-3-3 (SlTFT) genes and functional analysis of SlTFT6 under heat stress.

The plant 14-3-3 proteins are essential for many biological processes and responses to abiotic stress. We performed genome-wide identification and analysis of the 14-3-3 family genes in tomato. To explore the properties of the thirteen Sl14-3-3 found in the tomato genome, their chromosomal location, phylogenetic, and syntenic relationships were analyzed. The Sl14-3-3 promoters were found to have a number of growth-, hormone-, and stress-responsive cis-regulatory elements. Moreover, the qRT-PCR assay revealed that Sl14-3-3 genes are responsive to heat and osmotic stress. Subcellular localization experiments evidenced that the SlTFT3/6/10 proteins occur in the nucleus and cytoplasm Additional analysis on Sl14-3-3 putative interactor proteins revealed a number of prospective clients that potentially participate in stress reactions and developmental processes. Furthermore, overexpression of an Sl14-3-3 family gene, SlTFT6, improved tomato plants thermotolerance. Taken together, the study provides basic information on tomato 14-3-3 family genes in plant growth and abiotic stress response (high temperature stress), which can be helpful to further study the underlying molecular mechanisms.

Genome-Wide Analysis of MBF1 Family Genes in Five Solanaceous Plants and Functional Analysis of SlER24 in Salt Stress.

Multiprotein bridging factor 1 (MBF1) is an ancient family of transcription coactivators that play a crucial role in the response of plants to abiotic stress. In this study, we analyzed the genomic data of five Solanaceae plants and identified a total of 21 MBF1 genes. The expansion of MBF1a and MBF1b subfamilies was attributed to whole-genome duplication (WGD), and the expansion of the MBF1c subfamily occurred through transposed duplication (TRD). Collinearity analysis within Solanaceae species revealed collinearity between members of the MBF1a and MBF1b subfamilies, whereas the MBF1c subfamily showed relative independence. The gene expression of SlER24 was induced by sodium chloride (NaCl), polyethylene glycol (PEG), ABA (abscisic acid), and ethrel treatments, with the highest expression observed under NaCl treatment. The overexpression of SlER24 significantly enhanced the salt tolerance of tomato, and the functional deficiency of SlER24 decreased the tolerance of tomato to salt stress. SlER24 enhanced antioxidant enzyme activity to reduce the accumulation of reactive oxygen species (ROS) and alleviated plasma membrane damage under salt stress. SlER24 upregulated the expression levels of salt stress-related genes to enhance salt tolerance in tomato. In conclusion, this study provides basic information for the study of the MBF1 family of Solanaceae under abiotic stress, as well as a reference for the study of other plants.

Modulation of plant development and chilling stress responses by alternative splicing events under control of the spliceosome protein SmEb in Arabidopsis.

Cold stress resulting from chilling and freezing temperatures substantially inhibits plant growth and reduces crop production worldwide. Tremendous research efforts have been focused on elucidating the molecular mechanisms of freezing tolerance in plants. However, little is known about the molecular nature of chilling stress responses in plants. Here we found that two allelic mutants in a spliceosome component gene SmEb (smeb-1 and smeb-2) are defective in development and responses to chilling stress. RNA-seq analysis revealed that SmEb controls the splicing of many pre-messenger RNAs (mRNAs) under chilling stress. Our results suggest that SmEb is important to maintain proper ratio of the two COP1 splicing variants (COP1a/COP1b) to fine tune the level of HY5. In addition, the transcription factor BES1 shows a dramatic defect in pre-mRNA splicing in the smeb mutants. Ectopic expression of the two BES1 splicing variants enhances the chilling sensitivity of the smeb-1 mutant. Furthermore, biochemical and genetic analysis showed that CBFs act as negative upstream regulators of SmEb by directly suppressing its transcription. Together, our results demonstrate that proper alternative splicing of pre-mRNAs controlled by the spliceosome component SmEb is critical for plant development and chilling stress responses.

A new NLR gene for resistance to Tomato spotted wilt virus in tomato (Solanum lycopersicum).

KEY MESSAGE: A typical NLR gene, Sl5R-1, which regulates Tomato spotted wilt virus resistance, was fine mapped to a region less than 145 kb in the tomato genome. Tomato spotted wilt is a viral disease caused by Tomato spotted wilt virus (TSWV), which is a devastating disease that affects tomato (Solanum lycopersicum) production worldwide, and the resistance provided by the Sw-5 gene has broken down in some cases. In order to identify additional genes that confer resistance to TSWV, the F2 population was mapped using susceptible (M82) and resistant (H149) tomato lines. After 3 years of mapping, the main quantitative trait locus on chromosome 05 was narrowed to a genomic region of 145 kb and was subsequently identified by the F2 population, with 1971 plants in 2020. This region encompassed 14 candidate genes, and in it was found a gene cluster consisting of three genes (Sl5R-1, Sl5R-2, and Sl5R-3) that code for NBS-LRR proteins. The qRT-PCR and virus-induced gene silencing approach results confirmed that Sl5R-1 is a functional resistance gene for TSWV. Analysis of the Sl5R-1 promoter region revealed that there is a SlTGA9 transcription factor binding site caused by a base deletion in resistant plants, and its expression level was significantly up-regulated in infected resistant plants. Analysis of salicylic acid (SA) and jasmonic acid (JA) levels and the expression of SA- and JA-regulated genes suggest that SlTGA9 interacts or positively regulates Sl5R-1 to affect the SA- and JA-signaling pathways to resist TSWV. These results demonstrate that the identified Sl5R-1 gene regulates TSWV resistance by its own promoter interacting with the transcription factor SlTGA9.

Two Triacylglycerol Lipases Are Negative Regulators of Chilling Stress Tolerance in Arabidopsis.

Cold stress is one of the abiotic stress conditions that severely limit plant growth and development and productivity. Triacylglycerol lipases are important metabolic enzymes for the catabolism of triacylglycerols and, therefore, play important roles in cellular activities including seed germination and early seedling establishment. However, whether they play a role in cold stress responses remains unknown. In this study, we characterized two Arabidopsis triacylglycerol lipases, MPL1 and LIP1 and defined their role in cold stress. The expression of MPL1 and LIP1 is reduced by cold stress, suggesting that they may be negative factors related to cold stress. Indeed, we found that loss-of-function of MPL1 and LIP1 resulted in increased cold tolerance and that the mpl1lip1 double mutant displayed an additive effect on cold tolerance. We performed RNA-seq analysis to reveal the global effect of the mpl1 and lip1 mutations on gene expression under cold stress. The mpl1 mutation had a small effect on gene expression under both under control and cold stress conditions whereas the lip1 mutation caused a much stronger effect on gene expression under control and cold stress conditions. The mpl1lip1 double mutant had a moderate effect on gene expression under control and cold stress conditions. Together, our results indicate that MPL1 and LIP1 triacylglycerol lipases are negative regulators of cold tolerance without any side effects on growth in Arabidopsis and that they might be ideal candidates for breeding cold-tolerant crops through genome editing technology.

Genome-Wide Comparative Analysis of the R2R3-MYB Gene Family in Five Solanaceae Species and Identification of Members Regulating Carotenoid Biosynthesis in Wolfberry.

The R2R3-MYB is a large gene family involved in various plant functions, including carotenoid biosynthesis. However, this gene family lacks a comprehensive analysis in wolfberry (Lycium barbarum L.) and other Solanaceae species. The recent sequencing of the wolfberry genome provides an opportunity for investigating the organization and evolutionary characteristics of R2R3-MYB genes in wolfberry and other Solanaceae species. A total of 610 R2R3-MYB genes were identified in five Solanaceae species, including 137 in wolfberry. The LbaR2R3-MYB genes were grouped into 31 subgroups based on phylogenetic analysis, conserved gene structures, and motif composition. Five groups only of Solanaceae R2R3-MYB genes were functionally divergent during evolution. Dispersed and whole duplication events are critical for expanding the R2R3-MYB gene family. There were 287 orthologous gene pairs between wolfberry and the other four selected Solanaceae species. RNA-seq analysis identified the expression level of LbaR2R3-MYB differential gene expression (DEGs) and carotenoid biosynthesis genes (CBGs) in fruit development stages. The highly expressed LbaR2R3-MYB genes are co-expressed with CBGs during fruit development. A quantitative Real-Time (qRT)-PCR verified seven selected candidate genes. Thus, Lba11g0183 and Lba02g01219 are candidate genes regulating carotenoid biosynthesis in wolfberry. This study elucidates the evolution and function of R2R3-MYB genes in wolfberry and the four Solanaceae species.

Genome-Wide Identification and Analysis of the BBX Gene Family and Its Role in Carotenoid Biosynthesis in Wolfberry (Lycium barbarum L.).

The B-box proteins (BBXs) are a family of zinc-finger transcription factors with one/two B-Box domain(s) and play important roles in plant growth and development as well as stress responses. Wolfberry (Lycium barbarum L.) is an important traditional medicinal and food supplement in China, and its genome has recently been released. However, comprehensive studies of BBX genes in Lycium species are lacking. In this study, 28 LbaBBX genes were identified and classified into five clades by a phylogeny analysis with BBX proteins from Arabidopsis thaliana and the LbaBBXs have similar protein motifs and gene structures. Promoter cis-regulatory element prediction revealed that LbaBBXs might be highly responsive to light, phytohormone, and stress conditions. A synteny analysis indicated that 23, 20, 8, and 5 LbaBBX genes were orthologous to Solanum lycopersicum, Solanum melongena, Capsicum annuum, and Arabidopsis thaliana, respectively. The gene pairs encoding LbaBBX proteins evolved under strong purifying selection. In addition, the carotenoid content and expression patterns of selected LbaBBX genes were analyzed. LbaBBX2 and LbaBBX4 might play key roles in the regulation of zeaxanthin and antheraxanthin biosynthesis. Overall, this study improves our understanding of LbaBBX gene family characteristics and identifies genes involved in the regulation of carotenoid biosynthesis in wolfberry.

Abscisic acid homeostasis is mediated by feedback regulation of MdMYB88 and MdMYB124.

The phytohormone abscisic acid (ABA) is involved in various plant processes. In response to drought stress, plants quickly accumulate ABA, but the regulatory mechanism of ABA accumulation is largely unknown, especially in woody plants. In this study, we report that MdMYB88 and MdMYB124 are myeloblastosis (MYB) transcription factors critical for ABA accumulation in apple trees (Malus x domestica) following drought, and this regulation is negatively controlled by ABA. MdMYB88 and MdMYB124 positively regulate leaf water transpiration, photosynthetic capacity, and stress endurance in apple trees under drought conditions. MdMYB88 and MdMYB124 regulate the expression of biosynthetic and catabolic genes of ABA, as well as drought- and ABA- responsive genes. MdMYB88 associates with promoter regions of the ABA biosynthetic gene 9-cis-epoxycarotenoid dioxygenase 3 (NCED3). Finally, expression of MdMYB88 and MdMYB124 is repressed by ABA. Our results identify a feedback regulation of MdMYB88 and MdMYB124 in modulating ABA homeostasis in apple trees.

Identification, Classification, and Expression Analysis of the Triacylglycerol Lipase (TGL) Gene Family Related to Abiotic Stresses in Tomato.

Triacylglycerol Lipases (TGLs) are the major enzymes involved in triacylglycerol catabolism. TGLs hydrolyze long-chain fatty acid triglycerides, which are involved in plant development and abiotic stress responses. Whereas most studies of TGLs have focused on seed oil metabolism and biofuel in plants, limited information is available regarding the genome-wide identification and characterization of the TGL gene family in tomato (Solanum lycopersicum L.). Based on the latest published tomato genome annotation ITAG4.0, 129 SlTGL genes were identified and classified into 5 categories according to their structural characteristics. Most SlTGL genes were distributed on 3 of 12 chromosomes. Segment duplication appeared to be the driving force underlying expansion of the TGL gene family in tomato. The promoter analysis revealed that the promoters of SlTGLs contained many stress responsiveness cis-elements, such as ARE, LTR, MBS, WRE3, and WUN-motifs. Expression of the majority of SlTGL genes was suppressed following exposure to chilling and heat, while it was induced under drought stress, such as SlTGLa9, SlTGLa6, SlTGLa25, SlTGLa26, and SlTGLa13. These results provide valuable insights into the roles of the SlTGL genes family and lay a foundation for further functional studies on the linkage between triacylglycerol catabolism and abiotic stress responses in tomato.

Genome editing for plant research and crop improvement.

The advent of clustered regularly interspaced short palindromic repeat (CRISPR) has had a profound impact on plant biology, and crop improvement. In this review, we summarize the state-of-the-art development of CRISPR technologies and their applications in plants, from the initial introduction of random small indel (insertion or deletion) mutations at target genomic loci to precision editing such as base editing, prime editing and gene targeting. We describe advances in the use of class 2, types II, V, and VI systems for gene disruption as well as for precise sequence alterations, gene transcription, and epigenome control.

Genetic analysis implicates a molecular chaperone complex in regulating epigenetic silencing of methylated genomic regions.

DNA cytosine methylation confers stable epigenetic silencing in plants and many animals. However, the mechanisms underlying DNA methylation-mediated genomic silencing are not fully understood. We conducted a forward genetic screen for cellular factors required for the silencing of a heavily methylated p35S:NPTII transgene in the Arabidopsis thaliana rdm1-1 mutant background, which led to the identification of a Hsp20 family protein, RDS1 (rdm1-1 suppressor 1). Loss-of-function mutations in RDS1 released the silencing of the p35S::NPTII transgene in rdm1-1 mutant plants, without changing the DNA methylation state of the transgene. Protein interaction analyses suggest that RDS1 exists in a protein complex consisting of the methyl-DNA binding domain proteins MBD5 and MBD6, two other Hsp20 family proteins, RDS2 and IDM3, a Hsp40/DNAJ family protein, and a Hsp70 family protein. Like rds1 mutations, mutations in RDS2, MBD5, or MBD6 release the silencing of the transgene in the rdm1 mutant background. Our results suggest that Hsp20, Hsp40, and Hsp70 proteins may form a complex that is recruited to some genomic regions with DNA methylation by methyl-DNA binding proteins to regulate the state of silencing of these regions.

De novo assembly and analysis of the transcriptome of Ocimum americanum var. pilosum under cold stress.

BACKGROUND: Ocimum americanum var. pilosum is a chilling-sensitive, widely distributed plant that is consumed as a vegetable in central and southern China. To increase our understanding of cold stress responses in this species, we performed de novo transcriptome assembly for O. americanum var. pilosum and compared the transcriptomes of plants grown under normal and low temperatures. RESULTS: A total of 115,022,842 high quality, clean reads were obtained from four libraries (two replicates of control samples and two replicates of chilling-treated samples) and were used to perform de novo transcriptome assembly. After isoforms were considered, 42,816 unigenes were generated, 30,748 of which were similar to known proteins as determined by a BLASTx search (E-value < =1.0E-05) against NCBI non-redundant, Swiss-Prot, Gene Ontology, KEGG, and Cluster of COG databases. Comparative analysis of transcriptomes revealed that 5179 unigenes were differentially expressed (with at least 2-fold changes, FDR < 0.01) in chilling-treated samples, and that 2344 and 2835 unigenes were up- and down-regulated by chilling stress, respectively. Expression of the 10 most up-regulated and the five most down-regulated unigenes was validated by qRT-PCR. To increase our understanding of these differentially expressed unigenes, we performed Gene ontology and KEGG pathway enrichment analyses. The CBF-mediated transcriptional cascade, a well-known cold tolerance pathway, was reconstructed using our de novo assembled transcriptome. CONCLUSION: Our study has generated a genome-wide transcript profile of O. americanum var. pilosum and a de novo assembled transcriptome, which can be used to characterize genes related to diverse biological processes. This is the first study to assess the cold-responsive transcriptome in an Ocimum species. Our results suggest that cold temperature significantly affects genes related to protein translation and cellular metabolism in this chilling sensitive species. Although most of the CBF pathway genes have orthologs in O. americanum var. pilosum, none of the identified cold responsive (COR) gene orthologs was induced by cold, which is consistent with the lack of cold tolerance in this plant.

An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation.

DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

Arabidopsis proline-rich protein important for development and abiotic stress tolerance is involved in microRNA biogenesis.

MicroRNAs (miRNAs) are important for plant development and stress responses. However, factors regulating miRNA metabolism are not completely understood. SICKLE (SIC), a proline-rich protein critical for development and abiotic stress tolerance of Arabidopsis, was identified in this study. Loss-of-function sic-1 mutant plants exhibited a serrated, sickle-like leaf margin, reduced height, delayed flowering, and abnormal inflorescence phyllotaxy, which are common characteristics of mutants involved in miRNA biogenesis. The sic-1 mutant plants accumulated lower levels of a subset of miRNAs and transacting siRNAs but higher levels of corresponding primary miRNAs than the WT. The SIC protein colocalizes with the miRNA biogenesis component HYL1 in distinct subnuclear bodies. sic-1 mutant plants also accumulated higher levels of introns from hundreds of loci. In addition, sic-1 mutant plants are hypersensitive to chilling and salt stresses. These results suggest that SIC is a unique factor required for the biogenesis of some miRNAs and degradation of some spliced introns and important for plant development and abiotic stress responses.

Facile colorimetric smartphone-based biosensor for rapid detection of organophosphorus pesticides residues in environment using the aptamer-enhanced oxidase activity of octahedral Ag2O particles.

The long-term and excessive use of organophosphorus pesticides (OPs) leads to hazardous residues in the environment, which threatens human health to a considerable extent. Colorimetric methods can quickly and readily identify pesticide residue, but they still have various challenges in accuracy and stability. Herein, a smartphone-assisted and non-enzymatic colorimetric biosensor was constructed for rapid monitoring of multiple OPs based on the enhanced effect of aptamer on the catalytic ability of octahedral Ag2O. It was demonstrated that the aptamer sequence could enhance the affinity of colloidal Ag2O to chromogenic substrates, and accelerate the generation of oxygen radicals such as superoxide radical (·O2-) and singlet oxygen (1O2) from dissolved oxygen, thereby significantly increasing the oxidase activity of octahedral Ag2O. The color change of the solution can be easily converted to the corresponding RGB values by a smartphone for quantitative and rapid detection of multiple OPs. Hence, the smartphone-based and visual biosensor of multiple OPs was acquired with limit of detection of 10 µg L-1, 28 µg L-1 and 40 µg L-1 for isocarbophos, profenofos and omethoate, respectively. The colorimetric biosensor also exhibited good recoveries in several environmental and biological samples, showing that it may have broad application prospects for detecting OPs residues.

4-methylumbelliferone (4-MU) enhances drought tolerance of apple by regulating rhizosphere microbial diversity and root architecture.

The dwarfing rootstocks-mediated high-density apple orchard is becoming the main practice management. Currently, dwarfing rootstocks are widely used worldwide, but their shallow root system and drought sensitivity necessitate high irrigation requirements. Here, the root transcriptome and metabolome of dwarfing (M9-T337, a drought-sensitive rootstock) and vigorous rootstocks (Malus sieversii, a drought-tolerant species, is commonly used as a rootstock) showed that a coumarin derivative, 4-Methylumbelliferon (4-MU), was found to accumulate significantly in the roots of vigorous rootstock under drought condition. When exogenous 4-MU was applied to the roots of dwarfing rootstock under drought treatment, the plants displayed increased root biomass, higher root-to-shoot ratio, greater photosynthesis, and elevated water use efficiency. In addition, diversity and structure analysis of the rhizosphere soil microbial community demonstrated that 4-MU treatment increased the relative abundance of putatively beneficial bacteria and fungi. Of these, Pseudomonas, Bacillus, Streptomyces, and Chryseolinea bacterial strains and Acremonium, Trichoderma, and Phoma fungal strains known for root growth, or systemic resistance against drought stress, were significantly accumulated in the roots of dwarfing rootstock after 4-MU treatment under drought stress condition. Taken together, we identified a promising compound-4-MU, as a useful tool, to strengthen the drought tolerance of apple dwarfing rootstock.

Long noncoding RNA ARTA controls ABA response through MYB7 nuclear trafficking in Arabidopsis.

In eukaryotes, transcription factors are a crucial element in the regulation of gene expression, and nuclear translocation is the key to the function of transcription factors. Here, we show that the long intergenic noncoding RNA ARTA interacts with an importin ß-like protein, SAD2, through a long noncoding RNA-binding region embedded in the carboxyl terminal, and then it blocks the import of the transcription factor MYB7 into the nucleus. Abscisic acid (ABA)-induced ARTA expression can positively regulate ABI5 expression by fine-tuning MYB7 nuclear trafficking. Therefore, the mutation of arta represses ABI5 expression, resulting in desensitization to ABA, thereby reducing Arabidopsis drought tolerance. Our results demonstrate that lncRNA can hijack a nuclear trafficking receptor to modulate the nuclear import of a transcription factor during plant responses to environmental stimuli.

A mutation in SlCHLH encoding a magnesium chelatase H subunit is involved in the formation of yellow stigma in tomato (Solanum lycopersicum L.).

Chlorophylls are ubiquitous pigments responsible for the green color in plants. Changes in the chlorophyll content have a significant impact on photosynthesis, plant growth and development. In this study, we used a yellow stigma mutant (ys) generated from a green stigma tomato WT by using ethylmethylsulfone (EMS)-induced mutagenesis. Compared with WT, the stigma of ys shows low chlorophyll content and impaired chloroplast ultrastructure. Through map-based cloning, the ys gene is localized to a 100 kb region on chromosome 4 between dCAPS596 and dCAPS606. Gene expression analysis and nonsynonymous SNP determination identified the Solyc04g015750, as the potential candidate gene, which encodes a magnesium chelatase H subunit (CHLH). In ys mutant, a single base C to T substitution in the SlCHLH gene results in the conversion of Serine into Leucine (Ser92Leu) at the N-terminal region. The functional complementation test shows that the SlCHLH from WT can rescue the green stigma phenotype of ys. In contrast, knockdown of SlCHLH in green stigma tomato AC, observed the yellow stigma phenotype at the stigma development stage. Overexpression of the mutant gene Slys in green stigma tomato AC results in the light green stigma. These results indicate that the mutation of the N-terminal S92 to Leu in SlCHLH is the main reason for the formation of the yellow stigma phenotype. Characterization of the ys mutant enriches the current knowledge of the tomato chlorophyll mutant library and provides a novel and effective tool for understanding the function of CHLH in tomato.