The SlARF4-SlHB8 regulatory module mediates leaf rolling in tomato.

Leaf is the main photosynthetic organ in plants and the primary energy source all along the plant life. Given the beneficial role of leaf rolling in improving photosynthetic efficiency and yield in specific environmental conditions, a better understanding of the factors and molecular mechanisms underlying this process is highly suited. Previously, the SlARF4 knocking out mutant exhibited upward curly leaf showed higher resistance to water deficit which driving us to uncover the function of SlARF4 in regulating the curly leaf formation. In this study, we unraveled the unexplored role of the SlARF4-SlHB8 module of transcription factors in the development of leaf rolling. Both SlARF4 loss-of-function and SlHB8 overexpressing tomato plants exhibited upward-rolled leaves, reflecting the active role of the two genes in controlling leaf rolling. Dual-luciferase reporter assays and phenotypic analysis of hybrid progenies suggested that SlHB8 acts downstream of SlARF4 in curly leaf formation. SlARF4 and SlHB8 influence the development of leaf palisade tissues via modulating the expression of genes associated with curly leaf formation. SEM analysis revealed no significant differences in leaf epidermal cells between the two leaf-rolling mutants and the wild type, indicating that curly leaves of arf4 and SlHB8-OE do not result from the asymmetric leaf epidermal cell growth. Our data provide novel insight into the molecular mechanism of abaxial-adaxial determination involving SlARF4 and SlHB8 and reveals that leaf rolling operates via different regulation mechanisms in tomato and Arabidopsis model plant.

The SlHB8 acts as a negative regulator in tapetum development and pollen wall formation in Tomato.

Pollen development is crucial for the fruit setting process of tomatoes, but the underlying regulatory mechanism remains to be elucidated. Here, we report the isolation of one HD-Zip III family transcription factor, SlHB8, whose expression levels decreased as pollen development progressed. SlHB8 knockout using CRISPR/Cas9 increased pollen activity, subsequently inducing fruit setting, whereas overexpression displayed opposite phenotypes. Overexpression lines under control of the 35 s and p2A11 promoters revealed that SlHB8 reduced pollen activity by affecting early pollen development. Transmission electron microscopy and TUNEL analyses showed that SlHB8 accelerated tapetum degradation, leading to collapsed and infertile pollen without an intine and an abnormal exine. RNA-seq analysis of tomato anthers at the tetrad stage showed that SlHB8 positively regulates SPL/NZZ expression and the tapetum programmed cell death conserved genetic pathway DYT1-TDF1-AMS-MYB80 as well as other genes related to tapetum and pollen wall development. In addition, DNA affinity purification sequencing, electrophoretic mobility shift assay, yeast one-hybrid assay and dual-luciferase assay revealed SlHB8 directly activated the expression of genes related to pollen wall development. The study findings demonstrate that SlHB8 is involved in tapetum development and degradation and plays an important role in anther development.

VEGF to CITED2 ratio predicts the collateral circulation of acute ischemic stroke.

Objective: The research objective was to evaluate the predicting role of the vascular endothelial growth factor to CBP/P300-interacting transactivator with Glu/Asp-rich C-terminal domain 2 Ratio (VEGF/CITED2) from peripheral blood mononuclear cells (PBMCs) in the collateral circulation of acute ischemic stroke (AIS). Methods: In an observational study of patients with AIS, the western blot was applied to test the protein expression of VEGF and CITED2. Then, we calculated the VEGF/CITED2 and collected other clinical data. Binary logistic regression analysis between collateral circulation and clinical data was performed. Finally, receiver operating characteristic (ROC) curve analysis was used to explore the predictive value of VEGF/CITED2. Results: A total of 67 patients with AIS were included in the study. Binary logistic regression analysis indicated the VEGF/CITED2 (OR 165.79, 95%CI 7.25-3,791.54, P = 0.001) was an independent protective factor. The ROC analyses showed an area under the ROC curve of the VEGF/CITED2 was 0.861 (95%CI 0.761-0.961). The optimal cutoff value of 1.013 for VEGF/CITED2 had a sensitivity of 89.1% and a specificity of 85.7%. Conclusion: In patients with AIS, the VEGF/CITED2 was related to the establishment of collateral circulation. The VEGF/CITED2 is a potentially valuable biomarker for predicting collateral circulation. Clinical trial registration: ClinicalTrials.gov, identifier: NCT05345366.

Tomato SlBES1.8 Influences Leaf Morphogenesis by Mediating Gibberellin Metabolism and Signaling.

Leaf morphogenetic activity determines its shape diversity. However, our knowledge of the regulatory mechanism in maintaining leaf morphogenetic capacity is still limited. In tomato, gibberellin (GA) negatively regulates leaf complexity by shortening the morphogenetic window. We here report a tomato BRI1-EMS-suppressor 1 transcription factor, SlBES1.8, that promoted the simplification of leaf pattern in a similar manner as GA functions. OE-SlBES1.8 plants exhibited reduced sensibility to exogenous GA3 treatment whereas showed increased sensibility to the application of GA biosynthesis inhibitor, paclobutrazol. In line with the phenotypic observation, the endogenous bioactive GA contents were increased in OE-SlBES1.8 lines, which certainly promoted the degradation of the GA signaling negative regulator, SlDELLA. Moreover, transcriptomic analysis uncovered a set of overlapping genomic targets of SlBES1.8 and GA, and most of them were regulated in the same way. Expression studies showed the repression of SlBES1.8 to the transcriptions of two GA-deactivated genes, SlGA2ox2 and SlGA2ox6, and one GA receptor, SlGID1b-1. Further experiments confirmed the direct regulation of SlBES1.8 to their promoters. On the other hand, SlDELLA physically interacted with SlBES1.8 and further inhibited its transcriptional regulation activity by abolishing SlBES1.8-DNA binding. Conclusively, by mediating GA deactivation and signaling, SlBES1.8 greatly influenced tomato leaf morphogenesis.

The SlHB8 Acts as a Negative Regulator in Stem Development and Lignin Biosynthesis.

The stem is an important organ in supporting plant body, transporting nutrients and communicating signals for plant growing. However, studies on the regulation of stem development in tomato are rather limited. In our study, we demonstrated that SlHB8 negatively regulated tomato stem development. SlHB8 belongs to homeo domain-leucine zipper Class III gene family transcription factors and expressed in all the organs examined including root, stem, leaves, flower, and fruit. Among these tissues, SlHB8 showed stable high expression level during tomato stem development. Overexpression of SlHB8 gene decreased stem diameter with inhibited xylem width and xylem cell layers, while loss of function of SlHB8gene increased the stem diameter and xylem width. The contents of lignin were decreased both in leaves and stems of SlHB8 overexpression plants. RNA-seq analysis on the stems of wild type and SlHB8 transgenic plants showed that the 116 DEGs (differential expressed genes) with reversible expression profiles in SlHB8-ox and SlHB8-cr plants were significantly enriched in the phenylpropanoid biosynthesis pathway and plant-pathogen pathway which were related to lignin biosynthesis and disease resistance. Meanwhile, the key genes involved in the lignin biosynthesis pathway such as SlCCR (cinnamoyl-CoA reductase), SlCYP73A14/C4H (cinnamate 4-hydroxylase), SlC3H (coumarate 3-hydroxylase) and SlCAD (cinnamoyl alcohol dehydrogenase) were down-regulated in both stem and leaves of SlHB8 overexpression plants, indicating a negative regulatory role of SlHB8 in the lignin biosynthesis and stem development.

Rapid and user-friendly open-source CRISPR/Cas9 system for single- or multi-site editing of tomato genome.

CRISPR/Cas9-induced genome editing is a powerful tool for studying gene function in a variety of organisms, including plants. Using multi-sgRNAs to target one or more genes is helpful to improve the efficacy of gene editing and facilitate multi-gene editing. Here, we describe a CRISPR/Cas9 system which can be conveniently developed as a CRISPR kit. SgRNA expression cassettes can be rapidly generated by one-step PCR using our CRISPR kit. In our kit, there are two binary vectors pHNCas9 and pHNCas9HT. The binary vector pHNCas9 was constructed to allow to assemble up to eight sgRNA expression cassettes by one-step Golden Gate cloning. Another binary vector pHNCas9HT can be used to generate a large number of single target constructs by directly transforming ligation reactions products into A. tumefaciens without several procedures, such as PCR and plasmid extraction. The two binary vectors are designed according to the principles of standard BioBrick assembly to be used as an open-source tool. For example, we used BioBrick as a visual T-DNA tag. We also developed a primer design aid to complement the system. With this primer design aid, researchers can rapidly obtain primers and GC content, and sgRNA sequence of target site. Our CRISPR/Cas9 system can perform single- and multi-site editing and multiple gene editing to produce various types of mutations in tomato. This rapid and user-friendly CRISPR/Cas9 system for tomato can be potentially used for mutagenesis of important crop species for genetic improvement and is suitable for research into the function of genes.

Genome-wide identification, phylogenetic analysis, and expression profiling of polyamine synthesis gene family members in tomato.

Polyamines (PAs), including putrescine (Put), spermidine (Spd), spermine (Spm), and thermospermine (T-Spm), play key roles in plant development, including fruit setting and ripening, morphogenesis, and abiotic/biotic stress. Their functions appear to be intimately related to their synthesis, which occurs via arginine/ornithine decarboxylase (ADC/ODC), Spd synthase (SPDS), Spm synthase (SPMS), and Acaulis5 (ACL5), respectively. Unfortunately, the expression and function of these PA synthesis-relate genes during specific developmental process or under stress have not been fully elucidated. Here, we present the results of a genome-wide analysis of the PA synthesis genes (ADC, ODC, SPDS, SPMS, ACL5) in the tomato (Solanum lycopersicum). In total, 14 PA synthesis-related genes were identified. Further analysis of their structures, conserved domains, phylogenetic trees, predicted subcellular localization, and promoter cis-regulatory elements were analyzed. Furthermore, we also performed experiments to evaluate their tissue expression patterns and under hormone and various stress treatments. To our knowledge, this is the first study to elucidate the mechanisms underlying PA function in this variety of tomato. Taken together, these data provide valuable information for future functional characterization of specific genes in the PA synthesis pathway in this and other plant species. Although additional research is required, the insight gained by this and similar studies can be used to improve our understanding of PA metabolism ultimately leading to more effective and consistent plant cultivation.

Overexpression of SlGRAS40 in Tomato Enhances Tolerance to Abiotic Stresses and Influences Auxin and Gibberellin Signaling.

Abiotic stresses are major environmental factors that inhibit plant growth and development impacting crop productivity. GRAS transcription factors play critical and diverse roles in plant development and abiotic stress. In this study, SlGRAS40, a member of the tomato (Solanum lycopersicum) GRAS family, was functionally characterized. In wild-type (WT) tomato, SlGRAS40 was upregulated by abiotic stress induced by treatment with D-mannitol, NaCl, or H2O2. Transgenic tomato plants overexpressing SlGRAS40 (SlGRAS40-OE) were more tolerant of drought and salt stress than WT. SlGRAS40-OE plants displayed pleiotropic phenotypes reminiscent of those resulting from altered auxin and/or gibberellin signaling. A comparison of WT and SlGRAS40-OE transcriptomes showed that the expression of a large number of genes involved in hormone signaling and stress responses were modified. Our study of SlGRAS40 protein provides evidence of how another GRAS plays roles in resisting abiotic stress and regulating auxin and gibberellin signaling during vegetative and reproductive growth in tomato.

Cloning and Characterization of Argonaute Genes in Tomato.

Argonaute (AGO) proteins are core elements in plant posttranscriptional RNA silencing pathways. The identification and functional characterization of tomato (Solanum lycopersicum) AGOs will help to better understand RNA silencing-based posttranscriptional regulation in fleshy fruits. Here we describe how to identify and clone SlAGO genes, as well as the methodology for their functional characterization.

Silencing of SlPL, which encodes a pectate lyase in tomato, confers enhanced fruit firmness, prolonged shelf-life and reduced susceptibility to grey mould.

Pectate lyase genes have been documented as excellent candidates for improvement of fruit firmness. However, implementation of pectate lyase in regulating fruit postharvest deterioration has not been fully explored. In this report, 22 individual pectate lyase genes in tomato were identified, and one pectate lyase gene SlPL (Solyc03g111690) showed dominant expression during fruit maturation. RNA interference of SlPL resulted in enhanced fruit firmness and changes in pericarp cells. More importantly, the SlPL-RNAi fruit demonstrated greater antirotting and pathogen-resisting ability. Compared to wild-type, SlPL-RNAi fruit had higher levels of cellulose and hemicellulose, whereas the level of water-soluble pectin was lower. Consistent with this, the activities of peroxidase, superoxide dismutase and catalase were higher in SlPL-RNAi fruit, and the malondialdehyde concentration was lower. RNA-Seq results showed large amounts of differentially expressed genes involved in hormone signalling, cell wall modification, oxidative stress and pathogen resistance. Collectively, these data demonstrate that pectate lyase plays an important role in both fruit softening and pathogen resistance. This may advance knowledge of postharvest fruit preservation in tomato and other fleshy fruit.

Overexpression of a tomato miR171 target gene SlGRAS24 impacts multiple agronomical traits via regulating gibberellin and auxin homeostasis.

In Arabidopsis, the miR171-GRAS module has been clarified as key player in meristem maintenance. However, the knowledge about its role in fruit crops like tomato (Solanum lycopersicum) remains scarce. We previously identified tomato SlGRAS24 as a target gene of Sly-miR171. To study the role of this probable transcription factor, we generated transgenic tomato plants underexpressing SlGRAS24, overexpressing SlGRAS24, overexpressing Sly-miR171 and expressing ß-glucuronidase (GUS) under the SlGRAS24 promoter (proSlGRAS24-GUS). Plants overexpressing SlGRAS24 (SlGRAS24-OE) had pleiotropic phenotypes associated with multiple agronomical traits including plant height, flowering time, leaf architecture, lateral branch number, root length, fruit set and development. Many GA/auxin-related genes were down-regulated and altered responsiveness to exogenous IAA/NAA or GA3 application was observed in SlGRAS24-OE seedlings. Moreover, compromised fruit set and development in SlGRAS24-OE was also observed. These newly identified phenotypes for SlGRAS24 homologs in tomato were later proved to be caused by impaired pollen sacs and fewer viable pollen grains. At anthesis, the comparative transcriptome results showed altered expression of genes involved in pollen development and hormone signalling. Taken together, our data demonstrate that SlGRAS24 participates in a series of developmental processes through modulating gibberellin and auxin signalling, which sheds new light on the involvement of hormone crosstalk in tomato development.

Ectopic expression of SlAGO7 alters leaf pattern and inflorescence architecture and increases fruit yield in tomato.

ARGONAUTE7 (AGO7), a key regulator of the trans-acting small interfering RNAs (ta-siRNA) pathway, plays a conserved role in controlling leaf pattern among species. However, little is known about the ta-siRNA pathway in regulating inflorescence architecture and fruit yield. In this study, we characterized the expression pattern, subcellular localization and developmental functions of SlAGO7 in tomato (Solanum lycopersicum). Overexpressing SlAGO7 in tomato exhibited pleiotropic phenotypes, including improved axillary bud formation, altered leaf morphology and inflorescence architecture, and increased fruit yield. Cross-sectioning of leaves showed that the number of vascular bundles was significantly increased in 35:SlAGO7 lines. Overexpression of SlAGO7 increased the production of ta-siRNA, and repressed the expression ta-siRNA-targeted genes (SlARF2a, SlARF2b, SlARF3 and SlARF4). Further analysis showed that overexpression of SlAGO7 alters the expression of key genes implicated in leaf morphology, inflorescence architecture, auxin transport and signaling. In addition, the altered auxin response of 35:SlAGO7 lines were also investigated. These results suggested that SlAGO7 plays a positive role in determining inflorescence architecture and fruit yield though the ta-siRNA pathway. Therefore, SlAGO7 represents a useful gene that can be incorporated in tomato breeding programs for developing cultivars with yield potential.

Genome-wide identification, phylogeny and expression analysis of GRAS gene family in tomato.

BACKGROUND: GRAS transcription factors usually act as integrators of multiple growth regulatory and environmental signals, including axillary shoot meristem formation, root radial pattering, phytohormones, light signaling, and abiotic/biotic stress. However, little is known about this gene family in tomato (Solanum lycopersicum), the most important model plant for crop species with fleshy fruits. RESULTS: In this study, 53 GRAS genes were identified and renamed based on tomato whole-genome sequence and their respective chromosome distribution except 19 members were kept as their already existed name. Multiple sequence alignment showed typical GRAS domain in these proteins. Phylogenetic analysis of GRAS proteins from tomato, Arabidopsis, Populus, P.mume, and Rice revealed that SlGRAS proteins could be divided into at least 13 subfamilies. SlGRAS24 and SlGRAS40 were identified as target genes of miR171 using5'-RACE (Rapid amplification of cDNA ends). qRT-PCR analysis revealed tissue-/organ- and development stage-specific expression patterns of SlGRAS genes. Moreover, their expression patterns in response to different hormone and abiotic stress treatments were also investigated. CONCLUSIONS: This study provides the first comprehensive analysis of GRAS gene family in the tomato genome. The data will undoubtedly be useful for better understanding the potential functions of GRAS genes, and their possible roles in mediating hormone cross-talk and abiotic stress in tomato as well as in some other relative species.

Overexpression of SlREV alters the development of the flower pedicel abscission zone and fruit formation in tomato.

Versatile roles of REVOLUTA (REV), a Class III homeodomain-leucine zipper (HD-ZIP III) transcription factor, have been depicted mainly in Arabidopsis and Populus. In this study, we investigated the functions of its tomato homolog, namely SlREV. Overexpression of a microRNA166-resistant version of SlREV (35S::REV(Ris)) not only resulted in vegetative abnormalities such as curly leaves and fasciated stems, but also caused dramatic reproductive alterations including continuous production of flowers at the pedicel abscission zone (AZ) and ectopic fruit formation on receptacles. Microscopic analysis showed that meristem-like structures continuously emerged from the exodermises of the pedicel AZs and that ectopic carpels formed between the first and second whorl of floral buds in 35S::REV(Ris) plants. Transcriptional data suggest that SlREV may regulate genes related to meristem maintenance and cell differentiation in the development of the flower pedicel abscission zone, and modulate genes in homeodomain and MADS-box families and hormone pathways during fruit formation. Altogether, these results reveal novel roles of SlREV in tomato flower development and fruit formation.

miR168 influences phase transition, leaf epinasty, and fruit development via SlAGO1s in tomato.

In Arabidopsis thaliana, Argonaute1 (AGO1) interacts with miR168 to modulate the small RNA regulatory pathway. However, the underlying mechanism of regulation and relationship between AGO1 and miR168 is poorly understood in the cash crop Solanum lycopersicum (tomato). We previously found that SlAGO1A and SlAGO1B were cleaved by miR168 in tomato. In this study, we show that SlAGO1A and SlAGO1B accumulate in miR168-sponge transgenic plants, and that expression of miR168-resistant SlAGO1A (4m-SlAGO1A) and SlAGO1B (4m-SlAGO1B) in tomato results in a series of defects affecting growth rate, floral timing, leaves, and fruit. Accumulation of miR156 was found when 4m-SlAGO1A was at an early developmental stage compared to the wild type and original SlAGO1A transgenic plants, and miR172 was highly expressed in adult 4m-SlAGO1A compared to the controls. In addition, the expression of multiple small RNAs was altered in 4m-SlAGO1A. Taken together, our data provide novel insights into the interaction between SlAGO1s and miR168 in determining growth rate, phase change, leaf epinasty, fruit initiation and expansion, and other developmental processes in tomato.

Molecular cloning and characterisation of SlAGO family in tomato.

BACKGROUND: AGO (Argonaute) protein participates in plant developmental processes and virus defense as a core element of transcriptional regulator or/and post-transcriptional regulator in RNA induced silencing complex (RISC), which is guided by small RNAs to repress target genes expression. Previously, it was revealed that 15 putative AGO genes in tomato genome. RESULTS: In present study, out of 15 detected SlAGO genes, only SlAGO4C and SlAGO15 couldn't be detected in roots, stems, leaves, buds, flowers and fruit of tomato by 30 cycles of PCR. SlAGO7 could be detected in early stage of fruit (-2 dpa, 0 dpa and 4 dpa), but it was significantly down-regulated in fruit collected on the 6 days post anthesis. Moreover, SlAGO5 could only be detected in reproductive tissues and SlAGO4D was specifically detected in fruit. According to blast result with miRNA database, three SlAGO genes harbored complementary sequences to miR168 (SlAGO1A and SlAGO1B) or miR403 (SlAGO2A). 5' RACE (Rapid amplification of cDNA ends) mapping was used to detect the 3' cleavage products of SlAGO mRNAs. In addition, subcellular localization of SlAGO proteins was detected. Our results showed that most SlAGO proteins localized to nucleus and cytoplasm. Importantly, nuclear membrane localization of AGO proteins was observed. Furthermore, mutated miR168 complementary site of SlAGO1A resulted in expanded localization of SlAGO1A, indicating that miR168 regulated localization of SlAGO1A. CONCLUSIONS: Our results contribute to demonstration of potential roles of these newly isolated AGO family in tomato developmental processes and proved the conserved relationships between AGO genes and miRNAs in tomato, which might play important roles in tomato development and virus defense.