The SlGRAS9-SlZHD17 transcriptional cascade regulates chlorophyll and carbohydrate metabolism contributing to fruit quality traits in tomato.

Some essential components of fleshy fruits are dependent on photosynthetic activity and carbohydrate metabolism. Nevertheless, the regulatory mechanisms linking chlorophyll and carbohydrate metabolism remain partially understood. Here, we uncovered the role of SlGRAS9 and SlZHD17 transcription factors in controlling chlorophyll and carbohydrate accumulation in tomato fruit. Knockout or knockdown of SlGRAS9 or SlZHD17 resulted in marked increase in chlorophyll content, reprogrammed chloroplast biogenesis and enhanced accumulation of starch and soluble sugars. Combined genome-wide transcriptomic profiling and promoter-binding experiments unveiled a complex mechanism in which the SlGRAS9/SlZHD17 regulatory module modulates the expression of chloroplast and sugar metabolism either via a sequential transcriptional cascade or through binding of both TFs to the same gene promoters, or, alternatively, via parallel pathways where each of the TFs act on different target genes. For instance, the regulation of SlAGPaseS1 and SlSUS1 is mediated by SlZHD17 whereas that of SlVI and SlGLK1 occurs only through SlGRAS9 without the intervention of SlZHD17. Both SlGRAS9 and SlZHD17 can also directly bind the promoter of SlPOR-B to regulate its expression. Taken together, our findings uncover two important regulators acting synergistically to manipulate chlorophyll and carbohydrate accumulation and provide new potential breeding targets for improving fruit quality in fleshy fruits.

Identification of SRS transcription factor family in Solanum lycopersicum, and functional characterization of their responses to hormones and abiotic stresses.

The SHI RELATED SEQUENCE (SRS) family plays a vital role in the development of multiple plant organs such as floral meristem determinacy, organ morphogenesis, and signal transduction. Nevertheless, there is little understanding of the biological significance of tomato SRS family at this point. Our research identified eight SlSRS family members and classified them into three subfamilies based on phylogenetics, conserved motifs, and characteristic domain analysis. The intraspecies and interspecies collinearity analysis revealed clues of SRS family evolution. Many cis-elements related to hormones, stresses, and plant development can be found in the promoter region of SlSRS genes. All of eight SlSRS proteins were located in the nucleus and possessed transcriptional activity, half of which were transcriptional activators, and the other half were transcriptional repressors. Except for SlSRS1, which showed high transcript accumulation in vegetative organs, most SlSRS genes expressed ubiquitously in all flower organs. In addition, all SlSRS genes could significantly respond to at least four different plant hormones. Further, expression of SlSRS genes were regulated by various abiotic stress conditions. In summary, we systematically analyzed and characterized the SlSRS family, reviewed the expression patterns and preliminarily investigated the protein function, and provided essential information for further functional research of the tomato SRS genes in the determination of reproductive floral organs and the development of plants, and possibly other plants.

RIN enhances plant disease resistance via root exudate-mediated assembly of disease-suppressive rhizosphere microbiota.

The RIPENING-INHIBITOR (RIN) transcriptional factor is a key regulator governing fruit ripening. While RIN also affects other physiological processes, its potential roles in triggering interactions with the rhizosphere microbiome and plant health are unknown. Here we show that RIN affects microbiome-mediated disease resistance via root exudation, leading to recruitment of microbiota that suppress the soil-borne, phytopathogenic Ralstonia solanacearum bacterium. Compared with the wild-type (WT) plant, RIN mutants had different root exudate profiles, which were associated with distinct changes in microbiome composition and diversity. Specifically, the relative abundances of antibiosis-associated genes and pathogen-suppressing Actinobacteria (Streptomyces) were clearly lower in the rhizosphere of rin mutants. The composition, diversity, and suppressiveness of rin plant microbiomes could be restored by the application of 3-hydroxyflavone and riboflavin, which were exuded in much lower concentrations by the rin mutant. Interestingly, RIN-mediated effects on root exudates, Actinobacteria, and disease suppression were evident from the seedling stage, indicating that RIN plays a dual role in the early assembly of disease-suppressive microbiota and late fruit development. Collectively, our work suggests that, while plant disease resistance is a complex trait driven by interactions between the plant, rhizosphere microbiome, and the pathogen, it can be indirectly manipulated using "prebiotic" compounds that promote the recruitment of disease-suppressive microbiota.

Investigations of the distant metastatic non-small cell lung cancer without local lymph node involvement: Real world data from a large database.

INTRODUCTION: This study aimed to investigate the presentations and survival outcomes of the distant metastatic non-small cell lung cancer (NSCLC) without lymph node involvement to obtain a clearer picture of this special subgroup of metastatic NSCLC. METHOD: A least absolute shrinkage and selection operator (LASSO) penalized Cox regression analysis was used to select the prognostic variables. A nomogram and corresponding risk-classifying systems were constructed. The C-index and calibration curves were used to evaluate the performance of the model. Overall survival (OS) curves were plotted using the Kaplan-Meier method, and the log-rank test was used to compare OS differences between groups. Propensity score matching (PSM) was performed to reduce bias. RESULT: A total of 12 610 NSCLC patients with M1 category (N0 group: 3045 cases; N1-3 group: 9565 cases) were included. Regarding the N0 group, multivariate analysis demonstrated that age, sex, race, surgery, grade, tumor size, and M category were independent prognostic factors. A nomogram and corresponding risk-classifying systems were formulated. Favorable validation results were obtained from the C-index, calibration curves, and survival comparisons. Survival curves demonstrated that N0 NSCLC patients had better survival than N1-3 NSCLC patients both before and after PSM. Furthermore, the survival of resected N0M1 patients was superior to that of those without surgery. CONCLUSION: In this study, a prognostic nomogram and risk-classifying systems designed for the T1-4N0M1 NSCLC patients showed acceptable performance. Primary lung tumor resection might be a feasible treatment for this population subset. Additionally, we proposed that lymph node stage might have a place in the forthcoming tumor-node-metastasis (TNM) staging proposal for NSCLC patients with M1 category.

SlMYB99-mediated auxin and abscisic acid antagonistically regulate ascorbic acids biosynthesis in tomato.

Ascorbic acid (AsA) is a water-soluble antioxidant that plays important roles in plant development and human health. Understanding the regulatory mechanism underlying AsA biosynthesis is imperative to the development of high AsA plants. In this study, we reveal that the auxin response factor SlARF4 transcriptionally inhibits SlMYB99, which subsequently modulates AsA accumulation via transcriptional activation of AsA biosynthesis genes GPP, GLDH, and DHAR. The auxin-dependent transcriptional cascade of SlARF4-SlMYB99-GPP/GLDH/DHAR modulates AsA synthesis, while mitogen-activated protein kinase SlMAPK8 not only phosphorylates SlMYB99, but also activates its transcriptional activity. Both SlMYB99 and SlMYB11 proteins physically interact with each other, thereby synergistically regulating AsA biosynthesis by upregulating the expression of GPP, GLDH, and DHAR genes. Collectively, these results demonstrate that auxin and abscisic acid antagonistically regulate AsA biosynthesis during development and drought tolerance in tomato via the SlMAPK8-SlARF4-SlMYB99/11 module. These findings provide new insights into the mechanism underlying phytohormone regulation of AsA biosynthesis and provide a theoretical basis for the future development of high AsA plants via molecular breeding.

Brt9SIDA/IDALs as peptide signals mediate diverse biological pathways in plants.

As signal molecules, plant peptides play key roles in intercellular communication during growth and development, as well as stress responses. The 14-amino-acid (aa) INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) peptide was originally identified to play an essential role in the floral organ abscission of Arabidopsis. It is synthesized from its precursor, a small protein containing 77-aa residues with an N-terminal signal peptide sequence. Recently, the IDA/IDA-like (IDLs) genes are isolated in several angiosperms and are highly conserved in land plants. In addition, IDA/IDLs are not only involved in organ abscission but also function in multiple biological processes, including biotic and abiotic stress responses. Here, we summarize the post-translational modification and proteolytic processing, the evolutionary conservation, and the potential regulatory function of IDA/IDLs, and also present future perspectives to investigate the IDA/IDLs signaling pathway. We anticipate that this detailed knowledge will help to improve the understanding of the molecular mechanism of plant peptide signaling.

The auxin-responsive transcription factor SlDOF9 regulates inflorescence and flower development in tomato.

Understanding the mechanisms underlying differentiation of inflorescence and flower meristems is essential towards enlarging our knowledge of reproductive organ formation and to open new prospects for improving yield traits. Here, we show that SlDOF9 is a new modulator of floral differentiation in tomato. CRISPR/Cas9 knockout strategy uncovered the role of SlDOF9 in controlling inflorescence meristem and floral meristem differentiation via the regulation of cell division genes and inflorescence architecture regulator LIN. Tomato dof9-KO lines have more flowers in both determinate and indeterminate cultivars and produce more fruit upon vibration-assisted fertilization. SlDOF9 regulates inflorescence development through an auxin-dependent ARF5-DOF9 module that seems to operate, at least in part, differently in Arabidopsis and tomato. Our findings add a new actor to the complex mechanisms underlying reproductive organ differentiation in flowering plants and provide leads towards addressing the diversity of factors controlling the transition to reproductive organs.

Control of fruit softening and Ascorbic acid accumulation by manipulation of SlIMP3 in tomato.

Postharvest deterioration is among the major challenges for the fruit industry. Regulation of the fruit softening rate is an effective strategy for extending shelf-life and reducing the economic losses due postharvest deterioration. The tomato myoinositol monophosphatase 3 gene SlIMP3, which showed highest expression level in fruit, was expressed and purified. SlIMP3 demonstrated high affinity with the L-Gal 1-P and D-Ins 3-P, and acted as a bifunctional enzyme in the biosynthesis of AsA and myoinositol. Overexpression of SlIMP3 not only improved AsA and myoinositol content, but also increased cell wall thickness, improved fruit firmness, delayed fruit softening, decreased water loss, and extended shelf-life. Overexpression of SlIMP3 also increased uronic acid, rhamnose, xylose, mannose, and galactose content in cell wall of fruit. Treating fruit with myoinositol obtained similar fruit phenotypes of SlIMP3-overexpressed fruit, with increased cell wall thickness and delayed fruit softening. Meanwhile, overexpression of SlIMP3 conferred tomato fruit tolerance to Botrytis cinerea. The function of SlIMP3 in cell wall biogenesis and fruit softening were also verified using another tomato species, Ailsa Craig (AC). Overexpression of SlDHAR in fruit increased AsA content, but did not affect the cell wall thickness or fruit firmness and softening. The results support a critical role for SlIMP3 in AsA biosynthesis and cell wall biogenesis, and provide a new method of delaying tomato fruit softening, and insight into the link between AsA and cell wall metabolism.

Interaction of two MADS-box genes leads to growth phenotype divergence of all-flesh type of tomatoes.

All-flesh tomato cultivars are devoid of locular gel and exhibit enhanced firmness and improved postharvest storage. Here, we show that SlMBP3 is a master regulator of locular tissue in tomato fruit and that a deletion at the gene locus underpins the All-flesh trait. Intriguingly, All-flesh varieties lack the deleterious phenotypes reported previously for SlMBP3 under-expressing lines and which preclude any potential commercial use. We resolve the causal factor for this phenotypic divergence through the discovery of a natural mutation at the SlAGL11 locus, a close homolog of SlMBP3. Misexpressing SlMBP3 impairs locular gel formation through massive transcriptomic reprogramming at initial phases of fruit development. SlMBP3 influences locule gel formation by controlling cell cycle and cell expansion genes, indicating that important components of fruit softening are determined at early pre-ripening stages. Our findings define potential breeding targets for improved texture in tomato and possibly other fleshy fruits.

SlTPL1 Silencing Induces Facultative Parthenocarpy in Tomato.

Facultative parthenocarpy is of great practical value. However, the molecular mechanism underlying facultative parthenocarpy remains elusive. Transcriptional co-repressors (TPL) act as a central regulatory hub controlling all nine phytohormone pathways. Previously, we proved that SlTPLs participate in the auxin signaling pathway by interacting with auxin/indole acetic acid (Aux/IAAs) in tomato; however, their function in fruit development has not been studied. In addition to their high expression levels during flower development, the interaction between SlTPL1 and SlIAA9 stimulated the investigation of its functional significance via RNA interference (RNAi) technology, whereby the translation of a protein is prevented by selective degradation of its encoded mRNA. Down-regulation of SlTPL1 resulted in facultative parthenocarpy. Plants of SlTPL1-RNAi transgenic lines produced similar fruits which did not show any pleiotropic effects under normal conditions. However, they produced seedless fruits upon emasculation and under heat stress conditions. Furthermore, SlTPL1-RNAi flower buds contained higher levels of cytokinins and lower levels of abscisic acid. To reveal how SlTPL1 regulates facultative parthenocarpy, RNA-seq was performed to identify genes regulated by SlTPL1 in ovaries before and after fruit set. The results showed that down-regulation of SlTPL1 resulted in reduced expression levels of cytokinin metabolism-related genes, and all transcription factors such as MYB, CDF, and ERFs. Conversely, down-regulation of SlTPL1 induced the expression of genes related to cell wall and cytoskeleton organization. These data provide novel insights into the molecular mechanism of facultative tomato parthenocarpy and identify SlTPL1 as a key factor regulating these processes.

Histone posttranslational modifications rather than DNA methylation underlie gene reprogramming in pollination-dependent and pollination-independent fruit set in tomato.

Fruit formation comprises a series of developmental transitions among which the fruit set process is essential in determining crop yield. Yet, our understanding of the epigenetic landscape remodelling associated with the flower-to-fruit transition remains poor. We investigated the epigenetic and transcriptomic reprogramming underlying pollination-dependent and auxin-induced flower-to-fruit transitions in the tomato (Solanum lycopersicum) using combined genomewide transcriptomic profiling, global ChIP-sequencing and whole genomic DNA bisulfite sequencing (WGBS). Variation in the expression of the overwhelming majority of genes was associated with change in histone mark distribution, whereas changes in DNA methylation concerned a minor fraction of differentially expressed genes. Reprogramming of genes involved in processes instrumental to fruit set correlated with their H3K9ac or H3K4me3 marking status but not with changes in cytosine methylation, indicating that histone posttranslational modifications rather than DNA methylation are associated with the remodelling of the epigenetic landscape underpinning the flower-to-fruit transition. Given the prominent role previously assigned to DNA methylation in reprogramming key genes of the transition to ripening, the outcome of the present study supports the idea that the two main developmental transitions in fleshy fruit and the underlying transcriptomic reprogramming are associated with different modes of epigenetic regulations.

The MADS-Box Gene SlMBP21 Regulates Sepal Size Mediated by Ethylene and Auxin in Tomato.

Normal organ size is achieved by successful co-ordination of cell proliferation and cell expansion, which are modulated by multiple factors such as ethylene and auxin. In our work, SlMBP21-RNAi (RNA interference) tomato exhibited longer sepals and improved fruit set. Histological analysis indicated that longer sepals were attributed to cell expansion. To explore how SlMBP21 regulates sepal size, we compared the transcriptomes of sepals between SlMBP21-RNAi and the wild type by RNA sequencing and found that the differentially expressed genes were dominantly related to cell expansion, ethylene and auxin, and photosynthesis. Down-regulation of SlMBP21 affected ethylene production and the free IAA and IAA-Val intensity in sepals. Hormone treatment further indicated that SlMBP21 was involved in the ethylene and auxin pathways. As reported, ethylene and auxin were important factors for cell expansion. Hence, SlMBP21 negatively regulated cell expansion to control sepal size, and ethylene and auxin may mediate this process. Additionally, the contents of Chl and the activity of ribulose-1, 5-bisphosphate carboxylase/oxygenase, the key photosynthetic enzyme, were both increased in SlMBP21-RNAi sepals, which indicated that photosynthesis might be enhanced in transgenic longer sepals. Therefore, the longer sepal, with better protection and enhanced photosynthesis, may contribute to improve fruit set. Altogether, these results suggested that SlMBP21 was a novel factor involved in organ size control. Moreover, our study provided potential application value for improving fruit set.

Overexpression of the class D MADS-box gene Sl-AGL11 impacts fleshy tissue differentiation and structure in tomato fruits.

MADS-box transcription factors are key elements of the genetic networks controlling flower and fruit development. Among these, the class D clade gathers AGAMOUS-like genes which are involved in seed, ovule, and funiculus development. The tomato genome comprises two class D genes, Sl-AGL11 and Sl-MBP3, both displaying high expression levels in seeds and in central tissues of young fruits. The potential effects of Sl-AGL11 on fruit development were addressed through RNAi silencing and ectopic expression strategies. Sl-AGL11-down-regulated tomato lines failed to show obvious phenotypes except a slight reduction in seed size. In contrast, Sl-AGL11 overexpression triggered dramatic modifications of flower and fruit structure that include: the conversion of sepals into fleshy organs undergoing ethylene-dependent ripening, a placenta hypertrophy to the detriment of locular space, starch and sugar accumulation, and an extreme softening that occurs well before the onset of ripening. RNA-Seq transcriptomic profiling highlighted substantial metabolic reprogramming occurring in sepals and fruits, with major impacts on cell wall-related genes. While several Sl-AGL11-related phenotypes are reminiscent of class C MADS-box genes (TAG1 and TAGL1), the modifications observed on the placenta and cell wall and the Sl-AGL11 expression pattern suggest an action of this class D MADS-box factor on early fleshy fruit development.