Deciphering the regulatory network of the NAC transcription factor FvRIF, a key regulator of strawberry (Fragaria vesca) fruit ripening.

The NAC transcription factor ripening inducing factor (RIF) was previously reported to be necessary for the ripening of octoploid strawberry (Fragaria × ananassa) fruit, but the mechanistic basis of RIF-mediated transcriptional regulation and how RIF activity is modulated remains elusive. Here, we show that FvRIF in diploid strawberry, Fragaria vesca, is a key regulator in the control of fruit ripening and that knockout mutations of FvRIF result in a complete block of fruit ripening. DNA affinity purification sequencing coupled with transcriptome deep sequencing suggests that 2,080 genes are direct targets of FvRIF-mediated regulation, including those related to various aspects of fruit ripening. We provide evidence that FvRIF modulates anthocyanin biosynthesis and fruit softening by directly regulating the related core genes. Moreover, we demonstrate that FvRIF interacts with and serves as a substrate of MAP kinase 6 (FvMAPK6), which regulates the transcriptional activation function of FvRIF by phosphorylating FvRIF at Thr-310. Our findings uncover the FvRIF-mediated transcriptional regulatory network in controlling strawberry fruit ripening and highlight the physiological significance of phosphorylation modification on FvRIF activity in ripening.

Current insights into posttranscriptional regulation of fleshy fruit ripening.

Fruit ripening is a complicated process that is accompanied by the formation of fruit quality. It is not only regulated at the transcriptional level via transcription factors or DNA methylation but also fine-tuned after transcription occurs. Here, we review recent advances in our understanding of key regulatory mechanisms of fleshy fruit ripening after transcription. We mainly highlight the typical mechanisms by which fruit ripening is controlled, namely, alternative splicing, mRNA N6-methyladenosine RNA modification methylation, and noncoding RNAs at the posttranscriptional level; regulation of translation efficiency and upstream open reading frame-mediated translational repression at the translational level; and histone modifications, protein phosphorylation, and protein ubiquitination at the posttranslational level. Taken together, these posttranscriptional regulatory mechanisms, along with transcriptional regulation, constitute the molecular framework of fruit ripening. We also critically discuss the potential usage of some mechanisms to improve fruit traits.

Determination of the signaling proteins responsible for the antioxidant potential of the Yishenhuoxue formula for treating asthenospermia in rats.

Asthenospermia is a predominant cause of male infertility, and antioxidant supplements can be effective in treating asthenospermia. We demonstrate the antioxidant potential of traditional Chinese medicine, the Yishenhuoxue (YSHX) formula, in treating polyglycosides of Tripterygium wilfordii (GTW)-induced asthenospermia in rats. Fifty male rats were randomly divided into the normal, model, and treatment groups. HE staining was used to evaluate the improvement of spermatogenic function of rats, and TBA reaction, qRT-PCR, Western Blot and other methods were used to determine the changes of oxidative stress indicators and to evaluate the improvement of antioxidant capacity of rats by YSHX. Comparison with the model group showed significant improvement in pathological damage caused by GTW to seminiferous tubules. MDA and NO content in rat testes decreased, especially in middle- and high-dosage groups. No significant changes were observed in SOD and CAT activity or mRNA expression. GSH-Px activity and GSH mRNA expression were significantly higher in the low-dosage group than in the model group. Compared to the model group, GR activity was significantly lower in the middle and high dosage groups, while the mRNA expression was higher. The PKC-beta level increased, while p-ERK1/2, NF-κB, and the ratio of p-ERK1/2*(ERK1/2)-1 decreased significantly in the treatment groups. Therefore, YSHX can alleviate GTW-induced testicular damage, enhance GSH-Px activity, regulate GSH redox cycling, and mitigate oxidative stress injury. Furthermore, YSHX can promote PKC-beta expression and inhibit the phosphorylation of ERK1/2 and NF-κB. Using YSHX may be an effective way to increase sperm motility via the PKC-ERK1/2-NF-ĸB axis.

The pivotal ripening gene SlDML2 participates in regulating disease resistance in tomato.

Fruit ripening and disease resistance are two essential biological processes for quality formation and maintenance. DNA methylation, in the form of 5-methylcytosine (5mC), has been elucidated to modulate fruit ripening, but its role in regulating fruit disease resistance remains poorly understood. In this study, we show that mutation of SlDML2, the DNA demethylase gene essential for fruit ripening, affects multiple developmental processes of tomato besides fruit ripening, including seed germination, leaf length and width and flower branching. Intriguingly, loss of SlDML2 function decreased the resistance of tomato fruits against the necrotrophic fungal pathogen Botrytis cinerea. Comparative transcriptomic analysis revealed an obvious transcriptome reprogramming caused by SlDML2 mutation during B. cinerea invasion. Among the thousands of differentially expressed genes, SlßCA3 encoding a ß-carbonic anhydrase and SlFAD3 encoding a ω-3 fatty acid desaturase were demonstrated to be transcriptionally activated by SlDML2-mediated DNA demethylation and positively regulate tomato resistance to B. cinerea probably in the same genetic pathway with SlDML2. We further show that the pericarp tissue surrounding B. cinerea infection exhibited a delay in ripening with singnificant decrease in expression of ripening genes that are targeted by SlDML2 and increase in expression of SlßCA3 and SlFAD3. Taken together, our results uncover an essential layer of gene regulation mediated by DNA methylation upon B. cinerea infection and raise the possible that the DNA demethylase gene SlDML2, as a multifunctional gene, participates in modulating the trade-off between fruit ripening and disease resistance.

A receptor-like kinase SlFERL mediates immune responses of tomato to Botrytis cinerea by recognizing BcPG1 and fine-tuning MAPK signaling.

FERONIA (FER) is a receptor-like kinase showing versatile functions during plant growth, development, and responses to environmental stimuli. However, its functions during the interaction between fruit and necrotrophic fungal pathogens are still unclear. Combining reverse genetic approaches, physiological assays, co-immunoprecipitation, protein phosphorylation identification, and site-directed mutagenesis, we reported a tomato FER homolog SlFERL (Solanum lycopersicum FERONIA Like) involved in the immune responses to Botrytis cinerea invasion. The results indicated that SlFERL extracellular domain recognized and interacted with the secreted virulence protein BcPG1 from B. cinerea, further revealed that SlFERL triggered downstream signaling by phosphorylating SlMAP3K18 at Thr45, Ser49, Ser76, and Ser135. Moreover, we verified that SlMAP2K2 and SlMAP2K4 synergistically contributed to immune response of tomato to B. cinerea, in which SlFERL-SlMAP3K18 module substantially modulated protein level and/or kinase activity of SlMAP2K2/SlMAP2K4. These findings reveal a new pattern-triggered immune pathway, indicating that SlFERL participates in the immune responses to B. cinerea invasion via recognizing BcPG1 and fine-tuning MAPK signaling.

Global ubiquitinome analysis reveals the role of E3 ubiquitin ligase FaBRIZ in strawberry fruit ripening.

Ubiquitination is an important post-translational modification that mediates protein degradation in eukaryotic cells, participating in multiple biological processes. However, the profiling of protein ubiquitination and the function of this crucial modification in fruit ripening remain largely unknown. In this study, we found that suppression of proteasome by the inhibitor MG132 retarded strawberry fruit ripening. Using K-ɛ-GG antibody enrichment combined with high-resolution mass spectrometry, we performed a comprehensive ubiquitinome analysis in strawberry fruit. We identified 2947 ubiquitination sites for 2878 peptides within 1487 proteins, which are involved in a variety of cellular functions. The lysine at position 48 (K48)-linked poly-ubiquitin chains appeared to be the most prevalent type of modification among the identified ubiquitinated proteins. A large number of ubiquitination sites exhibited altered ubiquitination levels after proteasome inhibition, including those within ripening-related proteins associated with sugar and acid metabolism, cell wall metabolism, anthocyanin synthesis, and ABA biosynthesis and signalling. We further demonstrated that FaBRIZ, a RING-type E3 ligase, functions as a negative regulator of ripening in strawberry fruit. Our findings highlight the critical regulatory roles of protein ubiquitination in fruit ripening. The ubiquitinome data provide a basis for further exploration of the function of ubiquitination on specific proteins.

m6 A-mediated regulation of crop development and stress responses.

Dynamic chemical modifications in eukaryotic messenger RNAs (mRNAs) constitute an essential layer of gene regulation, among which N6 -methyladenosine (m6 A) was unveiled to be the most abundant. m6 A functionally modulates important biological processes in various mammals and plants through the regulation of mRNA metabolism, mainly mRNA degradation and translation efficiency. Physiological functions of m6 A methylation are diversified and affected by intricate sequence contexts and m6 A machineries. A number of studies have dissected the functional roles and the underlying mechanisms of m6 A modifications in regulating plant development and stress responses. Recently, it was demonstrated that the human FTO-mediated plant m6 A removal caused dramatic yield increases in rice and potato, indicating that modulation of m6 A methylation could be an efficient strategy for crop improvement. In this review, we summarize the current progress concerning the m6 A-mediated regulation of crop development and stress responses, and provide an outlook on the potential application of m6 A epitranscriptome in the future improvement of crops.

Four types of RNA modification writers predict the prognosis of prostate cancer.

RNA modification is an important part of epigenetic regulation. However, the relationship between RNA modification writers and prostate cancer (PCa) remains unclear. We obtained transcriptome data from The Cancer Genome Atlas; the expression of writers for four RNA modifications (N6-methyladenosine, N1-methyladenosine, alternative polyadenylation and adenosine-to-inosine RNA editing) was altered in PCa tissue when compared with normal tissue. RNA modification writers affect the expression of immune checkpoints. Gene ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the RNA modification was related to the cell cycle. Hub genes were screened using machine learning, and a risk score model was established using multivariate Cox analysis. Univariate and multivariate Cox analyses showed that a risk score model based on RNA modification writers could be an independent prognostic factor for PCa. In conclusion, our study showed that RNA modification writers are differentially expressed in PCa and might influence the development of PCa by regulating immune checkpoints and the cell cycle. The risk score model of RNA modification writers is predicted to be an independent prognostic factor for PCa. Thus, RNA modification writers might be used as potential indicators for the clinical diagnosis of PCa.

Jujing Zhuyu decoction inhibits apoptosis in rats with asthenozoospermia by regulating the mitochondrial apoptosis pathway.

Jujing Zhuyu decoction (JZD) is a traditional Chinese medicine that effectively improves sperm motility. However, the molecular mechanism of JZD on asthenozoospermia still remains unknown. In this study, we investigated the effect of JZD on the mitochondrial apoptosis pathway in an asthenozoospermia rat model. Sixty Sprague-Dawley rats were randomly divided into five groups-control, tripterygium glycosides (GTW) model, JZD-low (JZD-L), JZD-medium (JZD-M), and JZD-high (JZD-H) groups (n = 12/group). GTW was used to generate the asthenozoospermia model. The JZD-L, JZD-M, and JZD-H groups were administered 5, 10, or 15 g kg-1  day-1 of JZD granules respectively, for 4 weeks. Testicular tissue morphology was examined using histological staining, while sperm count was determined using manual and computer-aided semen analyses. Apoptosis of spermatogenic cells was detected with the TUNEL assay, and the expression of proteins and genes related to mitochondrial apoptosis was detected using western blotting and quantitative reverse transcription-polymerase chain reaction respectively. Histomorphological evaluation revealed superior seminiferous tubule structure and arrangement as well as improved spermatogenic cell morphology in the JZD-L, JZD-M, and JZD-H groups compared to those in the model group. Moreover, semen quality and the apoptotic index were significantly improved in the JZD-L, JZD-M, and JZD-H groups compared to those in the model group. Additionally, the mRNA expression and protein abundance of Apaf-1, Bax, Cyto-c, and caspase-3 was reduced, while those of Bcl-2 were increased in all JZD groups compared to those in the model group. JZD reduces the apoptosis rate of sperm cells and significantly promotes sperm survival by regulating the mitochondrial apoptosis pathway. This mechanism provides experimental support for the treatment of asthenozoospermia by JZD.

[Xianfang Huoming Decoction improves sperm quality in asthenospermia mice: An investigation based on the detoxification and sperm-nourishing method].

OBJECTIVE: To investigate the mechanism of Xianfang Huoming Decoction (XHD) improving sperm motility in mice with asthenospermia (AS). METHODS: Thirty normal BALB/c mice were randomly divided into six groups, blank control, AS model control, low-dose XHD, medium-dose XHD, high-dose XHD and levocarnitine + vitamin E (LC+VE). The AS model was established in the latter five groups by injection of methotrexate at 0.5 mg/kg once a week, and the mice in the blank control group were injected with the same volume of normal saline, all for 8 weeks. From the ninth week, the animals in the blank control and AS model control groups were treated with PBS at 0.1 ml/d, those in the low-, medium- and high-dose XHD groups with XHD at 7.13, 14,2 and 28.52 g/kg/ d respectively, and those in the LC+VE group with LC+VE (30:1) at 0.55 g/kg/d, all for 4 weeks. Then, the bilateral epididymides were harvested from all the mice for preparation of a sperm suspension and observation of the total numbers of sperm and motile sperm. The testis tissues were obtained for to determination of the expressions of Nrf-2- and HO-1-related mRNA and proteins by fluorescence staining, RT-PCR and Western blot. RESULTS: Compared with the AS model controls, the mice treated with low-, medium- and high-dose XHD showed dramatically increased sperm concentration (ï¼»22.36 ± 16.02ï¼½ vs ï¼»39.04 ± 4.50ï¼½, ï¼»40.76 ± 6.57ï¼½ and ï¼»41.04 ± 8.39ï¼½ ×106/ml, P < 0.01) and motility (ï¼»22.89 ± 14.96ï¼½% vs ï¼»47.98 ± 4.74ï¼½%, ï¼»48.53 ± 6.03ï¼½% and ï¼»49.31 ± 6.24ï¼½%, P< 0.01), decreased level of reactive oxygen species (ROS) (ï¼»16.82 ± 14.96ï¼½% vs ï¼»12.08 ± 3.26ï¼½%, ï¼»10.77 ± 2.21ï¼½% and ï¼»9.56 ± 2.08ï¼½%, P< 0.01), and up-regulated expressions of Nrf-2- and HO-1-related mRNA and proteins in the testis tissue (P < 0.05 or P < 0.01). CONCLUSION: Xianfang Huoming Decoction inhibits the development of oxidative stress by up-regulating the expressions of Nrf-2- and HO-1-related mRNA and proteins in the testis tissue, which has provided theoretical evidence for its clinical application in the treatment of asthenospermia.

SlFERL Interacts with S-Adenosylmethionine Synthetase to Regulate Fruit Ripening.

Fruit ripening is a complex and genetically programmed process modulated by transcription factors, hormones, and other regulators. However, the mechanism underlying the regulatory loop involving the membrane-protein targets of RIPENING-INHIBITOR (RIN) remains poorly understood. To unravel the function of tomato ( Solanum lycopersicum) FERONIA Like (SlFERL), a putative MADS-box transcription factor target gene, we investigated and addressed the significance of SlFERL in fruit ripening by combining reverse genetics, biochemical, and cytological analyses. Here, we report that RIN and Tomato AGAMOUS-LIKE1 (TAGL1) directly bind to the promoter region of SlFERL and further activate its expression transcriptionally, suggesting a potential role of SlFERL in fruit ripening. Overexpression of SlFERL significantly accelerated the ripening process of tomato fruit, whereas RNA interference knockdown of SlFERL resulted in delayed fruit ripening. Moreover, a surface plasmon resonance assay coupled with tandem mass spectrometry and a protein interaction assay revealed that SlFERL interacts with the key enzyme S-adenosyl-Met synthetase 1 (SlSAMS1) in the ethylene biosynthesis pathway, leading to increased S-adenosyl-Met accumulation and elevated ethylene production. Thus, SlFERL serves as a positive regulator of ethylene production and fruit ripening. This study provides clues to the molecular regulatory networks underlying fruit ripening.

SlREM1 Triggers Cell Death by Activating an Oxidative Burst and Other Regulators.

Programmed cell death (PCD), a highly regulated feature of the plant immune response, involves multiple molecular players. Remorins (REMs) are plant-specific proteins with varied biological functions, but their function in PCD and plant defense remains largely unknown. Here, we report a role for remorin in disease resistance, immune response, and PCD regulation. Overexpression of tomato (Solanum lycopersicum) REMORIN1 (SlREM1) increased susceptibility of tomato to the necrotrophic fungus Botrytis cinerea and heterologous expression of this gene triggered cell death in Nicotiana benthamiana leaves. Further investigation indicated that amino acids 173 to 187 and phosphorylation of SlREM1 played key roles in SlREM1-triggered cell death. Intriguingly, multiple tomato REMs induced cell death in N benthamiana leaves. Yeast two-hybrid, split luciferase complementation, and coimmunoprecipitation assays all demonstrated that remorin proteins could form homo- and heterocomplexes. Using isobaric tags for relative and absolute quantitative proteomics, we identified that some proteins related to cell death regulation, as well as N benthamiana RESPIRATORY BURST OXIDASE HOMOLOG B (which is essential for reactive oxygen species production), were notably upregulated in SlREM1-expressing leaves. Heterologous expression of SlREM1 increased reactive oxygen species accumulation and triggered other cell death regulators. Our findings indicate that SlREM1 is a positive regulator of plant cell death and provide clues for understanding the PCD molecular regulatory network in plants.

[Danhong Tongjing Prescription improves sperm quality in patients with bilateral varicocele after varicocelectomy].

OBJECTIVE: To investigate the effect of Danhong Tongjing Prescription (DTP) on sperm quality in patients with bilateral varicocele (VC) after microsurgical varicocelectomy. METHODS: We randomly assigned 68 patients with bilateral VC to receive microsurgical varicocelectomy (the control group, n = 34) or microsurgical varicocelectomy followed by oral administration of DTP for a course of 90 days (the DTP group, n = 34). Before and after treatment, we obtained the sperm concentration, total sperm count, total sperm motility, the percentage of progressively motile sperm (PMS), sperm acrosomal enzyme activity, inhibin B (Inh-B) level, and sperm DNA fragmentation index (DFI) from the patients and compared the parameters between the two groups. RESULTS: There were no statistically significant differences in sperm concentration, PMS, acrosomal enzyme activity or sperm DFI among the patients with different degrees of VC preoperatively. After 3 months of medication, sperm concentration, total sperm count, total sperm motility, PMS and acrosomal enzyme activity were all increased while DFI decreased in both the control and DTP groups, even more significantly in the DTP group than in the control, and the Inh-B level was also markedly elevated in the DTP group in comparison with the baseline. CONCLUSIONS: The severity of bilateral VC is not correlated with the reduction of semen quality. DTP can improve sperm quality by improving total sperm count, PMS and acrosomal enzyme activity and reducing DFI in VC patients after varicocelectomy. The underlying mechanisms of the prescription may be related to its anti-oxidative stress action and abilities of improving reproductive hypoxia, spermatogenic environment and the function of Sertoli cells, but the specific signaling pathway involved is not yet clear.

Regulatory network of fruit ripening: current understanding and future challenges.

Fruit ripening is a developmental process that is spatio-temporally tuned at multiple levels. Molecular dissections of the mechanisms underlying the ripening process have revealed a network encompassed by hormones, transcriptional regulators, epigenomic modifications and other regulatory elements that directly determine fruit quality and the postharvest commodity of fresh produce. Many studies have addressed the important roles of ethylene, abscisic acid (ABA) and other hormones in regulating fruit ripening. Recent studies have shown that some spontaneous mutants for tomato transcription factors (TFs) have resulted from loss-of-function or dominant-negative mutations. Unlike in DNA methylation variation, the histone mark H3K27me3 may be conserved and prevents the transcriptional feedback circuit from generating autocatalytic ethylene. These observations of a network of partially redundant component indicate the need to improve our current understanding. Here, we focussed on the recent advances and future challenges in investigations of the molecular mechanisms of fruit ripening. We also identified several issues that still need to be addressed in future studies.

Molecular basis and regulation of pathogenicity and patulin biosynthesis in Penicillium expansum.

Penicillium expansum is a necrotrophic plant pathogen with a wide range of fruit hosts. It causes blue mold rot during fruit storage, transport, and sale, resulting in huge economic losses to the fruit industry. Moreover, this pathogen is also the main producer of patulin, a toxic secondary metabolite that contaminates fruit and fruit-derived products and impairs human health. Therefore, understanding molecular basis of the pathogenicity and patulin biosynthesis of the fungal pathogen has important scientific significance and also plays an important guiding role in the research and development of new control technologies. Here, we comprehensively summarize the recent research progress, particularly regarding the molecular aspects of pathogenicity, patulin biosynthesis, and the related regulatory mechanisms, as well as control technologies for blue mold rot in the fruit industry.

NADPH Oxidase Is Crucial for the Cellular Redox Homeostasis in Fungal Pathogen Botrytis cinerea.

During interactions, both plants and pathogens produce reactive oxygen species (ROS). Plants generate ROS for defense induction, while pathogens synthesize ROS for growth, sporulation, and virulence. NADPH oxidase (NOX) complex in the plasma membrane represents a main protein complex for ROS production in pathogens. Although NOX plays a crucial role in pathogenicity of pathogens, the underlying molecular mechanisms of NOX, especially the proteins regulated by NOX, remain largely unknown. Here, we applied an iodoacetyl tandem mass tag-based redox proteomic assay to investigate the protein redox dynamics in deletion mutant of bcnoxR, which encodes a regulatory subunit of NOX in the fungal pathogen Botrytis cinerea. In total, 214 unique peptidyl cysteine (Cys) thiols from 168 proteins were identified and quantified in both the wild type and ∆bcnoxR mutant. The Cys thiols in the ∆bcnoxR mutant were generally more oxidized than those in the wild type, suggesting that BcNoxR is essential for maintaining the equilibrium of the redox state in B. cinerea. Site-specific thiol oxidation analysis indicated that 142 peptides containing the oxidized thiols changed abundance significantly in the ∆bcnoxR mutant. Proteins containing these differential peptides are classified into various functional categories. Functional analysis revealed that one of these proteins, 6-phosphate dehydrogenase, played roles in oxidative stress response and pathogenesis of B. cinerea. These results provide insight into the potential target proteins and the ROS signal transduction pathway regulated by NOX.

The RIN-MC Fusion of MADS-Box Transcription Factors Has Transcriptional Activity and Modulates Expression of Many Ripening Genes.

Fruit development and ripening is regulated by genetic and environmental factors and is of critical importance for seed dispersal, reproduction, and fruit quality. Tomato (Solanum lycopersicum) ripening inhibitor (rin) mutant fruit have a classic ripening-inhibited phenotype, which is attributed to a genomic DNA deletion resulting in the fusion of two truncated transcription factors, RIN and MC In wild-type fruit, RIN, a MADS-box transcription factor, is a key regulator of the ripening gene expression network, with hundreds of gene targets controlling changes in color, flavor, texture, and taste during tomato fruit ripening; MC, on the other hand, has low expression in fruit, and the potential functions of the RIN-MC fusion gene in ripening remain unclear. Here, overexpression of RIN-MC in transgenic wild-type cv Ailsa Craig tomato fruits impaired several ripening processes, and down-regulating RIN-MC expression in the rin mutant was found to stimulate the normal yellow mutant fruit to produce a weak red color, suggesting a distinct negative role for RIN-MC in tomato fruit ripening. By comparative transcriptome analysis of rin and rin 35S::RIN-MC RNA interference fruits, a total of 1,168 and 1,234 genes were identified as potential targets of RIN-MC activation and inhibition. Furthermore, the RIN-MC fusion gene was shown to be translated into a chimeric transcription factor that was localized to the nucleus and was capable of protein interactions with other MADS-box factors. These results indicated that tomato RIN-MC fusion plays a negative role in ripening and encodes a chimeric transcription factor that modulates the expression of many ripening genes, thereby contributing to the rin mutant phenotype.

Production, Signaling, and Scavenging Mechanisms of Reactive Oxygen Species in Fruit-Pathogen Interactions.

Reactive oxygen species (ROS) play a dual role in fruit-pathogen interaction, which largely depends on their different levels in cells. Fruit recognition of a pathogen immediately triggers an oxidative burst that is considered an integral part of the fruit defense response. ROS are also necessary for the virulence of pathogenic fungi. However, the accumulation of ROS in cells causes molecular damage and finally leads to cell death. In this review, on the basis of data regarding ROS production and the scavenging systems determining ROS homeostasis, we focus on the role of ROS in fruit defense reactions against pathogens and in fungi pathogenicity during fruit-pathogen interaction.

The mode of action of remorin1 in regulating fruit ripening at transcriptional and post-transcriptional levels.

Remorins are plant-specific and plasma membrane-associated proteins that display a variety of functions in plant growth, development, biotic and abiotic stresses, and signal transduction. However, little information is available for understanding their role in fruit ripening. Here, remorin 1 (SlREM1) is cloned from tomato and its localization is examined by co-localization analysis and immunoblotting. Functions of SlREM1 in fruit ripening are characterized based on gene expression, co-immunoprecipitation coupled with mass spectroscopy and split luciferase complementation imaging assays in SlREM1 overexpression and RNA interference (RNAi) lines. The results indicate that SlREM1 is localized at the plasma membrane. Overexpression of SlREM1 in tomato stimulates fruit ripening with an increase in ethylene production and lycopene accumulation as compared to the wild-type. Consistently, these genes involved in ethylene and lycopene biosynthesis and ripening regulators also are upregulated in SlREM1 overexpression lines. SlREM1 can interact with ethylene biosynthesis proteins SAM1, ACO1 and ACS2 and is degraded by ubiquitin-mediated proteolysis. Our findings reveal that SlREM1 serves as a positive regulator of fruit ripening and provide novel cues for understanding of the molecular regulation network of fruit ripening.

A Tomato Vacuolar Invertase Inhibitor Mediates Sucrose Metabolism and Influences Fruit Ripening.

Fruit ripening is a complex process that involves a series of physiological and biochemical changes that ultimately influence fruit quality traits, such as color and flavor. Sugar metabolism is an important factor in ripening, and there is evidence that it influences various aspects of ripening, although the associated mechanism is not well understood. In this study, we identified and analyzed the expression of 36 genes involved in Suc metabolism in ripening tomato (Solanum lycopersicum) fruit. Chromatin immunoprecipitation and gel mobility shift assays indicated that SlVIF, which encodes a vacuolar invertase inhibitor, and SlVI, encoding a vacuolar invertase, are directly regulated by the global fruit ripening regulator RIPENING INHIBITOR (RIN). Moreover, we showed that SlVIF physically interacts with SlVI to control Suc metabolism. Repression of SlVIF by RNA interference delayed tomato fruit ripening, while overexpression of SlVIF accelerated ripening, with concomitant changes in lycopene production and ethylene biosynthesis. An isobaric tags for relative and absolute quantification-based quantitative proteomic analysis further indicated that the abundance of a set of proteins involved in fruit ripening was altered by suppressing SlVIF expression, including proteins associated with lycopene generation and ethylene synthesis. These findings provide evidence for the role of Suc in promoting fruit ripening and establish that SlVIF contributes to fruit quality and the RIN-mediated ripening regulatory mechanisms, which are of significant agricultural value.