NAC transcription factor SlNOR-like1 plays a dual regulatory role in tomato fruit cuticle formation.

The plant cuticle is an important protective barrier on the plant surface, constructed mainly by polymerized cutin matrix and a complex wax mixture. Although the pathway of plant cuticle biosynthesis has been clarified, knowledge of the transcriptional regulation network underlying fruit cuticle formation remains limited. In the present work, we discovered that tomato fruits of the NAC transcription factor SlNOR-like1 knockout mutants (nor-like1) produced by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9] displayed reduced cutin deposition and cuticle thickness, with a microcracking phenotype, while wax accumulation was promoted. Further research revealed that SlNOR-like1 promotes cutin deposition by binding to the promoters of glycerol-3-phosphate acyltransferase6 (SlGPAT6; a key gene for cutin monomer formation) and CUTIN DEFICIENT2 (SlCD2; a positive regulator of cutin production) to activate their expression. Meanwhile, SlNOR-like1 inhibits wax accumulation, acting as a transcriptional repressor by targeting wax biosynthesis, and transport-related genes 3-ketoacyl-CoA synthase1 (SlKCS1), ECERIFERUM 1-2 (SlCER1-2), SlWAX2, and glycosylphosphatidylinositol-anchored lipid transfer protein 1-like (SlLTPG1-like). In conclusion, SlNOR-like1 executes a dual regulatory effect on tomato fruit cuticle development. Our results provide a new model for the transcriptional regulation of fruit cuticle formation.

Transcription factor SlBEL2 interferes with GOLDEN2-LIKE and influences green shoulder formation in tomato fruits.

Chloroplasts play a crucial role in plant growth and fruit quality. However, the molecular mechanisms of chloroplast development are still poorly understood in fruits. In this study, we investigated the role of the transcription factor SlBEL2 (BEL1-LIKE HOMEODOMAIN 2) in fruit of Solanum lycopersicum (tomato). Phenotypic analysis of SlBEL2 overexpression (OE-SlBEL2) and SlBEL2 knockout (KO-SlBEL2) plants revealed that SlBEL2 has the function of inhibiting green shoulder formation in tomato fruits by affecting the development of fruit chloroplasts. Transcriptome profiling revealed that the expression of chloroplast-related genes such as SlGLK2 and SlLHCB1 changed significantly in the fruit of OE-SlBEL2 and KO-SlBEL2 plants. Further analysis showed that SlBEL2 could not only bind to the promoter of SlGLK2 to inhibit its transcription, but also interacted with the SlGLK2 protein to inhibit the transcriptional activity of SlGLK2 and its downstream target genes. SlGLK2 knockout (KO-SlGLK2) plants exhibited a complete absence of the green shoulder, which was consistent with the fruit phenotype of OE-SlBEL2 plants. SlBEL2 showed an expression gradient in fruits, in contrast with that reported for SlGLK2. In conclusion, our study reveals that SlBEL2 affects the formation of green shoulder in tomato fruits by negatively regulating the gradient expression of SlGLK2, thus providing new insights into the molecular mechanism of fruit green shoulder formation.

A tomato NAC transcription factor, SlNAM1, positively regulates ethylene biosynthesis and the onset of tomato fruit ripening.

Fruit ripening in tomato (Solanum lycopersicum) is the result of selective expression of ripening-related genes, which are regulated by transcription factors (TFs). The NAC (NAM, ATAF1/2, and CUC2) TF family is one of the largest families of plant-specific TFs and members are involved in a variety of plant physiological activities, including fruit ripening. Fruit ripening-associated NAC TFs studied in tomato to date include NAC-NOR (non-ripening), SlNOR-like1 (non-ripening like1), SlNAC1, and SlNAC4. Considering the large number of NAC genes in the tomato genome, there is little information about the possible roles of other NAC members in fruit ripening, and research on their target genes is lacking. In this study, we characterize SlNAM1, a NAC TF, which positively regulates the initiation of tomato fruit ripening via its regulation of ethylene biosynthesis. The onset of fruit ripening in slnam1-deficient mutants created by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) technology was delayed, whereas fruit ripening in OE-SlNAM1 lines was accelerated compared with the wild type. The results of RNA-sequencing (RNA-seq) and promoter analysis suggested that SlNAM1 directly binds to the promoters of two key ethylene biosynthesis genes (1-aminocyclopropane-1-carboxylate synthase: SlACS2 and SlACS4) and activates their expression. This hypothesis was confirmed by electrophoretic mobility shift assays and dual-luciferase reporter assay. Our findings provide insights into the mechanisms of ethylene production and enrich understanding of the tomato fruit ripening regulatory network.

Sprout vacuum-infiltration: a simple and efficient agroinoculation method for virus-induced gene silencing in diverse solanaceous species.

UNLABELLED: Virus-induced gene silencing (VIGS) is a robust technique for identifying the functions of plant genes. Tobacco rattle virus (TRV)-mediated VIGS has been commonly used in many plants. In order to overcome the limitations of existing agroinoculation methods, we report an easy and effective method of agroinoculation for virus-induced gene silencing-sprout vacuum-infiltration (SVI). Using sprout vacuum-infiltration, we have successfully silenced the expression of phytoene desaturase and Mg-protoporphyrin chelatase genes in four important solanaceous crops, including tomato, eggplant, pepper, and Nicotiana benthamiana. The gene-silenced phenotypes are conspicuous in 1-week-old plants. The method is simple, low cost and rapid compared to other techniques such as leaf infiltration or agrodrench. It may be more practical for studying gene function in the early stages of plant growth. An important aspect of SVI is that it will be used for high-throughput VIGS screens in the future. SVI will be an effective tool to overcome the limitations of current inoculation methods and to facilitate large-scale VIGS analysis of cDNA libraries. KEY MESSAGE: SVI is a simple, low cost agroinoculation method for VIGS. It is practical for studying the function of genes expressed in early stages of plant growth and high-throughput VIGS screens.

Expression and DNA binding activity of the tomato transcription factor RIN (ripening inhibitor).

Recently, we have found that the accumulation of ripening inhibitor (RIN) protein increased gradually during tomato fruit ripening. Here, the recombinant protein was expressed in Escherichia coli and affinity-purified. The DNA binding activity of renatured RIN protein was tested by electrophoretic mobility shift assay. The results indicated that an optimal expression and purification system was suitable for obtaining active RIN with DNA binding activity.

Molecular cloning and characterization of ETHYLENE OVERPRODUCER 1-LIKE 1 gene, LeEOL1, from tomato (Lycopersicon esculentum Mill.) fruit.

Recently, ETHYLENE OVERPRODUCER 1 (ETO1) had been cloned and identified as a negative post-transcriptional regulator in the ethylene biosynthesis in Arabidopsis. However, little was known about the role of ETO1 in other species, especially in tomato, which was an ideal model for studying the biosynthesis of ethylene during tomato fruit ripening. In this study, a tomato ETHYLENE OVERPRODUCER 1-LIKE 1 (LeEOL1) was cloned. The LeEOL1 cDNA was 3,515 bp long and carried an ORF that putatively encoded a polypeptide of 886 amino acids with a predicted molecular mass of 95 kDa. It shared 74% identity in amino acid sequence with Arabidopsis EOL1 and had one BTB (Broad-complex, Tramtrack, Bric-à-brac) domain and two TPR (tetratricopeptide repeat) domains, which were also conserved domains in AtEOL1. RT-PCR analysis of the temporal expression of LeEOL1 showed that its transcript decreased companied with increase of ethylene production in tomato ripening. The level of LeEOL1 transcripts in wild type tomato fruit at mature green stage did not distinctively change when treated with exogenous ethylene.

Enhancement of virus-induced gene silencing in tomato by low temperature and low humidity.

Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studying gene function in plants. We showed that silencing of a phytoene desaturase (PDS) gene is maintained throughout TRV-PDS-inoculated tomato plants as well as in their flowers and fruit and is enhanced by low temperature (15 degrees C) and low humidity (30%). RT-PCR analysis of the PDS gene revealed a dramatic reduction in the level of PDS mRNA in leaves, flowers and fruits. Silencing of PDS results in the accumulation of phytoene, the desaturase substrate. In addition, the content of chlorophyll a, chlorophyll b and total chlorophyll in the leaves of PDS-silenced plants was reduced by more than 90%. We also silenced the LeEIN2 gene by infecting seedlings, and this suppressed fruit ripenning. We conclude that this VIGS approach should facilitate large-scale functional analysis of genes involved in the development and ripening of tomato.

Efficient Virus-Induced Gene Silencing in Solanum rostratum.

Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum.

Silencing of the SlNAP7 gene influences plastid development and lycopene accumulation in tomato.

Ripening is an important stage of fruit development. To screen the genes associated with pigment formation in tomato fruit, a suppression subtractive hybridization (SSH) cDNA library was constructed by using tomato fruit in the green ripe and break ripe stages, and 129 differential genes were obtained. Using redness as a screening marker, virus-induced gene silencing (VIGS) of the differential genes was performed with a sprout vacuum-infiltration system (SVI). The results showed that silencing the SlNAP7 gene affected the chloroplast development of tomato leaves, manifesting as a photo-bleaching phenotype, and silenced fruit significantly affected the accumulation of lycopene, manifested as a yellow phenotype. In our study, we found that silencing the SlNAP7 gene downregulates the expression of the POR and PORA genes and destroys the normal development of the chloroplast. The expression of related genes included in the lycopene biosynthesis pathway was not significantly changed, but lycopene accumulation was significantly reduced in tomato fruit. Perhaps it was caused by the destruction of the chromoplast, which leads to the oxidation of lycopene. The results show that the SlNAP7 gene influences chloroplast development and lycopene accumulation in tomato.

Transcriptome Analysis Provides a Preliminary Regulation Route of the Ethylene Signal Transduction Component, SlEIN2, during Tomato Ripening.

Ethylene is crucial in climacteric fruit ripening. The ethylene signal pathway regulates several physiological alterations such as softening, carotenoid accumulation and sugar level reduction, and production of volatile compounds. All these physiological processes are controlled by numerous genes and their expression simultaneously changes at the onset of ripening. Ethylene insensitive 2 (EIN2) is a key component for ethylene signal transduction, and its mutation causes ethylene insensitivity. In tomato, silencing SlEIN2 resulted in a non-ripening phenotype and low ethylene production. RNA sequencing of SlEIN2-silenced and wild type tomato, and differential gene expression analyses, indicated that silencing SlEIN2 caused changes in more than 4,000 genes, including those related to photosynthesis, defense, and secondary metabolism. The relative expression level of 28 genes covering ripening-associated transcription factors, ethylene biosynthesis, ethylene signal pathway, chlorophyll binding proteins, lycopene and aroma biosynthesis, and defense pathway, showed that SlEIN2 influences ripening inhibitor (RIN) in a feedback loop, thus controlling the expression of several other genes. SlEIN2 regulates many aspects of fruit ripening, and is a key factor in the ethylene signal transduction pathway. Silencing SlEIN2 ultimately results in lycopene biosynthesis inhibition, which is the reason why tomato does not turn red, and this gene also affects the expression of several defense-associated genes. Although SlEIN2-silenced and green wild type fruits are similar in appearance, their metabolism is significantly different at the molecular level.

Cloning of delta6-desaturase from Mucor circinelloides and its high expression in Saccharomyces cerevisiae.

The aim of this study is to obtain Saccharomyces cerevisiae engineering strain with high gamma-linolenic acid (GLA, gamma-C18:3), which is a nutritionally important fatty acid that plays a vital role in biological structure and cell functions. As the first step,we cloned gamma6-desaturase gene from fungus mucor circinelloides by RT-PCR; delta6-desaturase is responsible for the transformation of linoleic acid into GLA. The PCR product was subcloned into yeast expression vector pYES2 to generate a recombinant plasmid pYES412. Transformation of S. cerevisiae strain INVSc1 was done by the lithium acetate method and the recombinant yeast cells were selected on a uracil-deficient medium. On appropriate medium and temperature,linoleic acid was provided as a substrate to yeast cultures,and the level of gamma-linolenic acid reached 50.07%. So far,the result we obtained is the best in terms of the level of expression of delta6-desaturase gene in Saccharomyces cerevisiae.

[Relationship between ethylene and polygalacturonase in tomato fruits].

Ethylene and PG (polygalacturonase) are both key plant growth regulators in fruit ripening process. The expression of PG was markedly inhibited in either antisense ACS tomato (Lycopersicon esculentum cv. Lichun) where endogenous ethylene synthesis was suppressed, or in Nr mutant in which ethylene perception was severely damaged. Also, the PG activities in fruits of these mutants were significantly lower than that of wild-type tomato (Fig. 1B). PG gene expression was promoted in mature green tomato fruit by exogenous ethylene 100 microL/L treatment for 4 h, and was inhibited significantly in breaking tomato fruit after being treated with 1-MCP (1-methylcycloprane) 1 microL/L, a specific ethylene reception inhibitor. Ethylene production of antisense PG tomato fruit during 45-50 DAP was lower than that of wild-type tomato (Fig. 4), and the level of transcriptional expression of both the ethylene receptor gene LeETR4 and the ethylene response factor gene LeERF2 were lower in this transgenic tomato fruit (Fig. 5). Ethylene production and the expression of LeETR4 and LeERF2 were both promoted by treatments with D-GA 100 mg/L, a product of enzymatic degradation of PG, in immature tomato fruit (Fig. 6 and Fig. 7). The relationship of PG and ethylene in tomato fruit in this study provided forceful evidences to support the mechanism by which PG and ethylene synergistically regulated climacteric fruit ripening and softening.

Expression of a truncated ripening inhibitor (RIN) protein from tomato and production of an anti-RIN antibody.

The tomato ripening mutant, ripening inhibitor (rin), whose fruits fails to ripen, has been identified and widely studied. The RIN gene has been cloned. Here we present the expression of a truncated form of the RIN protein from tomato and the preparation of a polyclonal antibody against it. The resulting antibody recognized the RIN of crude protein extracts from different tomato tissues. The protein level of RIN in tomato was detected with this antibody by western blot, which suggested the accumulation of RIN protein increased gradually during tomato fruit ripening.

Virus-induced gene silencing in tomato fruit.

Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants. Here we report that either by syringe-infiltrating the tobacco rattle virus (TRV)-vector into the surface, stem or carpopodium of a tomato fruit attached to the plant or by vacuum-infiltrating into a tomato fruit detached from the plant, TRV can efficiently spread and replicate in the tomato fruit. Although VIGS can be performed in tomato fruit by all of the means mentioned above, the most effective method is to inject the TRV-vector into the carpopodium of young fruit attached to the plant about 10 days after pollination. Several reporter genes related to ethylene responses and fruit ripening, including LeCTR1 and LeEILs genes, were also successfully silenced by this method during fruit development. In addition, we found that the silencing of the LeEIN2 gene results in the suppression of tomato fruit ripening. The results of our study indicate that the application of VIGS techniques by the described methods can be successfully applied to tomato fruit and is a valuable tool for studying functions of the relevant genes during fruit developing.