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.

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.

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.

Role of the Tomato Non-Ripening Mutation in Regulating Fruit Quality Elucidated Using iTRAQ Protein Profile Analysis.

Natural mutants of the Non-ripening (Nor) gene repress the normal ripening of tomato fruit. The molecular mechanism of fruit ripening regulation by the Nor gene is unclear. To elucidate how the Nor gene can affect ripening and fruit quality at the protein level, we used the fruits of Nor mutants and wild-type Ailsa Craig (AC) to perform iTRAQ (isobaric tags for relative and absolute quantitation) analysis. The Nor mutation altered tomato fruit ripening and affected quality in various respects, including ethylene biosynthesis by down-regulating the abundance of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), pigment biosynthesis by repressing phytoene synthase 1 (PSY1), ζ-carotene isomerase (Z-ISO), chalcone synthase 1 (CHS1) and other proteins, enhancing fruit firmness by increasing the abundance of cellulose synthase protein, while reducing those of polygalacturonase 2 (PG2) and pectate lyase (PL), altering biosynthesis of nutrients such as carbohydrates, amino acids, and anthocyanins. Conversely, Nor mutation also enhanced the fruit's resistance to some pathogens by up-regulating the expression of several genes associated with stress and defense. Therefore, the Nor gene is involved in the regulation of fruit ripening and quality. It is useful in the future as a means to improve fruit quality in 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.

[Research on the Tomato Metacaspase Protein Interactions with Ca2+ by Spectroscopy and Molecular Probe].

Metacaspases are cysteine-dependent proteases found in protozoa, fungi and plants and are distantly related to metazoan caspases. Most of MCPs activation are the calcium dependent, but the mechanisms are still unknown. Based on the techniques of CD spectroscopy, fluorescence spectroscopy, and Terbium Stains-all probe, we selected three purified recombinant proteins from key residues mutated in tomato metacaspase (LeMCA1), including conserved catalytic site (C139A) mutant, N-sequenced cleaved site (K223G) mutant and the predicted Ca2+ binding sites (D116A/D117A) mutant, to explore the interaction mechanism of LeMCA1 and Ca2+. CD spectroscopy and Stains-all probe results suggested that the intense binding does not exist between LeMCA1 and Ca2+ as well as Ca2+ has little effect on the secondary structure of LeMCA1. However, fluorescence spectroscopy and Tb3+ probe results showed that Ca(2+)-induced the changes occur in the tertiary structure of LeMCA1, which contributes to the activation of zymogen. In addition, predicted Ca2+ binding residues, Asp-116 and Asp117, are the key sites resporisible for the Ca2+ interaction with LeMCA1, and the loss of these two residues resulted in decreased interaction. Our data firstly provided insight on the mechanism of the interaction between Ca2+ and recombinant purified Solanaceae type II metacaspase by spectroscopy and molecular probe techniques. Combined the results we got before from sequence-alignment and sites-mutation, the key residues Asp-116 and Asp117 affect the Ca(2+)-induced the changes of LeMCA1 tertiary structure. Our data provided information for the further biochemical and crystal assays of LeMCA1.

Biochemical evidence of key residues for the activation and autoprocessing of tomato type II metacaspase.

To investigate the autolysis pattern and activation of metacaspase in higher plants, the biochemical characteristics of purified recombinant type II metacaspase (LeMCA1) from tomato were explored. Western blotting analysis indicated that four cleaved bands were formed; two N-terminal fragments and two C-terminal fragments. N-terminal sequencing confirmed that LeMCA1 cleaves at Lys223 and Arg332. Site mutants indicated that catalytic Cys139, cleaved Lys223, Arg332 and predicted calcium binding Asp116/Asp117 are the key residues that are responsible for its Ca²âº and pH dependent activation. The cleavage of the full-size fragment seemed crucial for the activation of LeMCA1 in vitro.

The effect of genetically modified Lactobacillus plantarum 590 on the gut health of Sprague-Dawley rats.

Lp was a generally recognized as safe microorganism. Lactobacillus plantarum 590 was obtained by inserting nisI gene into Lp genome to help it tolerate higher concentration nisin. As the unintended effects of the genetically modified microorganism (GMM) are the most important barriers to the progress of GMM, we have performed a useful exploration to establish a new in vivo evaluation model for GMM from the point of view of intestinal health. In this study, Sprague-Dawley rats were orally administered with Lp 590 and Lp for 4 weeks. Fecal samples were collected to determine the number of beneficial bacteria Bifidobacterium and harmful bacteria Clostridium perfringens. Denaturing gradient gel electrophoresis was used to detect the bacterial profiles of every group. Fecal enzyme activities and short-chain fatty acids as main metabolites were also examined. Real time PCR (RT-PCR) and immunohistochemistry were used to analyze two proteins (ZO-1 and occludin) and secretory immunoglobulin A to detect intestinal permeability and mucosal immunity, gut permeability and gut mucosal immunity were analyzed to see whether GM Lp 590 can induce changes of the gut health when compared with non-GM Lp group, andeventually we concluded that there is no significant difference between GM Lp 590-fed group and non-GM Lp-fed group. The conclusion of gut health test was comparable withthat from traditional subchronic test. Evaluation of intestinal health will be a new approach of assessing the safety of GMM.

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.

Mycotoxin Ochratoxin A-induced cell death and changes in oxidative metabolism of Arabidopsis thaliana.

We evaluated the phytotoxicity of mycotoxin ochratoxin A (OTA) from Aspergillus and Penicillium strains on Arabidopsis thaliana. The results demonstrate that the growth of Arabidopsis thaliana on media containing OTA was inhibited significantly. Moreover, OTA induced necrotic lesions in detached leaves, which are reminiscent of hypersensitive response lesions that are activated during plant-pathogen interactions and other abiotic stress factors. From our study, we can see that OTA exposure stimulated a biphasic oxidative burst in the leaves, resulting in the generation of hydrogen peroxide (H2O2) and superoxide anion radicals (O2(.-)) and in the concomitant down-regulation of antioxidant enzyme defense responses and up-regulation of lipid peroxidation. These results suggested that OTA damage might result from reactive oxygen species pathways. Our experiments provide a useful model plant system for research on OTA-induced plant cell death.

Evidence for programmed cell death and activation of specific caspase-like enzymes in the tomato fruit heat stress response.

The tomato (Lycopersicon esculentum) fruit is the best available model to study the stress response of fleshy fruit. Programmed cell death (PCD) plays an important role in stress responses in mammals and plants. In this study, we provide evidence that PCD is triggered in the tomato fruit heat stress response by detection of the sequential diagnostic PCD events, including release of cytochrome c, activation of caspase-like proteases and the presence of TUNEL-positive nuclei. Investigating the time course of these events for 12 h after heat treatment indicated that cytochrome c release and caspase-like protease activation occurred rapidly and were consistent with the onset of DNA fragmentation. In addition, LEHDase and DEVDase enzymes were specifically activated in tomato fruit pericarp during the heat treatment and recovery time. There was no significant activation of YVADase or IETDase proteases. Preincubation of pericarp discs with the broad-spectrum, cell-permeable caspase inhibitor Z-VAD-FMK, suppressed heat-induced cell death measured by trypan blue, accompanied by a decrease in LEHDase and DEVDase activities.

A 90-day toxicology study of transgenic lysine-rich maize grain (Y642) in Sprague-Dawley rats.

The gene for a lysine-rich protein (sb401) obtained from potatoes (Solanum berthaultii) was inserted into maize seed to produce Y642 transgenic maize. Compositional analysis of Y642 grain demonstrated that the concentrations of lysine and total protein were higher than those observed in maize grain from a near-isogenic non-genetically modified (non-GM) commercially available control quality protein maize (Nongda 108). The safety of Y642 maize grain was assessed by comparison of toxicology response variables in Sprague-Dawley (SD) rats consuming diets containing Y642 maize grain with those containing Nongda 108 maize grain. Maize grains from Y642 or Nongda 108 were incorporated into rodent diets at low (30%) or high concentrations (76%) and administered to SD rats (n=10/sex/group) for 90 days. An additional group of negative control group of rats (n=10/sex/group) were fed AIN93G diets. No adverse diet-related differences in body weights, feed consumption/utilization, clinical chemistry, hematology, absolute and relative organ weights were observed. Further, no differences in gross or microscopic pathology were observed between rats consuming diets with Y642 maize grain compared with rats consuming diets containing Nongda 108 maize grain. These results demonstrated that Y642 lysine-rich maize is as safe and nutritious as conventional quality protein maize.

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.

[Long-term and acute toxicity of kallikrein from the venom of Agkistrodon hlays Pallas].

A novel serine protease with high purity was extracted from the venom of Agkistrodon hlays Pallas using monoclonal antibody affinity chromatography. This protease releases bradykinin and has arginine esterase activity without being activated. After purification, its hydrolytic activity exceeded 800 U/mg, far higher than its counterparts from mammalian sources. The purity of the kininogenase could exceed 95%. The acute toxicity and the long-term toxicity of this kallikrein was studied for its potential clinical application. The maximum tolerance dose in adult was 150,000 times greater than the maximum applied dose, and long-term administration at the dose 50 times of allowed clinical dose did not obviously after the animals' body weight, survival condition, liver function, renal function, or blood routines, suggesting the extremely low toxicity of the kallidrein.

[Application of ICP-MS to the detection of heavy metals in transgenic corn].

With the rapid development of the transgenic food, more and more transgenic food has been pouring into the market attracting much attention to the transgenic food's edible safety. Transgenic corns and its parents were studied by ICP-MS to detect the heavy metals. The results showed that the transgenic corn accumulated less heavy metals (Ni, Cu, Cd, As, Cr, Zn and Hg) than their own parents; and the contents of some heavy metals (V, Co and Pb) in transgenic corns were similar to their parents. All the data showed that the insertion of foreign gene (Bt) might change the absorbing dynamics of most heavy metals, especially some important heavy metals, which are disadvantageous to human health. The present paper indicated that the change in heavy metals absorption could harm the edible safety of transgenic plant. The cause of this change should be studied further.

[Application of ICP-MS to the detection of forty elements in wine].

With the rapid development of wine, more and more people begin to pay more attention to its ingredients. Four kinds of wine were studied by ICP-MS to detect the heavy metals and microelements. The results showed that the wine contained many elements necessary to human health: 7 kinds of macroelements and 29 kinds of microelements. The sequence of macroelements is K>P>Mg>Ca>Na>Al approximately Si. The concentration of K is more than 900 microg x mL(-1), sometimes reaching 2359 microg x mL(-1) (shelongzhu). Six kinds of elements (Rb, Mn, Sr, Zn, Fe and Ba) among 28 kinds of microelements are higher than 200 ng x g(-1), and some elements are more than 1000 ng x mL(-1) (Rb, Mn and Sr), which is important to human health. In addition to microelements, contents of heavy metals (As, Cr, Pb and Cd) are also an important standard to identify the quality of wine, and the results showed that wine contains little heavy metals, whose sequence is As (less than 50 ng x g(-1))>Cr>Pb>Cd. All the data showed that the wine meets the national hygiene standards.

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.

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.