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.

[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.

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.

[Distribution pattern of heavy metals in transgenic(Bt+CpTI) cotton].

With the rapid development of transgenic plant, more and more attention has been paid to its ecological safety. Dual-toxin transgenic (Bt+CpTI) cottons and regular cotton were studied by ICP-MS to detect the concentration of heavy metals. The results showed that the sequence of concentration of heavy metals is root > leaf > stem in cotton; the transgenic cotton accumulated less Cd and As than regular cotton in all the organs; and the content of Cr in leaf and stem of transgenic cotton was lower than that in regular cotton, while the content of Pb was lower only in stem. All the data showed that the insertion of foreign gene (Bt) might change the absorbing dynamics of most heavy metal. The reason for this change should be studied further.

[Distribution of six trace elements in Fuji apple].

Trace elements are important nutritional elements for human health. Six trace elements in Fuji apple skin and pulp were analyzed by the method of ICP-MS. The results showed that the concentrations of Ca, Mn, Fe, Se, Mo and I were 197 910, 1 623, 14 400, 2, 47 and 91 ng x g(-1) x FW in apple skin respectively; and 58 360, 281, 550, 4, 18 and 24 ng x g(-1) x FW respectively in apple pulp. The concentrations of most trace elements (Ca, Mn, Fe, Mo and I) in apple skin were several times higher than those in apple pulp, especially the concentrations of Fe, Mn and I in skin was 25.18, 4.78 and 2.79 times higher than those in pulp, except Se (2 ng x g(-1) x FW in skin and 4 ng x g(-1) x FW in pulp). So we should not peel apples before we eat them from regions where were not polluted by pesticides and heavy metals.

[Contents of nutrient elements in NH4(+)-N fertilizer and urea].

Fertilizer contains not only one compound or one element, so it is important to determine the contents of other elements necessitous and beneficial to plant. All the other nutrient elements for plant, including necessitous elements and beneficial elements in ammonia nitrogen fertilizer ((NH4)2SO4) and CO(NH2)2, were analyzed by method of ICP-MS. The results showed that ammonia nitrogen fertilizer ((NH4)2SO4) and CO(NH2)2 both contain many necessitous elements, Mg, P, K, Ca, Mn, Fe, Ni, Cu, Zn and Mo, thereinto the contents of Mg, P, K, Ca, Mn and Fe were on microg x g(-1) the level, and Ni, Cu, Zn and Mo were on the ng x g(-1) level; compared with CO(NH2)2, ammonia nitrogen fertilizer ((NH4)2SO4) contains more necessitous elements and beneficial elements except Mo and Si. All the above elements could influence the results of nitrogen fertilizer efficiency experiments, so pure fertilizer should be used in the future nitrogen fertilizer efficiency experiments and the comparative experiments of different form nitrogen fertilizer.

[Application of ICP-MS to detect rare elements in wild Cistanche deserticola Y. C. Ma].

Rare metals are important nutritional elements for human health. Fifteen rare metals in wild Cistanche deserticola Y. C. Ma were analyzed by the methods of ICP-MS. The results showed that concentrations of La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Yb and Lu in Wild Cistanche deserticola Y. C. Ma were 144.89, 259.75, 36.41, 133.25, 25.38, 6.10, 30.20, 3.85, 20.36, 3.42, 8.55, 6.42, 1.15, 20.02 and 29.77 ng x g(-1) respectively, the character of elements and the content of rare earth elements in soil should be responsible for the difference, but the mechanism of distribution should be further studied.

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.

Mechanisms of pseudolaric acid B-induced apoptosis in Bel-7402 cell lines.

Previous studies have shown that pseudolaric acid B (PB) would cause apoptosis in human tumor cell lines. However, the mechanisms of PB induced apoptosis are still unclear. In the present study, the mechanisms of PB induced apoptosis in the human hepatocellular carcinoma Bel-7402 cell line were investigated by measuring cell viability, rate of apoptosis, cell cycle, detecting DNA fragmentation, and measuring caspase-3 activation. The results indicated that PB inhibited Bel-7402 cell viability and induced cell death by causing DNA fragmentation, up regulating the early and late apoptotic rates, activating caspase-3 protein, and detaining the cell cycle in the G2/M phases. Additionally, PB-induced apoptosis was a dose- and time-dependent manner. These observations suggest that PB-induced apoptosis occurs through a caspase-dependent pathway and detains the cell cycle in the G2/M phase.

Microbial hydroxylation of cinobufagin by Mucor spinosus.

Mucor spinosus has been employed for the biotransformation of cinobufagin (1) to afford three metabolites. On the basis of their physico-chemical data, the structures of the transformation products have been characterized as 1beta-hydroxy-cinobufagin (2), 12beta-hydroxy-cinobufagin (3) and 1beta,12beta-dihydroxy-cinobufagin (4), of which metabolites 2 and 4 are new compounds. In vitro cytotoxic activities of the biotransformation products and the substrate-cinobufagin have been assayed against four tumor cell lines of Bel 7420, BGC 823, HeLa and HL 60; they all showed cytotoxic activities.