Fruit ripening in tomato (Solanum lycopersicum) is a complicated development process affected by both endogenous hormonal and genetic regulators and external signals. Although the role of NOR, a member of the NAC domain family, in mediating tomato fruit ripening has been established, its underlying molecular mechanisms remain unclear. To explore further the role of NAC transcription factors in fruit ripening, we characterized a new tomato NAC domain protein, named SlNAC4, which shows high accumulation in sepal and at the onset of fruit ripening. Various stress treatments including wounding, NaCl, dehydration and low temperature significantly increased the expression of SlNAC4. Reduced expression of SlNAC4 by RNA interference (RNAi) in tomato resulted in delayed fruit ripening, suppressed Chl breakdown and decreased ethylene synthesis mediated mainly through reduced expression of ethylene biosynthesis genes of system-2, and reduced carotenoids by alteration of the carotenoid pathway flux. Transgenic tomato fruits also displayed significant down-regulation of multiple ripening-associated genes, indicating that SlNAC4 functions as a positive regulator of fruit ripening by affecting ethylene synthesis and carotenoid accumulation. Moreover, we also noted that SlNAC4 could not be induced by ethylene and may function upstream of the ripening regulator RIN and positively regulate its expression. Yeast two-hybrid assay further revealed that SlNAC4 could interact with both RIN and NOR protein. These results suggested that ethylene-dependent and -independent processes are regulated by SlNAC4 in the fruit ripening regulatory network.
Pigment intensity and patterns are important factors that determine the nutritional and market values of tomato fruits. The acropetal manner of light-dependent anthocyanin accumulation with the highest levels at the stem end of the fruit makes Pro35S:BrTT8 tomato plants an ideal system for investigating the effects of light intensity on anthocyanin biosynthesis. Extensive transcript analyses indicate that anthocyanin pigmentation in Pro35S:BrTT8 plants under high light might be coordinately regulated by the exogenous protein BrTT8 and endogenous proteins SlAN2 and SlMYBL2. Furthermore, yeast two-hybrid assays showed that BrTT8 could interact efficiently with SlAN2, SlMYBL2, and SlAN11. Moreover, the physical interaction between BrTT8 and SlAN2 was validated by FRET. Simultaneous overexpression of SlAN2 and BrTT8 activated significant anthocyanin biosynthesis in infiltrated tobacco leaves. In addition, the ability of SlMYBL2 to suppress anthocyanin accumulation was also demonstrated in infiltrated tobacco leaves. Altogether, these results prove that tissue-specific assemblage of the heterogeneous MYB-bHLH-WD40 complex consisting of SlAN2, BrTT8 and SlAN11 triggers nonuniform anthocyanin accumulation in tomato fruit under high light. Additionally, it is proposed that a negative-feedback loop fulfilled by SlMYBL2 also participates in the regulation of anthocyanin production.
Background/Aims: Hyperlipidemia induces dysfunction in the smooth muscle cells (SMCs) of the blood vessels, and the vascular remodeling that ensues is a key proatherogenic factor contributing to cardiovascular events. Chemokines and chemokine receptors play crucial roles in vascular remodeling. Here, we examined whether the hyperlipidemia-derived chemokine CCL5 and its receptor CCR5 influence vascular SMC proliferation, phenotypic switching, and explored the underlying mechanisms. Methods: Thoracoabdominal aorta were isolated from wild-type, CCL5 and CCR5 double-knockout mice (CCL5–/–CCR5–/–) fed a high-fat diet (HFD) for 12 weeks. Expression of the contractile, synthetic, and proliferation markers were assayed using immunohistochemical and western blotting. The effects of CCL5 and palmitic acid on cultured SMC proliferation and phenotypic modulation were evaluated using flow cytometry, bromodeoxyuridine (BrdU), and western blotting. Results: Wild-type mice fed an HFD showed markedly increased total cholesterol, triglyceride, and CCL5 serum levels, as well as significantly increased CCL5 and CCR5 expression in the thoracoabdominal aorta vs. normal-diet-fed controls. HFD-fed CCL5-/-CCR5-/- mice showed significantly decreased expression of the synthetic phenotype marker osteopontin and the proliferation marker proliferating cell nuclear antigen, and increased expression of the contractile phenotype marker smooth muscle α-actin in the thoracoabdominal aorta vs. wild-type HFD-fed mice. Human aorta-derived SMCs stimulated with palmitic acid showed significantly increased expression of CCL5, CCR5, and synthetic phenotype markers, as well as increased proliferation. CCL5-treated SMCs showed increased cell cycle regulatory protein expression, paralleling increased synthetic and decreased contractile phenotype marker expression. Inhibition of CCR5 activity by the specific antagonist maraviroc or its expression using small interfering RNA significantly inhibited human aortic SMC proliferation and synthetic phenotype formation. Therefore, CCL5 induces SMC proliferation and phenotypic switching from a contractile to synthetic phenotype via CCR5. CCL5-mediated SMC stimulation activated ERK1/2, Akt/p70S6K, p38 MAPK, and NF-κB signaling. NF-κB inhibition significantly reduced CCR5 expression along with CCR5-induced SMC proliferation and synthetic phenotype formation. Conclusions: Hyperlipidemia-induced CCL5/CCR5 axis activation serves as a pivotal mediator of vascular remodeling, indicating that CCL5 and CCR5 are key chemokine-related factors in atherogenesis. SMC proliferation and synthetic phenotype transformation attenuation by CCR5 pharmacological inhibition may offer a new approach to treatment or prevention of atherosclerotic diseases associated with hyperlipidemia.
BackgroundLycopene is an important carotenoid pigment in red fruits and vegetables, especially in tomato. Although lycopene biosynthesis and catabolism have been found to be regulated by multiple factors including phytohormones, little is known about their regulatory mechanism. Cytokinins are crucial to various aspects of plant growth. Isopentenyltransferases (IPTs) catalyze the initial rate-limiting step of cytokinins biosynthesis, however, their roles in fruit ripening remain unclear.ResultsHere, the functions of SlIPT4, encoding an isopentenyltransferase, were characterized via RNAi-mediated gene silencing in tomato. As we expected, silencing of SlIPT4 expression resulted in accelerated leaf senescence. However, down-expression of SlIPT4 generated never-red orange fruits, corresponding with a dramatic reduction of lycopene. Among lycopene biosynthesis-related genes, the fact of remarkable decrease of ZISO transcript and upregulation of other genes, revealed that SlIPT4 regulates positively lycopene biosynthesis via directly affecting ZISO expression, and also supported the existence of regulatory loops in lycopene biosynthesis pathway. Meanwhile, the accumulation of abscisic acid (ABA) was reduced and the transcripts PSY1 were increased in SlIPT4-RNAi fruits, supporting the feedback regulation between ABA and lycopene biosynthesis.ConclusionThe study revealed the crucial roles of SlIPT4 in leaf senescence and the regulatory network of lycopene biosynthesis in tomato, providing a new light on the lycopene biosynthesis and fruit ripening.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1327-0) contains supplementary material, which is available to authorized users.
Rhodobacter sphaeroides has been intensively studied and provides an excellent model for studying both photosynthesis and membrane development. The photosynthetic apparatus (LH2 and LH1-RC complexes) can be synthesized in large scale and integrated into the intracytoplasmic membrane system under specific conditions, which thus provides us insight to utilize the puc or(and) puf operon to heterologously express recombinant proteins in the intracytoplasmic membrane using Rb. sphaeroides as a novel expression system. However, basal level of expression of puc and puf promoter is uncontrolled. We report the construction of LH2 polypeptide expression vector that contains a reengineered lacI(q)-puc promoter-lac operator hybrid promoter, which allows the puc operon to be regulated by both IPTG and low oxygen level. Synthesis of LH2 complexes was completely repressed in the absence of isopropyl beta-D-thiogalactoside (IPTG), and the degree of induction was controlled by varying the concentration of IPTG. The optimal concentration of IPTG was determined. SDS-PAGE and Western blot were employed for further analysis. Our results suggest that the reengineered hybrid promoter is efficient to tightly regulate the expression of the puc operon, and our strategy can open up a new approach in the study of the membrane protein expression system.
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