Involvement of an ethylene response factor in chlorophyll degradation during citrus fruit degreening.

Chlorophyll degradation naturally occurs during plant senescence. However, in fruit such as citrus, it is a positive characteristic, as degreening is an important colour development contributing to fruit quality. In the present work, Citrus sinensis Osbeck, cv. Newhall fruit was used as a model for chlorophyll degradation. An ethylene response factor, CitERF13, was isolated and its transcriptional changes were closely correlated with fruit peel degreening during development or in response to ethylene. Dual-luciferase and yeast one-hybrid assays, as well as motif mutation, indicated that CitERF13 directly binds to the CitPPH promoter and enhances its activity. Transient and stable over-expression of CitERF13 resulted in rapid chlorophyll degradation in Nicotiana tabacum leaves and led to accumulation of pheophorbide (Pheide) a, a metabolite of pheophorbide hydrolase (PPH). Similar results were observed from transient transformation of CitERF13 in citrus fruit peel. Moreover, this function of CitERF13 was conserved within Arabidopsis and tomato, as the homologs AtERF17 and SlERF16 similarly acted as activators of PPH genes and accelerators of chlorophyll degradation.

CitAP2.10 activation of the terpene synthase CsTPS1 is associated with the synthesis of (+)-valencene in 'Newhall' orange.

Aroma is a vital characteristic that determines the quality and commercial value of citrus fruits, and characteristic volatiles have been analyzed in different citrus species. In sweet orange, Citrus sinensis, the sesquiterpene (+)-valencene is a key volatile compound in the fruit peel. Valencene synthesis is catalyzed by the terpene synthase CsTPS1, but the transcriptional mechanisms controlling its gene expression are unknown. Here, the AP2/ERF (APETALA2/ethylene response factor) transcription factor, CitAP2.10, is characterized as a regulator of (+)-valencene synthesis. The expression pattern of CitAP2.10 was positively correlated with (+)-valencene content and CsTPS1 expression. Dual-luciferase assays indicated that CitAP2.10 could trans-activate the CsTPS1 promoter. Ethylene enhanced expression of CitAP2.10 and this effect was abolished by the ethylene antagonist 1-methylcyclopropene. The role and function of CitAP2.10 in (+)-valencene biosynthesis were confirmed using the Arabidopsis homolog (AtWRI1), which also transiently activated the CsTPS1 promoter. Furthermore, transient over-expression of CitAP2.10 triggered (+)-valencene biosynthesis in sweet orange fruit. These results indicate that CitAP2.10 regulates (+)-valencene synthesis via induction of CsTPS1 mRNA accumulation.

Isolation, classification and transcription profiles of the AP2/ERF transcription factor superfamily in citrus.

The AP2/ERF gene family encodes plant-specific transcription factors. In model plants, AP2/ERF genes have been shown to be expressed in response to developmental and environmental stimuli, and many function downstream of the ethylene, biotic, and abiotic stress signaling pathways. In citrus, ethylene is effective in regulation citrus fruit quality, such as degreening and aroma. However, information about the citrus AP2/ERF family is limited, and would enhance our understanding of fruit responses to environmental stress, fruit development and quality. CitAP2/ERF genes were isolated using the citrus genome database, and their expression patterns analyzed by real-time PCR using various orange organs and samples from a fruit developmental series. 126 sequences with homologies to AP2/ERF proteins were identified from the citrus genome, and, on the basis of their structure and sequence, assigned to the ERF family (102), AP2 family (18), RAV family (4) and Soloist (2). MEME motif analysis predicted the defining AP2/ERF domain and EAR repressor domains. Analysis of transcript accumulation in Citrus sinensis cv. 'Newhall' indicated that CitAP2/ERF genes show organ-specific and temporal expression, and provided a framework for understanding the transcriptional regulatory roles of AP2/ERF gene family members in citrus. Hierarchical cluster analysis and t tests identified regulators that potentially function during orange fruit growth and development.

Enantioselective iron-catalyzed intramolecular cyclopropanation reactions.

An iron-catalyzed asymmetric intramolecular cyclopropanation was realized in high yields and excellent enantioselectivity (up to 97% ee) by using the iron complexes of chiral spiro-bisoxazoline ligands as catalysts. The superiority of iron catalysts exhibited in this reaction demonstrated the potential abilities of this sustainable metal in asymmetric carbenoid transformation reactions.

Enantioselective palladium-catalyzed insertion of α-aryl-α-diazoacetates into the O-H bonds of phenols.

A palladium-catalyzed asymmetric O-H insertion reaction was developed. Palladium complexes with chiral spiro bisoxazoline ligands promoted the insertion of α-aryl-α-diazoacetates into the O-H bond of phenols with high yield and excellent enantioselectivity under mild reaction conditions. This palladium-catalyzed asymmetric O-H insertion reaction provided an efficient and highly enantioselective method for the preparation of synthetically useful optically active α-aryl-α-aryloxyacetates.

Copper-catalyzed B-H bond insertion reaction: a highly efficient and enantioselective C-B bond-forming reaction with amine-borane and phosphine-borane adducts.

A copper-catalyzed B-H bond insertion reaction with amine- and phosphine-borane adducts was realized with high yield and enantioselectivity under mild reaction conditions. The B-H bond insertion reaction provides a new C-B bond-forming methodology and an efficient approach to chiral organoboron compounds.

Enantioselective copper-catalyzed intramolecular phenolic O-H bond insertion: synthesis of chiral 2-carboxy dihydrobenzofurans, dihydrobenzopyrans, and tetrahydrobenzooxepines.

Efficient: A copper-catalyzed enantioselective intramolecular insertion of carbenoids into phenolic O-H bonds has been developed. This method can be used for the synthesis of the title compounds in high yields and excellent enantioselectivities under mild and neutral conditions. NaBAr(F)=sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate.

Formation of Fungal 2,18-Dioxo-2,18-seco Indole Diterpenes by Nonenzymatic Flavin-Catalyzed Oxidative Ring Expansion and Oxygen Incorporation from Solvent Water.

Most naturally occurring indole diterpenes share a 6/5/5/6/6/6 hexacyclic ring system, while a 6/8/6/6/6 pentacyclic skeleton is occasionally observed. In this study, we demonstrate the formation of an eight-membered C-N heteroring via nonenzymatic flavin-catalyzed oxidative indole ring opening. More interestingly, 18O-labeled experiments proved that the two incorporated oxygen atoms are predominantly originated from water instead of molecular oxygen. In this process, the oxidized form of flavin catalyzes two successive oxidations of amines to imines with involvement of hydrolysis for the ring expansion. The reduced flavin is then regenerated by oxidation with molecular oxygen to form H2O2.

Ergot alkaloid biosynthesis in Aspergillus fumigatus: Conversion of chanoclavine-I aldehyde to festuclavine by the festuclavine synthase FgaFS in the presence of the old yellow enzyme FgaOx3.

Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. A putative gene fgaFS has been identified in the biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine-type. The deduced gene product FgaFS comprises 290 amino acids with a molecular mass of about 32.1 kDa. The coding region of fgaFS consisting of three exons was amplified by PCR from a cDNA library of Aspergillus fumigatus, cloned into pQE70 and overexpressed in E. coli. The soluble monomeric His(6)-FgaFS was purified by affinity chromatography and used for enzyme assays. It has been shown that FgaFS is responsible for the conversion of chanoclavine-I aldehyde to festuclavine in the presence of the old yellow enzyme FgaOx3. The structure of festuclavine including the stereochemistry was unequivocally elucidated by NMR and MS analyses. Festuclavine formation was only observed when chanoclavine-I aldehyde was incubated with FgaOx3 and FgaFS simultaneously or as a tandem-reaction with a sequence of FgaOx3 before FgaFS. In the absence of FgaFS, two shunt products were formed and did not serve as substrates for FgaFS reaction.

Simultaneous C7- and N1-prenylation of cyclo-L-Trp-L-Trp catalyzed by a prenyltransferase from Aspergillus oryzae.

A putative prenyltransferase gene cTrpPT was amplified from Aspergillus oryzae DSM1147, cloned into pQE70 and overexpressed in Escherichia coli. The overproduced His(6)-CTrpPT was purified to near homogeneity and incubated with L-tryptophan or tryptophan-containing cyclic dipeptides in the presence of dimethylallyl diphosphate. The formation of the enzyme products was monitored with HPLC. It was shown that CTrpPT differed clearly from other known indole prenyltransferases in several aspects. This enzyme showed higher substrate specificity towards aromatic substrates, but lower regioselectivity regarding the prenylation position than other indole prenyltransferases. Cyclo-L-Trp-L-Trp was much better accepted than other cyclic dipeptides tested in this study. In comparison to other indole prenyltransferases with one dominant enzyme product, at least two product peaks were detected in the reaction mixtures of CTrpPT. (1)H- and (13)C-NMR analyses, including long-range (1)H-(13)C connectivities in Heteronuclear Multiple-Bond Correlation (HMBC) and Nuclear Overhauser Effect Spectroscopy (NOESY), proved the structures of the enzyme products as C7- and N1-prenylated derivatives with a ratio of 1:1.2 using cyclo-L-Trp-L-Trp as substrate. The K(M) values were determined at about 2.5 mM for dimethylallyl diphosphate and 0.3 mM for cyclo-L-Trp-L-Trp with a turnover number of 0.33 s(-1).

Preparation of pyrrolo[2,3-b]indoles carrying a beta-configured reverse C3-dimethylallyl moiety by using a recombinant prenyltransferase CdpC3PT.

Six beta-configured reversely C3-prenylated pyrrolo[2,3-b]indoles were successfully prepared by using a recombinant prenyltransferase from Neosartorya fischeri. For this purpose, the putative prenyltransferase gene NFIA_074280 (termed herewith cdpC3PT) was cloned into pQE60 and overexpressed in Escherichia coli. The overproduced His(6)-CdpC3PT was purified to near homogeneity and incubated with five cyclic tryptophan-containing dipeptides in the presence of dimethylallyl diphosphate (DMAPP). All of the substrates were accepted by CdpC3PT and converted to reversely C3-prenylated pyrrolo[2,3-b]indoles. Using cyclo-l-Trp-l-Trp as substrate, both mono- and diprenylated derivatives were obtained. The structures of the enzymatic products were confirmed by HR-ESI-MS, (1)H- and (13)C-NMR analyses as well as by long-range (1)H-(13)C connectivities in heteronuclear multiple-bond correlation (HMBC) spectra after preparative isolation. (1)H-(1)H spatial correlations in nuclear overhauser effect spectroscopy (NOESY) were used for determination of absolute configuration. The K(M) values were determined at about 1.5 mM for DMAPP and in the range from 0.22 to 5.5 mM for cyclic dipeptides. The turnover number k(cat) were found in the range of 0.023 to 0.098 s(-1) and specificity constants k(cat)/K(M) from 14.2 to 122.7 M(-1) s(-1). In contrast to the products of AnaPT bearing alpha-configured C3-dimethylallyl residues, the C3-prenyl moieties in the products of CdpC3PT have a beta-configuration. Discovery and characterisation of CdpC3PT expand the usage of the chemoenzymatic approach for stereospecific synthesis of C3-prenylated derivatives.

Reconstruction of pyrrolo[2,3-b]indoles carrying an alpha-configured reverse C3-dimethylallyl moiety by using recombinant enzymes.

Nine reversely C3-prenylated pyrrolo[2,3-b]indoles were successfully prepared by using two recombinant enzymes involved in the biosynthesis of acetylaszonalenin from Neosartorya fischeri. The prenyltransferase AnaPT catalysed the conversion of six tryptophan-containing cyclic dipeptides to reversely C3-prenylated indoline derivatives. Using cyclo-L-Trp-L-Trp as substrate, both mono- and diprenylated indolines were obtained. Two of the AnaPT products were acetylated at position N1 by the acetyltransferase AnaAT. The structures of the obtained compounds were characterised by HR-ESI-MS, (1)H- and (13)C-NMR analyses as well as by long-range (1)H-(13)C connectivities in heteronuclear multiple-bond correlation (HMBC) spectra after preparative isolation. Their absolute configurations were determined by analysing the (1)H-(1)H spatial correlations in rotating-frame nuclear overhauser effect spectroscopy (ROESY).

Auto- and mutual-regulation between two CitERFs contribute to ethylene-induced citrus fruit degreening.

Citrus fruit postharvest degreening is a critical stage in marketing, carried out by exposure to ethylene or ethephon. Genome-wide screening of the AP2/ERF superfamily indicated that a novel ERF-II (CitERF6) was shown to trans-activate the CitPPH promoter. Expression of CitERF6 is associated with both developmental and postharvest degreening in citrus fruit. Transient and stable over-expression of CitERF6 in Nicotiana tabacum leaves and 'Ponkan' fruit also results in rapid chlorophyll degradation. Auto- and mutual-regulation was also found between CitERF6 and the previously characterized CitERF13 using the dual-luciferase and yeast one-hybrid assays. Moreover, substitution of the 35S promoter for endogenous promoters showed that both pCitERF6::CitERF6 and pCitERF13::CitERF13 were effective in trans-activating their promoters or triggering chlorophyll degradation. It is proposed that ethylene is one of the triggers activating promoters of CitERF6 and CitERF13, and subsequent auto- and mutual-regulation between CitERF6 and CitERF13 might facilitate the effect of ethylene, leading to fruit degreening.

A novel ethylene responsive factor CitERF13 plays a role in photosynthesis regulation.

Ethylene responsive factors (ERFs) act as critical downstream components of the ethylene signalling pathway in regulating plant development and stress responses. However little is known about its role in regulation of photosynthesis. Here, we identified an ethylene-inducible ERF gene in citrus, CitERF13. Transient over-expression of CitERF13 in N. tabacum leaves, resulted in a significant decrease in net photosynthetic rate. Closer examination of photosynthetic activity of PSII and PSI indicated that CitERF13 overexpression led to declines of Fv/Fm, Y(II) and Y(I). However, change in NPQ was less pronounced. CitERF13 overexpression also significantly reduced Vc,max, Jmax and AQY, indicating inhibition of the Calvin cycle. The expression of photosynthesis-related genes was suppressed to a variable extent in leaf blades transiently over-expressing CitERF13. CitERF13 transient overexpression in tobacco or citrus both resulted in a decline of Chlorophyll content and CitERF13 overexpressing tobacco leaf disc was more susceptible to chlorosis in response to MV-mediated oxidative stress. The results suggest that CitERF13 is potentially involved in suppressing photosynthesis through multiple pathways, for instance, inhibiting photochemical activity of photosynthesis, CO2 carboxylation capacity and chlorophyll metabolism.

The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4.

Organic acids are essential to fruit flavor. The vacuolar H(+) transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties 'Ordinary Ponkan (OPK)' and an early maturing mutant 'Zaoshu Ponkan (ZPK)'. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis.

[Effects of TIMP-2 gene transfer on atherosclerotic plaque in rabbits].

OBJECTIVE: To evaluate the effects of TIMP-2 local gene transfer on atherosclerotic plaque. METHODS: Atherosclerosis models were induced by denuding femoral artery endothelium plus high lipid diet in rabbits. TIMP-2 gene was transferred locally by balloons eluted with pcDNA3-TIMP-2. RT-PCR and Western blot were performed to verify exogenous genes transfer. MMPs activity in atherosclerotic plaque was evaluated by zymography. HE and VG staining and automatic image analysis system were used for pathological analysis of atherosclerotic femoral arteries. The lumen area of the vessel and the collagen contents in the atherosclerotic plaque were measured. RESULTS: The expression of TIMP-2 gene in pcDNA3-TIMP-2 transferred group was significantly higher than control-vector transferred group at the end of week 2 after operation and reached the peak at the end of week 4. Comparing with the control group, the expression of TIMP-2 protein in treated group was also higher at the end of week 2, 4, and 8 after operation. Correspondingly, the MMP-2 and MMP-9 activities were lower in treated group. The thickness of fibrous cap of atherosclerotic plaque and the amount of collagen of the lesion were increased significantly in treated group compared with the control group, but there were no significant differences in vessel lumen area. CONCLUSION: TIMP-2 gene transfer locally in atherosclerotic plaque could inhibit the activities of MMP-2 and MMP-9 in the lesion, increase the thickness of fibrous cap and the amount of collagen of the lesion, but may have no effect on the degree of the stenosis.

[Effect of danshensu, protocatechualdehyde and danshen injection on calcium ion concentration in cytoplasm of human erythrocytes].

OBJECTIVE: To study the predominant calcium-antagonist components of Danshen injection. METHOD: The effects of danshensu, protocatechualdehyde and Danshen injection on calcium concentration in cytoplasm of erythrocytes were examined in vitro by the fluorescent Ca+ -chelator fura-2. RESULT: Either DS182 or PCAD can decrease in dose-dependent cytosolic free calcium concentration in human erythrocytes. They had additive effect when mixed, which was similar to Danshen injection. CONCLUSION: DS182 and PCAD may be predominant calcium-antagonist components of Danshen injection.