Adverse environmental conditions, such as drought, high salinity and extreme temperature, severely affect the growth and productivity of crop plants. MADS-box transcription factors have been described to participate in stress responses. In our study, a MADS-box transcription factor gene, SlMBP8, has been cloned from tomato. The expression of SlMBP8 was induced by Methyl-jasmonic acid (MeJA), high salinity, high temperature, wounding and dehydration. Whereas, the transcript of SlMBP8 was down-regulated by Abscisic acid (ABA), 1-aminocyclopropane-1-carboxylic acid (ACC) and Indole-3-acetic acid (IAA). To further elucidate the function of SlMBP8 gene in response to abiotic stress, plants by knockdown of SlMBP8 through RNA interference (RNAi) were used for investigating the effect of drought and salt stresses on tomato seedlings of wild type (WT) and SlMBP8-RNAi lines. Seedling growth of SlMBP8-RNAi plants was less inhibited by salt than WT at post-germination stage. Transgenic plants became more tolerant to drought and salt stress than WT plants in soil, which was demonstrated by higher levels of chlorophyll and water contents, lower water loss rate and malondialdehyde (MDA) contents. In addition, the expression of multiple stresses related genes were significantly up-regulated in the RNAi lines under control and abiotic stresses. Taken together, these results suggest that SlMBP8 function as a negative stress-responsive transcription factor in the drought and high salinity stress signaling pathways, and may have promising applications in the engineering of drought- and salt-tolerant tomato.

•Gene SlMBP8 plays an important role in fruit ripening.•A yeast two-hybrid assay revealed a clear interaction between SlMBP8 and SlMADS-RIN.•Silencing of SlMBP8 reduces the storability of fruits.MADS-box genes encode important transcription factors that are involved in many biological processes of plants, including fruit ripening. In our research, a MADS-box gene, SlMBP8, was identified, and its tissue-specific expression profiles were analysed. SlMBP8 was highly expressed in fruits of the B+4 stage, in senescent leaves and in sepals. To further characterize its function, an RNA interference (RNAi) expression vector of SlMBP8 was constructed and transferred into tomato. In the transgenic plants, the ripening of fruits was shortened by 2–4 days compared to that of wild type. At the same time, carotenoids accumulated to higher levels and the expression of phytone synthase 1 (PSY1), phytoene desaturase (PDS) and ς-carotene desaturase (ZDS) was enhanced in RNAi fruits. The transgenic fruits and seedlings showed more ethylene production compared with that of the wild type. Furthermore, SlMBP8-silenced seedlings displayed shorter hypocotyls due to higher endogenous ethylene levels, suggesting that SlMBP8 may modulates the ethylene triple response negatively. A yeast two-hybrid assay indicated that SlMBP8 could interact with SlMADS-RIN. Besides, the expression of ethylene-related genes, including ACO1, ACO3, ACS2, ERF1, E4 and E8, was simultaneously up-regulated in transgenic plants. In addition, SlMBP8-silenced fruits showed higher ethylene production, suggesting that suppressed expression of SlMBP8 promotes carotenoid and ethylene biosynthesis. In addition, the fruits of transgenic plants displayed more rapid water loss and decreased storability compared to wild type, which was due to the significantly induced expressions of cell wall metabolism genes such as PG, EXP, HEX, TBG4, XTH5 and XYL. These results suggest that SlMBP8 plays an important role in fruit ripening and softening.

•SlHDT3 influences carotenoid accumulation during tomato fruit ripening.•SlHDT3 act as an inhibitor influences ethylene biosynthesis.•SlHDT3 RNAi lines delays fruit ripening.•SlHDT3 RNAi lines fruits have a longer shelf life.The acetylation levels of histones on lysine residues are regulated by histone acetyltransferases and histone deacetylases, which play an important but understudied role in the control of gene expression in plants. There is an increasing research focus on histone deacetylation in crops, but to date, there is little information regarding tomato. With the aim of characterizing the tomato HD2 family of histone deacetylases, an RNA interference (RNAi) expression vector of SlHDT3 was constructed and transformed into tomato plants. The time of fruit ripening was delayed and the shelf life of the fruit was prolonged in SlHDT3 RNAi lines. The accumulation of carotenoid was decreased by altering of the carotenoid pathway flux. Ethylene content was also reduced and expression of ethylene biosynthetic genes (ACS2, ACS4 and ACO1, ACO3) and ripening-associated genes (RIN, E4, E8, PG, Pti4 and LOXB) was significantly down-regulated in SlHDT3 RNAi lines. The expression of genes involved in fruit cell wall metabolism (HEX, MAN, TBG4, XTH5 and XYL) was inhibited compared with wild type. These results indicate that SlHDT3 functions as a positive regulator of fruit ripening by affecting ethylene synthesis and carotenoid accumulation and that SlHDT3 lies upstream of SlMADS-RIN in the fruit ripening regulatory network.

Plant-specific NAC (NAM/ATAF/CUC) transcription factors play crucial roles in diverse development processes as well as biotic and abiotic stress responses. Nevertheless, to date only few reports regarding stress-related NAC genes are available in tomato. In this study, we isolated an abiotic stress-responsive NAC gene from tomato, here designated as SlNAC11. Expression analysis revealed that SlNAC11 was induced significantly by dehydration, cold, and heat. The functions of SlNAC11 in abiotic stress were further detected on SlNAC11-RNAi transgenic tomato plants. The results showed that SlNAC11-RNAi plants became less tolerant to drought and salt stress, which were demonstrated by lower level of chlorophyll content and seed germination rate, and higher level of MDA as compared to WT plants under stress conditions. Furthermore, transgenic seedlings exhibited less hypersensitive to ABA, demonstrated by longer hypocotyl and root as compared to WT plants. These results show that SlNAC11 functions as a stress-responsive transcription factor depicting positive response to abiotic stress tolerance and may hold promise for improving stress tolerance in transgenic tomato.

Anthocyanins, natural pigments with high antioxidant activities, are widely distributed in the plant kingdom and play important roles in various physiological processes. Much effort has been committed to enhancing the anthocyanin content of these health-promoting pigments in vegetables and grains, for their eye-catching colors and special nutrients. Previously, we reported that the SmMYB1 gene encoding a R2R3 MYB transcription factor participated in the regulation of anthocyanin biosynthesis in the peel of eggplant. In this work, we introduced SmMYB1 into a non-anthocyanin-accumulating eggplant cultivar (Solanum aethiopicum group Gilo) via Agrobacterium-mediated transformation. Genetically engineered plants exhibited high concentrations of anthocyanin in leaves, petals, stamens, and fruit peels under normal growth conditions, especially in fruit flesh. Furthermore, highly methylated anthocyanins, malvidin 3-(p-coumaroyl)rhamnoside(glucoside)-5-glucoside and malvidin 3-(feruloyl)rhamnoside(glucoside)-5-glucoside, were separated from the purple fruit flesh and identified by HPLC–ESI–MS/MS. qRT-PCR analysis revealed that most anthocyanin structural genes were dramatically up-regulated in the tissues of transgenic lines compared with non-transformed plants. In addition, the transgenic seedlings had greater tolerance to freezing stress and better recovery under rewarming conditions. These results provide a good foundation for the breeding of new eggplant cultivars with more healthy agronomic traits in future studies.

Kohlrabi (Brassica oleracea var. gongylodes L.) is an important dietary vegetable cultivated and consumed widely for the round swollen stem. Purple kohlrabi shows abundant anthocyanin accumulation in the leaf and swollen stem. Here, different kinds of anthocyanins were separated and identified from the purple kohlrabi cultivar (Kolibri) by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry. In order to study the molecular mechanism of anthocyanin biosynthesis in purple kohlrabi, the expression of anthocyanin biosynthetic genes and regulatory genes in purple kohlrabi and a green cultivar (Winner) was examined by quantitative PCR. In comparison with the colorless parts in the two cultivars, most of the anthocyanin biosynthetic genes and two transcription factors were drastically upregulated in the purple tissues. To study the effects of light shed on the anthocyanin accumulation of kohlrabi, total anthocyanin contents and transcripts of associated genes were analyzed in sprouts of both cultivars grown under light and dark conditions.

TomloxD is a lipoxygenase from Lycopersicon esculentum Mill, which has lipoxygenase activity. The expression of TomloxD can be stimulated by wounding, pathogen infection, jasmonate, and systemin. To investigate the function of TomloxD, transgenic tomato plants silencing tomloxD gene was produced. The suppression of TomloxD expression led to a marked reduction in the levels of lipoxygenase activity and endogenous jasmonic acid content, which suggested TomloxD can catalyze α-linolenic acids to produce (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT), and the 13-HPOT is metabolized further to synthesize jasmonic acid. Real-time RT-PCR revealed that the expression of defense genes LeHSP90, LePR1 and LePR6 was also less in the transformants than in the wild-type tomato plant. Resistance assay showed that the suppression of TomloxD in transgenic tomato plants reduced thermotolerance of tomato and increased its susceptibility to Cladosporium fulvum. Collectively, the data presented here suggest that the TomloxD plays a role as a component of the octadecanoid defense-signaling pathway and involved in the generation of endogenous jasmonic acid, and in turn regulates the expression of plant defense genes and resistance to high temperature and pathogen attack.

Viral infection seriously affects the yield and quality of potato tubers. Breeding of virus-resistant potato (Solanum tuberosum L.) varieties is essential. Introduction of resistance genes from other Solanaceae species into cultivated potato is likely to be a valuable method to achieve durable resistance to potato virus. Tm-22 resistance gene was cloned from tomato cv. KBV and introduced into the binary vector pBIN19 to yield Tm-22-constitutive expression vector under the control of the CaMV 35S promoter. The resultant vector was transferred into potato, and Tm-22 gene was successfully expressed in transgenic potato plants. After infection with TMV, ToMV, PVX, and PVY, transgenic potato plants showed the high level of resistance to these viruses, and potato tuber yield was significantly increased compared to wild type. qRT-PCR results showed that Tm-22 transgenic plants had much lower transcript levels of VIRUS-COAT genes than wild type, even no TMV- and ToMV-COAT RNAs were detected in upper non-inoculated systemic leaves of transgenic lines. These results indicate that Tm-22 confers different resistance responses against viruses depending on its expression level.

Tumorous stem mustard (Brassica juncea var. tumida Tsen et Lee) is an economically and nutritionally important dietary vegetable in Asian countries. Purple tumorous stem mustard contains inflated tumorous stem and abundant anthocyanin accumulation in leaves. Here, 20 anthocyanins were separated and identified from the purple tumorous stem mustard by high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MS/MS). In order to investigate the regulatory anthocyanin production in purple tumorous stem mustard, the expression of anthocyanin biosynthetic and regulatory genes in leaves from purple and green cultivars were examined. Regulatory gene BjTT8 and all biosynthetic genes were dramatically upregulated in the purple variety. Moreover, the transcript level of BjTT8 and all structural genes, except BjPAL, were all significantly higher in light-treated sprouts than in the dark. These results indicate that transcriptional activation of BjTT8 is associated with upregulation of most anthocyanin biosynthetic genes, to produce anthocyanins in purple tumorous stem mustard.

Eggplant (Solanum melongena L.) is an edible fruit vegetable cultivated and consumed worldwide. The purple eggplant is more eye-catching and popular for the health-promoting anthocyanins contained in the fruit skin. Two kinds of anthocyanin were separated and identified from purple cultivar (Zi Chang) by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry. To investigate the molecular mechanisms of anthocyanin accumulation in eggplant, the transcripts of anthocyanin biosynthetic and regulatory genes were analyzed in the fruit skin and the flesh of the purple cultivar and the white cultivar (Bai Xue). Compared with the other tissues, SmMYB1 and all anthocyanin biosynthetic genes except PAL were dramatically upregulated in the fruit skin of the purple cultivar. Overexpression of SmMYB1 activated abundant anthocyanin accumulation in the regenerating shoots of eggplant. These results prove that transcriptional activation of SmMYB1 accounts for constitutive upregulation of most anthocyanin biosynthetic genes and the onset of anthocyanin biosynthesis in the purple cultivar.

α-Linolenic and linoleic acids are essential fatty acids (EFAs) for humans and required for maintenance of optimal health, but they cannot be synthesized by the human body and must be obtained from dietary sources. Using TomloxC fragment, TomloxD fragment, and partial TomloxA sequence that is highly identical with TomloxB and TomloxE, a RNAi expression vector was constructed. The construct was used to transform tomato cotyledon explants with the Agrobacterium-mediated co-cultivation method. The real-time reverse transcription polymerase chain reaction analysis showed that the expression of TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE in transgenic tomato plants was drastically repressed, which led to a marked decrease in the levels of lipoxygenase activity. Finally, higher accumulations of the endogenous α-linolenic and linoleic acids were detected in the transgenic tomato fruits, which were 1.65–3.99 and 2.91–4.98 times that of the non-transformed tomato fruits, respectively.

NAC (NAM/ATAF1/2/CUC2) transcription factors comprise a large plant-specific gene family and play crucial roles in plant growth, development, and stress response. However, only little information regarding development- or stress-related NAC genes is available in tomato. In this study, six tomato NAC genes, designated as SlNAC5-SlNAC10, were characterized to encode NAC proteins that share high similarity with those known plant NACs, especially in the NAC domain. These SlNAC genes, except SlNAC10, have a conserved exon–intron structure despite their different intron length. Phylogenetic analysis suggested that SlNAC5, SlNAC7, and SlNAC10 may belong to the development-related NACs. Quantitative RT-PCR analysis revealed that the six SlNACs exhibited different tissue-specific (such as roots, leaves, and flowers) expression profiles, suggesting that they may have crucial and diverse roles during tomato growth and development. Varying degrees of induction were detected in the transcript level of these SlNACs except SlNAC6 when treated with multiple hormones including ABA, ACC, GA3, MeJA, and IAA. The expression of SlNAC5-SlNAC10 was also checked under various abiotic stresses including NaCl, wounding, dehydration, and high/low temperature; six SlNACs were induced by these stresses with differential/similar induction levels. SlNAC5 was the most induced gene by NaCl stress with a 950-fold induction. Collectively, the results in this study of tissue-specific and/or stress-responsive SlNAC genes provide valuable information for further exploring the functional roles of NAC genes during tomato development and in response to environmental stresses, and one or more of them may hold promise for improving stress tolerance in transgenic tomato.

Anthocyanins are ubiquitous plant secondary products that possess nutritive values and serve essential functions in plant defense. The purple kale (Brassica oleracea var. acephala f. tricolor) BoPAP1 gene, which encodes a MYB transcription factor associated with anthocyanin biosynthesis, was introduced into tomato (Solanum lycopersicum) under the control of the CaMV 35S promoter. Transgenic tomato plants exhibited restricted accumulation of anthocyanins in stamens under normal growth conditions. qPCR analysis revealed that the expression of the anthocyanin biosynthetic genes such as SlF3′5′H, SlDFR, and SlANS, as well as a bHLH gene SlTT8, was specifically induced in transgenic stamens. A yeast two-hybrid assay revealed that BoPAP1 was a transcription activator and could interact with SlTT8. Despite the absence of anthocyanins in seedlings under normal conditions, overexpression of BoPAP1 led to high levels of anthocyanins and chlorophylls in the transgenic seedlings that were exposed to low temperature for 2 weeks, which conferred chilling tolerance on transgenic tomato plants. The consistency of SlTT8 expression and anthocyanin accumulation was observed in both purple stamens and low temperature treated seedlings in transgenic lines. These results suggest that the endogenous bHLH gene SlTT8 could be a candidate bHLH gene involved in the tomato anthocyanin pathway. Considering that the inducible type of anthocyanins apparently does not interfere with the normal growth of the plant; it provides an optimized transgenic strategy for crop improvement.

Expression of the tomato gene encoding 13-lipoxygenase,TomloxD, is stimulated by wounding, pathogen infection, jasmonate, and systemin, but its role during growth and development of tomato (Lycopersicon Spp.) remains unclear. To assess the physiological role of TomloxD, we produced transgenic tomato plants with greatly increased TomloxD content using sense constructs under the control of the CaMV 35S promoter. Overexpression of TomloxD in transgenic tomatoes led to a marked increase in the levels of lipoxygenase activity and content of endogenous jasmonic acid (JA), which suggested that TomloxD can use α-linolenic acid as a substrate to produce (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT); the 13-HPOT produced appears to be metabolized further to synthesize JA. Real-time RT-PCR revealed that the expression levels of defense genes LeHSP90, LePR1, LePR6 and LeZAT in the transformants were higher than those in non-transformed plants. Assay for resistance to pathogenic fungus and high temperature stresses suggested that transgenic plants harboring TomloxD were more tolerant to Cladosporium fulvum and high temperature stress than non-transformed tomato plants. The data presented here indicate clearly that TomloxD is involved in endogenous JA synthesis and tolerance to biotic and abiotic stress. The tomloxD gene has potential applications in engineering cropping plants that are resistant to biotic and/or abiotic stress factors.

“White Dove” is a mutant in kale (Brassica oleracea var. acephala f. tricolor), which exhibits a mutant albino phenotype in the interior of the plant under low temperature conditions. Chlorophyll content in “White Dove” was dramatically reduced under low temperature conditions, while the content in “Green Dove” decreased slightly under the same conditions. The levels of five chlorophyll precursors suggested that chlorophyll biosynthesis in white kale was inhibited by low temperature stress at the step of Pchlide. However, Mg-Proto IX was not inhibited in white kale grown under low temperature conditions. The results of quantitative RT-PCR illustrated that the chlorophyll biosynthetic genes in the white cultivar were dramatically down-regulated by low temperature stress from the step of POR, while CISC and DBB1B in the white cultivar were dramatically induced under low temperature conditions. The results of transmission electron microscopy analysis showed that there were normal chloroplasts in the young leaves of white kale grown at 20 °C, whereas proplastids were observed in white kale grown at 5 °C. These results strongly suggested that low-temperature stress significantly inhibited plastid development in the young leaves of white kale, and repressed chlorophyll biosynthesis at the step of Pchlide by down-regulating the expression of downstream chlorophyll biosynthetic genes, resulting in undifferentiated proplastids and the albino phenotype observed in young leaves. Several genes associated with chlorophyll accumulation were also affected by low temperature conditions in white kale, especially CISC and DBB1B.

A novel member of the AP2/ERF transcription factor family, SlERF5, was identified from a tomato mature leaf cDNA library screen. The complete DNA sequence of SlERF5 encodes a putative 244-amino acid DNA-binding protein which most likely acts as a transcriptional regulator and is a member of the ethylene responsive factor (ERF) superfamily. Analysis of the deduced SlERF5 protein sequence showed that it contained an ERF domain and belonged to the class III group of ERFs proteins. Expression of SlERF5 was induced by abiotic stress, such as high salinity, drought, flooding, wounding and cold temperatures. Over-expression of SlERF5 in transgenic tomato plants resulted in high tolerance to drought and salt stress and increased levels of relative water content compared with wild-type plants. This study indicates that SlERF5 is mainly involved in the responses to abiotic stress in tomato.

The purple kale (Brassica Oleracea var. acephala f. tricolor) is a mutation in kales, giving the mutant phenotype of brilliant purple color in the interior. Total anthocyanin analysis showed that the amount of anthocyanins in the purple kale was up to 1.73 mg g−1 while no anthocyanin was detected in the white kale. To elucidate the molecular mechanism of the anthocyanin biosynthesis in the purple kale, we analyzed the expression of structural genes and some transcription factors associated with anthocyanin biosynthesis in the purple cultivar “Red Dove” and the white cultivar “White Dove”. The result showed that nearly all the anthocyanin biosynthetic genes showed higher expression levels in the purple cultivar than in the white cultivar, especially for DFR and ANS, they were barely detected in the white cultivar. Interestingly, the fact that a R2R3 MYB transcription factor named BoPAP1 was extremely up-regulated in the purple kale and induced by low temperature attracted our attention. Further sequence analysis showed that BoPAP1 shared high similarity with AtPAP1 and BoMYB1. In addition, the anthocyanin accumulation in the purple kale is strongly induced by the low temperature stress. The total anthocyanin contents in the purple kale under low temperature were about 50-fold higher than the plants grown in the greenhouse. The expression of anthocyanin biosynthetic genes C4H, F3H, DFR, ANS and UFGT were all enhanced under the low temperature. These evidences strongly suggest that BoPAP1 may play an important role in activating the anthocyanin structural genes for the abundant anthocyanin accumulation in the purple kale.

The full-length cDNA of LeSGR1 was cloned from tomato by RT-PCR and RACE. The cDNA encoded a protein of 272 amino acid residues and was deposited in GenBank (accession No. DQ100158). Northern analysis suggests that LeSGR1 gene specifically expresses in senescent leaves and mature fruits of tomatoes. Desiccation and flooding induce the expression of LeSGR1 in tomato leaves and stems. Both in ethylene-insensitive mutants (Nr) and ripening inhibitor mutants (rin), the expression of LeSGR1 is markedly decreased compared with that in the wild type. Alignment of the nucleotide sequence of SGR1 cloned from the tomato green flesh (gf) mutant with that from the wild type tomato shows a single nucleotide change leading to an amino acid substitution in gf mutant. Furthermore, LeSGR1 gene silencing by RNA interference results in inhibited chlorophyll degradation similar to the phenotype in gf mutant. Thus, we conclude that LeSGR1 is crucial to chlorophyll degradation and the mutation of SGR1 protein might be responsible for gf tomato properties.

FLOWERING LOCUS C (FLC), which encodes a MADS-box domain protein, is a flowering repressor involved in the key position of Arabidopsis (Arabidopsis thaliana) flowering network. In Brassica species, several FLC homologues are involved in flowering time like Arabidopsis FLC. Here, we report the analysis of splicing variation in BrpFLC1 and the expression of BrpFLC homologues associated with early flowering of Purple Flowering Stalk (Brassica campestris L. ssp. chinensis L. var. purpurea Bailey). It was indicated that a splice site mutation happened in intron 6 with G to A at the 5′ splice site. Three alternative splicing patterns of BrpFLC1, including the entire exon 6 excluded and 24 bp or 87 bp of intron 6 retained, were identified in Purple Flowering Stalk. But there was only one normal splicing pattern in Pakchoi (Brassica campestris ssp. chinensis var. communis). Northern blotting and semi-quantitative RT-PCR revealed that the expression levels of the three FLC homologues in Purple Flowering Stalk were lower than that in Pakchoi. However, the expression levels of downstream genes, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FLOWERING LOCUS T (FT), were higher in Purple Flowering Stalk. These results suggest that a natural splicing site mutation in BrpFLC1 gene and repressed expression of all BrpFLC genes contribute significantly to flowering time variation in Purple Flowering Stalk.

EIN2 (ethylene insensitive 2) is a very important component in the ethylene signal transduction pathway. Recently, the genomic DNA and full-length cDNA of LeEIN2 (tomato EIN2) have been isolated in our laboratory. To reveal the function of LeEIN2, transgenic tomato plants with reduced expression levels of LeEIN2 were produced. The fruit ripening and expressions of ripening-related genes encoding polygalacturonase and TomLoxB were inhibited in the LeEIN2-silenced transgenic plants compared to the wild-type Ailsa Craig. In the seedling ethylene response assay, the transgenic tomato plants with reduced LeEIN2 expression exhibited ethylene insensitivity. These results indicate that LeEIN2 plays a critical role in regulating tomato fruit ripening and is a positive regulator in ethylene signal transduction pathway.

Rhodobacter sphaeroides has been intensively studied and provides an excellent model for studying both photo-synthesis 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 lacIq-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 β-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.

The peripheral light-harvesting complex II (LHII) is an important component of the photosynthetic apparatus of Rhodobacter sphaeroides. In this study, genetic, biochemical, and spectroscopic approaches were applied to investigate the spectral properties and functions of LHII in which two amino acid residues Phe32 and Leu42 in the transmembrane helix domain of pucB-encoded β-apoprotein were replaced by Leu and Pro. The mutated LHII complex showed blue shift of absorbance peaks in the near infrared region at ∼801–845 nm in R. sphaeroides. It should be noted that the B800 peak was much lower than that of the native LHII, and transfer energy was efficient from the B800 to the B850 pigments in the LHII complex. The results suggest that the mutated pucB could be expressed in R. sphaeroides, and the functional LHII was assembled into the membrane of R. sphaeroides notwithstanding with the different spectral properties. These mutated residues were indeed critical for the modulation of characteristics and function of LHII complex.

In this study, three subfamily members of the human 12-transmembrane-domain cell-surface receptors GLUT1, GLUT2, and GLUT3 were heterologously expressed in the fission yeast Schizosaccharomyces pombe utilizing GST-GLUT fusion proteins. These fusion proteins were driven by the full-length nmt1 promoter (Pnmt1) derived from S. pombe. The transcription levels of the GST-GLUT fusion proteins were very high upon induction by removing thiamine from the media. One-step purification of the recombinant fusion proteins was achieved by GST-affinity chromatography. Approximately 300 µg of highly purified fusion protein were obtained from 3 g of wet cell paste (1 liter of cell culture), indicating that human membrane proteins can be efficiently expressed and purified in the fission yeast. With its available extensive genetic information and ease of genetic manipulation, the fission yeast is potentially a highly efficient host to express eukaryotic membrane proteins.

The light harvesting complexes, including LHII and LHI, are the important components of photosynthetic apparatus. Rhodovulum (Rdv.) sulfidophilum and Rhodobacter (R.) sphaeroides belong to two genera of photosynthetic bacteria, and they are very different in some physiological characteristics and light harvesting complexes structure. The LHII structural genes (pucBsAs) from Rdv. sulfidophilum and the LHI structural genes (pufBA) from R. sphaeroides were amplified, and cloned into an expression vector controlled by puc promoter from R. sphaeroides, which was then introduced into LHI and LHII-minus R. sphaeroides mutants; the transconjugant strains synthesized heterologous LHII and native LHI complexes, which played normal roles in R. sphaeroides. The Rdv. sulfidophilum LHII complex from pucBsAs had near-infrared absorption bands at ~801–853 nm in R. sphaeroides, and was able to transfer energy efficiently to the native LHI complex. The results show that the pucBsAs genes from Rdv. sulfidophilum could be expressed in R. sphaeroides, and the functional foreign LHII and native LHI were assembled into the membrane of R. sphaeroides.

Lablab pods, as dietary vegetable, have high nutritional values similar to most of edible legumes. Moreover, our studies confirmed that purple lablab pods contain the natural pigments of anthocyanins and flavonols. Compared to green pods, five kinds of anthocyanins (malvidin, delphinidin and petunidin derivatives) were found in purple pods by HPLC-ESI-MS/MS and the major contents were delphinidin derivatives. Besides, nine kinds of polyphenol derivatives (quercetin, myricetin, kaempferol and apigenin derivatives) were detected by UPLC-ESI-MS/MS and the major components were quercetin and myricetin derivatives. In order to discover their molecular mechanism, expression patterns of biosynthesis and regulatory gens of anthocyanins and flavonols were investigated. Experimental results showed that LpPAL, LpF3H, LpF3′H, LpDFR, LpANS and LpPAP1 expressions were significantly induced in purple pods compared to green ones. Meanwhile, transcripts of LpFLS were more abundant in purple pods than green or yellow ones, suggestind that co-pigments of anthocyanins and flavonols are accumulated in purple pods. Under continuously dark condition, no anthocyanin accumulation was detected in purple pods and transcripts of LpCHS, LpANS, LpFLS and LpPAP1 were remarkably repressed, indicating that anthocyanins and flavonols biosynthesis in purple pods was regulated in light-dependent manner. These results indicate that co-pigments of anthocyanins and flavonols contribute to purple pigmentations of pods.