The effect of endogenous mRNA levels on co-suppression in tomato

AbstractIntroduction of truncated polygalacturonase (PG) transgenes into tomato plants caused the production of small interfering RNAs (siRNAs) and co-suppression of both the endogenous and PG transgenes in ripening fruits by post-transcriptional gene silencing. In order to test the possible effect on co-suppression of the endogenous PG mRNA level, we transferred the PG transgenes from a PG-silenced line (wild type background) by crossing to two ripening regulatory mutants with reduced PG: Never-ripe (Nr, ∼10% endogenous PG mRNA compared to wild type) and ripening-inhibitor (rin, ∼1% endogenous PG mRNA) and to wild type (as a control). The PG transgenes caused strong co-suppression of the transgenes and the endogenous PG gene in cells with high PG mRNA background (wild type) and silencing appeared to be linked with higher transgene copy number and/or a particular transgene locus. In cells with low endogenous PG mRNA accumulation (Nr), the endogenous PG gene was preferentially suppressed compared to the transgenes, whose expression was not reduced significantly. The expression of the transgenes was also not reduced in the very low PG background (rin), in which endogenous PG was barely detectable. In all the analysed lines with all three PG background levels, siRNAs accumulated in leaves and green fruits, in which the endogenous PG gene is not transcribed. The relatively abundant production of siRNAs in most of the lines was linked with a particular transgene insert. These results suggest that a certain threshold level of endogenous PG mRNA is required for the co-suppression of the truncated PG transgenes and the endogenous PG gene or for extensive silencing of the transgenes

AtTRP1 encodes a novel TPR protein that interacts with the ethylene receptor ERS1 and modulates development in Arabidopsis

Arabidopsis AtTRP1 is an orthologue of SlTPR1, a tomato tetratricopeptide repeat protein that interacts with the tomato ethylene receptors LeETR1 and NR in yeast 2-hybrid assays and in vitro, and modulates plant development. AtTRP1 is encoded by a single copy gene in the Arabidopsis genome, and is related to TCC1, a human protein that competes with Raf-1 for Ras binding, and distantly related to the immunophilin-like FK-binding proteins TWD1 and PAS1. The former is involved in auxin transport and the latter is translocated to the nucleus in response to auxin. AtTRP1 interacted preferentially with the Arabidopsis ethylene receptor ERS1 in yeast two-hybrid assays. This association was confirmed by in vivo co-immunoprecipitation. AtTRP1 promoter–GUS was highly expressed in vascular tissue, mature anthers, the abscission zone, and was induced by ACC. Overexpression of AtTRP1 in wild-type Arabidopsis resulted in dwarf plants with reduced fertility, altered leaf/silique morphology, and enhanced expression of the ethylene responsive gene AtChitB. Exogenous GA did not reverse the dwarf habit. Etiolated transgenic seedlings overexpressing AtTRP1 displayed enhanced sensitivity to low ACC and this was correlated with the transgene expression. Seedlings overexpressing AtTRP1 at high levels exhibited shortened and swollen hypocotyls, inhibited root growth, and an altered apical hook. Plants overexpressing AtTRP1 also showed a reduced response to exogenous IAA and altered expression of a subset of auxin early responsive genes. These results indicated that overexpression of AtTRP1 affects cross-talk between ethylene and auxin signalling and enhances some ethylene responses and alters some auxin responses. A model for AtTRP1 action is proposed

Improved plant transformation vectors for fluorescent protein tagging

Fluorescent protein labelling technologies enable dynamic protein actions to be imaged in living cells and can also be used in conjunction with other methods such as Forster resonance energy transfer and biomolecular fluorescence complementation. In this report, we describe the generation of a series of 23 novel GATEWAY-compatible vectors based on pGreenII and pDH51 backbones with the latest fluorescent protein tags (Cerulean, EGFP and Venus) and the choice of three in planta selection markers. These vectors can be obtained from the Nottingham Arabidopsis Stock Centre (N9819-N9846) and should be a powerful tool box for transgenic research in plants

Altered patterns of senescence and ripening in gf, a stay-green mutant of tomato (Lycopersicon esculentum Mill.)

The gf tomato mutant, which retains chlorophyll during ripening, has been found to be affected in leaf senescence. The leaves of the gf mutant show an absolute stay-green phenotype. As leaf senescence and fruit ripening proceed, there is a marked difference in chlorophyll content between wild-type and gf. In both attached and detached leaf studies, or after treatment with ethylene, the leaves withered and abscised in gf with only slight loss of chlorophyll and carotenoids. Total protein content declined and free amino acids increased during leaf senescence in wild-type and gf, but Western analysis showed that LHCII polypeptides were retained at higher levels in gf. Expression of senescence-related mRNAs increased normally in gf whereas those for cab, rbcS and rbcL declined in both mutant and wild-type. The mutant possesses enzyme activity for chlorophyllase, the formation of phaeophorbide a by the action of Mg-dechelatase and the oxygenolytic opening of the porphyrin macrocycle. Analysis of chlorophyll breakdown products in fruit indicated that gf, like other stay-green mutants, accumulates chlorophyllides a and b, but phaeophorbide a does not accumulate in vivo. This may indicate that, in the mutant, in vivo the action of phaeophorbide a-oxygenase is somehow prevented, either by altered accessibility or transport of components required for thylakoid disassembly or the absence of another facto

Characterization of two cDNA clones for mRNAs expressed during ripening of melon (Cucumis melo L.) fruits

In vitro translation of mRNAs and polyacrylamide gel electrophoresis of proteins from melons revealed that several mRNAs increased in amount during ripening, indicating the existence of other ripening genes in addition to those cloned previously. To identify ripening-related genes we have screened a ripe melon cDNA library and isolated two novel cDNA clones (MEL2 and MEL7) encoding unidentified proteins. Southern analysis revealed that MEL2 and MEL7 are encoded by low-copy-number genes. The MEL2 cDNA clone is near full-length, corresponds to a 1600 nucleotide mRNA that accumulates during ripening and encodes a predicted protein rich in hydrophobic amino acids. The MEL7 cDNA clone is full-length, corresponds to a mRNA of 0.7 kb which accumulates during early ripening stages and is also present at low levels in other organs of the melon plant. The MEL7 predicted polypeptide is 17 kDa and shows significant homology with the major latex protein from opium-poppy. Wounding and ethylene treatment of unripe melon fruits 20 days after anthesis showed that MEL2 and MEL7 mRNAs are only induced by ethylene.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43441/1/11103_2004_Article_123153.pd

SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development

The gaseous hormone ethylene is perceived by a family of ethylene receptors which interact with the Raf-like kinase CTR1. SlTPR1 encodes a novel TPR (tetratricopeptide repeat) protein from tomato that interacts with the ethylene receptors NR and LeETR1 in yeast two-hybrid and in vitro protein interaction assays. SlTPR1 protein with a GFP fluorescent tag was localized in the plasmalemma and nuclear membrane in Arabidopsis, and SlTPR1-CFP and NR-YFP fusion proteins were co-localized in the plasmalemma and nuclear membrane following co-bombardment of onion cells. Overexpression of SlTPR1 in tomato resulted in ethylene-related pleiotropic effects including reduced stature, delayed and reduced production of inflorescences, abnormal and infertile flowers with degenerate styles and pollen, epinasty, reduced apical dominance, inhibition of abscission, altered leaf morphology, and parthenocarpic fruit. Similar phenotypes were seen in Arabidopsis overexpressing SlTPR1. SlTPR1 overexpression did not increase ethylene production but caused enhanced accumulation of mRNA from the ethylene responsive gene ChitB and the auxin-responsive gene SlSAUR1-like, and reduced expression of the auxin early responsive gene LeIAA9, which is known to be inhibited by ethylene and to be associated with parthenocarpy. Cuttings from the SlTPR1-overexpressors produced fewer adventitious roots and were less responsive to indole butyric acid. It is suggested that SlTPR1 overexpression enhances a subset of ethylene and auxin responses by interacting with specific ethylene receptors. SlTPR1 shares features with human TTC1, which interacts with heterotrimeric G-proteins and Ras, and competes with Raf-1 for Ras binding. Models for SlTPR1 action are proposed involving modulation of ethylene signalling or receptor levels

THE ANTIBACTERIAL ACTIVITY OF CLAUSENA ANISATA HOOK, A SOUTH AFRICAN MEDICINAL PLANT

Background: Clausena anisata Hook also known as Iperepesi in Xhosa language is a medicinal plant used traditionally for the treatment of various ailments and some opportunistic infections associated with tuberculosis (TB). Patients in South Africa based on the phytotherapeutic information on this species in the Eastern Cape, use this medicinal plant. Hence, the antibacterial activity of various solvent extracts of the leaves and barks were respectively, evaluated using selected bacterial strains. Method: The leaves and stem bark were tested against 10 selected strains of Gram - positive and Gram - negative bacteria through the agar dilution method. Acetone, dichloromethane and water extracts were used for the extraction. MIC was determined at different concentrations (0.1mg/ml, 0.5mg/ml, 1mg/ml and 5mg/ml) and the results obtained were compared to that of standard antibiotics. Result: The acetone extract of the leaves were more active against both Gram-positive and Gram –negative bacteria with MIC ranging from 0.1 mg/ml - 0.5 mg / ml. The dichloromethane extract of the bark showed appreciable activities against Staphylococcus aureus (ATCC 6538) (MIC: 0.1mg /ml) Escherichia coli and, Streptococcus pyogenes with an MIC of 5mg/ml respectively. On the other hand, the aqueous extract of the leaves showed no activity against the tested organisms with the exception of the aqueous bark extract which inhibited Staphylococcus aureus (MIC: 0.5mg/ml) and Pseudomonas aeruginosa (MIC: 5mg/ml). Conclusion: This study confirmed the antibacterial activities of acetone extract of the leaves of Clausena anisata. The capability of this extract to inhibit both Gram positive and negative bacteria is an indication that the extract is a potential broad spectrum antibacterial. The result of this study further justified its indigenous use for the treatment of bacteria commonly associated with TB especially among the people of Nkonkobe Municipality

PHYTOCHEMICAL AND ANTIOXIDANT INVESTIGATIONS OF A CLAUSENA ANISATA HOOK, A SOUTH AFRICAN MEDICINAL PLANT

Background: Clausena anisata (Willd). Hook (Rutaceae), also known as Iperepesi in Xhosa language is a medicinal plant widely used by herbalists for the treatment and/or management of several ailments such as chronic cough, tuberculosis and lung ulceration in Eastern Cape, South Africa. With reference to the information gathered in our previous study, we investigated the plant’s phyto-constituents, as well as its inhibitory effects using aqueous and two different organic solvent of extractions in order to justify its folkloric usage. Methods: Antioxidant activity of the plant was screened through 1,1- diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzthiazoline-6- sulfonic acid) (ABTS) diammonium salt, nitric oxide (NO), and ferric reducing power. Total phenols, flavonoids, flavonols, proanthocyanidins, tannins, alkaloids and saponins were investigated using spectroscopic techniques. Results: There were no significant differences in the flavonoid and proanthocyanidins contents between the leaves and bark extracts of C. anisata respectively, while the total phenolic content of the bark extract of C. anisata was significantly higher than that of the C. anisata leaf. The acetone extracts of both the leaf and bark indicated strong antioxidant activities. Conclusion: The observed activities of the plant extracts could be attributed to the high contents of the phenolics, alkaloids, flavonoids, saponins, proanthocyanidins and tannin. The acetone extracts of the plants have also exhibited strong antioxidant activities in vitro. It has been established scientifically that oxidative stress is linked with several degenerative conditions and diseases; the inhibitory effects of these plant extracts on the free radicals could logically justify the folkloric usage of C. anisata leaf and bark in the Eastern Cape for the treatment of respiratory infection diseases

Cloning and characterization of tomato leaf senescence-related cDNAs

Senescence-related cDNA clones designated SENU1, 4, 5 (senescence up-regulated) and SEND32, 33, 34, 35 and 36 (senescence down-regulated) isolated from a tomato leaf cDNA library [9] were characterized. Southern analysis showed that SEND32 is encoded by a single-copy gene while SEND33, 34, 35, 36 and SENU1 and SENU5 are members of small gene families. DNA and protein database searches revealed that SEND32, SEND35, SENU1 and SENU5 are novel cDNAs of unknown function. SEND33 encodes ferredoxin, SEND34 encodes a photosystem II 10 kDa polypeptide and SEND36 encodes catalase. The SENU4 sequence is identical to the P6 tomato protein previously reported to be pathogenesis-related [46]. The mRNA levels of SENU1, 4 and 5 increased during leaf senescence and SENU1 and SENU5 were also expressed at high levels during leaf development and in other plant organs. The SENU4 mRNA was associated more specifically with leaf senescence, although low expression was also detected in green fruit. The mRNAs for all SEND clones decreased during tomato leaf development and senescence and all except SEND32 were expressed at low levels in other plant organs. The accumulation of mRNA homologous to SENU4 and the decrease in abundance of SEND32 provide good molecular markers for leaf senescence.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43443/1/11103_2004_Article_125247.pd

Plastid structure and carotenogenic gene expression in red- and white-fleshed loquat (Eriobotrya japonica) fruits

Loquat (Eriobotrya japonica Lindl.) can be sorted into red- and white-fleshed cultivars. The flesh of Luoyangqing (LYQ, red-fleshed) appears red-orange because of a high content of carotenoids while the flesh of Baisha (BS, white-fleshed) appears ivory white due to a lack of carotenoid accumulation. The carotenoid content in the peel and flesh of LYQ was approximately 68 μg g−1 and 13 μg g−1 fresh weight (FW), respectively, and for BS 19 μg g−1 and 0.27 μg g−1 FW. The mRNA levels of 15 carotenogenesis-related genes were analysed during fruit development and ripening. After the breaker stage (S4), the mRNA levels of phytoene synthase 1 (PSY1) and chromoplast-specific lycopene β-cyclase (CYCB) were higher in the peel, and CYCB and β-carotene hydroxylase (BCH) mRNAs were higher in the flesh of LYQ, compared with BS. Plastid morphogenesis during fruit ripening was also studied. The ultrastructure of plastids in the peel of BS changed less than in LYQ during fruit development. Two different chromoplast shapes were observed in the cells of LYQ peel and flesh at the fully ripe stage. Carotenoids were incorporated in the globules in chromoplasts of LYQ and BS peel but were in a crystalline form in the chromoplasts of LYQ flesh. However, no chromoplast structure was found in the cells of fully ripe BS fruit flesh. The mRNA level of plastid lipid-associated protein (PAP) in the peel and flesh of LYQ was over five times higher than in BS peel and flesh. In conclusion, the lower carotenoid content in BS fruit was associated with the lower mRNA levels of PSY1, CYCB, and BCH; however, the failure to develop normal chromoplasts in BS flesh is the most convincing explanation for the lack of carotenoid accumulation. The expression of PAP was well correlated with chromoplast numbers and carotenoid accumulation, suggesting its possible role in chromoplast biogenesis or interconversion of loquat fruit