Characterisation of three ACC synthase gene family members during post-harvest-induced senescence in broccoli (Brassica oleracea L. var. italica)

We investigated the gene expression profiles of different members of the 1-aminocyclopropane-1-carboxilic acid (ACC) synthase (EC 4.4.1.14) gene family in broccoli (Brassica oleracea L. var. italica) during the post-harvest-induced senescence process. Using RT-PCR, three different cDNAs coding for ACC synthase (BROCACS1, BROCACS2 and BROCACS3) were amplified from floret tissue at the start of the senescence process. The three genes share relatively little homology, but have highly homologous sequences in Arabidopsis thaliana, and could be functionally related to these counterparts. Southern analyses suggest that BROCACS1 and BROCACS3 are present as single copy genes, while there are probably two copies of BROCACS2. All three genes showed different expression patterns: BROCACS1 is likely to be either wound - or mechanical stress-induced showing high transcript levels after harvesting, but no detectable expression afterwards. BROCACS2 shows steady expression throughout senescence, increasing at the latest stages, and BROCACS3 is almost undetectable until the final stages. Our results suggest that BROCACS1 could be required to initiate the senescence process, while BROCACS2 would be the main ACC synthase gene involved throughout the post-harvest-induced senescence. BROCACS3's expression pattern indicates that it is not directly involved in the initial stages of senescence, but in the final remobilization of cellular resources.

Distinct cis-elements in the Asparagus officinalis asparagine synthetase promoter respond to carbohydrate and senescence signals

The Asparagus officinalis L. asparagine (Asn) synthetase (AS) promoter was analysed for elements responding to carbohydrate and senescence signals. Transgenic Arabidopsis thaliana L. plants containing deletion constructs of the –1958 bp AS promoter linked to the β-glucuronidase (GUS) reporter gene (AS::GUS) were analysed by measuring GUS specific activity. Inclusion of sucrose (Suc), glucose (Glc) or fructose (Fru) in plant media repressed levels of GUS activity in –1958AS::GUS plants, regardless of the light environment, with increases in GUS found 1 d after incubation on Suc-lacking media. Hexokinase is likely to be involved in the signal pathway, as Suc, Glc, Fru, 2-deoxy-d-glucose and mannose were more effective repressors than 3-O-methylglucose, and the hexokinase inhibitor mannoheptulose reduced repression. Plants containing AS::GUS constructs with deletions that reduced the promoter to less than –405 bp did not show low sugar induction. AS::GUS activity was significantly higher in excised leaves induced to senesce by dark storage for 24 h, compared to fresh leaves, for lines containing at least –640 bp of the AS promoter but not those with –523 bp or smaller promoter fragments. Fusion of the –640 to –523 bp region to a –381AS::GUS construct generated a promoter that retained senescence induction but lacked low sugar induction. Alignment of this region to the 33-bp senescence-related sequence of the Arabidopsis and Brassica napus L. SAG12 promoters identified the sequence TTGCACG as being conserved in all the promoters, and which may be an important senescence-responsive element.

Development and senescence of Grevillea 'Sylvia' inflorescences, flowers and flower parts

To characterise the physiology of development and senescence for Grevillea 'Sylvia'. oral organs, respiration, ethylene production and ACC concentrations in harvested flowers and flower parts were measured. The respiration rate of harvested inflorescences decreased over time during senescence. In contrast, both ethylene production and ACC concentration increased. Individual flowers, either detached from cut inflorescences held in vases at 20degreesC or detached from in planta inflorescences at various stages of development, had similar patterns of change in ACC concentration and rates of respiration and ethylene production as whole inflorescences. The correlation between ACC concentration and ethylene production by individual flowers detached from cut inflorescences held in vases was poor (r(2)=0.03). The isolated complete gynoecium (inclusive of the pedicel) produced increasing amounts of ethylene during development. Further sub-division of flower parts and measurement of their ethylene production at various stages of development revealed that the distal part of the gynoecium (inclusive of the stigma) had the highest rate of ethylene production. In turn, anthers had higher rates of ethylene production and also higher ACC concentrations than the proximal part of the gynoecium (inclusive of the ovary). Rates of ethylene production and ACC concentrations for tepal abscission zone tissue and adjacent central tepal zone tissue were similar. ACC concentration in pollen was similar to that in senescing perianth tissue. Overall, respiration, ethylene and ACC content measurements suggest that senescence of G. 'Sylvia' is non-climacteric in character. Nonetheless, the phytohormone ethylene is produced and evidently mediates normal flower development and non-climacteric senescence processes.

The SEN1 gene of Arabidopsis is regulated by signals that link plant defence responses and senescence

Plant defence and senescence share many similarities as evidenced by extensive co-regulation of many genes during these responses. To better understand the nature of signals that are common to plant defence and senescence, we studied the regulation of SEN1 encoding a senescence-associated protein during plant defence responses in Arabidopsis. Pathogen inoculations and treatments with defence-related chemical signals, salicylic acid and methyl jasmonate induced changes in SEN1 transcript levels. Analysis of transgenic plants expressing the SEN1 promoter fused to uidA reporter gene confirmed the responsiveness of the SEN1 promoter to defence- and senescence-associated signals. Expression analysis of SEN1 in a number of defence signalling mutants indicated that activation of this gene by pathogen occurs predominantly via the salicylic and jasmonic acid signalling pathways, involving the functions of EDS5, NPR1 and JAR1 In addition, in the absence of pathogen challenge, the cpr5/hys1 mutant showed elevated SEN1 expression and displayed an accelerated senescence response following inoculation with the necrotrophic fungal pathogen Fusarhan oxysporum. Although the analysis of the sen1-1 knock-out mutant did not reveal any obvious role for this gene in defence or senescence-associated events, our results presented here show that SEN1 is regulated by signals that link plant defence and senescence responses and thus represents a useful marker gene to study the overlap between these two important physiological events. (c) 2005 Elsevier SAS. All rights reserved.

Systemic gene expression in arabidopsis during an incompatible interaction with Alternaria brassicicola

Pathogen challenge can trigger an integrated set of signal transduction pathways, which ultimately leads to a state of high alert, otherwise known as systemic or induced resistance in tissue remote to the initial infection. Although large-scale gene expression during systemic acquired resistance, which is induced by salicylic acid or necrotizing pathogens has been previously reported using a bacterial pathogen, the nature of systemic defense responses triggered by an incompatible necrotrophic fungal pathogen is not known. We examined transcriptional changes that occur during systemic defense responses in Arabidopsis plants inoculated with the incompatible fungal pathogen Alternaria brassicicola. Substantial changes (2.00-fold and statistically significant) were demonstrated in distal tissue of inoculated plants for 35 genes (25 up-regulated and 10 down-regulated), and expression of a selected subset of systemically expressed genes was confirmed using real-time quantitative polymerase chain reaction. Genes with altered expression in distal tissue included those with putative functions in cellular housekeeping, indicating that plants modify these vital processes to facilitate a coordinated response to pathogen attack. Transcriptional up-regulation of genes encoding enzymes functioning in the beta-oxidation pathway of fatty acids was particularly interesting. Transcriptional up-regulation was also observed for genes involved in cell wall synthesis and modification and genes putatively involved in signal transduction. The results of this study, therefore, confirm the notion that distal tissue of a pathogen-challenged plant has a heightened preparedness for subsequent pathogen attacks.

Effects of 6-benzylaminopurine treatments on the longevity of harvested Grevillea 'Sylvia' inflorescences

Exogenous treatments with cytokinins, such as 6-benzylaminopurine (BA), can delay senescence of some plant tissues. Grevillea 'Sylvia' inflorescences have a short vase life. BA supplied in vase solutions at up to 0.1 mM did not delay senescence of G. 'Sylvia' in florescences. However, BA applied by dipping at concentrations up to 10 mM extended their vase life (longevity). Senescence parameters of relative fresh weight, flower abscission, flower opening, flower discolouration and flower wilting were all suppressed by BA dips. Dip treatment with BA (1 mM) was effective on G. 'Sylvia' in florescences at three different maturity stages.

Effects of vase solution pH and abscisic acid on the longevity of cut 'Baccara' roses

Abscisic acid (ABA) supplied in the vase solution can induce stomatal closure in the leaves of cut flowers, including roses (Rosa hybrida L.). This effect may be beneficial in reducing water deficit stress. Extracellular pH can affect active ABA concentrations in the apoplast of guard cells, with sap alkalisation enhancing the physiological activity of ABA. Accordingly, it was hypothesized that vase solution pH may affect ABA-mediated stomatal closure of cut roses. Two experiments were conducted to study the interaction of vase solution pH and ABA. In the first, cut 'Baccara' roses were held in vase solutions with +/- 10(-5) M ABA at pH 6, pH 7 and pH 8. In the second experiment, roses were held with +/- 10(-5) M ABA at pH 6 and pH 8 in the presence and absence of 1 mg l(-1) AgNO3 as a bactericide. Supply of ABA increased vase life and reduced vase solution usage of flowers held in low pH 6 solutions, indicating induction of stomatal closure. Conversely, ABA supplied at pH 8 was associated with reduced vase life. This negative result was associated with enhanced development of vase solution microbes at high pH, which overrode any potential pH-mediated ABA efficacy effects.

Herpesvirus-specific CD8 T cell immunity in old age: Cytomegalovirus impairs the response to a coresident EBV infection

Aging in humans is associated with increased infections and the reduced proliferative capacity of T cells, part of the more global phenomenon termed immune senescence. The etiology of immune senescence is unknown but the accumulation of virus-specific memory T cells may be a contributory factor. We have examined CD8 T cell responses to two persistent herpesvirus infections, CMV and EBV, and to a recurrent virus infection, influenza, in different age cohorts of healthy donors using HLA-peptide tetramers and intracellular cytokine detection. Of these, CMV appears to be the most immunogenic, with the CD8 T cell response representing over 10% of the CD8 pool in many elderly donors. Interestingly, the effect of age upon EBV-specific responses depends upon donor CMV sero-status. In CMV seropositive donors, the magnitude of the EBV-specific immune response is stable with age, but in CMV seronegative donors, the response to EBV increases significantly with age. By contrast, the influenza-specific CD8 T cell immune response decreases with age, independent of CMV status. The functional activity of the herpesvirus-specific immune response decreases in elderly donors, although the characteristic phenotypes of CMV- and EBV-specific memory populations are retained. This demonstrates that aging is associated with a marked accumulation of CMV-specific CD8 T cells together with a decrease in immediate effector function. Moreover, infection with CMV can reduce prevailing levels of immunity to EBV, another persistent virus. These results suggest that carriage of CMV may be detrimental to the immunocompetent host by suppressing heterologous virus-specific immunity during aging.

Etr1-1 gene expression alters regeneration patterns in transgenic lettuce stimulating root formation

We have evaluated the transformation efficiency of two lettuce ( Lactuca sativa L.) cultivars, LE126 and Seagreen, using Agrobacterium tumefaciens- mediated gene transfer. Six- day- old cotyledons were co- cultivated with Agrobacterium cultures carrying binary vectors with two different genetic constructs. The first construct contained the beta- glucuronidase gene ( GUS) under the control of the cauliflower mosaic virus 35S promoter ( CaMV 35S), while the second construct contained the ethylene mutant receptor etr1- 1, which confers ethylene insensitivity, under the control of a leaf senescence- specific promoter ( sag12). Tissues co- cultivated with the GUS construct showed strong regeneration potential with over 90% of explants developing callus masses and 85% of the calli developing shoots. Histochemical GUS assays showed that 85.7% of the plants recovered were transgenic. Very different results were observed when cotyledon explants were co- cultivated with Agrobacteria carrying the etr1- 1 gene. There was a dramatic effect on the regeneration properties of the cultured explants with root formation taking place directly from the cotyledon tissue in 34% of the explants and no callus or shoots observed initially. Eventually callus formed in 10% of cotyledons and some organogenic shoots were obtained ( 2.86%). These results indicate that the ethylene insensitivity conferred by the etr1- 1 gene alters the normal pattern of regeneration in lettuce cotyledons, inhibiting the formation of shoots and stimulating root formation during regeneration.

Cell death and degeneration in the symbiotic dinoflagellates of the coral Stylophora pistillata during bleaching

Rising sea temperatures are increasing the incidences of mass coral bleaching (the dissociation of the coral-algal symbiosis) and coral mortality. In this study, the effects of bleaching (induced by elevated light and temperature) on the condition of symbiotic dinoflagellates (Symbiodinium sp.) within the tissue of the hard coral Stylophora pistillata (Esper) were assessed using a suite of techniques. Bleaching of S. pistillata was accompanied by declines in the maximum potential quantum yield of photosynthesis (F-v/F-m, measured using pulse amplitude modulated [PAM] fluorometry), an increase in the number of Sytox-green-stained algae (indicating compromised algal membrane integrity and cell death), an increase in 2',7'-dichlorodihydrofluroscein diacetate (H(2)DCFDA)stained algae (indicating increased oxidative stress), as well as ultrastructural changes (vacuolisation, losses of chlorophyll, and an increase in accumulation bodies). Algae expelled from S. pistillata exhibited a complete disorganisation of cellular contents; expelled cells contained only amorphous material. In situ samples taken during a natural mass coral bleaching event on the Great Barrier Reef in February 2002 also revealed a high number of Sytox-labelled algae cells in symbio. Dinoflagellate degeneration during bleaching seems to be similar to the changes resulting from senescence-phase cell death in cultured algae. These data support a role for oxidative stress in the mechanism of coral bleaching and highlight the importance of algal degeneration during the bleaching of a reef coral.

Hybrid 'Sinta' papaya exhibits unique ACC synthase 1 cDNA isoforms

Five ripening-related ACC synthase cDNA isoforms were cloned from 80% ripe papaya cv. 'Sinta' by reverse transcription-PCR using gene-specific primers. Clone 2 had the longest transcript and contained all common exons and three alternative exons. Clones 3 and 4 contained common exons and one alternative exon each, while clone 1, the most common transcript, contained only the common exons. Clone 5 could be due to cloning artifacts and might not be a unique cDNA fragment. Thus, there are only four isoforms of ACC synthase mRNA. Southern blot analysis indicates that all five clones came from only one gene existing as a single copy in the 'Sinta' papaya genome. Multiple sequence alignment indicates that the four isoforms arise from a single gene, possibly through alternative splicing mechanisms. All the putative alternative exons were present at the 5'-end of the gene comprising the N-terminal region of the protein. 'Sinta' ACC synthase cDNAs were of the capacs 1 type and are most closely related to a 1.4 kb capacs 1-type DNA (AJ277160) from Eksotika papaya. No capacs 2-type cDNAs were cloned from 'Sinta' by RT-PCR. This is the first report of possible alternative splicing mechanism in ripening-related ACC synthase genes in hybrid papaya, possibly to modulate or fine-tune gene expression relevant to fruit ripening.

RNA interference against human papillomavirus oncogenes in cervical cancer cells results in increased sensitivity to cisplatin

Targeted inhibition of oncogenes in tumor cells is a rational approach toward the development of cancer therapies based on RNA interference (RNAi). Tumors caused by human papillomavirus (HPV) infection are an ideal model system for RNAi-based cancer therapies because the oncogenes that cause cervical cancer, E6 and E7, are expressed only in cancerous cells. We investigated whether targeting HPV E6 and E7 oncogenes yields cancer cells more sensitive to chemotherapy by cisplatin, the chemotherapeutic agent currently used for the treatment of advanced cervical cancer. We have designed siRNAs directed against the HPV E6 oncogene that simultaneously targets both E6 and E7, which results in an 80% reduction in E7 protein and reactivation of the p53 pathway. The loss of E6 and E7 resulted in a reduction in cellular viability concurrent with the induction of cellular senescence. Interference was specific in that no effect on HPV-negative cells was observed. We demonstrate that RNAi against E6 and E7 oncogenes enhances the chemotherapeutic effect of cisplatin in HeLa cells. The IC50 for HeLa cells treated with cisplatin was 9.4 mu M, but after the addition of a lentivirus-delivered shRNA against E6, the IC50 was reduced almost 4-fold to 2.4 mu M. We also observed a decrease in E7 expression with a concurrent increase in p53 protein levels upon cotreatment with shRNA and cisplatin over that seen with individual treatment alone. Our results provide strong evidence that loss of E6 and E7 results in increased sensitivity to cisplatin, probably because of increased p53 levels.

Methyl jasmonate vapour treatment suppresses specking caused by Botrytis cinerea on cut Freesia hybrida L. flowers

Treatment of cut freesia var. Cote d'Azur flowers with methyl jasmonate (MeJA, 0.1 mu l MeJA l(-1)) vapour suppressed petal specking caused by Botrytis cinerea infection. MeJA efficacy was concentration and incubation temperature dependent. Disease severity, lesion numbers and lesion diameters decreased with increasing MeJA concentration from 0.025 to 0.1 mu l MeJA l(-1). However, there were no significant (P > 0.05) differences among MeJA concentrations examined. MeJA was more effective in reducing B. cinerea flower specking at 20 degrees C than at 12 degrees C. MeJA treatment was ineffective at 5 degrees C. At 20 degrees C, MeJA treatment at 0.1 mu l MeJA l(-1) reduced disease severity, lesion numbers and lesion diameters by 58, 50 and 48%, respectively, as compared to untreated controls. In a repeat experiment, disease severity, lesion numbers and lesion diameters on MeJA vapour treated flowers after 12 h of incubation were reduced by 68, 56 and 50%, respectively. MeJA did not exert direct antifungal activity in-vitro, suggesting that treatment in-vivo reduced B. cinerea-induced flower specking by induction of host defence responses. MeJA at 0.1 mu l MeJA l(-1) significantly (P < 0.05) increased vase life of cut freesia flowers and delayed senescence judged by lower wilt scores and higher fresh weights as compared to untreated controls. (c) 2005 Elsevier B.V. All rights reserved.

Coconut (Cocos nucifera) in vitro ecology: Modifications of headspace and medium additives can optimize somatic embryogenesis

Of those explants tested, immature zygotic embryo tissues proved to be the best for initiating callus with potential for somatic embryogenesis. Slicing of this tissue and use of the central sections (near to and including the meristematic tissue) gave the best embryogenic response. Slices that were placed under illumination necrosed more rapidly and to a greater degree than those incubated in the dark. Explant slice necrosis could be prevented or severely retarded by the addition of activated charcoal into the medium. Washing the explants for short periods of time prior to culture was also found to improve callus production. Prolonged washing resulted in low rates of callus production. In an attempt to prevent ethylene accumulation in the culture vessel headspace, AVG, an ethylene biosynthesis inhibitor and STS, a chemical which reduces the physiological action of ethylene, were successfully used to promote somatic embryogenesis. Spermidine, putrescine and spermine, polyamines that are known to delay plant senescence and promote somatic embryogenesis in some plant species, enhanced the rate of somatic embryogenesis when they were introduced into the callus induction medium. The use of polyethylene glycol in combination with abscisic acid helped promote somatic embryo formation and maturation as well as the subsequent formation of plantlets. The use of all of these improvements together has created a new and improved protocol for coconut somatic embryogenesis. This new protocol puts significant emphasis on improving the in vitro ecology of the explant, callus and somatic embryogenic tissues.