Later Stages Of Ripening Research Articles

Expression profiles of the MdACS3 gene suggest a function as an accelerator of apple ( Malus × domestica) fruit ripening

Ethylene plays an important role in apple fruit development and its biosynthesis is catalyzed by the two enzymes ACS (1-aminocyclopropane-1-carboxylate synthase) and ACO (1-aminocyclopropane-1-carboxylic acid oxidase). Within the apple ACS gene family, at least two members, MdACS1 and MdACS3, are expressed in apple fruit tissues. While MdACS1 expresses only at late ripening stages corresponding to a sudden increase of ethylene production, a typical feature of system-II ethylene biosynthesis, the expression of MdACS3 can be detected as early as 6 weeks before physiological maturity. The objective of this study was to characterize the cultivar-specific dynamics of MdACS3 expression at both preharvest and postharvest stages, its relationship with MdACS1 activation, and its roles in apple fruit ripening and quality. Based on the transcript profiles during 8-week on-tree maturation and ripening, two MdACS3 expression patterns could be clustered among 12 apple cultivars. Most of the cultivars in pattern 1 showed high level expression with a steadily increasing trend, and most of those in pattern 2 exhibited low level expression with a transient peak at or before physiological maturity. These two expression patterns appeared to correlate with fruit ripening season and fruit firmness change during ripening. Unlike MdACS1, the expression of MdACS3 was stimulated by 1-MCP treatment, indicating a negative feedback regulation mechanism.

Study of 'Redhaven' peach and its white-fleshed mutant suggests a key role of CCD4 carotenoid dioxygenase in carotenoid and norisoprenoid volatile metabolism.

BackgroundCarotenoids are plant metabolites which are not only essential in photosynthesis but also important quality factors in determining the pigmentation and aroma of flowers and fruits. To investigate the regulation of carotenoid metabolism, as related to norisoprenoids and other volatile compounds in peach (Prunus persica L. Batsch.), and the role of carotenoid dioxygenases in determining differences in flesh color phenotype and volatile composition, the expression patterns of relevant carotenoid genes and metabolites were studied during fruit development along with volatile compound content. Two contrasted cultivars, the yellow-fleshed 'Redhaven' (RH) and its white-fleshed mutant 'Redhaven Bianca' (RHB) were examined.ResultsThe two genotypes displayed marked differences in the accumulation of carotenoid pigments in mesocarp tissues. Lower carotenoid levels and higher levels of norisoprenoid volatiles were observed in RHB, which might be explained by differential activity of carotenoid cleavage dioxygenase (CCD) enzymes. In fact, the ccd4 transcript levels were dramatically higher at late ripening stages in RHB with respect to RH. The two genotypes also showed differences in the expression patterns of several carotenoid and isoprenoid transcripts, compatible with a feed-back regulation of these transcripts. Abamine SG - an inhibitor of CCD enzymes - decreased the levels of both isoprenoid and non-isoprenoid volatiles in RHB fruits, indicating a complex regulation of volatile production.ConclusionsDifferential expression of ccd4 is likely to be the major determinant in the accumulation of carotenoids and carotenoid-derived volatiles in peach fruit flesh. More in general, dioxygenases appear to be key factors controlling volatile composition in peach fruit, since abamine SG-treated 'Redhaven Bianca' fruits had strongly reduced levels of norisoprenoids and other volatile classes. Comparative functional studies of peach carotenoid cleavage enzymes are required to fully elucidate their role in peach fruit pigmentation and aroma.

Quantitative trait loci for seed dormancy in rice

Seed dormancy (SD) is controlled by its own complicated genetic factors and environmental factors. SD is an important trait affecting grain yield and quality in cereal crops. A population comprising 240 recombinant inbred lines (RIL) was used for detecting quantitative trait locus (QTL) for SD in rice. To minimize the effect of environment, data for lines for which the optimum temperature during the late ripening stage is either below 20°C or above 30°C were excluded from the analysis, which left 185 lines. In a dynamic germination test of the parents of the population, Minghui 63 showed clear SD and Zhenshan 97 showed none. The seeds of each RIL, harvested 32 days after heading, were divided into two lots: seeds in one lot were sown immediately, without any treatment to break their dormancy, whereas seeds in the other lot were sown after they had been exposed to dry heat (50°C) for 72 h. Composite interval mapping showed the presence of qDGE1, qDGR5a, qDGR5b, and qDGR7 in the first lot whereas only qDGR7 was detected in the second lot––which had been treated to break SD––indicating the strong influence of qDGR7 in controlling SD. A recently cloned Sdr4 is also located in the qDGR7 region. Coincidently, three rice homologues of Arabidopsis SD gene DOG1 were found in qDGE1 and qDGR5b regions where no SD QTL had been reported so far. These results indicate that the QTL found in this study are reliable, and that it would be worthwhile to clone qDGE1 and qDGR5b.

Enhanced polyamine accumulation alters carotenoid metabolism at the transcriptional level in tomato fruit over-expressing spermidine synthase

Polyamines are involved in crucial plant physiological events, but their roles in fruit development remain unclear. We generated transgenic tomato plants that show a 1.5- to 2-fold increase in polyamine content by over-expressing the spermidine synthase gene, which encodes a key enzyme for polyamine biosynthesis. Pericarp-columella and placental tissue from transgenic tomato fruits were subjected to 1H-nuclear magnetic resonance (NMR) for untargeted metabolic profiling and high-performance liquid chromatography-diode array detection for carotenoid profiling to determine the effects of high levels of polyamine accumulation on tomato fruit metabolism. A principal component analysis of the quantitative 1H NMR data from immature green to red ripe fruit showed a clear discrimination between developmental stages, especially during ripening. Quantification of 37 metabolites in pericarp-columella and 41 metabolites in placenta tissues revealed distinct metabolic profiles between the wild type and transgenic lines, particularly at the late ripening stages. Notably, the transgenic tomato fruits also showed an increase in carotenoid accumulation, especially in lycopene (1.3- to 2.2-fold), and increased ethylene production (1.2- to 1.6-fold) compared to wild-type fruits. Genes responsible for lycopene biosynthesis, including phytoene synthase, phytoene desaturase, and deoxy- d-xylulose 5-phosphate synthase, were significantly up-regulated in ripe transgenic fruits, whereas genes involved in lycopene degradation, including lycopene-epsilon cyclase and lycopene beta cyclase, were down-regulated in the transgenic fruits compared to the wild type. These results suggest that a high level of accumulation of polyamines in the tomato regulates the steady-state level of transcription of genes responsible for the lycopene metabolic pathway, which results in a higher accumulation of lycopene in the fruit.

Expression of Genes Associated with Aroma Formation Derived from the Fatty Acid Pathway during Peach Fruit Ripening

Changes in characteristic aroma volatiles, levels of fatty acids as aroma precursors, and expression patterns of related genes, including lipoxygenase (LOX), hydroperoxide lyase (HPL), alcohol dehydrogenase (ADH), alcohol acyltransferase (AAT), and fatty acid desaturase (FAD), were studied in peach ( Prunus persica L. Batsch., cv. Yulu) fruit during postharvest ripening at 20 degrees C. Concentrations of n-hexanal, (E)-2-hexenal, (E)-2-hexenol, and (Z)-3-hexenol decreased, whereas the production of (Z)-3-hexenyl acetate, gamma-hexalactone, gamma-octalactone, gamma-decalactone, and delta-decalactone increased with fruit ripening. Lactones showed a clear pattern concomitant with the climacteric rise in ethylene production, with gamma-decalactone being the principal volatile compound at the late ripening stage. Of the LOX family genes, PpLOX2 and PpLOX3 had relatively high transcript levels initially followed by a decline with fruit ripening, while levels of PpLOX1 and PpLOX4 transcripts were upregulated by accumulated ethylene production. Expression of PpHPL1, PpADH1, PpADH2, and PpADH3 showed similar decreasing patterns during ripening. Expression levels of PpAAT1 showed a rapid increase during the first 2 days of postharvest ripening followed by a gradual decrease. Contents of polyunsaturated linoleic and linolenic acids increased, and saturated palmitic acid levels tended to decline as the fruit ripened. The increased levels of unsaturated fatty acids closely paralleled increasing expression of PpFAD1 and PpFAD2. The significance of gene expression changes in relation to aroma volatile production is discussed.

Effect of prolonged root hypoxia on the antioxidant content of tomato fruit

Tomato fruit quality depends on its metabolite content, which in turn is determined by numerous metabolic changes occurring during fruit development and ripening. The aim of this work was to investigate whether flooding affects the nutritional quality of tomato fruit, focusing on compounds essential to human health: carotenoids and ascorbate. To this end, tomato plants ( Solanum lycopersicum L. cv Micro-Tom) were submitted to prolonged root hypoxia (1–2% O 2) at first flower anthesis. Fruits were harvested at five stages of the ripening process and analysed for their carotenoid and ascorbate contents. Our results showed that the ripening of fruits that developed on hypoxia treated plants was not inhibited. However, root hypoxia significantly limits carotenoid and ascorbate accumulation in pericarp during fruit ripening, the strongest effects being observed at late stages of ripening. Limitation of both carotenoids and ascorbate accumulation seems to be primarily mediated by the reduced level of expression of genes of the corresponding metabolic pathway.

Isolation of a Polygalacturonase Gene from Citrus sinensis Fruit and Its Expression Relative to Fruit Mastication Trait, Fruit Development, and Calcium or Boron Treatments

Polygalacturonase (PG) plays a significant role in pectin solubilization and depolymerization in vivo and is involved in numerous plant developmental processes. In this study, a polygalacturonase gene (CitPG, EF185420) was isolated from Citrus sinensis. It is 1,686 bp with a putative open reading frame of 1,338 bp encoding 445 amino acids. Phylogenetic analysis showed that CitPG belongs to clade B which includes PGs expressed in fruits or dehiscence zones. CitPG expression profiles during fruit ripening were compared between two pairs of varieties which differ in their mastication traits: C. sinensis cv. Fengjiewancheng (FJWC) vs. C. sinensis cv. Fengjie 72-1(FJ72-1) and Citrus reticulata cv. Kinokuni (NM) vs. C. reticulata cv. Miguang (MG), in which the mastication trait of “FJWC” and “NM” are inferior to their respective partner cultivars. Results indicated that the CitPG transcript level was significantly lower in the inferior mastication variety at the late ripening stage or at all ripening stages. Calcium or boron treatment resulted in a significant reduction in CitPG transcription level in fruit pulp at harvest time (235 days after anthesis). This was accompanied by an increase in protopectin content. In addition, the mRNA level of CitPG in the fruit peel and pulp increased during the fruit's rapid cell enlargement stage and was inversely correlated with protopectin levels. These results suggest that CitPG is associated with the favorable enhancement of the fruit mastication trait and is involved in dissolving the protopectin during cell enlargement.

Cloning and characterization of an expansin gene, RiEXP1, and a 1-aminocyclopropane-1-carboxylic acid synthase gene, RiACS1 in ripening fruit of raspberry ( Rubus idaeus L.)

An alpha-expansin ( RiEXPA1) gene was cloned and sequenced from raspberry. Expression of RiEXPA1 in different ripening stages of raspberry fruits has been detected. Northern blotting showed that the level of RiEXPA1 transcript accumulation increased with fruit ripening and the highest level of expression appeared at the later stages of ripening. This indicates that expression of RiEXPA1 is closely correlated with the ripening and softening of raspberry fruits. In order to determine whether expression of the RiEXPA1 was regulated by ethylene, we cloned and sequenced a 1-aminocyclo-propane-1-carboxylic acid synthase cDNA ( RiACS1) that encodes the rate-limiting enzyme for ethylene production. Transcript accumulation of RiACS1 was detected at a low level only in the later stage of fruit ripening. This reveals that ethylene is not a regulatory factor of RiEXPA1 expression in raspberry fruit. The results of this investigation suggest that the expression of RiACS1 gene during ripening resembles that of non-climacteric fruits.

Oxidative stress and antioxidant systems in Guava (Psidium guajava L.) fruits during ripening

Two varieties of guava viz., L-49 and Hisar Safeda differing in their shelf lives were analyzed for various components of oxidative stress and of enzymatic and non-enzymatic antioxidative system at different stages of fruit ripening. Indices of oxidative stress viz., lipoxygenase activity, malondialdehyde value and H2O2 content increased throughout during ripening in both the varieties. The extent of oxidative stress was more pronounced in Hisar Safeda (shelf life 3-4 days) than in L-49 (shelf life 7-8 days). Except for superoxide dismutase, activities of all other antioxidative enzymes viz., catalase, peroxidase, ascorbate peroxidase and glutathione reductase increased up to color turning stage and decreased thereafter. Superoxide dismutase activity, however, increased upto ripe stage followed by a decline. Contents of ascorbic acid and glutathione (total, oxidized and reduced) were found to be the maximum at turning and mature stage, respectively. It is inferred that ripening of guava fruit is accompanied by a progressive increase in oxidative/peroxidative stress which induces antioxidant system but not until later stages of ripening. Over-accumulation of ROS due to dysfunctioning of ROS scavenging system at later stages of fruit ripening appears to be responsible for loss of tissue structure as observed in ripened and over-ripened fruits.

Effect of freeze–dried kefir culture on proteolysis in feta-type and whey-cheeses

Abstract The effect of freeze–dried kefir culture on the proteolysis of feta-type and whey-cheese was investigated. All nitrogen fractions increased during ripening. Although no significant differences were observed in total nitrogen (TN), the levels of water-soluble nitrogen (WSN), pH 4.4-soluble nitrogen (SN), 12% trichloroacetic acid-soluble nitrogen (TCA-SN) and phosphotungstic acid-soluble nitrogen (PTA-SN) were significantly higher in cheeses produced by freeze–dried kefir culture during the later stages of ripening. Content of total free amino acids (FAA) was significantly affected by freeze–dried kefir starter culture and it was continuously increased in kefir-cheese while, in rennet-cheese it was increased up to 30 days of ripening and then slightly decreased. On the other hand, FAA content continuously decreased in kefir-whey-cheese whereas it increased in whey-cheese. The cheese samples produced by freeze–dried kefir as starter culture were characterised as high-quality products during the preliminary sensory evaluation and they were accepted by the panel. Overall, the use of freeze–dried kefir suggested acceleration of cheese ripening and resulted in improved sensory characteristics.

Water Flux ofVitis viniferaL. cv. Shiraz Bunches throughout Development and in Relation to Late-Season Weight Loss

<i>Vitis vinifera</i> (L.) cv. Shiraz commonly loses a significant amount of bunch water during late ripening, with attendant yield losses. The objective of the study was to quantitatively assess the hypothesis that this sustained weight loss was due to decreased vascular flow and increased transpiration. Transpiration, fresh and dry weights, and water content of whole attached grape bunches were measured from flowering to maturity on vines grown in controlled environments and in the vineyard. Seasonal changes in net water fluxes into and out of bunches were determined. A simple technique of weighing bunches in situ also provided independent measurement of daily rates of water gain and loss. Bunch transpiration rates were high just after flowering but declined to 0.2 g g (dry wt)⁻¹ d⁻¹ at about harvest date. Bunch net water import rates also showed a 90% decrease with development from 1.0 to 0.1 g g (dry wt)⁻¹ d⁻¹. Comparisons of these rates revealed net water import exceeded transpiration throughout early and midbunch development. However, at 60 to 80 days after flowering, import rates had declined to an extent that transpiration now exceeded import and an overall loss of water occurred. Quantitatively comparable rates of water gain and loss determined on Shiraz bunches over four growing seasons on vineyard-grown vines conformed closely with those rates determined as above. Changes in diurnal bunch water fluxes supported the conclusion that net water import exceeded transpiration losses throughout bunch development until the late stage of ripening. The hypothesis that sustained weight loss in late-ripening Shiraz grape bunches occurred because bunch water fluxes shifted from a net import to a net loss by transpiration was confirmed.

Expression patterns of cell wall-modifying genes from banana during fruit ripening and in relationship with finger drop.

Few molecular studies have been devoted to the finger drop process that occurs during banana fruit ripening. Recent studies revealed the involvement of changes in the properties of cell wall polysaccharides in the pedicel rupture area. In this study, the expression of cell-wall modifying genes was monitored in peel tissue during post-harvest ripening of Cavendish banana fruit, at median area (control zone) and compared with that in the pedicel rupture area (drop zone). To this end, three pectin methylesterase (PME) and seven xyloglucan endotransglycosylase/hydrolase (XTH) genes were isolated. The accumulation of their mRNAs and those of polygalaturonase, expansin, and pectate lyase genes already isolated from banana were examined. During post-harvest ripening, transcripts of all genes were detected in both zones, but accumulated differentially. MaPME1, MaPG1, and MaXTH4 mRNA levels did not change in either zone. Levels of MaPME3 and MaPG3 mRNAs increased greatly only in the control zone and at the late ripening stages. For other genes, the main molecular changes occurred 1–4 d after ripening induction. MaPME2, MaPEL1, MaPEL2, MaPG4, MaXTH6, MaXTH8, MaXTH9, MaEXP1, MaEXP4, and MaEXP5 accumulated highly in the drop zone, contrary to MaXTH3 and MaXTH5, and MaEXP2 throughout ripening. For MaPG2, MaXET1, and MaXET2 genes, high accumulation in the drop zone was transient. The transcriptional data obtained from all genes examined suggested that finger drop and peel softening involved similar mechanisms. These findings also led to the proposal of a sequence of molecular events leading to finger drop and to suggest some candidates.

Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera

Anthocyanins, flavan-3-ols, and flavonols are the three major classes of flavonoid compounds found in grape berry tissues. Several viticultural practices increase flavonoid content in the fruit, but the underlying genetic mechanisms responsible for these changes have not been completely deciphered. The impact of post-veraison sunlight exposure on anthocyanin and flavonol accumulation in grape berry skin and its relation to the expression of different transcriptional regulators known to be involved in flavonoid synthesis was studied. Treatments consisting of removing or moving aside the basal leaves which shade berry clusters were applied. Shading did not affect sugar accumulation or gene expression of HEXOSE TRANSPORTER 1, although in the leaf removal treatment, these events were retarded during the first weeks of ripening. Flavonols were the most drastically reduced flavonoids following shading and leaf removal treatments, related to the reduced expression of FLAVONOL SYNTHASE 4 and its putative transcriptional regulator MYB12. Anthocyanin accumulation and the expression of CHS2, LDOX, OMT, UFGT, MYBA1, and MYB5a genes were also affected. Other regulatory genes were less affected or not affected at all by these treatments. Non-transcriptional control mechanisms for flavonoid synthesis are also suggested, especially during the initial stages of ripening. Although berries from the leaf removal treatment received more light than shaded fruits, malvidin-3-glucoside and total flavonol content was reduced compared with the treatment without leaf removal. This work reveals that flavonol-related gene expression responds rapidly to field changes in light levels, as shown by the treatment in which shaded fruits were exposed to light in the late stages of ripening. Taken together, this study establishes MYB-specific responsiveness for the effect of sun exposure and sugar transport on flavonoid synthesis.

CDNA cloning and expression pattern of Cinnamate-4-Hydroxylase in the Korean black raspberry

Cinnamate-4-hydroxylase (C4H) is a key enzyme in the phenylpropanoid pathway, which is responsible for synthesizing a variety of secondary metabolites that participate in development and adaptation. In this study, we isolated a full-length cDNA of the C4H gene from the Korean black raspberry (Rubus sp.) and found that this gene existed as a single gene. By comparing the deduced amino acid sequence of Rubus sp. C4H with other sequences reported previously we determined that this sequence was highly conserved among widely divergent plant species. In addition, quantitative real time PCR studies indicated that the C4H gene had a differential expression pattern during fruit development, where gene expression was first detected in green fruit and was then remarkably reduced in yellow fruit, followed by an increase in red and black fruit. To investigate the two peaks in expression observed during fruit development and ripening, we measured the flavonoid content. The content of the major flavanol of Korean black raspberry fruits was determined to be highest at the beginning of fruit development, followed by a gradually decrease according to the developmental stages. In contrast, the content of anthocyanins during the progress of ripening was dramatically increased. Our results suggest that the C4H gene in Korean black raspberry plays a role during color development at the late stages of fruit ripening, whereas the expression of C4H gene during the early stages may be related to the accumulation of flavanols.

Direct measurement of hydraulic properties in developing berries of Vitis vinifera L. cv Shiraz and Chardonnay

Berries of Vitis vinifera L. cv Shiraz can undergo weight loss during later stages of ripening. Existing published views on how weight loss occurs are based on changes in capacity of the vascular system to import water during development (McCarthy and Coombe, Australian Journal of Grape and Wine Research, 5, 17–21, 1999). One important element of these views is the proposed cessation of water flow through the xylem after veraison. We have now measured the water flow into berries of Shiraz and Chardonnay as they develop using the pressure probe and the high pressure flow meter (HPFM). The pressure probe connected to the pedicel of individual berries provided measurements of single berry hydraulic conductance. By systematic excision of tissue segments of the berry and pedicel we determined where in the pathway hydraulic conductance changed during development. The HPFM was used on whole bunches showing that berries (including pedicels) represent parallel high hydraulic resistances and that the hydraulic resistance of the bunch axis was rather small. The hydraulic conductance per berry could be determined from excision experiments. There was close agreement between the pressure probe and HPFM measurements. Both showed a ten-fold reduction in hydraulic conductance of whole berries from veraison to full ripeness. Shiraz had hydraulic conductances that were 2- to 5-fold higher than those for Chardonnay. Shiraz maintained a higher hydraulic conductance past 90 days after flowering than Chardonnay. The decrease in hydraulic conductance occurred in both the distal and proximal parts of the berry for both varieties. The pressure probe also provided measurements of the xylem pressure that non-transpiring berries could develop. These pressures were –0.2 to –0.1 MPa until veraison and increased to zero when the juice osmotic potential reached about –3 MPa in Chardonnay and –4 MPa in Shiraz. The results suggest values of the reflection coefficient of the osmotic barrier around the xylem vessels of about 0.1–0.2 at veraison decreasing to 0 at harvest. It is suggested that in addition to changes in xylem anatomy, aquaporins in berry membranes may play a role in regulating hydraulic conductance. Water movement from the berry back to the parent vine via the xylem (backflow) may be an important component of berry weight loss in Shiraz, particularly if the phloem ceases functioning at high osmotic potentials near maximum weight. Backflow could account for a weight loss of 43 mg per day in Shiraz berries for a relatively small gradient of 0.1 MPa.

Cell death in grape berries: varietal differences linked to xylem pressure and berry weight loss.

Some varieties of Vitis vinifera L. can undergo berry weight loss during later stages of ripening. This defines a third phase of development in addition to berry formation and berry expansion. Berry weight loss is due to net water loss, but the component water flows through different pathways have remained obscure. Because of the very negative osmotic potential of the cell sap, the maintenance of semipermeable membranes in the berry is required for the berry to counter xylem and apoplast tensions that may be transferred from the vine. The transfer of tension is determined by the hydraulic connection through the xylem from the berry to the vine, which changes during development. Here we assess the membrane integrity of three varieties of V. vinifera berries (cvv. Shiraz, Chardonnay and Thompson seedless) throughout development using the vitality stains, fluorescein diacetate and propidium iodide, on fresh longitudinal sections of whole berries. We also measured the xylem pressure using a pressure probe connected to the pedicel of detached berries. The wine grapes, Chardonnay and Shiraz, maintained fully vital cells after veraison and during berry expansion, but began to show cell death in the mesocarp and endocarp at or near the time that the berries attain maximum weight. This corresponded to a change in rate of accumulation of solutes in the berry and the beginning of weight loss in Shiraz, but not in Chardonnay. Continuous decline in mesocarp and endocarp cell vitality occurred for both varieties until normal harvest dates. Shiraz grapes classified as high quality and sourced from a different vineyard also showed the same death response at the same time after anthesis, but they displayed a more consistent pattern of pericarp cell death. The table grape, Thompson seedless, showed near to 100% vitality for all cells throughout development and well past normal harvest date, except for berries with noticeable berry collapse that were treated with giberellic acid. The high cell vitality in Thompson seedless berries corresponded to negative xylem pressures that contrasted to the slightly positive pressures for Shiraz and Chardonnay. We hypothesise that two variety dependent strategies exist for grapevine berries late in development: (1) programmed cell death in the pericarp and loss of osmotically competent membranes that requires concomitant reduction in the hydraulic conductance via the xylem to the vine; (2) continued cell vitality and osmotically competent membranes that can allow high hydraulic conductance to the vine.

P-type H +-ATPases activity, membrane integrity, and apoplastic pH during papaya fruit ripening

The P-type H +-ATPase activity and related apoplastic pH were analysed in papaya fruit ( Carica papaya L. cv. Golden) at different stages of maturation. Postharvest ripening of papaya was characterized by ethylene emission, skin colouration, firmness, and titratable acidity (TA). The climacteric peak of ethylene production occurred on the 2nd day after harvest, along with a sharp decrease in ATP hydrolysis and a transient alkalization of the apoplast fluid. Such effects were consistent with progressive cell membrane deenergization occurring concomitantly with fruit softening and decrease of cell membrane integrity as manifest by electrolyte leakage. Nevertheless, it was possible to detect an expressive ATP-dependent H + gradient 2 days after the climacteric suggesting the maintenance of cellular function even in the late stages of the fruit ripening. The data provide new insights into the physiology of ripening in papaya and how that P-type H +-ATPase activity can be used as a biochemical marker for postharvest analysis and management of climacteric fruit.

포도 VIASR 유전자 프로모터의 분리 및 발현 분석

포도 ASR (VvMSA) 단백질은 hexose transporter 유전자 VvHT1의 전사를 조절하는 조절 인자 중의 하나로서 sugar 및 abscisic acid (ABA) 신호에 의해 발현이 유도된다. 본 연구진은 ACP RT-PCR (annealing control primer reverse transcriptase-polymerase chain reaction) 방법을 이용하여 포도 과실발달 과정에서 조절되는 유전자 중 VvMSA와 동일한 cDNA (VlASR)를 클로닝하였다. 이 유전자는 착과 시기에 발현되기 시작하여 과실이 발달하면서 점점 증가하여 착과 후 10 주에 가장 많이 발현되며, 숙기 후반에는 도리어 발현양이 감소하였다. 포도 asr 유전자의 조절기작을 밝히기 위해, 이 유전자의 genomic clone을 분리하였다. 총 1375 bp로 이루어진 이 유전자 절편에는 open reading frame과 100 bp의 intron을 포함하고 있다. 약 600 bp 길이의 프로모터 내에는 sugar 신호전달과 연관이 있는 것으로 알려진 sugar box(sucrose box 3 +sucrose response box 1)가 있다. 프로모터 절편을 reporter 유전자와 연결하여 Arabidopsis에 도입하고 형질전환체를 분석한 결과, reporter 유전자는 sucrose 처리와 상관없이 항상 발현되었다. 이러한 결과는 포도에서 보고된 ASR/VvHT1를 매개로 하는 sugar/ABA 신호전달계가 asr 유전자가 없는 Arabidopsis에서는 작동되지 않음을 시사하고 있다. VvMSA, a grapevine ASR which is highly inducible by sugar and abscisic acid signals was previously shown to be a transcription factor for a hexose transporter gene VvHT1. We isolated a cDNA clone, VlASR which is regulated temporally during the grape berry development by ACP RT-PCR (annealing control primer reverse transcriptase-polymerase chain reaction) and it proved identical to VvMSA. RT-PCR and real-time PCR analyses revealed that the VlASR gene was expressed in berries at fruit set and that its expression increased as berries aged but decreased at the late ripening stage. In order to understand the regulatory mechanism of the asr gene, a genomic fragment was cloned from grapevine. The genomic DNA was 1375 bp long and a sugar box (sucrose box 3 and sucrose responsive element 1) was identified in the 611 bp upstream region of the open reading frame. Analysis of the VlASR promoter::reporter gene fusion demonstrated that this promoter was expressed in transgenic Arabidopsis even without sucrose treatment. This result suggests that the ASR/VvHT1-mediated sugar/ABA signaling, previously reported in grapevine, may not function in Arabidopsis which has no ASR homologue.

Geography and Vintage Predicted by a Novel GIS Model of Wine δ18O

Wine hydrogen and oxygen stable isotopes record the climatic conditions experienced by the grape vine and the isotopic composition of the vine's source water during berry development. As such, stable isotopes have been explored extensively for use in detecting wine adulteration or for independently verifying claims of origin. We present the results of a study designed to evaluate the relationships between wine water delta18O and spatial climate and precipitation delta18O patterns across the winegrape-growing regions of Washington, Oregon, and California. Retail wines produced from typically small vineyards across these regions were obtained from the 2002 vintage, and the delta18O of wine water was analyzed using a CO2 equilibration method. Significant correlations were observed between the measured wine water delta18O from 2002 and the long-term average precipitation delta18O and late season 2002 climate, based on a spatial join with continuous geographic information system (GIS) maps of these drivers. We then developed a regression model that was implemented spatially in a GIS. The GIS model is the first of its kind and allows spatially explicit predictions of wine delta18O across the region. Because high spatial resolution monthly climate layers are now available for many years, wine delta18O could be modeled for previous years. We therefore tested the model by executing it for specific years and comparing the model predictions with previously published results for wine delta18O from seven vintages from Napa and Livermore Valleys, California. With the exception of one year, an anomaly potentially related to the effects of El Niño on precipitation isotopic composition, the model predicted well the wine delta18O for both locations for all vintages and generally reflected the consistent enrichment of wine from Napa relative to Livermore. Our results suggest that wine water delta18O records both source water delta18O and climate during the late stages of winegrape ripening and that GIS models of wine water delta18O are useful tools for independently verifying claims of regional origin and vintage.

Influence of ripening container on the lactic acid bacteria population in Tulum cheese

The objective of the present study was to investigate the influence of container material (plastic or goat-skin bag) on the growth of lactic acid bacteria in Tulum cheese during 9 months of ripening. The lactic acid bacteria in Tulum cheeses were periodically counted on MRS and M17 agars throughout ripening. Results showed that the highest counts of lactic acid bacteria on MRS or M17 were observed at the beginning of ripening and their counts decreased during later stages of ripening. The cheese samples ripened in plastic bags exhibited higher numbers of LAB on MRS and M-17 agars than those ripened in goat-skin bags. A total of 112 strains of lactic acid bacteria were isolated from Tulum cheeses ripened in plastic or goat-skin bags during ripening. The lactic acid bacteria present in the cheese were classified by Microbial Identification System (MIS) based on a comparison of the fatty acid methyl ester profiles. Different species including Enteroccocus, Lactobacillus, Streptococcus, Lactococcus and Pediococcus genera were found in unripened cheese. As ripening proceeded, the species Streptococcus and Lactococcus disappeared and the percentages of the species Enterococcus was unchanged in both containers. There were slight differences between the cheeses ripened in plastic or goat-skin bags in terms of the profiles of lactic acid bacteria isolated. Some species including L. brevis, L. mesenteroides subsp. dextranicum, P. damnosus and E. mundtii were isolated only in the cheeses ripened in plastic bags; however, L. coryniformis and L. malafermentans were isolated only in the cheeses ripened in goat-skin bags at 6 or 9 months of ripening. Also the numbers of E. faecalis isolates were higher in the cheeses ripened in plastic containers than cheeses ripened goat-skin bags at the 6 or 9 months of ripening. The results showed that Lactobacillus and Enterococcus were the predominant species in matured Tulum cheeses in both ripening containers. It seemed possible to produce Tulum cheese with similar characteristics from both the containers used.