Xuequn Pang

Gene-expression profiling of grape bud response to two alternative dormancy-release stimuli expose possible links between impaired mitochondrial activity, hypoxia, ethylene-ABA interplay and cell enlargement.

A grape-bud-oriented genomic platform was produced for a large-scale comparative analysis of bud responses to two stimuli of grape-bud dormancy release, hydrogen cyanamide (HC) and heat shock (HS). The results suggested considerable similarity in bud response to the stimuli, both in the repertoire of responding genes and in the temporary nature of the transcriptome reprogramming. Nevertheless, the bud response to HC was delayed, more condensed and stronger, as reflected by a higher number of regulated genes and a higher intensity of regulation compared to the response to HS. Integrating the changes occurring in response to both stimuli suggested perturbation of mitochondrial activity, development of oxidative stress and establishment of a situation that resembles hypoxia, which coincides with induction of glycolysis and fermentation, as well as changes in the interplay between ABA and ethylene metabolism. The latter is known to induce various growth responses in submerged plants and the possibility of a similar mechanism operating in the bud meristem during dormancy release is raised. The new link suggested between sub lethal stress, mitochondrial activity, hypoxic conditions, ethylene metabolism and cell enlargement during bud dormancy release may be instrumental in understanding the dormancy-release mechanism. Temporary increase of acetaldehyde, ethanol and ethylene in response to dormancy release stimuli demonstrated the predictive power of the working model, and its relevance to dormancy release was demonstrated by enhancement of bud break by exogenous ethylene and its inhibition by an ethylene signal inhibitor.

Similar mechanisms might be triggered by alternative external stimuli that induce dormancy release in grape buds

The detection of genes having similar expression profiles following the application of different stimuli that trigger bud break may constitute potent tools for the identification of pathways with a central role in dormancy release. We compared the effects of heat shock (HS) and hydrogen cyanamide (HC) and demonstrated that HS leads to earlier and higher bud-break levels. Changes in transcript levels of catalase, alcohol dehydrogenase and pyruvate decarboxylase were induced following both treatments. However, timing and extent of changes in transcript level differed. Changes occurred earlier in HS-treated buds and were more intense in HC-treated buds. The changes in transcript levels after both treatments were temporary. The rapid and short-lasting changes in gene expression following HS treatment correlated with the faster and higher level of bud-break that this treatment exerted. This correlation may propose that the reported molecular events are mechanistically involved in dormancy release. To test the hypothesis that temporary oxidative stress is part of the mechanism inducing dormancy release, we analyzed the effect of HS and HC treatments on the expression of ascorbate peroxidase, glutathione reductase, thioredoxin h, glutathione S-transferase and sucrose synthase genes and found that they were induced by both treatments in a similar pattern. Taken together, these findings propose that similar cellular processes might be triggered by different stimuli that lead to dormancy release, and are consistent with the hypothesis that temporary oxidative stress and respiratory stress might be part of the mechanism that leads to bud break.

Correlation of leaf senescence and gene expression/activities of chlorophyll degradation enzymes in harvested Chinese flowering cabbage (Brassica rapa var. parachinensis).

Chinese flowering cabbage is one of the main leafy vegetables produced in China. They have a rapid leaf yellowing due to chlorophyll degradation after harvest that limits their marketing. In the present study, leaf senescence of the cabbages was manipulated by ethylene and 6-benzyl aminopurine (6-BA) treatment to investigate the correlation of leaf senescence and chlorophyll degradation related to gene expression/activities in the darkness. The patterns of several senescence associated markers, including a typical marker, the expression of senescence-associated gene SAG(12), demonstrated that ethylene accelerated leaf senescence of the cabbages, while 6-BA retarded this progress. Similar to the trends of BrSAG(12) gene expression, strong activation in the expression of three chlorophyll degradation related genes, pheophytinase (BrPPH), pheophorbide a oxygenase (BrPAO) and red chlorophyll catabolite reductase (BrRCCR), was detected in ethylene treated and control leaves during the incubation, while no evident increase was recorded in 6-BA treated leaves. The overall dynamics of Mg-dechelatase activities in all treatments displayed increasing trends during the senescence process, and a delayed increase in the activities was observed for 6-BA treated leaves. However, chlorophyllase activity as well as the expression of BrChlase1 and BrChlase2 decreased with the incubation in all treatments. Taken together, the expression of BrPPH, BrPAO and BrRCCR, and the activity of Mg-dechelatase was closely associated with the chlorophyll degradation during the leaf senescence process in harvested Chinese flowering cabbages under dark conditions.

Accumulation of soluble sugars in peel at high temperature leads to stay-green ripe banana fruit

Bananas (Musa acuminata, AAA group) fail to develop a yellow peel and stay green when ripening at temperatures >24 degrees C. The identification of the mechanisms leading to the development of stay-green ripe bananas has practical value and is helpful in revealing pathways involved in the regulation of chlorophyll (Chl) degradation. In the present study, the Chl degradation pathway was characterized and the progress of ripening and senescence was assessed in banana peel at 30 degrees C versus 20 degrees C, by monitoring relevant gene expression and ripening and senescence parameters. A marked reduction in the expression levels of the genes for Chl b reductase, SGR (Stay-green protein), and pheophorbide a oxygenase was detected for the fruit ripening at 30 degrees C, when compared with fruit at 20 degrees C, indicating that Chl degradation was repressed at 30 degrees C at various steps along the Chl catabolic pathway. The repressed Chl degradation was not due to delayed ripening and senescence, since the fruit at 30 degrees C displayed faster onset of various ripening and senescence symptoms, suggesting that the stay-green ripe bananas are of similar phenotype to type C stay-green mutants. Faster accumulation of high levels of fructose and glucose in the peel at 30 degrees C prompted investigation of the roles of soluble sugars in Chl degradation. In vitro incubation of detached pieces of banana peel showed that the pieces of peel stayed green when incubated with 150 mM glucose or fructose, but turned completely yellow in the absence of sugars or with 150 mM mannitol, at either 20 degrees C or 30 degrees C. The results suggest that accumulation of sugars in the peel induced by a temperature of 30 degrees C may be a major factor regulating Chl degradation independently of fruit senescence.

An Intracellular Laccase Is Responsible for Epicatechin-Mediated Anthocyanin Degradation in Litchi Fruit Pericarp

Anthocyanin degradation by an intracellular laccase, mediated by epicatechin oxidation, causes the loss of red color in Lichi fruit peel. In contrast to the detailed molecular knowledge available on anthocyanin synthesis, little is known about its catabolism in plants. Litchi (Litchi chinensis) fruit lose their attractive red color soon after harvest. The mechanism leading to quick degradation of anthocyanins in the pericarp is not well understood. An anthocyanin degradation enzyme (ADE) was purified to homogeneity by sequential column chromatography, using partially purified anthocyanins from litchi pericarp as a substrate. The purified ADE, of 116 kD by urea SDS-PAGE, was identified as a laccase (ADE/LAC). The full-length complementary DNA encoding ADE/LAC was obtained, and a polyclonal antibody raised against a deduced peptide of the gene recognized the ADE protein. The anthocyanin degradation function of the gene was confirmed by its transient expression in tobacco (Nicotiana benthamiana) leaves. The highest ADE/LAC transcript abundance was in the pericarp in comparison with other tissues, and was about 1,000-fold higher than the polyphenol oxidase gene in the pericarp. Epicatechin was found to be the favorable substrate for the ADE/LAC. The dependence of anthocyanin degradation by the enzyme on the presence of epicatechin suggests an ADE/LAC epicatechin-coupled oxidation model. This model was supported by a dramatic decrease in epicatechin content in the pericarp parallel to anthocyanin degradation. Immunogold labeling transmission electron microscopy suggested that ADE/LAC is located mainly in the vacuole, with essential phenolic substances. ADE/LAC vacuolar localization, high expression levels in the pericarp, and high epicatechin-dependent anthocyanin degradation support its central role in pigment breakdown during pericarp browning.

The effect of delay between heat treatment and cold storage on alleviation of chilling injury in banana fruit

BACKGROUND To understand the mechanisms leading to the enhanced chilling resistance of banana by hot-water dipping (HWD, 52 °C for 3 min), we investigated the effect of a 0.5-24 h delay between HWD and cold storage on chilling resistance and the change related to the metabolism of reactive oxygen species (ROS). RESULTS The HWD-treated fruit with a delay of less than 6 h exhibited markedly less chilling injury than the non-heated control fruit, while a delay more than 6 h resulted in significant loss in chilling resistance. Increased hydrogen peroxide content and rate of superoxide radical production were detected in the fruit at 0.5-1.5 h after HWD treatment, and the levels declined with a longer delay, which may be correlated with the enhanced gene expression levels of the gene coding for a ROS-generating related enzyme, NADPH oxidase (MaNOX). Enhanced activities and gene expression of an ascorbate peroxidase (MaAPX) were recorded in the fruit at 1.5-6 h after the treatment, and after 6 h the ascorbate peroxidase levels decayed to the levels as the control fruit. The higher APX gene expression was maintained in the treated fruit with a 3 h delay during the subsequent cold storage at 7 °C, correlating with the enhanced chilling resistance. CONCLUSION The HWD-treated fruit left at ambient temperature up to 6 h prior to cold storage maintained the effect of heat treatment and transiently increased ROS content, and the ascorbate peroxidase activity that occurred 0.5-6 h after the treatment may be correlated with the elevated chilling resistance induced by HWD treatment.

Cytokinin-induced VvTFL1A expression may be involved in the control of grapevine fruitfulness

Grapevine bud fruitfulness is determined by the differentiation of uncommitted meristem (UCM) into either tendril or inflorescence. Since tendril and inflorescence differentiation have long been considered sequential steps in inflorescence development, factors that control the progression of floral meristem development may regulate the final outcome of UCM differentiation, and thus affect fruitfulness. A comparison of the expression profiles of the master regulators of floral meristem identity (FMI) during development of fruitful and non-fruitful buds along the same cane allowed associating the expression of a homolog of terminal flower 1 (TFL1, a negative regulator of FMI) to fruitful buds, and the expression of positive FMI regulators to non-fruitful buds. Combined with (a) cytokinin-induced upregulation of VvTFL1A expression in cultured tendrils, which accompanied cytokinin-derived tendril transformation into branched, inflorescence-like structures, (b) positive regulation of VvTFL1A expression by cytokinin, which was demonstrated in transgenic embryonic culture expressing GUS reporter under the control of VvTFL1A promoter, and (c) a significantly higher level of active cytokinins in fruitful positions, the data may support the assumption of cytokinin-regulated VvTFL1A activity’s involvement in the control of inflorescence development. Such activity may delay acquisition of FMI and allow an extended branching period for the UCM, resulting in the differentiation of inflorescence primordia.

Effects of high CO2 treatment on green-ripening and peel senescence in banana and plantain fruits

Banana fruit (Musa, AAA group, cv. Brazil) peel fails to fully degreen but the pulp ripens normally at temperatures above 24°C. This abnormal ripening, known as green-ripening, does not occur in plantains (Musa, ABB group, cv. Dajiao). Based on the fact that un-completely yellowing was also observed for bananas in poorly ventilated atmospheres, in the present study, the effect of high CO2 with regular O2 (21%) on banana ripening was investigated along with that on plantains at 20°C. The results showed that high CO2 conferred different effects on the color changing of bananas and plantains. After 6 d ripening in 20% CO2, plantains fully yellowed, while bananas retained high chlorophyll content and stayed green. In contrast to the differentiated color changing patterns, the patterns of the softening, starch degradation and soluble sugar accumulation in the pulp of 20% CO2 treated bananas and plantains displayed similarly as the patterns in the fruits ripening in regular air, indicating that the pulp ripening was not inhibited by 20% CO2, and the abnormal ripening of bananas in 20% CO2 can be considered as green ripening. Similar expression levels of chlorophyll degradation related genes, SGR, NYC and PaO, were detected in the peel of the control and treated fruits, indicating that the repressed degreening in 20% CO2 treated bananas was not due to the down-regulation of the chlorophyll degradation related genes. Compared to the effect on plantains, 20% CO2 treatment delayed the decline in the chlorophyll florescence (Fv/Fm) values and in the mRNA levels of a gene coding small subunit of Rubisco (SSU), and postponed the disruption of the ultrastructure of chloroplast in the peel tissue of bananas, indicating that the senescence of the green cells in the exocarp layer was delayed by 20% CO2, to more extent in bananas than in plantains. High CO2 reduced the ethylene production and the expression of the related biosynthesis gene, ACS, but elevated the respiration rates in both cultivars. The up-regulation of the expression of anaerobic respiration pathway genes, ADH and PDC, might be responsible for the subtle effect of high CO2 on the pulp ripening. Taken together, the atmosphere of high CO2 and regular O2, delayed the senescence of the green cells in the exocarp layer of the banana peel, but conferred no obvious inhibition on the pulp ripening, leading to a distinct green-ripening that was different from the phenomenon induced by high temperatures.

Reduction in Activity/Gene Expression of Anthocyanin Degradation Enzymes in Lychee Pericarp is Responsible for the Color Protection of the Fruit by Heat and Acid Treatment

Abstract Heat and acid treatments were reported to be a promising substitute for SO2 fumigation in color protection of postharvest lychee (Litchi chinensis Sonn.) fruits, but the mechanism was not clear. In the present study, hot water (70°C) dipping followed by immersion in 2% HCl (heat-acid) substantially protected the red color of the fruit during storage at 25°C and inhibited anthocyanin degradation while hot water dipping alone (heat) led to rapidly browning and about 90% loss in anthocyanin content. The pH values in the pericarp of the heat-acid treated fruit dropped to 3.2, while the values maintained around 5.0 in the heat-treated and control fruit. No significantly different pH values were detected among the arils of heat-acid, heat treated and control fruit. Heat-acid treatment dramatically reduced the activities of anthocyanin degradation enzyme (ADE), peroxidase (POD) and polyphenol oxidase in the pericarp. A marked reduction in LcPOD gene expression was also detected in heat-acid treated fruit, in contrast, induction was found in heat treated fruit. The pericarp of heat-acid treated fruit exhibited significantly lower respiration rate but faster water loss than that of the untreated or heat treated fruit. Taken together, heat treatment triggered quick browning and anthocyanin loss in lychee fruit, while heat-acid treatment protected the fruit color by a great reduction in the activities/gene expression of anthocyanin degradation enzymes and acidification of lychee pericarp.

B Type and Complex A/B Type Epicatechin Trimers Isolated from Litchi pericarp Aqueous Extract Show High Antioxidant and Anticancer Activity

Litchi (Litchi chinensis Sonn.) fruit is known for its rich source of phenolics. Litchi pericarp contains high levels of epicatechin that may form oligomers of various lengths. Except for several A or B type epicatechin dimers, other soluble oligomers have rarely been identified in the pericarp. Here, bioassay-guided column fractionation was applied to isolate bioactive phenolics from aqueous pericarp extract. A fraction (S3) was obtained by two rounds of Sephadex LH-20 column chromatography, and showed higher antioxidant activity and inhibition on the proliferation of human lung cancer cells (A549) than Litchi anthocyanins. S3 was further separated to isolate fractions P1–P4, which all showed higher antioxidant activity than vitamin C. P3 showed 32.9% inhibition on A549 cells at 30 μg/mL, higher than other fractions and cis-Dichlorodiamineplatinum (DDP, 0.5 μg/mL), but not as high as the combination of the four fractions. Using HPLC-Q-TOF-MS/MS, one B-type and complex A/B type epicatechin trimers were identified in P3; another B-type and two A/B-type trimers were identified in P4. P1 and P2, containing epicatechin and proanthocyanidin B2, respectively, showed no cell inhibition at 30 μg/mL. It is the first time that the two B type trimers of epicatechins (Litchitannin B1 and B2), have been found in Litchi species. The identified proanthocyanidins were detected in the pericarp of the young fruit, and the levels of the compounds decreased as the fruit developed, correlating to the decreasing patterns of the expression of LcLAR and LcANR, two key genes in the catechin biosynthesis pathway.