Pedro M. Civello

Effect of ethylene and 1-MCP treatments on strawberry fruit ripening.

BACKGROUND Strawberry is a soft fruit, considered as non-climacteric, being auxins the main hormones that regulate the ripening process. The role of ethylene in strawberry ripening is currently unclear and several studies have considered a revision of the possible role of this hormone. RESULTS Strawberry fruit were harvested at the white stage and treated with ethephon, an ethylene-releasing reagent, or 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene action. The effects of the treatments on fruit quality parameters and on the activity of enzymes related to anthocyanin synthesis and cell wall degradation were evaluated. Some aspects of ripening were accelerated (anthocyanin accumulation, total sugar content and increment of phenylalanine ammonia-lyase (PAL; EC 4.3.1.24) and beta-galactosidase (EC 3.2.1.23) activities), while others were repressed (chlorophyll levels and increment of endo-1,4-beta-glucanase (EC 3.2.1.4) and beta-xylosidase (EC 3.2.1.37) activities) or unchanged (reducing sugar content, pH, titratable acidity and alpha-L-arabinofuranosidase (EC 3.2.1.55) activity) by ethylene. 1-MCP treatment caused the opposite effect. However, its effects were more pronounced, particularly in anthocyanin accumulation, phenolics, PAL and polygalacturonase (EC 3.2.1.15 and EC 3.2.1.67) activities. CONCLUSION These observations probably indicate that strawberry produces low levels of ethylene that are sufficient to regulate some ripening aspects.

Chlorophyllase versus pheophytinase as candidates for chlorophyll dephytilation during senescence of broccoli

Degradation of chlorophylls during senescence is a highly regulated process which requires the concerted action of several enzymes. Traditionally, it has been stated that the dismantling process of the chlorophyll molecule begins with a dephytilation step, followed by Mg(2+) removal and other breakdown reactions. Recently, new evidence suggests the possibility of a rearrangement in the first two steps of this process, occurring Mg(2+) removal prior to the loss of the phytol side chain. With the purpose of approximating to the real sequential order of these reactions and to assess if dephytilation occurs on intact (catalyzed by chlorophyllase) or Mg-free (catalyzed by pheophytinase) chlorophyll, expression of both genes was analyzed in broccoli tissue during senescence. Samples of broccoli florets treated with plant hormones, such as cytokinin and ethylene were utilized, as to assess the effect of such compounds on the expression of these genes. Results showed that chlorophyllase expression did not correlate to typical expression patterns for genes related to senescence, since a decrease in expression during senescence was found for one of the two chlorophyllase genes analyzed, and the hormonal-treatment effects on gene expression did not match those observed on chlorophyll content for both chlorophyllase genes. Pheophytinase expression patterns, on the other hand, displayed an increase in the first 3 days of induced senescence, followed by lower expression values towards the end of the experiment. Samples subjected to postharvest treatments mostly showed an inhibition of pheophytinase expression, especially in samples in which degradation of chlorophylls had been delayed. These results suggest that pheophytinase expression correlates to the visual manifestation of postharvest treatments, supporting the possibility that this enzyme is responsible for the dephytilation step in chlorophyll breakdown.

Effect of visible light treatments on postharvest senescence of broccoli (Brassica oleracea L.).

BACKGROUND Broccoli (Brassica oleracea L.) is a rapidly perishable vegetable crop. Several postharvest treatments have been applied in order to delay de-greening. Since light has been shown to have an effect on pigment accumulation during development and darkness is known to induce senescence, the effect of continuous and periodic exposure to low-intensity white light at 22 °C on postharvest senescence of broccoli heads was assayed. RESULTS Exposure to a constant dose of 12 micromol m(-2) s(-1) was selected as the most suitable treatment and was employed for subsequent experiments. During the course of the treatments, hue and L* values as well as chlorophyll content and visual observation of florets indicated an evident delay in yellowing in treated samples compared with controls. No statistically significant differences in total protein content were found, but soluble protein content was higher in treated samples. Total and reducing sugar as well as starch levels decreased during postharvest senescence, with lower values in control samples. CONCLUSION The results of this study indicate that storage under continuous low-intensity light is an efficient and low-cost treatment that delays postharvest senescence while maintaining the quality of harvested broccoli florets.

α-L-arabinofuranosidase from strawberry fruit: cloning of three cDNAs, characterization of their expression and analysis of enzymatic activity in cultivars with contrasting firmness.

Softening of fleshy fruits during ripening is associated to catabolism of cell wall components. In strawberry, pectin degradation, as well as loss of neutral sugars (mainly arabinose), increases during ripening, and probably contributes to fruit softening. In this work, we report the activity of alpha-l-arabinofuranosidase (alpha-l-arafase) and the expression of related genes in strawberry. Activity of alpha-l-arafase was measured during ripening of cultivars with contrasting firmness. An important increment in the specific activity of alpha-l-arafase was detected during ripening in both cultivars. However, in the softest one (Toyonoka) the specific activities were higher than in the firmest (Camarosa). A combination of semi quantitative reverse transcriptase-PCR (RT-PCR) with degenerate primers and a screening of a cDNA library allowed the isolation and cloning of three cDNAs encoding putative alpha-l-arafases (FaAra1, FaAra2 and FaAra3). The deduced proteins revealed that FaAras belong to the glycoside hydrolase family 51 and not to glycoside hydrolase family 3. Expression studies, carried out by means of Northern-blot and semi quantitative RT-PCR, revealed that FaAras were predominantly expressed in fruit tissue and detected over the entire ripening process. Due to similarity of FaAras sequences, Northern-blot analysis probably grouped the expression of the three genes. The expression was high at small green stage, decreased at white stage and increased thereafter. The increment of the expression from white to 50% red stage was more evident in the softest cultivar (Toyonoka). Semi quantitative RT-PCR analysis allowed determining the expression of individual FaAras. The expression of the three genes was detected in all developmental and ripening stages. However, differences in expression levels could be detected between cultivars. In the softest cultivar, the expression of the three FaAras was higher at 50% and 75% red stages, and in the case of FaAra3 a higher expression was found also at 100% red stage. Overall, specific activity of alpha-l-arafase was higher in the softest cultivar; such activity reflects the expression of at least three putative FaAra genes.

Cloning of FaPAL6 gene from strawberry fruit and characterization of its expression and enzymatic activity in two cultivars with different anthocyanin accumulation.

The accumulation of anthocyanin pigments is one of the most important traits that turn strawberry fruit attractive to consumers. During ripening, strawberry fruit color development is associated to anthocyanin synthesis through the phenylpropanoid pathway. Phenylalanine ammonia-lyase (PAL) is a key enzyme in this pathway, having a determining role in strawberry fruit quality. In this work, we studied the level of anthocyanins during fruit ripening of two cultivars that differ in color development (Camarosa and Toyonoka). Toyonoka showed a lower anthocyanin accumulation that was limited to external fruit tissue, while Camarosa accumulated higher amount of anthocyanins in both internal and external sections. In addition, we cloned a full-length gene (FaPAL6) and analyzed its expression in different strawberry plant tissues. The expression of this gene is fruit specific, and increases during fruit ripening in both cultivars along with anthocyanin accumulation. The mRNA level of FaPAL6 was higher in Camarosa. PAL enzyme activity increased at similar rates in both cultivars at early ripening stages, but at the end of ripening PAL activity diminished in Toyonoka while it rose markedly in Camarosa. PAL activity was higher in internal fruit tissue, showing no correlation with anthocyanin level of the same section in both cultivars. The higher FaPAL6 expression and activity detected in Camarosa could be associated to the enhanced anthocyanin accumulation found in this cultivar.

Effects of ethylene, cytokinin and physical treatments on BoPaO gene expression of harvested broccoli

BACKGROUND Broccoli is a highly perishable vegetable that shows enhanced postharvest senescence and intense de-greening caused by chlorophyll degradation. One of the key steps of chlorophyll catabolism is the opening of chlorophyll tretrapyrrole catalysed by pheophorbide a oxygenase (PaO). In this study the expression of a gene encoding a putative PaO was characterised under several chemical and physical treatments. RESULTS A fragment of a gene encoding a PaO from broccoli (BoPaO) was cloned. The expression of BoPaO showed an important increment during postharvest senescence, in correlation with chlorophyll degradation. Furthermore, broccoli heads were treated with the hormones cytokinin and ethylene. Cytokinin delayed the increment in BoPaO expression, while ethylene accelerated the process. Also, several postharvest treatments were applied in order to evaluate their effect on BoPaO expression. Samples treated with modified atmosphere, hot air, UV-C or white light showed a delay in chlorophyll degradation and de-greening. In most cases the treatments also delayed the increment in BoPaO expression during senescence. CONCLUSION A close correlation between chlorophyll degradation and BoPaO expression was found during broccoli senescence. This relationship was corroborated in samples treated with different hormonal and physical applications.

Overexpression of the carbohydrate binding module of strawberry expansin2 in Arabidopsis thaliana modifies plant growth and cell wall metabolism.

Several cell wall enzymes are carbohydrate active enzymes that contain a putative Carbohydrate Binding Module (CBM) in their structures. The main function of these non-catalitic modules is to facilitate the interaction between the enzyme and its substrate. Expansins are non-hydrolytic proteins present in the cell wall, and their structure includes a CBM in the C-terminal that bind to cell wall polymers such as cellulose, hemicelluloses and pectins. We studied the ability of the Expansin2 CBM (CBMFaEXP2) from strawberry (Fragaria x ananassa, Duch) to modify the cell wall of Arabidopsis thaliana. Plants overexpressing CBMFaEXP2 were characterized phenotypically and biochemically. Transgenic plants were taller than wild type, possibly owing to a faster growth of the main stem. Cell walls of CBMFaEXP2-expressing plants were thicker and contained higher amount of pectins. Lower activity of a set of enzymes involved in cell wall degradation (PG, β-Gal, β-Xyl) was found, and the expression of the corresponding genes (AtPG, Atβ-Gal, Atβ-Xyl5) was reduced also. In addition, a decrease in the expression of two A. thaliana Expansin genes (AtEXP5 and AtEXP8) was observed. Transgenic plants were more resistant to Botrytis cinerea infection than wild type, possibly as a consequence of higher cell wall integrity. Our results support the hypothesis that the overexpression of a putative CBM is able to modify plant cell wall structure leading to modulation of wall loosening and plant growth. These findings might offer a tool to controlling physiological processes where cell wall disassembly is relevant, such as fruit softening.

Characterization of Mg-dechelating substance in senescent and pre-senescent Arabidopsis thaliana leaves

The removal of Mg2+ is an important step in the chlorophyll degradation pathway and extracts from senescent and presenescent Arabidopsis thaliana leaves were analyzed for Mg-dechelatase activity, using chlorophyllin, an artificial derivative of the natural substrate, chlorophyllide. The optimum temperature and pH for this reaction were determined to be at approximately 50 °C and 7.2, respectively. Mg-dechelatase activity was enhanced by addition of EDTA and inhibited by MgCl2, HgCl2 and reduced glutathione, indicating phenomenons such as retroinhibition by reaction products and dependence on the redox state of the mixture. Size exclusion chromatography was performed on Arabidopsis leaf extracts, and Mg-dechelatase activity was found in the fraction corresponding to molecular mass of about 42 kDa, which indicates that the Mg-dechelating compound in Arabidopsis is considerably larger than in other systems. During dark-induced senescence, the activity increased over time until reaching a maximum at day 4, and then decreased. The addition of plant growth regulators indicated that the accumulation of Mg-dechelatase was activated by ethylene and delayed by 6-benzylaminopurine.

Novel insights of ethylene role in strawberry cell wall metabolism.

Due to its organoleptic and nutraceutical qualities, strawberry fruit (Fragaria x ananassa, Duch) is a worldwide important commodity. The role of ethylene in the regulation of strawberry cell wall metabolism was studied in fruit from Toyonoka cultivar harvested at white stage, when most changes associated with fruit ripening have begun. Fruit were treated with ethephon, an ethylene-releasing reagent, or with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene action, maintaining a set of non-treated fruit as controls for each condition. Ethephon treated-fruit showed higher contents of hemicelluloses, cellulose and neutral sugars regarding controls, while 1-MCP-treated fruit showed a lower amount of those fractions. On the other hand, ethephon-treated fruit presented a lower quantity of galacturonic acid from ionically and covalently bound pectins regarding controls, while 1-MCP-treated fruit showed higher contents of those components. We also explored the ethylene effect over the mRNA accumulation of genes related to pectins and hemicelluloses metabolism, and a relationship between gene expression patterns and cell wall polysaccharides contents was shown. Moreover, we detected that strawberry necrotrophic pathogens growth more easily on plates containing cell walls from ethephon-treated fruit regarding controls, while a lower growth rate was observed when cell walls from 1-MCP treated fruit were used as the only carbon source, suggesting an effect of ethylene on cell wall structure. Around 60% of strawberry cell wall is made up of pectins, which in turns is 70% made by homogalacturonans. Our findings support the idea of a central role for pectins on strawberry fruit softening and a participation of ethylene in the regulation of this process.

Analysis of the carbohydrate-binding-module from Fragaria x ananassa α-L-arabinofuranosidase 1

α-L-arabinofuranosidases (EC 3.2.1.55) are enzymes involved in the catabolism of several cell-wall polysaccharides such as pectins and hemicelluloses, catalyzing the hydrolysis of terminal non-reducing α-L-arabinofuranosil residues. Bioinformatic analysis of the aminoacidic sequences of Fragaria x ananassa α-L-arabinofuranosidases predict a putative carbohydrate-binding-module of the family CBM_4_9, associated to a wide range of carbohydrate affinities. In this study, we report the characterization of the binding affinity profile to different cell wall polysaccharides of the putative CBM of α-L-arabinofuranosidase 1 from Fragaria x ananassa (CBM-FaARA1). The sequence encoding for the putative CBM was cloned and expressed in Escherichia coli, and the resultant recombinant protein was purified from inclusion bodies by a Nickel affinity chromatography under denaturing conditions. The refolded recombinant protein was then subjected to binding assays and affinity gel electrophoresis, which indicated its ability to bind cellulose and also high affinity for homogalacturonans.