María T. Sanchez-Ballesta

Anthocyanins: From plant to health

Anthocyanins are a group of natural occurring pigments responsible for the red-blue colour of many fruits and vegetables. Anthocyanins are of interest for two reasons because they cannot only be used in the technological field as natural colorants but also have important implications in the field of human health. Numerous studies indicate the potential effect that this family of flavonoids may have in reducing the incidence of cardiovascular disease, cancer, hyperlipidemias and other chronic diseases through the intake of anthocyanin-rich foods. This review examines existing literature in this area: from plant content and distribution to health implications, including the effect of agronomic and genetic modifications on the anthocyanin content of plants as well as other biotechnological factors and food processing. The bioavailability, metabolism, bioactivity, and epidemiology of anthocyanins will also be reviewed.

Dehydrin from citrus, which confers in vitro dehydration and freezing protection activity, is constitutive and highly expressed in the flavedo of fruit but responsive to cold and water stress in leaves.

A cDNA encoding a dehydrin was isolated from the flavedo of the chilling-sensitive Fortune mandarin fruit (Citrus clementina Hort. Ex Tanaka x Citrus reticulata Blanco) and designed as Crcor15. The predicted CrCOR15 protein is a K2S member of a closely related dehydrin family from Citrus, since it contains two tandem repeats of the unusual Citrus K-segment and one S-segment (serine cluster) at an unusual C-terminal position. Crcor15 mRNA is consistently and highly expressed in the flavedo during fruit development and maturation. The relative abundance of Crcor15 mRNA in the flavedo was estimated to be higher than 1% of total RNA. The high mRNA level remained unchanged during fruit storage at chilling (2 degrees C) and nonchilling (12 degrees C) temperatures, and it was depressed by a conditioning treatment (3 days at 37 degrees C) that induced chilling tolerance. Therefore, the expression of Crcor15 appears not to be related to the acquisition of chilling tolerance in mandarin fruits. However, Crcor15, which was barely detected in unstressed mandarin leaves, was rapidly induced in response to both low temperature and water stress. COR15 protein was expressed in Escherichia coli, and the purified protein conferred in vitro protection against freezing and dehydration inactivation. The potential role of Citrus COR15 is discussed.

PHENYLALANINE AMMONIA-LYASE AND ETHYLENE IN RELATION TO CHILLING INJURY AS AFFECTED BY FRUIT AGE IN CITRUS

Abstract Fruit of many citrus cultivars become injured when exposed to low, non-freezing temperatures. In this study we have determined changes in ethylene production and phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in fruit of three citrus cultivars, ‘Fortune’ mandarins, and ‘Navelina’ and ‘Valencia’ late oranges, with different tolerance to chilling injury (CI) and demonstrated the influence of fruit physiological stage on those stress responses. We have shown that the increase in ethylene production and PAL are cold-induced responses which are only stimulated in fruit of citrus cultivars showing chilling damage and that both responses may occur concomitantly with the development of chilling symptoms. However, the magnitude of these responses was not indicative of the degree of tolerance of a specific cultivar to chilling. The influence of fruit age on both responses was evaluated in the most (‘Navelina’) and the least (‘Fortune’) chilling tolerant cultivars. Chilling damage was not developed in ‘Navelina’ fruit at any physiological stage, but our results in ‘Fortune’ mandarins, which always developed chilling symptoms, indicated that the induction of PAL in response to chilling was dependent on the fruit physiological stage. Interestingly, increases in both PAL mRNA and activity were barely affected by cold stress in the youngest ‘Fortune’ fruit harvested in December in spite of its noticeable CI. For a similar CI index, the older the fruit, the higher was the shift in the levels of PAL transcript and in PAL activity in response to cold. In contrast, the cold-induced ethylene production was little affected by the physiological stage of the fruit.

Proteome Changes in Tomato Fruits Prior to Visible Symptoms of Chilling Injury are Linked to Defensive Mechanisms, Uncoupling of Photosynthetic Processes and Protein Degradation Machinery

A comparative proteomic analysis between tomato fruits stored at chilling and non-chilling temperatures was carried out just before the appearance of visible symptoms of chilling injury. At this stage of the stress period it was possible to discriminate between proteins involved in symptoms and proteins implicated in response. To investigate the changes in the tomato fruit proteome under this specific stressful condition, two-dimensional differential in-gel electrophoresis coupled with spot identification by mass spectrometry was applied. This proteomic approach allowed the identification of differentially expressed proteins which are involved in two main biological functions: (i) defensive mechanisms represented by small heat shock and late embryogenesis proteins; and (ii) reaction to the uncoupling of photosynthetic processes and the protein degradation machinery. One of the first changes observed in chilled fruits is the down-regulation of ATP synthase, 26S proteasome subunit RPN11 and aspartic proteinase, whereas the first responses in order to deal with the stress are mainly multifunctional proteins involved not only in metabolism but also in stress regulation such as glyceraldehyde phosphate dehydrogenase, 2-oxoglutarate dehydrogenase and invertase. In addition, our data seem to indicate a possible candidate to be used as a protein marker for further studies on cold stress: aldose-1-epimerase, which seems to have an important role in low temperature tolerance.

Understanding the mechanisms of chilling injury in bell pepper fruits using the proteomic approach.

In order to advance in the understanding of CI in pepper fruits, the cell ultrastructure alterations induced by CI and the physiological and metabolic changes have been studied along with the proteomic study. When stored at low temperatures bell pepper (Capsicum annuum) fruits exhibited visual CI symptoms and important alterations within the cell ultrastructure, since peroxisomes and starch grains were not detected and the structure of the chloroplast was seriously damaged in chilled tissues. Physiological and metabolic disorders were also observed in chilled fruits, such as higher ethylene production, increased MDA content, changes in sugar and organic acids and enzymatic activities. The comparative proteomic analysis between control and chilled fruits reveals that the main alterations induced by CI in bell pepper fruits are linked to redox homeostasis and carbohydrate metabolism. Thus, protein abundance in the ascorbate-glutathione cycle is altered and catalase is down-regulated. Key proteins from glycolysis, Calvin cycle and Krebs cycle are also inhibited in chilled fruits. Enolase and GAPDH are revealed as proteins that may play a key role in the development of chilling injury. This study also provides the first evidence at the protein level that cytosolic MDH is involved in abiotic stress.

Water status and quality improvement in high-CO2 treated table grapes

Unfreezable water (UFW) content in berry tissues (pulp, skin, seed) and rachis of table grape clusters stored at 0°C has been studied using differential scanning calorimetry. The effect of short exposure to high CO2 (20% CO2 for 3days) and the transfer to air were also studied. Water status of pulp tissues was related to the thawing behaviour and the structural characteristics, using low-temperature scanning electron microscopy (LT-SEM). The UFW content in all tissues increased rapidly in response to high CO2 while it remained stable or decreased in untreated clusters. The strong potential of this beneficial gaseous treatment for increasing the UFW content was also evident after transfer to air. The metabolic adjustment caused by exposure to high CO2, which reduced the amount of water available to be frozen, improved stored fruit quality, thus minimising structural damage and reducing water leakage associated with the freezing-thawing process.

Fructo-oligosaccharides in table grapes and response to storage.

Fructo-oligosaccharides (FOS) have been recognized as health food ingredients with a protective effect against environmental stresses in plants. We have analyzed the profiles of individual FOS in Cardinal table grape pulp, until now undetected, and quantified their changes in response to low temperature and high CO2 levels. FOS separation and quantification was carried out using anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and the glucose, fructose and sucrose content of the grapes was also determined. Five FOS were identified and quantified: 1-kestose, neokestose, nystose, nystose b and kestopentaose. While in non-treated table grapes the endogenous FOS remained at steady state levels during storage at 0°C, exposure to 20% CO2 for 3days significant increases the levels of 1-kestose and kestopentaose, members of the inulin series. Considering the competitive advantage afforded by CO2-treated grapes, this transitory FOS accumulation could provide protection against damage caused by low temperature storage.

Characterization of an antifungal and cryoprotective class i chitinase from table grape berries (Vitis vinifera Cv. Cardinal)

Gene expression of a class I chitinase (Vcchit1b) in the skin of table grapes was analyzed as a molecular marker for changes induced at low temperature and also to study the effect of high CO(2) levels modulating transcript levels at 0 degrees C. An active recombinant VcCHIT1b was overexpressed in Escherichia coli, and as the protein was produced as insoluble inclusion bodies, it was solubilized and refolded. The purified recombinant chitinase showed an optimum pH of 6.0 and a temperature of 50 degrees C, retaining activity at 0 and -10 degrees C. Purified chitinase exerted in vitro antifungal activity against Botrytis cinerea. Furthermore, recombinant chitinase was able to cryoprotect lactate dehydrogenase against freeze/thaw inactivation. However, the recombinant VcCHIT1b did not show any antifreeze activity when the thermal hysteresis activity was measured using differential scanning calorimetry.

Regulation of defense and cryoprotective proteins by high levels of CO2 in Annona fruit stored at chilling temperature

This study focuses on how the length of exposure to chilling temperature and atmosphere storage conditions regulate the hydrolytic activity and expression of chitinase (PR-Q) and 1,3-beta-glucanase (PR-2) isoenzymes in cherimoyas (Annona cherimola Mill.). Storage at 6 degrees C modified the expression of constitutive isoenzymes and induced the appearance of novel acidic chitinases, AChi26 and AChi24, at the onset of the storage period, and of a basic chitinase, BChi33, after prolonged storage. The induction of this basic isoenzyme was concomitant with the accumulation of basic constitutive 1,3-beta-glucanases. These low-temperature-induced chitinases modified the growth inhibition in vitro of Botrytis cinerea. Short-term high CO(2) treatment activated a coordinated response of acidic chitinases and 1,3-beta-glucanases after prolonged storage at chilling temperature. Moreover, the high in vitro cryoprotective activity of CO(2)-treated protein extracts was associated with the induction of two low molecular mass isoenzymes, AGlu19 and BChi14. Thus, exposure to high concentrations of CO(2) modified the response of fruit to low temperature, inducing the synthesis of cryoprotectant proteins such as specific pathogenesis-related isoenzymes that could be functionally associated with an increase in chilling tolerance in vivo.

Increasing Catechin and Procyanindin Accumulation in High-CO2-Treated Fragaria vesca Strawberries

This paper deals with the impact of low temperature and high CO2 levels on flavonols, proanthocyanidins, and anthocyanins, synthesized via branched pathways from common precursors, in strawberries (Fragaria vesca L.). Flavonoids were identified with Q-TOF equipment and quantified by HPLC-quadrupole. Proanthocyanins B1 and B3 accumulated in CO2-treated strawberries, whereas in untreated (air) fruit, flavonoid production was redirected toward anthocyanin accumulation with a sharp decrease in catechin and procyanidin B3 levels. Moreover, in CO2-treated fruit, mainly in those with 20% CO2, anthocyanin accumulation did not decline. Due to its antifungal activity, catechin induction in CO2-treated strawberries could explain the capacity of high CO2 treatments to reduce fungal decay. Ascorbic acid content increased in 40% CO2-treated fruits, whereas in those treated with 20% CO2 an increase in flavonol content was observed. Despite these differences, similar antioxidant capacities were found in untreated and CO2-treated Mara de Bois strawberries.