Structural and functional characterization of genes PYL-PP2C-SnRK2s in the ABA signalling pathway of Cucurbita pepo.

BACKGROUND: The core regulation of the abscisic acid (ABA) signalling pathway comprises the multigenic families PYL, PP2C, and SnRK2. In this work, we conducted a genome-wide study of the components of these families in Cucurbita pepo. RESULTS: The bioinformatic analysis of the C. pepo genome resulted in the identification of 19 CpPYL, 102 CpPP2C and 10 CpSnRK2 genes. The investigation of gene structure and protein motifs allowed to define 4 PYL, 13 PP2C and 3 SnRK2 subfamilies. RNA-seq analysis was used to determine the expression of these gene families in different plant organs, as well as to detect their differential gene expression during germination, and in response to ABA and cold stress in leaves. The specific tissue expression of some gene members indicated the relevant role of some ABA signalling genes in plant development. Moreover, their differential expression under ABA treatment or cold stress revealed those ABA signalling genes that responded to ABA, and those that were up- or down-regulated in response to cold stress. A reduced number of genes responded to both treatments. Specific PYL-PP2C-SnRK2 genes that had potential roles in germination were also detected, including those regulated early during the imbibition phase, those regulated later during the embryo extension and radicle emergence phase, and those induced or repressed during the whole germination process. CONCLUSIONS: The outcomes of this research open new research lines for agriculture and for assessing gene function in future studies.

The synergy of halotolerant PGPB and mauran mitigates salt stress in tomato (Solanum lycopersicum) via osmoprotectants accumulation.

Salinity stress is one of the major abiotic factors limiting sustainable agriculture. Halotolerant plant growth-promoting bacteria (PGPB) increased salt stress tolerance in plants, but the mechanisms underlying the tolerance are poorly understood. This study investigated the PGP activity of four halotolerant bacteria under salinity stress and the tomato salt-tolerance mechanisms induced by the synergy of these bacteria with the exopolysaccharide (EPS) mauran. All PGPB tested in this study were able to offer a significant improvement of tomato plant biomass under salinity stress; Peribacillus castrilensis N3 being the most efficient one. Tomato plants treated with N3 and the EPS mauran showed greater tolerance to NaCl than the treatment in the absence of EPS and PGPB. The synergy of N3 with mauran confers salt stress tolerance in tomato plants by increasing sodium transporter genes' expression and osmoprotectant content, including soluble sugars, polyols, proline, GABA, phenols and the polyamine putrescine. These osmolytes together with the induction of sodium transporter genes increase the osmotic adjustment capacity to resist water loss and maintain ionic homeostasis. These findings suggest that the synergy of the halotolerant bacterium N3 and the EPS mauran could enhance tomato plant growth by mitigating salt stress and could have great potential as an inductor of salinity tolerance in the agriculture sector.

Postharvest Treatment with Abscisic Acid Alleviates Chilling Injury in Zucchini Fruit by Regulating Phenolic Metabolism and Non-Enzymatic Antioxidant System.

Reports show that phytohormone abscisic acid (ABA) is involved in reducing zucchini postharvest chilling injury. During the storage of harvested fruit at low temperatures, chilling injury symptoms were associated with cell damage through the production of reactive oxygen species. In this work, we have studied the importance of different non-enzymatic antioxidants on tolerance to cold stress in zucchini fruit treated with ABA. The application of ABA increases the antioxidant capacity of zucchini fruit during storage through the accumulation of ascorbate, carotenoids and polyphenolic compounds. The quantification of specific phenols was performed by UPLC/MS-MS, observing that exogenous ABA mainly activated the production of flavonoids. The rise in all these non-enzymatic antioxidants due to ABA correlates with a reduction in oxidative stress in treated fruit during cold stress. The results showed that the ABA mainly induces antioxidant metabolism during the first day of exposure to low temperatures, and this response is key to avoiding the occurrence of chilling injury. This work suggests an important protective role of non-enzymatic antioxidants and polyphenolic metabolism in the prevention of chilling injury in zucchini fruit.

The ethylene receptor mutation etr2b reveals crosstalk between ethylene and ABA in the control of Cucurbita pepo germination.

The enhanced salt tolerance of squash ethylene-insensitive mutants during germination and early stages of seedling development suggested that abscisic acid (ABA) could mediate this tolerance. To gain insight into the crosstalk between ethylene and ABA in seed germination, the germination rate and early seedling growth of wild type (WT) and ethylene-insensitive etr2b mutant were compared in seeds germinated under water and exogenous ABA treatment. The etr2b seeds germinated earlier than WT under both water and ABA, and the effect of ABA on radicle length and seedling growth of etr2b was lower than in WT, indicating that etr2b is also insensitive to ABA. The comparison of ABA and ethylene contents and ABA and ethylene gene expression profiles in WT and etr2b dry and imbibed seeds in either water, NaCl or ABA demonstrated a clear crosstalk between ethylene and ABA in germination. The expression profiles of ethylene genes in WT and etr2b indicated that the role of ethylene in seed germination does not appear to follow the canonical ethylene signaling pathway. Instead, etr2b reduces ABA content during formation of the seeds (dry seeds) and in response to seed imbibition and germination, which means diminished dormancy in the ethylene mutant. The etr2b mutation downregulated the expression of ABA biosynthesis and signaling genes during germination, demonstrating the positive role of ethylene receptor gene CpETR2B on seed germination and early seedling growth in squash is mediated by ABA. The reduced effect of exogenous ABA on ethylene production and ethylene gene expression in etr2b seeds suggests that this regulation is also dependent on ethylene.

Jasmonate-deficient mutant lox3a reveals crosstalk between jasmonate and ethylene in the differential regulation of male and female flower opening and early fruit development in Cucurbita pepo.

Jasmonate (JA) has been found to be a relevant hormone in floral development in numerous species, but its function in cucurbit floral development and sex determination is unknown. Crosstalk between JA and ethylene (ET) in the differential regulation of male and female floral development was investigated by using the novel JA-deficient mutant lox3a, and the ET-deficient and -insensitive mutants, aco1a and etr2b, respectively, of Cucurbita pepo. The lox3a mutation suppresses male and female flower opening and induces the development of parthenocarpic fruit. A bulked-segregant analysis coupled with whole genome sequencing and fine mapping approach allowed the identification of lox3a mutation in CpLOX3A, a LIPOXYGENASE gene involved in JA biosynthesis. The reduced JA content and expression of JA-signalling genes in male and female flowers of lox3a, and the rescue of lox3a phenotype by external application of methyl jasmonate (MeJA), demonstrated that JA controls petal elongation and flower opening, as well as fruit abortion in the absence of fertilization. JA also rescued the phenotype of ET mutants aco1a and etr2b, which are both specifically defective in female flower opening and fruit abortion. ET, the sex determining hormone of cucurbits, is induced in female flowers towards anthesis, activating JA production and promoting the aperture of the female flower, and the abortion of the unfertilized ovary. Given the close association between flower closure and parthenocarpic fruit development, we propose that flower opening can act as a switch that triggers fruit set and development in fertilized ovaries, but may alternatively induce the abortion of the unfertilized ovary. Both ET and JA from mature and senescent petals can serve as remote signals that determine the alternative development of the ovary and fruit.

Changes in Morphology, Metabolism and Composition of Cuticular Wax in Zucchini Fruit During Postharvest Cold Storage.

Cuticle composition is an important economic trait in agriculture, as it is the first protective barrier of the plant against environmental conditions. The main goal of this work was to study the role of the cuticular wax in maintaining the postharvest quality of zucchini fruit, by comparing two commercial varieties with contrasting behavior against low temperatures; the cold-tolerant variety 'Natura', and the cold-sensitive 'Sinatra', as well as 'Sinatra' fruit with induced-chilling tolerance through a preconditioning treatment (15°C for 48 h). The freshly-harvested 'Natura' fruit had a well-detectable cuticle with a significant lower permeability and a subset of 15 up-regulated cuticle-related genes. SEM showed that zucchini epicuticular waxes mainly consisted of round-shaped crystals and clusters of them, and areas with more dense crystal deposition were found in fruit of 'Natura' and of preconditioned 'Sinatra'. The cuticular wax load per surface was higher in 'Natura' than in 'Sinatra' fruit at harvest and after 14 days at 4°C. In addition, total cuticular wax load only increased in 'Natura' and preconditioned 'Sinatra' fruit with cold storage. With respect to the chemical composition of the waxes, the most abundant components were alkanes, in both 'Natura' and 'Sinatra', with similar values at harvest. The total alkane content only increased in 'Natura' fruit and in the preconditioned 'Sinatra' fruit after cold storage, whereas the amount of total acids decreased, with the lowest values observed in the fruit that showed less chilling injury (CI) and weight loss. Two esters were detected, and their content also decreased with the storage in both varieties, with a greater reduction observed in the cold-tolerant variety in response to low temperature. Gene expression analysis showed significant differences between varieties, especially in CpCER1-like and CpCER3-like genes, involved in alkane production, as well as in the transcription factors CpWIN1-like and CpFUL1-like, associated with cuticle development and epidermal wax accumulation in other species. These results suggest an important role of the alkane biosynthetic pathway and cuticle morphology in maintaining the postharvest quality of zucchini fruit during the storage at low temperatures.

Consumer Acceptance of a Ready-to-Eat Meal during Storage as Evaluated with a Home-Use Test.

A home-use test (HUT) is one method that provides a measure of ecological validity as the product is consumed in home under common daily use circumstances. One product that benefits from being evaluated in-home are ready-to-eat (RTE) meals. This study determined consumer acceptance of microwave-thermally-pasteurized jambalaya, a multi-meat and vegetable dish from American Cajun cuisine, and a control (cooked frozen jambalaya) through an on-line home-use test (HUT) over a 12-week storage period. Paralleling the HUT, an online auction determined consumers' willingness to pay. The study also explored how the social environment may impact the liking of the meals when a partner of the participants joined the sensory evaluation of the meals. Consumers (n = 50) evaluated microwave-processed jambalaya stored at 2 °C and a control (cooked frozen jambalaya stored at -31 °C) after 2, 8 and 12 weeks of storage. Consumer liking of different sensory attributes was measured. Participants could choose to share the meals with a partner as a way to enhance ecological validity. The responses from 21 partners to the sensory-related questions were collected. After the sensory evaluation, the participants bid on the meal they had just sampled. Results showed that processing method (microwave vs. control) did not significantly influence the measured sensory attributes. Only flavor liking decreased over storage time (p < 0.05). The inclusion of partners significantly increased (p = 0.04) the liking of the appearance of the meals. The mean values of the bids for the meals ranged from $3.33-3.74, matching prices of commercially available jambalaya meals. This study found suggests that the shelf- life of microwave-processed meals could be extended up to 12 weeks without changing its overall liking. The study also shows the importance of exploring HUT methodology for the evaluation of consumers' acceptance of microwave-processed jambalaya and how including a partner could contribute to enhance ecological validity.

Does the order of presentation of extrinsic and intrinsic quality attributes matter when eliciting willingness to pay?

In this study, we estimate consumers' willingness to pay (WTP) for ready meals preserved using microwave assisted pasteurization systems (MAPS)-a novel pasteurization technology, compared to frozen, ready meals. We conducted a laboratory multi-round experimental auction for two samples of ready meal jambalaya in which appearance and sensory evaluation along with extrinsic information was sequentially disclosed to panelists. Our results suggest that when participants tasted the meals and formed an opinion from the meal itself, the liking of appearance and sensory attributes were the most impactful factors for participants' WTP regardless of other available extrinsic (name of the preservation technology and environmental impacts of each technology) attributes. The words "microwave" and "pasteurization" did not negatively impact the WTP. The order in which intrinsic and extrinsic attributes were evaluated and presented did not impact absolute WTP values, but the order did impact the weight of each attribute type on the WTP. PRACTICAL APPLICATION: The sensory perceptions had a larger effect than the name of the preservation technology and environmental impacts on the willingness to pay (WTP) for ready meals. The order of presented information to panelists did not impact absolute values of WTP. The words "microwave" and "pasteurization" did not have a negative effect on WTP.

The Ethylene Biosynthesis Gene CpACO1A: A New Player in the Regulation of Sex Determination and Female Flower Development in Cucurbita pepo.

A methanesulfonate-generated mutant has been identified in Cucurbita pepo that alters sex determination. The mutation converts female into hermaphrodite flowers and disrupts the growth rate and maturation of petals and carpels, delaying female flower opening, and promoting the growth rate of ovaries and the parthenocarpic development of the fruit. Whole-genome resequencing allowed identification of the causal mutation of the phenotypes as a missense mutation in the coding region of CpACO1A, which encodes for a type I ACO enzyme that shares a high identity with Cucumis sativus CsACO3 and Cucumis melo CmACO1. The so-called aco1a reduced ACO1 activity and ethylene production in the different organs where the gene is expressed, and reduced ethylene sensitivity in flowers. Other sex-determining genes, such as CpACO2B, CpACS11A, and CpACS27A, were differentially expressed in the mutant, indicating that ethylene provided by CpACO1A but also the transcriptional regulation of CpACO1A, CpACO2B, CpACS11A, and CpACS27A are responsible for determining the fate of the floral meristem toward a female flower, promoting the development of carpels and arresting the development of stamens. The positive regulation of ethylene on petal maturation and flower opening can be mediated by inducing the biosynthesis of JA, while its negative control on ovary growth and fruit set could be mediated by its repressive effect on IAA biosynthesis.

Two androecious mutations reveal the crucial role of ethylene receptors in the initiation of female flower development in Cucurbita pepo.

Ethylene is the key regulator of sex determination in monoecious species of the family Cucurbitaceae. This hormone determines which individual floral meristems develop as female or male flowers and the female flowering transition. The sex determination genes discovered so far code for ethylene biosynthesis enzymes, but little is known about the importance of ethylene signaling components. In this paper we characterize two novel ethylene-insensitive mutations (etr1a-1 and etr1b) which block the female flowering transition of Cucurbita pepo; this makes plants produce male flowers indefinitely (androecy). Two missense mutations in the ethylene-binding domain of the ethylene receptors CpETR1A or CpETR1B were identified as the causal mutations of these phenotypes by using whole-genome resequencing. The distinctive phenotypes of single and double mutants for four etr mutations have demonstrated that the final level of ethylene insensitivity depends upon the strength and dosage of mutant alleles for at least three cooperating ETR genes, and that the level of ethylene insensitivity determines the final sex phenotype of the plant. The sex phenotype ranges from monoecy in ethylene-sensitive wild-type plants to androecy in the strongest ethylene-insensitive ones, via andromonoecy in partially ethylene-insensitive plants. The induction of female flowering transition was found to be associated with upregulation of CpACS11, CpACO2 and CpACS27, three ethylene biosynthesis genes required for female flower development. A model is proposed herein, integrating both ethylene biosynthesis and receptor genes into the genetic network which regulates sex determination in C. pepo.

The ethylene receptors CpETR1A and CpETR2B cooperate in the control of sex determination in Cucurbita pepo.

High-throughput screening of an ethyl methanesulfonate-generated mutant collection of Cucurbita pepo using the ethylene triple-response test resulted in the identification of two semi-dominant ethylene-insensitive mutants: etr1a and etr2b. Both mutations altered sex determination mechanisms, promoting conversion of female into bisexual or hermaphrodite flowers, and monoecy into andromonoecy, thereby delaying the transition to female flowering and reducing the number of pistillate flowers per plant. The mutations also altered the growth rate and maturity of petals and carpels in pistillate flowers, lengthening the time required for flowers to reach anthesis, as well as stimulating the growth rate of ovaries and the parthenocarpic development of fruits. Whole-genome sequencing allowed identification of the causal mutation of the phenotypes as two missense mutations in the coding region of CpETR1A and CpETR2B, each one corresponding to one of the duplicates of ethylene receptor genes highly homologous to Arabidopsis ETR1 and ETR2. The phenotypes of homozygous and heterozygous single- and double-mutant plants indicated that the two ethylene receptors cooperate in the control of the ethylene response. The level of ethylene insensitivity, which was determined by the strength of each mutant allele and the dose of wild-type and mutant etr1a and etr2b alleles, correlated with the degree of phenotypic changes in the mutants.

Exogenous γ-aminobutyric acid treatment improves the cold tolerance of zucchini fruit during postharvest storage.

This work examines the effect of a treatment with 1 mM of γ-aminobutyric acid (GABA) on zucchini fruit during postharvest cold storage. Specifically, the effect of GABA on postharvest quality was measured, as well as its implication in the GABA shunt and other related metabolic pathways. The treatments were performed in Sinatra, a variety of zucchini highly sensitive to low-temperature storage. The application of GABA improved the quality of zucchini fruit stored at 4 °C, with a reduction of chilling-injury index, weight loss, and cell death, as well as a lower rate of electrolyte leakage. GABA content was significantly higher in the treated fruit than in the control fruit at all times analyzed. At the end of the storage period, GABA-treated fruit had higher contents of both proline and putrescine. The catabolism of this polyamine was not affected by exogenous GABA. Also, over the long term, the treatment induced the GABA shunt by increasing the activities of the enzymes GABA transaminase (GABA-T) and glutamate decarboxylase (GAD). GABA-treated fruit contained higher levels of fumarate and malate than did non-treated fruit, as well as higher ATP and NADH contents. These results imply that the GABA shunt is involved in providing metabolites to produce energy, reduce power, and help the fruit to cope with cold stress over the long term.

Phenomic and Genomic Characterization of a Mutant Platform in Cucurbita pepo.

The Cucurbita pepo genome comprises 263 Mb and 34,240 gene models organized in 20 different chromosomes. To improve our understanding of gene function we have generated an EMS mutant platform, consisting of 3,751 independent M2 families. The quality of the collection has been evaluated based on phenotyping and whole-genome re-sequencing (WGS) results. The phenotypic evaluation of the whole platform at seedling stage has demonstrated that the rate of variation for easily observable traits is more than 10%. The percentage of families with albino or chlorotic seedlings exceeded 3%, similar or higher to that found in other EMS collections of cucurbit crops. A rapid screening of the library for triple ethylene response in etiolated seedlings allowed the identification of four ethylene-insensitive mutants, that were found to be semidominant (ein1, ein2, and ein3) or dominant (EIN4). By evaluating 4 adult plants from 300 independent families more than 28% of apparent mutations were found for vegetative and reproductive traits, including plant vigor, leaf size and shape, sex expression and sex determination, and fruit set and development. Two pools of genomic DNA derived from 20 plants of two mutant families were subjected to WGS by using NGS methodology, estimating the density, spectrum, distribution and impact of EMS induced mutation. The number of EMS mutations in the genomes of families L1 and L2 was 1,704 and 859, respectively, which represents a density of 11.8 and 6 mutations per Mb, respectively. As expected, the predominant EMS induced mutations were C > T and G > A transitions (80.3% in L1, and 61% L2), that were found to be randomly distributed along the 20 chromosomes of C. pepo. The mutations were mostly affecting intergenic regions, but 7.9 and 6% of the identified EMS mutations in L1 and L2, respectively, were located in the exome, and 0.4 and 0.2% had a moderate and high putative impact on gene functions. These results provide information regarding the potential use of the obtained mutant platform in the discovery of novel alleles for both functional genomics and Cucurbita breeding by using direct- or reverse-genetic approaches.

Effect of a distal weight-bearing implant on visual analog scale scores in 23 transfemoral amputees.

The objective of this interrupted time series clinical trial was to evaluate the effect of a distal weight-bearing implant on well-being in patients with transfemoral amputations using the visual analog scale (VAS). A total of 29 patients from five hospitals with previous transfemoral amputations were surgically implanted with an osseoanchored implant with a distal spacer that allows a direct load on the residuum over the distal surface of the socket. Patients were followed for a 14-month period and assessed presurgically and postsurgically using the VAS. The Wilcoxon test was used to evaluate the differences between variables. VAS mean scores improved significantly after intervention. Significant and clinically meaningful improvements in the VAS score suggest overall improvement in well-being for patients after receiving a distal weight-bearing implant.

Oxidative Stress Associated with Chilling Injury in Immature Fruit: Postharvest Technological and Biotechnological Solutions.

Immature, vegetable-like fruits are produced by crops of great economic importance, including cucumbers, zucchini, eggplants and bell peppers, among others. Because of their high respiration rates, associated with high rates of dehydration and metabolism, and their susceptibility to chilling injury (CI), vegetable fruits are highly perishable commodities, requiring particular storage conditions to avoid postharvest losses. This review focuses on the oxidative stress that affects the postharvest quality of vegetable fruits under chilling storage. We define the physiological and biochemical factors that are associated with the oxidative stress and the development of CI symptoms in these commodities, and discuss the different physical, chemical and biotechnological approaches that have been proposed to reduce oxidative stress while enhancing the chilling tolerance of vegetable fruits.

Recomendaciones de rehabilitación en el paciente quemado: revisión de literatura / Recomendações de reabilitação para paciente queimado: revisão da literatura / Rehabilitation recommendations in the burn patient: literature review

OBJETIVO: Realizar una revisión bibliográfica acerca de la rehabilitación en el paciente quemado, sintetizando las recomendaciones expuestas por los documentos de consenso internacionales y la bibliografía reciente. Además, exponemos la manera en que estas recomendaciones se han aplicado en la Unidad de Grandes Quemados del Hospital La Fe, en forma de un protocolo multidisciplinar de rehabilitación intensiva. MÉTODO: Se realizó una búsqueda bibliográfica de la literatura existente y los consensos internacionales centrados en el ámbito de la rehabilitación en el paciente quemado. Seguidamente, se relata la forma en que estas recomendaciones fueron implementadas en la Unidad de Grandes Quemados del Hospital La Fe de Valencia; en forma de un protocolo de rehabilitación intensivo y multidisciplinar. RESULTADOS: La bibliografía revisada refuerza los argumentos en favor del desarrollo y aplicación de protocolos de rehabilitación intensiva en las unidades de quemados para la mejora de los resultados funcionales de los pacientes. La experiencia en nuestra unidad en la cual se aplica un protocolo de este tipo coincide con los buenos resultados reportados. CONCLUSIONES: La aplicación precoz de una terapia rehabilitadora intensiva resulta clave a la hora de prevenir y tratar las posibles complicaciones funcionales y secuelas producidas por las quemaduras. Este tratamiento rehabilitador interdisciplinar se ha de centrar en la prevención de problemas a largo plazo, como las contracturas, la cicatrización anómala, deformidades, atrofia muscular, limitación de movilidad y otros problemas que merman la función física. Su utilización en cualquier unidad de quemados, permitirá optimizar los resultados funcionales de nuestros pacientes.(AU)

Objetivo: Realizar uma revisão da literatura científica sobre a reabilitação do paciente queimado, resumindo as recomendações estabelecidas por documentos de consenso internacional e na literatura recente. Além disso, apresentamos como essas recomendações foram implementadas na Unidade de Grandes Queimados do Hospital La Fe, na forma de um protocolo de reabilitação intensivo multidisciplinar. Método: Pesquisa bibliográfica da literatura e do consenso internacional existente centrado no campo da reabilitação no paciente queimado. Foi relatado como as recomendações internacionais foram implementadas para a prática dentro da unidade para atendimento ao grande queimado, no Hospital La Fe, no formato de um protocolo de reabilitação intensivo multidisciplinar. Resultados: A literatura existente reforça o desenvolvimento e implementação de protocolos de reabilitação intensiva em unidades de queimados para melhorar os resultados funcionais dos pacientes. A experiência em nossa unidade, em que esse tipo de protocolo é aplicado, corresponde aos bons resultados descritos na literatura. Conclusões: A aplicação antecipada de terapia de reabilitação intensiva é fundamental para a prevenção e tratamento de complicações e possíveis consequências funcionais causadas por queimaduras. Este tratamento interdisciplinar de reabilitação tem de se concentrar na prevenção de problemas de longo prazo, tais como contraturas, cicatrizes anormais, deformidades, atrofia muscular, dificuldade de locomoção e outros problemas que prejudicam a função física. Seu uso em qualquer unidade de queimados irá otimizar os resultados funcionais dos nossos pacientes.(AU)

ABSTRACT: OBJECTIVE: To perform a bibliographic review about rehabilitation of the burn patient, summarizing recommendations provided by international consensus documents and the most recent evidence. Moreover, we present the method in which this recommendations have been applied to the Great Burns Unit of the Hospital La Fe, in the format of a interdisciplinary intensive rehabilitation protocol. METHOD: A bibliographic search was performed among existent literature and international consensus documents focused on the field of burn patient rehabilitation. Following, the way in which this recommendations were applied at the Great Burns Unit of the Hospital La Fe of Valencia are presented in the shape of an intensive and multidisciplinar rehabilitation protocol. RESULTS: Reviewed bibliography supports the development and application of intensive rehabilitation protocols in burn units for the improvement of the functional results of their patients. The experience in our unit in which this sort of protocol is applied, matches the good results described in literature. CONCLUSIONS: Early intensive rehabilitation therapy is a key stone when it comes to prevention and treatment of functional complications. This interdisciplinary approach must focus in prevention of long term complications, such as contractures, abnormal scarring, deformities, muscular atrophy, mobility limitations and other issues which can decrease physical function. Its utilization in a Burn Unit, will help optimize functional results of their patients.(AU)

Individual Shrink Wrapping of Zucchini Fruit Improves Postharvest Chilling Tolerance Associated with a Reduction in Ethylene Production and Oxidative Stress Metabolites.

We have studied the effect of individual shrink wrapping (ISW) on the postharvest performance of refrigerated fruit from two zucchini cultivars that differ in their sensitivity to cold storage: Sinatra (more sensitive) and Natura (more tolerant). The fruit was individually shrink wrapped before storing at 4°C for 0, 7 and 14 days. Quality parameters, ethylene and CO2 productions, ethylene gene expression, and oxidative stress metabolites were assessed in shrink wrapped and non-wrapped fruit after conditioning the fruit for 6 hours at 20°C. ISW decreased significantly the postharvest deterioration of chilled zucchini in both cultivars. Weight loss was reduced to less than 1%, pitting symptoms were completely absent in ISW fruit at 7 days, and were less than 25% those of control fruits at 14 days of cold storage, and firmness loss was significantly reduced in the cultivar Sinatra. These enhancements in quality of ISW fruit were associated with a significant reduction in cold-induced ethylene production, in the respiration rate, and in the level of oxidative stress metabolites such as hydrogen peroxide and malonyldialdehyde (MDA). A detailed expression analysis of ethylene biosynthesis, perception and signaling genes demonstrated a downregulation of CpACS1 and CpACO1 genes in response to ISW, two genes that are upregulated by cold storage. However, the expression patterns of six other ethylene biosynthesis genes (CpACS2 to CpACS7) and five ethylene signal transduction pathway genes (CpCTR1, CpETR1, CpERS1, CpEIN3.1 and CpEN3.2), suggest that they do not play a major role in response to cold storage and ISW packaging. In conclusion, ISW zucchini packaging resulted in improved tolerance to chilling concomitantly with a reduction in oxidative stress, respiration rate and ethylene production, as well as in the expression of ethylene biosynthesis genes, but not of those involved in ethylene perception and sensitivity.

Contribution of polyamines and other related metabolites to the maintenance of zucchini fruit quality during cold storage.

In order to investigate the contribution of polyamines and related amino acids in the maintenance of zucchini fruit quality during cold storage, two varieties of Cucurbita pepo with different degrees of chilling tolerance were used, Natura (more tolerant) and Sinatra (moresensitive). After harvest, free putrescine levels decreased during storage at 20 °C, whereas in fruit kept at 4 °C this polyamine accumulated in both varieties, but with higher levels in the sensitive variety (Sinatra). This behavior suggests that putrescine is accumulated as a response to low temperature in zucchini fruit by stress-induced chilling injury, and not due to the postharvest storage itself. ADC activity responds quickly to chilling but sharply decreases after 14 days, whereas its expression remains high in both varieties. ODC activity takes over when the cold stress is relatively severe, as this activity was found to be much higher in Sinatra. ODCexpression also correlated with ODC activity. DAO activity increased in Natura fruit, and conversely decreased in Sinatra fruit during storage at 4 °C, whereas the proline content was higher in Natura and lower in Sinatra. Therefore, we suggest that putrescine degradation and proline accumulation contribute to the acquisition of chilling tolerance in zucchini fruit. GABA content decreased in both varieties, with a greater reduction in Natura fruit and less in Sinatra fruit. In addition, GABA transaminase showed a higher activity in Natura fruit than in Sinatra fruit during cold storage, suggesting that GABA catabolism could be involved in the tolerance to postharvest cold storage in zucchini fruit.

Molecular and functional characterization of CpACS27A gene reveals its involvement in monoecy instability and other associated traits in squash (Cucurbita pepo L.).

A number of Cucurbita pepo genotypes showing instable monoecy or partial andromonoecy, i.e. an incomplete conversion of female into bisexual flowers, have been detected. Given that in melon and cucumber andromonoecy is the result of reduction of ethylene production in female floral buds, caused by mutations in the ethylene biosynthesis genes CmACS7 and CsACS2; we have cloned and characterized two related C. pepo genes, CpACS27A and CpACS27B. The molecular structure of CpACS27A and its specific expression in the carpels of female flowers during earlier stages of flower development suggests that this gene is the Cucurbita ortholog of CmACS7 and CsACS2. CpACS27B is likely to be a paralogous pseudogene since it has not been found to be expressed in any of the analyzed tissues. CpACS27A was sequenced in Bolognese (Bog) and Vegetable Spaghetti (Veg), two monoecious inbred lines whose F2 was segregating for partial andromonoecy. The Bog allele of CpACS27A carried a missense mutation that resulted in a substitution of the conserved serine residue in position 176 by an alanine. Segregation analysis indicated that this mutant variant is necessary but not sufficient to confer the andromonoecious phenotype in squash. In concordance with its involvement in stamen arrest, a reduction in CpACS27A expression has been found in bisexual flower buds at earlier stages of development. This reduction in CpACS27A expression was concomitant with a downregulation of other ethylene biosynthesis and signaling genes during earlier and later stages of ovary development. The role of CpACS27A is discussed regarding the regulation of ethylene biosynthesis and signaling genes in the control of andromonoecy-associated traits, such as the delayed maturation of corolla and stigma as well as the parthenocarpic development of the fruit.

Changes in carbohydrate content in zucchini fruit (Cucurbita pepo L.) under low temperature stress.

The postharvest handling of zucchini fruit includes low-temperature storage, making cold stress unavoidable. We have investigated the changes of soluble carbohydrates under this stress and its relation with weight loss and chilling injury in zucchini fruit during postharvest storage at 4 °C and 20 °C for up to 14 days. Two varieties with different degrees of chilling tolerance were compared: Natura, the more tolerant variety, and Sinatra, the variety that suffered more severe chilling-injury symptoms and weight loss. In both varieties, total soluble carbohydrates, reducing soluble carbohydrates and polyols content was generally higher during storage at 4 °C than at 20 °C, thus these parameters are related to the physiological response of zucchini fruit to cold stress. However, the raffinose content increased in Natura and Sinatra fruits during storage at 4 °C and 20 °C, although at 20 °C the increase in raffinose was more remarkable than at 4 °C in both varieties, so that the role of raffinose could be more likely related to dehydration than to chilling susceptibility of zucchini fruit. Glucose, fructose, pinitol, and acid invertase activity registered opposite trends in both varieties against chilling, increasing in Natura and decreasing in Sinatra. The increase in acid invertase activity in Natura fruit during cold storage could contribute in part to the increase of these reducing sugars, whose metabolism could be involved in the adaptation to postharvest cold storage.