Azacytidine arrests ripening in cultivated strawberry (Fragaria × ananassa) by repressing key genes and altering hormone contents.

BACKGROUND: Strawberry ripening involves a number of irreversible biochemical reactions that cause sensory changes through accumulation of sugars, acids and other compounds responsible for fruit color and flavor. The process, which is strongly dependent on methylation marks in other fruits such as tomatoes and oranges, is highly controlled and coordinated in strawberry. RESULTS: Repeated injections of the hypomethylating compound 5-azacytidine (AZA) into green and unripe Fragaria × ananassa receptacles fully arrested the ripening of the fruit. The process, however, was reversible since treated fruit parts reached full maturity within a few days after AZA treatment was stopped. Transcriptomic analyses showed that key genes responsible for the biosynthesis of anthocyanins, phenylpropanoids, and hormones such as abscisic acid (ABA) were affected by the AZA treatment. In fact, AZA downregulated genes associated with ABA biosynthetic genes but upregulated genes associated with its degradation. AZA treatment additionally downregulated a number of essential transcription factors associated with the regulation and control of ripening. Metabolic analyses revealed a marked imbalance in hormone levels, with treated parts accumulating auxins, gibberellins and ABA degradation products, as well as metabolites associated with unripe fruits. CONCLUSIONS: AZA completely halted strawberry ripening by altering the hormone balance, and the expression of genes involves in hormone biosynthesis and degradation processes. These results contradict those previously obtained in other climacteric and fleshly fruits, where AZA led to premature ripening. In any case, our results suggests that the strawberry ripening process is governed by methylation marks.

The fruit-specific transcription factor FaDOF2 regulates the production of eugenol in ripe fruit receptacles.

Only a few transcription factors have been described in the regulation of the strawberry (Fragaria x ananassa) fruit ripening process. Using a transcriptomic approach, we identified and functionally characterized FaDOF2, a DOF-type ripening-related transcription factor, which is hormonally regulated and specific to the receptacle, though high expression levels were also found in petals. The expression pattern of FaDOF2 correlated with eugenol content, a phenylpropanoid volatile, in both fruit receptacles and petals. When FaDOF2 expression was silenced in ripe strawberry receptacles, the expression of FaEOBII and FaEGS2, two key genes involved in eugenol production, were down-regulated. These fruits showed a concomitant decrease in eugenol content, which confirmed that FaDOF2 is a transcription factor that is involved in eugenol production in ripe fruit receptacles. By using the yeast two-hybrid system and bimolecular fluorescence complementation, we demonstrated that FaDOF2 interacts with FaEOBII, a previously reported regulator of eugenol production, which determines fine-tuning of the expression of key genes that are involved in eugenol production. These results provide evidence that FaDOF2 plays a subsidiary regulatory role with FaEOBII in the expression of genes encoding enzymes that control eugenol production. Taken together, our results provide new insights into the regulation of the volatile phenylpropanoid pathway in ripe strawberry receptacles.

An R2R3-MYB Transcription Factor Regulates Eugenol Production in Ripe Strawberry Fruit Receptacles.

Eugenol is a volatile phenylpropanoid that contributes to flower and ripe fruit scent. In ripe strawberry (Fragaria × ananassa) fruit receptacles, eugenol is biosynthesized by eugenol synthase (FaEGS2). However, the transcriptional regulation of this process is still unknown. We have identified and functionally characterized an R2R3 MYB transcription factor (emission of benzenoid II [FaEOBII]) that seems to be the orthologous gene of PhEOBII from Petunia hybrida, which contributes to the regulation of eugenol biosynthesis in petals. The expression of FaEOBII was ripening related and fruit receptacle specific, although high expression values were also found in petals. This expression pattern of FaEOBII correlated with eugenol content in both fruit receptacle and petals. The expression of FaEOBII was repressed by auxins and activated by abscisic acid, in parallel to the ripening process. In ripe strawberry receptacles, where the expression of FaEOBII was silenced, the expression of cinnamyl alcohol dehydrogenase1 and FaEGS2, two structural genes involved in eugenol production, was down-regulated. A subsequent decrease in eugenol content in ripe receptacles was also observed, confirming the involvement of FaEOBII in eugenol metabolism. Additionally, the expression of FaEOBII was under the control of FaMYB10, another R2R3 MYB transcription factor that regulates the early and late biosynthetic genes from the flavonoid/phenylpropanoid pathway. In parallel, the amount of eugenol in FaMYB10-silenced receptacles was also diminished. Taken together, these data indicate that FaEOBII plays a regulating role in the volatile phenylpropanoid pathway gene expression that gives rise to eugenol production in ripe strawberry receptacles.

Metabolic interaction between anthocyanin and lignin biosynthesis is associated with peroxidase FaPRX27 in strawberry fruit.

Plant phenolics have drawn increasing attention due to their potential nutritional benefits. Although the basic reactions of the phenolics biosynthetic pathways in plants have been intensively analyzed, the regulation of their accumulation and flux through the pathway is not that well established. The aim of this study was to use a strawberry (Fragaria × ananassa) microarray to investigate gene expression patterns associated with the accumulation of phenylpropanoids, flavonoids, and anthocyanins in strawberry fruit. An examination of the transcriptome, coupled with metabolite profiling data from different commercial varieties, was undertaken to identify genes whose expression correlated with altered phenolics composition. Seventeen comparative microarray analyses revealed 15 genes that were differentially (more than 200-fold) expressed in phenolics-rich versus phenolics-poor varieties. The results were validated by heterologous expression of the peroxidase FaPRX27 gene, which showed the highest altered expression level (more than 900-fold). The encoded protein was functionally characterized and is assumed to be involved in lignin formation during strawberry fruit ripening. Quantitative trait locus analysis indicated that the genomic region of FaPRX27 is associated with the fruit color trait. Down-regulation of the CHALCONE SYNTHASE gene and concomitant induction of FaPRX27 expression diverted the flux from anthocyanins to lignin. The results highlight the competition of the different phenolics pathways for their common precursors. The list of the 15 candidates provides new genes that are likely to impact polyphenol accumulation in strawberry fruit and could be used to develop molecular markers to select phenolics-rich germplasm.

MYB10 plays a major role in the regulation of flavonoid/phenylpropanoid metabolism during ripening of Fragaria x ananassa fruits.

This work characterized the role of the R2R3-MYB10 transcription factor (TF) in strawberry fruit ripening. The expression of this TF takes place mainly in the fruit receptacle and is repressed by auxins and activated by abscisic acid (ABA), in parallel to the ripening process. Anthocyanin was not produced when FaMYB10 expression was transiently silenced in fruit receptacles. An increase in FaMYB10 expression was observed in water-stressed fruits, which was accompanied by an increase in both ABA and anthocyanin content. High-throughput transcriptomic analyses performed in fruits with downregulated FaMYB10 expression indicated that this TF regulates the expression of most of the Early-regulated Biosynthesis Genes (EBGs) and the Late-regulated Biosynthesis Genes (LBGs) genes involved in anthocyanin production in ripened fruit receptacles. Besides, the expression of FaMYB10 was not regulated by FaMYB1 and vice versa. Taken together, all these data clearly indicate that the Fragaria × ananassa MYB10 TF plays a general regulatory role in the flavonoid/phenylpropanoid pathway during the ripening of strawberry.

The strawberry (Fragariaxananassa) fruit-specific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes an enzyme involved in the degradation of cell-wall middle lamellae.

Pectins are essential components of primary plant cell walls and middle lamellae, and are related to the consistency of the fruit and its textural changes during ripening. In fact, strawberries become soft as the middle lamellae of cortical parenchyma cells are extensively degraded during ripening, leading to the observed short post-harvest shelf life. Using a custom-made oligonucleotide-based strawberry microarray platform, a putative rhamnogalacturonate lyase gene (FaRGlyase1) was identified. Bioinformatic analysis of the FaRGlyase1 sequence allowed the identification of a conserved rhamnogalacturonate lyase domain, which was also present in other putative RGlyase sequences deposited in the databases. Expression of FaRGlyase1 occurred mainly in the receptacle, concurrently with ripening, and it was positively regulated by abscisic acid and negatively by auxins. FaRGLyase1 gene expression was transiently silenced by injecting live Agrobacterium cells harbouring RNA interference constructs into fruit receptacles. Light and electron microscopy analyses of these transiently silenced fruits revealed that this gene is involved in the degradation of pectins present in the middle lamella region between parenchymatic cells. In addition, genetic linkage association analyses in a strawberry-segregating population showed that FaRGLyase1 is linked to a quantitative trait loci linkage group related to fruit hardness and firmness. The results showed that FaRGlyase1 could play an important role in the fruit ripening-related softening process that reduces strawberry firmness and post-harvest life.

Textbook outcome in gastric surgery, what implications does it have on survival?

INTRODUCTION: The textbook outcome (TO) is a multidimensional measure to assess the quality of healthcare practice. This is reflected as the "ideal" surgical result, attending to a series of indicators or established reference points that are adapted depending on the surgical disease that we want to analyze. There are few references and series published about TO, all of them very recent. OBJECTIVE: We present a series of gastric surgery from the TO perspective and we analyze its impact on survival. METHOD: Retrospective observational study of all gastric neoplasms operated on in our center. PERIOD: January 2015-December 2020. The criteria for TO were: margins R0, >15 lymph nodes in the histological study, no Clavien-Dindo complications > IIIa, hospital stay < 21 days, no mortality or readmission in the 30 postoperative days. A comparative analysis was performed between the TO group versus the non-TO group. RESULTS: 91 patients were operated on. We reached the TO in 34.1% of the patients. The variable >15 lymph nodes was the one that most affected to achieve a TO. When performing the survival analysis, we obtained that the group in which the TO was obtained had a greater survival (p < 0.008). CONCLUSION: In our series, obtaining the TO has an impact on survival which 34,1% of degree of compliance.

Textbook Outcome in Bariatric Surgery: Evolution During 15 Years in a Referral Center.

BACKGROUND: Textbook outcome (TO) is a multidimensional measure used to assess the quality of care. It is the "ideal" surgical result, based on a series of established indicators. In the field of bariatric surgery (BS), only one publication on TO is available. OBJECTIVES: To determine TO in our BS unit and identify the factors linked to TO. SETTING: University public hospital in Alicante (Spain). METHODS: Retrospective observational study of all primary BS was performed. TO for BS was defined in relation to the following features: no major postoperative complications (Clavien-Dindo >II), hospital stay <75th percentile, and no mortality or readmissions within 30 days of surgery. Comparative analysis of the characteristics of the TO and non-TO groups was performed, as well as univariate and multivariate logistic regressions, to identify the independent factors associated with obtaining TO. RESULTS: In 970 patients, TO was achieved in 71.5%. The hospital stay was the one that most affected achievement of TO. Analysis according to the type of procedure (sleeve gastrectomy and gastric bypass) did not reveal any differences between both procedures in terms of obtaining TO (71.5 vs 71.26%). Logistic regression identified smoking, heart disease, operative time, and upper gastrointestinal bleeding as independent factors associated with obtaining TO (p<0.05). Analysis of the annual evolution of TO reveals a progressive increase in its achievement (7.7-86.4%). CONCLUSION: In our series, TO was obtained in 71.5% of patients. The standardization of the technique and the experience gained over the years has improved our TO results.

The fruit ripening-related gene FaAAT2 encodes an acyl transferase involved in strawberry aroma biogenesis.

Short-chain esters contribute to the blend of volatiles that define the strawberry aroma. The last step in their biosynthesis involves an alcohol acyltransferase that catalyses the esterification of an acyl moiety of acyl-CoA with an alcohol. This study identified a novel strawberry alcohol acyltransferase gene (FaAAT2) whose expression pattern during fruit receptacle growth and ripening is in accordance with the production of esters throughout strawberry fruit ripening. The full-length FaAAT2 cDNA was cloned and expressed in Escherichia coli and its activity was analysed with acyl-CoA and alcohol substrates. The semi-purified FaAAT2 enzyme had activity with C1-C8 straight-chain alcohols and aromatic alcohols in the presence of acetyl-CoA. Cinnamyl alcohol was the most efficient acyl acceptor. When FaAAT2 expression was transiently downregulated in the fruit receptacle by agroinfiltration, the volatile ester production was significantly reduced in strawberry fruit. The results suggest that FaAAT2 plays a significant role in the production of esters that contribute to the final strawberry fruit flavour.

Strawberry fruit FanCXE1 carboxylesterase is involved in the catabolism of volatile esters during the ripening process.

Volatile compounds produced during ripening of strawberry are key determinants of fruit quality and consumer preference. Strawberry volatiles are largely esters which are synthesized by alcohol acyltransferases (AATs) and degraded by carboxylesterases (CXEs). Although CXE activity can have a marked influence on volatile contents in ripe strawberry fruits, CXE function and regulation in them are poorly known. Here, we report the biochemical and functional characterization of the fruit receptacle-specific and ripening-related carboxylesterase FanCXE1. The expression of the corresponding gene was found to be antagonistically regulated by auxins and abscisic acid, key hormones that regulate fruit growth and ripening in strawberry. In vitro, FanCXE1 was able to hydrolyze artificial ester substrates similar to those produced by ripe strawberry fruits. Transient suppression of the FanCXE1 gene by RNAi resulted in an increase of important volatile esters such as methyl hexanoate, methyl butanoate and ethyl hexanoate as well as a decrease of the alcohols hexenol and linanool. The results of this work enhance our understanding of the molecular basis for volatile syntheses and facilitate production of better flavored strawberry fruits by introduction of the relevant alleles into common cultivars.

Early postoperative complications after gastric bypass revisional surgery in patients with previous sleeve gastrectomy versus primary gastric bypass.

BACKGROUND: Revisional bariatric surgery (RS) is indicated if there is weight regain or insufficient weight loss, no improvement or reappearance of co-morbidities, or previous bariatric surgery complications. It has been associated with higher postoperative morbidity. OBJECTIVE: To evaluate the early postoperative complications (<30 d) of Roux-en-Y gastric bypass RS (RYGB-RS) after primary sleeve gastrectomy (SG-1) compared with primary RYGB (RYGB-1) at a bariatric surgery referral center. SETTING: Department of General and Digestive Surgery of General Universitary Hospital of Alicante, Spain. METHODS: Retrospective cohort study comparing RYGB-RS after SG-1 and RYGB-1 between January 2008 and March 2021. Postoperative complications, hospital stay, mortality, and readmissions were analyzed. RESULTS: Six hundred and twenty-eight RYGB surgeries (48 RYGB-RS, 580 RYGB-1) were studied. The mean age of patients undergoing RYGB-RS was 50 years, compared with 46 years in the RYGB-1 group (P = .017). Mean initial body mass index was 44.2 kg/m2 (RYGB-RS) versus 47.6 kg/m2 (RYGB-1; P = .004). Cardiovascular risk factors were higher in the RYGB-1 group (P < .05). Indications for RS were weight regain or insufficient weight loss (72.9%), weight regain or insufficient weight loss plus gastroesophageal reflux disease (14.6%), and gastroesophageal reflux disease (12.5%). There were no differences in the frequency of complications (RYGB-RS 22.9% vs RYGB-1 20.5%) or in their severity (Clavien-Dindo ≥IIIa; RYGB-RS 10.4% vs RYGB-1 6.4%; P > .05). There were no differences in emergency room visits (RYGB-RS at 12.5% vs RYGB-1 at 14.9%) or in readmissions (RYGB-RS at 12.5% vs RYGB-1 at 9.4%). CONCLUSION: No differences were observed between primary RYGB and revisional RYGB in early morbidity, mortality, emergencies, or readmissions. Revisional bariatric surgery is a safe procedure at referral centers and must be done by expert hands.

cpFBPaseII, a novel redox-independent chloroplastic isoform of fructose-1,6-bisphosphatase.

A full-length FBPase cDNA has been isolated from Fragaria x ananassa (strawberry) corresponding to a novel putative chloroplastic FBPase but lacking the regulatory redox domain, a characteristic of the plastidial isoenzyme (cpFBPaseI). Another outstanding feature of this novel isoform, called cpFBPaseII, is the absence of the canonical active site. Enzymatic assays with cpFBPaseII evidenced clear Mg(2+)-dependent FBPase activity and a K(m) for fructose-1,6-bisphosphate (FBP) of 1.3 mM. Immunolocalization experiments and chloroplast isolation confirmed that the new isoenzyme is located in the stroma. Nevertheless, unlike cpFBPaseI, which is redox activated, cpFBPaseII did not increase its activity in the presence of either DTT or thioredoxin f (TRX f) and is resistant to H(2)O(2) inactivation. Additionally, the novel isoform was able to complement the growth deficiency of the yeast FBP1 deletion fed with a non-fermentable carbon source. Furthermore, orthologues are restricted to land plants, suggesting that cpFBPaseII is a novel and an intriguing chloroplastic FBPase that emerged late in the evolution of photosynthetic organisms, possibly because of a pressing need of land plants.

Genome-wide analysis of the NAC transcription factor family and their expression during the development and ripening of the Fragaria × ananassa fruits.

NAC proteins are a family of transcription factors which have a variety of important regulatory roles in plants. They present a very well conserved group of NAC subdomains in the N-terminal region and a highly variable domain at the C-terminus. Currently, knowledge concerning NAC family in the strawberry plant remains very limited. In this work, we analyzed the NAC family of Fragaria vesca, and a total of 112 NAC proteins were identified after we curated the annotations from the version 4.0.a1 genome. They were placed into the ligation groups (pseudo-chromosomes) and described its physicochemical and genetic features. A microarray transcriptomic analysis showed six of them expressed during the development and ripening of the Fragaria x ananassa fruit. Their expression patterns were studied in fruit (receptacle and achenes) in different stages of development and in vegetative tissues. Also, the expression level under different hormonal treatments (auxins, ABA) and drought stress was investigated. In addition, they were clustered with other NAC transcription factor with known function related to growth and development, senescence, fruit ripening, stress response, and secondary cell wall and vascular development. Our results indicate that these six strawberry NAC proteins could play different important regulatory roles in the process of development and ripening of the fruit, providing the basis for further functional studies and the selection for NAC candidates suitable for biotechnological applications.

Equations to estimate fetal age at the moment of death in the Mexican population.

Metric standards are presented for the estimation of fetal age at the time of death in the Mexican population. To obtain these standards, both metric and radiological studies were conducted on 97 fetuses and complete stillborn infants of both sexes, phenotypically normal between 10 and 38 weeks of morphological age. All the fetuses used were the product of spontaneous abortions in Mexico City between 1990 and 2000. Equations were obtained by calibrating quadratic linear regression models adjusted for the diaphyseal length of the humerus, radius, ulna, femur, tibia and fibula, characterized as the most adequate indicators to represent the growth of long bones in this age group, and verified by the evaluation of the model assumptions and the coefficient of determination (R(2)). To conclude, these models facilitate a more precise prediction in fetuses of the Mexican population, constituting the first metric standards of their type at a national level.

Discriminant function analysis for sex assessment in pelvic girdle bones: sample from the contemporary Mexican population.

Sex assessment of skeletal remains plays an important role in forensic anthropology. The pelvic bones are the most studied part of the postcranial skeleton for the assessment of sex. It is evident that a population-specific approach improves rates of accuracy within the group. The present study proposes a discriminant function method for the sex assessment of skeletal remains from a contemporary Mexican population. A total of 146 adult human pelvic bones (61 females and 85 males) from the skeletal series pertaining to the National Autonomous University of Mexico were evaluated. Twenty-four direct metrical parameters of coxal and sacral bones were measured and subsequently, sides and sex differences were evaluated, applying a stepwise discriminant function analysis. Coxal and sacra functions achieved accuracies of 99% and 87%, respectively. These analyses follow a population-specific approach; nevertheless, we consider that our results are applicable to any other Hispanic samples for purposes of forensic human identification.

Functional characterization of FaCCD1: a carotenoid cleavage dioxygenase from strawberry involved in lutein degradation during fruit ripening.

A gene encoding a carotenoid cleavage dioxygenase class 1 enzyme (FaCCD1) was identified among a strawberry fruit expressed sequence tag collection. The full-length cDNA was isolated, and the expression profiles along fruit receptacle development and ripening, determined by quantitative real time polymerase chain reaction, showed that FaCCD1 is a ripening-related gene that reaches its maximal level of expression in the red fully ripe stage. FaCCD1 was expressed in Escherichia coli, and the products formed by the recombinant protein through oxidative cleavage of carotenoids were identified by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry analyses. The FaCCD1 protein cleaves zeaxanthin, lutein, and beta-apo-8'-carotenal in vitro. Although beta-carotene is not a good substrate for FaCCD1 in vitro, the expression of FaCCD1 in an engineered carotenoid-producing E. coli strain caused the degradation of beta-carotene in vivo. Additionally, the carotenoid profile in strawberry was analyzed by high-performance liquid chromatography-photodiode detection, and a correlation between the increase of the expression level of FaCCD1 during ripening and the decrease of the lutein content suggests that lutein could constitute the main natural substrate of FaCCD1 activity in vivo.

FaQR, required for the biosynthesis of the strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone, encodes an enone oxidoreductase.

The flavor of strawberry (Fragaria x ananassa) fruit is dominated by an uncommon group of aroma compounds with a 2,5-dimethyl-3(H)-furanone structure. We report the characterization of an enzyme involved in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol), the key flavor compound in strawberries. Protein extracts were partially purified, and the observed distribution of enzymatic activity correlated with the presence of a single polypeptide of approximately 37 kD. Sequence analysis of two peptide fragments showed total identity with the protein sequence of a strongly ripening-induced, auxin-dependent putative quinone oxidoreductase, Fragaria x ananassa quinone oxidoreductase (FaQR). The open reading frame of the FaQR cDNA consists of 969 bp encoding a 322-amino acid protein with a calculated molecular mass of 34.3 kD. Laser capture microdissection followed by RNA extraction and amplification demonstrated the presence of FaQR mRNA in parenchyma tissue of the strawberry fruit. The FaQR protein was functionally expressed in Escherichia coli, and the monomer catalyzed the formation of HDMF. After chemical synthesis and liquid chromatography-tandem mass spectrometry analysis, 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone was confirmed as a substrate of FaQR and the natural precursor of HDMF. This study demonstrates the function of the FaQR enzyme in the biosynthesis of HDMF as enone oxidoreductase and provides a foundation for the improvement of strawberry flavor and the biotechnological production of HDMF.

Molecular analysis of the interaction between Olea europaea and the biotrophic fungus Spilocaea oleagina.

SUMMARY The mitosporic fungus Spilocaea oleagina is an obligate biotroph of olive (Olea europaea) causing a scab disease associated with leaf fall and substantial losses in production. Using differential display we have identified 162 cDNA fragments corresponding to transcripts that show altered abundance during the defence response of a resistant olive cultivar to S. oleagina. Detailed analyses of 21 selected genes by real-time quantitative RT-PCR revealed different kinetics of induction. Genes involved in signalling, transcriptional control, oxidative stress, biotic and abiotic stress, and several genes with unknown function were found to be induced rapidly after infection. In contrast, genes involved in metabolism and cellular maintenance showed delayed induction. The induction of the selected genes in a susceptible cultivar was delayed and/or reduced during the response to S. oleagina. Interestingly, the basal expression of some genes in the uninfected resistant cultivar was higher than in the susceptible one, suggesting a constitutive activation of defence responses. Expression of these genes in response to salicylic acid, methyl jasmonate, a mixture of both, ethephon, hydrogen peroxide, menadione and wounding was also investigated. The results are discussed in relation to the molecular bases and signalling events involved in this biotrophic interaction.

Ribonucleoside diphosphate reductase is a component of the replication hyperstructure in Escherichia coli.

Although the nrdA101 allele codes for a ribonucleoside diphosphate (rNDP) reductase that is essentially destroyed in less than 2 min at 42 degrees C, and chemical inhibition of the enzyme by hydroxyurea stops DNA synthesis at once, we found that incubation at 42 degrees C of an Escherichia coli strain containing this allele allows DNA replication for about 40min. This suggests that mutant rNDP reductase is protected from thermal inactivation by some hyperstructure. If, together with the temperature upshift, RNA or protein synthesis is inhibited, the thermostability time of the mutant rNDP reductase becomes at least as long as the replication time and residual DNA synthesis becomes a run-out replication producing fully replicated chromosomes. This suggests that cessation of replication in the nrdA101 mutant strain is not the result of inactivation of its gene product but of the activity of a protein reflecting the presence of a partially altered enzyme. The absence of Tus protein, which specifically stops the replication complex by inhibiting replicative helicase activity, allows forks to replicate for a longer time at the restrictive temperature in the nrdA101 mutant strain. We therefore propose that rNDP reductase is a component of the replication complex, and that this association with other proteins protects the protein coded by allele nrdA101 from thermal inactivation.

Identification of a strawberry gene encoding a non-specific lipid transfer protein that responds to ABA, wounding and cold stress.

cDNA and genomic clones encoding a strawberry (Fragariaxananassa cv. Chandler) non-specific lipid transfer protein (Fxaltp gene) were isolated and characterized. The spatio-temporal expression pattern and structural features of this gene were studied for the first time in strawberry, a non-climacteric fruit of agricultural importance. The architecture and the encoded amino acid sequence of this non-climacteric fruit ltp gene were similar to those of other plant LTPs previously reported, and presents the eight cysteine residues and other features characteristic of plant LTPs. In addition, the deduced protein posseses an N-terminal signal peptide and lacks the K/HDEL retention signal, indicating that the strawberry LTP protein would enter the secretory pathway. In situ studies have shown that the Fxaltp gene is expressed in the epidermal cell layer of the strawberry fruit receptacle and achenes, flowers, and within the cell layer surrounding the endosperm. These results suggest that this Fxaltp gene promoter could be used as an endogenous promoter for biotechnological purposes in strawberry. Computer analysis using the PLACE database predicted the presence of several putative cis-regulatory sequences in response to abscisic acid and cold or wounding stresses within the Fxaltp 5'-flanking region. Accordingly, the strawberry gene responds to ABA and SA, but not to salt and heat stresses. It is also reported that ltp gene expression in strawberry is stimulated by wounding and repressed by cold stresses.