Mandelonitrile lyase MDL2-mediated regulation of seed amygdalin and oil accumulation of Prunus Sibirica.

BACKGROUND: The Prunus sibirica seeds with rich oils has great utilization, but contain amygdalin that can be hydrolyzed to release toxic HCN. Thus, how to effectively reduce seed amygdalin content of P. sibirica is an interesting question. Mandelonitrile is known as one key intermediate of amygdalin metabolism, but which mandelonitrile lyase (MDL) family member essential for its dissociation destined to low amygdalin accumulation in P. sibirica seeds still remains enigmatic. An integration of our recent 454 RNA-seq data, amygdalin and mandelonitrile content detection, qRT-PCR analysis and function determination is described as a critical attempt to determine key MDL and to highlight its function in governing mandelonitrile catabolism with low amygdalin accumulation in Prunus sibirica seeds for better developing edible oil and biodiesel in China. RESULTS: To identify key MDL and to unravel its function in governing seed mandelonitrile catabolism with low amygdalin accumulation in P. sibirica. Global identification of mandelonitrile catabolism-associated MDLs, integrated with the across-accessions/developing stages association of accumulative amount of amygdalin and mandelonitrile with transcriptional level of MDLs was performed on P. sibirica seeds of 5 accessions to determine crucial MDL2 for seed mandelonitrile catabolism of P. sibirica. MDL2 gene was cloned from the seeds of P. sibirica, and yeast eukaryotic expression revealed an ability of MDL2 to specifically catalyze the dissociation of mandelonitrile with the ideal values of Km (0.22 mM) and Vmax (178.57 U/mg). A combination of overexpression and mutation was conducted in Arabidopsis. Overexpression of PsMDL2 decreased seed mandelonitrile content with an increase of oil accumulation, upregulated transcript of mandelonitrile metabolic enzymes and oil synthesis enzymes (involving FA biosynthesis and TAG assembly), but exhibited an opposite situation in mdl2 mutant, revealing a role of PsMDL2-mediated regulation in seed amygdalin and oil biosynthesis. The PsMDL2 gene has shown as key molecular target for bioengineering high seed oil production with low amygdalin in oilseed plants. CONCLUSIONS: This work presents the first integrated assay of genome-wide identification of mandelonitrile catabolism-related MDLs and the comparative association of transcriptional level of MDLs with accumulative amount of amygdalin and mandelonitrile in the seeds across different germplasms and developmental periods of P. sibirica to determine MDL2 for mandelonitrile dissociation, and an effective combination of PsMDL2 expression and mutation, oil and mandelonitrile content detection and qRT-PCR assay was performed to unravel a mechanism of PsMDL2 for controlling amygdalin and oil production in P. sibirica seeds. These findings could offer new bioengineering strategy for high oil production with low amygdalin in oil plants.

Cloning and characterization of the Cerasus humilis sucrose phosphate synthase gene (ChSPS1).

Sucrose is crucial to the growth and development of plants, and sucrose phosphate synthase (SPS) plays a key role in sucrose synthesis. To understand the genetic and molecular mechanisms of sucrose synthesis in Cerasus humilis, ChSPS1, a homologue of SPS, was cloned using RT-PCR. Sequence analysis showed that the open reading frame (ORF) sequence of ChSPS1 is 3174 bp in length, encoding a predicted protein of 1057 amino acids. The predicted protein showed a high degree of sequence identity with SPS homologues from other species. Real-time RT-PCR analysis showed that ChSPS1 mRNA was detected in all tissues and the transcription level was the highest in mature fruit. There is a significant positive correlation between expression of ChSPS1 and sucrose content. Prokaryotic expression of ChSPS1 indicated that ChSPS1 protein was expressed in E. coli and it had the SPS activity. Overexpression of ChSPS1 in tobacco led to upregulation of enzyme activity and increased sucrose contents in transgenic plants. Real-time RT-PCR analysis showed that the expression of ChSPS1 in transgenic tobacco was significantly higher than in wild type plants. These results suggested that ChSPS1 plays an important role in sucrose synthesis in Cerasus humilis.

Crosslinked enzyme aggregates of hydroxynitrile lyase partially purified from Prunus dulcis seeds and its application for the synthesis of enantiopure cyanohydrins.

Hydroxynitrile lyases are powerful catalysts in the synthesis of enantiopure cyanohydrins which are key synthons in the preparations of a variety of important chemicals. The response surface methodology including three-factor and three-level Box-Behnken design was applied to optimize immobilization of hydroxynitrile lyase purified partially from Prunus dulcis seeds as crosslinked enzyme aggregates (PdHNL-CLEAs). The quadratic model was developed for predicting the response and its adequacy was validated with the analysis of variance test. The optimized immobilization parameters were initial glutaraldehyde concentration, ammonium sulfate saturation concentration, and crosslinking time, and the response was relative activity of PdHNL-CLEA. The optimal conditions were determined as initial glutaraldehyde concentration of 25% w/v, ammonium sulfate saturation concentration of 43% w/v, and crosslinking time of 18 h. The preparations of PdHNL-CLEA were examined for the synthesis of (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, (R)-4-bromomandelonitrile, (R)-4-fluoromandelonitrile, and (R)-4-nitromandelonitrile from their corresponding aldehydes and hydrocyanic acid. After 96-h reaction time, the yield-enantiomeric excess values (%) were 100-99, 100-21, 100-99, 83-91, 100-99, 100-72, and 100-14%, respectively, for (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, (R)-4-bromomandelonitrile, (R)-4-fluoromandelonitrile, and (R)-4-nitromandelonitrile. The results show that PdHNL-CLEA offers a promising potential for the preparation of enantiopure (R)-mandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, and (R)-4-bromomandelonitrile with a high yield and enantiopurity.

Glucitol dehydrogenase from peach (Prunus persica) fruits is regulated by thioredoxin h.

Glucitol (Gol) is a major photosynthetic product in plants from the Rosaceae family. Herein we report the molecular cloning, heterologous expression and characterization of Gol dehydrogenase (GolDHase, EC 1.1.1.14) from peach (Prunus persica) fruits. The recombinant enzyme showed kinetic parameters similar to those reported for orthologous enzymes purified from apple and pear fruits. The activity of recombinant GolDHase was strongly inhibited by Cu(2+) and Hg(2+), suggesting that it might have cysteine residues critical for functionality. Oxidizing compounds (such as diamide, hydrogen peroxide and oxidized glutathione) inactivated the enzyme, whereas its activity was restored after incubation with reduced glutathione and thioredoxin from Escherichia coli. Recombinant thioredoxin h from peach fruits also recovered the activity of oxidized GolDHase. Our results suggest that peach fruit GolDHase could be redox regulated in vivo and this would be of relevance to determine carbon assimilation and partitioning in plants accumulating sugar alcohols.

Increased levels of IAA are required for system 2 ethylene synthesis causing fruit softening in peach (Prunus persica L. Batsch).

The fruit of melting-flesh peach (Prunus persica L. Batsch) cultivars produce high levels of ethylene caused by high expression of PpACS1 (an isogene of 1-aminocyclopropane-1-carboxylic acid synthase), resulting in rapid fruit softening at the late-ripening stage. In contrast, the fruit of stony hard peach cultivars do not soften and produce little ethylene due to low expression of PpACS1. To elucidate the mechanism for suppressing PpACS1 expression in stony hard peaches, a microarray analysis was performed. Several genes that displayed similar expression patterns as PpACS1 were identified and shown to be indole-3-acetic acid (IAA)-inducible genes (Aux/IAA, SAUR). That is, expression of IAA-inducible genes increased at the late-ripening stage in melting flesh peaches; however, these transcripts were low in mature fruit of stony hard peaches. The IAA concentration increased suddenly just before harvest time in melting flesh peaches exactly coinciding with system 2 ethylene production. In contrast, the IAA concentration did not increase in stony hard peaches. Application of 1-naphthalene acetic acid, a synthetic auxin, to stony hard peaches induced a high level of PpACS1 expression, a large amount of ethylene production and softening. Application of an anti-auxin, α-(phenylethyl-2-one)-IAA, to melting flesh peaches reduced levels of PpACS1 expression and ethylene production. These observations indicate that suppression of PpACS1 expression at the late-ripening stage of stony hard peach may result from a low level of IAA and that a high concentration of IAA is required to generate a large amount of system 2 ethylene in peaches.

Purification and chemical characterisation of a cell wall-associated ß-galactosidase from mature sweet cherry (Prunus avium L.) fruit.

Using four different chromatographic steps, ß-galactosidase was purified from the ripe fruit of sweet cherry to apparent electrophoretic homogeneity with approximately 131-fold purification. The Prunus avium ß-galactosidase showed an apparent molecular mass of about 100 kDa and consisted of four different active polypeptides with pIs of about 7.9, 7.4, 6.9 and 6.4 as estimated by native IEF and ß-galactosidase-activity staining. The active polypeptides were individually excised from the gel and subjected to SDS-PAGE. Each of the four native enzymes showing ß-galactosidase activity was composed of two polypeptides with an estimated mass of 54 and 33 kDa. Both of these polypeptides were subjected to N-terminal amino acid sequence analysis. The 54 kDa polypeptide of sweet cherry ß-galactosidase showed a 43% identity with the 44 kDa subunit of persimmon and apple ß-galactosidases and the 48 kDa subunit of carambola galactosidase I. The sweet cherry ß-galactosidase exhibited a strict specificity towards p-nitrophenyl ß-D-galactopyranoside, a pH optimum of 4.0 and K(m) and V(max) values of 0.42 mM and 4.12 mmol min(-1) mg(-1) of protein respectively with this substrate. The enzyme was also active towards complex glycans. Taken together the results of this study prompted a role for this class of enzymes on sweet cherry fruit ripening and softening.

Postharvest polyamine application alleviates chilling injury and affects apricot storage ability.

Fruit of two apricot cultivars 'Bagheri' and 'Asgarabadi' were treated with putrescine (Put) or spermidine (Spd) at 1 mM and then were stored at 1 °C for 21 days. Fruit were sampled weekly and stored 2 days at 20 °C for shelf-life study. The treatments reduced ethylene production and maintained the firmness and color of the fruit. Peroxidase (POX), catalase (CAT), superoxide dismutase (SOD), and polyphenol oxidase (PPO) activities and total phenol (TP) concentrations were measured during storage. Both cultivars showed chilling injury (CI) incidence, and the severity in control fruit was higher than either Put or Spd treatments. CI incidence in Spd-treated fruit was lower than that of Put-treated fruit. Polyamine (PA) treatment generally increased antioxidant enzyme activity of fruit during storage. PA treatments may help maintain the quality of apricot fruit during storage by inhibiting ripening and decreasing CI incidence.

Cloning of NAD-SDH cDNA from plum fruit and its expression and characterization.

A full-length cDNA consisting of 1444 bp for NAD dependent sorbitol dehydrogenase (NAD-SDH) was cloned from fruit of plum (Prunus salicina var. cordata cv. Younai) by means of RT-PCR and RACE. The cDNA containing an open reading frame (ORF) of 1101 bp encoded a polypeptide of 367 amino acid residues. The maltose binding protein fusion SDH (MBP-SDH) was expressed and partially purified from Escherichia coli cells, and biochemical properties of MBP-SDH and SDH cleaved from the fusion protein by factor Xa were characterized. The MBP-SDH had the specific affinity for NAD and was able to oxidize sorbitol, xylitol, l-ribitol and mannitol but not ethyl alcohol, arabitol and other polyols. The optimum pH for the oxidation of sorbitol and the reduction of fructose was 9.0 and 7.0, respectively; the maximum reaction rate occurred when temperature increased up to 50 °C in the presence of sorbitol. The MBP-SDH with a subunit of 80 kDa appears to be a hexamer. Its molecular weight was 478.6 kDa estimated by gel filtration and 493.2 kDa estimated using native linear gradient PAGE. The K(m) values for sorbitol, NAD, fructose and NADH were 95.86 mM, 0.31 mM, 1.04 M and 0.038 mM, respectively. However, when MBP was cleaved from the fusion enzyme, the SDH exists as a homotetramer with the native molecular weight of 164.8 kDa estimated by gel filtration. The K(m) values were 111.8 mM, 0.35 mM, 1.25 M and 0.048 mM for sorbitol, NAD, fructose and NADH, respectively. The MBP-SDH and the SDH were similar with respect to their kinetic characteristics despite their difference in quaternary structures.

Development and metabolism of the fruit and seed of the Japanese plum Ozark premier (Rosaceae).

The growth characteristics of some plums and their component parts have been previously studied, as have some aspects of their developmental anatomy and composition. However, little is known about either their metabolism or about the interactions between the metabolism of their component parts. In this study we investigated these aspects in the Japanese plum Ozark Premier. Throughout fruit and seed development, changes in sugar and organic acid contents, protein composition and abundance of selected enzymes were determined. In the stone, there was a transient accumulation of vegetative storage proteins. These were subsequently mobilized and this coincided with the onset of the lignification of the stone and the start of storage protein accumulation in the seed. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was present in the seeds, even though they lacked chlorophyll, and its presence may be related to limited gas exchange. In the flesh of some fruits, phosphoenolpyruvate carboxykinase (PEPCK) and NADP malic enzyme (NADP-ME) are thought to function in the dissimilation of malate and/or citrate during ripening. However, PEPCK and NADP-ME were present in plum flesh for most of its development, although there was no net dissimilation of malate until the latter stages of ripening. There is an interaction between the developing seed and endocarp with respect to the utilization of imported sugars and amino acids. An hypothesis is presented to account for the presence of PEPCK and NADP-ME enzyme in plum flesh when there was no net dissimilation of organic acids.

Metabolic profiling during peach fruit development and ripening reveals the metabolic networks that underpin each developmental stage.

Fruit from rosaceous species collectively display a great variety of flavors and textures as well as a generally high content of nutritionally beneficial metabolites. However, relatively little analysis of metabolic networks in rosaceous fruit has been reported. Among rosaceous species, peach (Prunus persica) has stone fruits composed of a juicy mesocarp and lignified endocarp. Here, peach mesocarp metabolic networks were studied across development using metabolomics and analysis of key regulatory enzymes. Principal component analysis of peach metabolic composition revealed clear metabolic shifts from early through late development stages and subsequently during postharvest ripening. Early developmental stages were characterized by a substantial decrease in protein abundance and high levels of bioactive polyphenols and amino acids, which are substrates for the phenylpropanoid and lignin pathways during stone hardening. Sucrose levels showed a large increase during development, reflecting translocation from the leaf, while the importance of galactinol and raffinose is also inferred. Our study further suggests that posttranscriptional mechanisms are key for metabolic regulation at early stages. In contrast to early developmental stages, a decrease in amino acid levels is coupled to an induction of transcripts encoding amino acid and organic acid catabolic enzymes during ripening. These data are consistent with the mobilization of amino acids to support respiration. In addition, sucrose cycling, suggested by the parallel increase of transcripts encoding sucrose degradative and synthetic enzymes, appears to operate during postharvest ripening. When taken together, these data highlight singular metabolic programs for peach development and may allow the identification of key factors related to agronomic traits of this important crop species.

Effect of jasmonates on ethylene biosynthesis and aroma volatile emission in Japanese apricot infected by a pathogen (Colletotrichum gloeosporioides).

The effects of the application of the jasmonic acid derivative n-propyl dihydrojasmonate (PDJ) on ethylene biosynthesis, volatile compounds, and endogenous jasmonic acid (JA) and methyl jasmonate (MeJA) were examined in Japanese apricot (Prunus mume Sieb.) infected by a pathogen (Colletotrichum gloeosporioides). The fruit were dipped into 0.4 mM PDJ solution before inoculation with the pathogen and stored at 25 °C for 6 days. The inoculation induced an increase in 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene, JA, and MeJA. In contrast, PDJ application reduced the endogenous JA, MeJA, and ethylene production and expression of the ACC oxidase gene (PmACO1) caused by the pathogen infection. The lesion diameter with C. gloeosporioides decreased upon PDJ application. The alcohol, ester, ketone, and lactone concentrations and alcohol acyltransferase (AAT) activity increased in the pathogen-infected fruit, but were decreased by PDJ application. These results suggest that PDJ application might influence ethylene production through PmACO1 and that aroma volatile emissions affected by pathogen infection can be correlated with the ethylene production, which is mediated by the levels of jasmonates.

Structure-activity relationships in a series of C2-substituted gluco-configured tetrahydroimidazopyridines as ß-glucosidase inhibitors.

Inhibition of glycoside hydrolases has widespread application in treatment of diabetes, viral infections, lysosomal storage diseases and cancers. Gluco-configured tetrahydroimidazopyridines are the most potent ß-glucosidase inhibitors reported to date. Using transition state mimic strategy, a series of C2-substituted gluco-configured tetrahydroimidazopyridines were designed and synthesized. Compounds 3 (K(i)=0.64 nM) and 5 (K(i)=0.58 nM) showed stronger inhibitory potency against ß-glucosidase. Maestro 9.1 was used to study the structure-activity relationships by docking the compounds into the ß-glucosidase active sites.

Physiological responses and differential gene expression in Prunus rootstocks under iron deficiency conditions.

Two Prunus rootstocks, the Myrobalan plum P 2175 and the interspecific peach-almond hybrid, Felinem, were studied to characterize their biochemical and molecular responses induced under iron-Deficient conditions. Plants of both genotypes were submitted to different treatments using a hydroponic system that permitted removal of Fe from the nutrient solution. Control plants were grown in 90 µM Fe (III)-EDTA, Deficient plants were grown in an iron free solution, and plants submitted to an Inductor treatment were resupplied with 180 µM Fe (III)-EDTA over 1 and 2 days after a period of 4 or 15 days of growth on an iron-free solution. Felinem increased the activity of the iron chelate reductase (FC-R) in the Inductor treatment after 4 days of iron deprivation. In contrast, P 2175 did not show any response after at least 15 days without iron. The induction of the FC-R activity in this genotype was coincident in time with the medium acidification. These results suggest two different mechanisms of iron chlorosis tolerance in both Strategy I genotypes. Felinem would use the iron reduction as the main mechanism to capture the iron from the soil, and in P 2175, the mechanism of response would be slower and start with the acidification of the medium synchronized with the gradual loss of chlorophyll in leaves. To better understand the control of these responses at the molecular level, the differential expression of PFRO2, PIRT1 and PAHA2 genes involved in the reductase activity, the iron transport in roots, and the proton release, respectively, were analyzed. The expression of these genes, estimated by quantitative real-time PCR, was different between genotypes and among treatments. The results were in agreement with the physiological responses observed.

Antioxidant system and protein pattern in peach fruits at two maturation stages.

Peach fruits were selected to study the protein pattern and antioxidant system as well as oxidative parameters such as superoxide radical and hydrogen peroxide accumulation, at two maturity stages, which were chosen for being suitable for the processing industry and fresh consumption. The proteins phosphoenolpyruvate carboxylase, sucrose synthase, and 1-aminocyclopropane-1-carboxylate oxidase, as well as the antioxidants glutathione synthetase and ascorbate peroxidase, appeared as new in the mature peach fruits. Activities of superoxide dismutase (SOD) and components of the ascorbate-glutathione cycle were also measured to explore their role in the two maturity stages studied. Changes in the SOD isoenzyme pattern and an increase in the activities of ascorbate peroxidase, monodehydroascorbate reductase, and glutathione reductase were observed in mature fruits, revealing an efficient system to cope with the oxidative process accompanying ripening.

Chitosan acetate as an active coating material and its effects on the storing of Prunus avium L.

In this article, chitosan acetate (CA) was prepared by the method of solid-liquid reaction. CA was a stable faint yellow powder with water solubility. CA kept the same backbone in the chemical structure as the raw material of chitosan, and it also had the similar antibacterial properties with chitosan. CA could form a coating film on the outside surface of the sweet cherries, could effectively retard the loss of the water, titratable acidity, and ascorbic acid of sweet cherries, and could induce a significant increase in the peroxidase and catalase activities in the fruit. The CA coating could also increase the ratio of the total soluble solids and titratable acidity in the fruit. The application of CA effectively maintained quality attributes and extended postharvest life of the sweet cherries. The results revealed that the CA salts had potential application in active edible coating materials in the storage of fresh fruit.

Molecular and genetic analyses of four nonfunctional S haplotype variants derived from a common ancestral S haplotype identified in sour cherry (Prunus cerasus L.).

Tetraploid sour cherry (Prunus cerasus) has an S-RNase-based gametophytic self-incompatibility (GSI) system; however, individuals can be either self-incompatible (SI) or self-compatible (SC). Unlike the situation in the Solanaceae, where self-compatibility accompanying polyploidization is often due to the compatibility of heteroallelic pollen, the genotype-dependent loss of SI in sour cherry is due to the compatibility of pollen containing two nonfunctional S haplotypes. Sour cherry individuals with the S(4)S(6)S(36a)S(36b) genotype are predicted to be SC, as only pollen containing both nonfunctional S(36a) and S(36b) haplotypes would be SC. However, we previously found that individuals of this genotype were SI. Here we describe four nonfunctional S(36) variants. Our molecular analyses identified a mutation that would confer loss of stylar S function for one of the variants, and two alterations that might cause loss of pollen S function for all four variants. Genetic crosses showed that individuals possessing two nonfunctional S(36) haplotypes and two functional S haplotypes have reduced self-fertilization due to a very low frequency of transmission of the one pollen type that would be SC. Our finding that the underlying mechanism limiting successful transmission of genetically compatible gametes does not involve GSI is consistent with our previous genetic model for Prunus in which heteroallelic pollen is incompatible. This provides a unique case in which breakdown of SI does not occur despite the potential to generate SC pollen genotypes.

Molecular cloning and expression analysis of an arginine decarboxylase gene from peach (Prunus persica).

Arginine decarboxylase (ADC), one of the enzymes responsible for putrescine (Put) biosynthesis, has been shown to be implicated in stress response. In the current paper attempts were made to clone and characterize a gene encoding ADC from peach (Prunus persica (L.) Batsch, 'Akatsuki'). Rapid amplification of cDNA ends (RACE) gave rise to a full-length ADC cDNA (PpADC) with a complete open reading frame of 2178 bp, encoding a 725 amino acid polypeptide. Homology search and sequence multi-alignment demonstrated that the deduced PpADC protein sequence shared a high identity with ADCs from other plants, including several highly conservative motifs and amino acids. Southern blotting indicated that PpADC existed in peach genome as a single gene. Expression levels of PpADC in different tissues of peach (P. persica 'Akatsuki') were spatially and developmentally regulated. Treatment of peach shoots from 'Mochizuki' with exogenous 5 mM Put, an indirect product of ADC, remarkably induced accumulation of PpADC mRNA. Transcripts of PpADC in peach leaves from 'Mochizuki' were quickly induced, either transiently or continuously, in response to dehydration, high salinity (200 mM NaCl), low temperature (4 degrees C) and heavy metal (150 microM CdCl(2)), but repressed by high temperature 37 degrees C) during a 2-day treatment, which changed in an opposite direction when the stresses were otherwise removed with the exception of CdCl(2) treatment. In addition, steady-state of PpADC mRNA could be also transiently up-regulated by abscisic acid (ABA) in 'Mochizuki' leaves. All of these, taken together, suggest that PpADC is a stress-responsive gene and can be considered as a potential target that is genetically manipulated so as to create novel germplasms with enhanced stress tolerance in the future.

Functional characterization of PaLAX1, a putative auxin permease, in heterologous plant systems.

We have isolated the cDNA of the gene PaLAX1 from a wild cherry tree (Prunus avium). The gene and its product are highly similar in sequences to both the cDNAs and the corresponding protein products of AUX/LAX-type genes, coding for putative auxin influx carriers. We have prepared and characterized transformed Nicotiana tabacum and Arabidopsis thaliana plants carrying the gene PaLAX1. We have proved that constitutive overexpression of PaLAX1 is accompanied by changes in the content and distribution of free indole-3-acetic acid, the major endogenous auxin. The increase in free indole-3-acetic acid content in transgenic plants resulted in various phenotype changes, typical for the auxin-overproducing plants. The uptake of synthetic auxin, 2,4-dichlorophenoxyacetic acid, was 3 times higher in transgenic lines compared to the wild-type lines and the treatment with the auxin uptake inhibitor 1-naphthoxyacetic acid reverted the changes caused by the expression of PaLAX1. Moreover, the agravitropic response could be restored by expression of PaLAX1 in the mutant aux1 plants, which are deficient in auxin influx carrier activity. Based on our data, we have concluded that the product of the gene PaLAX1 promotes the uptake of auxin into cells, and, as a putative auxin influx carrier, it affects the content and distribution of free endogenous auxin in transgenic plants.

Trans-specific S-RNase and SFB alleles in Prunus self-incompatibility haplotypes.

Self-incompatibility in the genus Prunus is controlled by two genes at the S-locus, S-RNase and SFB. Both genes exhibit the high polymorphism and high sequence diversity characteristic of plant self-incompatibility systems. Deduced polypeptide sequences of three myrobalan and three domestic plum S-RNases showed over 97% identity with S-RNases from other Prunus species, including almond, sweet cherry, Japanese apricot and Japanese plum. The second intron, which is generally highly polymorphic between alleles was also remarkably well conserved within these S-allele pairs. Degenerate consensus primers were developed and used to amplify and sequence the co-adapted polymorphic SFB alleles. Sequence comparisons also indicated high degrees of polypeptide sequence identity between three myrobalan and the three domestic plum SFB alleles and the corresponding Prunus SFB alleles. We discuss these trans-specific allele identities in terms of S-allele function, evolution of new allele specificities and Prunus taxonomy and speciation.

Partial purification of latent polyphenol oxidase from peach (Prunus persica L. Cv. Catherina). Molecular properties and kinetic characterization of soluble and membrane-bound forms.

This paper analyzes the kinetic and structural characteristics of polyphenol oxidase (PPO) from peach cv. Catherina. The PPO was obtained in a latent state in both the soluble and membrane-bound forms, and both forms were activated by acid shock and the detergent SDS. Plant defense is the main function assigned to PPO, which would be activated by the acid environment resulting from tissue damage. On the other hand, it has been suggested that, physiologically, the role played by SDS may be fulfilled by lipids. Native isoelectric focusing identified two acid isoforms of pI 5.7 and 5.8 for the soluble form and one isoform with pI 5.7 for the membrane-bound form. A partially denaturing SDS-PAGE revealed two very close bands of activity in both cases, but the Western blot performed on a totally denaturing SDS-PAGE, using polyclonal antibodies against bean PPO, revealed a single band in the membrane-bound fraction with a molecular mass of 60 kDa.