More than a spiny morphology: plastome variation in the prickly pear cacti (Opuntieae).

BACKGROUND: Plastid genomes (plastomes) have long been recognized as highly conserved in their overall structure, size, gene arrangement and content among land plants. However, recent studies have shown that some lineages present unusual variations in some of these features. Members of the cactus family are one of these lineages, with distinct plastome structures reported across disparate lineages, including gene losses, inversions, boundary movements or loss of the canonical inverted repeat (IR) region. However, only a small fraction of cactus diversity has been analysed so far. METHODS: Here, we investigated plastome features of the tribe Opuntieae, the remarkable prickly pear cacti, which represent one of the most diverse and important lineages of Cactaceae. We assembled de novo the plastome of 43 species, representing a comprehensive sampling of the tribe, including all seven genera, and analysed their evolution in a phylogenetic comparative framework. Phylogenomic analyses with different datasets (full plastome sequences and genes only) were performed, followed by congruence analyses to assess signals underlying contentious nodes. KEY RESULTS: Plastomes varied considerably in length, from 121 to 162 kbp, with striking differences in the content and size of the IR region (contraction and expansion events), including a lack of the canonical IR in some lineages and the pseudogenization or loss of some genes. Overall, nine different types of plastomes were reported, deviating in the presence of the IR region or the genes contained in the IR. Overall, plastome sequences resolved phylogenetic relationships within major clades of Opuntieae with high bootstrap values but presented some contentious nodes depending on the dataset analysed (e.g. whole plastome vs. genes only). Congruence analyses revealed that most plastidial regions lack phylogenetic resolution, while few markers are supporting the most likely topology. Likewise, alternative topologies are driven by a handful of plastome markers, suggesting recalcitrant nodes in the phylogeny. CONCLUSIONS: Our study reveals a dynamic nature of plastome evolution across closely related lineages, shedding light on peculiar features of plastomes. Variation of plastome types across Opuntieae is remarkable in size, structure and content and can be important for the recognition of species in some major clades. Unravelling connections between the causes of plastome variation and the consequences for species biology, physiology, ecology, diversification and adaptation is a promising and ambitious endeavour in cactus research. Although plastome data resolved major phylogenetic relationships, the generation of nuclear genomic data is necessary to confront these hypotheses and assess the recalcitrant nodes further.

Genetic diversity and relationships among Nopalea sp. and Opuntia spp. accessions revealed by RAPD, ISSR and ITS molecular markers.

BACKGROUND: Dactylopius opuntiae (Cockerell) (carmine cochineal) is an insect pest highly noxious that has spread through cactus pear crops in the Brazilian semiarid region. Knowledge of diversity and genetic relationships of the cactus pear accessions is fundamental to create new varieties resistance to carmine cochineal. Therefore, this investigation was undertaken to assess the genetic diversity and genetic relationships that existed among cactus pear accessions of Nopalea sp. and Opuntia spp. with contrasting resistance to D. opuntiae. METHODS AND RESULTS: We conducted a molecular analysis in seven cactus pear accessions from the "reference collection" of the Agronomic Institute of Pernambuco, Brazil using RAPD, ISSR and ITS molecular markers. A total of 242 bands were detected from 26 polymorphic primers. The high percentage of polymorphism by RAPD (89.8%), ISSR (81.2%) and ITS (75%) markers suggests that the cactus pear accessions have high genetic diversity. The combined analysis of markers systems enabled the accessions discrimination of about the genus and ploidy, but were incongruous in relation to resistance level to D. opuntiae. CONCLUSIONS: Genetic diversity, discrimination of about the genus and ploidy was confirmed by merging information from ISSR, RAPD and ITS markers systems. The IPA-200016, IPA-200149, IPA-100004, IPA-200205 accessions are genetically divergent, therefore could be potentially incorporated into any further breeding programs directed to create new varieties of cactus pear resistant to D. opuntiae.

Self-Repair in Cacti Branches: Comparative Analyses of Their Morphology, Anatomy, and Biomechanics.

Damage-repair is particularly important for the maintenance of the water-storing abilities of succulent plants such as cacti. Comparative morphological, anatomical, and biomechanical analyses of self-repair were performed on artificially wounded branches of Opuntia ficus-indica and Cylindropuntia bigelovii. Macroscopic observations, contrast staining, and lignin-proof staining were used to investigate morphological and anatomical responses after wounding at various time intervals. Two-point bending tests were repeatedly performed on the same branches under unwounded, freshly wounded, and healed conditions by using customized 3D-printed clamping jaws. Morphologically, both species showed a rolling-in of the wound edges, but no mucilage discharge. Anatomically, ligno-suberized peridermal layers developed that covered the wound region, and new parenchyma cells formed, especially in O. ficus-indica. In all samples, the wounding effect directly after damage caused a decrease between 18% and 37% in all the tested mechanical parameters, whereas a positive healing effect after 21 days was only found for C. bigelovii. Based on our data, we hypothesize a high selection pressure on the restoration of structural integrity in the wound area, with a focus on the development of efficient water-retaining mechanisms, whereas the concept of "sufficient is good enough" seems to apply for the restoration of the mechanical properties.

Resilience of cactus pear genotypes in a tropical semi-arid region subject to climatic cultivation restriction.

The cactus pear has demonstrated productive potential in arid and semi-arid regions due to its photosynthetic process of crassulacean acid metabolism. Thus, this study aimed to evaluate agronomic characteristics and chemical composition of three genotypes of cactus pear at different locations of a tropical semiarid region classified as non-suitable for cactus pear cultivation. A completely randomized design in a factorial arrangement (3 × 7) (three genotypes of cactus pear [Miúda, Baiana (Nopalea cochenillifera) and Orelha de Efefante Mexicana - OEM (Opuntia stricta)] and seven locations) was used. The climatic conditions characterized an environment that restricts the growth of cactus pear genotypes, mainly due to the air relative humidity values. All morphological characteristics of the cactus pear genotypes were influenced by the interaction genotype x location, with higher expression of the characteristics on the different genotypes under hot semi-arid climate and tropical wet and dry climate. An effect of the interaction genotype x location was observed (p < 0.05) on water use efficiency, water accumulation and carrying capacity, where the highest values were observed for genotype Baiana at location 1. Also, there was influence of the interaction genotype x location over the chemical composition of the cactus pear. The cultivation of cactus pear is recommended under restricted climatic conditions in semi-arid tropical regions, especially the genotype Baiana, based on growth factors, biomass production and chemical composition.

Molecular characterization and in vitro interaction analysis of Op14-3-3 µ protein from Opuntia ficus-indica: identification of a new client protein from shikimate pathway.

In plants, 14-3-3 proteins are important modulators of protein-protein interactions in response to environmental stresses. The aim of the present work was to characterize one Opuntia ficus-indica 14-3-3 and get information about its client proteins. To achieve this goal, O. ficus-indica 14-3-3 cDNA, named as Op14-3-3 µ, was amplified by 3'-RACE methodology. Op14-3-3 µ contains an Open Reading Frame of 786 bp encoding a 261 amino acids protein. Op14-3-3 µ cDNA was cloned into a bacterial expression system and recombinant protein was purified. Differential Scanning Fluorimetry, Dynamic Light Scattering, and Ion Mobility-Mass Spectrometry were used for Op14-3-3 µ protein characterization, and Affinity-Purification-Mass Spectrometry analysis approach was used to obtain information about their potential client proteins. Pyrophosphate-fructose 6-phosphate 1-phosphotransferase, ribulose bisphosphate carboxylase large subunit, and vacuolar-type H+-ATPase were identified. Interestingly chorismate mutase p-prephenate dehydratase was also identified. Op14-3-3 µ down-regulation was observed in Opuntia calluses when they were induced with Jasmonic Acid, while increased accumulation of Op14-3-3 µ protein was observed. The putative interaction of 14-3-3 µ with chorismate mutase, which have not been reported before, suggest that Op14-3-3 µ could be an important regulator of metabolites biosynthesis and responses to stress in Opuntia spp. SIGNIFICANCE: Opuntia species are important crops in arid and semiarid areas worldwide, but despite its relevance, little information about their tolerance mechanism to cope with harsh environmental conditions is reported. 14-3-3 proteins have gained attention due to its participation as protein-protein regulators and have been linked with primary metabolism and hormones responses. Here we present the characterization of the first Opuntia ficus-indica 14-3-3 (Op14-3-3) protein using affinity purification-mass spectrometry (AP-MS) strategy. Op14-3-3 has high homology with other 14-3-3 from Caryophyllales. A novel Op14-3-3 client protein has been identified; the chorismate mutase p-prephenate dehydratase, key enzyme that links the primary with secondary metabolism. The present results open new questions about the Opuntia spp. pathways mechanisms in response to environmental stress and the importance of 14-3-3 proteins in betalains biosynthesis.

Rapid, simple and potentially universal method for DNA extraction from Opuntia spp. fresh cladode tissues suitable for PCR amplification.

In Opuntia spp., the cladode tissues contain many polysaccharides and secondary metabolites that interfere with obtaining high-quality deoxyribonucleic acid (DNA), using currently available methods. To circumvent this problem, three commercial kits, three modified versions of the conventional cetyltrimethylammonium bromide method (CTAB) method and one combined method were tested in Opuntia ficus-indica, O. robusta, O. dillenii and O. elata species. We obtained a rapid and simple protocol that allows the extraction of DNA from all the tested species with good DNA yield and purity, namely, the combined method. With this method (DNeasy® Plant Mini Kit combined with the CTAB method), DNA yields from 13.2 ± 7.8 to 15.9 ± 11.3 µg g-1 of fresh tissue were obtained in the four Opuntia species. The purity, evaluated by the ratio A260/A280 ratio, ranged from 1.67 ± 0.12 to 2.01 ± 0.25, revealing low levels of problematic metabolites. The extracted DNA quality was confirmed by amplifying a set of nuclear microsatellites obtained for the genus. Reliable reproducible bands and electropherogram profiles were obtained. The combined method has potential to be universal for good-quality DNA extraction in cacti, particularly in the Opuntia genus and other difficult-to-extract species.

Genetic divergence between Mexican Opuntia accessions inferred by polymerase chain reaction-restriction fragment length polymorphism analysis.

Molecular methods are powerful tools in characterizing and determining relationships between plants. The aim of this study was to study genetic divergence between 103 accessions of Mexican Opuntia. To accomplish this, polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of three chloroplast intergenic spacers (atpB-rbcL, trnL-trnF, and psbA-trnH), one chloroplast gene (ycf1), two nuclear genes (ppc and PhyC), and one mitochondrial gene (cox3) was conducted. The amplified products from all the samples had very similar molecular sizes, and there were only very small differences between the undigested PCR amplicons for all regions, with the exception of ppc. We obtained 5850 bp from the seven regions, and 136 fragments were detected with eight enzymes, 37 of which (27.2%) were polymorphic. We found that 40% of the fragments from the chloroplast regions were polymorphic, 9.8% of the bands detected in the nuclear genes were polymorphic, and 20% of the bands in the mitochondrial locus were polymorphic. trnL-trnF and psbA-trnH were the most variable regions. The Nei and Li/Dice distance was very short, and ranged from 0 to 0.12; indeed, 77 of the 103 genotypes had the same genetic profile. All the xoconostle accessions (acidic fruits) were grouped together without being separated from three genotypes of prickly pear (sweet fruits). We assume that the genetic divergence between prickly pears and xoconostles is very low, and question the number of Opuntia species currently considered in Mexico.

Genetic diversity and phylogenetic relationship among Tunisian cactus species (Opuntia) as revealed by random amplified microsatellite polymorphism markers.

Opuntia ficus indica is one of the most economically important species in the Cactaceae family. Increased interest in this crop stems from its potential contribution to agricultural diversification, application in the exploitation of marginal lands, and utility as additional income sources for farmers. In Tunisia, O. ficus indica has been affected by drastic genetic erosion resulting from biotic and abiotic stresses. Thus, it is imperative to identify and preserve this germplasm. In this study, we focused on the use of random amplified microsatellite polymorphisms to assess genetic diversity among 25 representatives of Tunisian Opuntia species maintained in the collection of the National Institute of Agronomic Research of Tunisia. Seventy-two DNA markers were screened to discriminate accessions using 16 successful primer combinations. The high percentage of polymorphic band (100%), the resolving power value (5.68), the polymorphic information content (0.94), and the marker index (7.2) demonstrated the efficiency of the primers tested. Therefore, appropriate cluster analysis used in this study illustrated a divergence among the cultivars studied and exhibited continuous variation that occurred independently of geographic origin. O. ficus indica accessions did not cluster separately from the other cactus pear species, indicating that their current taxonomical classifications are not well aligned with their genetic variability or locality of origin.

miRNA expression during prickly pear cactus fruit development.

miRNAs are a class of small non-coding RNAs that regulate gene expression. They are involved in the control of many developmental processes, including fruit development. The increasing amount of information on miRNAs, on their expression, abundance, and conservation between various species, provides a new opportunity to study the role of miRNAs in non-model plant species. In this work, we used a combination of Northern blot and tissue print hybridization analysis to identify conserved miRNAs expressed during prickly pear cactus (Opuntia ficus indica) fruit development. Comparative profiling detected the expression of 34 miRNAs, which were clustered in three different groups that were associated with the different phases of fruit development. Variation in the level of miRNA expression was observed. Gradual expression increase of several miRNAs was observed during fruit development, including miR164. miR164 was selected for stem-loop RT-PCR and for a detailed spatial-temporal expression analysis. At early floral stages, miR164 was mainly localized in meristematic tissues, boundaries and fusion zones, while it was more homogenously expressed in fruit tissues. Our results provide the first evidence of miRNA expression in the prickly pear cactus and provide the basis for future research on miRNAs in Opuntia. Moreover, our analyses suggest that miR164 plays different roles during prickly pear cactus fruit development.

Chemical composition of volatiles from Opuntia littoralis, Opuntia ficus-indica, and Opuntia prolifera growing on Catalina Island, California.

The essential oils from the cladodes of Opuntia littoralis, Opuntia ficus-indica and Opuntia prolifera growing wild on Santa Catalina Island, California, were obtained by hydrodistillation and analysed by gas chromatography-mass spectrometry (GC-MS). Terpenoids were the dominant class of volatiles in O. littoralis, with the two main components being the furanoid forms of cis-linalool oxide (10.8%) and trans-linalool oxide (8.8%). Fatty acid-derived compounds dominated the essential oil of O. ficus-indica with linoleic acid (22.3%), palmitic acid (12.7%), lauric acid (10.5%) and myristic acid (4.2%) as major fatty acids. O. prolifera oil was composed of 46.6% alkanes and the primary hydrocarbon component was heptadecane (19.2%). Sixteen compounds were common to all the three Opuntia species.

Stem fasciation in cacti and succulent species--tissue anatomy, protein pattern and RAPD polymorphisms.

Fasciated and normal stem segments of Opuntia microdasys, Opuntia cylindrica, Huernia primulina and Euphorbia lactea were collected from the same plant and compared for their anatomy, water relations and genetic variations. Anatomical differences in terms of thickness of cuticle, vascular bundle, xylem and phloem were analyzed in both normal and fasciated stems. The mucilage cells were higher in the fasciated form of Opuntia microdasys than that in the normal form. Water status in terms of total water content (TWC), water deficit and relative water content (RWC) was influenced by fasciation. Genetic variations were tested in normal and fasciated stems using randomly amplified polymorphic DNA (RAPD) fingerprints and SDS-PAGE of soluble protein extracts. SDS-PAGE protein and RAPD analysis confirmed that normal and fasciated tissues were genetically different. Polymerase chain reaction (PCR) yielded different polymorphic banding patterns that were unique to each primer and distinguishable over all samples. The PCR results of normal and fasciated samples were significantly different in cases of primers P1, P2 and P3. These results indicate that occurrence of fasciation in Opuntia microdasys, Opuntia cylindrica, Huernia primulina and Euphorbia lactea is an epigenetic mutation of tissues.

The Opuntia streptacantha OpsHSP18 gene confers salt and osmotic stress tolerance in Arabidopsis thaliana.

Abiotic stress limits seed germination, plant growth, flowering and fruit quality, causing economic decrease. Small Heat Shock Proteins (sHSPs) are chaperons with roles in stress tolerance. Herein, we report the functional characterization of a cytosolic class CI sHSP (OpsHSP18) from Opuntia streptacantha during seed germination in Arabidopsis thaliana transgenic lines subjected to different stress and hormone treatments. The over-expression of the OpsHSP18 gene in A. thaliana increased the seed germination rate under salt (NaCl) and osmotic (glucose and mannitol) stress, and in ABA treatments, compared with WT. On the other hand, the over-expression of the OpsHSP18 gene enhanced tolerance to salt (150 mM NaCl) and osmotic (274 mM mannitol) stress in Arabidopsis seedlings treated during 14 and 21 days, respectively. These plants showed increased survival rates (52.00 and 73.33%, respectively) with respect to the WT (18.75 and 53.75%, respectively). Thus, our results show that OpsHSP18 gene might have an important role in abiotic stress tolerance, in particular in seed germination and survival rate of Arabidopsis plants under unfavorable conditions.

Functional characterization of an acidic SK(3) dehydrin isolated from an Opuntia streptacantha cDNA library.

Cactus pears are succulent plants of the Cactaceae family adapted to extremely arid, hot and cold environments, making them excellent models for the study of molecular mechanisms underlying abiotic stress tolerance. Herein, we report a directional cDNA library from 12-month-old cladodes of Opuntia streptacantha plants subjected to abiotic stresses. A total of 442 clones were sequenced, representing 329 cactus pear unigenes, classified into eleven functional categories. The most abundant EST (unigen 33) was characterized under abiotic stress. This cDNA of 905 bp encodes a SK(3)-type acidic dehydrin of 248 amino acids. The OpsDHN1 gene contains an intron inserted within the sequence encoding the S-motif. qRT-PCR analysis shows that the OpsDHN1 transcript is specifically accumulated in response to cold stress, and induced by abscisic acid. Over-expression of the OpsDHN1 gene in Arabidopsis thaliana leads to enhanced tolerance to freezing treatment, suggesting that OpsDHN1 participates in freezing stress responsiveness. Generation of the first EST collection for the characterization of cactus pear genes constitutes a useful platform for the understanding of molecular mechanisms of stress tolerance in Opuntia and other CAM plants.

Conserved and divergent rhythms of crassulacean acid metabolism-related and core clock gene expression in the cactus Opuntia ficus-indica.

The cactus Opuntia ficus-indica is a constitutive Crassulacean acid metabolism (CAM) species. Current knowledge of CAM metabolism suggests that the enzyme phosphoenolpyruvate carboxylase kinase (PPCK) is circadian regulated at the transcriptional level, whereas phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK) are posttranslationally controlled. As little transcriptomic data are available from obligate CAM plants, we created an expressed sequence tag database derived from different organs and developmental stages. Sequences were assembled, compared with sequences in the National Center for Biotechnology Information nonredundant database for identification of putative orthologs, and mapped using Kyoto Encyclopedia of Genes and Genomes Orthology and Gene Ontology. We identified genes involved in circadian regulation and CAM metabolism for transcriptomic analysis in plants grown in long days. We identified stable reference genes for quantitative polymerase chain reaction and found that OfiSAND, like its counterpart in Arabidopsis (Arabidopsis thaliana), and OfiTUB are generally appropriate standards for use in the quantification of gene expression in O. ficus-indica. Three kinds of expression profiles were found: transcripts of OfiPPCK oscillated with a 24-h periodicity; transcripts of the light-active OfiNADP-ME and OfiPPDK genes adapted to 12-h cycles, while transcript accumulation patterns of OfiPEPC and OfiMDH were arrhythmic. Expression of the circadian clock gene OfiTOC1, similar to Arabidopsis, oscillated with a 24-h periodicity, peaking at night. Expression of OfiCCA1 and OfiPRR9, unlike in Arabidopsis, adapted best to a 12-h rhythm, suggesting that circadian clock gene interactions differ from those of Arabidopsis. Our results indicate that the evolution of CAM metabolism could be the result of modified circadian regulation at both the transcriptional and posttranscriptional levels.

The influence of historical geneflow, bathymetry and distribution patterns on the population genetics of morphologically diverse Galápagos' Opuntia echios.

Throughout history, remote archipelagos have repeatedly been designated natural laboratories to study evolutionary processes. The extensive, geographically structured, morphological variation within Galápagos' Opuntia cacti has been presumed to be another example of how such processes shape diversity. However, recent genetic studies on speciation and potential effects of plasticity within this system failed to confirm earlier classification and hypothesized radiation on both global and single island levels. Detailed population genetic information, however, is crucial in conserving these semi-arid ecosystem keystone species. In this article, we re-evaluate the genetics of Opuntia echios inhabiting one of the most taxon rich places on the archipelago: Santa Cruz and its surrounding satellite islands, using microsatellite data. Our analysis revealed high genetic variability within all sampled locations, providing little support for the hypothesis of clonal reproduction. Inter-island gene flow patterns appear to be largely influenced by bathymetry and sea levels during last ice ages. Although O. echios from Seymour Norte are morphologically recognized as being a separate taxon, Daphné Major's cacti are the most differentiated. In addition, we found a potential barrier for gene flow along the ring-like distribution of Opuntias at the western side of Santa Cruz, suggesting potential links with geology.

Are fungi important for breaking seed dormancy in desert species? Experimental evidence in Opuntia streptacantha (Cactaceae).

Seeds of Opuntia spp. have physiological dormancy; they need a period of after-ripening to break dormancy, and the embryos have low growth potential. We evaluated the combined effects of seed age and presence of fungi on the testa on germination of Opuntia streptacantha, an abundant species in the Chihuahuan Desert (Mexico), assuming that older seeds have broken seed dormancy and fungi can reduce mechanical resistance to germination. In a preliminary experiment, we found no germination of 9-year-old (1998) and freshly collected (2007) seeds. However, we obtained 67% and 27% germination from 9-year-old and fresh non-sterilized seeds, respectively, and found fungi growing on the testa of all germinated seeds. Two fungal strains were isolated and identified using ribosomal internal transcribed spacer (ITS) sequence analysis: Penicillium chrysogenum and Phoma sp. In a second experiment, we inoculated seeds with strains of P. chrysogenum and Phoma sp., as well as Trichoderma koningii and binucleate Rhizoctonia (Gto17S2), to evaluate their ability to break seed dormancy. Seeds inoculated with P. chrysogenum, Phoma sp. and T. koningii had higher germination than controls for both seed ages, but germination was higher in older seeds. Scanning electron microscopy showed that these fungi eroded the funiculus, reducing its resistance. Binucleate Rhizoctonia did not lead to germination and controls had almost no germination. Our results strongly indicate that fungi are involved in breaking seed dormancy of O. streptacantha, and that the effect of fungi on seeds is species-specific.

The effect of variety and location on cactus pear (Opuntia ficus-indica) fruit quality.

Little is known about the performance of South African cactus pear varieties in different agro-ecological regions. Effects of locality on internal quality parameters of available cactus pear varieties were examined. With only one exception, no significant differences among the mean replication values for the different parameters between the different locations were observed. The differences between mean values for most individual parameters at the three localities were highly significant. Highly significant differences between the mean values for the measured characteristics were observed, not only among the locations (except for the pulp glucose values), but also for the influences of genotype and interaction between locality and genotype. Significant variations existed between mean values of the different characteristics between localities. Genotype x environmental interactions were noted. It was concluded that Meyers is the most appropriate cultivar for economical purposes in South Africa.

Salt stress increases the expression of p5cs gene and induces proline accumulation in cactus pear.

Proline (Pro) is one of the most accumulated osmolytes in salinity and water deficit conditions in plants. In the present study, we measured the Pro content, the activity and the expression level of delta 1-pyrroline-5-carboxylate synthetase (P5CS: gamma-glutamyl kinase, EC 2.7.2.11 and glutamate-5-semialdehyde dehydrogenase, EC 1.2.1.41), a key regulatory enzyme involved in the biosynthesis of Pro, in cactus pear (Opuntia streptacantha) subjected to 6, 9 and 11 days of salt stress. Treatment with NaCl of O. streptacantha young plants resulted in a decrease in the cladode thickness and root length, and in a significant and gradual accumulation of Pro in young cladodes, in a time- and concentration-dependent manner. P5CS activity, studied as gamma-glutamyl kinase, was reduced at all times as a consequence of salt treatment, except at the sixth day at 75 and 150mM of NaCl, where a slight increase was observed. We isolated an open reading frame (ORF) fragment of p5cs gene. The deduced amino acid sequence of the P5CS protein exhibited 90.4% of identity with the P5CS protein from Mesembryanthemum crystallinum. RT-PCR analysis revealed that the Osp5cs gene of O. streptacantha was induced by salt stress at 9 and 11 days of treatment. Furthermore, ABA-induced Osp5cs gene expression was observed in cladodes of cactus pear young plants. We observed an evident correlation between the transcript up-regulation and the Pro accumulation under salt stress; however, these results do not parallel with the changes in P5CS enzymatic activity. This Pro accumulation might function as an osmolyte for the intracellular osmotic adjustment and might be playing a critical role in protecting photosynthetic activity in O. streptacantha plants under salt stress.

Color, betalain pattern, and antioxidant properties of cactus pear (Opuntia spp.) clones.

Total phenolics, ascorbic acid, and betalain contents of differently colored cactus pear clones (nine Opuntia ficus-indica [L.] Mill. clones and one O. robusta Wendl. clone) were investigated and related to their respective antioxidant potential assessed by Trolox-equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays. TEAC and ORAC values were very highly correlated with each other and also with values for total phenolics, betalain contents, and ascorbic acid concentrations. Total phenolics had the greatest contribution to ORAC and TEAC values. High-performance liquid chromatography (HPLC)-diode array detector (DAD)-tandem mass spectrometry (MS/MS) measurements of cactus pear juices permitted the differentiation of the clones based on variations in pigment patterns and betalain concentrations. The red and yellow betalains were absent in lime green colored cactus fruits. The ratio and concentration of these pigments were responsible for the yellow, orange, red, and purple colors in the other clones. Progeny of purple and lime green colored parents were characterized by 12% and 88% of plants bearing lime green and purple fruit, respectively. This implies that the genes for betalain production were lacking in the lime green fruits but could be provided by a parent with a complete set of genes, that is, purple fruits. Besides known pigments typical of Cactaceae, two unexpected betalains were identified. Whereas gomphrenin I was found for the first time in tissues of cactus plants, methionine-betaxanthin has never been described before as a genuine betalain. In addition to their alleged health-promoting properties, various combinations of yellow betaxanthins and red-purple betacyanins may allow the development of new food products without using artificial colorants.

Expression of ripening-related genes in prickly pear (Opuntia sp.) fruits.

To throw light on the expression of ripening-related genes in prickly pear (Opuntia sp.) fruits and on the possible role of the gaseous hormone ethylene in nonclimacteric fruit ripening, cDNA fragments that showed high homologies with 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase cDNAs from other plants were cloned and partially characterized. Thus, the corresponding genes were accordingly named opaccs-1 and opacco-1, after Opuntia ACC synthase-1 and Opuntia ACC oxidase-1, respectively. Southern analysis suggests the presence of at least one copy of both genes, as well as other related homologous sequences in the Opuntia genome. Northern analysis of the opaccs-1 gene shows an enhanced expression in ripening fruit tissues, whereas opacco-1 expression is highly induced in ripe tissues with respect to the green fruits and mature cladodes. These results are in agreement with an active metabolic role of ethylene during nonclimacteric prickly pear fruit ripening. This is the first report on the analysis at the molecular level of ripening-related genes of the Opuntia genus.