Volatile metabolite profiling reveals the changes in the volatile compounds of new spontaneously generated loquat cultivars.

In recent years, the advantageous traits of three new loquat cultivars have drawn the attention of breeders and growers. All three have spontaneously arisen from the 'Algerie' cultivar: the new 'Xirlero' cultivar is a bud mutant of 'Algerie', while 'Amadeo' and 'Raúl' arose as chance seedlings. Following a non-targeted approach based on HS-SPME-GC-MS, the volatile compounds profile of the fruits from the new cultivars were obtained and compared to the original 'Algerie' cultivar. Carboxylic acids clearly dominated the volatile profile of all the loquat cultivars, but esters, aldehydes, ketones and alcohols were also predominant compounds. Interestingly when the bud mutant event did not lead to marked changes in the volatile compounds complement, pronounced changes in the volatile composition of chance seedling-generated cultivars 'Amadeo' and 'Raúl' were observed. 'Amadeo' fruits showed lower levels of 2-methyl butanoic acid and much higher levels of methylhexanoate, methylbutanoate and 2-hydroxy-5-methylacetophenone. The 'Raúl' cultivar also had a distinctive volatile profile characterised by high levels of C6-aldehydes, (E)-2-hexanal, 2-hexenal, (Z)-3-hexenal and hexanal, and several carotenoid-derived volatiles; e.g. 2-pentene-1,4-dione 1-(1,2,2-trimethylcyclopentyl), (S)-dihydroactinidiolide, isodurene, cis-geranyl acetone, ß-damascenone, ß-ionone, α-ionone and 3,4-dehydro-ß-ionone. These changes in volatiles were associated with a more intense flavour in cultivars 'Amadeo' and 'Raúl', according to the sensory evaluation of the flavour intensity carried out by a semi-trained panel. A metabolomic correlation network analysis provided insights as to how volatiles were regulated, and revealed that the compounds modified in 'Amadeo' were uncoupled from the rest of the volatilome, while the volatiles modified in 'Raul' changed according to specific groups. To conclude, this work provides a holistic view of how the loquat volatilome was affected, and this information was integrated with the physical-chemical-sensory attributes to understand the changes that occur in the new cultivars.

Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies.

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

Accumulation of PAL transcript and PAL activity as affected by heat-conditioning and low-temperature storage and its relation to chilling sensitivity in mandarin fruits.

The effects of different periods of heating at 37 degrees C on phenylalanine ammonia-lyase (PAL) and how this relates to chilling tolerance was investigated in fruits of the chilling-sensitive Fortune mandarin. All effective heat-conditioning treatments caused an early and transient increase in PAL mRNA and PAL activity. Conditioning fruits at 37 degrees C for 1 or 2 days prevented the manifestation of chilling symptoms but not the accumulation of PAL mRNA and PAL activity observed in untreated fruits. In fruits conditioned for 3 days, cold-induced damage and PAL activity were also suppressed but not the accumulation of PAL transcript upon subsequent storage at 2 degrees C. Storage of 3-day-heated fruits at a nonchilling temperature (12 degrees C) induced an early and transient increase in both PAL mRNA and PAL activity. High levels of PAL transcript and PAL activity were detected in freshly harvested fruits of a chilling-resistant mandarin (Hernandina) that decreased upon cold storage at 2 degrees C in heat-treated and nontreated fruits. These results indicate that sensitivity of mandarins to chilling correlates with low constitutive levels of PAL mRNA and PAL activity and with the inducibility of both upon exposure to low temperatures.

Programme of senescence in petals and carpels of Pisum sativum L. flowers and its control by ethylene.

The role of ethylene in the control of senescence of both petals and unpollinated carpels of pea was investigated. An increase in ethylene production accompanied senescence, and the inhibitors of ethylene action were effective in delaying senescence symptoms in different flower verticils. Pollination did not seem to trigger the senescence syndrome in the corolla as deduced from the observation that petals from pollinated and unpollinated flowers and from flowers whose carpels had been removed senesced at the same time. A cDNA clone encoding a putative ethylene-response sensor (psERS) was isolated from pea flowers, and the pattern of expression of its mRNA was studied during development and senescence of different flower tissues. The levels of psERS mRNA paralleled ethylene production (and also levels of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) mRNA) in both petals and styles. Silver thiosulfate treatments were efficient at preventing ACO and psERS mRNA induction in petals. However, the same inhibitor showed no ability to modify expression patterns in pea carpels around the anthesis stage, suggesting different controls for ethylene synthesis and sensitivity in different flower organs.

The plant homologue of the defender against apoptotic death gene is down-regulated during senescence of flower petals.

Petal senescence is an example of a highly reproducible cell death programme. In this programme, DNA is fragmented internucleosomally and cells with condensed nuclei containing an increased number of 5' ends can be detected with the TUNEL technique. The pea homologue of defender against apoptotic death (dad), a gene described to suppress endogenous programmed cell death in Caenorhabditis elegans and mammals was isolated. Expression studies show that dad declines dramatically upon flower anthesis disappearing in senescent petals, and is down-regulated by the plant hormone ethylene.

A non-photosynthetic ferredoxin gene is induced by ethylene in Citrus organs.

The sequence and expression of mRNA homologous to a cDNA encoding a non-photosynthetic ferredoxin (Fd1) from Citrus fruit was investigated. The non-photosynthetic nature of this ferredoxin was deduced from: (1) amino acid sequence alignments showing better scores with non-photosynthetic than with photosynthetic ferredoxins, (2) higher expression in tissues containing plastids other than chloroplast such as petals, young fruits, roots and peel of fully coloured fruits, and (3) the absence of light-dark regulation characteristic of photosynthetic ferredoxins. In a phylogenetic tree constructed with higher-plant ferredoxins, Citrus fruit ferredoxin clustered together with root ferredoxins and separated from the photosynthetic ferredoxins. Non photosynthetic (root and fruit) ferredoxins, but not the photosynthetic ferredoxins, have their closest homologs in cyanobacteria. Analysis of ferredoxin genomic organization suggested that non-photosynthetic ferredoxins exist in Citrus as a small gene family. Expression of Fd1 is developmentally regulated during flower opening and fruit maturation, both processes may be mediated by ethylene in Citrus. Exogenous ethylene application also induced the expression of Fd1 both in flavedo and leaves. The induction on non-photosynthetic ferredoxins could be related with the demand for reducing power in non-green, but biosynthetically active, tissues.

Evidence for the involvement of ethylene in the expression of specific RNAs during maturation of the orange, a non-climacteric fruit.

Twelve cDNAs corresponding to mRNAs inducible by ethylene were isolated by differential screening of a cDNA library from ethylene-treated Citrus sinensis fruits. Northern analysis of RNA extracted from flavedo of ethylene-treated fruits and from fruits at different maturation stages showed that some of the mRNAs corresponding to these cDNAs were regulated both by ethylene treatment and during fruit maturation. The effect of exogenous ethylene on leaves and of endogenous ethylene on flowers showed that gene induction was not restricted to the flavedo tissue. The possible role of ethylene during maturation of the non-climacteric Citrus fruit is discussed.

A putative vacuolar processing protease is regulated by ethylene and also during fruit ripening in Citrus fruit.

A putative citrus vacuolar processing thiolprotease cDNA (Cit-vac) was isolated from a cDNA library of Citrus fruits (Citrus sinensis L. Osbeck var Washington navel). The cDNA is 58 and 57% identical with vacuolar processing seed proteases from castor bean and soybean, respectively. The Citvac sequence shows a typical signal peptide for entering into the endoplasmic reticulum and two glycosylation signals. Using an in vitro transcription-translation system, we show that the Citvac precursor is able to enter a microsomal fraction and to undergo proteolytic processing and glycosylation. Transcript levels for the Citvac are developmentally regulated in the flavedo (outer colored part of the fruit peel) and increase during fruit ripening and in the flower during opening. Exogenous treatment with ethylene induces Citvac mRNA expression in both fruits and leaves. Citvac is encoded by one or two genes in the Citrus genome. The possible role of the Citvac gene product during fruit ripening and other ethylene-mediated processes is discussed.

Expression of arginine decarboxylase is induced during early fruit development and in young tissues of Pisum sativum (L.).

A cDNA coding for arginine decarboxylase (ADC, EC 4.1.1.19) has been isolated from a cDNA library of parthenocarpic young fruits of Pisum sativum (L.). The deduced aminoacid sequence is 74%, 46% and 35% identical to ADCs from tomato, oat and Escherichia coli, respectively. When the pea ADC cDNA was put under the control of the galactose inducible yeast promoter CYC1-GAL10 and introduced into Saccharomyces cerevisiae, it conferred galactose-regulated expression of the ADC activity. The ADC activity expressed in S. cerevisiae was inhibited 99% by alpha-DL-difluoromethylarginine (DFMA), a specific inhibitor of ADC activity. No activity was detected in the untransformed S. cerevisiae, nor when it was transformed with an antisense ADC construct. This provides direct evidence that the ADC cDNA from pea encoded a functional, specific ADC activity and that S. cerevisiae is able to process correctly the protein. In the pea plant, gene expression of the ADC is high in young developing tissues like shoot tips, young leaflets and flower buds. Fully expanded leaflets and roots have much lower, but still detectable, levels of the ADC transcript. In the ovary and fruit, they are developmentally regulated, showing high levels of expression during the early stages of fruit growth, which in pea is mainly due to cell expansion. The observed changes in the steady-state levels of ADC mRNA alone, however, cannot account for the differences in ADC activity suggesting that other regulatory mechanisms must be acting.

Floral development and expression of floral homeotic genes are influenced by cytokinins.

Tobacco plants that are somatic mosaics for the expression of a cytokinin-synthesizing gene have viviparous leaves. Epiphyllous buds can be either vegetative or floral. Floral adventitious buds can be either normal or abnormal. Abnormalities of floral development correlate with: (i) a local activation of the cytokinin-synthesizing gene, (ii) a drastic increase in floral cytokinin content, and (iii) a decrease in the steady-state levels of mRNA homologous of the homeotic genes DEFA, GLO and PLENA of Antirrhinum majus. Thus, these data show in planta that cytokinins, a class of phytohormones, are able to alter the development of floral organs and to decrease the expression of three homeotic floral genes.

Temporal and spatial expression of a thiolprotease gene during pea ovary senescence, and its regulation by gibberellin.

Clones encoding a thiolprotease (tpp) have been isolated from a cDNA library of unpollinated, senescent pea ovaries and its pattern of expression during both ovary senescence and parthenocarpic development have been studied. The sequence of the tpp cDNA displays a high similarity with other plant and animal thiolproteases of the papain group. The homology is highest around the Cys-His of the active centre; a 109 amino acid sequence at the carboxy terminus was found to be homologous only to thiolproteases of plant origin; this part of the mRNA is also present in another pea mRNA that exhibits similar patterns of induction. tpp mRNA shows a temporal pattern of accumulation that precedes that observed for proteolytic activity. Such accumulation did not occur when ovaries were induced to grow parthenocarpically by gibberellic acid (GA) treatment; furthermore the initial low level of expression present in ovaries decreased after GA treatment, indicating that the gene is down-regulated by gibberellins. Spatially, tpp mRNA is localized mainly within the ovule and ovary vascular elements, and transiently within the endocarp of senescent ovaries. This pattern of expression precedes the development of the cytopathogenic effects observed as unpollinated ovaries undergo senescence.

Nuclear factors binding to the extensin promoter exhibit differential activity in carrot protoplasts and cells.

The expression of the cell wall protein extensin, a hydroxyproline-rich glycoprotein, is induced by several different stimuli, including wounding. The process of protoplast preparation mimics the wounding effect and results in the induction of extensin. Using transient expression in protoplasts we analyzed several deletions of the extensin promoter. We identified an important transcriptional regulatory element located between the two TATA boxes that characterize the extensin promoter. Other regulatory elements, located further upstream between -719 to -658, are necessary for maximum level of expression. Employing electrophoretic mobility shift assays and methylation interference experiments, we demonstrate the interaction of nuclear factors with these upstream regulatory elements. In addition to the previously identified factors EGBF-1 and EGBF-2, which are mainly present in unwounded cells, we identified an additional novel DNA-binding activity that is present in extracts prepared from protoplasts but not in extracts from unwounded cells. This factor, designated EBF (extensin-binding protein), binds to a DNA fragment which when deleted results in a 48% reduction in expression.

Isolation and characterization of cDNAs from Lycopersicon esculentum and Arabidopsis thaliana encoding the 33 kDa protein of the photosystem II-associated oxygen-evolving complex.

We have identified and isolated cDNA clones of the 33 kDa protein of the oxygen-evolving complex (OEE1) from Lycopersicon esculentum (tomato) and Arabidopsis thaliana and determined their nucleotide sequences. The cDNA clones and antibodies prepared against OEE1 were used as probes to examine the expression of the oee1 gene with respect to regulation by light, organ specificity, and ripening stage of tomato fruit. The steady-state mRNA level is regulated by light, being present in light-grown plants and absent in etiolated seedlings. The oee1 transcripts that accumulate during growth in the light were reduced to non-detectable or low levels by a 3-day dark treatment. The oee1 gene also exhibits differential expression in various organs of the tomato plant. Steady-state mRNA level was highest in immature leaves and absent in roots while the protein was most abundant in mature leaves and absent in roots. During tomato fruit ripening oee1 mRNA decreases to a low level in the pericarp while the protein level decreases below detection. The expression of oee1 appears to be under the control of a complex mechanism regulating both the amount of RNA and its translation.

Fractionation with ethanol of nucleic acids from viroid-infected plants.

A combination of high salt and low ethanol concentration allowed the fractionation of nucleic acids extracted from viroid-infected leaves. By adding 0.4-0.5 vol of ethanol to 1 vol of a solution in 2 M LiCl of nucleic acids (containing mainly DNA, 4S, 5S, 7S, and viroid RNAs), 85% of the DNA and 75% of the 4S RNA remained in solution, from where they could be recovered by increasing the ethanol concentration, whereas almost all 5S, 7S, and viroid RNAs precipitated. When this process was repeated three times a 95% elimination of the initial DNA and 4S RNA was achieved. The method can be of special interest in viroid purification considering that DNA and 4S RNA are the most abundant contaminants in the starting solution of nucleic acids. It is suggested that the highly ordered secondary structure of viroid RNA may be responsible for its particular behavior in the ethanol fractionation of nucleic acids.