José M. Colmenero-Flores

Characterization of Phaseolus vulgaris cDNA clones responsive to water deficit: identification of a novel late embryogenesis abundant-like protein.

Six cDNA clones from Phaseolus vulgaris, whose expression is induced by water deficit and ABA treatment (rsP cDNAs) were identified and characterized. The sequence analyses of the isolated clones suggest that they encode two types of late-embryogenesis abundant (LEA) proteins, a class-1 cytoplasmic low-molecular-weight heat shock protein (lmw-HSP), a lipid transfer protein (LTP), and two different proline-rich proteins (PRP). One of the putative LEA proteins identified corresponds to a novel 9.3 kDa LEA-like protein. During the plant response to a mild water deficit (Ψw= −0.35 MPa) all genes identified present a maximal expression at around 16 or 24 h of treatment, followed by a decline in expression levels. Rehydration experiments revealed that those genes encoding PRPs and LTP transiently re-induce or maintain their expression when water is added to the soil after a dehydration period. This is not the case for the lea genes whose transcripts rapidly decrease, reaching basal levels a few hours after rehydration (4 h). Under water deficit and ABA treatments, the highest levels of expression for most of the genes occur in the root, excluding the ltp gene whose maximum expression levels are found in the aerial regions of the plant. This indicates that for these genes, both water deficit and ABA-dependent expression are under organ-specific control. The data presented here support the importance of these proteins during the plant response to water deficit.

Large changes in anatomy and physiology between diploid Rangpur lime (Citrus limonia) and its autotetraploid are not associated with large changes in leaf gene expression

Very little is known about the molecular origin of the large phenotypic differentiation between genotypes arising from somatic chromosome set doubling and their diploid parents. In this study, the anatomy and physiology of diploid (2x) and autotetraploid (4x) Rangpur lime (Citrus limonia Osbeck) seedlings has been characterized. Growth of 2x was more vigorous than 4x although leaves, stems, and roots of 4x plants were thicker and contained larger cells than 2x that may have a large impact on cell-to-cell water exchanges. Leaf water content was higher in 4x than in 2x. Leaf transcriptome expression using a citrus microarray containing 21 081 genes revealed that the number of genes differentially expressed in both genotypes was less than 1% and the maximum rate of gene expression change within a 2-fold range. Six up-regulated genes in 4x were targeted to validate microarray results by real-time reverse transcription-PCR. Five of these genes were apparently involved in the response to water deficit, suggesting that, in control conditions, the genome expression of citrus autotetraploids may act in a similar way to diploids under water-deficit stress condition. The sixth up-regulated gene which codes for a histone may also play an important role in regulating the transcription of growth processes. These results show that the large phenotypic differentiation in 4x Rangpur lime compared with 2x is not associated with large changes in genome expression. This suggests that, in 4x Rangpur lime, subtle changes in gene expression may be at the origin of the phenotypic differentiation of 4x citrus when compared with 2x.

Molecular Physiology of Development and Quality of Citrus

Abstract Citrus is the most economically important fruit crop in the world. Citrus fruits are classified as hesperidiums, berries of very special organization characterized by a juicy pulp made of vesicles within segments. Besides the typical fruit components, citrus fruit contain many organic compounds necessary for human diet and an extraordinary number of metabolites displaying valuable properties for health. In citrus, the concept of fruit quality comprises several other aspects intimately related to human health apart from physical attributes and diet components. Citrus also possess a rare combination of intriguing biological characteristics including an unusual reproductive biology, a non‐climacteric fruit ripening and several specific tree‐traits. The combination of these characteristics suggests that the study of fruit growth regulation in citrus may reveal original mechanisms based on explicit molecular differences and on exclusive genes. Citrus is, therefore, an excellent model to study fruit quality because of its peculiar fruiting, singular biochemistry and economical relevance. In this chapter, the progress that has been carried out in the research on the molecular determinants related to development and fruit quality of citrus is reviewed. The review also intends to provide a physiological frame for the implementation of the information generated during the past years. Molecular background is provided on the current status of principal reproductive processes related to fruit quality mainly flowering, fruiting, ripening, and abscission. We also have focused on main characteristic secondary bioactive compounds, as major contributors of aroma and flavour and finally, on the abiotic stresses influencing development and fruit growth.

Membrane transporters and carbon metabolism implicated in chloride homeostasis differentiate salt stress responses in tolerant and sensitive Citrus rootstocks

Salinity tolerance in Citrus is strongly related to leaf chloride accumulation. Both chloride homeostasis and specific genetic responses to Cl− toxicity are issues scarcely investigated in plants. To discriminate the transcriptomic network related to Cl− toxicity and salinity tolerance, we have used two Cl− salt treatments (NaCl and KCl) to perform a comparative microarray approach on two Citrus genotypes, the salt-sensitive Carrizo citrange, a poor Cl− excluder, and the tolerant Cleopatra mandarin, an efficient Cl− excluder. The data indicated that Cl− toxicity, rather than Na+ toxicity and/or the concomitant osmotic perturbation, is the primary factor involved in the molecular responses of citrus plant leaves to salinity. A number of uncharacterized membrane transporter genes, like NRT1-2, were differentially regulated in the tolerant and the sensitive genotypes, suggesting its potential implication in Cl− homeostasis. Analyses of enriched functional categories showed that the tolerant rootstock induced wider stress responses in gene expression while repressing central metabolic processes such as photosynthesis and carbon utilization. These features were in agreement with phenotypic changes in the patterns of photosynthesis, transpiration, and stomatal conductance and support the concept that regulation of transpiration and its associated metabolic adjustments configure an adaptive response to salinity that reduces Cl− accumulation in the tolerant genotype.

Shared and novel molecular responses of mandarin to drought

Drought is the most important stress experienced by citrus crops. A citrus cDNA microarray of about 6.000 genes has been utilized to identify transcriptomic responses of mandarin to water stress. As observed in other plant species challenged with drought stress, key genes for lysine catabolism, proline and raffinose synthesis, hydrogen peroxide reduction, vacuolar malate transport, RCI2 proteolipids and defence proteins such as osmotin, dehydrins and heat-shock proteins are induced in mandarin. Also, some aquaporin genes are repressed. The osmolyte raffinose could be detected in stressed roots while the dehydrin COR15 protein only accumulated in stressed leaves but not in roots. Novel drought responses in mandarin include the induction of genes encoding a new miraculin isoform, chloroplast β-carotene hydroxylase, oleoyl desaturase, ribosomal protein RPS13A and protein kinase CTR1. These results suggest that drought tolerance in citrus may benefit from inhibition of proteolysis, activation of zeaxanthin and linolenoyl synthesis, reinforcement of ribosomal structure and down-regulation of the ethylene response.

Actin expression in germinating seeds of Phaseolus vulgaris L.

Abstract. Actin was present at very low levels in the seeds of common bean (Phaseolus vulgaris L.) compared with those from other species, and was observed mostly in the embryo. A time-course of actin expression in germinating bean seeds revealed an induced expression of both the mRNA and protein. Initially, the actin mRNA in seeds was barely detectable by northern blot analysis. However, there was a substantial increase in the expression of the actin mRNA at 24, 48 and 72 h after imbibition, compared with an internal control consisting of a late-embryogenesis-abundant (LEA) type IV gene from P. vulgaris. An increase in the amount of actin in total seed extracts that parallelled that of the mRNA was detected by western blotting starting at 24 h after imbibition. This increase was more apparent when the embryo alone was analyzed. Two-dimensional western blots initially revealed three actin isoforms with isoelectric points (pIs) of approximately 5.6, 5.7 and 5.8, the amounts of which increased within a 48-h period, when a new minor isoform of pI approximately 5.5 appeared; however, after 72 h, the pI-5.8 isoform had almost disappeared and the pI-5.5 isoform had disappeared completely, indicating that these two minor isoforms are expressed transiently. These results indicate that actin is at very low levels in the dry seed but undergoes an increased and differential expression during imbibition, an event probably required to carry out all the necessary functions for germination.

Transcriptional profile analysis of young and mature leaves of citrus trees acclimated to salinity

11 paginas.-- 3 figuras.-- 2 tablas.-- 32 referencias.-- Articulo publicado en XII International Citrus Congress - International Society of Citriculture