Pedro Carrasco

Polyamines: molecules with regulatory functions in plant abiotic stress tolerance

Early studies on plant polyamine research pointed to their involvement in responses to different environmental stresses. During the last few years, genetic, transcriptomic and metabolomic approaches have unravelled key functions of different polyamines in the regulation of abiotic stress tolerance. Nevertheless, the precise molecular mechanism(s) by which polyamines control plant responses to stress stimuli are largely unknown. Recent studies indicate that polyamine signalling is involved in direct interactions with different metabolic routes and intricate hormonal cross-talks. Here we discuss the integration of polyamines with other metabolic pathways by focusing on molecular mechanisms of their action in abiotic stress tolerance. Recent advances in the cross talk between polyamines and abscisic acid are discussed and integrated with processes of reactive oxygen species (ROS) signalling, generation of nitric oxide, modulation of ion channel activities and Ca2+ homeostasis, amongst others.

Involvement of polyamines in plant response to abiotic stress

Environmental stresses are the major cause of crop loss worldwide. Polyamines are involved in plant stress responses. However, the precise role(s) of polyamine metabolism in these processes remain ill-defined. Transgenic approaches demonstrate that polyamines play essential roles in stress tolerance and open up the possibility to exploit this strategy to improve plant tolerance to multiple environmental stresses. The use of Arabidopsis as a model plant enables us to carry out global expression studies of the polyamine metabolic genes under different stress conditions, as well as genome-wide expression analyses of insertional-mutants and plants over-expressing these genes. These studies are essential to dissect the polyamine mechanism of action in order to design new strategies to increase plant survival in adverse environments.

Integration of polyamines in the cold acclimation response.

Temperature is one of the most important environmental factors limiting the geographical distribution of plants and accounts for significant reductions in the yield of agriculturally important crops. Low temperature damages many plant species, especially those adapted to tropical climates. In contrast, some species from temperate regions are able to develop freezing tolerance in response to low-non-freezing temperature, an adaptive process named cold acclimation. Numerous molecular, biochemical and physiological changes occur during cold acclimation, most of them being associated with significant changes in gene expression and metabolite profiles. During recent years, transcriptomic and metabolomic approaches have allowed the identification of cold-responsive genes and main metabolites which accumulate in plants exposed to cold. The obtained data support the previously held idea that polyamines (PAs) are involved in plant responses to cold, although their specific role is still not well understood. In this review, we synthesize published data regarding PA-responses to cold stress and integrate them with global transcriptional and metabolic changes. The potential of PA genetic engineering for the development of plants resistant to cold and freezing temperatures, and their plausible mechanisms of action are also discussed.

Aminopropyltransferases Involved in Polyamine Biosynthesis Localize Preferentially in the Nucleus of Plant Cells

Plant aminopropyltransferases consist of a group of enzymes that transfer aminopropyl groups derived from decarboxylated S-adenosyl-methionine (dcAdoMet or dcSAM) to propylamine acceptors to produce polyamines, ubiquitous metabolites with positive charge at physiological pH. Spermidine synthase (SPDS) uses putrescine as amino acceptor to form spermidine, whereas spermine synthase (SPMS) and thermospermine synthase (TSPMS) use spermidine as acceptor to synthesize the isomers spermine and thermospermine respectively. In previous work it was shown that both SPDS1 and SPDS2 can physically interact with SPMS although no data concerning the subcellular localization was reported. Here we study the subcellular localization of these enzymes and their protein dimer complexes with gateway-based Bimolecular Fluorescence Complementation (BiFC) binary vectors. In addition, we have characterized the molecular weight of the enzyme complexes by gel filtration chromatography with in vitro assembled recombinant enzymes and with endogenous plant protein extracts. Our data suggest that aminopropyltransferases display a dual subcellular localization both in the cytosol and nuclear enriched fractions, and they assemble preferably as dimers. The BiFC transient expression data suggest that aminopropyltransferase heterodimer complexes take place preferentially inside the nucleus.

Ecological and agronomic importance of the plant genus Lotus. Its application in grassland sustainability and the amelioration of constrained and contaminated soils

The genus Lotus comprises around 100 annual and perennial species with worldwide distribution. The relevance of Lotus japonicus as a model plant has been recently demonstrated in numerous studies. In addition, some of the Lotus species show a great potential for adaptation to a number of abiotic stresses. Therefore, they are relevant components of grassland ecosystems in environmentally constrained areas of several South American countries and Australia, where they are used for livestock production. Also, the fact that the roots of these species form rhizobial and mycorrhizal associations makes the annual L. japonicus a suitable model plant for legumes, particularly in studies directed to recognize the mechanisms intervening in the tolerance to abiotic factors in the field, where these interactions occur. These properties justify the increased utilization of some Lotus species as a strategy for dunes revegetation and reclamation of heavy metal-contaminated or burned soils in Europe.

New insights into the role of spermine in Arabidopsis thaliana under long-term salt stress ☆

Polyamines (putrescine, spermidine and spermine) are traditionally implicated in the response of plants to environmental cues. Free spermine accumulation has been suggested as a particular feature of long-term salt stress, and in the model plant Arabidopsis thaliana the spermine synthase gene (AtSPMS) has been reported as inducible by abscisic acid (ABA) and acute salt stress treatments. With the aim to unravel the physiological role of free spermine during salinity, we analyzed polyamine metabolism in A. thaliana salt-hypersensitive sos mutants (salt overlay sensitive; sos1-1, sos2-1 and sos3-1), and studied the salt stress tolerance of the mutants in spermine and thermospermine synthesis (acl5-1, spms-1 and acl5-1/spms-1). Results presented here indicate that induction in polyamine metabolism is a SOS-independent response to salinity and is globally over-induced in a sensitive background. In addition, under long-term salinity, the mutants in the synthesis of spermine and thermospermine (acl5-1, spms-1 and double acl5-1/spms-1) accumulated more Na(+) and performed worst than WT in survival experiments. Therefore, support is given to a role for these higher polyamines in salt tolerance mechanisms.

Putrescine accumulation in Arabidopsis thaliana transgenic lines enhances tolerance to dehydration and freezing stress

Polyamines have been globally associated to plant responses to abiotic stress. Particularly, putrescine has been related to a better response to cold and dehydration stresses. It is known that this polyamine is involved in cold tolerance, since Arabidopsis thaliana plants mutated in the key enzyme responsible for putrescine synthesis (arginine decarboxilase, ADC; EC 4.1.1.19) are more sensitive than the wild type to this stress. Although it is speculated that the over-expression of ADC genes may confer tolerance, this is hampered by pleiotropic effects arising from the constitutive expression of enzymes from the polyamine metabolism. Here, we present our work using A. thaliana transgenic plants harboring the ADC gene from oat under the control of a stress-inducible promoter (pRD29A) instead of a constitutive promoter. The transgenic lines presented in this work were more resistant to both cold and dehydration stresses, associated with a concomitant increment in endogenous putrescine levels under stress. Furthermore, the increment in putrescine upon cold treatment correlated with the induction of known stress-responsive genes, and suggested that putrescine may be directly or indirectly involved in ABA metabolism and gene expression.

Hormonal regulation of S-adenosylmethionine synthase transcripts in pea ovaries

Two cDNA clones coding for S-adenosyl-L-methionine synthase (SAMs, EC 2.5.1.6) have been isolated from a cDNA library of gibberellic acid-treated unpollinated pea ovaries. Both cDNAs were sequenced showing a high degree of identity but coding for different SAMs polypeptides. The presence of two SAMs genes in pea was further confirmed by Southern analysis. Expression of the SAMs genes in the pea plant was found at different levels in vegetative and reproductive tissues. We characterized the expression levels of SAMs genes during the development or senescence of pea ovaries. Northern analysis showed that transcription of SAMs genes in parthenocarpic fruits was upregulated by auxins in the same manner as in fruits from pollinated ovaries. In both pollinated and 2,4-dichlorophenoxyacetic acid-treated ovaries, an induction of SAMs mRNA levels was detected at the onset of fruit development. Gibberellic acid and benzyladenine, although able to induce parthenocarpic development, did not affect SAMs mRNA levels. These data are consistent with an active participation of auxins in the upregulation of SAMs during fruit setting in pea and suggest that, at the molecular level, parthenocarpic development of pea ovaries is different for gibberellin- and cytokinin-treated ovaries than for auxin-induced parthenocarpic fruits. In senescing ovaries, SAMs mRNA levels also increased, probably associated with ethylene biosynthesis since treatment of the ovaries with aminoethoxyvinylglycine resulted in a delay of senescence and prevention of SAMs mRNA accumulation. A possible mechanism for hormonal regulation of SAMs during ovary development is discussed.

Molecular Characterization and Evolution of the Protein Phosphatase 2A B′ Regulatory Subunit Family in Plants

Type 2A serine/threonine protein phosphatases (PP2A) are important components in the reversible protein phosphorylation events in plants and other organisms. PP2A proteins are oligomeric complexes constituted by a catalytic subunit and several regulatory subunits that modulate the activity of these phosphatases. The analysis of the complete genome of Arabidopsis allowed us to characterize four novel genes, AtB′ε, AtB′ζ,AtB′η, and AtB′θ, belonging to the PP2A B′ regulatory subunit family. Because four genes of this type had been described previously, this family is composed of eight members. Reverse transcriptase-polymerase chain reaction experiments showed thatAtB′ε mRNAs are present in all Arabidopsis tissues analyzed, and their levels do not respond significantly to heat stress. Expressed sequence tags corresponding to AtB′ζ,AtB′η, and AtB′θ have been identified, indicating that the new genes are actively transcribed. The genomic organization of this family of PP2A regulatory subunits is reported, as well as its chromosomal location. An extensive survey of the family has been carried out in plants, characterizing B′ subunits in a number of different species, and performing a phylogenetic study that included several B′ regulatory proteins from animals. Our results indicate that the animal and plant proteins have evolved independently, that there is a relationship between the number of B′ isoforms and the complexity of the organism, and that there are at least three main subfamilies of regulatory subunits in plants, which we have named α, η, and κ.

Developmental and organ-specific changes in DNA-protein interactions in the tomato rbcS3B and rbcS3C promoter regions

Sites of DNA-protein interaction were mapped in the promoter regions of two of the five genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) in tomato. The two genes, designated rbcS3B and -3C, are actively transcribed in cotyledons of light-grown seedlings and in leaves, but are transcriptionally inactive cotyledons of dark-grown seedlings, in young and mature tomato fruit, and in roots. The combination and order of conserved DNA sequence elements in the promoter regions of the two genes are essentially identical, but differ considerably from that found in the promoters of the other three tomato rbcS genes, which show different transcription patterns. Nuclear extracts from cotyledons of 7-day-old tomato seedlings, and from leaves and young tomato fruit of mature plants defined multiple DNase I-protected sites in the promoter regions of both genes. The protection patterns were organspecific, and encompassed previously identified conserved DNA sequence motifs as well as uncharacterized sequences. In contrast, nuclear extracts from mature tomato fruit and roots of 7-day-old seedlings failed to protect any of the promoter sequences, implying that DNA-binding proteins required for transcription of rbcS3B and -3C are inactive in these organs. These results are somewhat surprising since DNA-binding proteins from cotyledons of dark-grown seedlings and young fruit interact with the two promoters, although rbcS3B and -3C are not transcribed in these organs. The basis for transcriptional regulation of these two genes is discussed and the detailed pattern of DNase I protection in the promoter regions of the two genes is presented.