Alejandra A. Covarrubias

The Enigmatic LEA Proteins and Other Hydrophilins

Water limitation affects all types of organisms at some stage during their life cycle; therefore, many strategies have been selected through evolution to cope with water deficit, including changes in enzyme activities and in gene expression, among others. In plants, a group of very hydrophilic

Conserved and novel miRNAs in the legume Phaseolus vulgaris in response to stress.

MicroRNAs (miRNAs) are small RNA molecules recognized as important regulators of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data. We are interested in the identification of miRNAs in Phaseolus vulgaris (common bean) to uncover different plant strategies to cope with adverse conditions and because of its relevance as a crop in developing countries. Here we present the identification of conserved and candidate novel miRNAs in P. vulgaris present in different organs and growth conditions, including drought, abscisic acid treatment, and Rhizobium infection. We also identified cDNA sequences in public databases that represent the corresponding miRNA precursors. In addition, we predicted and validated target mRNAs amongst reported EST and cDNAs for P. vulgaris. We propose that the novel miRNAs present in common bean and other legumes, are involved in regulation of legume-specific processes including adaptation to diverse external cues.

Abscisic acid inhibits germination of mature Arabidopsis seeds by limiting the availability of energy and nutrients

Abstract. The addition of abscisic acid (ABA) to mature non-dormant seeds inhibits their germination. This effect of ABA might be related to its natural function as an endogenous inhibitor of precocious germination during seed formation. In this work, we studied how ABA affects the germination of mature seeds and the growth of nascent seedlings of Arabidopsisthaliana (L.) Heynh. Our findings were as follows: (i) inhibition by ABA was gradual, dose-dependent, and did not disappear after germination; (ii) inhibition of germination was relieved by the addition of metabolizable sugars or amino acids to the plating media; (iii) the effect of sugars and amino acids was cooperative, indicating that these two groups of metabolites relieve different deficiencies; (iv) ABA caused appreciable alterations in energy and nitrogen metabolism; and (v) ABA prevented the degradation of the seed storage proteins. In summary, ABA appears to inhibit seed germination by restricting the availability of energy and metabolites. This mechanism seems consistent with other known effects of ABA.

Three genes whose expression is induced by stress in Saccharomyces cerevisiae

In this work we report the isolation and characterization of three genes induced by different stress conditions in the yeast Saccharomyces cerevisiae. These genes, named GRE1, GRE2 and GRE3, were identified by the differential display technique using total RNAs obtained from yeast grown under hyperosmotic conditions. Northern analysis of RNA obtained from different growth conditions shows that their corresponding transcripts accumulate not only in response to osmotic stress but also to ionic, oxidative and heat stress. Analysis of the deduced amino acid sequences indicated that GRE1, GRE2 and GRE3 correspond to ORFs YPL223C, YOL151W and YHR104W, respectively. Additionally, it suggested that GRE1 encodes a hydrophilic polypeptide that it is not homologous to any known protein but has features resembling the late embryogenesis abundant (LEA) proteins characterized in higher plants; GRE2 encodes a putative reductase with similarity to plant dihydroflavonol‐4‐reductases; and GRE3 codifies for a keto‐aldose reductase highly related to fungal xylose‐reductases. The three genes are induced in the late growth phases in agreement with the presence of PDS elements in their promoter regions. The three of them are under the control of the HOG pathway, even though GRE1 and GRE2 promoter regions do not present the consensus core STRE sequence. In addition, GRE1 and GRE3 are regulated negatively by the cAMP–PKA transduction pathway and positively by the transcriptional factors Msn2p and Msn4p. Gene disruptions of the GRE genes did not show a phenotype in any of the tested stress conditions. Copyright

Post‐transcriptional gene regulation of salinity and drought responses by plant microRNAs

In the past few years, factors involved in abscisic acid signalling have been isolated and recognized as elements related to RNA metabolism, suggesting that post-transcriptional regulation of gene expression is required for abiotic stress responses. Some of these factors can be linked to the biogenesis of microRNAs (miRNAs), small RNA molecules that are important regulators of gene expression at the posttranscriptional level by repressing mRNA expression. Here, we review the role of miRNAs in stress responses, highlighting recent advances in elucidating the role of individual miRNAs and efforts to identify stress-responsive miRNAs at a genome-wide level in different model plants. Complete understanding of miRNA action depends on the identification of its target transcripts, and recent developments in miRNA research indicate that they will be uncovered in the near future.

Construction and characterization of new cloning vehicles V. Mobilization and coding properties of pBR322 and several deletion derivatives including pBR327 and pBR328

A DNA sequence essential for the R64drd11 + ColK-mediated conjugal transfer of pBR322 has been located in a 540 bp HaeIII fragment (HaeIII-2) between the vegetative origin of replication and the tetracycline resistance (Tcr) gene of this vector. The pBR322 derivatives pBR327 and pBR328 lack this DNA sequence and are not mobilized by conjugation. Two derivatives of pBR328 were constructed by re-inserting the HaeIII-2 fragment in both orientations into the chloramphenicol-resistance gene of the same vector. One orientation of the HaeIII-2 fragment permitted mobilization by conjugation while the opposite orientation prevented mobilization. Further examination of pBR322 and derivatives revealed that the region between the origin of replication and Tcr gene also plays a role in regulating plasmid copy number.

Functional Analysis of the Group 4 Late Embryogenesis Abundant Proteins Reveals Their Relevance in the Adaptive Response during Water Deficit in Arabidopsis

Late-Embryogenesis Abundant (LEA) proteins accumulate to high levels during the last stages of seed development, when desiccation tolerance is acquired, and in vegetative and reproductive tissues under water deficit, leading to the hypothesis that these proteins play a role in the adaptation of plants to this stress condition. In this work, we obtained the accumulation patterns of the Arabidopsis (Arabidopsis thaliana) group 4 LEA proteins during different developmental stages and plant organs in response to water deficit. We demonstrate that overexpression of a representative member of this group of proteins confers tolerance to severe drought in Arabidopsis plants. Moreover, we show that deficiency of LEA proteins in this group leads to susceptible phenotypes upon water limitation, during germination, or in mature plants after recovery from severe dehydration. Upon recovery from this stress condition, mutant plants showed a reduced number of floral and axillary buds when compared with wild-type plants. The lack of these proteins also correlates with a reduced seed production under optimal irrigation, supporting a role in fruit and/or seed development. A bioinformatic analysis of group 4 LEA proteins from many plant genera showed that there are two subgroups, originated through ancient gene duplication and a subsequent functional specialization. This study represents, to our knowledge, the first genetic evidence showing that one of the LEA protein groups is directly involved in the adaptive response of higher plants to water deficit, and it provides data indicating that the function of these proteins is not redundant to that of the other LEA proteins.

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.

Functional dissection of Hydrophilins during in vitro freeze protection

In plants, Late Embryogenesis Abundant (LEA) proteins typically accumulate in response to low water availability conditions imposed during development or by the environment. Analogous proteins in other organisms are induced when exposed to stress conditions. Most of this diverse set of proteins can be grouped according to properties such as high hydrophilicity and high content of glycine or other small amino acids in what we have termed hydrophilins. Previously, we showed that hydrophilins protect enzyme activities in vitro from low water availability effects. Here, we demonstrate that hydrophilins can also protect enzyme activities from the adverse effects induced by freeze-thaw cycles in vitro. We monitored conformational changes induced by freeze-thaw on the enzyme lactate dehydrogenase (LDH) using the fluorophore 1-anilinonaphthalene-8-sulfonate (ANS). Hydrophilin addition prevents enzyme inactivation and this effect is reflected in changes in the ANS-fluorescence levels determined for LDH. We further show that for selected plant hydrophilins, removal of certain conserved domains affects their protecting capabilities. Thus, we propose that hydrophilins, and in particular specific protein domains, have a role in protecting cell components from the adverse effects caused by low water availability such as those present during freezing conditions by preventing deleterious changes in protein secondary and tertiary structure.

Construction of a CUP1 promoter-based vector to modulate gene expression in Saccharomyces cerevisiae

We report the construction and characterization of a yeast shuttle-expression vector that can be used to express genes in yeast in a modulated form. This vector is centromeric, with a polylinker that optionally can provide an ATG start of translation. Expression features are based on the CUP1 yeast metallothionein gene promoter, which can be tightly modulated by copper.