Pedro M. Barros

Transcription Regulation of Abiotic Stress Responses in Rice: A Combined Action of Transcription Factors and Epigenetic Mechanisms

Plant growth and crop production are highly reduced by adverse environmental conditions and rice is particularly sensitive to abiotic stresses. Plants have developed a number of different mechanisms to respond and try to adapt to abiotic stress. Plant response to stress such as drought, cold, and high salinity, implies rapid and coordinated changes at transcriptional level of entire gene networks. During the last decade many transcription factors, belonging to different families, have been shown to act as positive or negative regulators of stress responsive genes, thus playing an extremely important role in stress signaling. More recently, epigenetic mechanisms have been also involved in the regulation of the stress responsive genes. In this review, we have performed a comprehensive analysis of the rice transcription factors reported so far as being involved in abiotic stress responses. The impact of abiotic stresses on epigenomes is also addressed. Finally, we update the connections made so far between DNA-binding transcription factors (TFs), and epigenetic mechanisms (DNA methylation and histones methylation or acetylation) emphasizing an integrative view of transcription regulation.

Seven zinc-finger transcription factors are novel regulators of the stress responsive gene OsDREB1B

Plants have evolved several mechanisms in order to cope with adverse environmental conditions. The transcription factors (TFs) belonging to the DREB1/CBF subfamily have been described as major regulators of the plant responses to different abiotic stresses. This study focused on the rice gene OsDREB1B, initially described as highly and specifically induced by cold. However, here it is shown that OsDREB1B is not only induced by low temperatures, but also by drought and mechanical stress. In order to identify novel TFs that bind to its promoter, a yeast one-hybrid system was used to screen a cold-induced cDNA expression library. Thereby seven novel Zn-finger TFs were identified that bind to the promoter of OsDREB1B. Among them, there were four Zn-finger homeodomain (ZF-HD) and three C(2)H(2)-type Zn-finger TFs. Gene expression studies showed that these TFs are differentially regulated at transcriptional level by different abiotic stress conditions, which is illustrative of the crosstalk between stress signalling pathways. Protein-protein interaction studies revealed the formation of homo- and heterodimers among the ZF-HD TFs identified, but not for the C(2)H(2)-type. Using a transactivation assay in Arabidopsis protoplasts, all the TFs identified repressed the expression of the reporter gene, driven by the promoter of OsDREB1B. This assay also showed that the dimerization observed between the ZF-HD TFs may play a role on their transactivation activity. The results here presented suggest a prominent role of Zn-finger TFs in the regulation of OsDREB1B.

Cold acclimation and floral development in almond bud break: insights into the regulatory pathways

In temperate fruit trees, seasonal dormancy and cold acclimation have a major impact on blooming time and, consequently, fruit production. To gain insight into the still unclear molecular processes underlying blooming, expression of genes putatively involved in the cold response was studied in almond (Prunus dulcis Mill.), which is the earliest fruit tree in the family Rosaceae to bloom. The transcript levels of two C-repeat binding factor (PdCBF) genes and one of their putative targets, PdDehydrin1 (PdDHN1), were analysed in flower buds and shoot internodes during seasonal dormancy up to bud break. In parallel, expression of candidate genes related to flower development was also followed. In a 2-year study, PdCBF2 showed a progressive increase in transcript abundance during the autumn in close correlation with cold acclimation, while high transcript levels of PdCBF1 and PdDHN1 were already found by summer. After bud break, with temperatures still within the chilling range, both PdCBF genes and PdDHN1 were found to sharply reduce transcription in flower buds and internodes, suggesting damping of CBF-mediated cold signalling during growth resumption, in correlation with cold hardiness decline. Flower bud break was also followed by a decrease in the expression of PdGA20OX, a candidate gene involved in gibberellin biosynthesis, and an increase in the expression of two homeotic genes related to floral organ development, PdMADS1 and -3. These genes may also be relevant players in the regulation of anthesis in this model Rosaceae species.

Functional characterization of two almond C-repeat-binding factors involved in cold response

Low temperature plays a crucial role in seasonal development of woody plants and may directly impact crop production, more particularly in temperate fruit trees. Given its high genetic variability and adaptability to different climatic conditions, almond (Prunus dulcis Mill.) is an interesting model to understand the mechanisms regulating low temperature sensing in fruit trees. In this paper, we report the cloning and characterization of two genes (PdCBF1 and PdCBF2) belonging to the C-repeat-binding factor (CBF) family of transcription factors. Southern blotting analysis showed that this family is composed of at least five members. In almond shoots propagated in vitro, transcription of these genes was rapidly induced by low temperature, suggesting an involvement in cold acclimation. Transactivation assays showed that PdCBF1 and PdCBF2 could bind to dehydration responsive element/C-repeat containing sequences, as activators of gene expression. In addition, induction of both PdCBFs by cold was higher towards the end of the day, which agreed with the expression pattern of PdDehydrin1, a predicted CBF target gene. Furthermore, PdCBF1 and PdCBF2 were also transiently induced by abscisic acid and drought treatments. Considering the bin mapping analysis that correlated PdCBFs and PdDHN1 (respectively in linkage groups 5 and 7) with two different quantitative trait locicontrolling blooming time, it is relevant to perform further association studies that may validate their effect on this trait.

Genomics of Drought

Foreseeable global environmental changes combine with a growing world population to increase pressure on water resources. In many regions, diminishing water availability for irrigation is leading to major constrains in crop productivity, posing a real threat and a challenge for food security in the next few years. Plants have evolved a myriad of adaptive mechanisms to cope with water scarcity. We can identify several different tolerance and resistance strategies, of multigenic nature and with different molecular backgrounds that may occur in the same or in different individuals. Therefore, in order to improve plant productivity under low water availability, we must understand the basic mechanisms of perception and molecular response to water deficit conditions. In this chapter, we revise the most relevant morphophysiological adaptations of plants, from roots to shoots, to resist water deficit, and we focus in more depth in the underneath molecular mechanisms. We discuss the perception and signal transduction mechanisms related to abscisic acid (ABA), a key phytohormone modulating several water deficit responses, as well as ABA-independent mechanisms. We also cover the molecular aspects leading to the accumulation of compatible solutes, aimed to reduce water loss or minimize its damaging effects, and analyze the impact of water scarcity on photosynthesis and regulation of stomatal gas exchanges. An overview of the epigenetic regulation of drought stress response is also provided. Although drought tolerance is a difficult trait to breed for, especially if based on a single-gene approach, we also highlight some genes described as positively affecting crop performance and productivity under water deficit regimes and that may aid in future breeding programs. Given the large extent of work already developed to address the drought effect on plants, this review is necessarily incomplete. However, it was designed to elucidate the main drought response strategies and to uncover the major findings and indicate ways for future progress.

Transcriptomics and physiological analyses reveal co-ordinated alteration of metabolic pathways in Jatropha curcas drought tolerance

Jatropha curcas, a multipurpose plant attracting a great deal of attention due to its high oil content and quality for biofuel, is recognized as a drought-tolerant species. However, this drought tolerance is still poorly characterized. This study aims to contribute to uncover the molecular background of this tolerance, using a combined approach of transcriptional profiling and morphophysiological characterization during a period of water-withholding (49 d) followed by rewatering (7 d). Morphophysiological measurements showed that J. curcas plants present different adaptation strategies to withstand moderate and severe drought. Therefore, RNA sequencing was performed for samples collected under moderate and severe stress followed by rewatering, for both roots and leaves. Jatropha curcas transcriptomic analysis revealed shoot- and root-specific adaptations across all investigated conditions, except under severe stress, when the dramatic transcriptomic reorganization at the root and shoot level surpassed organ specificity. These changes in gene expression were clearly shown by the down-regulation of genes involved in growth and water uptake, and up-regulation of genes related to osmotic adjustments and cellular homeostasis. However, organ-specific gene variations were also detected, such as strong up-regulation of abscisic acid synthesis in roots under moderate stress and of chlorophyll metabolism in leaves under severe stress. Functional validation further corroborated the differential expression of genes coding for enzymes involved in chlorophyll metabolism, which correlates with the metabolite content of this pathway.

The draft genome sequence of cork oak

Cork oak (Quercus suber) is native to southwest Europe and northwest Africa where it plays a crucial environmental and economical role. To tackle the cork oak production and industrial challenges, advanced research is imperative but dependent on the availability of a sequenced genome. To address this, we produced the first draft version of the cork oak genome. We followed a de novo assembly strategy based on high-throughput sequence data, which generated a draft genome comprising 23,347 scaffolds and 953.3 Mb in size. A total of 79,752 genes and 83,814 transcripts were predicted, including 33,658 high-confidence genes. An InterPro signature assignment was detected for 69,218 transcripts, which represented 82.6% of the total. Validation studies demonstrated the genome assembly and annotation completeness and highlighted the usefulness of the draft genome for read mapping of high-throughput sequence data generated using different protocols. All data generated is available through the public databases where it was deposited, being therefore ready to use by the academic and industry communities working on cork oak and/or related species.

Differential DNA Methylation Patterns Are Related to Phellogen Origin and Quality of Quercus suber Cork

DNA methylation is thought to influence Quercus suber cork quality, which is the main constraint for its economic valorisation. However, a deep knowledge of the cytosine methylation patterns disclosing the epigenetic variability of trees with different cork quality types is totally missing. This study investigates the hypothesis that variations in DNA methylation contribute to differences in cork cellular characteristics directly related to original or traumatic phellogen activity. We used MSAPs (Methylation Sensitive Amplified Polymorphism) to assess DNA methylation patterns of cork and leaf tissues of Q. suber adult trees growing in three cork oak stands. The relationship between the detected polymorphisms and the diversity of cork quality traits was explored by a marker-trait analysis focusing on the most relevant quality characteristics. Populations differed widely in cork quality, but only slightly in degree of epigenetic differentiation. Four MSAP markers (1.3% of the total) were significantly associated with the most noteworthy quality traits: wood inclusions (nails) and porosity. This evidence supports the potential role of cytosine methylation in the modulation of differential phellogen activity either involved in localized cell death or in pore production, resulting in different cork qualities. Although, the underlying basis of the methylation polymorphism of loci affecting cork quality traits remain unclear, the disclosure of markers statistically associated with cork quality strengthens the potential role of DNA methylation in the regulation of these traits, namely at the phellogen level.

The identification of almond GIGANTEA gene and its expression under cold stress, variable photoperiod, and seasonal dormancy

Seasonal growth is characteristic for many tree species including almond. Varying conditions during the season are responsible for growth cessation, bud set, dormancy entry, cold hardening, and bud burst. Here, we report the characterization of an almond homologue of the Arabidopsis GIGANTEA (AtGI) gene (designated as PdGI, GenBank accession No. KJ502316). We propose a role for this gene in the transition to dormancy and cold acclimation. The complementary DNA (cDNA) sequence of PdGI was 4 322 bp long and contained an open reading frame of 3 512 bp. The deduced amino acid sequence of PdGI shared 76 % identity with AtGI. The expression of PdGI at ambient day/night temperatures of 22/20 ºC was differentially regulated under a 16-h or 12-h photoperiod, increasing during the day and decreasing after dusk. However, this diurnal regulation was disrupted when plants were transferred to cold (12 ºC) conditions. In addition, we have assessed the expression of PdGI and putative almond homologues of the downstream target genes CONSTANS (PdCO-like) and FLOWERING LOCUS T (PdFT-like) in flower buds and shoots from adult trees during the bud break period in autumn and early winter. Our results show a clear increase in transcript abundance towards anthesis, suggesting a role of these genes in flower development.

Quality of life after elective cardiac surgery in elderly patients

OBJECTIVES Cardiac surgery has little effect on life expectancy in elderly patients. Thus, improving the quality of life should be the main factor affecting therapeutic decisions. Most studies on quality of life in elderly patients undergoing cardiac surgery report improvement but have limitations. Consequently, we assessed improvements in the quality of life of elderly patients undergoing elective cardiac surgery, identified influencing variables and established patterns of mental and physical health variations in the first year postoperatively. METHODS We conducted a prospective study of patients aged 65 or older who underwent elective cardiac surgery between September 2011 and August 2013. The 36-item Short Form (SF-36) surveys were obtained preoperatively and at 3, 6 and 12 months postoperatively. RESULTS The 430 preoperative patients with a mean age of 74 years (SD 5.5 years) comprised 220 men. Most physical health improvements occurred within 3 months and continued to improve significantly until 12 months. Predictive variables for patients showing less improvement were poor preoperative physical health, female sex, older age and longer length of hospital stay. Mental health improved significantly through the third postoperative month. The negative predictive variables were poor preoperative mental health and longer intensive care unit stay. CONCLUSIONS Most patients improved both physically and mentally after surgery, and most of the improvement occurred within 3 months post-surgery. These improvement patterns should be taken into account when creating rehabilitation programmes, and patients should be counselled on what improvements can be expected during the first 12 months after surgery.