Cell-type-specific alternative splicing in the Arabidopsis germline.

During sexual reproduction in flowering plants, the two haploid sperm cells (SCs) embedded within the cytoplasm of a growing pollen tube are carried to the embryo sac for double fertilization. Pollen development in flowering plants is a dynamic process that encompasses changes at transcriptome and epigenome levels. While the transcriptome of pollen and SCs in Arabidopsis (Arabidopsis thaliana) is well documented, previous analyses have mostly been based on gene-level expression. In-depth transcriptome analysis, particularly the extent of alternative splicing (AS) at the resolution of SC and vegetative nucleus (VN), is still lacking. Therefore, we performed RNA-seq analysis to generate a spliceome map of Arabidopsis SCs and VN isolated from mature pollen grains. Based on our de novo transcriptome assembly, we identified 58,039 transcripts, including 9,681 novel transcripts, of which 2,091 were expressed in SCs and 3,600 in VN. Four hundred and sixty-eight genes were regulated both at gene and splicing levels, with many having functions in mRNA splicing, chromatin modification, and protein localization. Moreover, a comparison with egg cell RNA-seq data uncovered sex-specific regulation of transcription and splicing factors. Our study provides insights into a gamete-specific AS landscape at unprecedented resolution.

The Combined Effect of Heat and Osmotic Stress on Suberization of Arabidopsis Roots.

The simultaneous occurrence of heat stress and drought is becoming more regular as a consequence of climate change, causing extensive agricultural losses. The application of either heat or osmotic stress increase cell-wall suberization in different tissues, which may play a role in improving plant resilience. In this work, we studied how the suberization process is affected by the combination of drought and heat stress by following the expression of suberin biosynthesis genes, cell-wall suberization and the chemical composition in Arabidopsis roots. The Arabidopsis plants used in this study were at the onset of secondary root development. At this point, one can observe a developmental gradient in the main root, with primary development closer to the root tip and secondary development, confirmed by the suberized phellem, closer to the shoot. Remarkably, we found a differential response depending on the root zone. The combination of drought and heat stress increased cell wall suberization in main root segments undergoing secondary development and in lateral roots (LRs), while the main root zone, at primary development stage, was not particularly affected. We also found differences in the overall chemical composition of the cell walls in both root zones in response to combined stress. The data gathered showed that, under combined drought and heat stress, Arabidopsis roots undergo differential cell wall remodeling depending on developmental stage, with modifications in the biosynthesis and/or assembly of major cell wall components.

Quercus suber Roots Activate Antioxidant and Membrane Protective Processes in Response to High Salinity.

Cork oak (Quercus suber) is a species native to Mediterranean areas and its adaptation to the increasingly prevalent abiotic stresses, such as soil salinization, remain unknown. In sequence with recent studies on salt stress response in the leaf, it is fundamental to uncover the plasticity of roots directly exposed to high salinity to better understand how Q. suber copes with salt stress. In the present study we aimed to unveil the antioxidants and key-genes involved in the stress-responses (early vs. later responses) of Q. suber roots exposed to high salinity. Two-month-old Q. suber plants were watered with 300 mM NaCl solution and enzymatic and non-enzymatic antioxidants, lipid peroxidation and the relative expression of genes related to stress response were analysed 8 h and 6 days after salt treatment. After an 8 h of exposure, roots activated the expression of QsLTI30 and QsFAD7 genes involved in stress membrane protection, and QsRAV1 and QsCZF1 genes involved in tolerance and adaptation. As a result of the continued salinity stress (6 days), lipid peroxidation increased, which was associated with an upregulation of QsLTI30 gene. Moreover, other protective mechanisms were activated, such as the upregulation of genes related to antioxidant status, QsCSD1 and QsAPX2, and the increase of the antioxidant enzyme activities of superoxide dismutase, catalase, and ascorbate peroxidase, concomitantly with total antioxidant activity and phenols. These data suggest a response dependent on the time of salinity exposure, leading Q. suber roots to adopt protective complementary strategies to deal with salt stress.

Spatiotemporal development of suberized barriers in cork oak taproots.

The longevity and high activity of the cork cambium (or phellogen) from Quercus suber L. (cork oak) are the cornerstones for the sustainable exploitation of a unique raw material. Cork oak is a symbolic model to study cork development and cell wall suberization, yet most genetic and molecular studies on these topics have targeted other model plants. In this study, we explored the potential of taproots as a model system to study phellem development and suberization in cork oak, thereby avoiding the time constraints imposed when studying whole plants. In roots, suberin deposition is found in mature endodermis cells during primary development and in phellem cells during secondary development. By investigating the spatiotemporal characteristics of both endodermis and phellem suberization in young seedling taproots, we demonstrated that secondary growth and phellogen activity are initiated very early in cork oak taproots (approx. 8 days after sowing). We further compared the transcriptomic profile of root segments undergoing primary (PD) and secondary development (SD) and identified multiple candidate genes with predicted roles in cell wall modifications, mainly lignification and suberization, in addition to several regulatory genes, particularly transcription factor- and hormone-related genes. Our results indicate that the molecular regulation of suberization and secondary development in cork oak roots is relatively conserved with other species. The provided morphological characterization creates new opportunities to allow a faster assessment of phellogen activity (as compared with studies using stem tissues) and to tackle fundamental questions regarding its regulation.

CorkOakDB-The Cork Oak Genome Database Portal.

Quercus suber (cork oak) is an evergreen tree native to the Mediterranean basin, which plays a key role in the ecology and economy of this area. Over the last decades, this species has gone through an observable decline, mostly due to environmental factors. Deciphering the mechanisms of cork oak's response to the environment and getting a deep insight into its biology are crucial to counteract biotic and abiotic stresses compromising the stability of a unique ecosystem. In the light of these setbacks, the publication of the genome in 2018 was a major step towards understanding the genetic make-up of this species. In an effort to integrate this information in a comprehensive, accessible and intuitive format, we have developed The Cork Oak Genome Database Portal (CorkOakDB). The CorkOakDB is supported by the BioData.pt e-infrastructure, the Portuguese ELIXIR node for biological data. The portal gives public access to search and explore the curated genomic and transcriptomic data on this species. Moreover, CorkOakDB provides a user-friendly interface and functional tools to help the research community take advantage of the increased accessibility to genomic information. A study case is provided to highlight the functionalities of the portal. CorkOakDB guarantees the update, curation and data collection, aiming to collect data besides the genetic/genomic information, in order to become the main repository in cork oak research. Database URL: http://corkoakdb.org/.

Molecular evolution and diversification of the GRF transcription factor family.

- Growth Regulating Factors (GRFs) comprise a transcription factor family with important functions in plant growth and development. They are characterized by the presence of QLQ and WRC domains, responsible for interaction with proteins and DNA, respectively. The QLQ domain is named due to the similarity to a protein interaction domain found in the SWI2/SNF2 chromatin remodeling complex. Despite the occurrence of the QLQ domain in both families, the divergence between them had not been further explored. Here, we show evidence for GRF origin and determined its diversification in angiosperm species. Phylogenetic analysis revealed 11 well-supported groups of GRFs in flowering plants. These groups were supported by gene structure, synteny, and protein domain composition. Synteny and phylogenetic analyses allowed us to propose different sets of probable orthologs in the groups. Besides, our results, together with functional data previously published, allowed us to suggest candidate genes for engineering agronomic traits. In addition, we propose that the QLQ domain of GRF genes evolved from the eukaryotic SNF2 QLQ domain, most likely by a duplication event in the common ancestor of the Charophytes and land plants. Altogether, our results are important for advancing the origin and evolution of the GRF family in Streptophyta.

Long-term somatic memory of salinity unveiled from physiological, biochemical and epigenetic responses in two contrasting rice genotypes.

Plants are constantly exposed to environmental fluctuations, that may occur in a single day or over longer periods. In many cases, abiotic stresses are transient and recurrent, impacting how plants respond in subsequent adverse conditions. Adaptation mechanisms may occur at the physiological, biochemical and molecular level, modifying transcriptional response, regulatory proteins, epigenetic marks or metabolites. Here, we aimed to uncover the different strategies that rice uses to respond to recurrent stress. We tested varieties with contrasting behavior towards salinity (tolerance or sensitivity) and imposed salt stress (150 mM NaCl) during 48 h at vegetative and/or reproductive stages. After 48 h of stress in reproductive stage, leaves and roots were harvested separately or otherwise the plants were submitted to a 24 h recovery, prior to sample harvesting. Plants submitted to a recurrent stress responded differently from those suffering a single stress event. In the case of the sensitive genotype, recurrent stress led to lower Na/K ratio in roots and lower hydrogen peroxide accumulation and lipid peroxidation in leaves, but maintenance of global DNA methylation levels. In the tolerant genotype, recurrent stress did neither affect the Na/K ratio nor the stomatal conductance, although the levels of superoxide anion and hydrogen peroxide accumulation were lower, as also observed for global levels of DNA methylation. Our work shows that a short pre-exposure to salt stress may improve rice tolerance to subsequent stress, trough biochemical, physiological and epigenetic processes, with more significant changes visible in the tolerant genotype.

More Transporters, More Substrates: The Arabidopsis Major Facilitator Superfamily Revisited.

The Major Facilitator Superfamily (MFS) is ubiquitous in living organisms and represents the largest group of secondary active membrane transporters. In plants, significant research efforts have focused on the role of specific families within the MFS, particularly those transporting macronutrients (C, N, and P) that constitute the vast majority of the members of this superfamily. Other MFS families remain less explored, although a plethora of additional substrates and physiological functions have been uncovered. Nevertheless, the lack of a systematic approach to analyzing the MFS as a whole has obscured the high diversity and versatility of these transporters. Here, we present a phylogenetic analysis of all annotated MFS domain-containing proteins encoded in the Arabidopsis thaliana genome and propose that this superfamily of transporters consists of 218 members, clustered in 22 families. In reviewing the available information regarding the diversity in biological functions and substrates of Arabidopsis MFS members, we provide arguments for intensified research on these membrane transporters to unveil the breadth of their physiological relevance, disclose the molecular mechanisms underlying their mode of action, and explore their biotechnological potential.

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.

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.

Synergistic Binding of bHLH Transcription Factors to the Promoter of the Maize NADP-ME Gene Used in C4 Photosynthesis Is Based on an Ancient Code Found in the Ancestral C3 State.

C4 photosynthesis has evolved repeatedly from the ancestral C3 state to generate a carbon concentrating mechanism that increases photosynthetic efficiency. This specialized form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world's most productive crops. The C4 cycle is accomplished through cell-type-specific accumulation of enzymes but cis-elements and transcription factors controlling C4 photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we tested whether mechanisms impacting on transcription in C4 plants evolved from ancestral components found in C3 species. Two basic Helix-Loop-Helix (bHLH) transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C4NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans-activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis-elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis-elements is found in both C3 and C4 Panicoid grass species of the PACMAD clade. Our analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C3 state. We conclude that C4 photosynthesis has co-opted an ancient C3 regulatory code built on G-box recognition by bHLH to regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C4 photosynthesis.

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.

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.

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.

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.

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.