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Rapid population growth and rising food demand present severe challenges to agroecosystems. One significant constraint to crop productivity worldwide is soil salinity.Rice (Oryza sativa L.) is an essential crop worldwide feeding half of the world population, but it is highly susceptible to abiotic stress conditions, including soil salinity.Seedborne PGPB (plant growth promoting bacteria) have been gaining attention since it constitutes a primary inoculum for the plant. Identifying salt-tolerant PGPB (ST-PGPB) can lead to novel mechanisms for coping with salt stress and providing sustainable solutions for agriculture.Inoculation of salt-sensitive rice plants with P. pabuli, B. subtilis, B. altitudinis show promising improvement in salt-tolerance. The genes OsHKT2;1 and OsCATA, seem to have the potential to be influenced by PGPB.

Thiamin (or thiamine), known as vitamin B1, represents an indispensable component of human diets, being pivotal in energy metabolism. Thiamin research depends on adequate vitamin quantification in plant tissues. A recently developed quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is able to assess the level of thiamin, its phosphorylated entities and its biosynthetic intermediates in the model plant Arabidopsis thaliana, as well as in rice. However, their implementation requires expensive equipment and substantial technical expertise. Microbiological assays can be useful in deter-mining metabolite levels in plant material and provide an affordable alternative to MS-based analysis. Here, we evaluate, by comparison to the LC-MS/MS reference method, the potential of a carefully chosen panel of yeast assays to estimate levels of total vitamin B1, as well as its biosynthetic intermediates pyrimidine and thiazole in Arabidopsis samples. The examined panel of Saccharomyces cerevisiae mutants was, when implemented in microbiological assays, capable of correctly assigning a series of wild-type and thiamin biofortified Arabidopsis plant samples. The assays provide a readily applicable method allowing rapid screening of vitamin B1 (and its biosynthetic intermediates) content in plant material, which is particularly useful in metabolic engineering approaches and in germplasm screening across or within species.

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.

Cork oak (Quercus suber), native to Mediterranean areas, is a plant of ecological and economical relevance, nevertheless, the effects of soil salinization on this species are currently unknown. We have investigated the physiological and biochemical impact of a high-salinity episode on young cork oak (Q. suber) plants. Besides the control (plants only irrigated with water), two experimental groups (irrigated once with a 300 mM NaCl solution) were analysed, one assessed at 24 h and the other at 6 days. Pigments (chlorophylls and carotenoids) were found increased at 24 h, but decreased at day 6 in salinity conditions. Sugars (glucose, sucrose, starch but not fructose) increased with stress (24 h and 6 days). Salinity conditions impaired photosystem II (PSII) photochemistry, mostly associated with decrease in Fv′/Fm′ and chlorophyll content (6 days). While hydrogen peroxide levels did not increase above control levels, lipid peroxidation increased, suggesting oxidative damage. In salinity conditions, superoxide dismutase and ascorbate peroxidase showed higher activity in the 24 h timepoint, whereas catalase activity increased at 24 h and 6 days. These observations reveal adaptations of Q. suber to high salinity, nevertheless, the decreased photosynthetic activity and oxidative damages observed suggest that additional studies are required to assess Q. suber adaptation to diverse salinity conditions. Moreover, these data provide more information for future programs of conservation and management of salinity areas in the Mediterranean region, and selection of salinity tolerant species.

Jatropha curcas is a drought-tolerant plant that maintains its photosynthetic pigments under prolonged drought, and quickly regains its photosynthetic capacity when water is available. It has been reported that drought stress leads to increased thermal dissipation in PSII, but that of PSI has been barely investigated, perhaps due to technical limitations in measuring the PSI absolute quantum yield. In this study, we combined biochemical analysis and spectroscopic measurements using an integrating sphere, and verified that the quantum yields of both photosystems are temporarily down-regulated under drought. We found that the decrease in the quantum yield of PSII was accompanied by a decrease in the core complexes of PSII while light-harvesting complexes are maintained under drought. In addition, in drought-treated plants, we observed a decrease in the absolute quantum yield of PSI as compared with the well-watered control, while the amount of PSI did not change, indicating that non-photochemical quenching occurs in PSI. The down-regulation of both photosystems was quickly lifted in a few days upon re-watering. Our results indicate, that in J. curcas under drought, the down-regulation of both PSII and PSI quantum yield protects the photosynthetic machinery from uncontrolled photodamage.

Rice (Oryza sativa) is one of the world's most important food crops, and is comprised largely of japonica and indica subspecies. Here, we reconstruct the history of rice dispersal in Asia using whole-genome sequences of more than 1,400 landraces, coupled with geographic, environmental, archaeobotanical and paleoclimate data. Originating around 9,000 yr ago in the Yangtze Valley, rice diversified into temperate and tropical japonica rice during a global cooling event about 4,200 yr ago. Soon after, tropical japonica rice reached Southeast Asia, where it rapidly diversified, starting about 2,500 yr BP. The history of indica rice dispersal appears more complicated, moving into China around 2,000 yr BP. We also identify extrinsic factors that influence genome diversity, with temperature being a leading abiotic factor. Reconstructing the dispersal history of rice and its climatic correlates may help identify genetic adaptations associated with the spread of a key domesticated species.

Unable to move, plants are physically restrained to the place where they grow. Remarkably, plants have developed a myriad of mechanisms to perceive the surrounding environment in order to maximize growth and survival. One of those mechanisms is the ability to perceive mechanical stimulus such as touch (thigmomorphogenesis), in order to adjust growth patterns (in different organs) to either attach to or surround an object. Roots are able to perceive several mechanical forces (e.g., gravity, touch). However, being the "hidden part" of a plant, it is difficult to assess their response to mechanical stimulation. In this chapter, our team presents a simple method to evaluate rice (Oryza sativa L.) root mechanosensing response that can be used to test different conditions (e.g., hormones) affecting rice root response to touch stimulus. This method is affordable to any lab and can be upgraded with a fully automated image recording system. We provide a detailed protocol with several notes for a more comprehensive application.

Rice (Oryza sativa L.) is the staple food for over half of the world population. However, most rice varieties are severely injured by abiotic stresses, with strong social and economic impacts. Understanding rice responses to stress may guide breeding for more tolerant varieties. However, the lack of consistency in the design of the stress experiments described in the literature limits comparative studies and output assessments. The use of identical setups is the only way to generate comparable data. This chapter comprises three sections, describing the experimental conditions established at the Genomics of Plant Stress (GPlantS) unit of ITQB NOVA to assess the response of rice plants to different abiotic stresses-high salinity, cold, drought, simulated drought, and submergence-and their recovery capacity when intended. All sections include a detailed description of the materials and methodology and useful notes gathered from our team experience. We use seedlings since rice plants at this stage show high sensitivity to abiotic stresses. For the salt, cold, and simulated drought (PEG, polyethylene glycol) stress assays, we grow rice seedlings in a hydroponic system, while for the drought assay, plants are grown in soil and subjected to water withholding. For submergence, we use water-filled Magenta boxes. All setups enable visual score determination and are suitable for sample collection during stress imposition and also recovery. The proposed methodologies are affordable and straightforward to implement in most labs, allowing the discrimination of several rice genotypes at the molecular and phenotypic levels.

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.

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.

The phellem is a specialized boundary tissue providing the first line of defense against abiotic and biotic stresses in organs undergoing secondary growth. Phellem cells undergo several differentiation steps, which include cell wall suberization, cell expansion and programmed cell death. Yet, the molecular players acting particularly in phellem cell differentiation remain poorly described, particularly in the widely used model plant Arabidopsis thaliana. Using specific marker lines we followed the onset and progression of phellem differentiation in A. thaliana roots, and further targeted the translatome of new developed phellem cells using Translating Ribosome Affinity Purification followed by mRNA sequencing (TRAP-SEQ). We showed that phellem suberization is initiated early after phellogen (cork cambium) division. The specific translational landscape was organized in three main domains related to energy production, synthesis and transport of cell wall components, and response to stimulus. Novel players in phellem differentiation, related to suberin monomer transport and assembly, as well as novel transcription regulators were identified. This strategy provided an unprecedented resolution of the translatome of developing phellem cells, giving a detailed and specific view on the molecular mechanisms acting on cell differentiation in periderm tissues of the model plant Arabidopsis.

Soil is a living ecosystem, the health of which depends on fine interactions among its abiotic and biotic components. These form a delicate equilibrium maintained through a multilayer network that absorbs certain perturbations and guarantees soil functioning. Deciphering the principles governing the interactions within soils is of critical importance for their management and conservation. Here, we focus on soil microbiota and discuss the complexity of interactions that impact the composition and function of soil microbiota and their interaction with plants. We discuss how physical aspects of soils influence microbiota composition and how microbiota-plant interactions support plant growth and responses to nutrient deficiencies. We predict that understanding the principles determining the configuration and functioning of soil microbiota will contribute to the design of microbiota-based strategies to preserve natural resources and develop more environmentally friendly agricultural practices.

Cynara cardunculus health benefits have aroused much interest, leading to the discovery of valuable bioactive compounds with a crucial role in plant defence. Guaianolides and hydroxycinnamic acids, mainly represented by cynaropicrin and chlorogenic acid, constitute the major secondary metabolites in leaves (9.5% and 10.4% dry weight, respectively). These compounds evidence biological activity, namely antioxidant, antitumoral, hepatoprotective, antimicrobial and anti-hyperlipidemic effects. Therefore, numerous efforts have been undertaken in this species to unveil the biosynthetic pathways of such compounds, by means of genomic and biochemical approaches, which could support advances, via breeding programs, in C. cardunculus chemical composition and, consequently, in the improvement of its extracts biological activity. Addressing this challenge, relevant genes in cynaropicrin biosynthesis, as well as in chlorogenic acid biosynthesis have been widely studied. The present review highlights the current knowledge on the biosynthesis and distribution of guaianolides and hydroxycinnamic acids, especially of cynaropicrin and chlorogenic acid in C. cardunculus, as well as their association with plant defence mechanisms and human health-promoting effects, prospecting the valorisation of this Mediterranean species as a potential source of bioactive compounds for food, nutraceutical and pharmaceutical purposes.

Plants are unable to physically escape environmental constraints and have, therefore, evolved a range of molecular and physiological mechanisms to maximize survival in an ever-changing environment. Among these, the post-translational modification of ubiquitination has emerged as an important mechanism to understand and improve the stress response. The ubiquitination of a given protein can change its abundance (through degradation), alter its localization, or even modulate its activity. Hence, ubiquitination increases the plasticity of the plant proteome in response to different environmental cues and can contribute to improve stress tolerance. Although ubiquitination is mediated by different enzymes, in this review, we focus on the importance of E3-ubiquitin ligases, which interact with the target proteins and are, therefore, highly associated with the mechanism specificity. We discuss their involvement in abiotic stress response and place them as putative candidates for ubiquitination-based development of stress-tolerant crops. This review covers recent developments in this field using rice as a reference for crops, highlighting the questions still unanswered.

SummaryThe phellem is a specialized boundary tissue providing the first line of defense against abiotic and biotic stresses in organs undergoing secondary growth. Phellem cells undergo several differentiation steps, which include cell wall suberization, cell expansion and programmed cell death. Yet, the molecular players acting particularly in phellem cell differentiation remain poorly described, particularly in the widely used model plantArabidopsis thaliana.Using specific marker lines we followed the onset and progression of phellem differentiation inA. thalianaroots, and further targeted the translatome of new developed phellem cells using Translating Ribosome Affinity Purification followed by mRNA sequencing (TRAP-SEQ).We showed that phellem suberization is initiated early after phellogen (cork cambium) division. The specific translational landscape was organized in three main domains related to energy production, synthesis and transport of cell wall components, and response to stimulus. Novel players in phellem differentiation, related to suberin monomer transport and assembly, as well as novel transcription regulators were identified.This strategy provided an unprecedented resolution of the transcriptome of developing phellem cells, giving a detailed and specific view on the molecular mechanisms controlling cell differentiation in periderm tissues of the model plantArabidopsis.Significance statementTo improve the understanding of phellem differentiation into a suberized protective layer, we followed the establishment of periderm inArabidopsisroots and sequenced the phellem-specific translatome. We found that phellem suberization occurs shortly after pericycle cell divisions with the induction of pivotal suberin biosynthesis genes. In parallel, we detected the activation of three central genetic modules acting throughout the phellem differentiation. This study provides a unique and targeted genetic resource for further functional studies of phellem tissues.

Calcium-dependent protein kinases (CDPKs) play essential roles in plant development and stress responses. CDPKs have a conserved kinase domain, followed by an auto-inhibitory junction connected to the calmodulin-like domain that binds Ca2+. These structural features allow CDPKs to decode the dynamic changes in cytoplasmic Ca2+ concentrations triggered by hormones and by biotic and abiotic stresses. In response to these signals, CDPKs phosphorylate downstream protein targets to regulate growth and stress responses according to the environmental and developmental circumstances. The latest advances in our understanding of the metabolic, transcriptional, and protein–protein interaction networks involving CDPKs suggest that they have a direct influence on plant carbon/nitrogen (C/N) balance. In this review, we discuss how CDPKs could be key signaling nodes connecting stress responses with metabolic homeostasis, and acting together with the sugar and nutrient signaling hubs SnRK1, HXK1, and TOR to improve plant fitness., This review discusses the possible links between Ca2+ signaling through calcium-dependent protein kinases and the regulation of carbon and nitrogen metabolism during stress.

Goji berries’ bioactive compounds, which allowed classifying them as superfruits, led to an enormous increase of its consumption in western countries. However, the potential risk of allergy is a concern. In this study, we aimed to characterise the proteome of goji berries (Lycium barbarum) and identify proteins with putative role in the allergic reaction (IgE-binding proteins). We firstly used twodimensional (2D) gel electrophoresis followed by mass spectrometry (MS) to characterise goji berries’ proteome, and then Immunoblot reactivity with plasma from tomato and potato (same botanical family, Solanaceae) allergic individuals was assessed to characterise goji berries IgE-binding proteins. An inhibition assay was further performed to evaluate cross-reactivity among potato, tomato and goji berries. We significantly identified 93 out of the 180 MS analysed spots, corresponding to 29 protein functions. From these, 11 could be identified as goji berries IgE-binding proteins. We further demonstratedcross-reactivity between goji berries, tomato and potato. This work was supported by research unit GREEN-IT “Bioresources for Sustainability” (UID/Multi/04551/2013). IML acknowledges FCT for funding her PhD fellowship (PD/BD/113982/2015) under MolBioS PhD-Program (PD/00133/2012), and IAA acknowledges the FCT Investigator funding (POPH-QREN - IF/00764/2014). info:eu-repo/semantics/publishedVersion

DELLA proteins modulate GA signalling and are major regulators of plant plasticity to endure stress. DELLAs are mostly regulated at the post-translational level, and their activity relies on the interaction with upstream regulators and transcription factors (TFs). SUMOylation is a post-translational modification (PTM) capable of changing protein interaction and found to influence DELLA activity in Arabidopsis. We determined that SUMOylation of the single rice DELLA SLENDER RICE1 (SLR1) occurs in a lysine residue different from the one previously identified in Arabidopsis REPRESSOR OF GA (RGA). Remarkably, artificially increasing SUMOylated SLR1 (SUMO1SLR1) levels attenuated the penalty of salt stress on plant yield. Gene expression analysis revealed that the overexpression of SUMOylated SLR1 regulates key dioxygenases that modulate active GA levels, namely GA20ox2 and GA2ox3, which could partially explain the sustained productivity upon salt stress imposition. Besides, SLR1 SUMOylation blocked the interaction with the growth regulator YAB4, which may fine-tune GA20ox2 expression. Mechanistically, we propose that SLR1 SUMOylation disrupts the interaction with members of several transcription factor families to modulate gene expression. We found that SLR1 SUMOylation represents a novel mechanism modulating DELLA activity, which attenuates the impact of stress on plant performance.One sentence summaryRice plants show increased yield under salt stress when its gibberellin transcriptional regulator DELLA protein is artificially SUMOylated.

C4 photosynthesis has evolved repeatedly from the ancestral C3 state to generate a carbon concentrating mechanism that increases photosynthetic efficiency. This specialised 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 aimed to better understand molecular mechanisms associated with the evolution of C4 photosynthesis. 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 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.

Deep Eutectic Solvents (DESs) have recently emerged as green alternatives to ionic liquids (ILs). Encouraged by the sustainability of this novel class of solvents, the aim of this work is to prepare DESs using three different hydrogen bond acceptors (HBA), such as cholinium chloride, acetylcholinium chloride and benzyldimethyl(2-hydroxyethyl)ammonium chloride and five naturally occurring carbohydrates, namely d -(+)-Xylose, d -(+)-Mannose, d -(−)-Fructose, d -(+)-Glucose and d -(−)-Ribose, which will act as hydrogen bond donors (HBD). NMR and FTIR were used to ascertain about the chemical structures and purities of these carbohydrates-based DESs, while their thermal properties were evaluated through DSC and TGA analysis. Due to the high viscosity of the carbohydrate-based DESs, and its high hydrophilic and hygroscopic nature, 10 wt% of water was added to mimic its use under normal atmosphere conditions and enable their use as solvents. Densities and viscosities at atmospheric pressure and in the temperature range between 293.15 K and 353.15 K were measured for the prepared DES aqueous solutions. Finally, the suitability of these carbohydrates-based DESs as solvents for the dissolution of rice straw was investigated.

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.

Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here, we test the hypothesis that OsCPK17 plays a role in rice cold stress response by analysing OsCPK17 knockout, silencing and overexpressing rice lines under low temperature. Altered OsCPK17 gene expression compromises cold tolerance performance, without affecting the expression of key cold stress-inducible genes. A comparative phosphoproteomic approach led to the identification of six potential in vivo OsCPK17 targets, which are associated with sugar and nitrogen metabolism, and with osmotic regulation. To test direct interaction, in vitro kinase assays were performed, showing that the sucrose-phosphate synthase OsSPS4 and the aquaporin OsPIP2;1/OsPIP2;6 are phosphorylated by OsCPK17 in a calcium-dependent manner. Altogether, our data indicates that OsCPK17 is required for a proper cold stress response in rice, likely affecting the activity of membrane channels and sugar metabolism.

Rice (Oryza sativa) is one of the world's most important food crops, and is comprised largely of japonica and indica subspecies. Here, we reconstruct the history of rice dispersal in Asia using whole-genome sequences of more than 1,400 landraces, coupled with geographic, environmental, archaeobotanical and paleoclimate data. Originating around 9,000 yr ago in the Yangtze Valley, rice diversified into temperate and tropical japonica rice during a global cooling event about 4,200 yr ago. Soon after, tropical japonica rice reached Southeast Asia, where it rapidly diversified, starting about 2,500 yr BP. The history of indica rice dispersal appears more complicated, moving into China around 2,000 yr BP. We also identify extrinsic factors that influence genome diversity, with temperature being a leading abiotic factor. Reconstructing the dispersal history of rice and its climatic correlates may help identify genetic adaptations associated with the spread of a key domesticated species.

Rice (Oryza sativa) is one of the world’s most important food crops. We reconstruct the history of rice dispersal in Asia using whole-genome sequences of >1,400 landraces, coupled with geographic, environmental, archaeobotanical and paleoclimate data. We also identify extrinsic factors that impact genome diversity, with temperature a leading abiotic factor. Originating ∼9,000 years ago in the Yangtze Valley, rice diversified into temperate and tropical japonica during a global cooling event ∼4,200 years ago. Soon after, tropical rice reached Southeast Asia, where it rapidly diversified starting ∼2,500 yBP. The history of indica rice dispersal appears more complicated, moving into China ∼2,000 yBP. Reconstructing the dispersal history of rice and its climatic correlates may help identify genetic adaptation associated with the spread of a key domesticated species.One sentence summaryWe reconstructed the ancient dispersal of rice in Asia and identified extrinsic factors that impact its genomic diversity.

Compartmentation of photosynthetic reactions between mesophyll and bundle sheath cells is a key feature of C4 photosynthesis and depends on the cell-specific accumulation of major C4 enzymes, such as phosphoenolpyruvate carboxylase 1. The ZmPEPC1 upstream region, which drives light-inducible and mesophyll-specific gene expression in maize, has been shown to keep the same properties when introduced into rice (C3 plant), indicating that rice has the transcription factors (TFs) needed to confer C4 -like gene expression. Using a yeast one-hybrid approach, we identified OsbHLH112, a rice basic Helix-Loop-Helix (bHLH) TF that interacts with the maize ZmPEPC1 upstream region. Moreover, we found that maize OsbHLH112 homologues, ZmbHLH80, and ZmbHLH90, also interact with the ZmPEPC1 upstream region, suggesting that these C4 regulators were co-opted from C3 plants. A transactivation assay in maize mesophyll protoplasts revealed that ZmbHLH80 represses, whereas ZmbHLH90 activates, ZmPEPC1 expression. In addition, ZmbHLH80 was shown to impair the ZmPEPC1 promoter activation caused by ZmbHLH90. We showed that ZmbHLH80 and ZmbHLH90 bind to the same cis-element within the ZmPEPC1 upstream region either as homodimers or heterodimers. The formation of homo- and heterodimers with higher oligomeric forms promoted by ZmbHLH80 may explain its negative effect on gene transcription. Gene expression analysis revealed that ZmbHLH80 is preferentially expressed in bundle sheath cells, whereas ZmbHLH90 does not show a clear cell-specific expression pattern. Altogether, our results led us to propose a model in which ZmbHLH80 contributes to mesophyll-specific ZmPEPC1 gene expression by impairing ZmbHLH90-mediated ZmPEPC1 activation in the bundle sheath cells.

Many flowering plants contain stylar S-RNases that are involved in self-incompatibility and S-like RNases of which the biological function is uncertain. This paper reports the deduced amino acid sequence of an S-like RNase gene (PD1) from the self-incompatible plant Prunus dulcis (almond). The amino acid sequence of PD1, which was derived from cDNA and genomic DNA clones, showed 34-86% identity to acidic plant S-like RNases reported so far, with the highest degree of similarity being to an S-like RNase from Japanese pear (Pyrus pyrifolia). Based on RNA hybridisation experiments it appears that, like for many other S-like RNases, the expression of PD1 is not pistil-specific. Analysis of the genomic structure revealed the presence of three introns, of which one is similar in location to that of the related S-RNase gene from Solanaceae and Rosaceae. At least four bands hybridising to PD1 were found upon Southern hybridisation, suggesting the presence of a multigene family of S-like RNase genes in almond. The putative biological function of PD1 is discussed.

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.

Increase in soil salinity levels is becoming a major cause of crop yield losses worldwide. Rice (Oryza sativa) is the most salt-sensitive cereal crop, and many studies have focused on rice salinity tolerance, but a global understanding of this crop's response to salinity is still lacking. We systematically analyzed phenotypic data previously collected for 56 rice genotypes to assess the extent to which rice uses three known salinity tolerance mechanisms: shoot-ion independent tolerance (or osmotic tolerance), ion exclusion, and tissue tolerance. In general, our analyses of different phenotypic traits agree with results of previous rice salinity tolerance studies. However, we also established that the three salinity tolerance mechanisms mentioned earlier appear among rice genotypes and that none of them is predominant. Against the pervasive view in the literature that the K(+) /Na(+) ratio is the most important trait in salinity tolerance, we found that the K(+) concentration was not significantly affected by salt stress in rice, which puts in question the importance of K(+) /Na(+) when analyzing rice salt stress response. Not only do our results contribute to improve our global understanding of salt stress response in an important crop, but we also use our results together with an extensive literature research to highlight some issues commonly observed in salinity stress tolerance studies and to propose solutions for future experiments.

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. Machine-accessible metadata file describing the reported data (ISA-Tab format)

Background SUMOylation is an essential eukaryotic post-translation modification that, in plants, regulates numerous cellular processes, ranging from seed development to stress response. Using rice as a model crop plant, we searched for potential regulatory points that may influence the activity of the rice SUMOylation machinery genes. Results We analyzed the presence of putative cis-acting regulatory elements (CREs) within the promoter regions of the rice SUMOylation machinery genes and found CREs related to different cellular processes, including hormone signaling. We confirmed that the transcript levels of genes involved in target-SUMOylation, containing ABA- and GA-related CREs, are responsive to treatments with these hormones. Transcriptional analysis in Nipponbare (spp. japonica) and LC-93-4 (spp. indica), showed that the transcript levels of all studied genes are maintained in the two subspecies, under normal growth. OsSUMO3 is an exceptional case since it is expressed at low levels or is not detectable at all in LC-93-4 roots and shoots, respectively. We revealed post-transcriptional regulation by alternative splicing (AS) for all genes studied, except for SUMO coding genes, OsSIZ2, OsOTS3, and OsELS2. Some AS forms have the potential to alter protein domains and catalytic centers. We also performed the molecular and phenotypic characterization of T-DNA insertion lines of some of the genes under study. Knockouts of OsFUG1 and OsELS1 showed increased SUMOylation levels and non-overlapping phenotypes. The fug1 line showed a dwarf phenotype, and significant defects in fertility, seed weight, and panicle architecture, while the els1 line showed early flowering and decreased plant height. We suggest that OsELS1 is an ortholog of AtEsd4, which was also supported by our phylogenetic analysis. Conclusions Overall, we provide a comprehensive analysis of the rice SUMOylation machinery and discuss possible effects of the regulation of these genes at the transcriptional and post-transcriptional level. We also contribute to the characterization of two rice SUMO proteases, OsELS1 and OsFUG1. Electronic supplementary material The online version of this article (10.1186/s12870-018-1547-3) contains supplementary material, which is available to authorized users.

International audience; Drought is a major environmental threat to agriculture sustainability. We have characterised the role of OsICE1, a basic helix-loop-helix (bHLH) transcription factor from rice (Otyza saliva L.), in response to drought. OsICEI gene is constitutively expressed in roots and shoots of rice seedlings grown under control conditions, as well as in response to low temperature, high salinity, and ABA. Nevertheless, in response to drought, OsICE1 is up-regulated in roots. Plants over-expressing OsICE1 (OsICEI-OX) show improved drought tolerance at both vegetative and reproductive stages, whereas OsICE1 silenced plants (OsICE1-RNA1) show decreased drought tolerance in the same phenological stages. Under drought, OsICE1-0X plants show improved net photosynthetic rates and stomatal conductance to water vapour, as well as higher photochemical efficiency of photosystem II and tolerance to photo-inhibition, as compared to the WT and OsICEI-RNAi. In addition, under severe drought, OsICEI-OX plants show lower grain yield losses than WT, while OsICE1-12NAi plants show the highest grain losses. OsICE1-OX and OSICE1-RNAi plants do not differentially respond to ABA, and drought-inducible genes are more responsive in OsICEI-RNAi and less in OsICE1-OX plants (as compared to WT), suggesting that OsICE1 has a central role modulating molecular responses to drought in an ABA-independent way. This role might be mediated by OsWsi18, a LEA encoding gene up regulated in OsICE1-OX rice plants and known to enhance membrane stability under drought.

A aprovação das plantas geneticamente modificadas é precedida de vários anos de investigação intensiva de forma a garantir a sua segurança para o homem e ambiente. Recentemente demonstrámos que o stress da cultura in vitro parece ser o fator que mais influencia as diferenças proteómicas encontradas entre as plantas geneticamente modificadas (GM) e os seus controlos. Neste trabalho tentámos avaliar quantas gerações são necessárias para os efeitos da “memória” desse stress serem apagados. Tentámos também comparar a relevância das alterações provocadas pelo stress da cultura in vitro quando comparadas com as provocadas por outros stresses ambientais. Para tal seguimos três linhas de arroz durante oito gerações após a transgénese combinando técnicas de análise transcritómica e proteómica. Na geração F6 as plantas foram sujeitas a stress salino. Os resultados deste trabalho demonstraram que: (a) as diferenças promovidas durante a modificação genética são maioritariamente diferenças fisiológicas de curta duração, que atenuam ao longo do tempo; (b) os stresses ambientais podem causar mais alterações proteómicas/trascritómicas que a engenharia genética. Com base nos dados obtidos questionamos quais os ensaios que são realmente relevantes e quais aqueles claramente excessivos na avaliação do risco das plantas GM para a saúde humana e animal, plantas e ambiente. The approval of genetically modified (GM) crops is preceded by years of intensive research to demonstrate safety to humans and environment. We recently showed that in vitro culture stress is the major factor influencing proteomic dif ferences of GM vs. non-GM plants. This made us question the number of generations needed to erase such "memor y". We also wondered about the relevance of alterations promoted by transgenesis as compared to environment-induced ones. Here we followed three rice lines throughout eight generations af ter transgenesis combining proteomics and transcriptomics, and fur ther analyzed their response to salinity stress on the F6 generation. Our results showed that: (a) dif ferences promoted during genetic modification are mainly shor t-term physiological changes, attenuating throughout generations, and (b) environmental stress may cause far more proteomic/transcriptomic alterations than transgenesis. Based on our data, we question what is really relevant in risk assessment design for GM food crops. info:eu-repo/semantics/publishedVersion

Background Cynara cardunculus L. offers a natural source of phenolic compounds with the predominant molecule being chlorogenic acid. Chlorogenic acid is gaining interest due to its involvement in various biological properties such as, antibacterial, antifungal, antioxidant, hepatoprotective, and anticarcinogenic activities. Results In this work we screened a Cynara cardunculus collection for new allelic variants in key genes involved in the chlorogenic acid biosynthesis pathway. The target genes encode p-coumaroyl ester 3′-hydroxylase (C3′H) and hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase (HQT), both participating in the synthesis of chlorogenic acid. Using high-resolution melting, the C3′H gene proved to be highly conserved with only 4 haplotypes while, for HQT, 17 haplotypes were identified de novo. The putative influence of the identified polymorphisms in C3′H and HQT proteins was further evaluated using bioinformatics tools. We could identify some polymorphisms that may lead to protein conformational changes. Chlorogenic acid content, antioxidant and antithrombin activities were also evaluated in Cc leaf extracts and an association analysis was performed to assess a putative correlation between these traits and the identified polymorphisms. Conclusion In this work we identified allelic variants with putative impact on C3′H and HQT proteins which are significantly associated with chlorogenic acid content and antioxidant activity. Further study of these alleles should be explored to assess putative relevance as genetic markers correlating with Cynara cardunculus biological properties with further confirmation by functional analysis. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3534-8) contains supplementary material, which is available to authorized users.

Identification of differences between genetically modified plants and their original counterparts plays a central role in risk assessment strategy. Our main goal was to better understand the relevance of transgene presence, genetic, and epigenetic changes induced by transgene insertion, and in vitro culture in putative unintended differences between a transgenic and its comparator. Thus, we have used multiplex fluorescence 2DE coupled with MS to characterize the proteome of three different rice lines (Oryza sativa L. ssp. japonica cv. Nipponbare): a control conventional line (C), an Agrobacterium-transformed transgenic line (Ta) and a negative segregant (NSb). We observed that Ta and NSb appeared identical (with only one spot differentially abundant--fold difference ≥ 1.5), contrasting with the control (49 spots with fold difference ≥ 1.5, in both Ta and NSb vs. control). Given that in vitro culture was the only event in common between Ta and NSb, we hypothesize that in vitro culture stress was the most relevant condition contributing for the observed proteomic differences. MS protein identification support our hypothesis, indicating that Ta and NSb lines adjusted their metabolic pathways and altered the abundance of several stress related proteins in order to cope with in vitro culture.

Thymus caespititius Brot. is an Iberian endemic species, whose essential oils possess high polymorphism. They consist mostly of mono- and sesquiterpene, some of them with interest for the pharmaceutical and food industries. The search for terpene synthase genes was performed in three in vitro T. caespititius genotypes. For these plants, the expression of a previously described γ-terpinene synthase gene, Tctps2 , was confirmed, occurring concomitantly with a new gene encoding an enzyme with similar activity, named Thymus caespititius terpene synthase 4 ( Tctps4 ). The two isogenes were isolated and functionally characterized in the three plant genotypes. Alignment of the two Tctps revealed a transit peptide much shorter in Tctps4 than in Tctps2 (3–4 amino acids instead of 47). The Tctps4 open reading frame is shorter than Tctps2 (1665 bp versus 1794 bp). The amino acid sequence of both γ-terpinene synthases shared an 88% pairwise identity. The fact that T. caespititius carries two isogenes for γ-terpinene synthases, suggests gene duplication along the evolutionary process, followed by mutations leading to the differentiation of both genes. These mutations didn’t compromise protein activity. A high accumulation of transcripts from both genes was found in shoots of in vitro plantlets, while in roots they could not be detected. Still, γ-terpinene levels in aerial parts were reduced, probably due to fast conversion into carvacrol and thymol, the main components from T. caespititius essential oils. This study is a contribution to the identification of terpene synthase genes in Lamiaceae.

Thymus caespititius Brot. is an Iberian endemic species, whose essential oils possess high diversity and some of them could be used in pharmaceutical and food industries. The effect of culture media composition and fungal extracts on essential oils composition and on the expression of terpene synthase (TPS) genes was evaluated in T. caespititius shoots cultures. Nutrient composition influenced quantitatively the essential oils content. A decrease in the acetate compounds in the essential oils was observed in SH medium as compared with MS. The TPS transcripts accumulation was higher in all genotypes grown in SH, except in sabinene/carvacrol (SC) genotype. B. cinerea extracts highly induced TPS gene expression in the first hours after treatment in SC genotype, while in carvacrol/thymol (CT) only a slight increase was observed. TPS down-regulation observed during the next time points could result from reallocation of energy resources to plants’ vital functions. Fungal extracts did not affect qualitatively and quantitatively the CT essential oils, however in SC, the sabinene relative content decreased and this was inversely related to the increase of two antifungal compounds, carvacrol and thymol, in the oil. T. caespititius in vitro cultures revealed to be a good system for terpene metabolism study.