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The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper describes methods optimized to isolate plant-derived polyribosomes and their associated mRNAs to be analyzed using RNA-sequencing. First, cytoplasmic lysates are obtained through homogenization in polysome- and RNA-preserving conditions from whole, frozen soybean nodules. Then, lysates are cleared by low-speed centrifugation, and 15% of the supernatant is used for total RNA (TOTAL) isolation. The remaining cleared lysate is used to isolate polysomes by ultracentrifugation through a two-layer sucrose cushion (12% and 33.5%). Polysome-associated mRNA (PAR) is purified from polysomal pellets after resuspension. Both TOTAL and PAR are evaluated by highly sensitive capillary electrophoresis to meet the quality standards of sequencing libraries for RNA-seq. As an example of a downstream application, after sequencing, standard pipelines for gene expression analysis can be used to obtain differentially expressed genes at the transcriptome and translatome levels. In summary, this method, in combination with RNA-seq, allows the study of the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.

Drought is the main cause of productivity losses in soybean plants, triggering physiological and biochemical responses, stomatal closure being essential to prevent water losses and thus mitigate the negative effects of drought. Abscisic acid (ABA) is the main molecule involved in stomatal closure under drought conditions along with nitric oxide (• NO). However, the role of • NO in this process is not yet fully understood and contrasting findings about its role have been reported. Most of the assays in the literature have been carried out under in vitro conditions using • NO donors or scavengers, but little is known about the effects of endogenously produced • NO under drought conditions. This study is aimed to determine the pattern of endogenous • NO accumulation from the establishment of water stress and how this relates to stomatal closure and other biochemical and physiological responses. The analysis of soybean plant responses to drought revealed no correlation between whole-leaf • NO accumulation and typical water-deficit stress markers. Moreover, • NO accumulation did not explain oxidative damage induced by drought. However, endogenous • NO content correlated with the early stomatal closure. Analysis of stomatal behavior and endogenous • NO content in guard cells through epidermal peel technique showed a stomatal population with high variation in stomatal opening and • NO content under the initial stages of water stress, even when ABA responses are activated. Our data suggest that upon early stress perception, soybean plants respond by accumulating • NO in the guard cells to inhibit stomatal closure, potentially through the inhibition of ABA responses.

The photosynthesis process is determined by the intensity level and spectral quality of the light; therefore, leaves need to adapt to a changing environment. The incident energy absorbed can exceed the sink capability of the photosystems, and, in this context, photoinhibition may occur in both photosystem II (PSII) and photosystem I (PSI). Quantum yield parameters analyses reveal how the energy is managed. These parameters are genotype-dependent, and this genotypic variability is a good opportunity to apply mapping association strategies to identify genomic regions associated with photosynthesis energy partitioning. An experimental and mathematical approach is proposed for the determination of an index which estimates the energy per photon flux for each spectral bandwidth (Δλ) of the light incident (QI index). Based on the QI, the spectral quality of the plant growth, environmental lighting, and the actinic light of PAM were quantitatively very similar which allowed an accurate phenotyping strategy of a rice population. A total of 143 genomic single regions associated with at least one trait of chlorophyll fluorescence were identified. Moreover, chromosome 5 gathers most of these regions indicating the importance of this chromosome in the genetic regulation of the photochemistry process. Through a GWAS strategy, 32 genes of rice genome associated with the main parameters of the photochemistry process of photosynthesis in rice were identified. Association between light-harvesting complexes and the potential quantum yield of PSII, as well as the relationship between coding regions for PSI-linked proteins in energy distribution during the photochemical process of photosynthesis is analyzed.

Brassinosteroids (BRs) form a group of steroidal hormones essential for plant growth, development, and stress responses. BRs are perceived extracellularly by plasma membrane receptor-like kinases that activate an interconnected signal transduction cascade, leading to the transcriptional regulation of BR-responsive genes. TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) genes are specific for land plants, and their encoded proteins are defined by the presence of protein–protein interaction motives, that is, an intrinsic disordered region at the N terminus, six tetratricopeptide repeat domains, and a C terminus with homology to thioredoxins. TTL proteins thus likely mediate the assembly of multiprotein complexes. Phenotypic, molecular, and genetic analyses show that TTL proteins are positive regulators of BR signaling in Arabidopsis (Arabidopsis thaliana). TTL3 directly interacts with a constitutively active BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor kinase, BRI1-SUPPRESSOR1 phosphatase, and the BRASSINAZOLE RESISTANT1 transcription factor and associates with BR-SIGNALING KINASE1, BRASSINOSTEROID INSENSITIVE2 kinases, but not with BRI1-ASSOCIATED KINASE1. A functional TTL3-green fluorescent protein (GFP) shows dual cytoplasmic plasma membrane localization. Depleting the endogenous BR content reduces plasma membrane localization of TTL3-GFP, while increasing BR content causes its plasma membrane relocalization, where it strengthens the association of BR signaling components. Our results reveal that TTL proteins promote BR responses and suggest that TTL proteins may function as scaffold proteins by bringing together cytoplasmic and plasma membrane BR signaling components.

Environmental and biotic stresses induce metabolic changes in plants which led to the accumulation of specific metabolites. For instance, the accumulation of gamma-aminobutyric acid (GABA) and proline is a conserved response of plants to a wide range of stresses. In particular, these amino acids accumulate the most under abiotic stress conditions and, to a lesser extent, under biotic stress conditions. Multiple shared roles have been assigned to these molecules, including osmoprotection, osmotic adjustment, carbon source, redox balance and antioxidant functions. For some of these roles, however, the physiological function of these molecules has been a matter of debate. Recent studies point to unexplored functions of these molecules. For instance, proline accumulation was suggested to contribute to sustaining photosynthesis under stress conditions and proline catabolism was suggested to be relevant to induce plant autophagy. Moreover, many enzymes of proline metabolism were suggested to be important for optimal response to biotic stress. This is even clearer for GABA, for which several mechanisms of action were already described to explain its capacity to protect the plant against pathogens. However, the role of GABA under biotic stress is highly dependent on the biotic stressor involved. Furthermore, GABA was recently demonstrated to enhance biotic stress tolerance when applied as a priming agent and its effect seems to be dependent on ethylene. Here we will summarize and integrate the current knowledge of GABA and proline accumulation in response to stress and discuss future perspectives.

Drought is the main cause of productivity losses in soybean plants, triggering physiological and biochemical responses, stomatal closure being essential to prevent water losses and thus mitigate the negative effects of drought. Abscisic acid (ABA) is the main molecule involved in stomatal closure under drought conditions along with nitric oxide (

The photosynthesis process is determined by the intensity level and spectral quality of the light; therefore, leaves need to adapt to a changing environment. The incident energy absorbed can exceed the sink capability of the photosystems, and, in this context, photoinhibition may occur in both photosystem II (PSII) and photosystem I (PSI). Quantum yield parameters analyses reveal how the energy is managed. These parameters are genotype-dependent, and this genotypic variability is a good opportunity to apply mapping association strategies to identify genomic regions associated with photosynthesis energy partitioning. An experimental and mathematical approach is proposed for the determination of an index which estimates the energy per photon flux for each spectral bandwidth (Δ

Biological membranes allow the regulation of numerous cellular processes, which are affected when unfavorable environmental factors are perceived. Lipids and proteins are the principal components of biological membranes. Each lipid has unique biophysical properties, and, therefore the lipid composition of the membrane is critical to maintaining the bilayer structure and functionality. Membrane composition and integrity are becoming the focus of studies aiming to understand how plants adapt to its environment. In this study, using a combination of di-4-ANEPPDHQ fluorescence and spectral phasor analysis, we report that the drought hypersensitive/squalene epoxidase (dry2/sqe1-5) mutant with reduced major sterols such as sitosterol and stigmasterol in roots presented higher membrane fluidity than the wild type. Moreover, analysis of endomembrane dynamics showed that vesicle formation was affected in dry2/sqe1-5. Further analysis of proteins associated with sterol rich micro domains showed that dry2/sqe1-5 presented micro domains function altered.

Drought is considered one of the abiotic stresses with significant implications on plant productivity. Previously, we have shown that water deficit produces a differential nitro-oxidative stress in roots and leaves of Lotus japonicus L. plants. Using this model legume, we studied the nitro-oxidative stress in drought-stressed roots by complementary biochemical, cellular and proteomic approaches. Cellular analyses of root cross-sections by confocal laser scanning microscopy (CLSM) using specific fluorescent probes for superoxide radical (O.-), nitric oxide (NO-), peroxynitrite (ONOO) and S-nitrosothiols (SNOs) showed that drought stress causes a differential cellular localization of these reactive species. Mainly, O.- and ONOO- had a wide distribution in almost all root cell types (xylem, parenchyma, and peridermis), whereas NO and SNOs accumulated in cortical cells (peridermis). Liquid chromatography-electrospray/mass spectrometry (LC-ES/MS) analyses showed that the content of ascorbate, S-nitrosoglutaathione (GSNO), and reduced glutathione (GSH) in drought-stressed roots was drastically diminished. Nitroproteome analysis by two-dimensional gel electrophoresis and mass spectrometry allowed to identify 13 tyrosine-nitrated proteins such as methionine synthase, Hsp70, adenosyl-homocysteinase, peroxidase, alcohol dehydrogenases, glutamine synthetase, fructokinase, 1,3-beta-glucanase, chitinases, endochitinase, among others which are directly (24%) or indirectly (74%) related to plant defense. Taken together, these results indicate that drought-stressed roots have an active metabolism of reactive oxygen and nitrogen species (ROS and RNS) characterized by an increase of protein nitration and accumulation of NO and SNOs in cortical cells. The possibility of autophagy taking place in the stressed roots is also discussed., LC-ES/MS was carried out at the Instrumental Technical Services of the Estación Experimental del Zaidín (CSIC), Granada, confocal laser-scanning microscopy (CLSM) analyses were carried out at the Technical Services of the University of Jaén, and proteomic analyses were made at the Proteomic Service (SCAI) at the University of Córdoba, Spain. Researchin FJC´s laboratory is supported by an European Regional Development Fund (ERDF) grant co-financed by the Ministry of Economy and Competitiveness (AGL2015-65104-P). SS, OB, and JM were partially supported by PEDECIBA (Programa de Desarrollo de Ciencias Básicas, Uruguay). SS, OB, and JM are associate and active members of the National System of Researchers (SNI, Uruguay).

La viticultura de precisión es una herramienta performante para la gestión del vigor intra parcelario. En una parcela de 1,1 hectáreas de un viñedo comercial de la variedad Tannat, implantado en clima sub- húmedo, en donde delimitó dos zonas de vigor (alto y bajo). Uno de los factores determinantes de dichas diferencias de vigor, sería la diferencia en el contenido de agua en el suelo. Para demostrar esta hipótesis se instalaron parcelas por cada nivel de vigor en las cuales se realizó un seguimiento del estado de hidratación de las plantas mediante el potencial hídrico foliar de base (Ψf). Se determinó la composición isotópica (δ18O) en agua de hojas y mosto. Se demostraron correlaciones significativas entre Ψf y OI. Las medidas de δ18O en hojas brindaron un dato puntual del contenido de agua, en tanto que las del mosto describieron la evolución del estado de hidratación de la planta durante el ciclo del cultivo. El δ18O podría ser una forma rápida de identificación de zonas de diferente vigor y que permitiría determinar a escala parcelaria las diferencias existentes en el suelo en el contenido hídrico y gestionar de manera más eficiente el recurso agua. Palabras clave: ratio isotópico, δ18O, estrés hídrico, viticultura de precisión, Vitis vinífera L.

One of the main limitations of rice yield in regions of high productive performance is the light-use efficiency (LUE). LUE can be determined at the whole-plant level or at the photosynthetic apparatus level (quantum yield). Both vary according to the intensity and spectral quality of light. The aim of this study was to analyze the cultivar dependence regarding LUE at the plant level and quantum yield using four rice cultivars and four light environments. To achieve this, two in-house Light Systems were developed: Light System I which generates white light environments (spectral quality of 400–700 nm band) and Light System II which generates a blue-red light environment (spectral quality of 400–500 nm and 600–700 nm bands). Light environment conditioned the LUE and quantum yield in PSII of all evaluated cultivars. In white environments, LUE decreased when light intensity duplicated, while in blue-red environments no differences on LUE were observed. Energy partition in PSII was determined by the quantum yield of three de-excitation processes using chlorophyll fluorescence parameters. For this purpose, a quenching analysis followed by a relaxation analysis was performed. The damage of PSII was only increased by low levels of energy in white environments, leading to a decrease in photochemical processes due to the closure of the reaction centers. In conclusion, all rice cultivars evaluated in this study were sensible to low levels of radiation, but the response was cultivar dependent. There was not a clear genotypic relation between LUE and quantum yield.

Brassinosteroids (BRs) form a group of steroidal hormones essential for plant growth, development and stress responses. BR perception at the plasma membrane initiates a series of phosphorylation events enabling the nuclear accumulation and activity of the key transcription factors BZR1 and BES1. Previous studies support that all BR signaling components form an interconnected pathway, although it is not known how these proteins are brought together for the prompt signal transduction from the plasma membrane to the nucleus. Here we report that plant-specific Tetratricopeptide Thioredoxin- Like (TTL) proteins are positive regulators of BR signaling that function as scaffold for the BR signaling components in Arabidopsis. TTL3 directly interacts with BZR1 and the phosphatase BSU1, and associates in vivo with most core components involved in BR signaling, with the exception of the BAK1 coreceptor. TTL3 is mainly localized in the cytoplasm, and BR treatment increases its localization at the plasma membrane. In addition, the expression of TTL3 strengthens the association of BR-signaling components BSK1 and BZR1 at the plasma membrane. Consistent with a role in BR signaling, mutations in TTL3, and related TTL1 and TTL4 genes cause reduced BR responses, and these defects that highly enhanced in a triple ttl1 ttl3 ttl4 mutant. We propose a novel mechanistic model for BR signaling, in which cytoplasmic/nuclear BR components bound to TTL proteins are recruited to the plasma membrane upon BR perception, which in turn allows the assembly of a BR signaling complex with the goal of ensuring dephosphorylation and nuclear accumulation of the transcription factors BZR1 and BES1. Interestingly, this novel TTL scaffold model for BR signaling resembles that of Wnt signaling in metazoans, in which TTL proteins would act similar to Axin1, optimizing signaling efficiency of the cascade by promoting the assembly of the signaling complex at the plasma membrane.

Water is usually the main limiting factor for soybean productivity worldwide and yet advances in genetic improvement for drought resistance in this crop are still limited. In the present study, we investigated the physiological and molecular responses to drought in two soybean contrasting genotypes, a slow wilting N7001 and a drought sensitive TJS2049 cultivars. Measurements of stomatal conductance, carbon isotope ratios and accumulated dry matter showed that N7001 responds to drought by employing mechanisms resulting in a more efficient water use than TJS2049. To provide an insight into the molecular mechanisms that these cultivars employ to deal with water stress, their early and late transcriptional responses to drought were analyzed by suppression subtractive hybridization. A number of differentially regulated genes from N7001 were identified and their expression pattern was compared between in this genotype and TJS2049. Overall, the data set indicated that N7001 responds to drought earlier than TJ2049 by up-regulating a larger number of genes, most of them encoding proteins with regulatory and signaling functions. The data supports the idea that at least some of the phenotypic differences between slow wilting and drought sensitive plants may rely on the regulation of the level and timing of expression of specific genes. One of the genes that exhibited a marked N7001-specific drought induction profile encoded a eukaryotic translation initiation factor iso4G (GmeIFiso4G-1a). GmeIFiso4G-1a is one of four members of this protein family in soybean, all of them sharing high sequence identity with each other. In silico analysis of GmeIFiso4G-1 promoter sequences suggested a possible functional specialization between distinct family members, which can attain differences at the transcriptional level. Conditional overexpression of GmeIFiso4G-1a in Arabidopsis conferred the transgenic plants increased tolerance to osmotic, salt, drought and low temperature stress, providing a strong experimental evidence for a direct association between a protein of this class and general abiotic stress tolerance mechanisms. Moreover, the results of this work reinforce the importance of the control of protein synthesis as a central mechanism of stress adaptation and opens up for new strategies for improving crop performance under stress.

Low temperature inhibits seedling emergence and early vegetative growth of rice (Oryza sativa L.), especially in cultivars of the subspecies indica, which are the most productive in the temperate region of South America. To improve breeding efficiency, we identified molecular markers associated with cold response related phenotypes, such as membrane integrity, photoinhibition and visual assessment of damage. A candidate gene approach was used to select genomic simple sequence repeat (gSSR) markers. For marker-trait association two strategies were used, candidate gene association mapping (CGAM) and single marker analysis (SMA) in a segregating F2:3 population. Thirty-six gSSR were identified but only 14 were polymorphic in the CGAM population. Using this approach, we found marker RM22034 associated with membrane integrity, RM6547 and RM14978 with photoinhibition, and RM144 with visual assessment of damage. The SMA strategy identified markers RM6651, RM14978, RM22034 and RM4 associated with membrane integrity and RM22034 with photoinhibition. All markers are currently available for marker assisted selection in rice breeding programs located in temperate regions.

The accumulation of proline (Pro) and overproduction of reactive oxygen species (ROS) by plants exposed to stress is well-documented. In vitro assays show that enzyme inactivation by hydroxyl radicals ((•)OH) can be avoided in the presence of Pro, suggesting this amino acid might act as a (•)OH scavenger. Although production of hydroxyproline (Hyp) has been hypothesized in connection with such antioxidant activity, no evidence on the detailed mechanism of scavenging has been reported. To elucidate whether and how Hyp might be produced, we used density functional theory calculations coupled to a polarizable continuum model to explore 27 reaction channels including H-abstraction by (•)OH and (•)OH/H2O addition. The structure and energetics of stable species and transition states for each reaction channel were characterized at the PCM-(U)M06/6-31G(d,p) level in aqueous solution. Evidence is found for a main pathway in which Pro scavenges (•)OH by successive H-abstractions (ΔG(‡,298) = 4.1 and 7.5 kcal mol(-1)) to yield 3,4-Δ-Pro. A companion pathway with low barriers yielding Δ(1)-pyrroline-5-carboxylate (P5C) is also supported, linking with 5-Hyp through hydration. However, this connection remains unlikely in stressed plants because P5C would be efficiently recycled to Pro (contributing to its accumulation) by P5C reductase, hypothesis coined here as the "Pro-Pro cycle".

Squalene epoxidase enzymes catalyse the conversion of squalene into 2,3-oxidosqualene, the precursor of cyclic triterpenoids. Here we report that the Arabidopsis drought hypersensitive/squalene epoxidase 1-5 (dry2/sqe1-5) mutant, identified by its extreme hypersensitivity to drought stress, has altered stomatal responses and root defects because of a point mutation in the SQUALENE EPOXIDASE 1 (SQE1) gene. GC-MS analysis indicated that the dry2/sqe1-5 mutant has altered sterol composition in roots but wild-type sterol composition in shoots, indicating an essential role for SQE1 in root sterol biosynthesis. Importantly, the stomatal and root defects of the dry2/sqe1-5 mutant are associated with altered production of reactive oxygen species. As RHD2 NADPH oxidase is de-localized in dry2/sqe1-5 root hairs, we propose that sterols play an essential role in the localization of NADPH oxidases required for regulation of reactive oxygen species, stomatal responses and drought tolerance.

In cyanobacteria both photosynthesis and nitrogen fixation can be affected by UV radiation. Two of the most abundant heterocystous cyanobacteria isolates from a temperate ricefield in Uruguay belonging to Anabaena and Calothrix genus were exposed for 1 or 3 hours to UV-B dosis similar to those to which they are exposed in summer. Anabaena survival after 1 h of UV-B exposure was 10% whereas in Calothrix’s was 30%. Both the quantum yields of photosybtem II fluorescence and O2 photoevolution decreased with time of UV-B exposure for Calothrix and only till 1 h for Anabaena. Only the Calothrix strain presented phycoerithryn as antenna pigment and constitutive UV-B screening mycosporine like aminoacids. In the Anabaena strain, nitrogenase activity was drastically reduced with UV-B irradiation but in Calothrix was not affected. Proline content and lipid peroxidation increased after 3 hours of UV-B exposure only in Anabaena sp. The antioxidant enzyme activities evaluated followed different trends for both isolates, with an increase in superoxide dismutase activity in the Calothrix isolate. These results show that the two nitrogen-fixing cyanobacteria studied have different responses to UV-B radiation and that cyanobacteria diversity may be considered when selecting strains to be used as biofertilizers.

Water use efficiency (WUE), defined as the ratio of biomass (B) produced per unit of water transpired (E) by plants, is an important determinant of plant productivity. A mathematical approach was developed with the purpose explaining the WUE of two forage legumes (clover and birdsfoot trefoil). The approach applied involved an interpolation method by cubic spline which results in the smoothest curve that fits the set of data points obtained experimentally. The results obtained show the importance of recognizing the WUE as a function of two variables, one represents the supply (θ) and the other represents the demand (D). It is important to note that these surfaces generated by the model allowed the estimation of the WUE value for any value of θ and any value of D, showing it is able to dissect the effect of both environmental variables on WUE. From the surface generated by the model, a scalar field plot (SFP) was created. Analysis of these SFPs allowed decomposing the environmental effect on B and E parameters defining WUE. The SFPs allow identify, in one picture, what are the environmental conditions and what variable are explaining a high WUE in both species. Spline application for generating SFP could have a significant impact on the quantitative understanding of the WUE and our study represents a first step towards an analytical and integrated view of this parameters.

Plants accumulate proline under diverse types of stresses, and it has been suggested that this α-amino acid has the capacity to protect against oxidative stress. However, it is still controversial whether its protection is due to the direct scavenging of reactive oxygen species (ROS). To solve this issue and considering that nitrosative stress is directly related with an oxidative stress condition, we evaluated whether proline can protect against nitrosative damage. Using proteins of Lotus japonicus (Regel) K.Larsen leaves exposed to a peroxynitrite (ONOO–/ONOOH) generator in presence and absence of 100mM proline, the potential of proline to protect was analysed by the protein nitration profile and NADP-dependent isocitrate dehydrogenase activity, which is inhibited by nitration. In both cases, the presence of proline did not diminish the peroxynitrite effects. Additionally, proline biosynthesis Arabidopsis knockout (KO) mutant plants of Δ(1)-pyrroline-5-carboxylate synthetase1 (P5CS1) gene, designated as Atp5cs1-1 and Atp5cs1-4, showed similar protein nitration levels as wild-type plants under salinity-induced oxidative stress, despite mutants having higher levels of lipid oxidation, H2O2 and superoxide (O2·–). Finally, by a fluorometric assay using specific fluorescent probes, it was determined that the presence of 100mM proline did not affect the time-course content of peroxynitrite or nitric oxide generation in vitro. Our results reveal the relevance of proline accumulation in vivo under stress, but unequivocally demonstrate that proline is not a direct scavenger of peroxynitrite, superoxide, ·NO and nitrogen dioxide (·NO2).

Two japonica rice genotypes, INIA Tacuarí and L2825CA, were analyzed for tolerance to low temperature during early vegetative growth. Effect on photosynthesis, energy dissipation, pigment content, xanthophyll-cycle pool conversion, hydrogen peroxide accumulation, oxidative damage and antioxidant enzyme activities were determined to better understand potential mechanisms for cold tolerance. Photoinhibition was measured using chlorophyll fluorescence and oxidative damage by lipid peroxidation and electrolyte leakage. Both genotypes were demonstrated to be cold tolerant which was consistent with their reduced levels of photoinhibition and oxidative damage compared with a cold-sensitive genotype during chilling stress. The strategy for cold tolerance differed between the two genotypes, and involved different mechanisms for disposal of excess energy. The presence of high lutein concentrations and the existence of active non-harmful energy dissipation processes through the xanthophyll cycle appeared to be responsible for chilling tolerance in INIA Tacuarí. On the other hand, increased cold tolerance of L2825CA relative to INIA Tacuarí was related to the higher constitutive superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11) and catalase (CAT, EC 1.11.1.6).

RNA interference, mediated by small interfering RNAs (siRNAs), is a conserved regulatory process that has evolved as an antiviral defense mechanism in plants and animals. It is not known whether host cells also use siRNAs as an antibacterial defense mechanism in eukaryotes. Here, we report the discovery of an endogenous siRNA, nat-siRNAATGB2, that is specifically induced by the bacterial pathogen Pseudomonas syringae carrying effector avrRpt2 . We demonstrate that the biogenesis of this siRNA requires DCL1, HYL1, HEN1, RDR6, NRPD1A, and SGS3. Its induction also depends on the cognate host disease resistance gene RPS2 and the NDR1 gene that is required for RPS2 -specified resistance. This siRNA contributes to RPS2 -mediated race-specific disease resistance by repressing PPRL, a putative negative regulator of the RPS2 resistance pathway.

SummaryIn higher eukaryotes, miRNAs and siRNAs guide translational inhibition, mRNA cleavage, or chromatin regulation. We found that the antisense overlapping gene pair of Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH), a stress-related gene, and SRO5, a gene of unknown function, generates two types of siRNAs. When both transcripts are present, a 24-nt siRNA is formed by a biogenesis pathway dependent on DCL2, RDR6, SGS3, and NRPD1A. Initial cleavage of the P5CDH transcript guided by the 24-nt siRNA establishes a phase for the subsequent generation of 21-nt siRNAs by DCL1 and further cleavage of P5CDH transcripts. The expression of SRO5 is induced by salt, and this induction is required to initiate siRNA formation. Our data suggest that the P5CDH and SRO5 proteins are also functionally related, and that the P5CDH-SRO5 gene pair defines a mode of siRNA function and biogenesis that may be applied to other natural cis-antisense gene pairs in eukaryotic genomes.

The tss1 tomato (Lycopersicon esculentum) mutant exhibited reduced growth in low K+ and hypersensitivity to Na+ and Li+. Increased Ca2+ in the culture medium suppressed the Na+ hypersensitivity and the growth defect on low K+ medium of tss1 seedlings. Interestingly, removing NH4 + from the growth medium suppressed all growth defects of tss1, suggesting a defective NH4 +-insensitive component of K+ transport. We performed electrophysiological studies to understand the contribution of the NH4 +-sensitive and -insensitive components of K+ transport in wild-type and tss1 roots. Although at 1 mm Ca2+ we found no differences in affinity for K+ uptake between wild type and tss1 in the absence of NH4 +, the maximum depolarization value was about one-half in tss1, suggesting that a set of K+ transporters is inactive in the mutant. However, these transporters became active by raising the external Ca2+ concentration. In the presence of NH4 +, a reduced affinity for K+ was observed in both types of seedlings, but tss1 at 1 mm Ca2+ exhibited a 2-fold higher K m than wild type did. This defect was again corrected by raising the external concentration of Ca2+. Therefore, membrane potential measurements in root cells indicated that tss1 is affected in both NH4 +-sensitive and -insensitive components of K+ transport at low Ca2+ concentrations and that this defective transport is rescued by increasing the concentration of Ca2+. Our results suggest that the TSS1 gene product is part of a crucial pathway mediating the beneficial effects of Ca2+ involved in K+ nutrition and salt tolerance.

Soil salinity is a major abiotic stress in plant agriculture strongly, influencing plant productivity world-wide. Classical breeding for salt tolerance in crop plants has been attempted to improve field performance without success. Therefore, an alternative strategy is to generate salt tolerant plants through genetic engineering. Several species and experimental approaches have been used in order to identify those genes that are important for salt tolerance. Due to high level of salt tolerance, halophytes are good candidates to identify salt tolerance genes. However, other species such as yeast and glycophytes have also been employed. Three approaches are commonly used to identify genes important for salt tolerance. The first approach is to identify genes involved in processes known to be critical for salt tolerance (osmolyte synthesis, ion homeostasis, etc.). The second approach is to identify genes whose expression is regulated by salt stress. This is relatively simply and applicable to any plant species. Genetic amenability of some species allows the third approach, which consists in the identification of salt tolerance determinants based on functionality. At the moment, there is a large number of reports in the literature claiming that plants with increased salt tolerance have been obtained. The main problem is that different plant species, stage of development, organs, promoters and salt conditions used it is difficult to compare the degree of salt tolerance conferred by different genes. In this review, we discuss progress made towards understanding the molecular elements involved in salt stress responses that have been used in transgenic approaches to improve salt tolerance.

�Summary Osmotic stress severely limits plant growth and agricultural productivity. We have used mutagenesis to identify plant genes that are required for osmotic stress tolerance in tomato. As a result, we have isolated a novel mutant in tomato (tos1) caused by a single recessive nuclear mutation that is hypersensitive to general osmotic stress. Growth measurements demonstrated that the tos1 mutant is less sensitive to intracellular abscisic acid (ABA) and this decreased ABA sensitivity of tos1 is a basic cellular trait expressed by the mutant at all developmental stages analysed. It is not caused by a deficiency in the synthesis of ABA because the tos1 seedlings accumulated more ABA than the wild type (WT) after osmotic stress. In contrast, the tss2 tomato mutant, which is also hypersensitive to osmotic stress, is hypersensitive to exogenous ABA. Comparative analysis of tos1 and tss2 indicates that appropriate ABA perception and signalling is essential for osmotic tolerance.

Lotus uliginosus (greater lotus, GL) and L. corniculatus (birdsfoot trefoil, BT) are species markedly different in their genetics, morphology and environmental adaptation. We evaluated the hybrids and parental species under differential conditions: in vitro culture, growth chamber and open field environments. The experimental evaluation included biochemical, physiological, developmental and productive parameters. Parental species exhibited significant differences in root growth under different osmotic potentials generated by polyethylene glycol, and hybrids exhibited variability in their response compared with their parents. Plants grown in pots and subjected to drought exhibited differences in biochemical parameters. Proline accumulation and oxidative damage measured by lipid peroxidation were higher in birdsfoot trefoil than any other genotype, and the lowest values were observed in greater lotus. The total phenolic content in shoot ranged from 147 to 279 mg gallic acid equivalents 100 g–1 dry mater. With regard to antioxidant capacity estimated by 2,2-diphenyl-1-picrylhydrazyl, greater lotus had the highest value and birdsfoot trefoil the lowest, at almost one-third, whereas hybrids had intermediate values. Under stress conditions, water-use efficiency showed significant differences between both parental species. In the field, under either irrigation or drought, the dry matter accumulated by the hybrids was higher than that of the parents. Hybrids express recombination of features, making them an interesting material to continue evaluation.

Drought brings about different biochemical responses in plants in order to minimize its deleterious effects. Drought induces an oxidative stress in Lotus corniculatus leaves, measured as an increment in lipid membrane peroxidation and in situ detected superoxide. As a result, total superoxide dismutase (SOD: E.C. 1.15.1.1) activity increases after a 4-h drought, when the hydric potential has decreased to −0.77 MPa. Assays with specific inhibitors suggest the presence of MnSOD, Cu/ZnSOD and FeSOD activity in L. corniculatus plants, contribute 60, 30 and 10% of total SOD activity, respectively. The Cu/ZnSOD isoform proved to be the most responsive to drought showing a remarkable increase in its activity as that corresponds to the induction of three different isoenzymes. Expression analysis of SOD isoforms revealed an increase in Cu/ZnSOD transcripts with a maximum accumulation 4 h after drought imposition. The possible role of SOD enzymes as an antioxidant protector system under water stress conditions in L. corniculatus is discussed. © 2001 Elsevier Science Ireland Ltd. All rights reserved.

Previous studies have shown that salicylic acid (SA) is an essential component of the plant resistance to pathogens. We now show that SA plays a role in the plant response to adverse environmental conditions, such as salt and osmotic stresses. We have studied the responses of wild-type Arabidopsis and an SA-deficient transgenic line expressing a salicylate hydroxylase (NahG) gene to different abiotic stress conditions. Wild-type plants germinated under moderate light conditions in media supplemented with 100 mmNaCl or 270 mm mannitol showed extensive necrosis in the shoot. In contrast, NahG plants germinated under the same conditions remained green and developed true leaves. The lack of necrosis observed in NahG seedlings under the same conditions suggests that SA potentiates the generation of reactive oxygen species in photosynthetic tissues during salt and osmotic stresses. This hypothesis is supported by the following observations. First, the herbicide methyl viologen, a generator of superoxide radical during photosynthesis, produced a necrotic phenotype only in wild-type plants. Second, the presence of reactive oxygen-scavenging compounds in the germination media reversed the wild-type necrotic phenotype seen under salt and osmotic stress. Third, a greater increase in the oxidized state of the glutathione pool under NaCl stress was observed in wild-type seedlings compared with NahG seedlings. Fourth, greater oxidative damage occurred in wild-type seedlings compared with NahG seedlings under NaCl stress as measured by lipid peroxidation. Our data support a model for SA potentiating the stress response of the germinating Arabidopsis seedling.

Summary Water stress induced proline accumulation in Lotus corniculatus regardless of the nitrogen source provided to plants, although this accumulation was higher in nitrogen fixing plants. Enzymes related to synthesis and oxidation of proline were analysed. Fd-GOGAT activity and Fd-GOGAT protein content increased in response to water stress in contrast to PDH activity, which decreased under such condition. However, rehydration for 6 h induced an increment in PDH activity. The differential effect of water deficit on enzymes involved in the proline pathway in L. corniculatus plants growing under different nitrogen regimes is discussed. The possible role of nitrate as an osmoprotectant in the place of proline is also considered.

In order to evaluate energy efficiency of nitrogen fixation by the Lotus corniculatus/Rhizobium loti symbiosis, Uruguayan R. loti strains were tested for hydrogen-uptake (Hup) status. Nodules induced in L. corniculatus by all eight R. loti strains tested evolved high amounts of hydrogen (2.0–8.7 μ mol H2/h.g nodule fresh weight). This production of hydrogen corresponds to 38–69% of total nitrogenase activity estimated as acetylene reduction, suggesting that hydrogen is not recycled within these nodules. This was confirmed by the lack of hydrogenase activity in bacteroid suspensions. Additionally, no hybridization signals were observed in total DNA restriction digests from these strains when a DNA fragment containing part of hydrogenase structural genes from Rhizobium leguminosarum bv. viciae was used as probe. Cosmid pHU52, containing the complete gene cluster required for hydrogen oxidation in Bradyrhizobium japonicum, was introduced into two R. loti strains. Transconjugants from only one of the strains were able to express hydrogenase activity in vegetative cells incubated under the derepression conditions described for B. japonicum. Bacteroids induced by both transconjugant strains in L. corniculatus and Lotus tenuis expressed hydrogenase activity in nodules. The level of hydrogenase activity induced in L. tenuis nodules was two-fold higher than those induced in L. corniculatus. This implies the existence of a strong host effect on hydrogenase expression in this symbiotic system.

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals., This work was supported by grants from Ministerio de Ciencia e Innovación (cofinanced by the European Regional Development Fund) to M.A.B. (BIO2011-23859 and CSD2007-00057), A.F. (BIO2009-06984 and CSD2007-00036), and O.B. from Universidad de la República-Comisión Sectorial de Investigación Científica (Grupo 418). V.G.D. was supported by a Formación del Personal Investigador fellowship from Ministerio de Educación y Ciencia (BIO2005-04733), and V.A.-S. was supported by FundaçÃo para a Ciência e a Tecnologia (FCT) (Grant SFRH/BD/38583/2007).

Identification of metabolic targets of environmental stress factors is critical to improve the stress tolerance of plants. Studying the biochemical and physiological responses of plants with different capacities to deal with stress is a valid approach to reach this objective. Lotus corniculatus (lotus) and Trifolium pratense (clover) are legumes with contrasting summer stress tolerances. In stress conditions, which are defined as drought, heat or a combination of both, we found that differential biochemical responses of leaves explain these behaviours. Lotus and clover showed differences in water loss control, proline accumulation and antioxidant enzymatic capacity. Drought and/or heat stress induced a large accumulation of proline in the tolerant species (lotus), whereas heat stress did not cause proline accumulation in the sensitive species (clover). In lotus, Mn-SOD and Fe-SOD were induced by drought, but in clover, the SOD-isoform profile was not affected by stress. Moreover, lotus has more SOD-isoforms and a higher total SOD activity than clover. The functionality and electrophoretic profile of photosystem II (PSII) proteins under stress also exhibited differences between the two species. In lotus, PSII activity was drastically affected by combined stress and, interestingly, was correlated with D2 protein degradation. Possible implications of this event as an adaption mechanism in tolerant species are discussed. We conclude that the stress-tolerant capability of lotus is related to its ability to respond to oxidative damage and adaption of the photosynthetic machinery. This reveals that these two aspects should be included in the evaluation of the tolerance of species to stress conditions.

Plants are commonly subjected to several environmental stresses that lead to an overproduction of reactive oxygen species (ROS). As plants accumulate proline in response to stress conditions, some authors have proposed that proline could act as a non-enzymatic antioxidant against ROS. One type of ROS aimed to be quenched by proline is singlet oxygen (1O2), molecular oxygen in its lowest energy electronically excited state, constitutively generated in oxygenic, photosynthetic organisms. In this study we clearly prove that proline cannot quench 1O2 in aqueous buffer, giving rise to a rethinking about the antioxidant role of proline against 1O2., This research has financial support from ANII and PEDECIBA(Uruguay), the Spanish Ministry of Economy and Competitiveness and the Research Council of Norway.

Water stress is one of the most severe problems for plant growth and productivity. Using the legume Lotus japonicus exposed to water stress, a comparative analysis of key components in metabolism of reactive nitrogen and oxygen species (RNS and ROS, respectively) were made. After water stress treatment plants accumulated proline 23 and 10-fold in roots and leaves respectively, compared with well-watered plants. Significant changes in metabolism of RNS and ROS were observed, with an increase in both protein tyrosine nitration and lipid peroxidation, which indicate that water stress induces a nitro-oxidative stress. In roots, ·NO content was increased and S-nitrosoglutathione reductase activity was reduced by 23%, wherein a specific protein nitration pattern was observed. As part of this response, activity of NADPH-generating dehydrogenases was also affected in roots resulting in an increase of the NADPH/NADP(+) ratio. Our results suggest that in comparison with leaves, roots are significantly affected by water stress inducing an increase in proline and NO content which could highlight multiple functions for these metabolites in water stress adaptation, recovery and signaling. Thus, it is proposed that water stress generates a spatial distribution of nitro-oxidative stress with the oxidative stress component being higher in leaves whereas the nitrosative stress component is higher in roots.

TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins are characterized by the presence of six tetratricopeptide repeats in conserved positions and a carboxyl-terminal region known as the thioredoxin-like domain with homology to thioredoxins. In Arabidopsis (Arabidopsis thaliana), the TTL gene family is composed by four members, and the founder member, TTL1, is required for osmotic stress tolerance. Analysis of sequenced genomes indicates that TTL genes are specific to land plants. In this study, we report the expression profiles of Arabidopsis TTL genes using data mining and promoter-reporter β-glucuronidase fusions. Our results show that TTL1, TTL3, and TTL4 display ubiquitous expression in normal growing conditions but differential expression patterns in response to osmotic and NaCl stresses. TTL2 shows a very different expression pattern, being specific to pollen grains. Consistent with the expression data, ttl1, ttl3, and ttl4 mutants show reduced root growth under osmotic stress, and the analysis of double and triple mutants indicates that TTL1, TTL3, and TTL4 have partially overlapping yet specific functions in abiotic stress tolerance while TTL2 is involved in male gametophytic transmission.