Instituto de Biología Molecular y Celular de Plantas

This paper reports the genome sequence of domesticated tomato, a major crop plant, and a draft sequence for its closest wild relative; comparative genomics reveal very little divergence between the two genomes but some important differences with the potato genome, another important food crop in the genus Solanum. Tomato (Solanum lycopersicum) is a major crop plant and a model system for fruit development. Solanum is one of the largest angiosperm genera1 and includes annual and perennial plants from diverse habitats. Here we present a high-quality genome sequence of domesticated tomato, a draft sequence of its closest wild relative, Solanum pimpinellifolium2, and compare them to each other and to the potato genome (Solanum tuberosum). The two tomato genomes show only 0.6% nucleotide divergence and signs of recent admixture, but show more than 8% divergence from potato, with nine large and several smaller inversions. In contrast to Arabidopsis, but similar to soybean, tomato and potato small RNAs map predominantly to gene-rich chromosomal regions, including gene promoters. The Solanum lineage has experienced two consecutive genome triplications: one that is ancient and shared with rosids, and a more recent one. These triplications set the stage for the neofunctionalization of genes controlling fruit characteristics, such as colour and fleshiness.

Abscisic acid (ABA) regulates numerous developmental processes and adaptive stress responses in plants. Many ABA signaling components have been identified, but their interconnections and a consensus on the structure of the ABA signaling network have eluded researchers. Recently, several advances have led to the identification of ABA receptors and their three-dimensional structures, and an understanding of how key regulatory phosphatase and kinase activities are controlled by ABA. A new model for ABA action has been proposed and validated, in which the soluble PYR/PYL/RCAR receptors function at the apex of a negative regulatory pathway to directly regulate PP2C phosphatases, which in turn directly regulate SnRK2 kinases. This model unifies many previously defined signaling components and highlights the importance of future work focused on defining the direct targets of SnRK2s and PP2Cs, dissecting the mechanisms of hormone interactions (i.e., cross talk) and defining connections between this new negative regulatory pathway and other factors implicated in ABA signaling.

ABA Receptor Rumbled? The plant hormone abscisic acid (ABA) is critical for normal development and for mediating plant responses to stressful environmental conditions. Now, two papers present analyses of candidate ABA receptors (see the news story by Pennisi ). Ma et al. (p. 1064; published online 30 April) and Park et al. (p. 1068, published online 30 April) used independent strategies to search for proteins that physically interact with ABI family phosphatase components of the ABA response signaling pathway. Both groups identified different members of the same family of proteins, which appear to interact with ABI proteins to form a heterocomplex that can act as the ABA receptor. The variety of both families suggests that the ABA receptor may not be one entity, but rather a class of closely related complexes, which may explain previous difficulties in establishing its identity.

Modern commercial tomato varieties are substantially less flavorful than heirloom varieties. To understand and ultimately correct this deficiency, we quantified flavor-associated chemicals in 398 modern, heirloom, and wild accessions. A subset of these accessions was evaluated in consumer panels, identifying the chemicals that made the most important contributions to flavor and consumer liking. We found that modern commercial varieties contain significantly lower amounts of many of these important flavor chemicals than older varieties. Whole-genome sequencing and a genome-wide association study permitted identification of genetic loci that affect most of the target flavor chemicals, including sugars, acids, and volatiles. Together, these results provide an understanding of the flavor deficiencies in modern commercial varieties and the information necessary for the recovery of good flavor through molecular breeding.

Plants undergoing the onslaught of wound-causing agents activate mechanisms directed to healing and further defence. Responses to mechanical damage are either local or systemic or both and hence involve the generation, translocation, perception, and transduction of wound signals to activate the expression of wound-inducible genes. Although the central role for jasmonic acid in plant responses to wounding is well established, other compounds, including the oligopeptide systemin, oligosaccharides, and other phytohormones such as abscisic acid and ethylene, as well as physical factors such as hydraulic pressure or electrical pulses, have also been proposed to play a role in wound signalling. Different jasmonic acid-dependent and -independent wound signal transduction pathways have been identified recently and partially characterized. Components of these signalling pathways are mostly similar to those implicated in other signalling cascades in eukaryotes, and include reversible protein phosphorylation steps, calcium/calmodulin-regulated events, and production of active oxygen species. Indeed, some of these components involved in transducing wound signals also function in signalling other plant defence responses, suggesting that cross-talk events may regulate temporal and spatial activation of different defences.

Significance We described the high-throughput identification of DNA-binding specificities of 63 plant transcription factors (TFs) and their relevance as cis- regulatory elements in vivo. Almost half of the TFs recognized secondary motifs partially or completely differing from their corresponding primary ones. Analysis of coregulated genes, transcriptomic data, and chromatin hypersensitive regions revealed the biological relevance of more than 80% of the binding sites identified. Our combined analysis allows the prediction of the function of a particular TF as activator or repressor through a particular DNA sequence. The data support the correlation between cis- regulatory elements in vivo and the sequence determined in vitro. Moreover, it provides a framework to explore regulatory networks in plants and contributes to decipher the transcriptional regulatory code.

ABSTRACT Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H 2 O 2 and O 2 ·− production was studied in leaves from pea plants growth for 2 weeks with 50 µ m Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl 3 and Mn/DAB staining for H 2 O 2 and O 2 ·− , respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µ m CdCl 2 a rise of six times in the H 2 O 2 content took place in comparison with control plants, and the accumulation of H 2 O 2 was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H 2 O 2 was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H 2 O 2 could be a NADPH oxidase. The subcellular localization of O 2 ·− production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd‐induced production of the ROS, H 2 O 2 and O 2 ·− , could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd‐grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca 2+ channels, and cGMP.

Robustness is the invariance of phenotypes in the face of perturbation. The robustness of phenotypes appears at various levels of biological organization, including gene expression, protein folding, metabolic flux, physiological homeostasis, development, and even organismal fitness. The mechanisms underlying robustness are diverse, ranging from thermodynamic stability at the RNA and protein level to behavior at the organismal level. Phenotypes can be robust either against heritable perturbations (e.g., mutations) or nonheritable perturbations (e.g., the weather). Here we primarily focus on the first kind of robustness--genetic robustness--and survey three growing avenues of research: (1) measuring genetic robustness in nature and in the laboratory; (2) understanding the evolution of genetic robustness: and (3) exploring the implications of genetic robustness for future evolution.

Little is known about the mutational fitness effects associated with single-nucleotide substitutions on RNA viral genomes. Here, we used site-directed mutagenesis to create 91 single mutant clones of vesicular stomatitis virus derived from a common ancestral cDNA and performed competition experiments to measure the relative fitness of each mutant. The distribution of nonlethal deleterious effects was highly skewed and had a long, flat tail. As expected, fitness effects depended on whether mutations were chosen at random or reproduced previously described ones. The effect of random deleterious mutations was well described by a log-normal distribution, with -19% reduction of average fitness; the effects distribution of preobserved deleterious mutations was better explained by a beta model. The fit of both models was improved when combined with a uniform distribution. Up to 40% of random mutations were lethal. The proportion of beneficial mutations was unexpectedly high. Beneficial effects followed a gamma distribution, with expected fitness increases of 1% for random mutations and 5% for preobserved mutations.

Abscisic acid (ABA) is a key phytohormone involved in adaption to environmental stress and regulation of plant development. Clade A protein phosphatases type 2C (PP2Cs), such as HAB1, are key negative regulators of ABA signaling in Arabidopsis. To obtain further insight into regulation of HAB1 function by ABA, we have screened for HAB1-interacting partners using a yeast two-hybrid approach. Three proteins were identified, PYL5, PYL6 and PYL8, which belong to a 14-member subfamily of the Bet v1-like superfamily. HAB1-PYL5 interaction was confirmed using BiFC and co-immunoprecipitation assays. PYL5 over-expression led to a globally enhanced response to ABA, in contrast to the opposite phenotype reported for HAB1-over-expressing plants. F(2) plants that over-expressed both HAB1 and PYL5 showed an enhanced response to ABA, indicating that PYL5 antagonizes HAB1 function. PYL5 and other members of its protein family inhibited HAB1, ABI1 and ABI2 phosphatase activity in an ABA-dependent manner. Isothermal titration calorimetry revealed saturable binding of (+)ABA to PYL5, with K(d) values of 1.1 mum or 38 nm in the absence or presence of the PP2C catalytic core of HAB1, respectively. Our work indicates that PYL5 is a cytosolic and nuclear ABA receptor that activates ABA signaling through direct inhibition of clade A PP2Cs. Moreover, we show that enhanced resistance to drought can be obtained through PYL5-mediated inhibition of clade A PP2Cs.

Abscisic acid (ABA) is a key hormone for plant growth, development, and stress adaptation. Perception of ABA through four types of receptors has been reported. We show here that impairment of ABA perception through the PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) branch reduces vegetative growth and seed production and leads to a severe open stomata and ABA-insensitive phenotype, even though other branches for ABA perception remain functional. An Arabidopsis thaliana sextuple mutant impaired in six PYR/PYL receptors, namely PYR1, PYL1, PYL2, PYL4, PYL5, and PYL8, was able to germinate and grow even on 100 μM ABA. Whole-rosette stomatal conductance (Gst) measurements revealed that leaf transpiration in the sextuple pyr/pyl mutant was higher than in the ABA-deficient aba3-1 or ABA-insensitive snrk2.6 mutants. The gradually increasing Gst values of plants lacking three, four, five, and six PYR/PYLs indicate quantitative regulation of stomatal aperture by this family of receptors. The sextuple mutant lacked ABA-mediated activation of SnRK2s, and ABA-responsive gene expression was dramatically impaired as was reported in snrk2.2/2.3/2.6. In summary, these results show that ABA perception by PYR/PYLs plays a major role in regulation of seed germination and establishment, basal ABA signaling required for vegetative and reproductive growth, stomatal aperture, and transcriptional response to the hormone.

Salinity is one of the major abiotic stresses which affects plant cell metabolism and reduces plant productivity. Variations in the antioxidant defence systems under salinity among two bean genotypes were investigated. Our results indicate that the difference between the genotypes in response to salinity is a quantitative trait rather than qualitative since they develop the same strategies with a significant variation in the rate of synthesis and accumulation, with the exemption of the antioxidant defence based on the synthesis of phenolic compounds. For both genotypes, salinity induced a marked reduction in dry matter gain in roots and shoots along with oxidative stress as indicated by the significant increase in malondialdehyde content. In addition, the photosynthetic pigments decreased with the increase of salinity. The only qualitative difference that we found among both genotypes was the decrease of total production of phenolic compounds in leaves that was only detectable in the low-yielding genotype under high salinity. The high-yielding genotype may have a better protection against oxidative damages by increasing the activity of antioxidant enzymes and the amounts of total flavonoids and ascorbic acid under high salinity, which allows maintaining higher yield even upon stress conditions. These results indicate that salt induced oxidative stress in bean is mainly counteracted by enzymatic defence systems, and that the metabolism of phenolic compounds is induced under very extreme conditions. The selection of genotypes for this trait will increase yield under stress conditions.

The plant hormone abscisic acid (ABA) orchestrates plant adaptive responses to a variety of stresses, including drought. This signaling pathway is regulated by reversible protein phosphorylation, and genetic evidence demonstrated that several related protein phosphatases 2C (PP2Cs) are negative regulators of this pathway in Arabidopsis thaliana. Here, we developed a protein phosphatase profiling strategy to define the substrate preferences of the HAB1 PP2C implicated in ABA signaling and used these data to screen for putative substrates. Interestingly, this analysis designated the activation loop of the ABA activated kinase OST1, related to Snf1 and AMPK kinases, as a putative HAB1 substrate. We experimentally demonstrated that HAB1 dephosphorylates and deactivates OST1 in vitro. Furthermore, HAB1 and the related PP2Cs ABI1 and ABI2 interact with OST1 in vivo, and mutations in the corresponding genes strongly affect OST1 activation by ABA. Our results provide evidence that PP2Cs are directly implicated in the ABA-dependent activation of OST1 and further suggest that the activation mechanism of AMPK/Snf1-related kinases through the inhibition of regulating PP2Cs is conserved from plants to human.

Growth of pea (Pisum sativum L.) plants with 50 microM CdCl2 for 15 d produced a reduction in the number and length of lateral roots, and changes in structure of the principal roots affecting the xylem vessels. Cadmium induced a reduction in glutathione (GSH) and ascorbate (ASC) contents, and catalase (CAT), GSH reductase (GR) and guaiacol peroxidase (GPX) activities. CuZn-superoxide dismutase (SOD) activity was also diminished by the Cd treatment, although Mn-SOD was slightly increased. CAT and CuZn-SOD were down-regulated at transcriptional level, while Mn-SOD, Fe-SOD and GR were up-regulated. Analysis of reactive oxygen species (ROS) and nitric oxide (NO) levels by fluorescence and confocal laser microscopy (CLM) showed an over-accumulation of O2*- and H2O2, and a reduction in the NO content in lateral and principal roots. ROS overproduction was dependent on changes in intracellular Ca+2 content, and peroxidases and NADPH oxidases were involved. Cadmium also produced an increase in salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) contents. The rise of ET and ROS, and the NO decrease are in accordance with senescence processes induced by Cd, and the increase of JA and SA could regulate the cellular response to cope with damages imposed by cadmium.

Mutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified during the course of two independent screenings and named salt resistant (sre) and salobreño (sañ). The sre and sañ mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay. Complementation analyses revealed that sre1-1, sre1-2, sañ3-1, and sañ3-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene. A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of four new aba2 alleles. The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD- or NADP-dependent oxidoreductases. Recombinant ABA2 protein produced in Escherichia coli exhibits a K(m) value for xanthoxin of 19 micro M and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC-mass spectrometry. The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress. The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin-->abscisic aldehyde-->ABA transition as the last steps of the major ABA biosynthetic pathway.

Public databases contain a planetary collection of nucleic acid sequences, but their systematic exploration has been inhibited by a lack of efficient methods for searching this corpus, which (at the time of writing) exceeds 20 petabases and is growing exponentially1. Here we developed a cloud computing infrastructure, Serratus, to enable ultra-high-throughput sequence alignment at the petabase scale. We searched 5.7 million biologically diverse samples (10.2 petabases) for the hallmark gene RNA-dependent RNA polymerase and identified well over 105 novel RNA viruses, thereby expanding the number of known species by roughly an order of magnitude. We characterized novel viruses related to coronaviruses, hepatitis delta virus and huge phages, respectively, and analysed their environmental reservoirs. To catalyse the ongoing revolution of viral discovery, we established a free and comprehensive database of these data and tools. Expanding the known sequence diversity of viruses can reveal the evolutionary origins of emerging pathogens and improve pathogen surveillance for the anticipation and mitigation of future pandemics. Serratus, an open-source cloud-computing infrastructure, can be used to screen millions of nucleic acid sequencing libraries at the petabase scale, and has enabled many new RNA viruses to be identified efficiently.

NADPH oxidases (Nox) have been characterized as higher eukaryotic enzymes used deliberately to produce reactive oxygen species (ROS). The recent discovery of new functional members of the Nox family in plants and animals has led to the recognition of the increasing importance of ROS as signals involved in regulation of diverse cellular processes such as defence, growth and signalling. Here, we address the role of NADPH oxidase-generated ROS in the biology of the filamentous fungus Aspergillus nidulans. We characterize the noxA gene and show that it encodes a member of a novel NADPH oxidase subfamily ubiquitous in lower eukaryotes. Deletion of noxA specifically blocks differentiation of sexual fruit bodies (cleistothecia), without affecting hyphal growth or asexual development. Accordingly, the noxA gene is induced during sexual development, peaking at the time of cleistothecia differentiation and in parallel with the hülle cell-associated catalase peroxidase gene cpeA. This expression pattern is not dependent on transcription factors SteA and StuA, which are essential for cleistothecia formation. In contrast, noxA-dependent premature sexual development correlates with noxA derepression in DeltasakA null mutants, connecting stress MAPK signalling to the regulated production of ROS. Using a nitroblue tetrazolium (NBT) assay to detect superoxide, we found that hülle cells and cleistothecia initials produce superoxide in a process inhibited by NADPH oxidase inhibitor DPI and markedly reduced in DeltanoxA mutants. Furthermore, using H2DCFDA, we detected that H2O2 and possibly other ROS are generated in a NoxA-dependent fashion, mainly in the external walls from cleistothecia initials. The essential role of NoxA-generated ROS in A. nidulans sexual differentiation and the presence of one or two noxA homologues in all analysed filamentous fungi suggest that NADPH oxidase-generated ROS play important roles in fungal physiology and differentiation.

The MADS-domain transcription factor APETALA1 (AP1) is a key regulator of Arabidopsis flower development. To understand the molecular mechanisms underlying AP1 function, we identified its target genes during floral initiation using a combination of gene expression profiling and genome-wide binding studies. Many of its targets encode transcriptional regulators, including known floral repressors. The latter genes are down-regulated by AP1, suggesting that it initiates floral development by abrogating the inhibitory effects of these genes. Although AP1 acts predominantly as a transcriptional repressor during the earliest stages of flower development, at more advanced stages it also activates regulatory genes required for floral organ formation, indicating a dynamic mode of action. Our results further imply that AP1 orchestrates floral initiation by integrating growth, patterning, and hormonal pathways.

Pretty or Sweet The grocery-store tomato that looks beautiful but tastes like tart cardboard arises from selection processes favoring phenotypes that make commercial production more reliable. Significant in that selection process was a mutation that reduced the mottled color variations of unripe green tomatoes, leaving them a uniform, pale, green. Powell et al. (p. 1711 ) analyzed the molecular biology of the mutation. The uniform ripening mutation turns out to disable a transcription factor called Golden 2-like ( GLK2 ). GLK2 expression increases the fruit's photosynthetic capacity, resulting in higher sugar content.

Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an evergrowing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects.