Transcriptome analysis of intraspecific competition in Arabidopsis thaliana reveals organ-specific signatures related to nutrient acquisition and general stress response pathways.

ABSTRACT: BACKGROUND: Plants are sessile and therefore have to perceive and adjust to changes in their environment. The presence of neighbours leads to a competitive situation where resources and space will be limited. Complex adaptive responses to such situation are poorly understood at the molecular level. RESULTS: Using microarrays, we analysed whole-genome expression changes in Arabidopsis thaliana plants subjected to intraspecific competition. The leaf and root transcriptome was strongly altered by competition. Differentially expressed genes were enriched in genes involved in nutrient deficiency (mainly N, P, K), perception of light quality, and responses to abiotic and biotic stresses. Interestingly, performance of the generalist insect Spodoptera littoralis on densely grown plants was significantly reduced, suggesting that plants under competition display enhanced resistance to herbivory. CONCLUSIONS: This study provides a comprehensive list of genes whose expression is affected by intraspecific competition in Arabidopsis. The outcome is a unique response that involves genes related to light, nutrient deficiency, abiotic stress, and defence responses.

Screening for wound-induced oxylipins in Arabidopsis thaliana by differential HPLC-APCI/MS profiling of crude leaf extracts and subsequent characterisation by capillary-scale NMR

A simple non-targeted differential HPLC-APCI/MS approach has been developed in order to survey metabolome modifications that occur in the leaves of Arabidopsis thaliana following wound-induced stress. The wound-induced accumulation of metabolites, particularly oxylipins, was evaluated by HPLC-MS analysis of crude leaf extracts. A generic, rapid and reproducible pressure liquid extraction procedure was developed for the analysis of restricted leaf samples without the need for specific sample preparation. The presence of various oxylipins was determined by head-to-head comparison of the HPLC-MS data, filtered with a component detection algorithm, and automatically compared with the aid of software searching for small differences in similar HPLC-MS profiles. Repeatability was verified in several specimens belonging to different series. Wound-inducible jasmonates were efficiently highlighted by this non-targeted approach without the need for complex sample preparation as is the case for the 'oxylipin signature' procedure based on GC-MS. Furthermore this HPLC-MS screening technique allowed the isolation of induced compounds for further characterisation by capillary-scale NMR (CapNMR) after HPLC scale-up. In this paper, the screening method is described and applied to illustrate its potential for monitoring polar and non-polar stress-induced constituents as well as its use in combination with CapNMR for the structural assignment of wound-induced compounds of interest

Aproximación de química genómica para el entendimiento de la repuesta defensiva vegetal frente a patógenos: Identificación de moléculas antagonistas y agonistas de la regulación transcripcional en el mutante ocp1 de Arabidopsis thaliana

La genètica química en plantes constitueix una poderosa ferramenta en auge. Per a profunditzar en l’enteniment dels mecanismes defensius vegetals front a l’atac de microorganismes patògens es va realitzar un rastreig de molècules moduladores de l’expressió del gen Ep5C, que s’activa en resposta a l’aplicació de H2O2 y a l’atac de Pseudomonas syringae. S’ha utilizat el mutant ocp1 d’ Arabidopsis thaliana, obtés per mutagènesi a partir de plantes transgèniques pEp5C::GUS. Coneguda la relació d’Ep5C amb la RdDM gràcies a la implicació funcional d’OCP1 i OCP11, s’identifica a MOD18, MOD2 i MOD1 com a potencials molècules implicades en aquesta ruta. A més a més, s’identifica a MOD1 com a molècula activadora de les defenses a través de la ruta mediada per àcid salicílic.

Gibberellic acid signaling is required for ambient temperature-mediated induction of flowering in Arabidopsis thaliana.

Distinct molecular mechanisms integrate changes in ambient temperature into the genetic pathways that govern flowering time in Arabidopsis thaliana. Temperature-dependent eviction of the histone variant H2A.Z from nucleosomes has been suggested to facilitate the expression of FT by PIF4 at elevated ambient temperatures. Here we show that, in addition to PIF4, PIF3 and PIF5, but not PIF1 and PIF6, can promote flowering when expressed specifically in phloem companion cells (PCC), where they can induce FT and its close paralog, TSF. However, despite their strong potential to promote flowering, genetic analyses suggest that the PIF genes seem to have only a minor role in adjusting flowering in response to photoperiod or high ambient temperature. In addition, loss of PIF function only partially suppressed the early flowering phenotype and FT expression of the arp6 mutant, which is defective in H2A.Z deposition. In contrast, the chemical inhibition of gibberellic acid (GA) biosynthesis resulted in a strong attenuation of early flowering and FT expression in arp6. Furthermore, GA was able to induce flowering at low temperature (15°C) independently of FT, TSF, and the PIF genes, probably directly at the shoot apical meristem. Together, our results suggest that the timing of the floral transition in response to ambient temperature is more complex than previously thought and that GA signaling might play a crucial role in this process.

Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.

The evolution of post-zygotic isolation barriers by immune-triggered hybrid incompatibilities in Arabidopsis thaliana

A long-standing question in evolutionary biology is what defines a species. The biological species concept considers a species as a population of individuals that interbreeds freely and produces viable offspring. Therefore, reproductive isolation is the essence of species. Hybrid necrosis is one form of post-zygotic reproductive isolation. In this chapter, we summarize what is known to date about this phenomenon and highlight progress made in the understanding of these immune-triggered hybrid incompatibilities through our research in the plant model Arabidopsis thaliana.

The evolution of post-zygotic isolation barriers by immune-triggered hybrid incompatibilities in Arabidopsis thaliana

A long-standing question in evolutionary biology is what defines a species. The biological species concept considers a species as a population of individuals that interbreeds freely and produces viable offspring. Therefore, reproductive isolation is the essence of species. Hybrid necrosis is one form of post-zygotic reproductive isolation. In this chapter, we summarize what is known to date about this phenomenon and highlight progress made in the understanding of these immune-triggered hybrid incompatibilities through our research in the plant model Arabidopsis thaliana.

Natural variability in Arabidopsis thaliana germplasm response to Xanthomonas campestris pv. campestris

This work aimed to study the interaction between the model plant Arabidopsis thaliana and Xanthomonas campestris pv. campestris (Xcc), the pathogen responsible for black rot of crucifers. The response of 32 accessions of A. thaliana to the Brazilian isolate of Xcc CNPH 17 was evaluated. No immunity-like response was observed. "CS1308", "CS1566" and "CS1643" grown in continuous light were among the accessions that showed strongest resistance when inoculated with 5 x 10(6) CFU/mL. In contrast, "CS1194" and "CS1492" were among the most susceptible accessions. Similar results were obtained when plants were grown under short-day conditions. To quantify the differences in disease symptoms, total chlorophyll was extracted from contrasting accessions at different time points after inoculation. Chlorophyll levels from controls and Xcc inoculated plants showed a similar reduction in resistant accessions, whereas Xcc-inoculated susceptible accessions showed a greater reduction compared to controls. To test the specificity of resistance, accessions CS1308, CS1566, CS1643 and CS1438 (which showed partial resistance to CNPH 17), were inoculated with a more aggressive isolate of Xcc (CNPH 77) and Ralstonia solanacearum. Among the accessions tested, "CS1566" was the most resistant to Xcc CNPH 77 and also displayed resistance to R. solanacearum. Accessions CS1308, CS1566 and CS1643 were also inoculated with a high titer of Xcc CNPH 17 (5 x 10(8) CFU/mL). No collapse of tissue was observed up to 48 h after inoculation, indicating that a hypersensitive response is not involved in the resistance displayed by these accessions.

Leaf-Targeted Ferredoxin-NADP+-Oxidoreductase (FNR): Electron Transfer Properties and Thylakoid Association in Arabidopsis thaliana

Photosynthetic reactions are divided in two parts: light-driven electron transfer reactions and carbon fixation reactions. Electron transfer reactions capture solar energy and split water molecules to form reducing energy (NADPH) and energy-carrying molecules (ATP). These end-products are used for fixation of inorganic carbon dioxide into organic sugar molecules. Ferredoxin-NADP⁺ oxidoreductase (FNR) is an enzyme that acts at the branch point between the electron transfer reactions and reductive metabolism by catalyzing reduction of NADP+ at the last step of the electron transfer chain. In this thesis, two isoforms of FNR from A rabidopsis thaliana, FNR1 and FNR2, were characterized using the reverse genetics approach. The fnr1 and fnr2 mutant plants resembled each other in many respects. Downregulation of photosynthesis protected the single fnr mutant plants from excess formation of reactive oxygen species (ROS), even without significant upregulation of antioxidative mechanisms. Adverse growth conditions, however, resulted in phenotypic differences between fnr1 and fnr2. While fnr2 plants showed downregulation of photosynthetic complexes and upregulation of antioxidative mechanisms under low-temperature growth conditions, fnr1 plants had the wild-type phenotype, indicating that FNR2 may have a specific role in redistribution of electrons under unfavorable conditions. The heterozygotic double mutant (fnr1xfnr2) was severely devoid of chloroplastic FNR, which clearly restricted photosynthesis. The fnr1xfnr2 plants used several photoprotective mechanisms to avoid oxidative stress. In wild-type chloroplasts, both FNR isoforms were found from the stroma, the thylakoid membrane, and the inner envelope membrane. In the absence of the FNR1 isoform, FNR2 was found only in the stroma, suggesting that FNR1 and FNR2 form a dimer, by which FNR1 anchors FNR2 to the thylakoid membrane. Structural modeling predicted formation of an FNR dimer in complex with ferredoxin. In this thesis work, Tic62 was found to be the main protein that binds FNR to the thylakoid membrane, where Tic62 and FNR formed high molecular weight complexes. The formation of such complexes was shown to be regulated by the redox state of the chloroplast. The accumulation of Tic62-FNR complexes in darkness and dissociation of complexes from the membranes in light provide evidence that the complexes may have roles unrelated to photosynthesis. This and the high viability of fnr1 mutant plants lacking thylakoid-bound FNR indicate that the stromal pool of FNR is photosynthetically active.

Characterization of Leaf-Type Ferredoxin-NADP+ Oxidoreductase (FNR) Isoforms in Arabidopsis thaliana

Life on earth is based on sunlight, which is captured in chemical form by photosynthetic reactions. In the chloroplasts of plants, light reactions of photosynthesis take place at thylakoid membranes, whereas carbon assimilation reactions occur in the soluble stroma. The products of linear electron transfer (LET), highly-energetic ATP molecules, and reducing power in the form of NADPH molecules, are further used in the fixation of inorganic CO2 molecules into organic sugars. Ferredoxin-NADP+ oxidoreductase (FNR) catalyzes the last of the light reactions by transferring electrons from ferredoxin (FD) to NADP+. In addition to LET, FNR has been suggested to play a role in cyclic electron transfer (CET), which produces ATP without the accumulation of reducing equivalents. CET is proposed to occur via two putative routes, the PGR5- route and the NDH-route. In this thesis, the leaf-type FNR (LFNR) isoforms LFNR1 and LFNR2 of a model organism, Arabidopsis thaliana, were characterized. The physiological roles of LFNRs were investigated using single and double mutant plants. The viability of the single mutants indicates functionality of both isoforms, with neither appearing to play a specific role in CET. The more severe phenotype of low-temperature adapted fnr2 plants compared to both wild-type (WT) and fnr1 plants suggests a specific role for LFNR2 under unfavorable growth conditions. The more severe phenotype of the fnr1 x fnr2 (F1 generation) plants compared to single mutants reflects down-regulated photosynthetic capacity, whereas slightly higher excitation pressure indicates mild over-excitation of electron transfer chain (ETC). However, induction of CET and various photoprotective mechanisms enable adaptation of fnr1 x fnr2 plants to scarcity of LFNR. The fnr1 fnr2 plants (F2 generation), without detectable levels of LFNR, were viable only under heterotrophic conditions. Moreover, drought stress induced acceleration of the rate of P700 + re-reduction in darkness was accompanied by a concomitant up-regulation of the PGR5-route specific components, PGR5 and PGRL1, demonstrating the induction of CET via the PGR5-route. The up-regulation of relative transcriptional expression of the FD1 gene indicates that the FD1 isoform may have a specific function in CET, while no such role could be defined for either of the LFNR isoforms. Both the membrane-bound and soluble LFNR1 and LFNR2 each appear as two distinct spots after 2D-PAGE with different isoelectric points (pIs), indicating the existence of post-translational modifications (PTMs) which do not determine the membrane attachment of LFNR. The possibility of phosphorylation and glycosylation PTMs were excluded, but all four LFNR forms were shown to contain acetylated lysine residues as well as alternative N-termini. N-terminal acetylation was shown to shift the pI of both LFNRs to be more acidic. In addition, all four LFNR forms were demonstrated to interact both with FD1 and FD2 in vitro

Xyloglucans from Hymenaea courbaril var. stilbocarpa seeds affect Arabidopsis thaliana seedling growth by enhancing lateral root development

The effect of crude xyloglucan (XG) preparations from jatobá (Hymenaea courbaril var. stilbocarpa (Hayne) Y. T. Lee & Langenh.) seeds on Arabidopsis thaliana (L.) Heynh. root system development was investigated. The XG extracts exerted a dual effect on root system development by slowing down root growth and improving lateral root formation. These observed morphological changes were not due to oligosaccharides that could be generated following hydrolysis of the XG polymers, since XG hydrolysate induced a drastic inhibition of the overall growth process of the Arabidopsis thaliana seedlings. Histochemical test of GUS gene expression assay performed on seven and 14-days-old transgenic Arabidopsis thaliana plants carrying the CycB1;1-GUS fusion indicated that the improvement of the lateral root development by jatobá XG extracts was not correlated with the expression of this cell cycle marker gene in the root system. A potential agricultural application of jatobá seeds XG extract is discussed.

Unique and unifying themes in the mechanisms regulating the expression of the arabidopsis thaliana PR-1 and Solanum tuberosum PR-10a inducible defense genes

Arabidopsis is a model plant used to study disease resistance; Solanum tuberosum or potato is a crop species. Both plants possess inducible defense mechanisms that are deployed upon recognition of pathogen invasion. Transcriptional reprogramming is crucial to the activation of defense responses. The Pathogenesis-Related (PR) genes are activated in these defense programs. Expression of Arabidopsis PR-l and potato PR-10a serve as markers for the deployment of defense responses in these plants. PR-l expression indicates induction of systemic acquired resistance (SAR). Activation of SAR requires accumulation of salicylic acid (SA), in addition to the interaction of the non-expressor of pathogenesis-related genes I (NPRI), with the TGA transcription factors. The PR-10a is activated in response to pathogen invasion, wounding and elicitor treatment. PR-10a induction requires recruitment of the Whirly I (Whyl) activator to the promoter. This locus is also negatively regulated by the silencer element binding factor (SEBF). We established that both the PR-l and PR-10a are occupied by repressors under non-inducing conditions. TGA2 was found to be a constitutive resident and repressor of PR-l, which mediates repression by forming an oligomeric complex on the promoter. The DNA-binding activity of this oligomer required the TGA2 N-terminus (NT). Under resting conditions we determined that the PR-10a is bound by a repressosome containing SEBF and curiously the activator Pto interacting protein 4 (Pti4). In the context of this repressosome, SEBF is responsible for PR-10a binding, yet rWe also showed that PR-l and PR-10a are activated by different means. In PR-l activation the NPRI NT domain alleviates TGA2-mediated repression by interacting with the TGA2 NT. TGA2 remains at the PR-l but adopts a dimeric conformation and forms an enhanceosome with NPRl. In contrast, the PR-10a is activated by evicting the repressosome and recruiting Why! to the promoter. These results advance our understanding of the mechanisms regulating PR-l and PR-10a expression under resting and inducing conditions. This study also revealed that the means of regulation for related genes can differ greatly between model and crop s

Regulation of Systemic Acquired Resistance through the Interaction of Arabidopsis thaliana Transcription factors TGAI and TGA2 with NPRI

Arabidopsis thaliana is an established model plant system for studying plantpathogen interactions. The knowledge garnered from examining the mechanism of induced disease resistance in this model system can be applied to eliminate the cost and danger associated with current means of crop protection. A specific defense pathway, known as systemic acquired resistance (SAR), involves whole plant protection from a wide variety of bacterial, viral and fungal pathogens and remains induced weeks to months after being triggered. The ability of Arabidopsis to mount SAR depends on the accumulation of salicylic acid (SA), the NPRI (non-expressor of pathogenesis related gene 1) protein and the expression of a subset of pathogenesis related (PR) genes. NPRI exerts its effect in this pathway through interaction with a closely related class of bZIP transcription factors known as TGA factors, which are named for their recognition of the cognate DNA motif TGACG. We have discovered that one of these transcription factors, TGA2, behaves as a repressor in unchallenged Arabidopsis and acts to repress NPRI-dependent activation of PRJ. TGA1, which bears moderate sequence similarity to TGA2, acts as a transcriptional activator in unchallenged Arabidopsis, however the significance of this activity is J unclear. Once SAR has been induced, TGAI and TGA2 interact with NPRI to form complexes that are capable of activating transcription. Curiously, although TGAI is capable of transactivating, the ability of the TGAI-NPRI complex to activate transcription results from a novel transactivation domain in NPRI. This transactivation domain, which depends on the oxidation of cysteines 521 and 529, is also responsible for the transactivation ability of the TGA2-NPRI complex. Although the exact mechanism preventing TGA2-NPRI interaction in unchallenged Arabidopsis is unclear, the regulation of TGAI-NPRI interaction is based on the redox status of cysteines 260 and 266 in TGAl. We determined that a glutaredoxin, which is an enzyme capable of regulating a protein's redox status, interacts with the reduced form of TGAI and this interaction results .in the glutathionylation of TGAI and a loss of interaction with NPRl. Taken together, these results expand our understanding of how TGA transcription factors and NPRI behave to regulate events and gene expression during SAR. Furthermore, the regulation of the behavior of both TGAI and NPRI by their redox status and the involvement of a glutaredoxin in modulating TGAI-NPRI interaction suggests the redox regulation of proteins is a general mechanism implemented in SAR.

The Molecular Consequences of CK2-mediated Phosphorylation of the TGA2 Transcription Factor within Systemic Acquired Resistance of Arabidopsis thaliana

During infection, the model plant Arabidopsis thaliana is capable of activating long lasting defence responses both in tissue directly affected by the pathogen and in more distal tissue. Systemic acquired resistance (SAR) is a type of systemic defence response deployed against biotrophic pathogens resulting in altered plant gene expression and production of antimicrobial compounds. One such gene involved in plant defence is called pathogenesis-related 1 (PR1) and is under the control of several protein regulators. TGA II-clade transcription factors (namely TGA2) repress PR1 activity prior to infection by forming large oligomeric complexes effectively blocking gene transcription. After pathogen detection, these complexes are dispersed by a mechanism unknown until now and free TGA molecules interact with the non-expressor of pathogenesis-related gene 1 (NPR1) protein forming an activating complex enabling PR1 transcription. This study elucidates the TGA2 dissociation mechanism by introducing protein kinase CK2 into this process. This enzyme efficiently phosphorylates TGA2 resulting in two crucial events. Firstly, the DNA-binding ability of this transcription factor is completely abolished explaining how the large TGA2 complexes are quickly evicted from the PR1 promoter. Secondly, a portion of TGA2 molecules dissociate from the complexes after phosphorylation which likely makes them available for the formation of the TGA2-NPR1 activating complex. We also show that phosphorylation of a multiserine motif found within TGA2’s N terminus is responsible for the change of affinity to DNA, while modification of a single threonine in the leucine zipper domain seems to be responsible for deoligomerization. Despite the substantial changes caused by phosphorylation, TGA2 is still capable of interacting with NPR1 and these proteins together form a complex on DNA promoting PR1 transcription. Therefore, we propose a change in the current model of how PR1 is regulated by adding CK2 which targets TGA2 displacing it’s complexes from the promoter and providing solitary TGA2 molecules for assembly of the activating complex. Amino acid sequences of regions targeted by CK2 in Arabidopsis TGA2 are similar to those found in TGA2 homologs in rice and tobacco. Therefore, the molecular mechanism that we have identified may be conserved among various plants, including important crop species, adding to the significance of our findings.