Comparative hormonal and metabolic profile analysis based on mass spectrometry provides information on the regulation of water-deficit stress response of sunflower (Helianthus annuus L.) inbred lines with different water-deficit stress sensitivity.

Water-deficit stress is the most important abiotic stress restricting plant growth, development and yield. The effects of this stress, however, depend on genotypes, among other factors. This study assembles morpho-physiological and metabolic approaches to assess hormonal and metabolic profile changes, upon water-deficit stress, in the shoot and roots of two contrasting sunflower inbred lines, B59 (water-deficit stress sensitive) and B71 (water-deficit stress tolerant). The analyses were carried out using mass spectrometry and performing a multivariate statistical analysis to identify relationships between the analyzed variables. Water-deficit stress reduced all morpho-physiological parameters, except for root length in the tolerant inbred line. The hormonal pathways were active in mediating the seedling performance to imposed water-deficit stress in both lines, although with some differences between lines at the organ level. B59 displayed a diverse metabolite battery, including organic acids, organic compounds as well as sugars, mainly in the shoot, whereas B71 showed primary amino acids, organic acids and organic compounds predominantly in its roots. The discrimination between control and water-deficit stress conditions was possible thanks to potential biomarkers of stress treatment, e.g., proline, maleic acid and malonic acid. This study indicated that the studied organs of sunflower seedlings have different mechanisms of regulation under water-deficit stress. These findings could help to better understand the physio-biochemical pathways underlying stress tolerance in sunflower at early-growth stage.

TuMV triggers stomatal closure but reduces drought tolerance in Arabidopsis.

Compatible plant viral infections are a common cause of agricultural losses worldwide. Characterization of the physiological responses controlling plant water management under combined stresses is of great interest in the current climate change scenario. We studied the outcome of TuMV infection on stomatal closure and water balance, hormonal balance and drought tolerance in Arabidopsis. TuMV infection reduced stomatal aperture concomitantly with diminished gas exchange rate, daily water consumption and rosette initial dehydration rate. Infected plants overaccumulated salicylic acid and abscisic acid and showed altered expression levels of key ABA homeostasis genes including biosynthesis and catabolism. Also the expression of ABA signalling gene ABI2 was induced and ABCG40 (which imports ABA into guard cells) was highly induced upon infection. Hypermorfic abi2-1 mutant plants, but no other ABA or SA biosynthetic, signalling or degradation mutants tested abolished both stomatal closure and low stomatal conductance phenotypes caused by TuMV. Notwithstanding lower relative water loss during infection, plants simultaneously subjected to drought and viral stresses showed higher mortality rates than mock-inoculated drought stressed controls, alongside downregulation of drought-responsive gene RD29A. Our findings indicate that despite stomatal closure triggered by TuMV, additional phenomena diminish drought tolerance upon infection.

Biochemical and molecular approach of oxidative damage triggered by water stress and rewatering in sunflower seedlings of two inbred lines with different ability to tolerate water stress.

Water stress accelerates the generation of reactive oxygen species, which trigger a cascade of antioxidative defence mechanisms comprising enzymatic and nonenzymatic antioxidants. The aim of this study was to investigate the oxidative damage and the antioxidative defence systems in seedlings of the water stress-tolerant (B71) and the sensitive (B59) inbred lines of sunflower (Helianthus annuus L.) in response to water stress and rewatering. In addition, we characterised the transcriptomic profile associated with enzymatic antioxidative defence. An elevated electrolyte leakage in B59 indicated increased plasmatic membrane permeability, which correlated with greater sensitivity to water stress. In response to water stress, both lines showed an increase in malondialdehyde and H2O2 content but these increases were more noticeable in the sensitive line. In both lines, an increase in enzymatic activity (e.g. peroxidase and ascorbate peroxidase) was not sufficient to overcome the H2O2 accumulation triggered by water stress. Upon water stress, the overall expression level of genes associated with the enzymatic antioxidant system increased in B71 and decreased in B59, which showed downregulated levels of most genes in the shoots. The general profile of phenolic compounds was clearly different between organs and between inbred lines. The B59 line activated nonenzymatic antioxidant responses to counteract the oxidative stress caused by water stress. The tolerance of B71 to water stress could be associated with compensatory antioxidant mechanisms based on the expression of genes encoding enzyme components of the ascorbate-glutathione and redoxin cycles, which contributed to explaining, at least partly, the response of this line.

Spatiotemporal variations in salicylic acid and hydrogen peroxide in sunflower seeds during transition from dormancy to germination.

Phytohormones and reactive oxygen species mediate processes such as germination and dormancy. The elucidation of the physiological and biochemical events implicated in the transition from dormancy to germination in different plant species such as sunflower becomes a topic of interest. In this study, we investigated the spatiotemporal variation of salicylic acid (SA), hydrogen peroxide (H2 O2 ) and the activity of two antioxidant enzymes (catalase, CAT - EC 1.11.1.6 and ascorbate peroxidase - EC 1.11.1.11) in embryonic axis and cotyledons of dry and imbibed seeds of dormant (B123) and non-dormant (B91) sunflower lines. The results showed that embryonic axis had higher level of SA and H2 O2 than cotyledons in both lines. In dry seeds, B123 embryo (embryonic axis + cotyledons) showed a higher SA content than B91. After dry storage at room temperature, SA decreased in B123 embryos to a value close to that registered in B91 embryos. B123 embryonic axis of dry seeds presented higher H2 O2 levels than B91. Dry storage led to an increase of H2 O2 levels and a decrease of CAT activity in B123 embryonic axis. During imbibition, B123 seeds stored for 33 days displayed an increase in SA level in the embryonic axis (3 h of imbibition) and this lower level correlated with a decrease in H2 O2 (6 h of imbibition). Thus, the embryo-imposed dormancy in B123 dry seeds was associated with high levels of SA and low H2 O2 , whereas the dormancy release was linked with SA decrease and increase of H2 O2 as a consequence of lower CAT activity.

Interactions between soybean, Bradyrhizobium japonicum and Soybean mosaic virus: the effects depend on the interaction sequence.

The symbiotic interaction between soybean and nitrogen-fixing rhizobia can lead to plant growth promotion and induced systemic responses. Symbiotic interactions may increase tolerance/resistance to abiotic/biotic stress conditions, but are also sensitive to environmental conditions. Soybean mosaic virus (SMV), which is transmitted by seed and aphids, severely affects crop yields in many areas of the world, consequently virus infection may precede rhizobium infection or vice versa in the field. With the hypothesis that sequence of interaction is a key determinant of the resulting responses; growth, primary metabolism and defence responses were evaluated in different interaction sequences. Results showed that vegetative growth was promoted by Bradyrhizobium japonicum (Bj) inoculation and drastically impaired by SMV infection. The negative effect of SMV single infection on soybean growth parameters was correlated with photosynthesis decrease, sugar accumulation, oxidative damage, and increases in salicylic acid levels. Bj inoculation partially reversed virus-induced symptoms, mainly at Bj-SMV sequence. However, this symptom attenuation did not correlate with less virus accumulation. Nodulation was negatively affected by SMV, particularly when virus infection was previous to Bj inoculation (SMV-Bj). Defence related hormones (salicylic acid (SA)/jasmonic acid (JA)) and the expression of defence-related genes were dependent on the sequence of tripartite interaction. The present study showed that the sequence of the tripartite interaction among soybean, Bj and SMV determinates the tolerance/susceptibility to SMV infection, through changes in the defence mechanism and metabolic alteration.

The mitochondrial oxidation resistance protein AtOXR2 increases plant biomass and tolerance to oxidative stress.

This study demonstrates the existence of the oxidation resistance (OXR) protein family in plants. There are six OXR members in Arabidopsis that contain the highly conserved TLDc domain that is characteristic of this eukaryotic protein family. AtOXR2 is a mitochondrial protein able to alleviate the stress sensitivity of a yeast oxr1 mutant. It was induced by oxidative stress and its overexpression in Arabidopsis (oeOXR2) increased leaf ascorbate, photosynthesis, biomass, and seed production, as well as conferring tolerance to methyl viologen, antimycin A, and high light intensities. The oeOXR2 plants also showed higher ABA content, changes in ABA sensitivity, and modified expression of ABA- and stress-regulated genes. While the oxr2 mutants had a similar shoot phenotype to the wild-type, they exhibited increased sensitivity to stress. We propose that by influencing the levels of reactive oxygen species (ROS), AtOXR2 improves the efficiency of photosynthesis and elicits basal tolerance to environmental challenges that increase oxidative stress, allowing improved plant growth and biomass production.

Resistance to citrus canker induced by a variant of Xanthomonas citri ssp. citri is associated with a hypersensitive cell death response involving autophagy-associated vacuolar processes.

Xanthomonas citri ssp. citri (X. citri) is the causal agent of Asiatic citrus canker, a disease that seriously affects most commercially important Citrus species worldwide. We have identified previously a natural variant, X. citri AT , that triggers a host-specific defence response in Citrus limon. However, the mechanisms involved in this canker disease resistance are unknown. In this work, the defence response induced by X. citri AT was assessed by transcriptomic, physiological and ultrastructural analyses, and the effects on bacterial biofilm formation were monitored in parallel. We show that X. citri AT triggers a hypersensitive response associated with the interference of biofilm development and arrest of bacterial growth in C. limon. This plant response involves an extensive transcriptional reprogramming, setting in motion cell wall reinforcement, the oxidative burst and the accumulation of salicylic acid (SA) and phenolic compounds. Ultrastructural analyses revealed subcellular changes involving the activation of autophagy-associated vacuolar processes. Our findings show the activation of SA-dependent defence in response to X. citri AT and suggest a coordinated regulation between the SA and flavonoid pathways, which is associated with autophagy mechanisms that control pathogen invasion in C. limon. Furthermore, this defence response protects C. limon plants from disease on subsequent challenges by pathogenic X. citri. This knowledge will allow the rational exploitation of the plant immune system as a biotechnological approach for the management of the disease.

Differential hormonal and gene expression dynamics in two inbred sunflower lines with contrasting dormancy level.

Seed germination and dormancy are tightly regulated by hormone metabolism and signaling pathway. We investigated the endogenous content of abscisic acid (ABA), its catabolites, and gibberellins (GAs), as well as the expression level of certain ABA and GAs metabolic and signaling genes in embryo of dry and imbibed cypselas of inbred sunflower (Helianthus annuus L., Asteraceae) lines: B123 (dormant) and B91 (non-dormant). Under our experimental conditions, the expression of RGL2 gene might be related to the ABA peak in B123 line at 3 h of imbibition. Indeed, RGL2 transcripts are absent in dry and early embedded cypselas of the non-dormant line B91. ABA increase was accompanied by a significant ABA-Glucosyl ester (ABA-GE) and phaseic acid (PA) (two ABA catabolites) decrease in B123 line (3 h) which indicates that ABA metabolism seems to be more active in this line, and that it would be involved in the imposition and maintenance of sunflower seed dormancy, as it has been reported for many species. Finally, an increase of bioactive GAs (GA1 and GA3) occurs at 12 h of imbibition in both lines after a decrease in ABA content. This study shows the first report about the RGL2 tissue-specific gene expression in sunflower inbred lines with contrasting dormancy level. Furthermore, our results provide evidence that ABA and GAs content and differential expression of metabolism and signaling genes would be interacting in seed dormancy regulation through a mechanism of action related to embryo itself.

Early perception of stink bug damage in developing seeds of field-grown soybean induces chemical defences and reduces bug attack.

BACKGROUND: Southern green stink bugs (Nezara viridula L.) invade field-grown soybean crops, where they feed on developing seeds and inject phytotoxic saliva, which causes yield reduction. Although leaf responses to herbivory are well studied, no information is available about the regulation of defences in seeds. RESULTS: This study demonstrated that mitogen-activated protein kinases MPK3, MPK4 and MPK6 are expressed and activated in developing seeds of field-grown soybean and regulate a defensive response after stink bug damage. Although 10-20 min after stink bug feeding on seeds induced the expression of MPK3, MPK6 and MPK4, only MPK6 was phosphorylated after damage. Herbivory induced an early peak of jasmonic acid (JA) accumulation and ethylene (ET) emission after 3 h in developing seeds, whereas salicylic acid (SA) was also induced early, and at increasing levels up to 72 h after damage. Damaged seeds upregulated defensive genes typically modulated by JA/ET or SA, which in turn reduced the activity of digestive enzymes in the gut of stink bugs. Induced seeds were less preferred by stink bugs. CONCLUSION: This study shows that stink bug damage induces seed defences, which is perceived early by MPKs that may activate defence metabolic pathways in developing seeds of field-grown soybean. © 2015 Society of Chemical Industry.

TCP15 modulates cytokinin and auxin responses during gynoecium development in Arabidopsis.

We studied the role of Arabidopsis thaliana TCP15, a member of the TEOSINTE BRANCHED1-CYCLOIDEA-PCF (TCP) transcription factor family, in gynoecium development. Plants that express TCP15 from the 35S CaMV promoter (35S:TCP15) develop flowers with defects in carpel fusion and a reduced number of stigmatic papillae. In contrast, the expression of TCP15 fused to a repressor domain from its own promoter causes the development of outgrowths topped with stigmatic papillae from the replum. 35S:TCP15 plants show lower levels of the auxin indoleacetic acid and reduced expression of the auxin reporter DR5 and the auxin biosynthesis genes YUCCA1 and YUCCA4, suggesting that TCP15 is a repressor of auxin biosynthesis. Treatment of plants with cytokinin enhances the developmental effects of expressing TCP15 or its repressor form. In addition, treatment of a knock-out double mutant in TCP15 and the related gene TCP14 with cytokinin causes replum enlargement, increased development of outgrowths, and the induction of the auxin biosynthesis genes YUCCA1 and YUCCA4. A comparison of the phenotypes observed after cytokinin treatment of plants with altered expression levels of TCP15 and auxin biosynthesis genes suggests that TCP15 modulates gynoecium development by influencing auxin homeostasis. We propose that the correct development of the different tissues of the gynoecium requires a balance between auxin levels and cytokinin responses, and that TCP15 participates in a feedback loop that helps to adjust this balance.

Endophytic bacteria improve seedling growth of sunflower under water stress, produce salicylic acid, and inhibit growth of pathogenic fungi.

Endophytic bacterial strains SF2 (99.9% homology with Achromobacter xylosoxidans), and SF3 and SF4 (99.9% homology with Bacillus pumilus) isolated from sunflower grown under irrigation or drought were selected on the basis of plant growth-promoting bacteria (PGPB) characteristics. Aims of the study were to examine effects of inoculation with SF2, SF3, and SF4 on sunflower cultivated under water stress, to evaluate salicylic acid (SA) production by these strains in control medium or at Ψa = -2.03 MPa, and to analyze effects of exogenously applied SA, jasmonic acid (JA), bacterial pellets, and bacterial supernatants on growth of pathogenic fungi Alternaria sp., Sclerotinia sp., and Verticillum sp. Growth response to bacterial inoculation was studied in two inbred lines (water stress-sensitive B59 and water stress-tolerant B71) and commercial hybrid Paraiso 24. Under both water stress and normal conditions, plant growth following inoculation was more strongly enhanced for Paraiso 24 and B71 than for B59. All three strains produced SA in control medium; levels for SF3 and SF4 were higher than for SF2. SA production was dramatically higher at Ψa = -2.03 MPa. Exogenously applied SA or JA caused a significant reduction of growth for Sclerotinia and a lesser reduction for Alternaria and Verticillum. Fungal growth was more strongly inhibited by bacterial pellets than by bacterial supernatants. Our findings indicate that these endophytic bacteria enhance growth of sunflower seedlings under water stress, produce SA, and inhibit growth of pathogenic fungi. These characteristics are useful for formulation of inoculants to improve growth and yield of sunflower crops.