Raúl Cassia

An increase in the concentration of abscisic acid is critical for nitric oxide-mediated plant adaptive responses to UV-B irradiation

Here, the link between UV-B stimulus and the abscisic acid (ABA)-induced nitricoxide (NO) synthesis pathway was studied in leaves of maize (Zea mays).The ABA concentration increased by 100% in UV-B irradiated leaves. Leaves of viviparous 14 (vp14), a mutant defective in ABA synthesis, were more sensitive to UV-B-induced damage than those of the wild type (wt). ABA supplementation attenuated UV-B-induced damage in both the wt and vp14. The hydrogen peroxide(H2O2) concentration increased in the irradiated wt, but changed only slightly in vp14. This increase was prevented by diphenylene iodonium (DPI), an inhibitor of NADPH oxidase (pNOX).NO was detected using the fluorophore 4,5-diamino-fluorescein diacetate(DAF-2DA). DAF-2DA fluorescence increased twofold in UV-B-irradiated wt leaves but not in vp14 leaves. H2O2 and NO production was restored in vp14 plants supplied with 100 μM ABA. Catalase, DPI and the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) partially blocked UV-B-induced NO accumulation, suggesting that H2O2 as well as NOS-like activity is required for a full plant response to UV-B. NO protects against UV-B-induced cell damage.Our results suggest that UV-B perception triggers an increase in ABA concentration,which activates pNOX and H2O2 generation, and that an NOS-like-dependent mechanism increases NO production to maintain cell homeostasis and attenuate UV-B-derived cell damage.

Accumulation of cytosolic glyceraldehyde-3-phosphate dehydrogenase RNA under biological stress conditions and elicitor treatments in potato

Plants respond to pathogen infection and environmental stress by regulating the coordinate expression of many stress-related genes. In plants, the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced under environmental stress. This work was aimed at investigating whether the expression pattern of cytosolic GAPDH is also modulated upon infection of potato plants (Solanum tuberosum L.) with the late blight fungal agent Phytophthora infestans. Northern blot analysis showed the accumulation of the GAPDH gene transcripts in leaves and stems of inoculated potato plants. When tuber discs were treated with eicosapentaenoic acid (EPA), an elicitor found in P. infestans, GAPDH gene transcripts level increased. This increase was parallel to that of the hydroxymethyl glutharyl coenzyme A reductase (HMGR), an enzyme involved in pathogen defense reactions. Glucans obtained from P. infestans cell wall acts sinergistically with EPA on GAPDH and HMGR gene induction. Salicylic acid, an endogenous signal for inducing systemic acquired resistance, was also effective in stimulating the GAPDH transcript accumulation in potato leaves. These experiments suggest that related multi-component factors, which are part of both primary and secondary metabolism, are probably regulated by similar signal transduction pathways when they are induced under biotic or abiotic stress conditions.

Nitric oxide enhances plant ultraviolet‐B protection up‐regulating gene expression of the phenylpropanoid biosynthetic pathway

The link between ultraviolet (UV)-B, nitric oxide (NO) and phenylpropanoid biosynthetic pathway (PPBP) was studied in maize and Arabidopsis. The transcription factor (TF) ZmP regulates PPBP in maize. A genetic approach using P-rr (ZmP+) and P-ww (ZmP⁻) maize lines demonstrate that: (1) NO protects P-rr leaves but not P-ww from UV-B-induced reactive oxygen species (ROS) and cell damage; (2) NO increases flavonoid and anthocyanin content and prevents chlorophyll loss in P-rr but not in P-ww and (3) the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) blocks the UV-B-induced expression of ZmP and their targets CHS and CHI suggesting that NO plays a key role in the UV-B-regulated PPBP. Involvement of endogenous NO was studied in Arabidopsis nitric oxide dioxygenase (NOD) plants that express a NO dioxygenase gene under the control of a dexamethasone (DEX)-inducible promoter. Expression of HY5 and MYB12, TFs involved in PPBP regulation, was induced by UV-B, reduced by DEX in NOD plants and recovered by subsequent NO treatment. C4H regulates synapate esters synthesis and is UV-B-induced in a NO-independent pathway. Data indicate that UV-B perception increases NO concentration, which protects plant against UV-B by two ways: (1) scavenging ROS; and (2) up-regulating the expression of HY5, MYB12 and ZmP, resulting in the PPBP activation.

ABA says NO to UV-B: a universal response?

Abscisic acid (ABA) signaling pathways have been widely characterized in plants, whereas the function of ABA in animals is less well understood. However, recent advances show ABA production by a wide range of lower animals and higher mammals. This enables a new evaluation of ABA signaling pathways in different organisms in response to common environmental stress, such as ultraviolet (UV)-B. In this opinion article, we propose that the induction of common signaling components, such as ABA, nitric oxide (NO) and Ca(2+), in plant and animal cells in response to high doses of UV-B, suggests that the evolution of a general mechanism activated by UV-B is conserved in divergent multicellular organisms challenged by a changing common environment.

Nitric oxide and flavonoids are systemically induced by UV-B in maize leaves

Flavonoids are UV-B absorbing compounds whose concentration, increase in plant cells stimulated by UV-B irradiation. In this work, we characterized the systemic accumulation of flavonoids in maize seedlings irradiated with 3.3 W m(-2) UV-B. Results indicate that both nitric oxide (NO) and flavonoids are systemically induced in UV-B-irradiated maize seedlings. Maize leaves pre-treated with the specific NO scavenger cPTIO, do not accumulate NO and flavonoids in response to UV-B. Whereas NO and flavonoids are accumulated in the mesophyll cells near to the leaf side receiving the UV-B irradiation, they are distributed in all tissues displaying the systemic response. Flavonoids and NO co-localize in UV-B irradiated maize leaves analyzed by images from epifluorescence microscopy. Chalcone synthase (CHS) and chalcone isomerase (CHI) genes are involved in the flavonoid biosynthetic pathway and their expression is systemically induced by UV-B in a NO dependent pathway. Finally, a functional approach demonstrates that maize leaves expressing the systemic response to UV-B show low cellular damage measured as ion leakage when they are challenged by a second round of irradiation.

Ultraviolet-B-Induced Stomatal Closure in Arabidopsis Is Regulated by the UV RESISTANCE LOCUS8 Photoreceptor in a Nitric Oxide-Dependent Mechanism

The Arabidopsis UV RESISTANCE LOCUS8 (UVR8) photoreceptor increases nitric oxide in response to UV-B, thus promoting stomatal closure. UV RESISTANCE LOCUS8 (UVR8) signaling involves CONSTITUTIVELY PHOTOMORPHOGENIC1, the ELONGATED HYPOCOTYL5 (HY5) transcription factor, and the closely related HY5 HOMOLOG. Some UV-B responses mediated by UVR8 are also regulated by nitric oxide (NO), a bioactive molecule that orchestrates a wide range of processes in plants. In this study, we investigated the participation of the UVR8 pathway and its interaction with NO in UV-B-induced stomatal movements in Arabidopsis (Arabidopsis thaliana). Stomata in abaxial epidermal strips of Arabidopsis ecotype Landsberg erecta closed in response to increasing UV-B fluence rates, with maximal closure after 3-h exposure to 5.46 μmol m–2 s–1 UV-B. Both hydrogen peroxide (H2O2) and NO increased in response to UV-B, and stomatal closure was maintained by NO up to 24 h after the beginning of exposure. Stomata of plants expressing bacterial NO dioxygenase, which prevents NO accumulation, did not close in response to UV-B, although H2O2 still increased. When the uvr8-1 null mutant was exposed to UV-B, stomata remained open, irrespective of the fluence rate. Neither NO nor H2O2 increased in stomata of the uvr8-1 mutant. However, the NO donor S-nitrosoglutathione induced closure of uvr8-1 stomata to the same extent as in the wild type. Experiments with mutants in UVR8 signaling components implicated CONSTITUTIVELY PHOTOMORPHOGENIC1, HY5, and HY5 HOMOLOG in UV-B-induced stomatal closure. This research provides evidence that the UVR8 pathway regulates stomatal closure by a mechanism involving both H2O2 and NO generation in response to UV-B exposure.

S-nitrosylation influences the structure and DNA binding activity of AtMYB30 transcription factor from Arabidopsis thaliana

MYB proteins are a family of transcription factors that play an important role in plant development and regulatory defense processes. Arabidopsis thaliana MYB30 (AtMYB30), a member of this protein family, is involved in cell death processes during the hypersensitive response (HR) of plants. HR is characterized by a vast production of reactive oxygen species (ROS) and nitric oxide (NO). NO may thus influence the binding of AtMYB30 to DNA. In this work we evaluated the effect of NO on AtMYB30 DNA binding activity, and also in the protein structural properties. A fully active minimal DNA-binding domain (DBD) of AtMYB30 (residues 11-116) containing two cysteine residues (C49 and C53) was overexpressed and purified. Site-directed mutagenesis was used to obtain AtMYB30 DBD mutants C49A and C53A. The DNA binding activity of AtMYB30 DBD, and Cys single mutants is clearly inhibited upon incubation with a NO donor, and S-nitrosylation was confirmed by the biotin switch assay. Finally, in order to understand the mechanism of NO effect on AtMYB30 DNA binding activity we performed circular dichroism analysis, to correlate the observed protein function inhibition and a potential structural impairment on AtMYB30 DBD. Indeed, NO modification of C49 and C53 residues promotes a subtle modification on the secondary structure of this transcription factor. We thus demonstrated, using various techniques, the in vitro effect of NO on AtMYB30 DBD, and thus the potential consequences of NO activity on plant metabolism influenced by this transcription factor.

Autocrine abscisic acid mediates the UV-B-induced inflammatory response in human granulocytes and keratinocytes.

UV‐B is an abiotic environmental stress in both plants and animals. Abscisic acid (ABA) is a phytohormone regulating fundamental physiological functions in plants, including response to abiotic stress. We previously demonstrated that ABA is an endogenous stress hormone also in animal cells. Here, we investigated whether autocrine ABA regulates the response to UV‐B of human granulocytes and keratinocytes, the cells involved in UV‐triggered skin inflammation. The intracellular ABA concentration increased in UV‐B‐exposed granulocytes and keratinocytes and ABA was released into the supernatant. The UV‐B‐induced production of NO and of reactive oxygen species (ROS), phagocytosis, and cell migration were strongly inhibited in granulocytes irradiated in the presence of a monoclonal antibody against ABA. Moreover, presence of the same antibody strongly inhibited release of NO, prostaglandin E2 (PGE2), and tumor necrosis factor‐α (TNF‐α) by UV‐B irradiated keratinocytes. Lanthionine synthetase C‐like protein 2 (LANCL2) is required for the activation of the ABA signaling pathway in human granulocytes. Silencing of LANCL2 in human keratinocytes by siRNA was accompanied by abrogation of the UV‐B‐triggered release of PGE2, TNF‐α, and NO and ROS production. These results indicate that UV‐B irradiation induces ABA release from human granulocytes and keratinocytes and that autocrine ABA stimulates cell functions involved in skin inflammation. J. Cell. Physiol. 227: 2502–2510, 2012.

Apocynin-induced nitric oxide production confers antioxidant protection in maize leaves

The effect of apocynin on nitric oxide (NO) synthesis and oxidative stress was studied in corn (Zea mays) seedlings. After treatment with 100 microM apocynin, strongly increased amounts of NO were detected in the leaves. This NO production was reduced by more than 70% by N(G)-nitro-l-arginine methyl ester (L-NAME), a NO synthase (NOS) inhibitor, but there was no reduction in NO production when apocynin was applied in combination with diphenylene iodonium (a plant NOX inhibitor). When maize seedlings were UV-B-irradiated, cellular damage occurred and reactive oxygen species (ROS) were found widely distributed in chloroplasts and mesophyll cells. Pre-treatment with apocynin and coinciding NO accumulation prevented this damage. However, the protective effect was averted by L-NAME application. Leaf discs placed in 1M H(2)O(2) for 24h showed a reduction in chlorophyll content that could also be avoided by apocynin treatment. Our results show that apocynin induces the accumulation of NO in leaves of maize seedlings through a NOS-like activity, a mechanism alternative to NOX inhibition, and confers an augmented tolerance to different types of abiotic oxidative stress. Indeed, we propose the use of apocynin as an alternative approach to study NO functionality in plants.

Involvement of Iron and Ferritin in the Potato–Phytophthora infestans Interaction

This work shows that the infection of potato (Solanum tuberosum) detached leaves by the late blight pathogen Phytophthora infestans, was drastically reduced by adding deferoxamine, an exogenous iron chelator. Reactive oxygen species in leaves inoculated with P. infestans were also reduced after adding deferoxamine. A leaf ferritin cDNA fragment was obtained by PCR and used as probe for screening a tuber cDNA library. A cDNA (named StF1) encoding the iron-storing potato ferritin was cloned. StF1 is 915 bp in length and has an open reading frame of 230 amino acids that contains the information for the mature 28 kDa subunit of potato ferritin. StF1 was used as probe in northern blot hybridizations to analyze expression of the ferritin gene. In leaves, ferritin mRNA accumulated in response to pathogen attack. In tubers, ferritin mRNA increased upon treatment with the elicitor eicosapentaenoic acid. These results suggest that iron plays a role in the potato-P. infestans interaction.