S-Nitrosoglutathione is a component of wound- and salicylic acid-induced systemic responses in Arabidopsis thaliana.

S-Nitrosoglutathione (GSNO) is a bioactive, stable, and mobile reservoir of nitric oxide (NO), and an important player in defence responses to herbivory and pathogen attack in plants. It has been demonstrated previously that GSNO reductase (GSNOR) is the main enzyme responsible for the in vivo control of intracellular levels of GSNO. In this study, the role of S-nitrosothiols, in particular of GSNO, in systemic defence responses in Arabidopsis thaliana was investigated further. It was shown that GSNO levels increased rapidly and uniformly in injured Arabidopsis leaves, whereas in systemic leaves GSNO was first detected in vascular tissues and later spread over the parenchyma, suggesting that GSNO is involved in the transmission of the wound mobile signal through the vascular tissue. Moreover, GSNO accumulation was required to activate the jasmonic acid (JA)-dependent wound responses, whereas the alternative JA-independent wound-signalling pathway did not involve GSNO. Furthermore, extending previous work on the role of GSNOR in pathogenesis, it was shown that GSNO acts synergistically with salicylic acid in systemic acquired resistance activation. In conclusion, GSNOR appears to be a key regulator of systemic defence responses, in both wounding and pathogenesis.

A role for protein kinase CK2 in plant development: evidence obtained using a dominant-negative mutant.

Protein kinase CK2 is an evolutionary conserved Ser/Thr phosphotransferase composed of two distinct subunits, alpha (catalytic) and beta (regulatory), that combine to form a tetrameric complex. Plant genomes contain multiple genes for each subunit, the expression of which gives rise to different active holoenzymes. In order to study the effects of loss of function of CK2 on plant development, we have undertaken a dominant-negative mutant approach. We generated an inactive catalytic subunit by site-directed mutagenesis of an essential lysine residue. The mutated open reading frame was cloned downstream of an inducible promoter, and stably transformed Arabidopsis thaliana plants and tobacco BY2 cells were isolated. Continuous expression of the CK2 kinase-inactive subunit did not prevent seed germination, but seedlings exhibited a strong phenotype, affecting chloroplast development, cotyledon expansion, and root and shoot growth. Prolonged induction of the transgene was lethal. Moreover, dark-germinated seedlings exhibited an apparent de-etiolated phenotype that was not caused by disruption of the light-signalling pathways. Short-term induction of the CK2 kinase-inactive subunit allowed plant survival, but root growth and lateral root formation were significantly affected. The expression pattern of CYCB1;1::GFP in the root meristems of mutant plants demonstrated an important decrease of mitotic activity, and expression of the CK2 kinase-inactive subunit in stably transformed BY2 cells provoked perturbation of the G1/S and G2 phases of the cell cycle. Our results are consistent with a model in which CK2 plays a key role in cell division and cell expansion, with compelling effects on Arabidopsis development.

In situ hybridization analysis of protein kinase CK2 expression during plant development.

Protein kinase CK2 (EC 2.7.1.37) is a ubiquitous Ser/Thr kinase composed of two subunits, a subunit (catalytic) and a subunit (regulatory). Although CK2 is one of the components of the network that controls the eukaryotic cell cycle, very little is known about the developmental control of its expression, particularly in plants. Using in situ hybridization we have determined the pattern of expression of its two subunits in relation to plant morphogenesis. Our results show a cell-specific mRNA accumulation, equivalent for both subunits, which suggests a co-ordinate regulation during development. The spatial and temporal pattern of expression correlates with the appearance of the meristems, and high levels of transcripts are also present in differentiated tissues with high mitotic activity. These results confirm the transcriptional regulation of CK2 and establish a general correlation with cell proliferation in a developmental context. They also provide suggestive evidence for the requirement of CK2 expression for the initiation and organization of meristem activity.

S-nitrosoglutathione reductase affords protection against pathogens in Arabidopsis, both locally and systemically.

Nitric oxide and S-nitrosothiols (SNOs) are widespread signaling molecules that regulate immunity in animals and plants. Levels of SNOs in vivo are controlled by nitric oxide synthesis (which in plants is achieved by different routes) and by S-nitrosoglutathione turnover, which is mainly performed by the S-nitrosoglutathione reductase (GSNOR). GSNOR is encoded by a single-copy gene in Arabidopsis (Arabidopsis thaliana; Martínez et al., 1996; Sakamoto et al., 2002). We report here that transgenic plants with decreased amounts of GSNOR (using antisense strategy) show enhanced basal resistance against Peronospora parasitica Noco2 (oomycete), which correlates with higher levels of intracellular SNOs and constitutive activation of the pathogenesis-related gene, PR-1. Moreover, systemic acquired resistance is impaired in plants overexpressing GSNOR and enhanced in the antisense plants, and this correlates with changes in the SNO content both in local and systemic leaves. We also show that GSNOR is localized in the phloem and, thus, could regulate systemic acquired resistance signal transport through the vascular system. Our data corroborate the data from other authors that GSNOR controls SNO in vivo levels, and shows that SNO content positively influences plant basal resistance and resistance-gene-mediated resistance as well. These data highlight GSNOR as an important and widely utilized component of resistance protein signaling networks conserved in animals and plants.

Modification of intracellular levels of glutathione-dependent formaldehyde dehydrogenase alters glutathione homeostasis and root development.

Glutathione (GSH)-dependent formaldehyde dehydrogenase (FALDH) is a highly conserved medium-chain dehydrogenase reductase and the main enzyme that metabolizes intracellular formaldehyde in eukaryotes. It has been recently shown that it exhibits a strong S-nitrosoglutathione (GSNO) reductase activity and could be a candidate to regulate NO-signalling functions. However, there is a lack of knowledge about the tissue distribution of this enzyme in plants. Here, we have studied the localization and developmental expression of the enzyme using immunolocalization and histochemical activity assay methods. We conclude that FALDH is differentially expressed in the organs of Arabidopsis thaliana mature plants, with higher levels in roots and leaves from the first stages of development. Spatial distribution of FALDH in these two organs includes the main cell types [epidermis (Ep) and cortex (Cx) in roots, and mesophyll in leaves] and the vascular system. Arabidopsis thaliana mutants with modified levels of FALDH (both by over- and under-expression of the FALDH-encoding gene) show a significant reduction of root length, and this phenotype correlates with an overall decrease of intracellular GSH levels and alteration of spatial distribution of GSH in the root meristem. Tansgenic roots are partially insensitive to exogenous GSH, suggesting an inability to detect reduction-oxidation (redox) changes of the GSH pool and/or maintain GSH homeostasis.

Differential expression of genes encoding protein kinase CK2 subunits in the plant cell cycle.

Protein kinase CK2 is a ubiquitous Ser/Thr/Tyr kinase essential for cell viability in eukaryotes. It comprises alpha catalytic and beta regulatory subunits, which combine to form the classical tetrameric structure, alpha2beta2. Although CK2 is a component of the network that controls the eukaryotic cell cycle, very little is known about the expression patterns of genes encoding its constituent subunits, especially in plants. A study of the complexity of CK2alpha- and CK2beta-encoding genes in BY-2 cells was undertaken in this work, and cloning of the different members of the gene families was performed. The expression of the individual members of each family in relation to cell proliferation was measured by real time RT-PCR. The data obtained provide an accurate understanding of the transcriptional regulation of CK2 in relation to the cell cycle and cell proliferation.