Phospholipase PLA(1)-I gamma 2 or otherwise DAD1-LIKE LIPASE 3 (DALL3) is a member of class I phospholipases and has a role in JA biosynthesis. AtDALL3 was previously identified in a yeast two... Show morePhospholipase PLA(1)-I gamma 2 or otherwise DAD1-LIKE LIPASE 3 (DALL3) is a member of class I phospholipases and has a role in JA biosynthesis. AtDALL3 was previously identified in a yeast two-hybrid screening as an interacting protein of the Arabidopsis Selenium Binding Protein 1 (SBP1). In this work, we have studied AtDALL3 as an interacting partner of the Arabidopsis Selenium Binding Protein 1 (SBP1). Phylogenetic analysis showed that DALL3 appears in the PLA1-Igamma1, 2 group, paired with PLA1-Igammmal. The highest level of expression of AtDALL3 was observed in 10-day-old roots and in flowers, while constitutive levels were maintained in seedlings, cotyledons, shoots and leaves. In response to abiotic stress, DALL3 was shown to participate in the network of genes regulated by cadmium, selenite and selenate compounds. DALL3 promoter driven GUS assays revealed that the expression patterns defined were overlapping with the patterns reported for AtSBP1 gene, indicating that DALL3 and SBP1 transcripts co-localize. Furthermore, quantitative GUS assays showed that these compounds elicited changes in activity in specific cells files, indicating the differential response of DALL3 promoter. GFP::DALL3 studies by confocal microscopy demonstrated the localization of DALL3 in the plastids of the root apex, the plastids of the central root and the apex of emerging lateral root primordia. Additionally, we confirmed by yeast two hybrid assays the physical interaction of DALL3 with SBP1 and defined a minimal SBP1 fragment that DALL3 binds to. Finally, by employing bimolecular fluorescent complementation we demonstrated the in planta interaction of the two proteins. Show less
During abiotic stress the primary symptom of phytotoxicity can be ROS production which is strictly regulated by ROS scavenging pathways involving enzymatic and non-enzymatic antioxidants.... Show moreDuring abiotic stress the primary symptom of phytotoxicity can be ROS production which is strictly regulated by ROS scavenging pathways involving enzymatic and non-enzymatic antioxidants. Furthermore, ROS are well described secondary messengers of cellular processes, while during the course of evolution, plants have accomplished high degree of control over ROS and used them as signalling molecules. Glutaredoxins (GRXs) are small and ubiquitous glutathione (GSH) -or thioredoxin reductase (TR)-dependent oxidoreductases belonging to the thioredoxin (TRX) superfamily which are conserved in most eukaryotes and prokaryotes. In Arabidopsis thaliana GRXs are subdivided into four classes playing a central role in oxidative stress responses and physiological functions. In this work, we describe a novel interaction of AtGRXS14 with the Selenium Binding Protein 1 (AtSBP1), a protein proposed to be integrated in a regulatory network that senses alterations in cellular redox state and acts towards its restoration. We further show that SBP protein family interacts with AtGRXS16 that also contains a PICOT domain, like AtGRXS14. Show less
KNOTTED1-LIKE HOMEOBOX (KNOX) genes are important regulators of meristem function, and a complex network of transcription factors ensures tight control of their expression. Here, we show that... Show moreKNOTTED1-LIKE HOMEOBOX (KNOX) genes are important regulators of meristem function, and a complex network of transcription factors ensures tight control of their expression. Here, we show that members of the GROWTH-REGULATING FACTOR (GRF) family act as players in this network. A yeast (Saccharomyces cerevisiae) one-hybrid screen with the upstream sequence of the KNOX gene Oskn2 from rice (Oryza sativa) resulted in isolation of OsGRF3 and OsGRF10. Specific binding to a region in the untranslated leader sequence of Oskn2 was confirmed by yeast and in vitro binding assays. ProOskn2:β-glucuronidase reporter expression was down-regulated by OsGRF3 and OsGRF10 in vivo, suggesting that these proteins function as transcriptional repressors. Likewise, we found that the GRF protein BGRF1 from barley (Hordeum vulgare) could act as a repressor on an intron sequence in the KNOX gene Hooded/Barley Knotted3 (Bkn3) and that AtGRF4, AtGRF5, and AtGRF6 from Arabidopsis (Arabidopsis thaliana) could repress KNOTTED-LIKE FROM ARABIDOPSIS THALIANA2 (KNAT2) promoter activity. OsGRF overexpression phenotypes in rice were consistent with aberrant meristematic activity, showing reduced formation of tillers and internodes and extensive adventitious root/shoot formation on nodes. These effects were associated with down-regulation of endogenous Oskn2 expression by OsGRF3. Conversely, RNA interference silencing of OsGRF3, OsGRF4, and OsGRF5 resulted in dwarfism, delayed growth and inflorescence formation, and up-regulation of Oskn2. These data demonstrate conserved interactions between the GRF and KNOX families of transcription factors in both monocot and dicot plants. Show less
The metabolic role and regulation of selenium, particularly in plants, is poorly understood. One of the proteins probably involved in the metabolic regulation of this element is the selenium... Show moreThe metabolic role and regulation of selenium, particularly in plants, is poorly understood. One of the proteins probably involved in the metabolic regulation of this element is the selenium-binding protein (SBP) with homologues present across prokaryotic and eukaryotic species. The high degree of conservation of SBP in different organisms suggests that this protein may play a role in fundamental biological processes. In order to gain insight into the biochemical function of SBP in plants we used the yeast two-hybrid system to identify proteins that potentially interact with an Arabidopsis thaliana (L.) Heynh. homologue. Among the putative binding partners of SBP, a NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a fructose-bisphosphate aldolase (FBA) were found as reliable positive candidates. The interaction of these proteins with SBP was confirmed by in vitro binding assays. Previous findings in Escherichia coli, demonstrated the direct binding of selenium to both GAPDH and aldolase. Therefore our results reveal the interaction, at least in pairs, of three proteins that are possibly linked to selenium and suggest the existence of a protein network consisting of at least SBP, GAPDH and FBA, triggered by or regulating selenium metabolism in plant cells. Show less
In the Arabidopsis genome there are three highly conserved homologues of the mammalian 56-kD selenium-binding protein (SBP). To study the function of SBP in this model plant, we used a transgenic... Show moreIn the Arabidopsis genome there are three highly conserved homologues of the mammalian 56-kD selenium-binding protein (SBP). To study the function of SBP in this model plant, we used a transgenic approach by constitutively overexpressing and down-regulating the endogenous Atsbp1 gene. In the latter case, we employed both a conventional antisense method and gene silencing by intron-containing hairpin RNAs. Atsbp1-overexpressing and silenced plants were phenotypically normal, under standard growth conditions, when compared with wild type plants. Transgenic plants exhibited different growth responses to exogenously supplied selenite, which correlated with the expression levels of Atsbp1. Plants with increased Atsbp1 transcript levels showed enhanced tolerance to selenite, while plants with reduced levels were more sensitive. Our results indicate that, although Atsbp1 does not play a detectable role in the regulation of developmental processes under normal growth conditions, it appears to be involved in processes controlling tolerance of Arabidopsis to selenium toxicity. Show less
Immunolocalisation and in situ hybridisation allow the detection of proteins and RNA respectively, in individual cells of different tissue types. Hybridisation on serial 5–8 μm sections is most... Show moreImmunolocalisation and in situ hybridisation allow the detection of proteins and RNA respectively, in individual cells of different tissue types. Hybridisation on serial 5–8 μm sections is most frequently performed with tissue embedded in paraffin wax but it is often difficult to obtain high-resolution sections from soft tissue with this embedding process. To overcome this, alternative localisation protocols have been developed utilising plastic resins. We have used plastic embedded tissue from Lotus japonicus roots and young nodules successfully in immunolocalisation experiments and developed a protocol that can also be adapted for in situ RNA localisation studies. The different parameters tested are described, as well as the use of alkaline phosphatase- or fluorescently-conjugated secondary antibodies. Show less
Flemetakis, E.; Agalou, A.; Kavroulakis, N.; Dimou, M.; Martsikovskaya, A.; Slater, A.; ... ; Katinakis, P. 2002
We have isolated and characterized a Lotus japonicus gene (Ljsbp) encoding a putative polypeptide with striking homology to the mammalian 56-kDa selenium-binding protein (SBP). cDNA clones... Show moreWe have isolated and characterized a Lotus japonicus gene (Ljsbp) encoding a putative polypeptide with striking homology to the mammalian 56-kDa selenium-binding protein (SBP). cDNA clones homologous to LjSBP were also isolated from soybean, Medicago sativa, and Arabidopsis thaliana. Comparative expression studies in L japonicus and A. thaliana showed that sbp transcripts are present in various tissues and at different levels. Especially in L. japonicus nodules and seedpods and A. thaliana siliques, sbp expression appears to be developmentally up-regulated. sbp Gene transcripts were localized by in situ hybridization in the infected cells and vascular bundles of young nodules, while in mature nodules, low levels of expression were only detected in the parenchymatous cells. Expression of sbp transcripts in young seedpods and siliques was clearly visible in vascular tissues and embryos, while in embryos, low levels of expression were detected in the root epidermis and the vascular bundles. Polyclonal antibodies raised against a truncated LjSBP recombinant protein recognized a polypeptide of about 60 kDa in nodule extracts. Immunohistochemical experiments showed that accumulation of LjSBP occurred in root hairs, in the root epidermis above the nodule primordium, in the phloem of the vasculature, and abundantly in the infected cells of young nodules. Irrespective of the presence of rhizobia, expression of SBP was also observed in root tips, where it was confined in the root epidermis and protophloem cells. We hypothesize that LjSBP may have more than one physiological role and can be implicated in controlling the oxidation/reduction status of target proteins, in vesicular Golgi transport, or both. Show less
