APRF1 Interactome Reveals HSP90 as a New Player in the Complex That Epigenetically Regulates Flowering Time in Arabidopsis thaliana.

WD40 repeat proteins (WDRs) are present in all eukaryotes and include members that are implicated in numerous cellular activities. They act as scaffold proteins and thus as molecular "hubs" for protein-protein interactions, which mediate the assembly of multifunctional complexes that regulate key developmental processes in Arabidopsis thaliana, such as flowering time, hormonal signaling, and stress responses. Despite their importance, many aspects of their putative functions have not been elucidated yet. Here, we show that the late-flowering phenotype of the anthesis promoting factor 1 (aprf1) mutants is temperature-dependent and can be suppressed when plants are grown under mild heat stress conditions. To gain further insight into the mechanism of APRF1 function, we employed a co-immunoprecipitation (Co-IP) approach to identify its interaction partners. We provide the first interactome of APRF1, which includes proteins that are localized in several subcellular compartments and are implicated in diverse cellular functions. The dual nucleocytoplasmic localization of ARRF1, which was validated through the interaction of APRF1 with HEAT SHOCK PROTEIN 1 (HSP90.1) in the nucleus and with HSP90.2 in the cytoplasm, indicates a dynamic and versatile involvement of APRF1 in multiple biological processes. The specific interaction of APRF1 with the chaperon HSP90.1 in the nucleus expands our knowledge regarding the epigenetic regulation of flowering time in A. thaliana and further suggests the existence of a delicate thermoregulated mechanism during anthesis.

Conserved structured domains in plant non-coding RNA enod40, their evolution and recruitment of sequences from transposable elements.

Plant long noncoding RNA enod40 is involved in the regulation of symbiotic associations with bacteria, in particular, in nitrogen-fixing root nodules of legumes, and with fungi in phosphate-acquiring arbuscular mycorrhizae formed by various plants. The presence of enod40 genes in plants that do not form such symbioses indicates its other roles in cell physiology. The molecular mechanisms of enod40 RNA function are poorly understood. Enod40 RNAs form several structured domains, conserved to different extents. Due to relatively low sequence similarity, identification of enod40 sequences in plant genomes is not straightforward, and many enod40 genes remain unannotated even in complete genomes. Here, we used comparative structure analysis and sequence similarity searches in order to locate enod40 genes and determine enod40 RNA structures in nitrogen-fixing clade plants and in grasses. The structures combine conserved features with considerable diversity of structural elements, including insertions of structured domain modules originating from transposable elements. Remarkably, these insertions contain sequences similar to tandem repeats and several stem-loops are homologous to microRNA precursors.

Evolutionary Aspects of Selenium Binding Protein (SBP).

Selenium-binding proteins represent a ubiquitous protein family and recently SBP1 was described as a new stress response regulator in plants. SBP1 has been characterized as a methanethiol oxidase, however its exact role remains unclear. Moreover, in mammals, it is involved in the regulation of anti-carcinogenic growth and progression as well as reduction/oxidation modulation and detoxification. In this work, we delineate the functional potential of certain motifs of SBP in the context of evolutionary relationships. The phylogenetic profiling approach revealed the absence of SBP in the fungi phylum as well as in most non eukaryotic organisms. The phylogenetic tree also indicates the differentiation and evolution of characteristic SBP motifs. Main evolutionary events concern the CSSC motif for which Acidobacteria, Fungi and Archaea carry modifications. Moreover, the CC motif is harbored by some bacteria and remains conserved in Plants, while modified to CxxC in Animals. Thus, the characteristic sequence motifs of SBPs mainly appeared in Archaea and Bacteria and retained in Animals and Plants. Our results demonstrate the emergence of SBP from bacteria and most likely as a methanethiol oxidase.

The SAH7 Homologue of the Allergen Ole e 1 Interacts with the Putative Stress Sensor SBP1 (Selenium-Binding Protein 1) in Arabidopsis thaliana.

In this study, we focused on a member of the Ole e 1 domain-containing family, AtSAH7, in Arabidopsis thaliana. Our lab reports for the first time on this protein, AtSAH7, that was found to interact with Selenium-binding protein 1 (AtSBP1). We studied by GUS assisted promoter deletion analysis the expression pattern of AtSAH7 and determined that the sequence 1420 bp upstream of the transcription start can act as a minimal promoter inducing expression in vasculature tissues. Moreover, mRNA levels of AtSAH7 were acutely increased under selenite treatment in response to oxidative stress. We confirmed the aforementioned interaction in vivo, in silico and in planta. Following a bimolecular fluorescent complementation approach, we determined that the subcellular localization of the AtSAH7 and the AtSAH7/AtSBP1 interaction occur in the ER. Our results indicate the participation of AtSAH7 in a biochemical network regulated by selenite, possibly associated with responses to ROS production.

GUS Reporter-Aided Promoter Deletion Analysis of A. thaliana POLYAMINE OXIDASE 3.

Polyamine oxidases (PAOs) have been correlated with numerous physiological and developmental processes, as well as responses to biotic and abiotic stress conditions. Their transcriptional regulation is driven by signals generated by various developmental and environmental cues, including phytohormones. However, the inductive mechanism(s) of the corresponding genes remains elusive. Out of the five previously characterized Arabidopsis PAO genes, none of their regulatory sequences have been analyzed to date. In this study, a GUS reporter-aided promoter deletion approach was used to investigate the transcriptional regulation of AtPAO3 during normal growth and development as well as under various inductive environments. AtPAO3 contains an upstream open reading frame (uORF) and a short inter-cistronic sequence, while the integrity of both appears to be crucial for the proper regulation of gene expression. The full-length promoter contains several cis-acting elements that regulate the tissue-specific expression of AtPAO3 during normal growth and development. Furthermore, a number of TFBS that are involved in gene induction under various abiotic stress conditions display an additive effect on gene expression. Taken together, our data indicate that the transcription of AtPAO3 is regulated by multiple environmental factors, which probably work alongside hormonal signals and shed light on the fine-tuning mechanisms of PAO regulation.

Selenium-binding Protein 1 (SBD1): A stress response regulator in Chlamydomonas reinhardtii.

Selenium-binding proteins (SBPs) represent a ubiquitous protein family implicated in various environmental stress responses, although the exact molecular and physiological role of the SBP family remains elusive. In this work, we report the identification and characterization of CrSBD1, an SBP homolog from the model microalgae Chlamydomonas reinhardtii. Growth analysis of the C. reinhardtii sbd1 mutant strain revealed that the absence of a functional CrSBD1 resulted in increased growth under mild oxidative stress conditions, although cell viability rapidly declined at higher hydrogen peroxide (H2O2) concentrations. Furthermore, a combined global transcriptomic and metabolomic analysis indicated that the sbd1 mutant exhibited a dramatic quenching of the molecular and biochemical responses upon H2O2-induced oxidative stress when compared to the wild-type. Our results indicate that CrSBD1 represents a cell regulator, which is involved in the modulation of C. reinhardtii early responses to oxidative stress. We assert that CrSBD1 acts as a member of an extensive and conserved protein-protein interaction network including Fructose-bisphosphate aldolase 3, Cysteine endopeptidase 2, and Glutaredoxin 6 proteins, as indicated by yeast two-hybrid assays.

Investigation of the interaction of a papain-like cysteine protease (RD19c) with selenium-binding protein 1 (SBP1) in Arabidopsis thaliana.

AtRD19c is a member of the papain-like cysteine proteases known for its participation in anther development after its maturation by ßVPE (vacuolar processing enzyme). This papain-like cysteine protease was identified as an interacting protein of AtSBP1 (selenium binding protein 1) in a yeast two-hybrid screening. To confirm this interaction, we studied AtRD19c with respect to its expression and ability to interact with AtSBP1. The highest gene expression levels of AtRD19c were observed in the roots of 10-day-old seedlings, whereas minimum levels appeared in the hypocotyls of 10-day-old seedlings and flowers. AtRD19c expression was upregulated by selenium, and analysis of its promoter activity showed colocalization of a reporter gene (GUS) with AtSBP1. Additionally, the AtRD19c expression pattern was upregulated in the presence of selenite, indicating its participation in the Se response network. Confocal fluorescence microscopy revealed that AtRD19c localizes in the root tip, lateral roots, and leaf trichomes. Finally, we confirmed the physical interaction between AtRD19c and AtSBP1 and showed the importance of the first 175 aa of the AtSBP1 polypeptide in this interaction. Importantly, the AtRD19c-AtSBP1 interaction was also demonstrated in planta by employing bimolecular fluorescent complementation (BiFC) in a protoplast system.

RNAi silencing of the Arabidopsis thaliana ULCS1 gene results in pleiotropic phenotypes during plant growth and development.

WD40-repeat-containing proteins (WDRs) are highly abundant in all eukaryotes. Several have been implicated as subunits of multi-protein CRL E3 ligase complexes that regulate ubiquitination mediated protein degradation and thus various cellular and developmental processes. Impairment of the WDR protein ULCS1 from Arabidopsis causes pleiotropic phenotypes during plant development, including reduced lignification, anther indehiscence, and sterility. Here we show that RNAi-mediated downregulation of ULCS1 results in a fast-growing phenotype during vegetative development. Due to accelerated growth, ulcs1i mutants reach their vegetative to reproductive transition point earlier than WT plants. However, their comparable germination rate and their similar number of secondary branches and rosette leaves at bolting indicate that ulcs1i is not an early flowering time mutant. GUS staining of progeny, obtained from crosses between ulcs1i and CYCB1::GUS plants, revealed an increased number of mitotic cell divisions in the root meristems of ulcs1i compared to WT. Immunolabeling of homogalacturonans (HGAs) epitopes showed significant fluorescent signal differences at the cell walls and the mucilage of the seeds between ulcs1i and WT. Furthermore, we demonstrate that ULCS1 interacts with the UBA-like protein in a yeast two-hybrid assay, suggesting a direct or indirect physical coupling of these proteins in Arabidopsis.

Investigation of the interaction of DAD1-LIKE LIPASE 3 (DALL3) with Selenium Binding Protein 1 (SBP1) in Arabidopsis thaliana.

Phospholipase PLA1-Iγ2 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-Igammma1. 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.

Novel interactions of Selenium Binding Protein family with the PICOT containing proteins AtGRXS14 and AtGRXS16 in Arabidopsis thaliana.

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. 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.

Molecular and functional characterization of Arabidopsis thaliana VPNB1 gene involved in plant vascular development.

Armadillo (ARM) repeat containing proteins constitute a large family in plants and are involved in diverse cellular functions, like signal transduction, proliferation and differentiation. In animals, ARM repeat proteins have been implicated in cancer development. In this study, we aimed in characterizing the VPNB1 gene from Arabidopsis thaliana and its role in plant development, by implementing a number of genetic and molecular approaches. AtVPNB1 encodes for an ARM repeat protein of unknown function, exclusively expressed in the cambium as well as in the differentiating xylem and phloem cells of the vascular system. Subcellular localization experiments showed that VPNB is confined in nucleoplasmic speckle-like structures unrelated to cajal bodies. Transgenic VPNB-impaired plants exhibit a slower growing phenotype and a non-canonical pattern of xylem tissue. On the contrary, VPNB overexpression lines display an inverted phenotype of increased growth, accompanied by an increased deposition of phloem and xylem cell layers. In line with the above data, qPCR analysis revealed a deregulation of several key master genes of secondary wall biosynthesis, underlining the involvement of VPNB1 in the regulation and differentiation of the root and shoot vascular tissue.

Promoter analysis and functional implications of the selenium binding protein (SBP) gene family in Arabidopsis thaliana.

Selenium Βinding Protein (SBP, originally termed SBP56) was identified in mouse liver as a cytosolic protein that could bind radioactive selenium. SBPs are highly conserved proteins present in a wide array of species across all kingdoms and are likely to be involved in selenium metabolism. In Arabidopsis, the selenium binding protein (SBP) gene family comprises three genes (AtSBP1, AtSBP2 and AtSBP3). AtSBP1 and AtSBP2 are clustered in a head-to-tail arrangement on chromosome IV, while AtSBP3 is located on chromosome III. In this work, we studied the promoter activity of the Arabidopsis SBP genes, determined their tissue specificity and showed that they are differentially regulated by sodium selenite and sodium selenate. All three SBP genes are upregulated in response to externally applied selenium compounds and the antioxidant NAC selectively downregulates SBP2. Although the effect on SBP2 levels was the most prominent, in all cases, the concurrent exposure of plants to selenite and the antioxidant supressed the expression of the SBP genes. We provide evidence that (at least) SBP1 expression is tightly linked to detoxification processes related to oxidative stress, since it is downregulated in the presence of NAC in selenium-treated plants. Furthermore, our results suggest that SBP genes may participate in the mechanisms that sense redox imbalance.

Investigation of the Phaseolus vulgaris circadian clock and the repressive role of the PvTOC1 factor by a newly established in vitro system.

The circadian clock is crucial for the synchronization of an organism's physiology and metabolism with the geophysical time. In plants, previous work on the common bean (Phaseolus vulgaris) has identified various differing aspects of clock function compared to the widely studied Arabidopsis thaliana clock. However, transformation of legumes for the study of the circadian clock regulatory mechanisms is extremely laborious. In the present work, we describe an easy-to-follow and rapid method of preparing bean leaf protoplasts with high transformation potential and a functional circadian clock. In this system, we show that application of trichostatin A differentially changes the expression levels of several clock genes. More importantly, we investigate the effect of the clock protein PvTOC1 (Phaseolus vulgaris TIMING OF CAB EXPRESSION 1) on the activity of bean circadian promoters. We present new evidence on the function of PvTOC1 as a repressor of the promoter activity of its own gene, mediated by its conserved CCT (CONSTANS, CO-LIKE and TOC1) domain. Using our protoplast system we were able to uncover functions of the bean circadian clock and to identify an additional target of the PvTOC1clock transcription factor, not previously reported.

APRF1 promotes flowering under long days in Arabidopsis thaliana.

Arabidopsis thaliana flowering time mutants revealed the function of numerous genes that regulate the transition from vegetative to reproductive growth. Analyses of their loci have shown that many of them act as chromatin modifiers. In this study, a combination of molecular and genetic approaches have been implemented, to characterize the function of APRF1 (ANTHESIS POMOTING FACTOR 1) gene in A. thaliana and to investigate its role in plant development. APRF1 encodes for a low molecular weight nuclear WDR protein which displays functional homology to the Swd2 protein, an essential subunit of the yeast histone methylation COMPASS complex. Compared to WT plants, total loss-of-function aprf1 mutants exhibited shoot apical meristem (SAM) alterations and increased growth rates. However, the vegetative phase of aprf1 plants was prolonged and bolting was delayed, indicating an impairment in flowering under long days (LD). On the contrary, overexpression of APRF1 accelerates flowering. Consistent with the late flowering phenotype, the molecular data confirmed that FLC and SOC1 expression were significantly altered in the aprf1 mutants. Our data suggest that APRF1 acts upstream of FLC and promotes flowering under LD.

An NADPH-Oxidase/Polyamine Oxidase Feedback Loop Controls Oxidative Burst Under Salinity.

The apoplastic polyamine oxidase (PAO) catalyzes the oxidation of the higher polyamines spermidine and spermine, contributing to hydrogen peroxide (H2O2) accumulation. However, it is yet unclear whether apoplastic PAO is part of a network that coordinates the accumulation of reactive oxygen species (ROS) under salinity or if it acts independently. Here, we unravel that NADPH oxidase and apoplastic PAO cooperate to control the accumulation of H2O2 and superoxides (O2·-) in tobacco (Nicotiana tabacum). To examine to what extent apoplastic PAO constitutes part of a ROS-generating network, we examined ROS accumulation in guard cells of plants overexpressing or down-regulating apoplastic PAO (lines S2.2 and A2, respectively) or down-regulating NADPH oxidase (line AS-NtRbohD/F). The H2O2-specific probe benzene sulfonyl-H2O2 showed that, under salinity, H2O2 increased in S2.2 and decreased in A2 compared with the wild type. Surprisingly, the O2·--specific probe benzene sulfonyl-So showed that O2·- levels correlated positively with that of apoplastic PAO (i.e. showed high and low levels in S2.2 and A2, respectively). By using AS-NtRbohD/F lines and a pharmacological approach, we could show that H2O2 and O2·- accumulation at the onset of salinity stress was dependent on NADPH oxidase, indicating that NADPH oxidase is upstream of apoplastic PAO. Our results suggest that NADPH oxidase and the apoplastic PAO form a feed-forward ROS amplification loop, which impinges on oxidative state and culminates in the execution of programmed cell death. We propose that the PAO/NADPH oxidase loop is a central hub in the plethora of responses controlling salt stress tolerance, with potential functions extending beyond stress tolerance.

Silencing S-Adenosyl-L-Methionine Decarboxylase (SAMDC) in Nicotiana tabacum Points at a Polyamine-Dependent Trade-Off between Growth and Tolerance Responses.

Polyamines (PAs) are nitrogenous molecules that are indispensable for cell viability and with an agreed-on role in the modulation of stress responses. Tobacco plants with downregulated SAMDC (AS-SAMDC) exhibit reduced PAs synthesis but normal levels of PA catabolism. We used AS-SAMDC to increase our understanding on the role of PAs in stress responses. Surprisingly, at control conditions AS-SAMDC plants showed increased biomass and altered developmental characteristics, such as increased height and leaf number. On the contrary, during salt stress AS-SAMDC plants showed reduced vigor when compared to the WT. During salt stress, the AS-SAMDC plants although showing compensatory readjustments of the antioxidant machinery and of photosynthetic apparatus, they failed to sustain their vigor. AS-SAMDC sensitivity was accompanied by inability to effectively control H2O2 levels and concentrations of monovalent and divalent cations. In accordance with these findings, we suggest that PAs may regulate the trade-off between growth and tolerance responses.

RNAi-mediated silencing of the Arabidopsis thaliana ULCS1 gene, encoding a WDR protein, results in cell wall modification impairment and plant infertility.

Ubiquitin mediated protein degradation constitutes one of the most complex post translational gene regulation mechanisms in eukaryotes. This fine-tuned proteolytic machinery is based on a vast number of E3 ubiquitin ligase complexes that mark target proteins with ubiquitin. The specificity is accomplished by a number of adaptor proteins that contain functional binding domains, including the WD40 repeat motif (WDRs). To date, only few of these proteins have been identified in plants. An RNAi mediated silencing approach was used here to functionally characterize the Arabidopsis thaliana ULCS1 gene, which encodes for a small molecular weight WDR protein. AtULCS1 interacts with the E3Cullin Ring Ligase subunit DDB1a, regulating most likely the degradation of specific proteins involved in the manifestation of diverse developmental events. Silencing of AtULCS1 results in sterile plants with pleiotropic phenotypes. Detailed analysis revealed that infertility is the outcome of anther indehiscence, which in turn is due to the impairment of the plants to accomplish secondary wall modifications. Furthermore, IREGULAR XYLEM gene expression and lignification is diminished in anther endothecium and the stem vascular tissue of the silenced plants. These data underline the importance of AtULCS1 in plant development and reproduction.

Identification of conserved secondary structures and expansion segments in enod40 RNAs reveals new enod40 homologues in plants.

enod40 is a plant gene that participates in the regulation of symbiotic interaction between leguminous plants and bacteria or fungi. Furthermore, it has been suggested to play a general role in non-symbiotic plant development. Although enod40 seems to have multiple functions, being present in many land plants, the molecular mechanisms of its activity are unclear; they may be determined though, by short peptides and/or RNA structures encoded in the enod40 genes. We utilized conserved RNA structures in enod40 sequences to search nucleotide sequence databases and identified a number of new enod40 homologues in plant species that belong to known, but also, to yet unknown enod40-containing plant families. RNA secondary structure predictions and comparative sequence analysis of enod40 RNAs allowed us to determine the most conserved structural features, present in all known enod40 genes. Remarkably, the topology and evolution of one of the conserved structural domains are similar to those of the expansion segments found in structural RNAs such as rRNAs, RNase P and SRP RNAs. Surprisingly, the enod40 RNA structural elements are much more stronger conserved than the encoded peptides. This finding suggests that some general functions of enod40 gene could be determined by the encoded RNA structure, whereas short peptides may be responsible for more diverse functions found only in certain plant families.

Reliable and controllable antibody fragment selections from Camelid non-immune libraries for target validation.

With the completion of the sequence of the human genome, emphasis is now switching to the human proteome. However, the number of proteins is not only larger than mRNAs in the transcriptome, proteins need often to be in complex with other proteins to be functional. A favourable option to study proteins in their natural context is with a combination of biochemical and microscopic techniques using specific antibodies. Therefore, we designed a fast, reliable and controllable selection and screening of single-domain antibody fragments (VHH) from a Camelid non-immune library. We isolated VHH for four muscle disease related proteins; emerin, actin, tropomyosin-1, and nuclear poly(A)-binding protein. Important features of antibodies for target validation studies are recognition of the antigen in natural conformations and biologically relevant complexes. We show that selected antibody fragments are functional in various immunological techniques and prove useful in diagnostic applications. Our selection strategy is amenable to automation and to the establishment of proteomics platforms. It opens the way to quickly and cost-effectively obtain multiple antibody fragments for many antigens that can detect changes in their localization, level, and modification as well as subtle changes in supramolecular structures, which often associate with disease.

Novel interaction of selenium-binding protein with glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase of Arabidopsis thaliana.

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-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.