Anna Wawrzyńska

Identification and functional analysis of Joka2, a tobacco member of the family of selective autophagy cargo receptors.

Two main mechanisms of protein turnover exist in eukaryotic cells: the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Autophagy is an emerging important constituent of many physiological and pathological processes, such as response to nutrient deficiency, programmed cell death and innate immune response. In mammalian cells the selectivity of autophagy is ensured by the presence of cargo receptors, such as p62/SQSTM1 and NBR1, responsible for sequestration of the ubiquitinated proteins. In plants no selective cargo receptors have been identified yet. The present report indicates that structural and functional homologs of p62 and NBR1 proteins exist in plants. The tobacco protein, named Joka2, has been identified in yeast two-hybrid search as a binding partner of a small coiled-coil protein, a member of UP9/LSU family of unknown function, encoded by the UP9C gene strongly and specifically induced during sulfur deficiency. The typical domains of p62 and NBR1 are conserved in Joka2. Similarly to p62, Joka2-YFP has dual localization (cytosolic speckles and the nucleus); it forms homodimers and interacts with a member of the ATG8 family. Increased expression of Joka2 and ATG8f was observed in roots of tobacco plants grown for two days in nutrient-deficient conditions. Constitutive ectopic expression of Joka2-YFP in tobacco resulted in attenuated response (manifested by lesser yellowing of the leaves) to nutrient deficiency. In conclusion, Joka2, and presumably the process of selective autophagy, might constitute an important part of plant response to environmental stresses.

Activity of the AtMRP3 promoter in transgenic Arabidopsis thaliana and Nicotiana tabacum plants is increased by cadmium, nickel, arsenic, cobalt and lead but not by zinc and iron.

Characterization of the function, regulation and metal-specificity of metal transporters is one of the basic steps needed for the understanding of transport and accumulation of toxic metals and metalloids by plants. In this work GUS was used as a reporter for monitoring the activity of the promoter of the AtMRP3 gene from Arabidopsis thaliana, a gene encoding an ABC-transporter, expression of which is induced by heavy metals. The AtMRP3 promoter-GUS fusion expression cassette was introduced into the genome of two model plants, A. thaliana and Nicotiana tabacum. The promoter induces GUS activity in the roots as well as in the shoots upon metal exposure. Similar responses of the AtMRP3 promoter to the presence of the selected metals was observed in both plant species. Cadmium, nickel, arsenic, cobalt and lead strongly activated the transcription of the reporter gene, while zinc and iron had no impact. The AtMRP3 promoter thus seems to be a useful new tool in designing plants that can be used for biomonitoring of environmental contaminations.

A Contribution to Identification of Novel Regulators of Plant Response to Sulfur Deficiency: Characteristics of a Tobacco Gene UP9C, Its Protein Product and the Effects of UP9C Silencing

Extensive changes in plant transcriptome and metabolome have been observed by numerous research groups after transferring plants from optimal conditions to sulfur (S) deficiency. Despite intensive studies and recent important achievements, like identification of SLIM1/EIL3 as a major transcriptional regulator of the response to S-deficiency, many questions concerning other elements of the regulatory network remain unanswered. Investigations of genes with expression regulated by S-deficiency stress encoding proteins of unknown function might help to clarify these problems. This study is focused on the UP9C gene and the UP9-like family in tobacco. Homologs of these genes exist in other plant species, including a family of four genes of unknown function in Arabidopsis thaliana (LSU1-4), of which two were reported as strongly induced by S-deficit and to a lesser extent by salt stress and nitrate limitation. Conservation of the predicted structural features, such as coiled coil region or nuclear localization signal, suggests that these proteins might have important functions possibly mediated by interactions with other proteins. Analysis of transgenic tobacco plants with silenced expression of UP9-like genes strongly argues for their significant role in regulation of plant response to S-deficit. Although our study shows that the UP9-like proteins are important components of such response and they might be also required during other stresses, their molecular functions remain a mystery.

Links Between Ethylene and Sulfur Nutrition—A Regulatory Interplay or Just Metabolite Association?

Multiple reports demonstrate associations between ethylene and sulfur metabolisms, however the details of these links have not yet been fully characterized; the links might be at the metabolic and the regulatory levels. First, sulfur-containing metabolite, methionine, is a precursor of ethylene and is a rate limiting metabolite for ethylene synthesis; the methionine cycle contributes to both sulfur and ethylene metabolism. On the other hand, ethylene is involved in the complex response networks to various stresses and it is known that S deficiency leads to photosynthesis and C metabolism disturbances that might be responsible for oxidative stress. In several plant species, ethylene increases during sulfur starvation and might serve signaling purposes to initiate the process of metabolism reprogramming during adjustment to sulfur deficit. An elevated level of ethylene might result from increased activity of enzymes involved in its synthesis. It has been demonstrated that the alleviation of cadmium stress in plants by application of S seems to be mediated by ethylene formation. On the other hand, the ethylene-insensitive Nicotiana attenuata plants are impaired in sulfur uptake, reduction and metabolism, and they invest their already limited S into methionine needed for synthesis of ethylene constitutively emitted in large amounts to the atmosphere. Regulatory links of EIN3 and SLIM1 (both from the same family of transcriptional factors) involved in the regulation of ethylene and sulfur pathway, respectively, is also quite probable as well as the reciprocal modulation of both pathways on the enzyme activity levels.

Tobacco LSU-like protein couples sulphur-deficiency response with ethylene signalling pathway

Most genes from the plant-specific family encoding Response to Low Sulphur (LSU)-like proteins are strongly induced in sulphur (S)-deficient conditions. The exact role of these proteins remains unclear; however, some data suggest their importance for plants’ adjustment to nutrient deficiency and other environmental stresses. This work established that the regulation of ethylene signalling is a part of plants’ response to S deficiency and showed the interaction between UP9C, a tobacco LSU family member, and one of the tobacco isoforms of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO2A). Increase in ethylene level induced by S deficiency does not take place in tobacco plants with UP9C expressed in an antisense orientation. Based on transcriptomics data, this work also demonstrated that the majority of tobacco’s response to S deficiency is misregulated in plants expressing UP9C-antisense. A link between response to S deficiency, ethylene sensing, and LSU-like proteins was emphasized by changes in expression of the genes encoding ethylene receptors and F-box proteins specific for the ethylene pathway.

The family of LSU-like proteins.

The plant response to sulfur deficiency includes extensive metabolic changes which can be monitored at various levels (transcriptome, proteome, metabolome) even before the first visible symptoms of sulfur starvation appear. Four members of the plant-specific LSU (response to Low SUlfur) gene family occur in Arabidopsis thaliana (LSU1-4). Variable numbers of LSU genes occur in other plant species but they were studied only in Arabidopsis and tobacco. Three out of four of the Arabidopsis LSU genes are induced by sulfur deficiency. The LSU-like genes in tobacco were characterized as UP9 (UPregulated by sulfur deficit 9). LSU-like proteins do not have characteristic domains that provide clues to their function. Despite having only moderate primary sequence conservation they share several common features including small size, a coiled–coil secondary structure and short conserved motifs in specific positions. Although the precise function of LSU-like proteins is still unknown there is some evidence that members of the LSU family are involved in plant responses to environmental challenges, such as sulfur deficiency, and possibly in plant immune responses. Various bioinformatic approaches have identified LSU-like proteins as important hubs for integration of signals from environmental stimuli. In this paper we review a variety of published data on LSU gene expression, the properties of lsu mutants and features of LSU-like proteins in the hope of shedding some light on their possible role in plant metabolism.

To control and to be controlled: understanding the Arabidopsis SLIM1 function in sulfur deficiency through comprehensive investigation of the EIL protein family.

Sulfur limitation 1 (SLIM1), a member of the EIN3-like (EIL) family of transcription factors in Arabidopsis, is the regulator of many sulfur deficiency responsive genes. Among the five other proteins of the family, three regulate ethylene (ET) responses and two have unassigned functions. Contrary to the well-defined ET signaling, the pathway leading from sensing sulfate status to the activation of its acquisition via SLIM1 is completely unknown. SLIM1 binds to the 20 nt-long specific UPE-box sequence; however, it also recognizes the shorter TEIL sequence, unique for the whole EIL family. SLIM1 takes part in the upregulation and downregulation of various sulfur metabolism genes, but also it controls the degradation of glucosinolates under sulfur deficient conditions. Besides facilitating the increased flux through the sulfate assimilation pathway, SLIM1 induces microRNA395, specifically targeting ATP sulfurylases and a low-affinity sulfate transporter, SULTR2;1, thus affecting sulfate translocation to the shoot. Here, we briefly review the identification, structural characteristics, and molecular function of SLIM1 from the perspective of the whole EIL protein family.

EIN3 interferes with the sulfur deficiency signaling in Arabidopsis thaliana through direct interaction with the SLIM1 transcription factor

Sulfur deficiency in plants leads to metabolic reprogramming through changes of gene expression. SLIM1 is so far the only characterized transcription factor associated strictly with sulfur deficiency stress in Arabidopsis thaliana. It belongs to the same protein family as EIN3, a major positive switch of ethylene signaling pathway. It binds to the specific cis sequence called UPE-box. Here we show that SLIM1 interacts with UPE-box as a homodimer. Interestingly, the same region of the protein is used for heterodimerization with EIN3; however, the heterodimer is not able to recognize UPE-box. Expression of several SLIM1-dependent genes is enhanced in sulfur deficiency grown Arabidopsis ein3-1 seedlings (with mutated EIN3 protein). This implies a possible regulatory mechanism of ethylene in sulfur metabolism through direct EIN3-SLIM1 interaction.

Intronic T-DNA insertion in Arabidopsis NBR1 conditionally affects wild-type transcript level

Abstract The SALK_135513 line of Arabidopsis thaliana is annotated by GenBank to have the T-DNA insertion in the fourth exon of NBR1 (At4g24690). Careful molecular analyses of the homozygous plants of SALK_135513 line indicated the place of T-DNA insertion in the fourth intron. Unexpectedly, 2 kinds of NBR1 transcripts, the wild-type and the mutated, resulting from alternative splicing events, were detected in those plants. Our findings explain the problems encountered by us with phenotypic evaluation of this line and emphasize the necessity for independent verification of the exact insertion site followed by careful expression studies when working with Arabidopsis T-DNA insertional mutants.

Direct targeting of Arabidopsis cysteine synthase complexes with synthetic polypeptides to selectively deregulate cysteine synthesis

Biosynthesis of cysteine is one of the fundamental processes in plants providing the reduced sulfur for cell metabolism. It is accomplished by the sequential action of two enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they constitute the hetero-oligomeric cysteine synthase (CS) complex through specific protein-protein interactions influencing the rate of cysteine production. The aim of our studies was to deregulate the CS complex formation in order to investigate its function in the control of sulfur homeostasis and optimize cysteine synthesis. Computational modeling was used to build a model of the Arabidopsis thaliana mitochondrial CS complex. Several polypeptides based on OAS-TL C amino-acid sequence found at SAT-OASTL interaction sites were designed as probable competitors for SAT3 binding. After verification of the binding in a yeast two-hybrid assay, the most strongly interacting polypeptide was introduced to different cellular compartments of Arabidopsis cell via genetic transformation. Moderate increase in total SAT and OAS-TL activities, but not thiols content, was observed dependent on the transgenic line and sulfur availability in the hydroponic medium. Though our studies demonstrate the proof of principle, they also suggest more complex interaction of both enzymes underlying the mechanism of their reciprocal regulation.