Control of ABA Signaling and Crosstalk with Other Hormones by the Selective Degradation of Pathway Components

A rapid and appropriate genetic and metabolic acclimation, which is crucial for plants’survival in a changing environment, is maintained due to the coordinated action of plant hormones and cellular degradation mechanisms influencing proteostasis. The plant hormone abscisic acid (ABA) rapidly accumulates in plants in response to environmental stress and plays a pivotal role in the reaction to various stimuli. Increasing evidence demonstrates a significant role of autophagy in controlling ABA signaling. This field has been extensively investigated and new discoveries are constantly being provided. We present updated information on the components of the ABA signaling pathway, particularly on transcription factors modified by different E3 ligases. Then, we focus on the role of selective autophagy in ABA pathway control and review novel evidence on the involvement of autophagy in different parts of the ABA signaling pathway that are important for crosstalk with other hormones, particularly cytokinins and brassinosteroids

Overexpression of the selective autophagy cargo receptor NBR1 modifies plant response to sulfur deficit

Plants exposed to sulfur deficit elevate the transcription of NBR1 what might reflect an increased demand for NBR1 in such conditions. Therefore, we investigated the role of this selective autophagy cargo receptor in plant response to sulfur deficit (-S). Transcriptome analysis of the wild type and NBR1 overexpressing plants pointed out differences in gene expression in response to -S. Our attention focused particularly on the genes upregulated by -S in roots of both lines because of significant overrepresentation of cytoplasmic ribosomal gene family. Moreover, we noticed overrepresentation of the same family in the set of proteins co-purifying with NBR1 in -S. One of these ribosomal proteins, RPS6 was chosen for verification of its direct interaction with NBR1 and proven to bind outside the NBR1 ubiquitin binding domains. The biological significance of this novel interaction and the postulated role of NBR1 in ribosomes remodeling in response to starvation remain to be further investigated. Interestingly, NBR1 overexpressing seedlings have significantly shorter roots than wild type when grown in nutrient deficient conditions in the presence of TOR kinase inhibitors. This phenotype probably results from excessive autophagy induction by the additive effect of NBR1 overexpression, starvation, and TOR inhibition

Similar but Not Identical—Binding Properties of LSU (Response to Low Sulfur) Proteins From Arabidopsis thaliana

Members of the plant-specific LSU (RESPONSE TO LOW SULFUR) family are strongly induced during sulfur starvation. The molecular functions of these proteins are unknown; however, they were identified as important stress-related hubs in several studies. In Arabidopsis thaliana, there are four members of the LSU family (LSU1–4). These proteins are small (approximately 100 amino acids), with coiled-coil structures. In this work, we investigated interactions between different monomers of LSU1–4. Differences in homo and heterodimer formation were observed. Our structural models of LSU1–4 homo- and heterodimers were in agreement with our experimental observations and may help understand their binding properties. LSU proteins are involved in multiple protein–protein interactions, with the literature suggesting they can integrate abiotic and biotic stress responses. Previously, LSU partners were identified using the yeast two hybrid approach, therefore we sought to determine proteins co-purifying with LSU family members using protein extracts isolated from plants ectopically expressing TAP-tagged LSU1–4 constructs. These experiments revealed 46 new candidates for LSU partners. We tested four of them (and two other proteins, CAT2 and NBR1) for interaction with LSU1–4 by other methods. Binding of all six proteins with LSU1–4 was confirmed by Bimolecular Fluorescence Complementation, while only three of them were interacting with LSUs in yeast-two-hybrid. Additionally, we conducted network analysis of LSU interactome and revealed novel clues for the possible cellular function of these proteins

Molecular characterization of central cytoplasmic loop in Aspergillus nidulans AstA transporter

AstA (alternative sulfate transporter) belongs to a large, but poorly characterized, Dal5 family of allantoate permeases of the Major Facilitator Superfamily. The astA gene has been cloned from an IAM 2006 Japanese strain of Aspergillus nidulans by complementation of a sulfate permease-deficient mutant. In this study we show that conserved lysine residues in Central Cytoplasmic Loop (CCL) of the AstA protein may participate in anion selectivity, and control kinetic properties of the AstA transporter. A three-dimensional model containing four clustered lysine residues was created, showing a novel substrate-interacting structure in Major Facilitator Superfamily transporters. The assimilation constant (Kτ) of wild type AstA protein is 85 μM, while Vmax/mg of DW of AstA is twice that of the main sulfate transporter SB per mg of dry weight (DW) of mycelium (1.53 vs. 0.85 nmol/min, respectively). Amino acid substitutions in CCL did not abolish sulfate uptake, but affected its kinetic parameters. Mutants affected in the lysine residues forming the postulated sulfate-interacting pocket in AstA were able to grow and uptake sulfate, indicating that CCL is not crucial for sulfate transportation. However, these mutants exhibited altered values of Kτ and Vmax, suggesting that CCL is involved in control of the transporter activity

Novel mutations reveal two important regions in Aspergillus nidulans transcriptional activator MetR

Expression of the sulfur assimilation pathway in Aspergillus nidulans is under control of sulfur metabolite repression, which is composed of scon genes encoding subunits of ubiquitin ligase and the metR gene coding for a transcriptional activator. In this paper we report three dominant suppressors of methionine requirement isolated from a metB3 diploid strain. All three mutations lead to the substitution of phenylalanine 48 by serine or leucine in the conserved N-terminal region of the MetR protein. Strains carrying the dominant suppressor mutations exhibit increased activities of homocysteine synthase and sulfur assimilation enzymes as well as elevated levels of the corresponding transcripts. These changes are observed even under conditions of methionine repression, which suggests that the mutated MetR protein may be resistant to inactivation or degradation mediated by sulfur metabolite repression. We also found that a mutant impaired in sulfite reductase activity, known until now as sG8, has a frameshift which changes 41 C-terminal amino acids. Therefore, it is now designated metR18. This mutant has elevated levels of MetR-regulated transcripts and of activities of sulfur assimilation enzymes (except sulfite reductase), which can be repressed to the wild type level by exogenous methionine. Thus, metR18 and the three dominant suppressors represent new types of mutations affecting different parts of the A. nidulans MetR protein

The Aspergillus nidulans metZ gene encodes a transcription factor involved in regulation of sulfur metabolism in this fungus and other Eurotiales

In Aspergillus nidulans, expression of sulfur metabolism genes is activated by the MetR transcription factor containing a basic region and leucine zipper domain (bZIP). Here we identified and characterized MetZ, a new transcriptional regulator in Aspergillus nidulans and other Eurotiales. It contains a bZIP domain similar to the corresponding region in MetR and this similarity suggests that MetZ could potentially complement the MetR deficiency. The metR and metZ genes are interrupted by unusually long introns. Transcription of metZ, unlike that of metR, is controlled by the sulfur metabolite repression system (SMR) dependent on the MetR protein. Overexpression of metZ from a MetR-independent promoter in a metR background, activates transcription of genes encoding sulfate permease, homocysteine synthase and methionine permease, partially complementing the phenotype of the metR mutation. Thus, MetZ appears to be a second transcription factor involved in regulation of sulfur metabolism genes

A selective autophagy cargo receptor NBR1 modulates abscisic acid signalling in Arabidopsis thaliana

The plant selective autophagy cargo receptor neighbour of breast cancer 1 gene (NBR1) has been scarcely studied in the context of abiotic stress. We wanted to expand this knowledge by using Arabidopsis thaliana lines with constitutive ectopic overexpression of the AtNBR1 gene (OX lines) and the AtNBR1 Knock-Out (KO lines). Transcriptomic analysis of the shoots and roots of one representative OX line indicated differences in gene expression relative to the parental (WT) line. In shoots, many differentially expressed genes, either up- or down-regulated, were involved in responses to stimuli and stress. In roots the most significant difference was observed in a set of downregulated genes that is mainly related to translation and formation of ribonucleoprotein complexes. The link between AtNBR1 overexpression and abscisic acid (ABA) signalling was suggested by an interaction network analysis of these differentially expressed genes. Most hubs of this network were associated with ABA signalling. Although transcriptomic analysis suggested enhancement of ABA responses, ABA levels were unchanged in the OX shoots. Moreover, some of the phenotypes of the OX (delayed germination, increased number of closed stomata) and the KO lines (increased number of lateral root initiation sites) indicate that AtNBR1 is essential for fine-tuning of the ABA signalling pathway. The interaction of AtNBR1 with three regulatory proteins of ABA pathway (ABI3, ABI4 and ABI5) was observed in planta. It suggests that AtNBR1 might play role in maintaining the balance of ABA signalling by controlling their level and/or activity