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 physio...

Transgenic tobacco (Nicotiana tabacum cv. LA Burley 21) lines expressing three genes encoding enzymes thought to be critical for the efficient production of phytochelatins, (i) serine acetyltransferase (EC 2.3.1.30) involved in the production of O-acetylserine, the cysteine precursor, (ii) γ-glutamylcysteine synthetase (EC 6.3.2.2) involved in the production of γ-glutamylcysteine, the precursor of glutathione, and (iii) phytochelatin synthase (EC 2.3.2.15), were obtained and analysed for non-protein thiol content and cadmium accumulation. After a 3 week exposure to 15 μM CdCl₂, plants expressing transgenes (either separately or in combination) had increased cadmium concentration in roots but not in shoots compared with the wild type. Nearly all transgenic lines analysed had more non-protein thiols than the wild type. The greatest effects (about 8-fold elevation of thiols) were found in one of the lines simultaneously expressing the three transgenes. Despite the fact that a multi-transgene strategy described in this work resulted in a strong increase in the levels of several classes of non-protein thiols in transgenic plants, other factors appeared to restrict cadmium accumulation in shoots.

Loss-of-function mutations in the Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced resistance to infection by powdery mildew (Golovinomyces cichoracearum). EDR1 encodes a protein kinase, but its substrates and the pathways regulated by EDR1 are unknown. To identify components of the EDR1 signal transduction pathway(s), we conducted a forward genetic screen for mutations that suppressed edr1-mediated disease resistance. Genetic mapping and cloning of one of these suppressor mutations revealed a recessive missense mutation in the KEEP ON GOING gene (KEG; At5g13530), which we designated keg-4. KEG encodes a multidomain protein that includes a RING E3 ligase domain, a kinase domain, ankyrin repeats, and HERC2-like repeats. The KEG protein has previously been shown to have ubiquitin ligase activity and to negatively regulate protein levels of the transcription factor ABCISIC ACID INSENSITIVE5. KEG mRNA levels were found to be 3-fold higher in edr1 mutant plants compared to wild type. Loss-of-function mutations in KEG are seedling lethal and are hypersensitive to glucose and abscisic acid (ABA). The keg-4 mutation, in contrast, conferred resistance to 6% glucose and suppressed edr1-mediated hypersensitivity to ABA, suggesting that the keg-4 mutation suppresses ABA signaling by altering KEG function. Several ABA-responsive genes were found to be further up-regulated in the edr1 mutant following ABA treatment, and this up-regulation was suppressed by the keg-4 mutation. We conclude that edr1-mediated resistance to powdery mildew is mediated, in part, by enhanced ABA signaling.

Sulphur deficiency severely affects plant growth and their agricultural productivity leading to diverse changes in development and metabolisms. Molecular mechanisms regulating gene expression under low sulphur conditions remain largely unknown. AtSLIM1, a member of the EIN3-like (EIL) family was reported to be a central transcriptional regulator of the plant sulphur response, however, no direct interaction of this protein with any sulphur-responsive promoters was demonstrated. The focus of this study was on the analysis of a promoter region of UP9C, a tobacco gene strongly induced by sulphur limitation. Cloning and subsequent examination of this promoter resulted in the identification of a 20-nt sequence (UPE-box), also present in the promoters of several Arabidopsis genes, including three out of four homologues of UP9C. The UPE-box, consisting of two parallel tebs sequences (TEIL binding site), proved to be necessary to bind the transcription factors belonging to the EIL family and of a 5-nt conserved sequence at the 3′-end. The yeast one-hybrid analysis resulted in the identification of one transcription factor (NtEIL2) capable of binding to the UPE-box. The interactions of NtEIL2, and its homologue from Arabidopsis, AtSLIM1, with DNA were affected by mutations within the UPE-box. Transient expression assays in Nicotiana benthamiana have further shown that both factors, NtEIL2 and AtSLIM1, activate the UP9C promoter. Interestingly, activation by NtEIL2, but not by AtSLIM1, was dependent on the sulphur-deficiency of the plants.

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.