An autophagy-associated Atg8 protein is involved in the responses of Arabidopsis seedlings to hormonal controls and abiotic stresses

Eukaryotes contain a ubiquitous family of autophagy-associated Atg8 proteins. In animal cells, these proteins have multiple functions associated with growth, cancer, and degenerative diseases, but their functions in plants are still largely unknown. To search for novel functions of Atg8 in plants, the present report tested the effect of expression of a recombinant AtAtg8 protein, fused at its N-terminus to green fluorescent protein (GFP) and at its C-terminus to the haemagglutinin epitope tag, on the response of Arabidopsis thaliana plants to the hormones cytokinin and auxin as well as to salt and osmotic stresses. Expression of this AtAtg8 fusion protein modulates the effect of cytokinin on root architecture. Moreover, expression of this fusion protein also reduces shoot anthocyanin accumulation in response to cytokinin feeding to the roots, implying the participation of AtAtg8 in cytokinin-regulated root-shoot communication. External application of cytokinin leads to the formation of novel GFP-AtAtg8-containing structures in cells located in the vicinity of the root vascular system, which are clearly distinct in size and dynamic movement from the GFP-AtAtg8-containing autophagosome-resembling structures that were observed in root epidermis cells. Expression of the AtAtg8 fusion construct also renders the plants more sensitive to a mild salt stress and to a lesser extent to a mild osmotic stress. This sensitivity is also associated with various changes in the root architecture, which are morphologically distinct from those observed in response to cytokinin. The results imply multiple functions for AtAtg8 in different root tissues that may also be regulated by different mechanisms.     

Selective autophagy in the aid of plant germination and response to nutrient starvation

Selective autophagy, mediated by Atg8 binding proteins, has not been extensively studied in plants. Plants possess a large gene family encoding multiple isoforms of the Atg8 protein. We have recently reported the identification of two new, closely homologous Arabidopsis thaliana plant proteins that bind the Arabidopsis Atg8f protein isoform. These two proteins are specific to plants and have no homologs in nonplant organisms. The expression levels of the genes encoding these proteins are elevated during carbon starvation and also during late stages of seed development. Exposure of young seedlings to carbon starvation induces the production of a newly identified compartment decorated by these Atg8-binding proteins. This compartment dynamically moves along the endoplasmic reticulum membrane and is also finally transported into the vacuole. Enhanced or suppressed expression of these Atg8-binding proteins respectively enhances or suppresses seed germination under suboptimal germination conditions, indicating that they contribute to seed germination vigor.     

Selective autophagy in the aid of plant germination and response to nutrient starvation

Selective autophagy, mediated by Atg8 binding proteins, has not been extensively studied in plants. Plants possess a large gene family encoding multiple isoforms of the Atg8 protein. We have recently reported the identification of two new, closely homologous Arabidopsis thaliana plant proteins that bind the Arabidopsis Atg8f protein isoform. These two proteins are specific to plants and have no homologs in nonplant organisms. The expression levels of the genes encoding these proteins are elevated during carbon starvation and also during late stages of seed development. Exposure of young seedlings to carbon starvation induces the production of a newly identified compartment decorated by these Atg8-binding proteins. This compartment dynamically moves along the endoplasmic reticulum membrane and is also finally transported into the vacuole. Enhanced or suppressed expression of these Atg8-binding proteins respectively enhances or suppresses seed germination under suboptimal germination conditions, indicating that they contribute to seed germination vigor.     

Physiological,biochemical and molecular analysis of sugar-starvation responses in tomato roots

Two-month-old tomato plants were submitted to day/night cycles and to prolonged darkness in order to investigate the physiological and biochemical response to sugar starvation in sink organs. Roots appeared particularly sensitive to the cessation of photosynthesis, as revealed by the reduction of the growth rate and the decline of the carbohydrate and protein content. Therefore, excised tomato roots were used as a model to deepen the characterization of sugar starvation symptoms. In excised roots, the endogenous sugars were rapidly exhausted and significant degradation of protein was observed. Glutamine and asparagine accounted for most of the nitrogen released by protein breakdown. Respiration declined and proliferation- and growth-associated genes were repressed soon after the beginning of the sugar depletion. Among the genes studied, only the gene encoding asparagine synthetase was strongly induced. All the starvation symptoms were reversible when the roots were resupplied with sugar. When the culture conditions deteriorated, the metabolic and molecular changes led to the triggering of apoptosis of the root cells.     

Functional characterization of the thi1 promoter region from Arabidopsis thaliana

The Arabidopsis thaliana THI1 protein is involved in thiamine biosynthesis and is targeted to both chloroplasts and mitochondria by N-terminal control regions. To investigate thi1 expression, a series of thi1 promoter deletions were fused to the beta-glucuronidase (GUS) reporter gene. Transgenic plants were generated and expression patterns obtained under different environmental conditions. The results show that expression derived from the thi1 promoter is detected early on during development and continues throughout the plant's life cycle. High levels of GUS expression are observed in both shoots and roots during vegetative growth although, in roots, expression is restricted to the vascular system. Deletion analysis of the thi1 promoter region identified a region that is responsive to light. The smallest fragment (designated Pthi322) encompasses 306 bp and possesses all the essential signals for tissue specificity, as well as responsiveness to stress conditions such as sugar deprivation, high salinity, and hypoxia.     

A new type of compartment, defined by plant-specific Atg8-interacting proteins, is induced upon exposure of Arabidopsis plants to carbon starvation

Atg8 is a central protein in bulk starvation-induced autophagy, but it is also specifically associated with multiple protein targets under various physiological conditions to regulate their selective turnover by the autophagy machinery. Here, we describe two new closely related Arabidopsis thaliana Atg8-interacting proteins (ATI1 and ATI2) that are unique to plants. We show that under favorable growth conditions, ATI1 and ATI2 are partially associated with the endoplasmic reticulum (ER) membrane network, whereas upon exposure to carbon starvation, they become mainly associated with newly identified spherical compartments that dynamically move along the ER network. These compartments are morphologically distinct from previously reported spindle-shaped ER bodies and, in contrast to them, do not contain ER-lumenal markers possessing a C-terminal HDEL sequence. Organelle and autophagosome-specific markers show that the bodies containing ATI1 are distinct from Golgi, mitochondria, peroxisomes, and classical autophagosomes. The final destination of the ATI1 bodies is the central vacuole, indicating that they may operate in selective turnover of specific proteins. ATI1 and ATI2 gene expression is elevated during late seed maturation and desiccation. We further demonstrate that ATI1 overexpression or suppression of both ATI1 and ATI2, respectively, stimulate or inhibit seed germination in the presence of the germination-inhibiting hormone abscisic acid.     

An Arabidopsis homolog of yeast ATG6/VPS30 is essential for pollen germination

Yeast (Saccharomyces cerevisiae) Atg6/Vps30 is required for autophagy and the sorting of vacuolar hydrolases, such as carboxypeptidase Y. In higher eukaryotes, however, roles for ATG6/VPS30 homologs in vesicle sorting have remained obscure. Here, we show that AtATG6, an Arabidopsis (Arabidopsis thaliana) homolog of yeast ATG6/VPS30, restored both autophagy and vacuolar sorting of carboxypeptidase Y in a yeast atg6/vps30 mutant. In Arabidopsis cells, green fluorescent protein-AtAtg6 protein localized to punctate structures and colocalized with AtAtg8, a marker protein of the preautophagosomal structure. Disruption of AtATG6 by T-DNA insertion resulted in male sterility that was confirmed by reciprocal crossing experiments. Microscopic analyses of AtATG6 heterozygous plants (AtATG6/atatg6) crossed with the quartet mutant revealed that AtATG6-deficient pollen developed normally, but did not germinate. Because other atatg mutants are fertile, AtAtg6 likely mediates pollen germination in a manner independent of autophagy. We propose that Arabidopsis Atg6/Vps30 functions not only in autophagy, but also plays a pivotal role in pollen germination.     

Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy

Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. Thus far, plant autophagy has been studied primarily using morphological analyses. A recent genome-wide search revealed significant conservation among autophagy genes (ATGs) in yeast and plants. It has not been proved, however, that Arabidopsis thaliana ATG genes are required for plant autophagy. To evaluate this requirement, we examined the ubiquitination-like Atg8 lipidation system, whose component genes are all found in the Arabidopsis genome. In Arabidopsis, all nine ATG8 genes and two ATG4 genes were expressed ubiquitously and were induced further by nitrogen starvation. To establish a system monitoring autophagy in whole plants, we generated transgenic Arabidopsis expressing each green fluorescent protein-ATG8 fusion (GFP-ATG8). In wild-type plants, GFP-ATG8s were observed as ring shapes in the cytoplasm and were delivered to vacuolar lumens under nitrogen-starved conditions. By contrast, in a T-DNA insertion double mutant of the ATG4s (atg4a4b-1), autophagosomes were not observed, and the GFP-ATG8s were not delivered to the vacuole under nitrogen-starved conditions. In addition, we detected autophagic bodies in the vacuoles of wild-type roots but not in those of atg4a4b-1 in the presence of concanamycin A, a V-ATPase inhibitor. Biochemical analyses also provided evidence that autophagy in higher plants requires ATG proteins. The phenotypic analysis of atg4a4b-1 indicated that plant autophagy contributes to the development of a root system under conditions of nutrient limitation.     

Arabidopsis homologues of the autophagy protein Atg8 are a novel family of microtubule binding proteins

Autophagy is the non-selective transport of proteins and superfluous organelles destined for degradation to the vacuole in fungae, or the lysosome in animal cells. Some of the genes encoding components of the autophagy pathway are conserved in plants, and here we show that Arabidopsis homologues of yeast Atg8 (Apg8/Aut7) and Atg4 (Apg4/Aut2) partially complement the yeast deletion strains. The yeast double mutant, a deletion strain with respect to both Atg8 and Atg4, could not be complemented by Arabidopsis Atg8, indicating that Arabidopsis Atg8 requires Atg4 for its function. Moreover, Arabidopsis Atg8 and Arabidopsis Atg4 interact directly in a two-hybrid assay. Interestingly, Atg8 shows significant homology with the microtubule binding light chain 3 of MAP1A and B, and here we show that Arabidopsis Atg8 binds microtubules. Our results demonstrate that a principle component of the autophagic pathway in plants is similar to that in yeast and we suggest that microtubule binding plays a role in this process.     

Macroautophagy is required for multicellular development of the social amoeba Dictyostelium discoideum

Macroautophagy is a mechanism employed by eukaryotic cells to recycle non-essential cellular components during starvation, differentiation, and development. Two conjugation reactions related to ubiquitination are essential for autophagy: Apg12p conjugation to Apg5p, and Apg8p conjugation to the lipid phosphatidylethanolamine. These reactions require the action of the E1-like enzyme, Apg7p, and the E2-like enzymes, Apg3p and Apg10p. In Dictyostelium, development is induced by starvation, conditions under which autophagy is required for survival in yeast and plants. We have identified Dictyostelium homologues of 10 budding yeast autophagy genes. We have generated mutations in apg5 and apg7 that produce defects typically associated with an abrogation of autophagy. Mutants are not grossly affected in growth, but survival during nitrogen starvation is severely reduced. Starved mutant cells show little turnover of cellular constituents by electron microscopy, whereas wild-type cells show significant cytoplasmic degradation and reduced organelle number. Bulk protein degradation during starvation-induced development is reduced in the autophagy mutants. Development is aberrant; the autophagy mutants do not aggregate in plaques on bacterial lawns, but they do proceed further in development on nitrocellulose filters, forming defective fruiting bodies. The autophagy mutations are cell autonomous, because wild-type cells in a chimaera do not rescue development of the autophagy mutants. We have complemented the mutant phenotypes by expression of the cognate gene fused to green fluorescent protein. A green fluorescent protein fusion of the autophagosome marker Apg8 mislocalizes in the two autophagy mutants. We show that the Apg5-Apg12 conjugation system is conserved in Dictyostelium.     

Alteration of nutrient allocation and transporter genes expression in rice under N,P, K,and Mg deficiencies

Nitrogen (N), phosphorus (P), potassium (K), and magnesium (Mg) have essential physiological functions in plants. Their interactions in plants are not fully understood especially at the molecular level. In this study, we detected the physiological and molecular responses of rice plants at the vegetative growth phase to N, P, K, and Mg starvations. Deficiencies of N and P resulted in accumulation of soluble sugar and starch in the leaves. The root to shoot ratio increased under N and P deficiencies, but decreased under K and Mg deficiencies. In addition, deficiency of either K or Mg resulted in accumulation of the other cation in shoots. Moreover, K starvation decreased both K and soluble sugar contents in the roots pronouncedly. RT-PCR analysis showed that several sugar transporter genes in the leaves orchestrated with sugar accumulation induced by the nutrient shortages. Expression of a high affinity K transporter gene (OsHAK1) and a putative Mg transporter gene (OsMGT) showed opposite down- and up-regulation in the roots by K starvation. These findings suggest that deficiencies of the major nutrients suppressed the export of carbohydrates from source leaves. The regulated sugar and nutrient transporter genes investigated in this study could be used for elucidating the molecular mechanism of plants in their adaptation to varied nutrient supply.     

Arabidopsis TCH3 encodes a novel Ca2+ binding protein and shows environmentally induced and tissue-specific regulation.

The Arabidopsis touch (TCH) genes are up-regulated in response to various environmental stimuli, including touch, wind, and darkness. Previously, it was determined that TCH1 encodes a calmodulin; TCH2 and TCH3 encode calmodulin-related proteins. Here, we present the sequence and genomic organization of TCH3. TCH3 is composed of three repeats; remarkably, the first two repeats share 94% sequence identity, including introns that are 99% identical. The conceptual TCH3 product is 58 to 60% identical to known Arabidopsis calmodulins; however, unlike calmodulin, which has four Ca2+ binding sites, TCH3 has six potential Ca2+ binding domains. TCH3 is capable of binding Ca2+, as demonstrated by a Ca(2+)-specific shift in electrophoretic mobility. 5' Fragments of the TCH3 locus, when fused to the beta-glucuronidase (GUS) reporter gene, are sufficient to confer inducibility of expression following stimulation of plants with touch or darkness. These TCH3 sequences also direct expression to growing regions of roots, vascular tissue, root/shoot junctions, trichomes, branch points of the shoot, and regions of siliques and flowers. The pattern of expression of the TCH3/GUS reporter genes most likely reflects expression of the native TCH3 gene, because immunostaining of the TCH3 protein shows similar localization. The tissue-specific expression of TCH3 suggests that expression may be regulated not only by externally applied mechanical stimuli but also by mechanical stresses generated during development. Consequently, TCH3 may perform a Ca(2+)-modulated function involved in generating changes in cells and/or tissues that result in greater strength or flexibility.     

Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response

An autophagy-related gene Atg8 was cloned for the first time from wild emmer wheat, named as TdAtg8, and its role on autophagy under abiotic stress conditions was investigated. Examination of TdAtg8 expression patterns indicated that Atg8 expression was strongly upregulated under drought stress, especially in the roots when compared to leaves. LysoTracker(?) red marker, utilized to observe autophagosomes, revealed that autophagy is constitutively active in Triticum dicoccoides. Moreover, autophagy was determined to be induced in plants exposed to osmotic stress when compared to plants grown under normal conditions. Functional studies were executed in yeast to confirm that the TdATG8 protein is functional, and showed that the TdAtg8 gene complements the atg8?::kan MX yeast mutant strain grown under nitrogen deficiency. For further functional analysis, TdATG8 protein was expressed in yeast and analyzed using Western immunoblotting. Atg8-silenced plants were exposed to drought stress and chlorophyll and malondialdehyde (MDA) content measurements demonstrated that Atg8 plays a key role on drought stress tolerance. In addition, Atg8-silenced plants exposed to osmotic stress were found to have decreased Atg8 expression level in comparison to controls. Hence, Atg8 is a positive regulator in osmotic and drought stress response.