AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana

Vacuolar autophagy is a major pathway by which eukaryotic cells degrade macromolecules, either to remove damaged or unnecessary proteins, or to produce respiratory substrates and raw materials to survive periods of nutrient deficiency. During autophagy, a double membrane forms around cytoplasmic components to generate an autophagosome, which is transported to the vacuole. The outer membrane fuses with the vacuole or lysosome, and the inner membrane and its contents are degraded by vacuolar or lysosomal hydrolases. We have identified a small gene family in Arabidopsis thaliana, members of which show sequence similarity to the yeast autophagy gene ATG18. Members of the AtATG18 gene family are differentially expressed in response to different growth conditions, and one member of this family, AtATG18a, is induced both during sucrose and nitrogen starvation and during senescence. RNA interference was used to generate transgenic lines with reduced AtATG18a expression. These lines show hypersensitivity to sucrose and nitrogen starvation and premature senescence, both during natural senescence of leaves and in a detached leaf assay. Staining with the autophagosome-specific fluorescent dye monodansylcadaverine revealed that, unlike wild-type plants, AtATG18a RNA interference plants are unable to produce autophagosomes in response to starvation or senescence conditions. We conclude that the AtATG18a protein is likely to be required for autophagosome formation in Arabidopsis.     

Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-AtATG8e fusion protein

Autophagy is a process that is thought to occur in all eukaryotes in which cells recycle cytoplasmic contents when subjected to environmental stress conditions or during certain stages of development. Upon induction of autophagy, double membrane-bound structures called autophagosomes engulf portions of the cytoplasm and transfer them to the vacuole or lysosome for degradation. In this study, we have characterized two potential markers for autophagy in plants, the fluorescent dye monodansylcadaverine (MDC) and a green fluorescent protein (GFP)-AtATG8e fusion protein, and propose that they both label autophagosomes in Arabidopsis. Both markers label the same small, apparently membrane-bound structures found in cells under conditions that are known to induce autophagy such as starvation and senescence. They are usually seen in the cytoplasm, but occasionally can be observed within the vacuole, consistent with a function in the transfer of cytoplasmic material into the vacuole for degradation. MDC-staining and the GFP-AtATG8e fusion protein can now be used as very effective tools to complement biochemical and genetic approaches to the study of autophagy in plant systems.     

Biosynthesis of unusual acyclic isoprenoids in the Alga Botryococcus braunii

A ‘resting state’ isolate of the hydrocarbon-producing alga Botryococcus braunii photoassimilated sodium [¹⁴C]bicarbonate at rates comparable to fast growing algae, such as Chlorella (> 1.50 μg atoms ¹⁴C/mg chlorophyll·hr). Early in the reaction (up to several min), most of the radioactivity was associated with water-soluble metabolites. However, labelling of hexane-soluble compounds steadily from ca 3% at 15 sec to over 50% of the total incorporated ¹⁴C at 60 min. The purified hexane fraction, which consisted of a series of botryococcenes and squalene, constituted a relatively constant proportion (40–45%) of the total hexane-soluble radioactivity at all but the earliest time points (< 60 sec). This fraction initially consisted almost exclusively of a C₃₀ botryococcene (ca 91%) and squalene (ca 8%); however, small amounts of radioactivity sequentially appeared in the C₃₁, C₃₂ and C₃₄ botryococcenes. The results of pulse-chase experiments implicated the C₃₀ botryococcene as the precursor of the higher homologues; during the chase, loss of radioactivity from the C₃₀ compound was accompanied by a concomitant increase in the labelling of the C₃₁ and C₃₂ compounds. This study provides further evidence that the relatively slow growth of Botryococcus in culture may result, in part, from the diversion of a large proportion of reduced carbon into energetically expensive compounds and that the slower growth rate in the ‘resting state’ cannot be totally attributed to an impaired or intrinsically slow metabolism.     

The stromal processing peptidase activities from Chlamydomonas reinhardtii and Pisum sativum: unexpected similarities in reaction specificity

We have partially purified the stromal processing peptidase from Chlamydomonas reinhardtii and compared the properties of this activity with those of the pea counterpart. Whereas previous studies have suggested that the two enzymes may have significantly different reaction specificities, we find that they are in fact very similar. Both enzymes process precursors of two higher-plant thylakoid lumen proteins, and one C. reinhardtii lumenal protein, to similar intermediate-size forms. However, whereas the algal enzyme processes the precursor of C. reinhardtii Rubisco small subunit to the correct mature size, this precursor is cleaved only to an intermediate size by the pea enzyme. The small subunit precursor from pea appears to be cleaved by both enzymes in a similar manner. In terms of sensitivity to inhibitors, the two activities are notably different; the pea enzyme has previously been shown to be inhibited by several types of heavy-metal chelator, but we have found that none of these compounds affect the algal activity.     

Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis

Upon encountering oxidative stress, proteins are oxidized extensively by highly reactive and toxic reactive oxidative species, and these damaged, oxidized proteins need to be degraded rapidly and effectively. There are two major proteolytic systems for bulk degradation in eukaryotes, the proteasome and vacuolar autophagy. In mammalian cells, the 20S proteasome and a specific type of vacuolar autophagy, chaperone-mediated autophagy, are involved in the degradation of oxidized proteins in mild oxidative stress. However, little is known about how cells remove oxidized proteins when under severe oxidative stress. Using two macroautophagy markers, monodansylcadaverine and green fluorescent protein-AtATG8e, we here show that application of hydrogen peroxide or the reactive oxidative species inducer methyl viologen can induce macroautophagy in Arabidopsis (Arabidopsis thaliana) plants. Macroautophagy-defective RNAi-AtATG18a transgenic plants are more sensitive to methyl viologen treatment than wild-type plants and accumulate a higher level of oxidized proteins due to a lower degradation rate. In the presence of a vacuolar H(+)-ATPase inhibitor, concanamycin A, oxidized proteins were detected in the vacuole of wild-type root cells but not RNAi-AtATG18a root cells. Together, our results indicate that autophagy is involved in degrading oxidized proteins under oxidative stress conditions in Arabidopsis.     

GROWTH AND BRANCHED HYDROCARBON PRODUCTION IN A STRAIN OF BOTRYOCOCCUS BRAUNII (CHLOROPHYTA)1

A strain Botryococcus braunii Kütz. that produces high levels of branched hydrocarbons (botryococcenes) was grown under different environmental conditions to investigate the relationship between growth and hydrocarbon production. Carbon dioxide concentration had the most significant influence on growth; 0.3% CO₂-enriched cultures demonstrated a minimum mass doubling time of ca. 40 h, compared to ca. 6 days for ambient air cultures grown on the same buffered growth medium. The botryococcene fraction, which consisted of 10 identified compounds (C_nH_2n-10; n = 30–34), usually constituted ca. 25–40% of the culture dry weight under various growth regimes, including nitrogen- and/or phosphate-deficiencies. CO₂ enrichment initially favored the production of the lower botryococcenes (C₃₀–C₃₂), whereas relatively slow-growing ambient air cultures accumulated C₃₃ and C₃₄ compounds. Colony color changed in response to different light intensities. High light increased the carotenoid/chlorophyll ratio, which resulted in orange colonies. Cultures exposed to low light intensity appeared green. This change in coloration was reversible over a period of a few days, and at no time were the linear hydrocarbons characteristic of the other form of the alga detected. Ostensible colony color is not, therefore, a reliable indicator of qualitative hydrocarbon content. Sequential solvent extraction experiments indicated that up to ca. 7% of the botryococcene fraction was intracellular and that the remainder was located within the colonial matrix. The internal (cellular) pool principally consisted of C₃₀–C₃₂ botryococcenes, whereas the external (colonial matrix) pool contained >99% of the C₃₃ and C₃₄ compounds, in addition to large amounts of the lower botryococcenes. These results, taken in conjunction with other data, are compatible with the hypothesis that the C₃₀ botryococcene is the precursor, presumably via methylation, of the higher botryococcenes.     

Regulation of ribulose 1,5-diphosphate carboxylase by substrates and other metabolites: further evidence for several types of binding sites

Ribulose 1,5-diphosphate carboxylase (RuDPCase, EC 4.1.1.39) isolated from spinach leaves is metabolically regulated at 10 mm Mg(2+) and low CO(2) concentrations by its substrates (RuDP and CO(2)) and by effectors which include 6-phosphogluconate (6-PGluA), NADPH, and fructose 1,6-diphosphate (FDP), but not fructose 6-phosphate. Physiological concentrations of RuDP severely inhibit the enzyme activity when the enzyme has not been preincubated with HCO(3) (-) and Mg(2-), and this inactivity persists for 20 minutes or longer after 1 mm HCO(3) (-) and 10 mm Mg(2+) are added. Maximum activity requires that the preincubation mixture also include either 0.01 mm 6-PGluA or 0.5 mm NADPH.When the enzyme, following preincubation with HCO(3) (-) and Mg(2+), is presented with RuDP plus either 6-PGluA or FDP, competitive inhibition is observed with respect to RuDP. The Ki value for 6-PGluA is 0.02 mm and the Ki value for FDP is 190 mum. NADPH or 3-phosphoglycerate (PGA) at physiological concentrations does not have any effect when presented simultaneously with RuDP. Other studies on the order of addition of substrates and effectors, concentration effects, and kinetics provide additional information that serves as a basis for a proposed model of allosteric regulation combined with competitive inhibition.In this model, there are catalytic sites at which the substrates and 6-PGluA and FDP can bind, and at least four allosteric regulatory sites, which we designate I, A(1), A(2), and A(3). RuDP binds very tightly to site I (in the absence of Mg(2+) or HCO(3) (-)), causing a conformational change in the protein to an inactive form which persists for as long as 20 minutes in the subsequent presence of Mg(2+) and 1 mm HCO(3) (-). Mg(2+) and HCO(3) (-) (or CO(2)) bind to site A(3) (in the absence of RuDP), holding the enzyme in an active form which has a much lower affinity for RuDP at site I, so that when physiological levels of RuDP are then added, only part of the enzyme activity is lost. This active form of the enzyme can bind 6-PGluA or FDP at site A(1) and NADPH at site A(2) during preincubation with Mg(2+) and HCO(3) (-). With optimal levels of bound effectors, 6-PGluA or NADPH, enzyme activity is fully maintained, even when RuDP is subsequently added. Without one of these effectors present, addition of RuDP following preincubation reduces enzyme activity to about 40% at the levels of substrates and effectors studied. FDP is a much poorer effector, and this is ascribed to a possible binding of FDP at site I, as well as at site A(1).The physiological role of this regulation is discussed, particularly with respect to protection of "C-3" plants against oxidation of RuDP to phosphoglycolate.     

Efficiency of Energy Conversion by Aerobic Glucose Metabolism in Aphanocapsa 6714

Efficiency of adenosine triphosphate (ATP) formation from glucose oxidation in Aphanocapsa 6714 was estimated by quantitative measurement of phosphorylated intermediary metabolites and glycogen (polyglucose) formed from (14)C-glucose. P/2e ratios based on oxygen uptake ranged from 2.62 to 3.08, whereas those based on (14)CO(2) evolution ranged from 1.66 to 1.72. The synthesis of glycogen, which is the dominant energy-consuming process in resting cells exposed to exogenous glucose, was almost totally inhibited under anaerobic conditions, and the cellular concentration of ATP decreased steadily. Thus, both net synthesis of ATP and the steady-state concentration of ATP are obligatorily linked to respiration in this heterotrophic unicellular blue-green alga.     

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly regulated by the ratio of NADPH/NADP⁺, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP⁺ ratio. With a ratio of NADPH/NADP⁺ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited.This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP⁺ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions.Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP⁺ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP⁺.It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP⁺ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolite unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP⁺ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.