Evidence for autophagy-dependent pathways of rRNA turnover in Arabidopsis

Ribosomes account for a majority of the cell''s RNA and much of its protein and represent a significant investment of cellular resources. The turnover and degradation of ribosomes has been proposed to play a role in homeostasis and during stress conditions. Mechanisms for the turnover of rRNA and ribosomal proteins have not been fully elucidated. We show here that the RNS2 ribonuclease and autophagy participate in RNA turnover in Arabidopsis thaliana under normal growth conditions. An increase in autophagosome formation was seen in an rns2–2 mutant, and this increase was dependent on the core autophagy genes ATG9 and ATG5. Autophagosomes and autophagic bodies in rns2–2 mutants contain RNA and ribosomes, suggesting that autophagy is activated as an attempt to compensate for loss of rRNA degradation. Total RNA accumulates in rns2–2, atg9–4, atg5–1, rns2–2 atg9–4, and rns2–2 atg5–1 mutants, suggesting a parallel role for autophagy and RNS2 in RNA turnover. rRNA accumulates in the vacuole in rns2–2 mutants. Vacuolar accumulation of rRNA was blocked by disrupting autophagy via an rns2–2 atg5–1 double mutant but not by an rns2–2 atg9–4 double mutant, indicating that ATG5 and ATG9 function differently in this process. Our results suggest that autophagy and RNS2 are both involved in homeostatic degradation of rRNA in the vacuole.     

Regulatory effects of ammonia on carbon metabolism in photosynthesizing Chlorella pyrenoidosa

Addition of ammonia to Chlorella pyrenoidosa, photosynthesizing under steady-state conditions, causes changes in the metabolism which are due not only to the increased availability of NH₄⁺ for reductive amination but also to regulation of controlled enzymes. One such effect is an increased rate of the reaction which converts phosphoenolpyruvate to pyruvate in vivo. This regulatory effect was revealed by kinetic tracer studies with ¹⁴CO₂, paper chromatography and radioautographic analysis, which showed that upon addition of NH₄⁺ (1) the levels of both 3-phosphoglycerate and phosphoenolpyruvate drop, with the ratio of 3-phosphoglycerate/phosphoenolpyruvate increasing, (2) the level of labeled pyruvic acid increases and the rate of formation of alanine increases rapidly, while the rate of formation of serine is unaffected, (3) the rate of flow of carbon into the tricarboxylic acid cycle acids, malate and citrate, increases along with the increased rates of formation of glutamate, glutamine and aspartate and (4) the rate of labeling of lipids increases. The increased flow of carbon into amino acids is mostly at the expense of sucrose synthesis; starch synthesis decreases only slightly. The interruption of sucrose synthesis apparently is due to stopping the reaction between UDP-glucose and fructose 6-phosphate. The rate of conversion of fructose 1,6-diphosphate to fructose 6-phosphate is also decreased upon NH₄⁺ addition.     

Appearance and accumulation of c(4) carbon pathway enzymes in developing wheat leaves

Soluble protein has been extracted from sections of wheat leaves, from base to tip, and the content of several key enzymes of photosynthetic carbon assimilation in each section has been determined by the protein blot method. In the first leaf, ribulose 1,5-bisphosphate carboxylase (RuBPC) (EC 4.1.1.39) in the basal 0 to 1 centimeter section is about 12% the level in the tip section, whereas phosphoenolpyruvate carboxylase (EC 4.1.1.31) is present in small amounts in the basal section and does not change much in the tip. Pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) first appears in the 4 to 6 centimeter section and increases gradually with development to 10-fold in the tip. Malic enzyme, NADP-dependent (EC 1.3.1.37) also appears in the 4 to 6 centimeter section but remains low to the tip.

Fixation of ¹⁴CO₂ by wheat leaf base sections resulted in 42% of total incorporation into malate and aspartate, indicating β-carboxylation, whereas in the tip section these labeled compounds were only 8% of the total. Although the amount of PPDK in wheat leaves is only 1 to 3% of that in maize leaves, this C₃ PPDK may have a limited role in photosynthesis leading to formation of C₄ compounds. The possibility of a further role, similar to that in C₄ plants, but for intracellular carbon transport in wheat leaves is discussed. The presence of malic dehydrogenase, NADP-specific (EC 1.1.1.82) in wheat leaf chloroplasts was shown, a necessary though not sufficient condition for such a proposed role. Assuming each of the four enzymes associated with C₄ carbon transport were fully active in vivo during photosynthesis, PPDK would still be rate limiting, even in the leaf tip where its activity is maximal. Possible evolutionary and breeding implications are discussed.

     

Variation in amounts of pyruvate, orthophosphate dikinase, and some other enzymes of the c(4) pathway in some wheat species

First leaves and flag leaves of the wheat species Triticum aestivum cv Anza (6×), T. boeoticum Boiss (2×) L. were examined for content of pyruvate, orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and ribulose 1,5-bisphosphate carboxylase (RuBPC) by protein blot analyses using antibodies to maize leaf enzymes and by activity assays. In agreement with previous reports, the amount of RuBPC per mesophyll cell was about 3 times more in the hexaploid species, T. aestivum, than in the diploid species, T. boeoticum, both in first leaves and in flag leaves. In contrast, the level of PPDK polypeptide was nearly 3-fold higher per unit leaf area in the first leaf and 63% higher in the flag leaf of this diploid species compared to this hexaploid species. There was no significant difference in the levels of polypeptide and enzyme activity of PEPC between diploid and hexaploid wheat. Despite this significantly greater level of PPDK in the diploid species, the actual amount of PPDK could still supply only a limited amount of the enzyme activity necessary to provide phosphoenolpyruvate (PEP) for any putative intracellular C₄ carbon shuttle providing carbon to RuBPC. Thus, this difference in enzyme amount could not by itself account for the reported high rates of net photosynthesis at high light intensity in T. boeoticum. Together with reported anatomical differences between the diploid and hexaploid species, however, this biochemical difference may be of physiological importance.     

Pyruvate orthophosphate dikinase: intracellular site of synthesis in maize leaf cells

Pyruvate orthophosphate dikinase is synthesized in non-green leaf cells of the maize mutant iojap. Since iojap plastids lack ribosomes, it is concluded that the site of synthesis of pyruvate orthophosphate dikinase in maize leaf cells is on ribosomes in the cytoplasm.     

Photosynthate supply and utilization in alfalfa : a developmental shift from a source to a sink limitation of photosynthesis

Long-term carbon dioxide enrichment, ¹⁴CO₂ feeding, and partial defoliation were employed as probes to investigate source/sink limitations of photosynthesis during the development of symbiotically grown alfalfa. In the mature crop, long-term CO₂ enrichment does not affect the rates of net photosynthesis, relative growth, ¹⁴C export to nonphotosynthetic organs, or the rates of ¹⁴C label incorporation into leaf sucrose, starch, or malate. The rate of glycolate labeling is, however, substantially reduced under these conditions. When the mature crop was partially defoliated, a considerable increase in net photosynthesis occurred in the remaining leaves. In the seedling crop, long-term CO₂ enrichment increased dry matter accumulation, primarily as a result of increases in leaf starch content. Although the higher rates of starch synthesis are not maintained, the growth enhancement of the enriched plants persisted throughout the experimental period. These results imply a source limitation of seedling photosynthesis and a sink limitation of photosynthesis in more mature plants. Consequently, both the supply and the utilization of photosynthate may limit seasonal photosynthesis in alfalfa.     

Glycolic Acid Labeling During Photosynthesis with CO(2) and Tritiated Water

Chlorella pyrenoidosa were allowed to photosynthesize for short periods of time in the presence of ¹⁴CO₂ and HTO. Analysis of tritium and ¹⁴C labeling of photosynthetic intermediate compounds showed that the T/¹⁴C ratio of glycolic acid was comparable to that of intermediate compounds of the photosynthetic carbon reduction cycle when photosynthesis was performed in nearly 100% oxygen and only slightly higher under steady-state conditions. It is concluded that formation of labeled glycolic acid as a consequence of its proposed hydrogen transport role in photosynthesis is quantitatively of limited importance compared to the net synthesis of glycolic acid from CO₂.     

Kinetics of Glucose Incorporation by Aphanocapsa 6714

Photoautotrophic metabolism of CO(2) was compared with glucose metabolism in the facultative unicellular blue-green alga, Aphanocapsa 6714. Glucose-fed cells incorporated more (14)C into phosphorylated sugar intermediates of the reductive and oxidative pentose phosphate cycles than autotrophic cells. The relative increases were: 140-fold in dark cells; 32-fold in dichlorophenylmethylurea (DCMU)-inhibited cells; and 16-fold in cells assumilating glucose during photosynthetic carbon reduction. On the other hand, incorporation of (14)C from glucose into 3-phosphoglycerate and the amino acid pools of glutamate and aspartate was reduced in dark cells. Rates of protein synthesis in dark and DCMU-inhibited cells were reduced 50 and 80% compared to photoautotrophic cells. In cells assimilating glucose during photosynthesis, rates of (14)C incorporation into the two amino acids and protein were the same as in photoautotrophic cells. Chase experiments, using an excess of (12)C-glucose and CO(2), revealed slow turnover of carbon in dark cells and intermediate turnover rates in DCMU-inhibited cells, when compared to cells assimilating glucose during photosynthesis.     

Sequence of Formation of Phosphoglycolate and Glycolate in Photosynthesizing Chlorella pyrenoidosa

In Chlorella pyrenoidosa which have been photosynthesizing in either 1.5% ¹⁴CO₂ or 0.05% ¹⁴CO₂ in air, gassing with 100% O₂ results in rapid formation of phosphoglycolate which is apparently converted to glycolate. However, only about one-third to one-half of the rate of glycolate formation can be accounted for by this route. The remaining glycolate formation may be the result of the oxidation of sugar monophosphates. The rates of formation of both glycolate and phosphoglycolate are about four times greater with algae that have been photosynthesizing in 1.5% ¹⁴CO₂ than with algae which have been photosynthesizing with air, when the algae are then gassed with 100% O₂.     

Activation and inhibition of ribulose 1,5-diphosphate carboxylase by 6-phosphogluconate

Ribulose 1,5-diphosphate carboxylase, when activated by preincubation with 1 mm bicarbonate and 10 mm MgCl₂ in the absence of ribulose 1,5-diphosphate, remains activated for 20 minutes or longer after reaction is initiated by addition of ribulose diphosphate. If as little as 50 μm 6-phosphogluconate is added during this preincubation period, 5 minutes before the start of the reaction, a further 188% activation is observed. However, addition of 6-phosphogluconate at the same time or later than addition of ribulose diphosphate, or at any time with 50 mm bicarbonate, gives inhibition of the enzyme activity. Possible relevance of these effects in vivo regulatory effects is discussed.