The review emphasizes the essentiality of mitochondrial oxidative metabolism for photosynthetic carbon assimilation. Photosynthetic activity in chloroplasts and oxidative metabolism in mitochondria interact with each other and stimulate their activities. During light, the partially modified TCA cycle supplies oxoglutarate to cytosol and chloroplasts. The marked stimulation of O2 uptake after few minutes of photosynthetic activity, termed as light enhanced dark respiration (LEDR), is now a well-known phenomenon. Both the cytochrome and alternative pathways of mitochondrial electron transport are important in such interactions. The function of chloroplast is optimized by the complementary nature of mitochondrial metabolism in multiple ways: facilitation of export of excess reduced equivalents from chloroplasts, shortening of photosynthetic induction, maintenance of photorespiratory activity, and supply of ATP for sucrose biosynthesis as well as other cytosolic needs. Further, the mitochondrial oxidative electron transport and phosphorylation also protects chloroplasts against photoinhibition. Besides mitochondrial respiration, reducing equivalents (and ATP) are used for other metabolic phenomena, such as sulfur or nitrogen metabolism and photorespiration. These reactions often involve peroxisomes and cytosol. The beneficial interaction between chloroplasts and mitochondria therefore extends invariably to also peroxisomes and cytosol. While the interorganelle exchange of metabolites is the known basis of such interaction, further experiments are warranted to identify other biochemical signals between them. The uses of techniques such as on-line mass spectrometric measurement, novel mutants/transgenics, and variability in metabolism by growth conditions hold a high promise to help the plant biologist to understand this

The possible role of L-ascorbate (AsA) as a biochemical signal during the interactions between photosynthesis and respiration was examined in leaf discs of Arabidopsis thaliana. AsA content was either decreased as in AsA-deficient vtc1 mutants or increased by treatment with L-galactono-1, 4-lactone (L-GalL, a precursor of AsA; EC 1.3.2.3). In mutants, photosynthesis was extremely sensitive to both antimycin A (inhibitor of the cytochrome c oxidase pathway [COX pathway]) and salicylhydroxamic acid (SHAM, inhibitor of the alternative pathway [AOX pathway]), particularly at high light conditions. Mitochondrial inhibitors lowered the ratio of reduced AsA to total AsA, at high light, indicating oxidative stress in leaf discs. Elevation of AsA by L-GalL decreased the sensitivity of photosynthesis at high light to antimycin A or SHAM, sustained photosynthesis at supraoptimal light and relieved the extent of photoinhibition. High ratios of reduced AsA to total AsA in L-GalL-treated leaf discs suggests that L-GalL lowers oxidative stress. The protection by L-GalL of photosynthesis against the mitochondrial inhibitors and photoinhibition was quite pronounced in vtc1 mutants. Our results suggest that the levels and redox state of AsA modify the pattern of modulation of photosynthesis by mitochondrial metabolism. The extent of the AOX pathway as a percentage of the total respiration in Arabidopsis mesophyll protoplasts was much higher in vtc1 than in wild type. We suggest that the role of AsA becomes pronounced at high light and/or when the AOX pathway is inhibited. While acknowledging the importance of the COX pathway, we hypothesize that AsA and the AOX pathway may complement each other to protect photosynthesis against photoinhibition.

The transcription factor MYC regulates cell survival and growth, and its level is tightly controlled in normal cells. We report that serine pyrophosphorylation – a posttranslational modification triggered by inositol pyrophosphate signaling molecules – controls MYC levels via regulated protein degradation. We find that endogenous MYC is stabilized and less polyubiquitinated in cells with reduced inositol pyrophosphates. We show that the inositol pyrophosphate 5-IP7 transfers its high-energy beta phosphate moiety to pre-phosphorylated serine residues in the central PEST domain of MYC. Loss of serine pyrophosphorylation in the PEST domain lowers the extent of MYC polyubiquitination and increases its stability. Fusion to the MYC PEST domain lowers the stability of GFP, but this effect is dependent on the extent of PEST domain pyrophosphorylation. The E3 ubiquitin ligase FBW7 can bind directly to the PEST domain of MYC, and this interaction is exclusively dependent on serine pyrophosphorylation. A stabilized, pyrophosphorylation-deficient form of MYC increases cell death during growth stress in untransformed cells. Splenocytes from mice lacking IP6K1, a kinase responsible for the synthesis of 5-IP7, have higher levels of MYC, and show increased cell proliferation in response to mitogens, compared with splenocytes from wild type mice. Thus, control of MYC stability through a novel pyro-phosphodegron provides unexpected insight into the regulation of cell survival in response to environmental cues.

Protein Charge Transfer Spectra (ProCharTS) originate when charged amino/carboxylate groups in the side chains of Lys/Glu act as electronic charge acceptors/donors for photoinduced charge transfer either from/to the polypeptide backbone or to each other. The absorption band intensities in ProCharTS at wavelengths of 250-800 nm are dependent on the 3D spatial proximity of these charged functional groups across the protein. Intrinsically disordered proteins (IDPs) are an important class of proteins involved in signalling and regulatory functions in the eukaryotic cell. IDPs are rich in charged amino acids, but lack structure-promoting intrinsic spectral probes like Tyr or Trp in their sequences, making their structural characterisation difficult. Here, we exploit the richness of charged amino acid populations among IDPs (like the PEST fragment of human c-Myc, its mutant and dehydrin from maize) to sense structural transitions in IDPs using ProCharTS absorption spectra. Conformational changes induced in the protein by altering the pH and temperature of the aqueous medium were monitored by ProCharTS and confirmed by CD spectra. Further, the utility of ProCharTS to detect protein aggregation was examined using Hen Egg-White Lysozyme (HEWL) protein. The results revealed that in the presence of Trp/Tyr, ProCharTS absorbance was substantially reduced, specifically at wavelengths where the absorption by Trp or Tyr was near its maximum. Significant changes in the ProCharTS spectra were observed with changing pH in the range of 3-11, which correlated with changes in the secondary structure of the PEST fragment. Importantly, the absorbance at 280 nm, which is often employed as a measure of protein concentration, was profoundly altered by changes in ProCharTS intensity in response to changing the pH in dehydrin. The ProCharTS intensity was sensitive to temperature-induced changes in the secondary structures of the PEST fragments between 25-85 °C. The presence of 0.25 M NaCl or KCl in the medium also altered the ProCharTS spectrum. Finally, an increase in ProCharTS absorbance with time in HEWL at pH 2 directly correlated with the growth of HEWL aggregates and amyloid fibrils, as confirmed by the increasing thioflavin T fluorescence. Taken together, our work highlights the utility of ProCharTS as a label-free intrinsic probe to monitor changes in protein charge, structure and oligomeric state.

A simple and quick method is described for rapid isolation of metabolically active mesophyll protoplasts from leaves of Arabidopsis thaliana. The optimal composition of the digestion medium, period of digestion and stability of protoplast preparation were examined. A large number of protoplasts could be prepared within an hour. The isolated protoplasts were intact, stable and metabolically very active, as indicated by their high rates of photosynthetic oxygen evolution. The important factors during the preparation of protoplasts are short time of digestion, composition of medium, use of nylon nets for filtration, centrifugation at low speed and use of pH 7.0 for storage. The highest rate of photosynthesis obtained in these experiments was 130 ± 4 μmol O2 evolved mg−1 Chl h−1, at 1 mM sodium bicarbonate and at a light intensity of 600 μE m−2 s−1. The present technique of isolation can be very useful for making Arabidopsis protoplasts for studies on not only metabolic processes, such as photosynthesis, but also metabolomics, proteomics and genomics.