Metabolism of starch is a major biological integrator of plant growth supporting nocturnal energy dynamics by transitory starch degradation as well as periods of dormancy, re-growth, and reproduction by utilization of storage starch. Especially, the extraordinarily well-tuned and coordinated rate of transient starch biosynthesis and degradation suggests the presence of very sophisticated regulatory mechanisms. Together with the circadian clock, land plants (being autotrophic and sessile organisms) need to monitor, sense, and recognize the photosynthetic rate, soil mineral availability as well as various abiotic and biotic stress factors. Currently it is widely accepted that post-translational modifications are the main way by which the diel periodic activity of enzymes of transient starch metabolism are regulated. Among these mechanisms, thiol-based redox regulation is suggested to be of fundamental importance and in chloroplasts, thioredoxins (Trx) are tightly linked up to photosynthesis and mediate light/dark regulation of metabolism. Also, light independent NADP-thioredoxin reductase C (NTRC) plays a major role in reactive oxygen species scavenging. Moreover, Trx and NTRC systems are interconnected at several levels and strongly influence each other. Most enzymes involved in starch metabolism are demonstrated to be redox-sensitive in vitro. However, to what extent their redox sensitivity is physiologically relevant in synchronizing starch metabolism with photosynthesis, heterotrophic energy demands, and oxidative protection is still unclear. For example, many hydrolases are activated under reducing (light) conditions and the strict separation between light and dark metabolic pathways is now challenged by data suggesting degradation of starch during the light period.
Water shortage is an increasing problem affecting crop yield. Accumulation of compatible osmolytes is a typical plant response to overcome water stress. Sucrose synthase 1 (SUS1), and glucan, water dikinase 2 (GWD2) and δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1) are members of small protein families whose role in the response of Arabidopsis thaliana plants to mild osmotic stress has been studied in this work. Comparative analysis between wild-type and single loss-of-function T-DNA plants at increasing times following exposure to drought showed no differences in the content of water-insoluble carbohydrate (i.e., transitory starch and cell wall carbohydrates) and in the total amount of amino acids. On the contrary, water-soluble sugars and proline contents were significantly reduced compared to wild-type plants regardless of the metabolic pathway affected by the mutation. The present results contribute to assigning a physiological role to GWD2, the least studied member of the GWD family; strengthening the involvement of SUS1 in the response to osmotic stress; showing a greater contribution of soluble sugars than proline in osmotic adjustment of Arabidopsis in response to drought. Finally, an interaction between proline and soluble sugars emerged, albeit its nature remains speculative and further investigations will be required for complete comprehension.
In land plants and algae, the Calvin–Benson (CB) cycle takes place in the chloroplast, a specialized organelle in which photosynthesis occurs. Thioredoxins (TRXs) are small ubiquitous proteins, known to harmonize the two stages of photosynthesis through a thiol-based mechanism. Among the 11 enzymes of the CB cycle, the TRX target phosphoribulokinase (PRK) has yet to be characterized at the atomic scale. To accomplish this goal, we determined the crystal structures of PRK from two model species: the green alga Chlamydomonas reinhardtii (CrPRK) and the land plant Arabidopsis thaliana (AtPRK). PRK is an elongated homodimer characterized by a large central β-sheet of 18 strands, extending between two catalytic sites positioned at its edges. The electrostatic surface potential of the catalytic cavity has both a positive region suitable for binding the phosphate groups of substrates and an exposed negative region to attract positively charged TRX-f. In the catalytic cavity, the regulatory cysteines are 13 Å apart and connected by a flexible region exclusive to photosynthetic eukaryotes—the clamp loop—which is believed to be essential for oxidation-induced structural rearrangements. Structural comparisons with prokaryotic and evolutionarily older PRKs revealed that both AtPRK and CrPRK have a strongly reduced dimer interface and an increased number of random-coiled regions, suggesting that a general loss in structural rigidity correlates with gains in TRX sensitivity during the molecular evolution of PRKs in eukaryotes.
31In land plants and algae, the Calvin-Benson (CB) cycle takes place in the chloroplast, a specialized 32 organelle in which photosynthesis occurs. Thioredoxins (TRXs) are small ubiquitous proteins, 33 known to harmonize the two stages of photosynthesis through a thiol-based mechanism. Among the 34 11 enzymes of the CB cycle, the TRX target phosphoribulokinase (PRK) has yet to be characterized 35 at the atomic scale. To accomplish this goal, we determined the crystal structures of PRK from two 36 model species: the green alga Chlamydomonas reinhardtii (CrPRK) and the land plant Arabidopsis 37 thaliana (AtPRK). PRK is an elongated homodimer characterized by a large central β-sheet of 18 38 strands, extending between two catalytic sites positioned at its edges. The electrostatic surface 39 potential of the catalytic cavity has both a positive region suitable for binding the phosphate groups 40 of substrates and an exposed negative region to attract positively charged TRX-f. In the catalytic 41 cavity, the regulatory cysteines are 13 Å apart and connected by a flexible region exclusive to 42 photosynthetic eukaryotes-the clamp loop-which is believed to be essential for oxidation-43 induced structural rearrangements. Structural comparisons with prokaryotic and evolutionarily older 44 PRKs revealed that both AtPRK and CrPRK have a strongly reduced dimer interface and increased 45 number of random coiled regions, suggesting that a general loss in structural rigidity correlates with 46 gains in TRX sensitivity during the molecular evolution of PRKs in eukaryotes. 47 48 In chloroplasts, five enzymes of the Calvin-Benson (CB) cycle are regulated by thioredoxins 53 (TRXs). These enzymes have all been structurally characterized with the notable exception of 54 phosphoribulokinase (PRK). Here, we determined the crystal structure of chloroplast PRK from two 55 model photosynthetic organisms. Regulatory cysteines appear distant from each other and are 56 4 linked by a long loop that is present only in plant-type PRKs and allows disulfide bond formation 57 and subsequent conformational rearrangements. Structural comparisons with ancient PRKs indicate 58that the presence of flexible regions close to regulatory cysteines is a unique feature that is shared 59 by TRX-dependent CB cycle enzymes, suggesting that the evolution of the PRK structure has 60 resulted in a global increase in protein flexibility for photosynthetic eukaryotes. 61 62 235 flexibility required to approach the substrates (8). Finally, we report that the regulatory pair of 236 cysteines in PRK is embedded in the substrate-binding site and that oxidation can be achieved by 237 the presence of the flexible clamp loop. 238 11We conclude that a common strategy has allowed the acquisition of TRX dependence of the CB 239 cycle enzymes during evolution-the introduction of flexible regions. This strategy is in sharp 240 contrast to the evolutionary rigidity of the TRX structure. 241 242 MATERIALS AND METHODS 243 Protein Expression and Purification 244 Recombinant A...
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