Aspartic-acid synthesis in C3 plants

In a previous study (Melzer and O'Leary, 1987, Plant Physiol. 84, 58–60), we used isotopic methods to show that a substantial fraction of protein-bound aspartic acid in tobacco is derived from anaplerotic synthesis via phosphoenolpyruvate (PEP) carboxylase. Similar studies in soybean (Glycine max L.) and spinach (Spinacia oleracea L.) showed a similar pattern, and this pattern persists with age because of slow protein turnover. A more quantitative analysis indicates that about 40% of protein-bound aspartate is derived in this manner. Analyses of free aspartic and malic acids show that contribution of PEP carboxylase to the synthesis of these acids decreases with increasing age. The C₄ plant Zea mays L. did not show this pattern.Abbreviations and Symbols RuBP ribulose bisphosphate - PEP phosphoenolpyruvate - OAA oxaloacetic acid - PGA 3-phosphoglyceric acid - ¹³C carbon-13 - isotopic content [R(sample)/R(standard)-1] × 1000, where R = [¹³CO₂]/[¹²CO₂] This work was supported by contract DE-ACO₂-83ER 13076 and grant DE-FGO₂-86ER13534 from the U.S. Department of Energy. E. M. was supported by a fellowship from Deutsche Forschungsgemeinschaft. We are grateful to Isabel Treichel for assistance with isotopic analyses.     

Disruption of microtubules in plants suppresses macroautophagy and triggers starch excess-associated chloroplast autophagy

Microtubules, the major components of cytoskeleton, are involved in various fundamental biological processes in plants. Recent studies in mammalian cells have revealed the importance of microtubule cytoskeleton in autophagy. However, little is known about the roles of microtubules in plant autophagy. Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana. Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy. Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts. Our findings reveal that an intact microtubule network is important for efficient macroautophagy and leaf starch degradation.     

A possible rate-limiting function of chloroplast hexosemonophosphate isomerase in starch synthesis of leaves

Levels of fructose 6-phosphate and glucose 6-phosphate were measured in chloroplasts which had been isolated non-aqueously from leaves of various plants. a large decrease in the ratio of glucose 6-phosphate to fructose 6-phosphate in the light indicated considerable displacement of the hexosephosphate isomerase reaction from equilibrium in leaves of spinach and red beet which were photosynthesizing at high rates. The decrease in the ratio of glucose 6-phosphate to fructose 6-phosphate was correlated with an increase in the chloroplastic level of 3-phosphoglyceric acid, which proved to be a competitive inhibitor of chloroplast hexosephosphate isomerase. Other metabolites, especially the product of the reaction, glucose 6-phosphate, and ions in concentrations as present in the stroma under natural conditions, cause a further reduction in the rate of the forward reaction of the hexosemonophosphate isomerase. When the concentration of O₂ in air was decreased from 21 to 2%, both the rate of leaf photosynthesis and the ratio of glucose 6-phosphate to fructose 6-phosphate increased, whereas the concentration of 3-phosphoglyceric acid and starch synthesis decreased. The results are explained in terms of activation of ADPglucose pyrophosphorylase and of inhibition of hexosephosphate isomerase by 3-phosphoglyceric acid. Hexosephosphate isomerase appears to assume a rate-limiting function in starch synthesis in the light when ADPglucose pyrophosphorylase is activated.     

Mutations affecting starch synthase III in Arabidopsis alter leaf starch structure and increase the rate of starch synthesis

The role of starch synthase (SS) III (SSIII) in the synthesis of transient starch in Arabidopsis (Arabidopsis thaliana) was investigated by characterizing the effects of two insertion mutations at the AtSS3 gene locus. Both mutations, termed Atss3-1 and Atss3-2, condition complete loss of SSIII activity and prevent normal gene expression at both the mRNA and protein levels. The mutations cause a starch excess phenotype in leaves during the light period of the growth cycle due to an apparent increase in the rate of starch synthesis. In addition, both mutations alter the physical structure of leaf starch. Significant increases were noted in the mutants in the frequency of linear chains in amylopectin with a degree of polymerization greater than approximately 60, and relatively small changes were observed in chains of degree of polymerization 4 to 50. Furthermore, starch in the Atss3-1 and Atss3-2 mutants has a higher phosphate content, approximately two times that of wild-type leaf starch. Total SS activity is increased in both Atss3 mutants and a specific SS activity appears to be up-regulated. The data indicate that, in addition to its expected direct role in starch assembly, SSIII also has a negative regulatory function in the biosynthesis of transient starch in Arabidopsis.     

Overexpression of 3-ketoacyl-acyl-carrier protein synthase IIIs in plants reduces the rate of lipid synthesis

A cDNA coding for 3-ketoacyl-acyl-carrier protein (ACP) synthase III (KAS III) from spinach (Spinacia oleracea; So KAS III) was used to isolate two closely related KAS III clones (Ch KAS III-1 and Ch KAS III-2) from Cuphea hookeriana. Both Ch KAS IIIs are expressed constitutively in all tissues examined. An increase in the levels of 16:0 was observed in tobacco (Nicotiana tabacum, WT-SR) leaves overexpressing So KAS III when under the control of the cauliflower mosaic virus-35S promoter and in Arabidopsis and rapeseed (Brassica napus) seeds overexpressing either of the Ch KAS IIIs driven by napin. These data indicate that this enzyme has a universal role in fatty acid biosynthesis, irrespective of the plant species from which it is derived or the tissue in which it is expressed. The transgenic rapeseed seeds also contained lower levels of oil as compared with the wild-type levels. In addition, the rate of lipid synthesis in transgenic rapeseed seeds was notably slower than that of the wild-type seeds. The results of the measurements of the levels of the acyl-ACP intermediates as well as any changes in levels of other fatty acid synthase enzymes suggest that malonyl-ACP, the carbon donor utilized by all the 3- ketoacyl-ACP synthases, is limiting in the transgenic plants. This further suggests that malonyl-coenzyme A is a potential limiting factor impacting the final oil content as well as further extension of 16:0.     

Characterization of starch breakdown in the intact spinach chloroplast

Starch degradation with a rate of 1 to 2 microgram-atom carbon per milligram chlorophyll per hour was monitored in the isolated intact spinach (Spinacia oleracea) chloroplast which had been preloaded with ¹⁴C-starch photosynthetically from ¹⁴CO₂. Starch breakdown was dependent upon inorganic phosphate and the ¹⁴C-labeled intermediates formed were principally those of the Embden-Meyerhof pathway from glucose phosphate to glycerate 3-phosphate. In addition, isotope was found in ribose 5-phosphate and in maltose and glucose. The appearance of isotope in the intermediates of the Embden-Meyerhof pathway but not in the free sugars was dependent upon the inorganic phosphate concentration. Dithiothreitol shifted the flow of ¹⁴C from triose-phosphate to glycerate 3-phosphate. Iodoacetic acid inhibited starch breakdown and caused an accumulation of triose-phosphate. This inhibition of starch breakdown was overcome by ATP. The inhibitory effect of ionophore A 23187 on starch breakdown was reversed by the addition of magnesium ions. The formation of maltose but not glucose was impaired by the ionophore. The inhibition of starch breakdown by glycerate 3-phosphate was overcome by inorganic phosphate. Fructose 1,6-bisphosphate and ribose 5-phosphate did not affect the rate of polysaccharide metabolism but increased the flow of isotope into maltose. Starch breakdown was unaffected by the uncoupler (trifluoromethoxyphenylhydrazone), electron transport inhibitors (rotenone, cyanide, salicylhydroxamic acid), or anaerobiosis. Hexokinase and the dehydrogenases of glucose 6-phosphate and gluconate 6-phosphate were detected in the chloroplast preparations. It was concluded (a) that chloroplastic starch was degraded principally by the Embden-Meyerhof pathway and by a pathway involving amylolytic cleavage; (b) ATP required in the Embden-Meyerhof pathway is generated by substrate phosphorylation in the oxidation of glyceraldehyde 3-phosphate to glycerate 3-phosphate; and (c) the oxidative pentose phosphate pathway is the probable source of ribose 5-phosphate.     

Control of PAR-saturated CO2 exchange rate in some C3 and CAM plants

We measured PAR-saturated CO₂ exchange rate (CER), and leaf N, P and chlorophyll (Chl) concentrations in 21 plant species, selected to encompass as broad a range in specific leaf area (SLA) as possible, and encompassing non-succulent C3 as well as succulent CAM plants. We worked with plants growing under uniform conditions in the facilities of a biological research station to ensure that any correlations found were due to inherent, genetically controlled, relationships between the measured parameters and not due to variations in resource availability in different habitats. We found CER to be strongly correlated to SLA, leaf N concentration and Chl concentration. CER increased much faster with increasing leaf N concentration (CER ≈ N^3.1) than with increasing SLA (CER ≈ SLA^1.2). CER also increased much faster with leaf N concentration than with increasing Chl concentration (CER ≈ Chl^1.3), indicating the photosynthetic N-use efficiency (NUE) to be higher for plants with high N concentration than for plants with low N concentration (NUE ≈ N^2.1). Analysis of covariance showed that these relationships exist even when comparing plants of widely different growth forms - succulent or non-succulent, and of different photosynthetic pathways, as the C3 and CAM plants compared here. Testing against scaling coefficients calculated using dimensional analysis, showed that the scaling of N, Chl and CER against SLA was not merely a result of diluting N and Chl with carbon in thicker leaves but that SLA, probably through influencing light absorptio and/or CO2 diffusion pathway, played an independent role in controlling CER. This revised version was published online in July 2006 with corrections to the Cover Date.     

Localisation of sucrose-phosphate synthase and starch in leaves of C4 plants

The activity and intercellular distribution of sucrose-phosphate synthase (SPS; EC 2.4.1.14) were determined in fully expanded leaves from a range of C₄ plants. In Zea mays L. and Atriplex spongiosa F. Muell., SPS was located almost exclusively in the mesophyll cells. In other species, SPS was found in both cell types, with the activity in the bundle sheath cells ranging from 5% of the total leaf activity in Echinochloa crus-galli (L.) Beauv. to 35% in Sorghum bicolor Moench. At the end of the light period, starch was found only in the bundle sheath cells in all of the species examined. There appears to be little correlation between C₄-acid decarboxylation type and the location of sucrose and starch synthesis in the leaves of C₄ plants. Received: 18 October 1996 / Accepted: 20 November 1996     

Role of procollagen mRNA levels in controlling the rate of procollagen synthesis.

Two factors must be present for primary avian tendon cells to commit 50% of their total protein production to procollagen: ascorbate and high cell density. Scorbutic primary avian tendon cells at high cell density (greater than 4 X 10(4) cells per cm2) responded to the addition of ascorbate by a sixfold increase in the rate of procollagen synthesis. The kinetics were biphasic, showing a slow increase during the first 12 h followed by a more rapid rise to a maximum after 36 to 48 h. In contrast, after ascorbate addition, the level of accumulated cytoplasmic procollagen mRNA (alpha 2) showed a 12-h lag followed by a slow linear increase requiring 60 to 72 h to reach full induction. At all stages of the induction process, the relative increase in the rate of procollagen synthesis over the uninduced state exceeded the relative increase in the accumulation of procollagen mRNA. A similar delay in mRNA induction was observed when the cells were grown in an ascorbate-containing medium but the cell density was allowed to increase. In all cases, the rate of procollagen synthesis peaked approximately 24 h before the maximum accumulation of procollagen mRNA. The kinetics for the increase in procollagen synthesis are not, therefore, in agreement with the simple model that mRNA levels are the rate-limiting factor in the collagen pathway. We propose that the primary control point is at a later step. Further support for this idea comes from inhibitor studies, using alpha, alpha'-dipyridyl to block ascorbate action. In the presence of 0.3 mM alpha, alpha'-dipyridyl there was a specific two- to threefold decrease in procollagen production after 4 h, but this was unaccompanied by a drop in procollagen mRNA levels. Therefore, inhibitor studies give further support to the idea that primary action of ascorbate is to release a post-translational block.     

Photosynthesis in intact leaves of C3 plants: Physics,physiology and rate limitations

The instantaneous rate of photosynthetic CO₂ assimilation in C₃ plants has generally been studied in model systems such as isolated chloroplasts and algae. From these studies and from theoretical analyses of gas exchange behavior it is now possible to study the biochemistry of photosynthesis in intact leaves using a combination of methods, most of which are nondestructive. The limitations to the rate of photosynthesis can be divided among three general classes: (1) the supply or utilization of CO₂, (2) the supply or utilization of light, and (3) the supply or utilization of phosphate. The first limitation is most readily studied by determining how the CO₂ assimilation rate varies with the partial pressure of CO₂ inside the leaf. The second limitation can be studied by determining the quantum requirement of photosynthesis. The third limitation is most easily detected as a loss of O₂ sensitivity of photosynthesis. Measurement of fluorescence from intact leaves can give additional information about the various limitations. These methods are all non-destructive and so can be observed repeatedly as the environment of a leaf is changed. In addition, leaves can be quick-frozen and metabolite concentrations then measured to give more information about the limitations to intact leaf photosynthesis rates. In this review the physics and biochemistry of photosynthesis in intact C₃ leaves, and the interface between physiology and photosynthesis—triose phosphate utilization—are discussed.     

Biochar amendment boosts photosynthesis and biomass in C3 but not C4 plants: A global synthesis

Biochar is a carbon (C)‐rich solid produced from the thermochemical pyrolysis of biomass. Its amendment to soils has been proposed as a promising mean to mitigate greenhouse gas emissions and simultaneously benefit agricultural crops. However, how biochar amendment affects plant photosynthesis and growth remains unclear, especially on a global scale. In this study, we conducted a global synthesis of 74 publications with 347 paired comparisons to acquire an overall tendency of plant photosynthesis and growth following biochar amendment. Overall, we found that biochar amendment significantly increased photosynthetic rate by 27.1%, and improved stomatal conductance, transpiration rate, water use efficiency, and chlorophyll concentration by 19.6%, 26.9%, 26.8%, and 16.1%, respectively. Meanwhile, plant total biomass, shoot biomass, and root biomass increased by 25.4%, 22.1%, and 34.4%, respectively. Interestingly, plant types (C₃ and C₄ plants) showed greater control over plant photosynthesis and biomass than a broad suite of soil and biochar factors. Biochar amendment largely boosted photosynthesis and biomass on C₃ plants, but had a limited effect on C₄ plants. Our results highlight the importance of the differential response of plant types to biochar amendment with respect to plant growth and photosynthesis, providing a scientific foundation for making reasonable strategies towards an extensive application of biochar for agricultural production management.     

Variable silicon accumulation in plants affects terrestrial carbon cycling by controlling lignin synthesis

Current climate and land‐use changes affect regional and global cycles of silicon (Si), with yet uncertain consequences for ecosystems. The key role of Si in marine ecology by controlling algae growth is well recognized but research on terrestrial ecosystems neglected Si since not considered an essential plant nutrient. However, grasses and various other plants accumulate large amounts of Si, and recently it has been hypothesized that incorporation of Si as a structural plant component may substitute for the energetically more expensive biosynthesis of lignin. Herein, we provide evidence supporting this hypothesis. We demonstrate that in straw of rice (Oryza sativa) deriving from a large geographic gradient across South‐East Asia, the Si concentrations (ranging from 1.6% to 10.7%) are negatively related to the concentrations of carbon (31.3% to 42.5%) and lignin‐derived phenols (32 to 102 mg/g carbon). Less lignin may explain results of previous studies that Si‐rich straw decomposes faster. Hence, Si seems a significant but hardly recognized factor in organic carbon cycling through grasslands and other ecosystems dominated by Si‐accumulating plants.     

Engineering the chloroplast targeted malarial vaccine antigens in Chlamydomonas starch granules

Background

Malaria, an Anopheles-borne parasitic disease, remains a major global health problem causing illness and death that disproportionately affects developing countries. Despite the incidence of malaria, which remains one of the most severe infections of human populations, there is no licensed vaccine against this life-threatening disease. In this context, we decided to explore the expression of Plasmodium vaccine antigens fused to the granule bound starch synthase (GBSS), the major protein associated to the starch matrix in all starch-accumulating plants and algae such as Chlamydomonas reinhardtii.

Methods and Findings

We describe the development of genetically engineered starch granules containing plasmodial vaccine candidate antigens produced in the unicellular green algae Chlamydomonas reinhardtii. We show that the C-terminal domains of proteins from the rodent Plasmodium species, Plasmodium berghei Apical Major Antigen AMA1, or Major Surface Protein MSP1 fused to the algal granule bound starch synthase (GBSS) are efficiently expressed and bound to the polysaccharide matrix. Mice were either immunized intraperitoneally with the engineered starch particles and Freund adjuvant, or fed with the engineered particles co-delivered with the mucosal adjuvant, and challenged intraperitoneally with a lethal inoculum of P. Berghei. Both experimental strategies led to a significantly reduced parasitemia with an extension of life span including complete cure for intraperitoneal delivery as assessed by negative blood thin smears. In the case of the starch bound P. falciparum GBSS-MSP1 fusion protein, the immune sera or purified immunoglobulin G of mice immunized with the corresponding starch strongly inhibited in vitro the intra-erythrocytic asexual development of the most human deadly plasmodial species.

Conclusion

This novel system paves the way for the production of clinically relevant plasmodial antigens as algal starch-based particles designated herein as amylosomes, demonstrating that efficient production of edible vaccines can be genetically produced in Chlamydomonas.     

Cyclic electron flow in C3 plants

This paper summarized our present view on the mechanism of cyclic electron flow in C3 plants. We propose that cyclic and linear pathways are in competition for the reoxidation of the soluble primary PSI acceptor, Ferredoxin (Fd), that freely diffuses in the stromal compartment. In the linear mode, Fd binds ferredoxin-NADP-reductase and electrons are transferred to NADP+ and then to the Benson and Calvin cycle. In the cyclic mode, Fd binds a site localized on the stromal side of the cytochrome b6f complex and electrons are transferred to P700 via a mechanism derived from the Q-cycle. In dark-adapted leaves, the cyclic flow operates at maximum rate, owing to the partial inactivation of the Benson and Calvin cycle. For increasing time of illumination, the activation of the Benson and Calvin cycle, and thus, that of the linear flow, is associated with a subsequent decrease in the rate of the cyclic flow. Under steady-state conditions of illumination, the contribution of cyclic flow to PSI turnover increases as a function of the light intensity (from 0 to approximately 50% for weak to saturating light, respectively). Lack of CO2 is associated with an increase in the efficiency of the cyclic flow. ATP concentration could be one of the parameters that control the transition between linear and cyclic modes.     

Introns in chloroplast protein-coding genes of land plants

Several protein-coding genes from land plant chloroplasts have been shown to contain introns. The majority of these introns resemble the fungal mitochondrial group II introns due to considerable nucleotide sequence homology at their 5 and 3 ends and they can readily be folded to form six hairpins characteristic of the predicted secondary structure of the mitochondrial group II introns. Recently it has been demonstrated that some mitochondrial group II introns are capable of self-splicing in vitro in the absence of protein co-factors. However evidence presented in this overview suggests that this is probably not the case for chloroplast introns and that trans-acting factors are almost certainly involved in their processing reactions.Abbreviations kop kilobase pairs - ORF Open Reading Frame - pre-RNA precursor ribonucleic acid     

Genetic analysis of chloroplast biogenesis in higher plants

The biogenesis of chloroplasts is genetically complex, involving hundreds of genes distributed between the nucleus and organelle. In higher plants, developmental parameters confer an added layer of complexity upon the genetic control of chloroplast biogenesis: the properties of plastids differ dramatically between different cell types. While the biochemistry and structure of different plastid types have been described in detail, factors that determine the timing and localization of chloroplast development and that mediate chloroplast assembly have remained elusive. To identify nuclear genes that play novel roles in chloroplast biogenesis, we are exploiting nuclear mutations that block the accumulation of subsets of chloroplast proteins. Detailed study of the mutant phenotypes provides clues concerning the primary defect in each mutant. Mutants with defects in chloroplast translation and mRNA metabolism have been identified. Other mutants defective in the accumulation of multiple thylakoid complexes show no apparent defect in the synthesis of the missing proteins. These may identify factors involved in the integration of proteins into the thylakoid membrane and their assembly into functional complexes.