Characterization of C₃--C₄ intermediate species in the genus Heliotropium L. (Boraginaceae): anatomy, ultrastructure and enzyme activity

Photosynthetic pathway characteristics were studied in nine species of Heliotropium (sensu lato, including Euploca), using assessments of leaf anatomy and ultrastructure, activities of PEP carboxylase and C₄ acid decarboxylases, and immunolocalization of ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco) and the P‐subunit of glycine decarboxylase (GDC). Heliotropium europaeum, Heliotropium calcicola and Heliotropium tenellum are C₃ plants, while Heliotropium texanum and Heliotropium polyphyllum are C₄ species. Heliotropium procumbens and Heliotropium karwinskyi are functionally C₃, but exhibit ‘proto‐Kranz’ anatomy where bundle sheath (BS) cells are enlarged and mitochondria primarily occur along the centripetal (inner) wall of the BS cells; GDC is present throughout the leaf. Heliotropium convolvulaceum and Heliotropium greggii are C₃–C₄ intermediates, with Kranz‐like enlargement of the BS cells, localization of mitochondria along the inner BS wall and a loss of GDC in the mesophyll (M) tissue. These C₃–C₄ species of Heliotropium probably shuttle photorespiratory glycine from the M to the BS tissue for decarboxylation. Heliotropium represents an important new model for studying C₄ evolution. Where existing models such as Flaveria emphasize diversification of C₃–C₄ intermediates, Heliotropium has numerous C₃ species expressing proto‐Kranz traits that could represent a critical initial phase in the evolutionary origin of C₄ photosynthesis.     

Growth at elevated CO2: photosynthetic responses mediated through Rubisco

Abstract. The global uptake of CO₂ in photosynthesis is about 120 gigatons (Gt) of carbon per year. Virtually all passes through one enzyme, ribulose bisphosphate carboxylase/oxygenase (rubisco), which initiates both the photosynthetic carbon reduction, and photorespiratory carbon oxidation, cycles. Both CO₂ and O₂ are substrates; CO₂ also activates the enzyme. In C₃ plants, rubisco has a low catalytic activity, operates below its K_m (CO₂), and is inhibited by O₂. Consequently, increases in the CO₂/O₂ ratio stimulate C₃ photosynthesis and inhibit photorespiration. CO₂ enrichment usually enhances the productivity of C₃ plants, but the effect is marginal in C₄ species. It also causes acclimation in various ways: anatomically, morphologically, physiologically or biochemically. So, CO₂ exerts secondary effects in growth regulation, probably at the molecular level, that are not predictable from its primary biochemical role in carboxylation. After an initial increase with CO₂ enrichment, net photosynthesis often declines. This is a common acclimation phenomenon, less so in field studies, that is ultimately mediated by a decline in rubisco activity, though the RuBP/P_i-regeneration capacities of the plant may play a role. The decline is due to decreased rubisco protein, activation state, and/or specific activity, and it maintains the rubisco fixation and RuBP/P_i regeneration capacities in balance. Carbohydrate accumulation is sometimes associated with reduced net photosynthesis, possibly causing feedback inhibition of the RuBP/P_iregeneration capacities, or chloroplast disruption. As exemplified by field-grown soybeans and salt marsh species, a reduction in net photosynthesis and rubisco activity is not inevitable under CO₂ enrichment. Strong sinks or rapid translocation may avoid such acclimation responses. Over geological time, aquatic autotrophs and terrestrial C₄ and CAM plants have genetically adapted to a decline in the external CO₂/O₂ ratio, by the development of mechanisms to concentrate CO₂ internally; thus circumventing O₂ inhibition of rubisco. Here rubisco affinity for CO₂ is less, but its catalytic activity is greater, a situation compatible with a high-CO₂ internal environment. In aquatic autotrophs, the CO₂ concentrating mechanisms acclimate to the external CO₂, being suppressed at high-CO₂. It is unclear, whether a doubling in atmospheric CO₂ will be sufficient to cause a de-adaptive trend in the rubisco kinetics of future C₃ plants, producing higher catalytic activities.     

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.     

On the significance of C3—C4 intermediate photosynthesis to the evolution of C4 photosynthesis

Abstract Evidence is drawn from previous studies to argue that C₃—C₄ intermediate plants are evolutionary intermediates, evolving from fully-expressed C₃ plants towards fully-expressed C₄ plants. On the basis of this conclusion, C₃—C₄ intermediates are examined to elucidate possible patterns that have been followed during the evolution of C₄ photosynthesis. An hypothesis is proposed that the initial step in C₄-evolution was the development of bundle-sheath metabolism that reduced apparent photorespiration by an efficient recycling of CO₂ using RuBP carboxylase. The CO₂-recycling mechanism appears to involve the differential compartmentation of glycine decarboxylase between mesophyll and bundle-sheath cells, such that most of the activity is in the bundlesheath cells. Subsequently, elevated phosphoenolpyruvate (PEP) carboxylase activities are proposed to have evolved as a means of enhancing the recycling of photorespired CO₂. As the activity of PEP carboxylase increased to higher values, other enzymes in the C₄-pathway are proposed to have increased in activity to facilitate the processing of the products of C₄-assimilation and provide PEP substrate to PEP carboxylase with greater efficiency. Initially, such a ‘C₄-cycle’ would not have been differentially compartmentalized between mesophyll and bundlesheath cells as is typical of fully-expressed C₄ plants. Such metabolism would have limited benefit in terms of concentrating CO₂ at RuBP carboxylase and, therefore, also be of little benefit for improving water- and nitrogen-use efficiencies. However, the development of such a limited C₄-cycle would have represented a preadaptation capable of evolving into the leaf biochemistry typical of fully-expressed C₄ plants. Thus, during the initial stages of C₄-evolution it is proposed that improvements in photorespiratory CO₂-loss and their influence on increasing the rate of net CO₂ assimilation per unit leaf area represented the evolutionary ‘driving-force’. Improved resourceuse efficiency resulting from an efficient CO₂-concentrating mechanism is proposed as the driving force during the later stages.     

Metabolism of CO2 by leaves of different photosynthetic types of Neurachne species

Following a series of continuous exposures to ¹⁴CO₂ for different lengths of time, leaves from Neurachne munroi (C₄), N. minor (C₃-C₄) and N. tenuifolia (C₃| were estimated to assimilate 100%, 9% and 2–4%, respectively, of atmospheric CO₂ by the C₄ pathway. The percentage of ¹⁴C-label appearing in malate and aspartate in leaves of N. minor progressively increased with longer exposure times indicating that a significant proportion of its C₄ acids are formed as secondary products. In ¹⁴CO₂/¹²CO₂ pulse/chase experiments, the ¹⁴C-label in leaves of N. munroi was rapidly transferred from C₄ acids to sugar monophosphates plus sugar diphosphates, and finally to sucrose. In leaves of N. minor, the ¹⁴C-label was slowly metabolized from the C-4 carboxyl of malate and asparate (apparent half-time = 250 s), and the formation of C₄ acids as secondary products was again evident. ¹⁴C-label in serine/glycine accumulated to comparable magnitudes in both N. minor and in N. tenuifolia, but there was an initial lag phase in the accumulation of label in N. minor. C₄ photosynthesis is apparently of minimal importance in reducing photorespiration in N. minor, but leaf anatomical specializations and a possible compartmentation of photorespiratory metabolism may be of considerable importance.     

Crassulacean acid metabolism contributes significantly to the in situ carbon budget in a population of the invasive aquatic macrophyte Crassula helmsii

1. The ecophysiological significance of Crassulacean acid metabolism (CAM) in the invasive aquatic macrophyte Crassula helmsii was studied in an English soft‐water lake. The extent and the contribution of CAM to the carbon budget was examined in spring (April) and summer (July) along a depth gradient (0.5–2.2 m), covering the growth range of C. helmsii in the lake. 2. Significant in situ CAM activity (30–80 meq kg⁻¹ FW) was present in all specimens, although it decreased with depth and hence correlated with the decline in photon irradiance. Potential CAM activity (60–161 meq kg⁻¹ FW), measured after exposure to low concentrations of CO₂ in the day and high concentrations at night, were on average 2.7‐times greater than in situ CAM activity. Overall CAM activity increased from April to July, which is consistent with higher potential carbon limitation caused by increased temperature and light availability. 3. CAM activity in C. helmsii appeared to be carbon‐limited at night because night‐time carbon‐fixation increased at raised, compared to ambient, concentrations of CO₂. 4. The high in situ CAM activity in C. helmsii was reflected in the contribution of CAM to the total carbon budget which, independent of depth and season, ranged from 18% to 42%. The amount of CO₂ taken up in the night via CAM was 0.74 to 2.94 times the amount of CO₂ lost in respiration, thus emphasizing the importance of CAM in refixation of potentially lost respiratory CO₂. 5. The onset of decarboxylation in the morning appeared to be under circadian control as there was a delay of up to 5.5 h between the start of the light period and a decline in cell acidity level. 6. There was little variation in δ¹³C content (−21.69 to 23.49‰) with season or depth suggesting, along with the estimated contribution to the carbon‐budget, that CAM is important for the whole population of C. helmsii. CAM may confer a competitive advantage in relation to growth, which may be one of the reasons for the invasiveness of this species.     

Inhibitory effect of aspirin on cholera toxin-induced phospholipase and cyclo-oxygenase activity

Cholera toxin (CT) stimulated phospholipase activity and caused [3H]arachidonic acid (3H-AA) release in a murine macrophage/monocyte cell line. Pretreatment of cells with dexamethasone, a phospholipase A2 (PLA2) inhibitor, did not affect CT-induced 3H-AA release. In contrast, aspirin, which is an inhibitor of phospholipase C (PLC), blocked CT-induced 3H-AA release and subsequent prostaglandin (PC) synthesis. The inhibitory effect of aspirin was dose dependent, with 4 mM reducing the CT response by approximately 50%. Similarly, inhibition was time dependent, occurring when the drug was added to the culture medium as late as 30 min after CT. Brief exposure (30 min) of the cells to aspirin did not alter their subsequent response to CT, but 3H-AA release from cells exposed to aspirin for 2.5 h was irreversibly inhibited. The data suggested that CT stimulation of AA metabolism may involve increased PLC activity.     

The photosynthesis of young Panicum C4 leaves is not C3-like

Evidence is presented contrary to the suggestion that C₄ plants grow larger at elevated CO₂ because the C₄ pathway of young C₄ leaves has C₃-like characteristics, making their photosynthesis O₂ sensitive and responsive to high CO₂. We combined PAM fluorescence with gas exchange measurements to examine the O₂ dependence of photosynthesis in young and mature leaves of Panicum antidotale (C₄, NADP-ME) and P. coloratum (C₄, NAD-ME), at an intercellular CO₂ concentration of 5 Pa. P. laxum (C₃) was used for comparison. The young C₄ leaves had CO₂ and light response curves typical of C₄ photosynthesis. When the O₂ concentration was gradually increased between 2 and 40%, CO₂ assimilation rates (A) of both mature and young C₄ leaves were little affected, while the ratio of the quantum yield of photosystem II to that of CO₂ assimilation (Φ_PSII/Φ_CO2) increased more in young (up to 31%) than mature (up to 10%) C₄ leaves. A of C₃ leaves decreased by 1·3 and Φ_PSII/Φ_CO2 increased by 9-fold, over the same range of O₂ concentrations. Larger increases in electron transport requirements in young, relative to mature, C₄ leaves at low CO₂ are indicative of greater O₂ sensitivity of photorespiration. Photosynthesis modelling showed that young C₄ leaves have lower bundle sheath CO₂ concentration, brought about by higher bundle sheath conductance relative to the activity of the C₄ and C₃ cycles and/or lower ratio of activities of the C₄ to C₃ cycles.     

干旱、半干旱区土壤蚯蚓稳定性碳同位素组成与轮作模式的关系

对10个不同耕地生境中蚯蚓(Lumbricus terrestris)的稳定性碳同位素进行了分析,研究了豫西洛阳干旱、半干旱地区土地轮作模式和耕作历史。结果表明,耕地表层土壤(0～30cm)中蚯蚓的δ13C介于-18.3‰和-25.6‰之间,变化幅度较大。经稳定性同位素质量平衡模式计算,10个生境中蚯蚓取食C3作物的比例在40.1%和99.4%之间波动,蚯蚓的取食生态受到土地上C3/C4作物轮作模式的影响。C4作物轮作频率与蚯蚓稳定性碳同位素比值之间呈正相关。土壤动物的稳定性碳同位素比值可较客观地反映出耕作制度和轮作模式。     

Experimental Evidence for the Isotope Effect in Photorespiration

Recent data on carbon isotope fractionation in photosynthesis are reviewed. Analysis of the carbon isotope composition in photosynthates and derivative products supports the hypothesis of the isotope effect in photorespiration. This hypothesis envisages the existence in a photosynthesizing cell of two carbon flows differing in isotope composition. One of these flows is enriched in ¹²C and associated with the assimilation pathway of photosynthesis. The other flow enriched in ¹³C circulates in the photorespiratory pathway. The relation between stimulated photorespiration and the carbon isotope composition of biomass supports this view. Our hypothesis of two interrelated isotope effects in photosynthesis leads to the conclusion that photosynthesis and photorespiration are coupled processes subject to periodical oscillations, where Rubisco acts as a main switch regulating these two pathways.