Root release and metabolism of organic acids in tea plants in response to phosphorus supply

Self-rooted, 10-month-old, uniform tea [Camellia sinensis (L.) O. Kuntze cv. Huangguanyin] plants were supplied for 17 weeks with 0, 40, 80, 160, 400, or 1000μM phosphorus (P) to investigate the effects of P supply on root citrate and malate release, the concentrations of malate and citrate and the activities of acid-metabolizing enzymes in leaves and roots. Root malate release and accumulation was induced by both 0 and 40μM P, while root citrate release and accumulation was induced only by 0μM P. Phosphorus-deficiency-induced malate and citrate release coincided with higher concentrations of root malate and citrate. The higher concentrations of malate and citrate were accompanied by increased activities of phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate phosphatase (PEPP), citrate synthase (CS) and NAD-malic enzyme (NAD-ME) and decreased activities of pyruvate kinase (PK), NADP-ME and NADP-isocitrate dehydrogenase (NADP-IDH) in roots. In contrast to roots, malate accumulated in the leaves only in response to 0μM P, and no change was observed in citrate levels. The P-deficiency-induced leaf malate accumulation coincided with increased activities of NADP-ME, NAD-ME and PK. Overall, the P-deficiency-induced changes in organic acid (OA) metabolism differed between roots and leaves. The high tolerance of tea plants to P-deficiency might be involved in two major processes: (a) increasing the availability of P by inducing root release of OA anions; and (b) improving the ability to use P efficiently by inducing bypass enzymes involved in tissue P economy.     

Proteomic analysis of Citrus sinensis roots and leaves in response to long-term magnesium-deficiency

Background

Magnesium (Mg)-deficiency is frequently observed in Citrus plantations and is responsible for the loss of productivity and poor fruit quality. Knowledge on the effects of Mg-deficiency on upstream targets is scarce. Seedlings of ‘Xuegan’ [Citrus sinensis (L.) Osbeck] were irrigated with Mg-deficient (0 mM MgSO₄) or Mg-sufficient (1 mM MgSO₄) nutrient solution for 16 weeks. Thereafter, we first investigated the proteomic responses of C. sinensis roots and leaves to Mg-deficiency using two-dimensional electrophoresis (2-DE) in order to (a) enrich our understanding of the molecular mechanisms of plants to deal with Mg-deficiency and (b) understand the molecular mechanisms by which Mg-deficiency lead to a decrease in photosynthesis.

Results

Fifty-nine upregulated and 31 downregulated protein spots were isolated in Mg-deficient leaves, while only 19 upregulated and 12 downregulated protein spots in Mg-deficient roots. Many Mg-deficiency-responsive proteins were involved in carbohydrate and energy metabolism, followed by protein metabolism, stress responses, nucleic acid metabolism, cell wall and cytoskeleton metabolism, lipid metabolism and cell transport. The larger changes in leaf proteome versus root one in response to Mg-deficiency was further supported by our observation that total soluble protein concentration was decreased by Mg-deficiency in leaves, but unaffected in roots. Mg-deficiency had decreased levels of proteins [i.e. ribulose-1,5-bisphosphate carboxylase (Rubisco), rubisco activase, oxygen evolving enhancer protein 1, photosynthetic electron transfer-like protein, ferredoxin-NADP reductase (FNR), aldolase] involved in photosynthesis, thus decreasing leaf photosynthesis. To cope with Mg-deficiency, C. sinensis leaves and roots might respond adaptively to Mg-deficiency through: improving leaf respiration and lowering root respiration, but increasing (decreasing) the levels of proteins related to ATP synthase in roots (leaves); enhancing the levels of proteins involved in reactive oxygen species (ROS) scavenging and other stress-responsive proteins; accelerating proteolytic cleavage of proteins by proteases, protein transport and amino acid metabolism; and upregulating the levels of proteins involved in cell wall and cytoskeleton metabolism.

Conclusions

Our results demonstrated that proteomics were more affected by long-term Mg-deficiency in leaves than in roots, and that the adaptive responses differed between roots and leaves when exposed to long-term Mg-deficiency. Mg-deficiency decreased the levels of many proteins involved in photosynthesis, thus decreasing leaf photosynthesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1462-z) contains supplementary material, which is available to authorized users.     

Adaptation responses in C4 photosynthesis of maize under salinity

The effect of salinity on C(4) photosynthesis was examined in leaves of maize, a NADP-malic enzyme (NADP-ME) type C(4) species. Potted plants with the fourth leaf blade fully developed were treated with 3% NaCl solution for 5d. Under salt treatment, the activities of pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPCase), NADP-dependent malate dehydrogenase (NADP-MDH) and NAD-dependent malate dehydrogenase (NAD-MDH), which are derived mainly from mesophyll cells, increased, whereas those of NADP-ME and ribulose-1,5-bisphosphate carboxylase, which are derived mainly from bundle sheath cells (BSCs), decreased. Immunocytochemical studies by electron microscopy revealed that PPDK protein increased, while the content of ribulose-1,5-bisphosphate carboxylase/oxygenase protein decreased under salinity. In salt-treated plants, the photosynthetic metabolites malate, pyruvate and starch decreased by 40, 89 and 81%, respectively. Gas-exchange analysis revealed that the net photosynthetic rate, the transpiration rate, stomatal conductance (g(s)) and the intercellular CO(2) concentration decreased strongly in salt-treated plants. The carbon isotope ratio (δ(13)C) in these plants was significantly lower than that in control. These findings suggest that the decrease in photosynthetic metabolites under salinity was induced by a reduction in gas-exchange. Moreover, in addition to the decrease in g(s), the decrease in enzyme activities in BSCs was responsible for the decline of C(4) photosynthesis. The increase of PPDK, PEPCase, NADP-MDH, and NAD-MDH activities and the decrease of NADP-ME activity are interpreted as adaptation responses to salinity.     

Physiological impacts of magnesium-deficiency in Citrus seedlings: photosynthesis, antioxidant system and carbohydrates

Magnesium (Mg)-deficiency affects productivity and quality in agriculture, yet at a physiological level it is not well understood. Citrus grandis and Citrus sinensis seedlings were irrigated for 12?weeks with 0, 50, 500 or 2,000?μM MgSO₄. Thereafter, Mg-deficiency-induced changes in photosynthesis, antioxidant system and carbohydrates were investigated. Mg-deficiency affected CO₂ assimilation more in C. grandis leaves than in C. sinensis ones, but Mg-deficiency-induced accumulation of sugars was not higher in the former except for sucrose. Mg-deficiency-induced photoinhibitory impairment occurring on the whole photosynthetic electron transport chain was more severe in C. grandis leaves than in C. sinensis ones. Mg-deficient leaves had higher or similar activities of antioxidant enzymes and contents of antioxidant metabolites except for catalase (CAT) activity and reduced glutathione (GSH) content. However, Mg-deficiency increased leaf malondialdehyde (MDA) content. In conclusion, the greater decrease in CO₂ assimilation in Mg-deficient C. grandis leaves may be caused by the greater decrease in the photosynthetic electron transport capacity. Mg-deficiency-induced up-regulation in leaf antioxidant system does not provide enough protection to Mg-deficient leaves against the oxidative damage.     

Organic acid exudation induced by phosphorus deficiency and/or aluminium toxicity in two contrasting soybean genotypes

Experiments in nutrient solution were conducted to investigate the exudation of organic acids (OAs) induced by phosphorus deficiency (–P) and/or aluminium toxicity (+Al) in two contrasting soybean genotypes as related to internal OA concentration and related enzyme activities. Baxi 10 (BX10), a known P‐efficient soybean (Glycine max[L] Merr.) genotype, was shown to be more resistant to +Al than a P‐inefficient genotype Bendi 2 (BD2), indicating the potential of selecting soybean cultivars with dual resistance to –P and +Al. The two contrasting genotypes were further characterized for root exudation and formation of oxalate, malate and citrate and their related enzyme activities in response to –P, +Al or both combined. –P significantly induced malate and oxalate exudation from both soybean genotypes, although the P‐efficient BX10 tended to excrete much more oxalate than the P‐inefficient BD2. The +Al treatment triggered citrate efflux from both genotypes, with BX10 having a much greater efflux rate than BD2. Interestingly, –P did not appear to induce citrate exudation, whereas +Al had no obvious effect on malate or oxalate exudation from the two genotypes. The exudation of OAs was generally diminished under the coupled stress of –P and +Al in comparison with either single stress, implying a possible antagonistic effect of the two stresses on OA exudation. Root malate content was negatively correlated with its exudation in BX10 but positively in BD2. A similar tendency was observed for oxalate content and exudation only with less magnitude. Determination of six related enzymes, phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate phosphatase (PEPP), malate enzyme (ME), isocitrate dehydrogenase (ICDH), malate dehydrogenase (MDH), and pyruvate kinase (PK), in the root tips showed that their activities were not significantly altered during the early stage of treatments (2 and 4 days) whereas at 14 days after stress imposition, the activities of PEPC, PEPP, ME and ICDH were generally enhanced for both genotypes. However, the activity of these enzymes did not appear to be correlated with OA exudation or formation. This study clearly demonstrates that OA exudation is differentially induced by –P and +Al in soybean plants, with specific induction of oxalate and malate by –P and citrate by +Al. The lack of a close relationship between OA exudation and internal concentration or enzyme activities may suggest that the regulation of OA formation and exudation by –P and/or +Al could be imposed at different stages.     

Metabolic responses in iron deficient tomato plants

The effects of Fe deficiency on different metabolic processes were characterized in roots, xylem sap and leaves of tomato. The total organic acid pool increased significantly with Fe deficiency in xylem sap and leaves of tomato plants, whereas it did not change in roots. However, the composition of the pool changed with Fe deficiency, with major increases in citrate concentrations in roots (20-fold), leaves (2-fold) and xylem sap (17-fold). The activity of phosphoenolpyruvate carboxylase, an enzyme leading to anaplerotic C fixation, increased 10-fold in root tip extracts with Fe deficiency, whereas no change was observed in leaf extracts. The activities of the organic acid synthesis-related enzymes malate dehydrogenase, citrate synthase, isocitrate dehydrogenase, fumarase and aconitase, as well as those of the enzymes lactate dehydrogenase and pyruvate carboxylase, increased with Fe deficiency in root extracts, whereas only citrate synthase increased significantly with Fe deficiency in leaf extracts. These results suggest that the enhanced C fixation capacity in Fe-deficient tomato roots may result in producing citrate that could be used for Fe xylem transport. Total pyridine nucleotide pools did not change significantly with Fe deficiency in roots or leaves, although NAD(P)H/NAD(P) ratios were lower in Fe-deficient roots than in controls. Rates of O(2) consumption were similar in Fe-deficient and Fe-sufficient roots, but the capacity of the alternative oxidase pathway was decreased by Fe deficiency. Also, increases in Fe reductase activity with Fe deficiency were only 2-fold higher when measured in tomato root tips. These values are significantly lower than those found in other plant species, where Fe deficiency leads to larger increases in organic acid synthesis-related enzyme activities and flavin accumulation. These data support the hypothesis that the extent of activation of different metabolic pathways, including carbon fixation via PEPC, organic acid synthesis-related enzymes and oxygen consumption is different among species, and this could modulate the different levels of efficiency in Strategy I plants.     

Boron-aluminum interactions affect organic acid metabolism more in leaves than in roots of <Emphasis Type="Italic">Citrus grandis</Emphasis> seedlings

Sour pummelo (Citrus grandis) seedlings were irrigated with nutrient solution containing four boron concentrations (i.e., 2.5, 10, 25 and 50 μM H₃BO₃) and two aluminum concentrations [i.e., 0 (-Al) and 1.2 mM AlCl₃ · 6 H₂O (+Al)]. It was found that B did not affect, but Al increased, the Al content in the roots. The Al and citrate contents in the -Al leaves either did not change or slightly increased with increasing B concentration. On the other hand, the Al and citrate contents in the +Al leaves rapidly decreased as B concentration increased from 2.5 to 50 μM, then decreased at the highest B concentration. The Al and citrate contents were higher in the +Al than in the -Al leaves, except for at 25 μM B when they were similar. The leaf malate content did not change in response to B or Al, except for an increase in the +Al leaves and a decrease in the -Al leaves at 2.5 μM B. Similarly, the root malate and citrate contents did not change in response to B with or without Al, except for a decrease in the malate and citrate contents in the +Al roots at 50 μM B and an increase in the citrate content in the -Al roots at 50 μM B. The activities of acid-metabolizing enzymes were less affected by B-Al interactions in the roots than in the leaves.     

Effects of biomin and algae suspensions on the activities of carboxylating and decarboxylating enzymes in cadmium-treated pea plants

Cadmium-treated pea plants exhibited PEPC and NADP-ME activities, titratable acidity, and malate concentration in the leaves similar to controls. The PEPC activity in the roots of Cd-treated plants decreased by about 40 %, and NADP-ME increased more than twice. The titratable acidity remained similar as in the leaves, but the malate content diminished by about 30 %. The application of 500 g(d.m.) m-3 biomin and a combination of biomin and algae suspensions to Cd-treated plants brought about an increase in the titratable acidity and in the malate concentrations in the leaves and the roots. This revised version was published online in August 2006 with corrections to the Cover Date.     

Activity of biochemical pH-stat enzymes in cereal root tips under oxygen deficiency

The activity of the enzymes of alcoholic and lactic-acid fermentation: pyruvate decarboxylase (PDC, EC 4.1.1.1), alcohol dehydrogenase (ADH, EC 1.1.1.1), lactate dehydrogenase (LDH, EC 1.1.1.27) and the enzymes of malic acid metabolism: phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.23), NAD-dependent malate dehydrogenase (NAD-MDH, EC 1.1.1.37), and NADP-dependent malic enzyme (NADP-ME, EC 1.1.1.40) involved in the operation of biochemical pH-stat was investigated in the root tips of wheat (Triticum aestivum L.) and rice (Oryza sativa L.) under hypoxia and anoxia. Exposures lasted for 6, 12, and 18 h. The most pronounced response was detected for the enzymes of alcoholic fermentation. The activation of ADH and PDC in wheat occurred only under hypoxia, whereas in rice it was detected both under hypoxia and anoxia. The activation of LDH in wheat occurred under hypoxia, and in rice, the activity of this enzyme was slightly enhanced. The activity of the enzymes of malic acid metabolism did not change except in wheat root tips under hypoxia when PEPC activity decreased and NADP-ME activity simultaneously rose. The role of biochemical pH-stat in the regulation of cytoplasmic pH in plant cells under oxygen deficit and the mechanisms for regulating the activities of enzymes involved in biochemical pH-stat are discussed as well as the interaction between biochemical pH-stat and other mechanisms maintaining pH of plant cells. The results are analyzed within a context of intracellular pH regulation.     

Ribulose-1,5-bisphosphate carboxylase-oxygenase,other Calvin-cycle enzymes,and chlorophyll decrease when glucose is supplied to mature spinach leaves via the transpiration stream

The inhibition of photosynthesis after supplying glucose to detached leaves of spinach (Spinacia oleracea L.) was used as a model system to search for mechanisms which potentially contribute to the sink regulation of photosynthesis. Detached leaves were supplied with 50 mM glucose or water for 7 d through the transpiration stream, holding the leaves in low irradiance (16 mol photons · m^–2 · s^–1) and a cycle of 9 h light/15 h darkness to prevent any endogenous accumulation of carbohydrate. Leaves supplied with water only showed marginal changes of photosynthesis, respiration, enzyme levels or metabolites. When leaves were supplied with 50 mM glucose, photosynthesis was gradually inhibited over several days. The inhibition was most marked when photosynthesis was measured in saturating irradiance and ambient CO₂, less marked in saturating irradiance and saturating CO₂, and least marked in limiting irradiance. There was a gradual loss of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) protein, fructose-1,6-bisphosphatase, NADP-glyceraldehyde-3-phosphate dehydrogenase and chlorophyll. The inhibition of photosynthesis was accompanied by a large decrease of glycerate-3-phosphate, an increase of triose-phosphates and fructose-1,6-bisphospate, and a small decrease of ribulose-1,5-bisphosphate. The stromal NADPH/NADP ratio increased (as indicated by increased activation of NADP-malate dehydrogenase), and the ATP/ADP ratio increased. Chlorophyll-fluorescence analysis indicated that thylakoid energisation was increased, and that the acceptor side of photosystem II was more reduced. Similar results were obtained when glucose was supplied by floating leaf discs in low irradiance on glucose solution, and when detached spinach leaves were held in high light to produce an endogenous accumulation of carbohydrate. Feeding glucose also led to an increased rate of respiration. This was not accompanied by any changes of pyruvate kinase, phosphofructokinase, or pyrophosphate: fructose-6-phosphate phosphotransferase activity. There was a decrease of phosphoenolpyruvate, glycerate-3-phosphate and glycerate-2-phosphate, an increase of pyruvate and triose-phosphates, and an increased ATP/ADP ratio. These results show (i) that accumulation of carbohydrate can inhibit photosynthesis via a long-term mechanism involving a decrease of Rubisco and other Calvin-cycle enzymes and (ii) that respiration is stimulated due to an unknown mechanism, which increases the utilisation of phosphoenolpyruvate.Abbreviations and Symbols C_i CO₂ concentration in the air space within the leaf - F_m fluorescence yield with a saturating pulse in dark-adapted material - F_o ground level of fluorescence using a weak non-actinic modulated beam in the dark - Fru1,6bisP fructose-1,6-bisphosphate - Fru1,6Pase fructose-1,6-bisphosphatase - Fru2,6bisP fructose-2,6-bisphosphate - IRGA infrared gas analyser - NAD-MDH NAD-dependent malate dehydrogenase - NADP-MDH NADP-dependent malate dehydrogenase - NADP-GAPDH NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - PEP phosphoenolpyruvate - PFK phospho-fructokinase - PFP pyrophospate: fructose-6-phosphate-phosphotransferase - 3-PGA glycerate-3-phospate - P_i inorganic phosphate - Ru1,5bisP ribulose 1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - triose-phosphates sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate This research was supported by the Deutsche Forschungsgemeinschaft (SFB 137).     

Characterization of NAD-dependent malate dehydrogenases from spinach leaves

Summary. Spinach leaves were used to extract isoforms of NAD-dependent malate dehydrogenase (NAD-MDH) (EC 1.1.1.37), either soluble or bound to microsomal, plasma, or chloroplast envelope membranes. All fractions were subjected to isoelectric focusing analysis, which showed that purified chloroplast envelopes contain an NAD-MDH isoform tightly bound to the membranes, since treatment with 0.5 or 1% Triton X-100 was not able to release the enzyme from the envelopes. In contrast, plasma membranes released an isoform with a pI of 3.5 following treatment with 0.5% Triton X-100. The most abundant soluble leaf isoform had a pI of 9, while the chloroplast stroma contained an isoform with a pI of 5.3. Kinetic analysis of oxaloacetate (OAA)-dependent NADH oxidation in different fractions gave different K _m values for both substrates, the envelope- and plasma membrane-bound NAD-MDH exhibiting the highest affinities for OAA. Leaf plasma membrane-bound MDH exhibited a high capacity for both reaction directions (malate oxidation and OAA reduction), while the two chloroplast isoforms (stromal and envelope-bound) preferentially reduced OAA. Our results indicate that the chloroplast envelope contains a specifically attached NAD-MDH isoform that could provide direct coupling between chloroplast and cytosol adenylate pools. Correspondence: T. Cvetić, Institute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia.     

Evidence for the expression of morphological and biochemical characteristics of C3-photosynthesis in chlorohyllous callus cultures of Zea mays

A Zea mays callus culture containing chlorophyll was established and grown photomixotrophically. Cell chloroplast structure, and pigment and soluble protein contents were examined. Expression of some key enzymes of C₄ carbon metabolism was compared with that of etiolated (heterotrophic) and green photoautotrophic leaves. Chlorophyll content of the callus was 15–20% that of green leaves. Soluble protein content of callus was half that of leaf cells. Electron microscopic observations showed that green callus cells contained only typical granal chloroplasts. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.38) activities in green callus were ca 30% those of green leaves but 2–3 times higher than in etiolated leaves. Quantitative enzyme protein determination, using antibodies specific to maize leaf Rubisco showed that the chloroplastic carboxylase represented about 7% of total soluble protein in green callus, in parallel to its low chlorophyll content. The specific activity of Rubisco in callus and leaves was unchanged. Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activity in green callus was about 20% that of green leaves and similar to that measured in etiolated leaves. Apparent K_m (PEP) values (0.08 mM) for PEPC isolated from green callus and etiolated leaves were very different from values (0.5 mM) obtained with PEPC from green leaves. These kinetic characteristics together with the absence of inhibition by malate and activation by glucose-6-phosphate suggest that the properties of PEPC isolated from green callus and etiolated maize leaves are very similar to those of PEPPC from C₃ plants. Using PEPC antibodies specific to green maize leaf enzyme, immunotitration of PEPC preparations containing identical enzyme units allowed complete precipitation of the green leaf enzyme with increasing antibody volumes. In contrast, 60–70% of the activity of PEPC from etiolated and green callus was inhibited, suggesting low affinity for the maize green leaf PEPC antiserum (typical C₄ form). Ouchterlony double diffusion tests revealed only partial recognition of PEPC in green callus and etiolated leaves. NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) activity in callus was 2 and 3 times higher, respectively, than in etiolated and green leaves. NADP-malic enzyme (NADP-ME, EC 1.1.1.40) activity in callus cultures was much lower than in green leaves. All our data support the hypothesis that cultures of fully dedifferentiated chlorophyllous tissues of Zea mays possess a C₃-like metabolism.     

Al-induced efflux of organic acid anions is poorly associated with internal organic acid metabolism in triticale roots

The secretion of organic acid anions from roots has been identified as a mechanism of resistance to Al. However, the process leading to the secretion of organic acid anions is poorly understood. The effect of Al on organic acid metabolism was investigated in two lines of triticale (xTriticosecale Wittmark) differing in Al-induced secretion of malate and citrate and in Al resistance. The site of Al-induced secretion of citrate and malate from a resistant line was localized to the root apices (terminal 5 mm). The levels of citrate (root apices and mature root segments) and malate (mature segments only) in roots increased during exposure to Al, but similar changes were observed in both triticale genotypes. The in vitro activities of four enzymes involved in malate and citrate metabolism (citrate synthase, phosphoenolpyruvate carboxylase, malate dehydrogenase, and NADP-isocitrate dehydrogenase) were similar for sensitive and resistant lines in both root apices and mature root segments. The response of these enzymes to pH did not differ between tolerant and sensitive lines or in the presence and absence of Al. Moreover, cytoplasmic and vacuolar pH were not affected by exposure to Al in either line. Together, these results indicate that the Al-dependent efflux of organic acid anions from the roots of triticale is not regulated by their internal levels in the roots or by the capacity of the root cells to synthesize malate and citrate.     

镁缺乏和过量胁迫对纽荷尔脐橙叶绿素荧光特性的影响

以2龄枳砧纽荷尔脐橙为材料,研究了镁缺乏和过量胁迫对叶片叶绿素含量和叶绿素荧光特性的影响。结果表明,镁缺乏胁迫导致老叶叶绿素含量显著降低,而新叶叶绿素含量无显著下降;镁过量胁迫显著抑制老叶叶绿素含量的下降,而促进了新叶叶绿素含量的降低。镁缺乏和过量均导致不同叶龄叶片的光化学效率(Fv/Fm)和相对电子传递速率(rETR)降低,但镁缺乏胁迫的影响显著大于镁过量胁迫。缺镁第4个月时,2龄秋梢、1龄春梢、1龄夏梢和晚夏梢叶的Fv/Fm分别比对照低了13.9%、12.6%、2.9%和2.0%,rETRmax值分别比对照低35.7%、56.2%、32.6%和15.2%;而镁过量胁迫叶片的Fv/Fm分别比对照低了0.5%、2.2%、3.4%和1.5%,rETRmax分别为对照的110.1%、68.8%、87.2%和84.5%。缺镁2龄秋梢、1龄春梢和1龄夏梢叶片的非光化学淬灭系数(NPQ)先升后降,热耗散能力显著下降,且显著低于镁过量胁迫。因此,在夏季高光照条件下缺镁胁迫对纽荷尔脐橙光合作用的影响显著,且大于镁过量胁迫,缺镁纽荷尔脐橙叶片易发生光抑制,产生光伤害。     

Phosphorus Stress-Induced Proteoid Roots Show Altered Metabolism in Lupinus albus

Proteoid roots develop in Lupinus albus L. in response to nutrient stress, especially P. Proteoid roots excrete citrate and thus increase the availability of P, Fe, and Mn in the rhizosphere. In an effort to understand citrate synthesis and organic acid metabolism in proteoid roots of lupin, we have evaluated in vitro enzyme activities of citrate synthase (CS), malate dehydrogenase (MDH), and phosphoenolpyruvate carboxylase (PEPC) in proteoid and normal roots of plants grown with or without P. Organic acid concentrations, respiration rates, and dark 14CO2-labeling patterns were also determined. The in vitro specific activities of CS, MDH, and PEPC and in vivo dark 14CO2 fixation were higher in proteoid roots compared to normal roots, particularly under P stress. Western blot analysis showed that PEPC enzyme protein was more highly expressed in -P proteoid roots compared to other tissues. The majority of the fixed 14C was found in organic acids, predominantly malate and citrate. A larger fraction of citrate was labeled in P- stressed proteoid roots compared to other root tissue. Respiration rates of proteoid roots were 31% less than those of normal roots. The data provide evidence for increased synthesis of citrate in proteoid roots compared to normal roots, particularly under P stress. A portion of the carbon for citrate synthesis is derived from nonautotrophic CO2 fixation via PEPC in proteoid roots.     

Role of magnesium in carbon partitioning and alleviating photooxidative damage

Magnesium (Mg) deficiency exerts a major influence on the partitioning of dry matter and carbohydrates between shoots and roots. One of the very early reactions of plants to Mg deficiency stress is the marked increase in the shoot-to-root dry weight ratio, which is associated with a massive accumulation of carbohydrates in source leaves, especially of sucrose and starch. These higher concentrations of carbohydrates in Mg-deficient leaves together with the accompanying increase in shoot-to-root dry weight ratio are indicative of a severe impairment in phloem export of photoassimilates from source leaves. Studies with common bean and sugar beet plants have shown that Mg plays a fundamental role in phloem loading of sucrose. At a very early stage of Mg deficiency, phloem export of sucrose is severely impaired, an effect that occurs before any noticeable changes in shoot growth, Chl concentration or photosynthetic activity. These findings suggest that accumulation of carbohydrates in Mg-deficient leaves is caused directly by Mg deficiency stress and not as a consequence of reduced sink activity. The role of Mg in the phloem-loading process seems to be specific; resupplying Mg for 12 or 24 h to Mg-deficient plants resulted in a very rapid recovery of sucrose export. It appears that the massive accumulation of carbohydrates and related impairment in photosynthetic CO2 fixation in Mg-deficient leaves cause an over-reduction in the photosynthetic electron transport chain that potentiates the generation of highly reactive O2 species (ROS). Plants respond to Mg deficiency stress by marked increases in antioxidative capacity of leaves, especially under high light intensity, suggesting that ROS generation is stimulated by Mg deficiency in chloroplasts. Accordingly, it has been found that Mg-deficient plants are very susceptible to high light intensity. Exposure of Mg-deficient plants to high light intensity rapidly induced leaf chlorosis and necrosis, an outcome that was effectively delayed by partial shading of the leaf blade, although the Mg concentrations in different parts of the leaf blade were unaffected by shading. The results indicate that photooxidative damage contributes to development of leaf chlorosis under Mg deficiency, suggesting that plants under high-light conditions have a higher physiological requirement for Mg. Maintenance of a high Mg nutritional status of plants is, thus, essential in the avoidance of ROS generation, which occurs at the expense of inhibited phloem export of sugars and impairment of CO2 fixation, particularly under high-light conditions.     

Light activation of NADP malic enzyme in leaves of maize: marginal increase in activity,but marked change in regulatory properties of enzyme

This article reports the characteristics of light activation of NADP-malic enzyme (NADP-ME, EC 1.1.1.40) in leaf discs of maize (Zea mays cv. VMH 404) for the first time. The leaf discs were illuminated in the presence of 2 mmol/L bicarbonate, as light activation increases in the presence of bicarbonate. Upon illumination, the Vmax of NADP-ME increased by about 30%. Although small, the increase was consistent and significant. The changes in regulatory properties of NADP-ME were quite pronounced. The extent of light activation was similar when substrate (malate) concentration was either 4 mmol/L (saturating) or 0.01 mmol/L (limiting). There was only a marginal change in the Km for malate, but there was marked change in the response of NADP-ME to activators or inhibitors. The Ki for pyruvate and oxalate increased by 100 and 67% respectively, while the Ka for the citrate and succinate increased by 36 and 32% respectively. These results suggest that the NADP-ME becomes less sensitive to feedback inhibition on illumination. The light-induced change seems to be due, at least partially, to the reduction of dithiols, as incubation of leaf extracts with DTE dampened light activation of NADP-ME. We conclude that the properties of NADP-ME do change on illumination. Although there was only a marginal increase in the activity of the enzyme on illumination of leaf discs, the changes in regulatory properties of NADP-ME were marked.     

Diurnal modulation of phosphoenolpyruvate carboxylation in pea leaves and roots as related to tissue malate concentrations and to the nitrogen source

Phosphoenolpyruvate (PEP) carboxylation was measured as dark ¹⁴CO₂ fixation in leaves and roots (in vivo) or as PEP carboxylase (PEPCase) activity in desalted leaf and roof extracts (in vitro) from Pisum sativum L. cv. Kleine Rheinländerin. Its relation to the malate content and to the nitrogen source (nitrate or ammonium) was investigated. In tissue from nitrate-grown plants, PEP carboxylation varied diurnally, showing an increase upon illumination and a decrease upon darkening. Diurnal variations in roots were much lower than in leaves. Fixation rates in leaves remained constantly low in continuous darkness or high in continuous light. Dark CO₂ fixation of leaf slices also decreased when leaves were preilluminated for 1 h in CO₂-free air, suggesting that the modulation of dark CO2 fixation was related to assimilate availability in leaves and roots. Phosphoenolpyruvate carboxylase activity was also measured in vitro. However, no difference in maximum enzyme activity was found in extracts from illuminated or darkened leaves, and the response to substrate and effectors (PEP, malate, glucose-6-phosphate, pH) was also identical. The serine/threonine protein kinase inhibitors K252b, H7 and staurosporine, and the protein phosphatase 2A inhibitors okadaic acid and cantharidin, fed through the leaf petiole, did not have the effects on dark CO₂ fixation predicted by a regulatory system in which PEPCase is modulated via reversible protein phosphorylation. Therefore, it is suggested that the diurnal modulation of PEP carboxylation in vivo in leaves and roots of pea is not caused by protein phosphorylation, but rather by direct allosteric effects. Upon transfer of plants to ammonium-N or to an N-free nutrient solution, mean daily malate levels in leaves decreased drastically within 4–5 d. At that time, the diurnal oscillations of PEP carboxylation in vivo disappeared and rates remained at the high light-level. The coincidence of the two events suggests that PEPCase was de-regulated because malate levels became very low. The drastic decrease of leaf malate contents upon transfer of plants from nitrate to ammonium nutrition was apparently not caused by increased amino acid or protein synthesis, but probably by higher decarboxylation rates.Abbreviations CAM crassulacean acid metabolism - PEP Phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PP protein phosphatase - PK protein kinase This work was supported by the Deutsche Forschungsgemeinschaft. B. Baur was a recipient of a doctoral grant, and L. Leport recipient of a post-doctoral grant of the DFG. The skilled technical assistance of Eva Wirth and Maria Lesch is gratefully acknowledged.     

CAM induction in Clusia minor L. during the transition from wet to dry season in Trinidad: the role of organic acid speciation and decarboxylation

The interrelationships between the induction of CAM and the turnover of malate and citrate in the dicotyledenous tree Clusia minor were compared with seasonal changes in rainfall, leaf water status, PFD and photoinhibitory responses during the transition from wet to dry season in Trinidad. Over a period of 8 weeks, as rainfall declined from a maximum observed around week 3, leaf xylem tensions measured at dusk and dawn reflected the concurrent reduction in day-time carbon gain and an increase in the diel turnover of malate (exposed leaves) and citrate (shaded leaves). Clear seasonal trends were observed in the turnover of malate and citrate during the transition from wet to dry season. In contrast to the declining back-ground concentrations of citrate during the wet-dry season transition, malate accumulation was markedly enhanced and the ratio of malalc:citrate accumulated overnight increased as the dry season advanced. Photo-inhibitory responses, assessed by chlorophyll fluorescence, indicated that photochemistry was largely determined by the diurnal course of PFD incident on leaves, regardless of the magnitude of internal CO₂ release from malate and citrate decarboxylation. In the long term, photochemical efficiency in both shaded and exposed leaves appeared to decline as the dry season progressed. Although there was a clear linear relationship between integrated PFD and overnight accumulation of malate, no such correlation was found for citrate. However, citrate breakdown during the day showed a much closer correlation with PFD as compared to malate, with levels of citrate measured at dusk declining in response to higher daily light intensities. Moreover, enhanced citrate decarboxylation during the day was strongly correlated with increased CAM and overnight accumulation of both malate and citrate.