氮肥对2个杨树品种碳氮代谢的影响

研究以‘丹红’杨(美洲黑杨)和‘通辽1号’杨(小叶杨)为材料,在田间进行施氮肥和不施氮肥处理,分析2个杨树品种的生长性状、碳氮相关代谢物和发育木质部转录组的变化特征,探讨不同杨树品种氮肥利用的生理机制,为杨树的氮高效利用遗传育种奠定基础。结果表明:(1)‘丹红’杨和‘通辽1号’杨的总生物量在施氮处理后分别比不施氮处理提高了1.69倍和1.10倍;‘丹红’杨的总生物量在施氮肥和不施氮肥条件下分别是‘通辽1号’杨的13倍和10倍。(2)施氮处理显著抑制‘丹红’杨和‘通辽1号’杨树皮和木质部中总氮和多种水解氨基酸的含量,但是没有明显影响木质部的总碳、纤维素、半纤维素和木质素含量。(3)施氮处理显著影响了2个杨树品种发育木质部碳固定、糖代谢、氨基酸合成等碳氮代谢途径的基因的高表达,从而促进了植株生物量的积累。研究发现,施氮处理可以显著促进杨树发育木质部碳氮代谢途径相关基因的高表达,从而促进了杨树生物量的积累和生长;‘丹红’杨的木材产量在不同的氮素环境下都远远高于‘通辽1号’杨,更加适合人工林的大面积推广和种植。     

Phosphoenolpyruvate Carboxylase in Arabidopsis Leaves Plays a Crucial Role in Carbon and Nitrogen Metabolism

Phosphoenolpyruvate carboxylase (PEPC) is a crucial enzyme that catalyzes an irreversible primary metabolic reaction in plants. Previous studies have used transgenic plants expressing ectopic PEPC forms with diminished feedback inhibition to examine the role of PEPC in carbon and nitrogen metabolism. To date, the in vivo role of PEPC in carbon and nitrogen metabolism has not been analyzed in plants. In this study, we examined the role of PEPC in plants, demonstrating that PPC1 and PPC2 were highly expressed genes encoding PEPC in Arabidopsis (Arabidopsis thaliana) leaves and that PPC1 and PPC2 accounted for approximately 93% of total PEPC activity in the leaves. A double mutant, ppc1/ppc2, was constructed that exhibited a severe growth-arrest phenotype. The ppc1/ppc2 mutant accumulated more starch and sucrose than wild-type plants when seedlings were grown under normal conditions. Physiological and metabolic analysis revealed that decreased PEPC activity in the ppc1/ppc2 mutant greatly reduced the synthesis of malate and citrate and severely suppressed ammonium assimilation. Furthermore, nitrate levels in the ppc1/ppc2 mutant were significantly lower than those in wild-type plants due to the suppression of ammonium assimilation. Interestingly, starch and sucrose accumulation could be prevented and nitrate levels could be maintained by supplying the ppc1/ppc2 mutant with exogenous malate and glutamate, suggesting that low nitrogen status resulted in the alteration of carbon metabolism and prompted the accumulation of starch and sucrose in the ppc1/ppc2 mutant. Our results demonstrate that PEPC in leaves plays a crucial role in modulating the balance of carbon and nitrogen metabolism in Arabidopsis.Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is a crucial enzyme that functions in primary metabolism by irreversibly catalyzing the conversion of phosphoenolpyruvate (PEP) and HCO₃⁻ to oxaloacetate (OAA) and inorganic phosphate. PEPC is found in all plants, green algae, and cyanobacteria, and in most archaea and nonphotosynthetic bacteria, but not in animals or fungi (Chollet et al., 1996; O’Leary et al., 2011a). Several isoforms of PEPC are present in plants, including plant-type PEPCs and one bacterium-type PEPC (Sánchez and Cejudo, 2003; Sullivan et al., 2004; Mamedov et al., 2005; Gennidakis et al., 2007; Igawa et al., 2010). Arabidopsis (Arabidopsis thaliana) possesses three plant-type PEPC genes, AtPPC1, AtPPC2, and AtPPC3, and one bacterium-type PEPC gene, AtPPC4. Unlike plant-type PEPCs, bacterium-type PEPCs lack a seryl-phosphorylation domain near the N terminus, a typical domain conserved in plant-type PEPCs (Sánchez and Cejudo, 2003). Plant-type PEPCs form class 1 PEPCs, which exist as homotetramers. Recently, bacterium-type PEPCs have been reported to interact with plant-type PEPCs to form heterooctameric class 2 PEPCs in several species, including unicellular green algae (Selenastrum minutum), lily (Lilium longiflorum), and castor bean (Ricinus communis; O’Leary et al., 2011a).Because of the irreversible nature of the enzymatic reactions catalyzed by PEPC isoforms, they are strictly regulated by a variety of mechanisms. PEPC is an allosteric enzyme and is activated by its positive effector, Glc-6-P, and inhibited by its negative effectors, malate, Asp, and Glu (O’Leary et al., 2011a). Control by reversible phosphorylation is another important mechanism that regulates the activity of PEPC. In this reaction, phosphorylation catalyzed by PEPC kinase changes the sensitivity of PEPC to its allosteric effectors (Nimmo, 2003). In addition, monoubiquitination may also regulate plant-type PEPC activity (Uhrig et al., 2008). Recent research in castor oil seeds suggests that bacterium-type PEPC is a catalytic and regulatory subunit of class 2 PEPCs, as class 1 and class 2 PEPCs show significant differences in their sensitivity to allosteric inhibitors (O’Leary et al., 2009, 2011b).A number of studies on PEPC function have been performed in a variety of organisms (O’Leary et al., 2011a). The best described function of PEPC is in fixing photosynthetic CO₂ during C4 and Crassulacean acid metabolism photosynthesis. However, in most nonphotosynthetic tissues and the photosynthetic tissues of C3 plants, the fundamental function of PEPC is to anaplerotically replenish tricarboxylic acid cycle intermediates (Chollet et al., 1996). PEPC also functions in malate production in guard cells and legume root nodules (Chollet et al., 1996). A chloroplast-located PEPC isoform in rice (Oryza sativa) was recently found to be crucial for ammonium assimilation (Masumoto et al., 2010). In addition, previous work in Arabidopsis suggested that AtPPC4 might play a role in drought tolerance (Sánchez et al., 2006).Transgenic plants expressing ectopic PEPC forms with diminished feedback inhibition showed an increase in overall organic nitrogen content at the expense of starch and soluble sugars (Rademacher et al., 2002; Chen et al., 2004; Rolletschek et al., 2004). However, the in vivo function of PEPC in carbon and nitrogen metabolism has not been reported previously.To further investigate the function of PEPC in higher plants, we isolated and characterized mutants of Arabidopsis deficient in the expression of the PEPC-encoding genes PPC1 and PPC2. We demonstrated that PPC1 and PPC2 were the most highly expressed PEPC genes in the leaves. To further define their role, we produced a double mutant (ppc1/ppc2) deficient in the expression of the PPC1 and PPC2 genes. We then conducted a detailed molecular, biochemical, and physiological characterization of this double mutant.     

水肥耦合下夏玉米不同生育时期的高光谱特性与反演模型研究

本文通过整个光谱范围内一阶导数光谱反射率与叶绿素、可溶性糖和可溶性蛋白的相关系数显著的波段,建立高光谱预监测水肥耦合条件下的夏玉米光合特性以及碳氮代谢,进而为玉米高产提供依据。在玉米拔节期和大喇叭口期选择596、1025和924nnl,吐丝期和乳熟期选择638、1068和965nm这几个显著性波段的实测值来建立估测模型。研究结果表明,拔节和大喇叭口期叶片叶绿素SPAD值的估测模型为y=28832．45p596＋39．34,可溶性糖含量的估测模型为y=640．54p1025＋7．92,可溶性蛋白含量一阶导数光谱估测模型为y=4092．90p924＋5．63,而吐丝期和乳熟期叶片叶绿素SPAD值的估测模型为y=134151．00p638＋129．92,可溶性糖含量的估测模型为y=524．80p1068＋9．20,可溶性蛋白含量一阶导数光谱估测模型为y=7321．61lp965＋36．64。所建立的高光谱预测模型在本试验所属的时空范围内能很好地预测和反演玉米生长状况。     

氮磷配施量对黑果枸杞叶片非结构性碳动态的影响

非结构性碳水化合物(NSC)是光合作用的主要产物和植物合成各种物质的基础,叶片不同时期的非结构性碳含量表征着植物体的碳摄入与碳需求特征。该研究以黑果枸杞为对象,设置4个氮磷配施(N∶P₂O₅为15∶1)量(按施纯N量计)水平,分别为13.5 g/株(高肥)、4.5 g/株(中肥)和1.5 g/株(低肥),以不施肥为对照,探讨叶片非结构性碳水化合物含量的日变化与生育期的动态变化特征以及对施肥的响应。结果表明：(1)叶片非结构性碳水化合物及其组分含量呈先升高后降低的日动态变化特征,在正午13:00时达到最高值;可溶性糖(SS)与淀粉(S)的比值呈先下降后上升的趋势,在正午13:00达到最低值。(2)随着生育期的变化,叶片非结构性碳水合物及其组分含量在生长季呈现先上升后下降的趋势,并在果实发育期(FDP)达到最高值,而可溶性糖与淀粉含量的比值逐渐降低。(3)叶片N含量与P含量呈极显著正相关关系(P<0.01),与NSC和S含量呈显著负相关关系(P<0.05);叶片P含量与S含量呈极显著负相关关系(P<0.01)。研究发现,在氮磷比为15∶1,纯N量为1.5 ~13.5 g/株之间的条件下,施肥能够显著提高黑果枸杞叶片非结构性碳总含量、可溶性糖含量、淀粉含量以及可溶性糖与淀粉的转换效率,促进了氮磷含量的消耗,并且随着氮磷配施量的增加影响效果愈加显著,故西北地区可通过施肥措施增强植物对逆境的适应性以达到生态恢复的目的。     

Effects of Nitrogen Fertilizer on the Bt Protein Content in Transgenic Rice and Nitrogen Metabolism Mechanism

Journal of Plant Growth Regulation - Transgenic rice with Bacillus thuringiensis (Bt) genes has been successfully cultivated in recent years. The stable and sustainable expression of Bt protein is...     

施氮时期对酿酒葡萄叶片氮代谢酶及相关基因表达的影响

以10年生‘蛇龙珠’葡萄为材料,在萌芽期(S1)、新梢旺长期(S2)、开花期(S3)、果实第一次膨大期(S4)、副梢生长旺期(S5)和果实第二次膨大期(S6)分别一次性施入尿素300kg·hm-2,以不施氮肥为对照(CK),分析了花前5d(DBF5)、花后25d(DAF25)、花后55d(DAF55)和花后85d(DAF85)叶片的各项指标,以明确氮素施用时期对葡萄叶片氮代谢的调控与影响。结果表明:(1)S1和S2处理葡萄叶片中总氮及可溶性蛋白含量在DAF25时显著增加。(2)S3和S4处理的NR及GS活性在DAF85时显著高于其他处理;在DAF25时,S1和S2处理的GOGAT活性,以及S3和S4处理的GDH活性均显著高于同期对照和其他施肥处理。(3)各施肥处理叶片VvNR表达水平在不同时期均高于同期对照,S3处理VvNR表达水平在DAF25和DAF85时分别为对照的3.4倍和2.7倍;S3和S4处理的VvGS表达水平分别在DAF55和DAF85时达到最高值,S3处理的VvGOGAT和S4处理的VvGDH表达水平在DAF55和DAF85均显著高于其他处理,S3处理的VvGDH表达水平在DAF55和DAF85仅次于S4处理。研究表明,氮素通过诱导叶片氮素代谢基因的响应,从而调控叶片中氮素代谢酶活性增加,促进了氮素的积累,S3和S4处理在不同时期氮代谢酶活性和对应的基因表达水平均较高,更有利于叶片中氮素的转化和代谢。     

Effects of Different Inorganic Nitrogen Sources on Photosynthetic Carbon Metabolism in Primary Leaves of Non-nodulated Phaseolus vulgaris L

Young bean plants (Phaseolus vulgaris L. var Saxa) were fed with three different types of inorganic nitrogen, after being grown on nitrogen-free nutrient solution for 8 days. The pattern of ¹⁴CO₂ fixation was investigated in photosynthesizing primary leaf discs of 11-day-old plants (3 days with nitrogen source) and in a pulse-chase experiment in 13-day-old plants (5 days with nitrogen source).

Ammonium caused, in contrast to nitrate nutrition, a higher level of ¹⁴C incorporation into sugar phosphates but a lower incorporation of label into malate, glycolate, glycerate, aspartate, and alanine. The labeling kinetics of glycine and serine were little changed by the nitrogen source. Ammonium feeding also produced an increase in the ratio of extractable activities of ribulose-1,5-bisphosphate carboxylase to phosphoenolpyruvate carboxylase and an increase in dark respiration and the CO₂ compensation concentration. Net photosynthesis was higher in plants assimilating nitrate.

The results point to stimulated turnover of the photosynthetic carbon reduction cycle metabolites, reduced phosphoenolpyruvate carboxylation, and altered turnover rates within the photosynthetic carbon oxidation cycle in ammonium-fed plants. Mechanisms of the regulation of primary carbon metabolism are proposed and discussed.

     

Effects of Carbon Dioxide on Metabolism by the Glycollate Pathway in Leaves

When solutions of [¹⁴C]glycollate, glycine, serine, glycerate,or glucose were supplied to segments of wheat leaves throughtheir cut bases in the light, most of the ¹⁴C was incorporatedinto sucrose in air but in CO₂-free air less sucrose was made.The synthesis of sucrose was decreased because metabolism ofserine was partly blocked. Sucrose synthesis from glucose andglycerate in CO₂-free air was decreased but to a smaller extent;relatively more CO₂ was evolved and serine accumulated. Theeffects of DCMU and light of different wavelengths on metabolismby leaves of L-[U-¹⁴C]serine confirmed that simultaneous photosyntheticassimilation of carbon was necessary for the conversion of serineto sucrose. Of various products of photosynthesis fed exogenouslyto the leaves -keto acids were the most effective in promotingphotosynthesis of sucrose and release of ¹⁴CO₂ from ¹⁴C-labelledserine. This suggests that in CO₂-free air the metabolism ofserine may be limited by a shortage of -keto acid acceptorsfor the amino group. In CO₂-free air added glucose stimulatedproduction of CO₂ and sucrose from D-[U-¹⁴C]- glycerate andno competitive effects were evident even though glucose is convertedrapidly to sucrose under these conditions. In addition to asupply of keto acid, photosynthesis may also provide substratesthat can be degraded and provide energy in the cytoplasm forthe conversion of glycerate to sugar and phosphates and sucrose.     

Soil Carbon and Nitrogen Responses to Nitrogen Fertilizer and Harvesting Rates in Switchgrass Cropping Systems

The environmental sustainability of bioenergy cropping systems depends upon multiple factors such as crop selection, agricultural practices, and the management of carbon (C), nitrogen (N), and water resources. Perennial grasses, such as switchgrass (Panicum virgatum L.), show potential as a sustainable bioenergy source due to high yields on marginal lands with low fertilizer inputs and an extensive root system that may increase sequestration of C and N in subsurface soil horizons. We quantified the C and N stocks in roots, free particulate, and mineral-associated soil organic matter pools in a 4-year-old switchgrass system following conversion from row crop agriculture at the W.K. Kellogg Biological Station in southwest Michigan. Crops were fertilized with nitrogen at either 0, 84, or 196 kg N ha^?1 and harvested either once or twice annually. Twice-annual harvesting caused a reduction of C and N stocks in the relatively labile roots and free-particulate organic matter pools. Nitrogen fertilizer significantly reduced total soil organic C and N stocks, particularly in the stable, mineral-associated C and N pools at depths greater than 15 cm. The largest total belowground C stocks in biomass and soil occurred in unfertilized plots with annual harvesting. These findings suggest that fertilization in switchgrass agriculture moderates the sequestration potential of the soil C pool.     

不同氮素水平下施硫对高产小麦碳氮运转和产量的影响

在大田栽培条件下,以2个不同穗型的高产冬小麦品种为试验材料,在施240 kg·hm-2(N240)和330 kg·hm-2(N330)纯氮水平下,分别施纯硫60 kg·hm-2(S60)和0 kg·hm-2(S0),研究了施硫对小麦不同器官碳氮运转及其对籽粒产量和蛋白质产量的影响.结果显示:(1)2个供氮水平下,施硫(S60)均比对照(S0)增加了2个小麦品种的叶片、茎、鞘、颖壳和穗轴等营养器官花前贮藏干物质、氮素的运转量和运转率以及总运转量和总运转率,提高了转运干物质、氮素对籽粒重和籽粒氮素的贡献率,极显著提高了籽粒和蛋白质产量.(2)施硫对豫农949品种籽粒和蛋白质产量提高幅度显著高于兰考矮早八;与对照(S0)相比,豫农949的N240S60和N330S60处理使籽粒产量分别增加12.74%和16.41%,蛋白质产量分别增加16.84%和16.14%.结果表明,中氮和高氮水平下施硫均可明显促进高产小麦植株的C-N运转,提高植株对氮的吸收利用和碳物质的积累,从而增加籽粒产量,但不同品种间的施硫效应存在差异.     

Effect of Methionine Sulfoximine on Photosynthetic Carbon Metabolism in Wheat Leaves

Methionine sulfoximine (MSO) greatly reduced the carbon dioxideexchange rate (CER) of detached wheat (Triticum aestivvm L.cv Roland) leaves in 21% O₂, but only slightly reduced it in2% O₂. A supply of 50 mM NH₄Cl had little effect on the CERirrespective of the O₂ concentration. A simultaneous additionof glutamine and MSO protected against the inhibition of photosynthesisto a considerable extent and caused the accumulation of moreNH₃ than did the addition of MSO alone. Fixation of ¹⁴CO₂ in wheat leaves was inhibited by MSO treatmentin 22% O₂, and there was decreased incorporation of ¹⁴G intoamino acids and sugars and increased label into acid fractions.The addition of MSO and glutamine together eliminated the effectof MSO on the photosynthetic ¹⁴CO₂ fixation pattern. NH₄Cl stimulatedthe synthesis of amino acids from ¹⁴CO₂, especially the synthesisof serine in 22% O₂. Our observations show that factors other than the uncouplingof photophosphorylation by accumulated NH₃ may be responsiblefor the early stage of photosynthesis inhibition by MSO underphotorespiratory conditions. ¹Present address: Department of Agricultural Chemistry, KyushuUniversity, Fukuoka 812 Japan. ²Also at U.S. Department of Agriculture, Agricultural ResearchService, Urbana, Illionois 61801, U.S.A. (Received September 13, 1983; Accepted February 2, 1984)     

施氮对冬小麦旗叶RuBP羧化酶活性及叶绿素荧光参数的影响

以大穗型小麦品种'兰考矮早8'和多穗型品种'豫麦49-198'为材料,采用盆栽试验研究了不同施氮量对两种穗型冬小麦品种旗叶RuBP(1,5二磷酸核酮糖)羧化酶和PEPC(磷酸烯醇式丙酮酸羧化酶)活性及叶绿素a荧光动力学参数的影响.结果表明,在本试验条件下,随着花后天数的增加,两小麦品种旗叶RuBP羧化酶和PEPC活性总体呈下降趋势;随着施氮量的增加,RuBP羧化酶和PEPC活性呈增加趋势,其中RuBP羧化酶活性多数以N4(N 4.8 g/盆)处理最高,PEPC活性多数以N3(N 3.6 g/盆)处理最高.随着施氮量的增加,两小麦品种旗叶Fv/F0、Fv/Fm和qP均呈增加趋势,且以N4 (N 4.8 g/盆)处理的值最高,并且处理之间的差异达显著水平(P<0.05).研究发现,本试验条件下,适量施用氮肥有利于小麦旗叶RuBP羧化酶和PEPC活性的增加及叶绿素a荧光动力学参数Fv/F0和Fv/Fm的提高,从而有助于光合同化物的积累和小麦穗粒重的提高.     

Glyoxylate Induced Changes in the Carbon and Nitrogen Metabolism of the Cyanobacterium Anabaena cylindrica

Addition of millimolar concentrations of glyoxylate to nitrogen-fixing cultures of Anabaena cylindrica, grown aerobically in the light, caused the following effects: an increase in the number of glycogen granules and in the excretion of carbohydrates; a decreased phycocyanin concentration, but an increase in the chlorophyll a to phycocyanin ratio. Also, an enhancement in the carbon to nitrogen ratio was noted, but this was restored if NH₄⁺ was added simultaneously. The most pronounced effect of glyoxylate addition was a 20-fold increase in the glycine pool. The effect of glyoxylate on N₂ fixation (acetylene reduction) was enhanced at high light intensities, but it did not affect the in vitro ribulose-1,5-bisphosphate carboxylase activity. However, addition of millimolar concentrations of glycolate did not cause changes in nitrogenase activity, CO₂ fixation, and NH₃ release comparable to those caused by glyoxylate. The primary mechanism of action of glyoxylate appears to be within the glycolate pathway of the vegetative cells and metabolically downstream from glycolate.     

Nitrogen, Carbon, and Sulfur Metabolism in Natural Thioploca Samples

Filamentous sulfur bacteria of the genus Thioploca occur as dense mats on the continental shelf off the coast of Chile and Peru. Since little is known about their nitrogen, sulfur, and carbon metabolism, this study was undertaken to investigate their (eco)physiology. Thioploca is able to store internally high concentrations of sulfur globules and nitrate. It has been previously hypothesized that these large vacuolated bacteria can oxidize sulfide by reducing their internally stored nitrate. We examined this nitrate reduction by incubation experiments of washed Thioploca sheaths with trichomes in combination with ¹⁵N compounds and mass spectrometry and found that these Thioploca samples produce ammonium at a rate of 1 nmol min⁻¹ mg of protein⁻¹. Controls showed no significant activity. Sulfate was shown to be the end product of sulfide oxidation and was observed at a rate of 2 to 3 nmol min⁻¹ mg of protein⁻¹. The ammonium and sulfate production rates were not influenced by the addition of sulfide, suggesting that sulfide is first oxidized to elemental sulfur, and in a second independent step elemental sulfur is oxidized to sulfate. The average sulfide oxidation rate measured was 5 nmol min⁻¹ mg of protein⁻¹ and could be increased to 10.7 nmol min⁻¹ mg of protein⁻¹ after the trichomes were starved for 45 h. Incorporation of ¹⁴CO₂ was at a rate of 0.4 to 0.8 nmol min⁻¹ mg of protein⁻¹, which is half the rate calculated from sulfide oxidation. [2-¹⁴C]acetate incorporation was 0.4 nmol min⁻¹ mg of protein⁻¹, which is equal to the CO₂ fixation rate, and no ¹⁴CO₂ production was detected. These results suggest that Thioploca species are facultative chemolithoautotrophs capable of mixotrophic growth. Microautoradiography confirmed that Thioploca cells assimilated the majority of the radiocarbon from [2-¹⁴C]acetate, with only a minor contribution by epibiontic bacteria present in the samples.