小麦磷酸丙糖转运器的特性及其对同化物分配的调节

磷酸丙糖转运器(tnose phosphate/phosphatetranslocator,TPT)是源、库间光合产物分配的第一调控部位,研究TPT的特性及其对同化物分配的调节,对于提高光合作用同化物利用效率有着重要意义.我们首先采用Percoll密度梯度离心从小麦(Triticum aestivum L.)叶片中分离制备了完整性达91%以上、具有较高纯度的完整叶绿体.利用TPT不可逆抑制剂[H3]2-DIDS标记和SDS-PAGE,以及小麦TPT抗体进行Western blotting分析,证明TPT蛋白仅存在于叶绿体被膜中,约占被膜总蛋白的15%,其分子量为35 kD,而在液泡膜和线粒体膜上不存在.采用硅油离心法研究TPT对磷酸二羟丙酮(dihydroxyacetone phosphate,DHAP)、磷酸烯醇式丙酮酸(phosphoenolpyruvate,PEP)、葡萄糖-6-磷酸(glucose-6-phosphate,G6P)与Pi的反向运输动力学的结果表明,DHAP/Pi的最大运输活性最高,PEP/Pi次之,G6P/Pi最低.TPT与这些运输底物的Km值由小至大,分别为DHAP、Pi、PEP和G6P,证明TPT的最适运输底物为DHAP.用DIDS处理时,TPT对DHAP运输活性的抑制达95%.TPT运输活性受到抑制时,可导致叶绿体内大量积累淀粉.TPT在调控小麦叶绿体同化产物的分配中起着重要作用,在保证卡尔文循环正常运转的前提下,通过TPT外运到胞质中参与蔗糖合成和其他代谢活动的磷酸丙糖(triose phosphate,TP)约占93.6%,而用于叶绿体内合成淀粉的TP仅占6.4%.生理条件下其功能是高效率地把大部分光合同化产物TP及时运出叶绿体到胞质中,用于合成蔗糖并运输到其他库器官的需要.     

Characteristics of Triose Phosphate／Phosphate Translocator from Wheat and Its Role in the Distribution of Assimilates

In plants, triose phosphate/phosphate translocator (TPT) is the first regulation point forpartitioning of photosynthate between source and sink. Studies on the characteristic of TPT and itsregulation on the distribution of assimilates are critical for improving the utilization rate of photosyntheticassimilates. Chloroplasts with intactness of more than 91% and high purity were isolated from wheat( Triticurn aestivurn L. cv. Jing 411) leaves. Analysis of SDS-PAGE and labeling with an irreversible specificinhibitor, [H3]2^-DIDS (4, 4‘-diisothiocyano-2, 2‘-stilbenedisulfonate, DIDS) demonstrated that wheat TPTwas a chloroplast membrane protein with a 35 kD molecular weight, which comprised about 15% of the totalmembrane proteins of chloroplasts. Western blotting analysis showed that wheat TPT is uniquelydistributed in the envelope membrane of chloroplasts, but not detected in the membranes of vacuoles andmitochondria. The silicone-oil-layer centrifugation system was employed to study the kinetic properties ofTPT. The results showed that the maximal transport activity of TPT was the highest for dihydroxyacetonephosphate (DHAP)/inorganic phosphate (Pi), then for phosphoenolpyruvate (PEP)/Pi and glucose-6-phosphate (G6P)/Pi. The Km value of TPT was the lowest for DHAP, followed by Pi, PEP and G6P,therefore the most preferred substrate of TPT is DHAP. The transport of wheat TPT to DHAP was stronglyinhibited by DIDS with a degree of 95%. Inhibition of TPT transport activity led to an obvious accumulationof starch in chloroplasts, therefore the TPT protein of wheat controls the export of TP out of chloroplastsinto cytosol. Except for the need of participating in the Calvin cycle, the ratio of TP exported out ofchloroplast to the one used for synthesizing starch was at least 93.6:6.4. The TPT protein from wheat hasmuch high transport efficiency, which plays an important role in the regulation of the distribution ofassimilates in wheat chloroplasts.     

小麦磷酸丙糖转运器cDNA的克隆及其基因表达分析

利用RT_PCR方法以及RACE(rapidamplificationofcDNAends)策略 ,从小麦 (TriticumaestivumL .)幼苗叶片中克隆了编码磷酸丙糖转运器 (TPT)的全长cDNA。序列分析结果表明 ,小麦TPTcDNA编码 40 2个氨基酸的前体蛋白 ,其中信号肽含有 78个氨基酸。成熟蛋白部分与玉米 (ZeamaysL .)TPT有很高的同源性 (89% )。推测小麦TPT成熟蛋白有 8个跨膜区 ,形成双亲α_螺旋的跨膜结构。位于第 7个跨膜区的Arg_2 74和Lys_2 75可能是底物结合位点。比较TPT基因在小麦幼苗的根、胚芽鞘、叶片和种子中的表达差异表明 :TPT基因在叶片、胚芽鞘中均有表达 ,但在胚芽鞘中的表达量较低 ,在种子和根中未见有表达。由此看来 ,小麦TPT的基因可能只局限在绿色组织中表达。还就C3 和C4植物TPT不同的底物特异性问题进行了讨论     

小麦磷酸丙糖转运器cDNA的克隆及其基因表达分析

利用RT-PCR方法以及RACE(rapid amplification of cDNA ends)策略,从小麦(Triticum aestivum L.) 幼苗叶片中克隆了编码磷酸丙糖转运器(TPT)的全长cDNA.序列分析结果表明,小麦TPT cDNA编码402个氨基酸的前体蛋白,其中信号肽含有78个氨基酸.成熟蛋白部分与玉米(Zea mays L.)TPT有很高的同源性(89%).推测小麦TPT成熟蛋白有8个跨膜区,形成双亲α-螺旋的跨膜结构.位于第7个跨膜区的Arg-274和Lys-275可能是底物结合位点.比较TPT基因在小麦幼苗的根、胚芽鞘、叶片和种子中的表达差异表明:TPT基因在叶片、胚芽鞘中均有表达,但在胚芽鞘中的表达量较低,在种子和根中未见有表达.由此看来,小麦TPT的基因可能只局限在绿色组织中表达.还就C3和C4植物TPT不同的底物特异性问题进行了讨论.     

第十八讲 叶绿体代谢物运转与光合产物的调节

内容一、引言二、叶绿体代谢物的运转 1.叶绿体被膜对物质的透性 (1)“Pi运转器” (2)“双羧酸运转器” 2.ATP及还原力的运转 (1)3PGA—DHAP穿梭运转 (2)Mal—OAA穿梭运转     

马齿苋叶片磷酸烯醇式丙酮酸羧化酶活性及调节特性的光/暗变化

在光照条件下C_4植物马齿黄金苋叶片PEPC的提取活性高于在黑暗中的。PEPC的光/暗活性比率与测定系统的pH及底物PEP浓度有关。pH升高及PEP浓度增加均可使光/暗活性比值下降。日间提取的PEPC与夜间提取的PEPC对于激活剂G6P及抑制剂Mal的敏感性有明显差异。日型PEPC的敏感性低于夜型PEPC的。G6P对PEPC的激活作用表现为增加酶对底物PEP的亲和性,Mal的抑制作用表现为既降低酶对底物PEP的亲和性,又降低酶促反应的最大速度。G6P、Mal对于日型和夜型PEPC的动力学参数的影响是不同的。     

叶绿体内被膜上的磷酸丙糖转运器

叶绿体内膜上存在有磷酸丙糖转运器。本文着重对该转运器的结构和功能、转运特性及其对光合作用的调节等做一介绍。磷酸丙糖转运器能够催化磷、磷酸丙糖和3 磷酸甘油酸的反向交换运输,从而使光合初级产物从叶绿体转运到胞质。在生理条件下,这种转运严格遵循1∶1的反向交换原则,并且转运活性受光的调节。目前,已经从一些植物中分离到磷酸丙糖转运器蛋白,并克隆了它们的cDNA。近年来,利用基因工程手段对磷酸丙糖转运器功能的研究也取得了很重要的进展。     

光合碳在叶片淀粉和蔗糖间分配的调节

叶片光合作用中产生的三碳糖在淀粉和蔗糖之间的分配受许多因素控制,蔗糖形成速率是决定性因素。蔗糖形成的调节酶是果糖1,6—二磷酸酯酶(F1,6P_2ase)和磷酸蔗糖合成酶(SPS),调节作用是通过无机磷(Pi)、磷酸二羟丙酮(DHAP)、磷酸己糖(己糖—P)、果糖1,6—二磷酸(F1,6P_2)和果糖2,6—二磷酸(F2,6P_2)之间的复杂的调节关系进行的。其中F2,6P_2起着关键作用,它以极低的浓度调节生糖和酵解作用,既参与蔗糖合成又参与反馈抑制。     

叶绿体内被膜上的磷酸丙糖转动器

叶绿体内膜上存在有磷酸丙糖转动器。本文着重转运器的结构和功能、转运特性及其对光合作用的调节等做一介绍。磷酸丙糖转运器能够催化磷、磷酸丙糖和3－磷酸甘油酸的反向交换运输,从而使光合初级产物从叶绿体转运到胞质。在生理条件下,这种转动严格遵循1：1的反向交换原则,并且转动活性受光的调节。目前,已经从一些植物中分离到磷酸丙糖转运器蛋白,并克隆了它们的cDNA。近年来,利用基因工程手段对磷酸丙糖转运器功能的研究也取得了很重要的进展。     

渗透压在葡萄酒酵母代谢中的调控作用

以磷酸丙糖异构酶部分缺失突变株做对比,研究了渗透压对葡萄酒酵母发酵过程中甘油合成与挥发酸生成的调节作用.结果表明:渗透压对野生型葡萄酒酵母中存在的磷酸二羟丙酮(DHAP)与3-磷酸甘油醛(GA3P)平衡具有调节作用,能使平衡向磷酸二羟丙酮方向迁移以合成更多的甘油,而当磷酸丙糖异构酶部分缺失时渗透压对这一平衡基本不起作用...     

Functional characterization of the plastidic phosphate translocator gene family from the thermo-acidophilic red alga Galdieria sulphuraria reveals specific adaptations of primary carbon partitioning in green plants and red algae

In chloroplasts of green plants and algae, CO₂ is assimilated into triose-phosphates (TPs); a large part of these TPs is exported to the cytosol by a TP/phosphate translocator (TPT), whereas some is stored in the plastid as starch. Plastidial phosphate translocators have evolved from transport proteins of the host endomembrane system shortly after the origin of chloroplasts by endosymbiosis. The red microalga Galdieria sulphuraria shares three conserved putative orthologous transport proteins with the distantly related seed plants and green algae. However, red algae, in contrast to green plants, store starch in their cytosol, not inside plastids. Hence, due to the lack of a plastidic starch pool, a larger share of recently assimilated CO₂ needs to be exported to the cytosol. We thus hypothesized that red algal transporters have distinct substrate specificity in comparison to their green orthologs. This hypothesis was tested by expression of the red algal genes in yeast (Saccharomyces cerevisiae) and assessment of their substrate specificities and kinetic constants. Indeed, two of the three red algal phosphate translocator candidate orthologs have clearly distinct substrate specificities when compared to their green homologs. GsTPT (for G. sulphuraria TPT) displays very narrow substrate specificity and high affinity; in contrast to green plant TPTs, 3-phosphoglyceric acid is poorly transported and thus not able to serve as a TP/3-phosphoglyceric acid redox shuttle in vivo. Apparently, the specific features of red algal primary carbon metabolism promoted the evolution of a highly efficient export system with high affinities for its substrates. The low-affinity TPT of plants maintains TP levels sufficient for starch biosynthesis inside of chloroplasts, whereas the red algal TPT is optimized for efficient export of TP from the chloroplast.     

Influence of Varying CO(2) and Orthophosphate Concentrations on Rates of Photosynthesis, and Synthesis of Glycolate and Dihydroxyacetone Phosphate by Wheat Chloroplasts

Intact chloroplasts of wheat (Triticum aestivum) were isolated from mesophyll protoplasts. With decreasing concentrations of bicarbonate from 10 to 0.3 millimolar (pH 8.0), the optimal concentration of orthophosphate (Pi) for photosynthetic O₂ evolution decreased from a value of 0.1 to 0.2 millimolar to 0 to 0.025 millimolar. The extremely low Pi optimum for photosynthesis at the low bicarbonate levels of 0.3 millimolar was increased by lowering the O₂ concentration from 253 (21% gas phase) to 72 micromolar (6% gas phase). The relative amount of glycolate and dihydroxyacetone phosphate (DHAP) synthesized under high and low levels of bicarbonate and varying levels of Pi was determined. At low levels of bicarbonate, glycolate was the main product, whereas at high bicarbonate levels, DHAP was the main product. Most of the DHAP and glycolate was found in the extrachloroplastic fraction.     

Transport of inorganic phosphate and C3- and C6-sugar phosphates across the envelope membranes of potato tuber amyloplasts

Amyloplasts have been isolated from tubers of potato plants (Solarium tuberosum. cv. Desirée). As it is difficult to isolate amyloplasts that have a high starch content, we used transformed plants in which the content of starch was reduced. This was achieved by decreasing the activity of ADP-glucose pyrophosphorylase by antisense techniques (Müller-Röber et al., 1992, EMBO. 11, 1229–1238). In the isolated plastids the activity of glutamine-oxoglutarate-aminotransferase (glutamate synthase, EC 2.6.1.53) was dependent upon the intactness of the plastids. For the supply of redox equivalents the addition of glucose-6-phosphate (Glc6P) was required. Glucose-1-phosphate (Glc1P) did not support glutamate synthesis. Plastids were treated with Triton X-100 and the solubilized proteins reconstituted into liposomes. Transport measurements with these liposomes revealed that inorganic phosphate (Pi), dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate and Glc6P are transported in a counter-exchange mode. Transport of phosphoenolpyruvate was low and Glc1P was virtually not transported in exchange for Pi. Kinetic constants were determined for the Pi/Pi and Glc6P/Pi counter exchanges. For comparison, proteins of mitochondria from potato tubers and pea leaves were reconstituted into liposomes. As expected, the Pi/Pi exchange across the mitochondrial membrane was not affected by DHAP and Glc6P. Kinetic constants of the Pi/Pi counter exchange were determined for potato tuber mitochondria.Abbreviations DHAP dihydroxyacetone phosphate - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP Phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - Pi inorganic phosphate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl] glycine This work was supported by Deutsche Forschungsgemeinschaft.     

Characterization of 4,4'-Diisothiocyano-2,2'-disulfonic Acid Stilbene Inhibition of 3-Phosphoglycerate-Dependent O(2) Evolution in Isolated Chloroplasts : Evidence for a Common Binding Site on the C(4) Phosphate Translocator for 3-Phosphoglycerate, Phosphoenolpyruvate, and Inorganic Phosphate

3-Phosphoglycerate (PGA)-dependent O₂ evolution by mesophyll chloroplasts of the C₄ plant, Digitaria sanguinalis L. Scop. (crabgrass), was inhibited by micromolar levels of 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS). As little as 1.8 micromolar DIDS added to the assay medium (containing 0.7 millimolar PGA) resulted in 80 to 100% inhibition of O₂ evolution. The extent of inhibition of O₂ evolution observed was dependent on various factors including: pH, concentration of DIDS to relative chlorophyll, concentration of PGA, and the time of addition of DIDS to the chloroplasts relative to addition of PGA.

Preincubation of crabgrass chloroplasts with micromolar levels of DIDS, followed by washing to remove any nonirreversibly bound DIDS, inhibited PGA-dependent O₂ evolution. Protection against this inhibition was afforded by preincubating the chloroplasts with various substrates before adding DIDS. For example, if the chloroplasts were first incubated with 8.3 millimolar PGA, phosphoenolpyruvate (PEP) or inorganic phosphate before adding 42 micromolar DIDS, the percentage of inhibition was decreased from 100% (without any substrate) to 0, 54, and 67%, respectively. 2-Phosphoglycerate caused a slight decrease in the inhibition (about 10%) and glucose-6-phosphate had no protective effect. If the chloroplasts were pretreated with DIDS initially, the inhibition could not be overcome by PGA, suggesting that DIDS acts as an irreversible inhibitor. Micromolar levels of DIDS also inhibited PGA dependent O₂ evolution by isolated chloroplasts of the C₃ plant barley. As with crabgrass, preincubation with PGA or inorganic phosphate resulted in a decrease in the DIDS inhibition, but PEP was very ineffective compared to the C₄ chloroplasts.

Oxalacetate-dependent O₂ evolution and its stimulation by the uncoupler, NH₄Cl, were unaffected by the addition of DIDS to crabgrass mesophyll chloroplasts. Furthermore, preincubation of the chloroplasts with DIDS (up to 65 micromolar) had no inhibitory effect on the extractable activity of NADP glyceraldehyde-3-P dehydrogenase and phosphoglycerate kinase. Inhibition by DIDS was interpreted to be at the substrate binding site of the phosphate translocator. The data further suggest that in C₄ crabgrass chloroplasts, PEP is transported on a carrier which also transports PGA.