Photosynthesis and starch metabolism of chloroplasts during prolonged illumination

Summary The starch metabolism of the chloroplasts in the leaves of Stellaria media was studied by means of electron microscopy. During the night the starch grains diminished in size but did not disappear entirely. In the light they grew due to photosynthesis. After prolonged illumination of the plant the grains almost filled up the chloroplasts. However, after an illumination of 26–27 hr a sudded disintegration took place. This was apparently caused by the increased activity of -amylase observed in an earlier investigation to occur at this time in the chloroplasts. After the disintegration the starch grains of the chloroplasts showed irregular changes.The rate of photosynthesis and respiration was measured by an infra-red gas analyser. During prolonged illumination Stellaria media showed a rather intensive and constant rate of assimilation. The role of starch disintegration and -amylase synthesis in making possible this constant assimilation has been discussed.     

A starch-accumulating mutant of Arabidopsis thaliana deficient in a chloroplastic starch-hydrolysing enzyme

The aim of this work was to identify enzymes that participate in the degradation of transitory starch in Arabidopsis . A mutant line was isolated by screening leaves at the end of the night for the presence of starch. The mutant had a higher starch content than the wild-type throughout the diurnal cycle. This accumulation was due to a reduction in starch breakdown, leading to an imbalance between the rates of synthesis and degradation. No reduction in the activity of endo-amylase (α-amylase), β-amylase, starch phosphorylase, maltase, pullulanase or D-enzyme could be detected in crude extracts of leaves of the mutant. However, native PAGE in gels containing amylopectin revealed that a starch-hydrolysing activity, putatively identified as an endo-amylase and present in wild-type chloroplasts, was absent or appreciably reduced in the mutant. This is the first time that a specific enzyme required for starch degradation has been identified in leaves.     

类根瘤对烟草叶片中叶绿体及其淀粉粒含量的影响

目的：探讨类根瘤对烟草叶片中的叶绿体及其淀粉粒含量的影响。方法：无菌培养烟草再生植株,其中一部分经过一定浓度的2,4－D与豇豆根瘤菌512快生型突变株菌液诱导处理,另外一部分为对照。运用常规电镜技术制作叶片厚切片,通过显微观察,对两组烟草叶片细胞中的叶绿体及其淀粉粒含量进行对比分析。结果：结瘤植株叶片细胞内的叶绿体含量虽然与对照之间无明显差异,但叶片栅栏组织和海绵组织细胞内淀粉粒的含量却均较对照减少,其中前者减少更为明显（P＜0.05)。结论：结瘤烟草叶片内光合产物的大量同化可能与类根瘤的形成有关。     

Maltose metabolism by pea chloroplasts

The aim of this work was to investigate the origin of maltose formed during starch breakdown in the dark by chloroplasts of Pisum sativum. The maximum catalytic activities of maltose phosphorylase and maltase in pea leaves were shown to be low, relative to those of enzymes known to be involved in starch breakdown. Fractionation of pea leaves indicated that the chloroplasts lack maltase but have enough maltose phosphorylase to synthesize the amounts of maltose formed when isolated chloroplasts breakdown starch. The absence of exogenous phosphate markedly reduced starch breakdown and maltose accumulation by isolated chloroplasts. When [¹⁴C]glucose was supplied to chloroplasts that were breaking down starch in the dark, maltose was labelled and most of the label was in the glucose moeity. It is suggested that maltose phosphorylase, using glucose-1-phosphate formed from starch by α-glucan phosphorylase, is responsible for, at least some of, the synthesis of maltose during starch breakdown by pea chloroplasts in vitro.     

Studies on the Activity of Beta-Analyses of the Chloroplasts of Stellaria media during Prolonged Illumination

The changes in the activity of enzymes hydrolyzing starch were studied. Enzymes were extracted from the leaves of Stellaria media. Enzyme activity seems to be concentrated in the chloroplasts. Beta-amylase has been identified as the main enzyme hydrolyzing starch in these leaves. When Stellaria media plants are left in continuous illumination after a normal light-dark-rhythm), beta-amylase activity remains essentially unchanged for some time. Alter an Illumination of 24–27 hours the activity suddenly increases. This phenomenon occurs at about the same time as the rapid disintegration of starch previously accumulated in the chloroplasts. In the light of present-day knowledge the increase in the activity cannot be explained.     

Regreening of senescent Nicotiana leaves. II. Redifferentiation of plastids

Single senescent leaves attached to decapitated shoots of Nicotiana rustica L. regreened, especially when treated with cytokinin. Regreening caused an increase in leaf thickness, due to cell expansion. Senescent leaf plastids (gerontoplasts) were smaller than green chloroplasts, with degenerated membrane systems and stroma, and larger plastoglobuli. At advanced senescence, micrographs showed disintegrating gerontoplasts, reduced numbers of plastids were counted, and regreening became variable. The redevelopment of grana and stroma in regreening plastids was accelerated by cytokinin. All plastids in regreening leaves were identifiable as redifferentiating gerontoplasts because of their content of plastoglobuli and starch. Immunogold labelling showed significant association of POR with etioplasts in cotyledons, but with mature plastids in regreening leaves. No proplastids or dividing chloroplasts were observed in regreening leaves. Plastids numbers declined during senescence and did not increase again during regreening. It is concluded that the chloroplasts of regreening leaves arose by redifferentiation of gerontoplasts.Keywords: Chloroplasts, cytokinin, Nicotiana, senescence, regreening.      

菊花黄绿叶突变体不同类型叶片的叶绿素含量和结构特征比较

以菊花黄绿叶突变体-NAu04-1-31为试验材料,测定了黄叶、黄绿叶和绿叶3种不同类型叶片的叶绿素含量,并观察比较了叶片的显微与超微解剖结构.叶绿素含量测定表明:黄叶、黄绿叶的叶绿素含量显著低于绿叶,而黄叶叶绿素a与叶绿素b的比值大于绿叶.叶绿体显微与超微结构观察发现:黄叶细胞内叶绿体形状不规1则,缺乏正常的叶绿体膜结构,无类囊体,无淀粉粒,嗜锇颗粒较多;黄绿叶叶片栅栏组织绿色部分与绿叶的栅栏组织相似,黄色部分与黄叶的栅栏组织棚似,黄色部分的海绵组织中有类似于绿色叶片的海绵组织结构,而绿色部分含有类似于黄叶的海绵组织的结构特征.绿叶细胞内叶绿体较多,形状规则,基粒片层清晰,其内淀粉粒多而大,嗜锇颗粒较少.     

The Effect of 6-Benzylaminopurine on the Starch Metabolism of Stellaria media

The effect of 6-benzylaminopurine (BAP) was studied by measuring the amylase activity of leaf extracts and the starch content of chloroplasts in Stellaria media-plants cultivated for five days in solutions containing different quantities of BAP. Small amounts of BAP decreased amylase activity in weak-growing plants. In strong-growing ones the effect was variable. However, in all experiments very high amount of this growth regulator caused a great increase in amylase activity which was accompanied by a relatively high concentration of starch in the chloroplasts. The induction of amylase activity was thus not high enough to diminish the starch content. This is supposed to be due to the lack of a sink organ since the growth of roots and shoots was greatly inhibited in concentrated BAP-solutions (10^-5M or more). In these cases the plants usually died during the first week. In some cases small amounts of BAP increased the growth of S. media, and high concentrations inhibited it causing characteristic morphological changes, such as creeping growth pattern, small, thick leaves and the turning up of the leaf margins. Benzylaminopurine also caused increased resistance to aging, especially in low concentrations (10^–7–10^–6M).     

Control of starch granule numbers in Arabidopsis chloroplasts

The aim of this work was to investigate starch granule numbers in Arabidopsis (Arabidopsis thaliana) leaves. Lack of quantitative information on the extent of genetic, temporal, developmental, and environmental variation in granule numbers is an important limitation in understanding control of starch degradation and the mechanism of granule initiation. Two methods were developed for reliable estimation of numbers of granules per chloroplast. First, direct measurements were made on large series of consecutive sections of mesophyll tissue obtained by focused ion beam-scanning electron microscopy. Second, average numbers were calculated from the starch contents of leaves and chloroplasts and estimates of granule mass based on granule dimensions. Examination of wild-type plants and accumulation and regulation of chloroplast (arc) mutants with few, large chloroplasts provided the following new insights. There is wide variation in chloroplast volumes in cells of wild-type leaves. Granule numbers per chloroplast are correlated with chloroplast volume, i.e. large chloroplasts have more granules than small chloroplasts. Mature leaves of wild-type plants and arc mutants have approximately the same number of granules per unit volume of stroma, regardless of the size and number of chloroplasts per cell. Granule numbers per unit volume of stroma are also relatively constant in immature leaves but are greater than in mature leaves. Granule initiation occurs as chloroplasts divide in immature leaves, but relatively little initiation occurs in mature leaves. Changes in leaf starch content over the diurnal cycle are largely brought about by changes in the volume of a fixed number of granules.     

Effect of Elevated CO2 Concentration on the Starch Grain Accumulation in Chloroplasts from Soybean Leaves at Different Nodes

Soybean ( Glycine max (L.) Merr. ) plants were grown under ambient and elevated CO2 (plus 350 μL/L) concentration in cylindrical open-top chamber to examine their effects on the ultra- structure of chloroplasts. The upper, lower and mid-node leaves were harvested after 7 days full expansion under different CO2 concentrations and ultrathin section were prepared for transmission electron microscopy. In general, the average content of starch grains and thylakoid membranes in the chloroplasts under the elevated CO2 concentration were always higher than the control. Under higher CO2 concentration, there were smaller and less starch grains in the chloroplasts from upper-node leaves than those from mid-node leaves. The shape of their starch grains changed from elliptical to oval,and their thylakoid membranes and grana remained normal. At lower-node leaves, one or two oval, or three timer starch grains accumulated in the chloroplasts. In the mid-node leaves,however, some chloroplasts under higher CO2 concentration had rather large tim elliptical starch grains which could consequently cause disruption of grana and stroama thylakoids in the chloroplasts, whereas in other chloroplasts, the thylakoid membranes and grana were not deformed as the starch grains were smaller and elliptical. On the other hand, under higher CO2 concentration, the stacking degree of thylakoid membranes and starch grains accumulation in the mid-node leaves were significantly higher than those in the lower-node leaves,and slightly higher than the upper-node leaves. These results, in agreement with the chlorophyll contents and photosynthetic rate which reported by other authors in the past, indicated that the ultrastmcture response of the chloroplasts from different leaf nodes of soybeen under elevated CO2 coneentration were different. The seed yield of soybean at different nodes was decreased gradually from mid-nodes towards both upper- and lower-nodes. The greatest effect of elevated CO2 eoneentrafion on seed yeild was at the mid-node leaves. The variation of seed yields of soybean at different nodes under elevated CO2 concentration was in eoneert with the change in the ultrastmcture of chloroplasts and in turn the change in their photosynthetic rates of leaves at different nodes.     

Die Bedeutung des Glucose-1-Phosphates für die Stärkesynthese in den Bündelscheidenzellen von Zea mays

Summary After illumination intact leaves of Zea mays contain sucrose and starch. The latter is located mainly in the bundle sheath cells. When 0.5 mm wide leaf strips are incubated with sucrose solution, the starch deposit in the bundle-sheath chloroplasts is greatly increased by light. When isolated bundle sheath cells are suspended in water or solutions of sucrose and various metabolites they are not capable of synthesizing starch. An appreciable production of starch in the chloroplasts of isolated bundle sheath cells can be observed only in the presence of glucose-1-phosphate.     

Salt tolerance in the halophyte Suaeda maritima (L.) Dum. The influence of the salinity of the culture solution on leaf starch and phosphate content

Abstract. The starch concentration in mature leaves of the halophyte Suaeda maritima increased from 4.7 to 7.3 mg mg⁻¹ chlorophyll when sodium chloride (680 mol M⁻³) was added to the solution in which the plants were grown. This effect of salinity on the starch: chlorophyll ratio was greater in young than in old leaves. Electron micrographs showed the starch to be in the chloroplasts and this was confirmed by measurements on isolated chloroplasts. Total phosphorus concentration (mg mg⁻¹ chlorophyll) in leaves of all ages from plants of S. maritima decreased on salinization of the growth medium suggesting an inverse relationship between phosphorus and starch concentrations. However, although leaf starch concentration varied with leaf age, phosphorus concentration did not. The cause of starch accumulation in chloroplasts at salinities which are optimal for growth (340 mol m⁻³) remains unclear.     

Changes in the plastid ultrastructure duringSedum rotundifolium leaf development

The ultrastructure of plastids was investigated in succulent leaves ofSedum rotundifolium to examine their changes during development. Leaves were categorized as etiolated, immature, young, and mature, based on their developmental stage and size. Of particular interest were the features of the tubular inclusion bodies (TIBs) and starch grains. These, along with vacuole size, showed remarkable changes over time. Etioplasts of unexposed leaves had prolamellar bodies, abundant starch grains, large TIB, few plastoglobuli, and thylakoid systems. Membranes of the thylakoids were still continuous with those of the prolamellar body. The plastids were often influenced by the presence and profile of inclusion bodies and starch grains throughout the early stages. Morphology was highly variable in the etioplasts but consistently hemispherical or ovoid in mature chloroplasts. TIB was most abundant in the etiolated leaves, but disappeared completely with development. Starch grains also became significantly reduced in size. Both young and mature mesophyll cells exhibited a normal chloroplast ultra-structure and huge central vacuoles, with an extremely thin peripheral cytoplasm. Grana were extensive and comprised a large portion of the chloroplasts. Traces of peripheral reticulum were also discovered in the chloroplasts of expanded leaves. The implications of these ultrastructural changes in the tubular inclusions and starch grains are discussed with relevance to Crassulacean acid metabolism (CAM).     

高CO_2浓度对大豆不同叶位叶片叶绿体淀粉粒积累的效应

对高CO_2浓度下生长的大豆(Glycine max(L.)Merr.)不同叶位的叶片进行了电镜观察,揭示出大豆不同叶位叶片的叶绿体对倍增的CO_2浓度反应不一。其显著的超微结构差异特征是:1.叶位居中的叶片叶绿体积累的淀粉粒不仅很大,而且最多,有的叶绿体中的淀粉粒可达20个,几乎充满着叶绿体的基质空间。2.下位叶叶绿体的淀粉粒积累较多,通常为2～5个;3.上位叶叶绿体所含淀粉粒既小又少,虽然有的叶绿体中也积累有3～4个淀粉粒,但大多数叶绿体中所含淀粉粒仅有1～2个。以上结果联系到大豆中位叶的光合作用速率较高及对籽粒产量起作用最大来讨论是很有意义的。     

Anatomy, chloroplast structure and compartmentation of enzymes relative to photosynthetic mechanisms in leaves and cotyledons of species in the tribe Salsoleae (Chenopodiaceae)

Certain members of the family Chenopodiaceae are the dominant species of the deserts of Central Asia; many of them are succulent halophytes which exhibit C4-type CO2 fixation of the NAD- or NADP-ME (malic enzyme) subgroup. In four C4 species of the tribe Salsoleae, the Salsoloid-type Kranz anatomy in leaves or stems was studied in relation to the diversity in anatomy which was found in cotyledons. Halocharis gossypina, has C4 NAD-ME Salsoloid-type photosynthesis in leaves and C3 photosynthesis in dorsoventral non-Kranz cotyledons; Salsola laricina has C4 NAD-ME Salsoloid-type leaves and C4 NAD-ME Atriplicoid-type cotyledons; Haloxylon persicum, has C4 NADP-ME Salsoloid-type green stems and C3 isopalisade non-Kranz cotyledons; and S. richteri has C4 NADP-ME Salsoloid-type leaves and cotyledons. Immunolocalization studies on Rubisco showed strong labelling in bundle sheath cells of leaves and cotyledons of organs having Kranz anatomy. The C4 pathway enzyme phosphoenolpyruvate carboxylase was localized in mesophyll cells, while the malic enzymes were localized in bundle sheath cells of Kranz-type tissue. Immunolocalization by electron microscopy showed NAD-ME is in mitochondria while NADP-ME is in chloroplasts of bundle sheath cells in the respective C4 types. In some C4 organs, it was apparent that subepidermal cells and water storage cells also contain some chloroplasts which have Rubisco, store starch, and thus perform C3 photosynthesis. In non-Kranz cotyledons of Halocharis gossypina and Haloxylon persicum, Rubisco was found in chloroplasts of both palisade and spongy mesophyll cells with the heaviest labelling in the layers of palisade cells, whereas C4 pathway proteins were low or undetectable. The pattern of starch accumulation correlated with the localization of Rubisco, being highest in the bundle sheath cells and lowest in the mesophyll cells of organs having Kranz anatomy. In NAD-ME-type Kranz organs (leaves and cotyledons of S. laricina and leaves of H. gossypina the granal index (length of appressed membranes as a percentage of total length of all membranes) of bundle sheath chloroplasts is 1.5 to 2.5 times higher than that of mesophyll chloroplasts. In contrast, in the NADP-ME-type Kranz organs (S. richteri leaves and cotyledons and H. persicum stems) the granal index of mesophyll chloroplasts is 1.5 to 2.2 times that of the bundle sheath chloroplasts. The mechanism of photosynthesis in these species is discussed in relation to structural differences.     

Ultrastructural aspects of kranz anatomy inDigitaria sanguinalis andSetaria viridis (poaceae)

Structural differentiation of Kranz anatomy has been investigated in leaf cross sections of two C-4 Poaceae:Digitaria sanguinalis andSetaria viridis. The study mainly focused on cellular and interfacial features of bundle sheath (BS) and mesophyll (MS) cells of the C-4 structure. Prominent BS, spaced by only two MS cells apart, were surrounded concentrically by a layer of MS cells. BS cells ofS. viridis had centrifugally arranged relatively large chloroplasts containing much starch, but the chloroplasts had agrana to rudimentary grana. Structural and size dimorphisms, when starch was present, were detected between BS and MS chloroplasts. Loosely arranged MS cells had peripherally displaced smaller chloroplasts containing little to none starch. BS chloroplasts ofD. sanguinalis were similar to those ofS. viridis, but had very little starch and well-developed long agranal stroma lamella. Features of MS cells were similar in both species, but well-defined peripheral reticulum (PR) was easily recognized in MS chloroplasts ofS. viridis. Virtually no PR was developed in BS chloroplasts examined. BS cells contained more mitochondria and microbodies, but no structural dimorphism was noticed. The electron-dense suberized lamella were often observed between BS and MS cells, especially in the primary wall of BS cells. It was most frequently found at the BS and MS cell interfaces and terminated in radial walls of the adjacent BS cells. Prominent pits with plasmodesmata (pd) were seen in the walls of both cells. There also were numerous pd in outer tangential walls of the BS cells. The number of pd ranged from 20 to 60. The pd trasversed a segment of cell wall much thinner than the adjacent wall. The current cellular data have been compared to the ultrastructural features known in leaves of other C-4 plants, especially NADP-ME species.     

NaCl胁迫对宁夏枸杞叶和幼根显微及超微结构的影响

以宁夏枸杞为材料,采用超薄切片技术制备样品,应用光学显微镜和透射电镜分析了不同浓度NaCl胁迫条件下宁夏枸杞叶和幼根显微及超微结构的变化。结果表明：随着NaCl胁迫的加重,(1)叶片上表皮细胞增厚,栅栏组织细胞出现缩短现象,排列疏松且紊乱;幼根的初生结构无明显变化。(2)叶片栅栏组织中叶绿体不再紧靠在细胞膜上,叶绿体双层膜破坏,基粒片层松散排列,杂乱无章,出现膨胀和空泡现象,淀粉粒和嗜锇颗粒增多,叶肉细胞中线粒体发生轻微变化;幼根中皮层薄壁细胞线粒体形状发生改变,结构破坏,内膜和外膜模糊甚至破裂,大多数嵴模糊,出现空泡现象;细胞核解体,基质外溢。研究表明, 不同浓度的NaCl胁迫对宁夏枸杞叶片和幼根细胞的显微及超微结构影响不同,NaCl浓度大于200 mmol/L时,宁夏枸杞叶片和幼根细胞的显微及超微结构发生了明显变化,且叶肉细胞中线粒体的变化没有叶绿体的变化显著,推测叶肉细胞中线粒体的耐盐性比叶绿体强。     

Chloroplast ultrastructure changes in Pisum sativum associated with supplementary ultraviolet (UV-B) radiation

Pea plants (Pisum sativum L. cv. Greenfeast) were grown and exposed to supplementary UV-B radiation from day 17 after planting under growth cabinet conditions. The effects of this exposure on the ultrastructure of chloroplasts and the total soluble sugar and starch concentrations were estimated. Supplementary UV-B radiation was shown to damage the structure of chloroplasts, as manifested by dilation of thylakoid membranes, a progressive disruption of the thylakoid structure and disintegration of the double membrane envelope surrounding the chloroplast, accompanied by the accumulation of large starch grains. Diurnal changes observed in starch concentration suggest that the higher concentration of starch in supplementary UV-B-treated leaves is due to its immobilization, rather than to any increase in starch synthesis: soluble sugars accumulated and remained at a higher level and then later declined.     

Seasonal dimorphism and winter chilling stress in Thymus sibthorpii

Seasonal dimorphism (summer/winter) has been so far studied only in a few plants and has been focused on summer drought stress. However, Thymus sibthorpii in the study area appears to be affected by winter chilling stress and not by summer drought stress. Thus, the winter leaves were thicker and more compact compared to the summer leaves and they had more stomata and peltate hairs, more sclerenchymatous fibers, vacuoles with phenolics, and chloroplasts than the summer leaves. In addition, their chloroplasts possessed large grana and starch grains. In the summer leaves, cell vacuoles in mesophyll did not contain phenolics, and chloroplasts were devoid of starch grains and had large plastoglobuli. Physiological measurements revealed higher net photosynthetic rate and chlorophyll content in the winter leaves than in the summer leaves. Proline and soluble sugar content along with antioxidative enzyme (superoxide dismutase, peroxidase, ascorbate peroxidase, glutathione reductase) activities were increased in the winter leaves.     

New insights on sucrose metabolism: evidence for an active A/N-Inv in chloroplasts uncovers a novel component of the intracellular carbon trafficking

The presence of sucrose (Suc) in plastids was questioned for several decades. Although it was reported some decades ago, neither Suc transporters nor Suc metabolizing enzymes were demonstrated to be active in those organelles. By biochemical, immunological, molecular and genetic approaches we show that alkaline/neutral invertases (A/N-Invs) are also localized in chloroplasts of spinach and Arabidopsis. A/N-Inv activity and polypeptide content were shown in protein extracts from intact chloroplasts. Moreover, we functionally characterized the Arabidopsis At-A/N-InvE gene coding for a chloroplast-targeted A/N-Inv. The At-A/N-InvE knockout plants displayed a lower total A/N-Inv activity in comparison with wild-type plants. Furthermore, neither A/N-Inv activity nor A/N-Inv polypeptides were detected in protein extracts prepared from chloroplasts of mutant plants. Also, the measurement of carbohydrate content, in leaves harvested either at the end of the day or at the end of the night period, revealed that the knockout plants showed a decrease in starch accumulation but no alteration in Suc levels. These are the first results demonstrating the presence of a functional A/N-Inv inside chloroplasts and its relation with carbon storage in Arabidopsis leaves. Taken together our data and recent reports, we conclude that the participation of A/N-Invs in the carbon flux between the cytosol and the plastids may be a general phenomenon in plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.