Maize F<Subscript>1</Subscript> hybrid differs from its maternal parent in the development of chloroplasts in bundle sheath,but not in mesophyll cells: Quantitative analysis of chloroplast ultrastructure and dimensions in different parts of leaf blade at the beginning of its senescence

The quantitative changes of chloroplast ultrastructure and dimensions in mesophyll (MC) and bundle sheath (BSC) cells, associated with the onset of leaf senescence, were followed along the developmental leaf blade gradient of the third leaf of maize (Zea mays L.). To ascertain whether the rapidity of structural changes associated with the transition of chloroplasts from mature to senescent state is a heritable trait, the parental and the first filial generations of plants were used. The heterogeneity of leaf blade, associated with the development of maize leaf (with the oldest regions at the apex and the youngest ones at the base) was clearly discernible in the ultrastructure and dimensions of chloroplasts; however, there were differences in the actual pattern of chloroplast development between both genotypes as well as between both cell types examined. While the course of MC chloroplasts’ development at the onset of leaf senescence in maize hybrid followed that of its parent rather well, this did not apply for the BSC chloroplasts. In this case, each genotype was characterized by its own distinguishable developmental pattern, particularly as regards the accumulation of starch inclusions and the associated changes of the size and shape of BSC chloroplasts.     

The Ultrastructure of Chloroplasts,Content of Photosynthetic Pigments,and Photochemical Activity of Maize (Zea mays L.) as Influenced by Different Concentrations of the Herbicide Amitrole

The effect of three different concentrations of amitrole (AM), a bleaching herbicide affecting carotenogenesis, on chloroplast ultrastructure, photosynthetic pigment contents, and photochemical activity was studied in two maize genotypes differing in photosynthetic characteristics. The content of photosynthetic pigments in leaves of plants treated with low (20 M) AM concentration was similar to control plants and no damaging effect of the herbicide on the ultrastructure of either mesophyll (MC) or bundle-sheath (BSC) cell chloroplasts was observed. Higher (60 and 120 M) concentrations of AM caused a significant decrease in the content of carotenoids (especially xanthophylls), which was followed by photooxidative destruction of chlorophylls and some alterations of chloroplast ultrastructure. MC chloroplasts appeared more sensitive to the damaging effect of AM compared to BSC chloroplasts. A significant decrease in the amount of both granal and intergranal thylakoids in MC chloroplasts was observed with the increasing concentration of AM. As regards BSC chloroplasts, rapid decrease in the volume density of starch inclusions was found in plants treated with higher concentrations of AM. When 120 M AM was used, both MC and BSC chloroplasts contained just a few thylakoid membranes that were strongly altered. The changes in the ultrastructure of MC chloroplasts were accompanied by the changes in their photochemical activity. The formation of chloroplast protrusions after treatment of plants with AM as well as in control plants was also observed.     

Ultrastructure and Dimensions of Chloroplasts in Leaves of Three Maize (Zea mays L.) Inbred Lines and their F1 Hybrids Grown Under Moderate Chilling Stress

Influence of moderate chilling stress on vascular bundle sheath cell (BSC) and especially mesophyll cell (MC) chloroplasts of mature maize leaves was studied by electron microscopy and stereology. Plants of two inbred lines of maize, differing in their photosynthetic activity, and their F₁ hybrids were cultivated during autumn in heated or unheated glasshouse. Generally, chilling temperatures resulted mainly in the decrease in stereological volume density (VD) of both granal and intergranal thylakoids of MC chloroplasts, while the ratio of granal to all thylakoids (granality) was less affected. The VD of peripheral reticulum and plastoglobuli usually increased after cold treatment of plants. The volume of MC chloroplasts usually increased under chilling stress, the shape of the chloroplasts changed only slightly. The ultra-structure of chloroplasts differed between individual genotypes; chilling-stressed hybrid plants showed positive heterosis particularly in the granal thylakoids' VD of MC chloroplasts.     

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.     

Ultrastructural localization of photosynthetic and photorespiratory enzymes in epidermal, mesophyll, bundle sheath, and vascular bundle cells of the C4 dicot Amaranthus viridis.

In the leaves of the NAD-malic enzyme (NAD-ME)-type C4 dicot Amaranthus viridis L., there are chloroplasts in the vascular parenchyma cells (VPC), companion cells (CC), ordinary epidermal cells (EC), and guard cells (GC), as well as in the mesophyll cells (MC) and the bundle sheath cells (BSC). However, the chloroplasts of the VPC, CC, EC, and GC are smaller than those of the MC and BSC. In this study, the accumulation of photosynthetic and photorespiratory enzymes in these leaf cell types was investigated by immunogold labelling and electron microscopy. Strong labelling for phosphoenolpyruvate carboxylase was found in the MC cytosol. Weak labelling was observed in the CC and GC cytosol. Labelling for pyruvate, Pi dikinase occurred to varying degrees in the chloroplasts of all cell types except CC. Labelling for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase was detected in the chloroplasts of all cell types except MC. For both NAD-ME and the P-protein of glycine decarboxylase, intense labelling was found in the BSC mitochondria; weaker labelling was recognized in the VPC mitochondria. These data indicate that when not only the MC and BSC but also the other leaf cell types are included, the cell-specific expression of the enzymes in C4 leaves becomes more complex than has been known previously. These findings are discussed in relation to the metabolic function of epidermal and vascular bundle cells.     

The Development of Chloroplast Ultrastructure and Hill Reaction Activity During Leaf Ontogeny in Different Maize (Zea Mays L.) Genotypes

Changes in Hill reaction activity (HRA) and ultrastructure of mesophyll cell (MC) chloroplasts were studied during the ontogeny of third leaf of maize plants using polarographic oxygen evolution measurement, transmission electron microscopy, and stereology. The chloroplast ultrastructure was compared in young (actively growing), mature, and senescing leaves of two different inbreds and their reciprocal F1 hybrids. Statistically significant differences in both HRA and MC chloroplast ultrastructure were observed between different stages of leaf ontogeny. Growth of plastoglobuli was the most striking characteristic of chloroplast maturation and senescence. The chloroplasts in mature and senescing leaves had a more developed system of thylakoids compared to the young leaves. Higher HRA was usually connected with higher thylakoid volume density of MC chloroplasts.     

Light Modulation and Localization of Sucrose Phosphate Synthase Activity between Mesophyll Cells and Bundle Sheath Cells in C(4) Species

Experiments were conducted with several Panicum species (representing the different C₄ subtypes) to examine the light modulation of sucrose phosphate synthase (SPS) activity and the effect of illumination on the distribution of SPS activity between mesophyll cells (MC) and bundle sheath cells (BSC). Activity of SPS in the light decreased in the order: C₄ > C₃-C₄ intermediate > C₃. In illuminated leaves, SPS activities were similar among the three C₄ subtypes, but SPS activity was higher for NAD-malic enzyme (NAD-ME) species with centripetal chloroplasts in BSC (NAD-ME(P) species) than for NAD-ME species with centrifugal chloroplasts in BSC (NAD-ME(F) species). Transfer of plants into darkness for 30 minutes resulted in decreased SPS activity for all species tested except Panicum bisulcatum (C₃ species) and Panicum virgatum (NAD-ME(P) species) which showed little or no change. All C₄ subtypes had some SPS activity both in MC and BSC. In the light, SPS activity was mainly in the MC for NADP-ME, NAD-ME(F) and phosphoenolpyruvate carboxykinase species, while it was mainly in the BSC for NAD-ME(P) species. In the dark, for all C₄ subtypes, SPS activity in the MC was decreased to a greater extent than that in the BSC. It is intriguing that NAD-ME(F) and NAD-ME(P) species differed in the activity and distribution of SPS activity between MC and BSC, although they are otherwise identical in the photosynthetic carbon assimilation pathway. Diurnal changes in SPS activity in the MC and BSC were also examined in maize leaves. SPS activity in the MC in maize leaves was high and relatively constant throughout the middle of the light period, dropped rapidly after sunset and increased again prior to the light period. On the other hand, SPS activity in the BSC was lower and changed more coincidently with light intensity than that in the MC. The results suggested that light activation of SPS activity located in the BSC may require higher irradiance for saturation than the SPS in the MC. We conclude that SPS may function in both MC and BSC for sucrose synthesis in the light, particularly at high light intensity, while in the dark, the major function may be in the BSC during starch degradation.     

The Development of Chloroplast Ultrastructure and Hill Reaction Activity During Leaf Ontogeny in Different Maize (Zea Mays L.) Genotypes

Changes in Hill reaction activity (HRA) and ultrastructure of mesophyll cell (MC) chloroplasts were studied during the ontogeny of third leaf of maize plants using polarographic oxygen evolution measurement, transmission electron microscopy, and stereology. The chloroplast ultrastructure was compared in young (actively growing), mature, and senescing leaves of two different inbreds and their reciprocal F1 hybrids. Statistically significant differences in both HRA and MC chloroplast ultrastructure were observed between different stages of leaf ontogeny. Growth of plastoglobuli was the most striking characteristic of chloroplast maturation and senescence. The chloroplasts in mature and senescing leaves had a more developed system of thylakoids compared to the young leaves. Higher HRA was usually connected with higher thylakoid volume density of MC chloroplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Degradation of Glucan Primers in the Absence of Starch Synthase 4 Disrupts Starch Granule Initiation in Arabidopsis

Arabidopsis leaf chloroplasts typically contain five to seven semicrystalline starch granules. It is not understood how the synthesis of each granule is initiated or how starch granule number is determined within each chloroplast. An Arabidopsis mutant lacking the glucosyl-transferase, STARCH SYNTHASE 4 (SS4) is impaired in its ability to initiate starch granules; its chloroplasts rarely contain more than one large granule, and the plants have a pale appearance and reduced growth. Here we report that the chloroplastic α-amylase AMY3, a starch-degrading enzyme, interferes with granule initiation in the ss4 mutant background. The amy3 single mutant is similar in phenotype to the wild type under normal growth conditions, with comparable numbers of starch granules per chloroplast. Interestingly, the ss4 mutant displays a pleiotropic reduction in the activity of AMY3. Remarkably, complete abolition of AMY3 (in the amy3 ss4 double mutant) increases the number of starch granules produced in each chloroplast, suppresses the pale phenotype of ss4, and nearly restores normal growth. The amy3 mutation also restores starch synthesis in the ss3 ss4 double mutant, which lacks STARCH SYNTHASE 3 (SS3) in addition to SS4. The ss3 ss4 line is unable to initiate any starch granules and is thus starchless. We suggest that SS4 plays a key role in granule initiation, allowing it to proceed in a way that avoids premature degradation of primers by starch hydrolases, such as AMY3.     

N4‐methylcytidine ribosomal RNA methylation in chloroplasts is crucial for chloroplast function,development, and abscisic acid response in Arabidopsis

Although the essential role of messenger RNA methylation in the nucleus is increasingly understood, the nature of ribosomal RNA (rRNA) methyltransferases and the role of rRNA methylation in chloroplasts remain largely unknown. A recent study revealed that CMAL (for Chloroplast mr aW‐ Like) is a chloroplast‐localized rRNA methyltransferase that is responsible for N4‐methylcytidine (m⁴C) in 16S chloroplast rRNA in Arabidopsis thaliana. In this study, we further examined the role of CMAL in chloroplast biogenesis and function, development, and hormone response. The cmal mutant showed reduced chlorophyll biosynthesis, photosynthetic activity, and growth‐defect phenotypes, including severely stunted stems, fewer siliques, and lower seed yield. The cmal mutant was hypersensitive to chloroplast translation inhibitors, such as lincomycin and erythromycin, indicating that the m⁴C‐methylation defect in the 16S rRNA leads to a reduced translational activity in chloroplasts. Importantly, the stunted stem of the cmal mutant was partially rescued by exogenous gibberellic acid or auxin. The cmal mutant grew poorer than wild type, whereas the CMAL‐overexpressing transgenic Arabidopsis plants grew better than wild type in the presence of abscisic acid. Altogether, these results indicate that CMAL is an indispensable rRNA methyltransferase in chloroplasts and is crucial for chloroplast biogenesis and function, photosynthesis, and hormone response during plant growth and development.     

Starch phosphorylase 2 is essential for cellular carbohydrate partitioning in maize

Carbohydrate partitioning is essential for plant growth and development, and its hindrance will result in excess accumulation of carbohydrates in source tissues. Most of the related mutants in maize(Zea mays L.) display impaired whole-plant sucrose transport, but other mechanisms affecting carbohydrate partitioning have seldom been reported. Here, we characterized chlorotic leaf3(chl3), a recessive mutation causing leaf chlorosis with starch accumulation excessively in bundle sheath chloroplasts...     

Starch Accumulation and Photosynthetic Activity in Primary Leaves of Bean (Phaseolus vulgaris L.)

The effects of starch accumulation on photosynthesis and chloroplastultrastructure were studied in primary leaves of bean (Phaseolusvulgaris L. cv. Bulgarian). De-topping the shoot above the primaryleaf node, caused over an 8-day period, a considerable increasein the photosynthetic activity of the primary leaves, despitethe fact that a large quantity of starch had accumulated intheir chloroplasts. The accumulation of starch was greater inthe chloroplasts of spongy cells in comparison with that ofthe palisade cells. Initiation of starch grains was observedmainly in the peripheral part of the chloroplast, distant fromthe cell wall. As a result, most of the starch was accumulatedclose to the inner part of the cell, leaving a considerablemass of the chloroplast near the cell wall free of starch. Theaccumulation of starch was accompanied by the destruction, deformationand disorientation of grana and thylakoids. It is concludedthat the accumulation of starch is not inevitably a limitingfactor in photosynthesis and the results cast doubt on the hypothesisthat starch accumulation or dissipation is the main factor involvedin the regulation of photosynthesis. Phaseolus vulgaris L, bean, photosynthesis, starch accumulation, chloroplast ultrastructure     

小麦黄化突变体叶绿体超微结构研究

利用透射电镜对小麦自然黄化突变体及其突变亲本(西农1718)叶片细胞叶绿体的数目、形态及超微结构进行比较分析。结果发现:(1)3种不同黄化程度突变体的叶绿体分布、数目、形状及大小与突变亲本无明显差异;(2)突变体叶绿素含量为野生型58%的黄绿植株与其突变亲本叶绿体超微结构无明显差异,基质类囊体与基粒类囊体高度分化,基粒数目以及基粒片层数目较多;(3)突变体金黄和绿黄植株的叶绿素含量分别为野生型的17%、24%,其叶绿体超微结构与突变亲本明显不同,突变体的叶绿体发育存在明显缺陷,其中突变体金黄植株的叶绿体内无基粒、基质片层清晰可见,有淀粉粒,嗜锇颗粒较多,而突变体绿黄植株的叶绿体内有基粒,但明显少于突变亲本,且基粒片层较少,基质类囊体较发达。结果表明该黄化突变体叶绿体超微结构的改变,是由于叶绿素含量降低造成,推测,该黄化突变是由于叶绿素合成受阻导致的。     

Chloroplast fine structure in the japonica-2 maize mutant exposed to continuous illumination. 1. The green tissues.

Exposure to continuous illumination causes the appearance of numerous plastoglobuli in the stroma of both the mesophyll and bundle sheath chloroplasts of the green tissues of the leaves of the japonica-2 mutant of maize. In the pale green tissues the thylakoids have markedly swollen membranes. Another feature of the plastids exposed to continuous illumination is the heavy accumulation of starch. The japonica-2 chloroplasts show a different sensitivity to light, the chloroplasts of the pale green tissues being affected more markedly than the ones of the dark green tissues, and the bundle sheath chloroplasts more than those of the mesophyll. The effects of continuous illumination may be interpreted as an acceleration of chloroplast ontogenesis.     

Chloroplast protein phosphorylation and chlorophyll fluorescence quenching. Activation by tetramethyl-p-hydroquinone, an electron donor to plastoquinone

When tetramethyl-p-benzoquinone (TMQ) is reduced to tetramethyl-p-hydroquinone (TMQH₂) by NaBH₄, TMQH₂ will act as an electron donor in isolated chloroplasts. The resulting electron transport is highly sensitive to inhibition by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), and the site of donation is inferred to be plastoquinone, in agreement with previous findings. In contrast, when TMQ is added to chloroplasts with ascorbate as reductant, the resulting electron transport is relatively insensitive to DBMIB, and so plastoquinone is assumed not to be involved. In darkness, TMQH₂ activates the chloroplast protein kinase that phosphorylates the light-harvesting chlorophyll a/b-protein complex (LHCP), while TMQ with ascorbate does not. TMQH₂ also activates ATP-dependent chlorophyll fluorescence quenching to a much greater extent than does TMQ with ascorbate. These findings are explained by the recent proposal that phosphorylation of LHCP is activated by reduced plastoquinone. They are therefore evidence for plastoquinone-regulated protein phosphorylation as a mechanism for self-adjustment of distribution of excitation between the two light reactions of photosynthesis.     

Observations on Photosystem II mutants of Scenedesmus: Pigments and proteinaceous components of the chloroplasts

Nine mutants of the green alga, Scenedesmus obliquus, which are blocked in the Photosystem II portion of photosynthesis were analyzed for possible deletion or alteration of (1) various components of the photosynthetic electron transport system, (2) of chloroplast lipids, (3) of total chlorophyll or of the chlorophyll a/chlorophyllb ratio, and (4) of their content of carotenes and carotenoids. No changes in content or activity of ferredoxin, ferredoxin-NADP⁺ reductase, plastocyanin, cytochrome c-552, and the membrane-bound b-type or c-type cytochromes were observed. The most consistent differences noted between the mutant strains and the wild-type strain were in the molar ratio of chlorophyll/plastoquinone A, the total chlorophyll content, and a decreased content of - and β-carotene with a concomitant increase of carotenoids. The loss of Photosystem II activity in these mutant strains, as observed either with whole cells or with isolated chloroplast fragments, may be accounted for by their decreased content of plastoquinone A. Their decreased chlorophyll content and altered carotene/xanthophyll ratio also suggests possible alteration of chloroplast membrances resulting in increased internal oxidation of the photosynthetic pigments.