Somatic instability of carotenoid biosynthesis in the tomato ghost mutant and its effect on plastid development

The tomato (Lycopersicon esculentum (L.) Mill.) ghost plant is a mutant of the San Marzano cultivar affected in carotenoid biosynthesis. ghost plants exhibit a variable pattern of pigment biosynthesis during development. Cotyledons are green but true leaves are white. Green sectors, which appear to be clonal in origin, are frequently observed in the white tissue. Because of the lack of photosynthesis ghost plants have a very low viability in soil. We have developed a strategy for propagating ghost plants that employs organ culture to generate variegated green-white plants which, supported by the photosynthetic green areas, develop in soil to almost wild-type size. These plants were used to analyze the pigment content of the different tissues observed during development and plastid ultrastructure. Cotyledons and green leaves contain both colored carotenoids and chlorophyll but only the colorless carotenoid phytoene accumulates in white leaves. the plastids in the white tissue of ghost leaves lack internal membrane structures but normal chloroplasts can be observed in the green areas. The chromoplasts of white fruits are also impaired in their ability to form thylakoid membranes.     

The DCL gene of tomato is required for chloroplast development and palisade cell morphogenesis in leaves.

The defective chloroplasts and leaves-mutable (dcl-m) mutation of tomato was identified in a Ds mutagenesis screen. This unstable mutation affects both chloroplast development and palisade cell morphogenesis in leaves. Mutant plants are clonally variegated as a result of somatic excision of Ds and have albino leaves with green sectors. Leaf midribs and stems are light green with sectors of dark green tissue but fruit and petals are wild-type in appearance. Within dark green sectors of dcl-m leaves, palisade cells are normal, whereas in albino areas of dcl-m leaves, palisade cells do not expand to become their characteristic columnar shape. The development of chloroplasts from proplastids in albino areas is apparently blocked at an early stage. DCL was cloned using Ds as a tag and encodes a novel protein of approximately 25 kDa, containing a chloroplast transit peptide and an acidic alpha-helical region. DCL protein was imported into chloroplasts in vitro and processed to a mature form. Because of the ubiquitous expression of DCL and the proplastid-like appearance of dcl-affected plastids, the DCL protein may regulate a basic and universal function of the plastid. The novel dcl-m phenotype suggests that chloroplast development is required for correct palisade cell morphogenesis during leaf development.     

Plastid Development and Ultrastructure in Yellowish Mutants of Sugar Cane

The comparisons of the ultrastructures of plastids in yellowish mutant and yellowish-green striped mutant and in normal green plant from tissue culture of sugar cane were made. There was no difference found in the structure and development of chloroplasts between the normal green leaves and the green tissue of striped leaves, but the plastid in the yellowish tissue of two kinds of mutants were anomalous. They had not a fully developed system of grana and stroma lameilae as in the normal green leaves. This aberrant plastid contained only some vesicles, a few lamellae and more or less clearly defined ribosome particles and DNA-like mierofibrils, while some stacking and swelling of thylakoids were often observed. In some sections of this aberrant plastid a bunchy lamellae and cross connective fibrils between parallel lamellae were often found too. However, the mixed cell which contains both of normal chloroplast and defective plastid together was never found in the leaves of the mutant plants. It was suggested that yellowish and yellowish-green striped leaves from tissue culture of sugar cane might be caused by nuclear gene mutation.     

DEVELOPMENT OF THE DIMORPHIC CHLOROPLASTS OF SUGAR CANE

The vascular bundle sheath cells of sugar cane contain starch-storing chloroplasts lacking grana, whereas the adjacent mesophyll cells contain chloroplasts which store very little starch and possess abundant grana. This study was undertaken to determine the ontogeny of these dimorphic chloroplasts. Proplastids in the two cell types in the meristematic region of light-grown leaves cannot be distinguished morphologically. Bundle sheath cell chloroplasts in tissue with 50% of its future chlorophyll possess grana consisting of 2-8 thylakoids/granum. Mesophyll cell chloroplasts of the same age have better developed grana and large, well structured prolamellar bodies. A few grana are still present in bundle sheath cell chloroplasts when the leaf tissue has 75% of its eventual chlorophyll, and prolamellar bodies are also found in mesophyll cell chloroplasts at this stage. The two cell layers in mature dark-grown leaves contain morphologically distinct etio-plasts. The response of these two plastids to light treatment also differs. Plastids in tissue treated with light for short periods exhibit protrusions resembling mitochondria. Plastids in bundle sheath cells of dark-grown leaves do not go through a grana-forming stage. It is concluded that the structure of the specialized chloroplasts in bundle sheath cells of sugar cane is a result of reduction, and that the development of chloroplast dimorphism is related in some way to leaf cell differentiation.     

Significance of structural variation in thylakoid membranes in maintaining functional photosystems during reproductive growth

Structural variation in the stroma‐grana (SG) arrangement of the thylakoid membranes, such as changes in the thickness of the grana stacks and in the ratio between grana and inter‐grana thylakoid, is often observed. Broadly, such alterations are considered acclimation to changes in growth and the environment. However, the relation of thylakoid morphology to plant growth and photosynthesis remains obscure. Here, we report changes in the thylakoid during leaf development under a fixed light condition. Histological studies on the chloroplasts of fresh green Arabidopsis leaves have shown that characteristically shaped thylakoid membranes lacking the inter‐grana region, referred to hereafter as isolated‐grana (IG), occurred adjacent to highly ordered, large grana layers. This morphology was restored to conventional SG thylakoid membranes with the removal of bolting stems from reproductive plants. Statistical analysis showed a negative correlation between the incidences of IG‐type chloroplasts in mesophyll cells and the rates of leaf growth. Fluorescence parameters calculated from pulse‐amplitude modulated fluorometry measurements and CO₂ assimilation data showed that the IG thylakoids had a photosynthetic ability that was equivalent to that of the SG thylakoids under moderate light. However, clear differences were observed in the chlorophyll a/b ratio. The IG thylakoids were apparently an acclimated phenotype to the internal condition of source leaves. The idea is supported by the fact that the life span of the IG thylakoids increased significantly in the later developing leaves. In conclusion, the heterogeneous state of thylakoid membranes is likely important in maintaining photosynthesis during the reproductive phase of growth.     

Transformation of plastids in the leaves of Acer negundo L. var. odessanum (H. Rothe).

The leaves of Acer negundo L. var. odessanum (H. Rothe), if permanently exposed to strong sunlight, do not green, but remain yellow and finally become bleached. In yellow leaves the plastids contain single thylakoids and no grana. In plastids of bleached leaves, however, only vesicles are present. The concentration of chlorophylls and photosynthetic activity are much lower in those leaves than in the green ones. If the illumination is reduced (e.g. by shading) both the yellow and the bleached leaves become greenish, and even fully green after a few days at a sufficiently low light intensity. The plastids of yellow-green leaves contain small grana. In dark green leaves the thylakoid system of the chloroplasts is normally developed forming true grana, regardless of whether the leaves were originally green, or became green by shading the yellow or bleached ones. Their pigment concentration and photosynthetic activity are also normal. If green leaves are exposed to sunlight they do not yellow or bleach. During a 3-week period the structure of the thylakoid system did not perceivably change, with the exception that large plastoglobules formed in the stroma.     

Comparative ultrastructural properties of pallisade tissue and spongy tissue chloroplasts from normal and mutant leaves of Pisum sativum L.*

Abstract. The ultrastructure of chloroplasts from palisade and spongy tissue was studied in order to analyse the adaptation of chloroplasts to the light gradient within the bifacial leaves of pea. Chloroplasts of two nuclear gene mutants of Pisum sativum (chlorotica-29 and chlorophyll b-less 130A), grown under normal light conditions, were compared with the wild type (WT) garden-pea cv. ‘Dippes Gelbe Viktoria’. The differentiation of the thylakoid membrane system of plastids from normal pea leaves exhibited nearly the same degree of grana formation in palisade and in spongy tissue. Using morphometrical measurements, only a slight increase in grana stacking capacity was found in chloroplasts of spongy tissue. In contrast, chloroplasts of mutant leaves differed in grana development in palisade and spongy tissue, respectively. Their thylakoid systems appeared to be disorganized and not developed as much as in chloroplasts from normal pea leaves. Grana contained fewer lamellae per granum, the number of grana per chloroplast section was reduced and the length of appressed thylakoid regions was decreased. Nevertheless, chloroplasts of the mutants were always differentiated into grana and stroma thylakoids. The structural changes observed and the reduction of the total chlorophyll content correlated with alterations in the polypeptide composition of thylakoid membrane preparations from mutant chloroplasts. In sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), polypeptide bands with a relative molecular mass of 27 and 26 kilodalton (kD) were markedly reduced in mutant chloroplasts. These two polypeptides represented the major apoproteins of the light harvesting chlorophyll a/b complex from photosystem II (LHC-II) as inferred from a comparison with the electrophoretic mobility of polypeptides isolated from the LHC-II.     

Isolation and characterisation of developing chloroplasts from light-grown barley leaves

Developing chloroplasts were isolated from the basal region of green barley ( Hordeum vulgare L. cv. Menuet) leaves and their ultrastructure and biochemical composition were compared to those of mature chloroplasts from the tip of the same leaves, using two methods of purification on sucrose and Percoll gradients.
When examined and compared to mature chloroplasts, the developing chloroplasts showed well-developed grana stacks, but these last organelles were 2-fold smaller and contained lower amounts of chlorohylls and polar lipids. Only traces of trans -3-hexadecenoic acid could be detected in phosphatidylglycerol of developing plastids. The protein content of these plastids was higher than in mature plastids and showed an increased proportion of polypeptides linked to P-700 chlorophyll α-protein. The photosynthetic activity of these plastids was about 2-fold lower and their photosystem 1/photosystem II ratio higher than in mature chloroplasts.     

Effect of kinetin on early stages of chlorophyll biosynthesis in streptomycin-treated barley seedlings

Treatment of barley seeds (Hordeum vulgare L.) with streptomycin, an inhibitor of plastid protein synthesis, resulted in growth of the albino phenotype seedlings with ribosome-deficient undifferentiated plastids and chlorophyll (Chl) level as low as 0.1% of that in control plant leaves. A major effect of the antibiotic was almost complete suppression of the ability of plants to synthesize 5-aminolevulinic acid (ALA) intended for Chl biosynthesis. The activity of synthesis of ALA intended for heme porphyrin biosynthesis in etiolated and greening seedlings and in light-grown albinophenotype plants was insensitive to light and cytokinins. In the upper parts of leaves of streptomycin-treated plants, exhibiting 60% Chl deficit, the cells with three types of chloroplasts could be observed: normally developed chloroplasts, chloroplasts composed of single thylakoids and grana, and completely undifferentiated plastids. In this Chl-deficient tissue, ALA synthesis was found to be stimulated by kinetin but much less than in leaves of the control plants. The endogenous cytokinin content in etiolated and greening seedlings treated with streptomycin was almost the same as it was in untreated control seedlings. The cytokinin level in the white tissue of plants grown in the light was on average twice as high as that in green leaves of the control plants. The capability of kinetin to stimulate the synthesis of ALA used for Chl biosynthesis was found to correlate with the Chl content and organization of the chloroplast internal structure. This correlation confirms the hypothesis that the normally developed internal structure of plastids is essential for the adequate phytohormone response in plants.     

甜菊组织培养物中叶绿体的超微结构与脱分代

含有叶绿体的甜菊（Ｓｔｅｖｉａｒｅｂａｕｄｉａｎａ）愈伤组织细胞转移至新鲜培养基后,导致光合片层的逐渐减少或消失,最后叶绿体脱分化形成原质体样的结构。超微结构观察表明,光合片层的减少或消失与降解及叶绿体分裂特别是不均等缢缩分裂而致基质组分和类囊体膜稀释有关。这一过程并不完全同步,一些质体含有少量正常的片展而另一些质体含有退化的片层甚至片展结构完全消失。细胞的一个明显特点是细胞器大多聚集在细胞核附近,细胞质增加并向细胞中央伸出细胞质丝。同时可观察到原质体。培养７ｄ后,许多细胞呈分生状态,细胞质富含细胞器,充满了细胞的大部分空间。此时细胞中的质体大多呈原质体状态。在细胞生长的稳定期,质体内膜组织成基质基粒片层,同时质体核糖体增加。文中讨论了高度液泡化细胞脱分化与细胞中叶绿体脱分化的关系。     

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.      

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.     

AN ULTRASTRUCTURAL STUDY OF CHLOROPLAST STRUCTURE AND DEDIFFERENTIATION IN TISSUE CULTURES OF STREPTANTHUS TORTUOSUS (CRUCIFERAE)

Cells of Streptanthus tortuosus callus tissue contain chloroplasts when cultured in a liquid medium in the light. Similar cells grown in the dark contain proplastids that fail to develop prolamellar bodies but do contain a complex of loosely-associated membranes. When green, light-grown cultures are cut into small pieces and subcultured to a fresh culture medium, they become bleached even though maintained under the same illumination. The fine structure of the chloroplasts and the chlorophyll content of the cells indicate a dedifferentiation of the chloroplasts to a proplastid state during the early culture period. The changes in the ultrastructure of the plastids are paralleled by a dedifferentiation of the vacuolate cells to a less differentiated, meristematic state. Subsequent growth in the light results in a re-formation of chloroplasts and an increase in the chlorophyll content of the cells. The period of chloroplast redevelopment is associated with the re-formation of large central vacuoles in the cultured cells. Invaginations of the inner membrane of the plastid envelope occur at all stages of plastid development and are not lost during the period of grana degeneration. The proplastids formed from the dedifferentiation of the chloroplasts contain a large number of these invaginations and the redevelopment of grana is associated with a change in the electron density of the invaginating membranes. The degradation of the chlorophyll-containing membranes of the grana occurs during a period of rapid cytoplasmic synthesis induced by the fresh supply of nutrients in the culture medium. These results suggest that the high levels of nutrients may act directly on the chloroplasts and cause their dedifferentiation or that the rapid cell growth induced by the nutrients may cause a degradation of the membrane proteins in the grana of the chloroplasts and an incorporation of the released amino acids into non-plastid components of the cytoplasm.     

In vivo import of plastocyanin and a fusion protein into developmentally different plastids of transgenic plants

Transgenic tomato plants that constitutively express a foreign plastocyanin gene were used to study protein transport in different tissues. Normally expression of endogenous plastocyanin genes in plants is restricted to photosynthetic tissues only, whereas this foreign plastocyanin protein is found to be present in all tissues examined. The protein is transported into the local plastids in these tissues and it is processed to the mature size. We conclude that plastids of developmentally different tissues are capable of importing precursor proteins that are normally not found in these tissues. Most likely such plastids, though functionally and morphologically differentiated, have similar or identical protein import mechanisms when compared to the chloroplasts in green tissue.     

The YELLOW VARIEGATED (VAR2) locus encodes a homologue of FtsH, an ATP-dependent protease in Arabidopsis

Variegated leaves are often caused by a nuclear recessive mutation in higher plants. Characterization of the gene responsible for variegation has shown to provide several pathways involved in plastid differentiation. Here we describe an Arabidopsis variegated mutant isolated by T-DNA tagging. The mutant displayed green and yellow sectors in all green tissues except for cotyledons. Cells in the yellow sector of the mutant contained both normal-appearing and mutant chloroplasts. The isolated mutant was shown to be an allele of the previously reported mutant, yellow variegated (var2). Cloning and molecular characterization of the VAR2 locus revealed that it potentially encodes a chloroplastic homologue of FtsH, an ATP-dependent metalloprotease that belongs to a large protein family involved in various cellular functions. ftsH-like genes appear to comprise a small gene family in Arabidopsis genome, since at least six homologues were found in addition to VAR2. Dispensability of VAR2 was therefore explained by the redundancy of genes coding for FstHs. In the yellow regions of the mutant leaves, accumulation of photosynthetic protein components in the thylakoid membrane appeared to be impaired. Based on the role of FtsH in a protein degradation pathway in plastids, we propose a possibility that VAR2 is required for plastid differentiation by avoiding partial photooxidation of developing chloroplasts.     

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.     

Changes in the protein and activity levels of the plastid NADH–plastoquinone–oxidoreductase complex during fruit development

Plastids contain an NADH dehydrogenase complex (Ndh complex) homologous to the mitochondrial complex I (EC 1.6.5.3). In this work, we have analysed the changes in the Ndh complex during ripening of pepper (Capsicum annum L., cv. Maor) and tomato (Lycopersicon esculentum Mill., cv. Marglobe) fruits. The Ndh complex was mainly present in the outer pericarp of tomato fruits, whereas it was evenly distributed in the pericarp of pepper. In both kinds of fruit we observed a decrease in the total amount of Ndh complex from the green to the red stage of development. This decrease corresponds to parallel decreases in the content and activity of the complex in plastids during the transition from chloroplasts to chromoplasts. Levels of plastidial quinol peroxidase activity were also higher during the first stages of tomato fruit development than during the latter stages of ripening. However, when referred to total plastid protein, the amount and activity of the Ndh complex in chloroplasts isolated from green fruits was higher than in chloroplasts isolated from leaves. These results strongly suggest that function of the Ndh complex, probably related to a plastidial electron transport chain, can be important during the first stages of fruit development.     

The history of research on white-green variegated plants

Plinius the Elder (23–79 A.D.) noted the existence of variegated varieties of ivy. But only subsequent to the middle of the last century, when botanists detected chloroplasts and began to study their origin, did variegation of leaves begin to be investigated scientifically. The white, yellow, or yellow-green parts of the many species of variegated leaves contain either leucoplasts, plastids in various stages of degeneration leading to their complete disappearance, or plastids containing only carotene or xanthophyll. In some cases normal and abnormal plastids are found together in “mixed” cells. In some cases the variegation is temperature and/or light sensitive.