Disposal of chloroplasts with abnormal function into the vacuole in Arabidopsis thaliana cotyledon cells

Summary. Autophagy is a process in which cell membrane rearrangement allows for the sequestration and degradation of part of the cytoplasm. Many protein components of the autophagic mechanism and their corresponding genes have been identified in yeast cells by molecular genetics, and this has enabled researchers to identify homologues of these genes in mammalian and plant systems. Autophagy is involved in the starvation response in which part of the cytoplasm is degraded in order to produce essential substrates to allow the cell to survive during extreme substrate-limiting conditions. However, autophagy may also be important as a quality control mechanism in normal cells. By screening Arabidopsis thaliana T-DNA insert mutants, we isolated an A. thaliana mutant that lacks the AtTIC40 gene and found that the cotyledon cells of this mutant contained undeveloped plastids. Moreover, many toluidine-stained particulate structures were found in the vacuoles of these mutant cells. The images from electron microscopy suggested that some of these particulate structures were partially degraded chloroplasts. Furthermore, oil bodies were found in the cotyledon cells of mutant and wild-type plants, which suggests that the mutant seedlings were not starved under the experimental conditions. These results may indicate that under nutrient-sufficient conditions, plant cells remove abnormal plastids by autophagy and that this mechanism is involved in the quality control of organelles.Present address: BioResource Center, Tsukuba Institute, Institute of Physical and Chemical Research (RIKEN), Tsukuba, Japan.Present address: Genomics Sciences Center, Yokohama Institute, Institute of Physical and Chemical Research (RIKEN), Yokohama, Japan.Correspondence and reprints: School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan.     

Plastids and stromules interact with the nucleus and cell membrane in vascular plants

The various metabolic activities of plastids require continuous exchange of reactants and products with other organelles of the plant cell. Physical interactions between plastids and other organelles might therefore enhance the efficiency of plant metabolism. We have observed a close apposition of plastids and nuclei in various organs of Nicotiana tabacum and Arabidopsis thaliana. In hypocotyl epidermal cells, plastids and stromules, stroma-filled tubular extensions of the plastid envelope membrane, were observed to reside in grooves and infoldings of the nuclear envelope, indicating a high level of contact between the two organelle membranes. In a number of non-green tissues, including suspension-cultured cells, perinuclear plastids were frequently associated with long stromules that extended from the cell center to the cell membrane. In cotyledon petioles, cells lying adjacent to one another frequently contained stromules that met on either side of the shared cell wall, suggesting a means of intercellular communication. Our results therefore suggest that stromules have diverse roles within plant cells, perhaps serving as pathways between nuclei and more distant regions of the cell and possibly even other cells.     

Preferential degradation of plastid DNA with preservation of mitochondrial DNA in the sperm cells ofPelargonium zonale during pollen development

Summary In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen ofPelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and by immunogold electron microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by immunogold electron microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells ofP. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of plastids inP. zonale.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system     

Molecular evidence for the origin of plastids from a cyanobacterium-like ancestor

Summary The origin of plastids by either a single or multiple endosymbiotic event(s) and the nature of the progenitor(s) of plastids have been the subjects of much controversy. The sequence of the small subunit rRNA (Ssu rRNA) from the plastid of the chlorophyllc-containing algaCryptomonas is presented, allowing for the first time a comparison of this molecule from all of the major land plant and algal lineages. Using a distance matrix method, the phylogenetic relationships among representatives of these lineages have been inferred and the results indicate a common origin of plastids from a cyanobacterium-like ancestor. Within the plastid line of descent, there is a deep dichotomy between the chlorophyte/land plant lineage and the rhodophyte/chromophyte lineage, with the cyanelle ofCyanophora paradoxa forming the deepest branch in the latter group. Interestingly,Euglena gracilis and its colorless relativeAstasia longa are more related to the chromophytes than to the chlorophytes, raising once again the question of the origin of the euglenoid plastids.     

The foundation of extranuclear inheritance: plastid and mitochondrial genetics

In 1909 two papers by Correns and by Baur published in volume 1 of Zeitschrift für induktive Abstammungs- und Vererbungslehre (now Molecular Genetics and Genomics) reported on the non-Mendelian inheritance of chlorophyll deficiencies. These papers, reporting the very first cases of extranuclear inheritance, laid the foundation for a new field: non-Mendelian or extranuclear genetics. Correns observed a purely maternal inheritance (in Mirabilis), whereas Baur found a biparental inheritance (in Pelargonium). Correns suspected the non-Mendelian factors in the cytoplasm, while Baur believed that the plastids carry these extranuclear factors. In the following years, Baur’s hypothesis was proved to be correct. Baur subsequently developed the theory of plastid inheritance. In many genera the plastids are transmitted only uniparentally by the mother, while in a few genera there is a biparental plastid inheritance. Commonly there is random sorting of plastids during ontogenetic development. Renner and Schwemmle as well as geneticists in other countries added additional details to this theory. Pioneering studies on mitochondrial inheritance in yeast started in 1949 in the group of Ephrussi and Slonimski; respiration-deficient cells (petites in yeast, poky in Neurospora) were demonstrated to be due to mitochondrial mutations. Electron microscopical and biochemical studies (1962–1964) showed that plastids and mitochondria contain organelle-specific DNA molecules. These findings laid the molecular basis for the two branches of extranuclear inheritance: plastid and mitochondrial genetics.     

Origins of the secondary plastids of Euglenophyta and Chlorarachniophyta as revealed by an analysis of the plastid‐targeting,nuclear‐encoded gene psbO1

Because the secondary plastids of the Euglenophyta and Chlorarachniophyta are very similar to green plant plastids in their pigment composition, it is generally considered that ancestral green algae were engulfed by other eukaryotic host cells to become the plastids of these two algal divisions. Recent molecular phylogenetic studies have attempted to resolve the phylogenetic positions of these plastids; however, almost all of the studies analyzed only plastid‐encoded genes. This limitation may affect the results of comparisons between genes from primary and secondary plastids, because genes in endosymbionts have a higher mutation rate than the genes of their host cells. Thus, the phylogeny of these secondary plastids must be elucidated using other molecular markers. Here, we compared the plastid‐targeting, nuclear‐encoded, oxygen‐evolving enhancer (psbO) genes from various green plants, the Euglenophyta and Chlorarachniophyta. A phylogenetic analysis based on the PsbO amino acid sequences indicated that the chlorarachniophyte plastids are positioned within the Chlorophyta (including Ulvophyceae, Chlorophyceae, and Prasinophyceae, but excluding Mesostigma). In contrast, plastids of the Euglenophyta and Mesostigma are positioned outside the Chlorophyta and Streptophyta. The relationship of these three phylogenetic groups was consistent with the grouping of the primary structures of the thylakoid‐targeting domain and its adjacent amino acids in the PsbO N‐terminal sequences. Furthermore, the serine‐X‐alanine (SXA) motif of PsbO was exactly the same in the Chlorarachniophyta and the prasinophycean Tetraselmis. Therefore, the chlorarachniophyte secondary plastids likely evolved from the ancestral Tetraselmis‐like alga within the Chlorophyta, whereas the Euglenophyte plastids may have originated from the unknown basal lineage of green plants.     

The 5S ribosomal RNA sequences of a red algal rhodoplast and a gymnosperm chloroplast. Implications for the evolution of plastids and cyanobacteria

Summary The 5S ribosomal RNA sequences have been determined for the rhodoplast of the red algaPorphyra umbilicalis and the chloroplast of the coniferJuniperus media. The 5S RNA sequence of theVicia faba chloroplast is corrected with respect to a previous report. A survey of the known sequences and secondary structures of 5S RNAs from plastids and cyanobacteria shows a close structural similarity between all 5S RNAs from land plant chloroplasts. The algal plastid 5S RNAs on the other hand show much more structural diversity and have certain structural features in common with bacterial 5S RNAs. A dendrogram constructed from the aligned sequences by a clustering algorithm points to a common ancestor for the present-living cyanobacteria and the land plant plastids. However, the algal plastids branch off at an early stage within the plastid-cyanobacteria cluster, before the divergence between cyanobacteria and land plant chloroplasts. This evolutionary picture points to the occurrence of multiple endosymbiotic events, with the ancestors of the present algal plastids already established as photosynthetic endosymbionts at a time when the ancestors of the present land plant chloroplasts were still free-living cells.     

Die Plastidenverteilung bei der Mitose der Schließzellenmutterzellen von haploidem Schwedenklee (Trifolium hybridum L.)

Summary In order to investigate whether during mitosis of guard cell mother cells the plastids are distributed to the daughter cells at random, a haploid of Trifolium hybridum, a species with only three to four chloroplasts in one diploid guard cell, was searched for and found. As expected, the guard cell mother cells in this plant contained only about two plastids. If distribution to the daughter cells would occur strictly at random, among the guard cell pairs with two chloroplasts the pairs with 1/1 and those with 2/0 chloroplasts should appear in equal amounts. However, 159 pairs of type 1/1 and only 35 pairs of type 2/0 were found, i.e., 18% of type 2/0 (upper limit of 99% confidence interval: 25%), indicating that the plastids have been apportioned to a fairly great degree. The result may be understood by considering that the plastids in guard cell mother cells are not scattered at random throughout the cell space, but are more regularly spread as are the chloroplasts in adult cells.     

Acid phosphatase activity in plastids (plastolysomes) of senescing embryo-suspensor cells

Autolysis of the suspensor, an embryonal haustorium, starts in the basal cells and proceeds in the direction of the embryo. InPhaseolus vulgaris, acid phosphatase activity is first found in transforming plastids, similar to the acid phosphatase activity inPh. coccineus [Nagl (1977) Z. Pflanzenphysiol.85, 45–51], although the ultrastructural details are different. InTropaeolum majus, autolysis begins in the most distal part of the suspensor, i.e., the chalazal or carpel haustorium. First the endoplasmic reticulum shows acid phosphatase activity, but neither the mitochondria, which undergo transformation similar to that observed in plastids ofPhaseolus, nor the leucoplasts show such activity. Later, however, the plastids exhibit low activity. Contrarily, the plastids in the suspensor cells adjacent to the embryo show increasing activity during senescence of the suspensor. During final autolysis, activity is found in all cytoplasmic membranes, while it is reduced in plastids. The visible ultrastructural transformations of various organelles into cytolysomes does not necessarily coincide with acid phosphatase activity. Our findings are a further indication of the high diversification and specialization of plastids during plant embryogenesis.     

Composition and fine structure of minor veins inTetragonia leaf

Summary Mesophyll containing the minor veins from leaves ofTetragonia expansa Murr. was examined in preparation for a study of effects of beet yellows virus on the leaf tissues of this plant. The sieve elements throughout the minor veins exhibit the characteristics commonly found in this type of cell in dicotyledons. The cells are connected with one another by sieve plates and with contiguous parenchyma cells by branched plasmodesmata. Mature sieve elements are enucleate and lack ribosomes. No tonoplast was discerned in these cells. Mitochondria, plastids, and sparse endoplasmic reticulum are retained in mature cells. The plastids, which contain a massive fibrous ring of proteinaceous material, resemble the sieve element plastids ofBeta. The P-protein in the sieve elements is occasionally composed of tubules; more commonly it is represented by loose helices. The tracheary elements have scalariform secondary thickenings. In regions free of these thickenings, the primary wall largely disintegrates; only some loosely arranged fibrils remain. The mesophyll and vascular parenchyma cells contain the various organelles characteristic of living plant cells but vary in degree of vacuolation and in density of cytoplasm. Some vascular parenchyma cells have particularly dense protoplasts. They contain numerous ribosomes and their background matrix consists of a dense population of fine fibrils. Occasional vascular parenchyma cells contain the tubular spiny cell component first recognized inBeta. This work was supported in part by National Science Foundation grant GB-5506.     

Tentoxin effects onSorghum: The role of polyphenol oxidase

Summary In an ultrastructural and cytochemical study of tentoxin-treatedSorghum bicolor (L.) Moench, both bundle sheath and mesophyll plastids were severely affected, Plastids from chlorotic leaf areas lacked most internal membranes yet had plastid ribosomes and large fibrillar areas of plastid DNA. In recovered areas (mottled yellow and green), cells were found that had plastids of near-normal ultrastructure as well as the severely affected plastid-types found in chlorotic leaf areas. Polyphenol oxidase (PPO) cytochemistry of these mottled leaf areas indicated that all recovered mesophyll plastids had PPO whereas all the abnormal mesophyll plastids showed no activity. Because bundle sheath plastids ofSorghum have no PPO activity at any developmental stage, yet are affected by tentoxin, PPO cannot be uniquely affected by this toxin. We suggest that tentoxin may affect the transport of cytosolic proteins into the plastid.     

Plastid number and plastid structural changes associated with tobacco mesophyll protoplast culture and plant regeneration

Mesophyll protoplasts were isolated from Nicotiana tabacum L. cv. Xanthi, and cell-colony formation induced in liquid culture. The plastid changes associated with the morphogenetic sequence from mesophyll protoplast to whole plant were examined. Minor ultrastructural changes in the plastids were evident after 1 d of culture, but by 8 d (four-to-eight-cell stage) the plastids were small, there was much less thylakoid membrane appression, and many prominent plastoglobuli were also present. Plastid-division figures were evident at this point of time and it was common to find plastids clustered around the nucleus. A typical proplastid was the dominant plastid type in the cultured cells from about 11 d until about five weeks when large amyloplasts and pregranal plastids were observed. Normally structured chloroplasts were present in the regenerated plant. There was no plastid division until the four-cell stage, with plastid numbers per cell approximately halving at each cell division, then stabilising around 12 per cell during cell-colony development, a number typical of meristematic cells. Though nucleoids were always present, their numbers in the plastids were reduced by the eight-cell stage.     

Ultrastructure of the stem of the aquatic plantCallitriche stagnalis L.

Summary The submerged stem ofCallitriche stagnalis L. has been studied by light and electron microscopy. The presence of plastids with an opaque body are noted in the parenchymatic cells near the conducting elements as well as many microtubules associated with polyribosomes and vesicles near the cell wall.It is of particular interest to note that the tracheids are connected to the pith lacuna and each other to form only one conducting cylinder, so that functionally theCallitriche stele seems to be a protostele. This reduction of the stele is probably due to the environment conditions, since by floral morphology this aquatic plant is considered an evolved species.     

RESPONSIVENESS OF YEAST CELLS TO AUXIN, ANIMAL SEX HORMONES AND YEAST SEXUAL HORMONES IN RELATION TO SEX CONTROLLING GENES

Genetic control of the ability of yeast cells to respond to cell-expanding action of animal, plant and yeast hormones was studied. Mating type specificity in the ability to respond to yeast sexual hormones was changed by introduction of homothallism-controlling genes, D and HM. Auxin, a plant hormone can expand cells of most homothallic strains, but not those of heterothallic strains as far as tested. Animal sex hormones showed the action similar to that of yeast sexual hormones.     

Two newly identified membrane-associated and plastidic tomato HXKs: characteristics, predicted structure and intracellular localization

Two new tomato hexokinase genes, LeHXK3 and LeHXK4, were cloned and characterized, placing tomato as the first plant with four characterized HXK genes. Based on their sequence, LeHXK3 is the third membrane-associated (type-B) and LeHXK4 is the first plastidic (type-A) HXK identified in tomato. Expression of HXK-GFP fusion proteins in protoplasts indicated that the LeHxk3 enzyme is associated with the mitochondria while LeHxk4 is localized in plastids. Furthermore, LeHxk4::GFP fusion protein is found within stromules, suggesting transport of LeHxk4 between plastids. Structure prediction of the various plant HXK enzymes suggests that unlike the plastidic HXKs, the predicted membrane-associated HXKs are positively charged near their putative N-terminal membrane anchor domain, which might enhance their association with the negatively charged membranes. LeHxk3 and LeHxk4 were analyzed following expression in yeast. Both enzymes have higher affinity for glucose relative to fructose and are inhibited by ADP. Yet, unlike the other HXKs, the stromal HXK has higher Vmax with glucose than with fructose. Expression analysis of the four HXK genes in tomato tissues demonstrated that LeHXK1 and LeHXK4 are the dominant HXKs in all tissues examined. Notably, the plastidic LeHXK4 is expressed in all tissues including starchless, non-photosynthetic sink tissues, such as pink and red fruits, implying phosphorylation of imported hexoses in plastids. It has been suggested that trehalose 6-phosphate (T6P) might inhibit HXK activity. However, none of the yeast-expressed tomato HXK genes was sensitive either to T6P or to trehalose, suggesting that unlike fungi HXKs, plant HXKs are not regulated by T6P.The nucleotide sequence data of LeHXK3 and LeHXK4 appear in the GenBank Nucleotide Sequence Database under accession numbers DQ056861 and DQ056862, respectively.M. Kandel-Kfir and H. Damari-Weissler contributed equally to this work.     

Paternal inheritance of plastids in Medicago sativa

Summary Plastids are plant cellular organelles that are generally inherited from the maternal parent in the angiosperms. Many species exhibit biparental inheritance of plastids, but usually with a predominantly maternal influence. In contrast to this, we report strong paternal inheritance of plastids in reciprocal crosses of alfalfa, Medicago sativa, by following restriction fragment length polymorphisms for plastid DNA in two normal green plastids. Mitochondrial inheritance remained exclusively maternal.     

Visualisation of plastid degradation in sperm cells of wheat pollen

Like most angiosperms, wheat (Triticum aestivum) shows maternal inheritance of plastids. It is thought that this takes place by cytoplasmic stripping at fertilisation rather than the absence of plastids in sperm cells. To determine the fate of plastids during sperm cell development, plastid-targeted green fluorescent protein was used to visualise these organelles in nuclear transgenic wheat lines. Fewer than thirty small 1–2-μm plastids were visible in early uninucleate pollen cells. These dramatically increased to several hundred larger (4 μm) plastids during pollen maturation and went through distinct morphological changes. Only small plastids were visible in generative cells (n?=?25) and young sperm cells (n?=?9). In mature sperm cells, these green fluorescent protein (GFP)-tagged plastids were absent. This is consistent with maternal inheritance of plastids resulting from their degradation in mature sperm cells in wheat.     

In situ plastid and mitochondrial DNA determination: Implication of the observed MINIMAL PLASTID GENOME NUMBER

The total loss of plastid DNA has never been reported for any alga or plant cell line, with the sole exception of the protozoan Euglena, yet plastid distribution at mitosis is apparently stochastric (Birky and Skavaril, Journal of Theoretical Biology, vol. 106, pp. 441–447, 1984) and accidental loss might be expected. It is not obvious how stem cells of photosynthetic eukaryotes avoid this problem. The chrysophyte alga Ochromonas danica, described as having but one or two plastids, can proliferate indefinitely without the benefit of photosynthesis. Under such conditions its plastid genome copy number per cell might drop to the absolute minimum compatible with maintaining its inheritance. In situ quantitation of Ochromonas plastid DNA in both photosynthetic and enriched mixotrophic growth, and in heterotrophic growth in prolonged darkness, suggests that plastids are capable of very wide variation (7 to >;200 genomes/plastid) in their DNA content, and likewise, cells can vary from one to >;8 plastids per cell, with total genomes numbers from 7 to >;1,000 per cell. Among many growth conditions tested, the smallest plastids were found in rapidly dividing cells grown in the dark, many of which contained but one plastid. The inability to find plastids with fewer than seven plastid genome equivalents of DNA, even in these rapidly multiplying cells grown in total darkness for months, suggests that multiple copies of the plastid genome may be very carefully maintained, even in the prolonged absence of photosynthesis. This implies that multiple copies are important for reasons other than photosynthetic capability; two possibilities are the biosynthetic steps necessary for eukaryote cell survival known to occur solely within a plastid, and/or the potential that multiple plastid genome copies provide to escape the effects of Muller's ratchet.     

Zea mays Fe deficiency‐related 4 (ZmFDR4) functions as an iron transporter in the plastids of monocots

Iron (Fe)‐homeostasis in the plastids is closely associated with Fe transport proteins that prevent Fe from occurring in its toxic free ionic forms. However, the number of known protein families related to Fe transport in the plastids (about five) and the function of iron in non‐green plastids is limited. In the present study, we report the functional characterization of Zea mays Fe deficiency‐related 4 (ZmFDR4), which was isolated from a differentially expressed clone of a cDNA library of Fe deficiency‐induced maize roots. ZmFDR4 is homologous to the bacterial FliP superfamily, coexisted in both algae and terrestrial plants, and capable of restoring the normal growth of the yeast mutant fet3fet4, which possesses defective Fe uptake systems. ZmFDR4 mRNA is ubiquitous in maize and is inducible by iron deficiency in wheat. Transient expression of the 35S:ZmFDR4–eGFP fusion protein in rice protoplasts indicated that ZmFDR4 maybe localizes to the plastids envelope and thylakoid. In 35S:c‐Myc‐ZmFDR4 transgenic tobacco, immunohistochemistry and immunoblotting confirmed that ZmFDR4 is targeted to both the chloroplast envelope and thylakoid. Meanwhile, ultrastructure analysis indicates that ZmFDR4 promotes the density of plastids and accumulation of starch grains. Moreover, Bathophenanthroline disulfonate (BPDS) colorimetry and inductively coupled plasma mass spectrometry (ICP‐MS) indicate that ZmFDR4 is related to Fe uptake by plastids and increases seed Fe content. Finally, 35S:c‐Myc‐ZmFDR4 transgenic tobacco show enhanced photosynthetic efficiency. Therefore, the results of the present study demonstrate that ZmFDR4 functions as an iron transporter in monocot plastids and provide insight into the process of Fe uptake by plastids.     

Streptomycin and lincomycin resistances are selective plastid markers in cultured Nicotiana cells

Summary Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.