Preferential synthesis of plastid DNA and increased replication of plastids in cultured tobacco cells following medium renewal

During the culture of tobacco BY 2 cells derived from Nicotiana tabacum L. cv. Bright Yellow 2, morphological changes of plastid (pt) nucleoids and their replication were examined by fluorescence microscopy after staining with 46-diamidino-2-phenylindole. Upon transfer to fresh medium, the fluorescence intensity originating from pt nucleoids increased markedly. Copy numbers of ptDNA per cell calculated from the quantitative data by super-sensitive microspectroscopy increased 11-fold within 1 d of culture to reach 11 000, then decreased gradually to 1 000 after one week of culture. Autoradiography by labelling with [³H]thymidine showed that DNA synthesis in plastids occurred exclusively during the first day of culture, whereas nuclear DNA synthesis was observed from the first to the sixth day of culture. Replication of plastids was most frequently observed on the second day. Thereafter the formation of starch granules predominated in plastids up to the fifth day of culture, but the starch granules disappeared in the stationary-phase cells. The meaning of such preferential synthesis of ptDNA upon transfer to fresh medium is discussed in relation to the interaction between plastids and nuclei.Abbreviations pt plastid - DAPI 4,6-diamidino-2-phenylindole     

DNA synthesis in isolated mitochondrial nucleoids from plasmodia ofPhysarum polycephalum

Summary A mitochondrion contains multiple copies of mitochondrial DNA (mtDNA) in the mitochondrial nucleoid (mt-nucleoid, synonym for mitochondrial nuclei). Replicaton of mtDNA in the mtnucleoids appears to be regulated within groups of adjacent mtDNA molecules, known as mitochondrial replicon clusters (MRCs). In this study, we isolated structurally intact mt-nucleoids from the plasmodia ofPhysarum polycephalum and characterized DNA synthesis in the isolated mt-nucleoids. The mt-nucleoids were isolated by dissolving the membranes of highly purified mitochondria with 0.5% Nonidet P-40. The structural integrity of the isolated mt-nucleoid was determined by observing the rod shape of the mt-nucleoid and the structure of the MRC. The isolated mt-nucleoids required four deoxyribonucleoside triphosphates and MgCl₂ for DNA synthesis. The DNA synthesis was resistant to aphidicolin and showed only low sensitivity to N-ethylmaleimide and to ddTTP, suggesting that the DNA synthesis is catalyzed by plant-type mitochondrial DNA polymerase. The capacity for DNA synthesis in the isolated mt-nucleoids was similar to that in the isolated mitochondria, despite removal of most of the mitochondrial matrix and membrane. Furthermore, visualization of sites of DNA synthesis in vitro revealed that DNA synthesis in the isolated mt-nucleoids occurred in each MRC. These results suggest that the isolated mt-nucleoids are capable of efficient and systematic DNA synthesis in vitro. Therefore, the use of isolated mt-nucleoids should permit in vitro characterization of the molecular mechanism of mtDNA replication in the MRC.Abbreviations BrdU 5-bromodeoxyuridine - BrdUTP 5-bromo-deoxyuridine triphosphate - DAPI 4,6-diamidino-2-phenylindole - dNTP deoxyribonucleoside triphosphate - ddCTP dideoxycytidine triphosphate - NEM N-ethylmaleimide - MRC mitochondrial replicon cluster; mt mitochondrial - NP-40 Nonidet P-40 - PBS phosphatebuffered saline - PMSF phenylmethanesulfonyl fluoride - rNTP ribonucleoside triphosphate - VIMPCS video-intensified microscope photon-counting system     

Variation of plastid and mitochondrial DNAs in the genus Hedysarum

Summary Plastid and mitochondrial DNAs from Hedysarum species of the western Mediterranean basin, H. spinosissimum ssp eu-spinosissimum, H. spinosissimum ssp capitatum, H. carnosum, H. coronarium and H. flexuosum, were compared by restriction endonuclease fragment analysis. ctDNA fragment patterns for ssp eu-spinosissimum and ssp capitatum were indistinguishable in different enzyme digests. An identical ctDNA variation was found in Hpa II digests with two Sardinian populations of ssp capitatum. Each of the two subspecies was characterized by specific mt DNA patterns with Pst I, Bam HI, Sma I and EcoRI. No variation was detected in populations of different geographical origins for a given subspecies. H. carnosum, H. coronarium and H. flexuosum generated specific ct and mt DNA patterns. Comparison of mitochondrial fragments indicated: — a strong homology between the two subspecies, — a closer homology among the three other diploids, each being closer to the other two than to H. spinosissimum subspecies — as was also the case for the plastid genomes.     

Homologies to plastid DNA in the nuclear and mitochondrial genomes of potato

Summary Potato plastid DNA clones, representing onefourth of the potato plastome complexity and containing sequences of the 16SrRNA, rps16, atpA, atpE, psaA, psaB, trnK, trnV, and trnG genes, were used as hybridization probes on nuclear- and mitochondrial-enriched DNAs. Each probe hybridized to multiple nuclear restriction fragments distinct from the plastid cleavage products generated by the same endonucleases. The nuclear hybridizable fragments are highly methylated at their Hpall target sequences (C/CGG). In some instances, the transfer seemed to involve plastid regions of several kilobase pairs, as reflected by the co-integration in the nucleus of restriction sites that are distant in the plastome. Three clones hybridized additionally to distinct mitochondrial fragments. These results indicate that extensive DNA transfers did occur between plastids and other organelles in potato.     

Structural changes in the plastid DNA of rice (Oryza sativa L.) during tissue culture

To investigate the rearrangement of the plastid genome during tissue culture, DNA from rice callus lines, which had been derived individually from single protoplasts isolated from seed or pollen callus (protoclones), was analyzed by Southern hybridization with rice chloroplast DNA (ctDNA) clones as probes. Among 44 long-term cultured protoclones, maintained for 4, 8 or 11 years, 28 contained plastid DNA (ptDNA) from which portions had been deleted. The ptDNA of all protoclones that had been maintained for 11 years had a deletion that covered a large region of the plastid genome. The deletions could be classified into 15 types from their respective sizes and positions. By contrast, no deletions were found in the ptDNA of 38 protoclones that had been maintained for only 1 month. These results indicate that long-term culture causes deletions in the plastid genome. Detailed hybridization experiments revealed that plastid genomes with deletions in several protoclones were organized as head-to-head or tail-to-tail structures. Furthermore, ptDNAs retained during long-term culture all had a common terminus at one end, where extensive rearrangement is known to have occurred during the speciation of rice and tobacco. Morphological analysis revealed the accumulation of starch granules in plastids and amyloplasts in protoclones in which the plastid genome had undergone deletion. Our observations indicated that novel structural changes in the plastid genome and morphological changes in the plastid had occurred in rice cells during long-term tissue culture. Moreover, the morphological changes in plastids were associated with deletions in the plastid genome.     

Disappearance of plastid and mitochondrial nucleoids during the formation of generative cells of higher plants revealed by fluorescence microscopy

Summary The fate of plastid and mitochondrial nucleoids (pt and mt nucleoids) ofTriticum aestivum was followed during the reproductive organ formation using fluorescence microscopy after staining with 4'6-diamidino-2-phenylindole (DAPI). This investigation showed a drastic morphological change of pt nucleoids during the differentiation of reproductive organs from the shoot apex. Dot-shaped pt nucleoids grew into ring-shaped ones, which divided into small pieces in the monocellular pollen grain, as observed in this plant's earlier stage of leaf development. During the development of mature pollen grain from monocellular pollen grain, pt and/or mt nucleoids disappeared through the division of the male generative cell ofT. aestivum. Cytologically, this observation is direct evidence of the maternal inheritance of higher plants. Thus far, cytological evidence of this phenomenon has been found mostly by morphological criteria using electron microscopy, which admits some ambiguity. In the plants exemplified byLilium longiflorum, pt and/or mt nucleoids disappeared after the first pollen grain mitosis, which precededT. aestivum. In the plants exemplified byTrifolium repens, pt and/or mt nucleoids existed in the generative cells of the mature pollen grain.The significance of these observations was discussed in relation to the interaction between nuclear and organelle genomes during plant development.Abbreviations DAPI 4'6 diamidino-2-phenylindole - Mt DNA Mitochondrial DNA - Mt nucleoid Mitochondrial nucleoid - Pt DNA Plastid DNA - Pt nucleoid Plastid nucleoid On leave from Department of Biology, Nagoya University, Furocho, Chikusaku, Nagoya 464, Japan.     

Differential patterns of mitochondrial,chloroplastic and nuclear DNA synthesis in the synchronous cell cycle of Chlamydomonas reinhardtii

Nuclear DNA (ncDNA) synthesis in Chlamydomonas reinhardtii was measured by both ³²P[or-thophosphoric acid] (³²P) and [¹⁴C]adenine incorporation and found to be highly synchronous. Ca. 85% of incorporation was confined to the first 6 h of the dark period of a synchronized regime consisting of an alternating light-dark period of 12 h each. In contrast, no such synchronous incorporation pattern was found for chloroplast (cp) and mitochondrial (mt) DNAs in the same cell population. These two organellar DNAs also exhibited different ³²P-incorporation patterns in the cell cycle. Considerable amounts of ³²P were incorporated into cpDNA throughout the light-dark synchronous cycle under both mixo- and phototrophic growth conditions, although the second 6-h light period under phototrophy showed an increase not apparent under mixotrophy. This change in growth conditions did not affect ³²P incorporation into mtDNA, which was found throughout the cell cycle, with a modest peak in the first 6-h of the dark period. The pattern of [³H]thymidine incorporation into cpDNA was also determined. Under synchronous phototrophic conditions, this pattern was quite different from that obtained with ³²P. Most [³H]thymidine incorporation occurred during the light period of the synchronous cycle; this period had been shown previously by density transfer experiments to be the time of cpDNA duplication. Such preferential [³H]thymidine incorporation into cpDNA in the light period was not observed under mixotrophic synchronous growth conditions; in these, [³H]thymidine incorporation was detected throughout the cell cycle. This lack of coincidence between the patterns of ³²P- and of [³H]thymidine incorporation into cpDNA during the synchronous cell cycle indicates that in addition to replication, the considerably reiterated organelle-DNA molecules may also regularly undergo an extensive repair process during each cell cycle.     

Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells

Summary The molecular size of mitochondrial DNA (mtDNA) molecules and the number of copies of mtDNA per mitochondrion were evaluated from cultured cells of the tobacco BY-2 line derived fromNicotiana tabacum L. cv. Bright Yellow-2. To determine the DNA content per mitochondrion, protoplasts of cultured cells were stained with 4,6-diamidino-2-phenylindole (DAPI), and the intensity of the fluorescence emitted from the mitochondrial nuclei (mt-nuclei) was measured with a video-intensified photon counting microscope system (VIM system). Each mitochondrion except for those undergoing a division contained one mt-nucleus. The most frequently measured size of the DNA in the mitochondria was between 120 and 200 kilobase pairs (kbp) throughout the course of culture of the tobacco cells. Mitochondria containing more than 200 kbp of DNA increased significantly in number 24 h after transfer of the cells into fresh medium but their number fell as the culture continued. Because division of mitochondria began soon after transfer of the cells into fresh medium and continued for 3 days, the change of the DNA content per mitochondrion during the culture must correspond to DNA synthesis of mitochondria in the course of mitochondrial division. By contrast, the analyses of products of digestion by restriction endonucleases indicated that the genome size of the mtDNA was at least 270 kbp. Electron microscopy revealed that mtDNAs were circular molecules and their length ranged from 1 to 35 m, and 60% of them ranged from 7 to 11 rn. These results indicate that the mitochondrial genome in tobacco cells consists of multiple species of mtDNA molecules, and mitochondria do not contain all the mtDNA species. Therefore, mitochondria are heterogeneous in mtDNA composition.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - mtDNA mitochondrial DNA - mt-genome mitochondrial genome - mt-nucleus mitochondrial nucleus - ptDNA proplastid DNA - pt-nucleus proplastid nucleus - VIM system video-intensified photon counting microscope system     

Fragments of plastid DNA in the nuclear genome of tomato: prevalence, chromosomal location, and possible mechanism of integration

Summary We have undertaken a systematic search for plastid DNA sequences integrated in the tomato nuclear genome, using heterologous probes taken from intervals of a plastid DNA region spanning 58 kb. A total of two short integrates (202 and 141 nucleotides) were isolated and mapped to chromosomes 9 and 5, respectively. The nucelotide sequence of the integrates and that of the flanking regions were determined. The integration sites contain direct repeat elements similar in position (but not in length or sequence) to the direct repeats previously observed with another plastid integrate in the tomato nuclear genome. Based on these results, a model for the process of movement and integration of plastid sequences into the nuclear genome is discussed.     

Cytoplasmic DNA apportionment and plastid differentiation during male gametophyte development in Pelargonium zonale

In the male gametophyte of Pelargonium zonale, generative and sperm cells contain cytoplasmic DNA in high density compared to vegetative cells. Cytoplasmic DNA was examined using the DNA fluorochrome DAPI (4'6-diamidino-2-phenylindole) and observed with epifluorescence and electron microscopy. The microspore cell contains a prominent central vacuole before mitosis; mitochondria and plastids are randomly distributed throughout the cytoplasm. Following the first pollen grain mitosis, neither the vegetative cell nor the early generative cell display a distributional difference in cytoplasmic DNA, nor is there in organelle content at this stage. During the maturation of the male gametophyte, however, a significant discrepancy in plastid abundance develops. Plastids in the generative cell return to proplastids and do not contain large starch grains, while those in the vegetative cell develop starch grains and differentiate into large amyloplasts. Plastid nucleoids in generative and sperm cells in a mature male gametophyte are easily discriminated after DAPI staining due to their compactness, while those in vegetative cells stained only weakly. The utility of the hydrophilic, non-autofluorescent resin Technovit 7100 in observing DAPI fluorescence is also demonstrated.     

Presence of plastid and absence of mitochondrial DNA in male reproductive cells as evidence for cytoplasmic inheritance in Turnera ulmifolia and Zantedeschia aethiopica

Summary. Epifluorescence microscopy of mature pollen grains of Turnera ulmifolia and Zantedeschia aethiopica stained with 4,6-diamidino-2-phenylindole demonstrated the presence of fluorescent cytoplasmic DNA aggregates in the male reproductive cells of both species. Double staining of the cells with 4,6-diamidino-2-phenylindole and 3,3-dihexyloxacarbocyanine iodide in Technovit resin sections showed that the mitochondria of these cells did not correspond to the fluorescent cytoplasmic DNA aggregates. Electron microscopy studies revealed both plastids and mitochondria in the cells of these species. In addition, immunoelectron microscopy using an anti-DNA monoclonal antibody showed clear labeling of plastids but not mitochondria. These data provide cytological evidence for biparental plastid inheritance and maternal mitochondrial inheritance in these species.Correspondence and reprints: College of Life Sciences, Peking University, Beijing 100871, Peoples Republic of China.     

The discovery and phylogenetic implications of a novel 41 bp plastid DNA deletion in wild potatoes

Insertions and deletions (indels) are common in intergenic spacer regions of plastid DNA and can provide important phylogenetic characters for closely related species. For example, a 241-bp plastid DNA deletion in the trnV-UAC/ndhC intergenic spacer region has been shown to have major phylogenetic importance in determining the origin of the cultivated potato. As part of a phylogenetic study of the wild potato Solanum series Piurana group we screened 199 accessions of 38 wild potato species in nine of the 19 tuber-bearing (Solanum section Petota) series that have not been examined before for indels in the trnV-UAC/ndhC intergenic spacer region. A novel 41 bp deletion (but no 241 bp deletion) was discovered for 30 accessions of three species: S. chiquidenum (5 of 10 accessions), S. chomatophilum (19 of 28), and S. jalcae (6 of 6). Accessions with and without this deletion are found throughout much of the north-south range of all three species in northern and central Peru, but not east of the Marañón River. Multivariate morphological analyses of these 44 accessions showed no morphological associations to the deletion. The results suggest extensive interspecific gene flow among these three species, or a common evolutionary history among species that have never been suggested to be interrelated.     

Achlya mitochondrial DNA: gene localization and analysis of inverted repeats

Summary Mitochondrial DNA from four strains of the oomycete Achlya has been compared and nine gene loci mapped, including that of the ribosomal protein gene, var1. Examination of the restriction enzyme site maps showed the presence of four insertions relative to a map common to all four strains. All the insertions were found in close proximity to genic regions. The four strains also cotained the inverted repeat first observed in A. ambisexualis (Hudspeth et al. 1983), allowing an examination by analysis of retained restriction sites of the evolutionary stability of repeated DNA sequences relative to single copy sequences. Although the inverted repeat is significantly more stable than single copy sequences, more detailed analysis indicated that this stability is limited to the portion encoding the ribosomal RNA genes. Thus, the apparent evolutionary stability of the repeat does not appear to derive from the inverted repeat structure per se.Abbreviations ATPase 6, 9 genes for ATPase subunits 6 and 9 - COI, II, III genes for cytochrome oxidase subunits 1, 2, and 3 - COB gene for apocytochrome b - L-, S-RNA genes for the mitochondrial large and small ribosomal RNAs - mtDNA mitochondrial DNA - var1 gene for the S. cerevisiae mitochondrially, encoded ribosomal protein - m.u. map units - bp base pairs - kb kilobase pairs     

Dynamics of localization and protein composition of plastid nucleoids in light-grown pea seedlings

Summary Localization and protein composition of plastid nucleoids was analyzed in light-grown pea seedlings at various stages of leaf development. In young plastids of unopened leaf buds, nucleoids were abundant and localized in the periphery of plastids, whereas, in mature leaves, chloroplasts contained nucleoids within narrow spaces restricted by thylakoids or grana. The migration of nucleoids into the interior of plastids preceded the formation of grana, and hence, the maturation of the photosynthetic apparatus. The protein composition of nucleoids was considerably different in young plastids and mature chloroplasts. Polypeptides with a molecular mass of 70–100 kDa predominated in the nucleoids of young plastids, whereas polypeptides with molecular mass of 20–30 kDa were abundant in the nucleoids of mature chloroplasts. Immuno-blot analysis with antibodies against the nucleoids of young plastids identified various polypeptides that were significantly more abundant in the nucleoids of young plastids than in the nucleoids of mature chloroplasts. These results demonstrate that plastid nucleoids are subject to dynamic changes in both localization and composition during the normal development of chloroplasts in the light.Abbreviations DAPI 4,6-diamidino-2-phenylindol - DiOC₆ 3,3-dihexyloxacarbocyanine iodide     

Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.

The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.     

Inheritance of chloroplast and mitochondrial DNA in alloplasmic forms of the genus Daucus

The inheritance of mitochondrial (mt) and chloroplast (ct) DNA in the progeny from interspecific crosses between the cultivated carrot (Daucus carota sativus) and wild forms of the genus Daucus was investigated by analysis of mt and ct RFLPs in single plants of the parental and filial generations. We observed a strict maternal inheritance of the organellar DNAs in all interspecific crosses examined. Previous studies on putative F₂ plants from a cross between Daucus muricatus x D. carota sativus suggested paternal inheritance of ctDNA. Our reinvestigation of this material revealed that the mtDNA of the putative F₂ plants differed from the mtDNA of both putative parents. Therefore, our data suggest that the investigated material originated from other, not yet identified, parents. Consequently, the analysis of this material cannot provide evidence for a paternal inheritance of ctDNA.     

Involvement of colchicine-sensitive cytoplasmic element in premitotic nuclear positioning ofAdiantum protonemata

Summary When the red-light grown protonema ofAdiantum capillus-veneris was transferred to the dark, the nucleus ceased its migration ca. 5 hours before cell plate formation (Mineyuki andFuruya 1980). To see whether the nucleus was held by some cytoplasmic structure during nuclear positioning, protonemata were treated with various centrifugal forces at different stages of the cell cycle. Nuclei of G₁ phase were easily displaced by centrifugation at 360×g for 15 minutes, but those of G₂ or M phase were not displaced by it, suggesting that the nuclei were held by some cytoplasmic elements in G₂ or M phase. This nuclear anchoring was not detectable in protonemata that were treated with 5mM colchicine. With this treatment, the nucleus did not stop its migration at late G₂ and moved even in prophase. And the retardation of organelle movement which was observed in cytoplasm on the lateral side of the nucleus after the cessation of premitotic nuclear migration (Mineyuki andFuruya 1984) was not observed in the presence of colchicine. Thus the nuclei appear to be held by colchicine-sensitive structure in cytoplasm between the lateral surface of the nucleus and cell wall during the premitotic nuclear positioning. Electron micrographs showing cytoplasmic microtubules were consistent with the idea.Abbreviations PPN Premitotic positioning of the nucleus - L region Cytoplasm between the lateral surface of the nucleus and cell wall (seeMineyuki et al. 1984)     

In vivo homologous recombination intermediates of yeast mitochondrial DNA analyzed by electron microscopy

Summary To study the structure of in vivo mitochondrial DNA recombination intermediates in Saccharomyces cerevisiae, we used a deletion mutant of the wild type mitochondrial genome. The mtDNA of this petite is composed of a direct tandem repetition of an 4,600 pb monomer repeat unit with a unique HhaI restriction enzyme site per repeat. The structure of native mtDNA isolated from log phase cells, and mtDNA crosslinked in vivo with trioxsalen plus UVA irradiation, was studied by electron microscopy. Both populations contained crossed strand Holliday type recombination intermediates. Digestion of both non-crosslinked and crosslinked and mtDNA with the enzyme HhaI released X and H shaped structures composed of two monomers. Electron microscopic analysis revealed that these structures had pairs of equal length arms as required for homologous recombination intermediates and that junctions could occur at points along the entire monomer length. The percentage of recombining monomers in both non-crosslinked and trioxsalen crosslinked mtDNA was calculated by quantitative analysis of all the structures present in an HhaI digest. The relationship between these values and the apparent dispersive replication of mtDNA in density-shift experiments and mtDNA fragility during isolation is discussed.     

Sequence rearrangements at theori 7 region ofSaccharomyces cerevisiae mitochondrial DNA

Summary Threeori elements (ori 2,ori 5, andori 7) have been sequenced inSaccharomyces cerevisiae strain Dip 2 and compared to the equivalentori elements of a second strain (B). Bothori 2 andori 5 exhibit 98% base matching between strains Dip 2 and B. In contrast, the thirdori element (ori 7) exhibits extensive sequence rearrangements whereby a segment located downstream in the consensus strain occurs within theori structure in Dip 2. This represents a novel polymorphic form of the yeast mitochondrial genome.     

The use of mitochondrial DNA polymorphism in the classification of individual onion plants by cytoplasmic genotypes

Summary Individual plants of a Japanese onion variety Sapporo-ki, which is characterized by the occasional occurrence of male-sterile plants, have been investigated for mitochondrial (mt) DNA polymorphism. Male-fertile and the Jones' cytoplasmic male-sterile (CMS) onions were also included for comparison. Southern blot hybridization with rrn26, cox-I, cox-II, cob, atpA and atp9 genes as probes revealed the two classes of mtDNA variation within a population of Sapporo-ki: Out of the 41 plants examined 19 contained mtDNA typical of malefertile plants, and 22 individuals contained mtDNA typical of the Jones' CMS genotype. Our results thus indcate that the use of the mitochondrial gene probes may greatly facilitate the classification of individual plants by cytoplasmic genotypes.