Isolation and characterization of paracrystalline structures from transgenic P<Emphasis Type="Italic">ssu-ipt</Emphasis> tobacco

Distinct crystalloids were found in chloroplasts of transgenic Pssu-ipt tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) overproducing endogenous cytokinins. They were present both in rooted (T) and grafted (TC) transgenic plants contrary to control tobacco (C). The fractions enriched by crystalloids were isolated from chloroplasts using a continuous or a discontinuous Percoll gradient. Chlorophyll (Chl) fluorescence emission spectra at 77 K indicated the presence of aggregates of light-harvesting complex proteins (LHC2) that was not connected to reaction centres of photosystem 2 both in isolated chloroplasts and in the fraction of 80 % Percoll gradient from both types of transgenic tobacco. Further analyses, i.e. pigment contents, polypeptide composition by SDS-PAGE, and immunoblotting support our hypothesis that crystalloids inside chloroplasts of transgenic tobacco are formed by LHC2 aggregates. Treatment with two distinct detergents, chosen with respect to their effects (i.e. β-dodecyl maltoside or Triton X-100), resulted in different degree of disintegration of Chl a/b proteins in transgenic plants compared to the control. Electron microscopic observations and immunogold labelling with specific LHC2 antibodies carried on the resin embedded leaf sections or free suspensions of chloroplasts showed that gold particles were bound preferentially on the outer surface of crystalloids. Three-dimensional reconstruction of chloroplasts and crystalloids proved that paracrystalline structures varied moderately in their size and took up a significant portion of total chloroplast volume.     

Changes in Chloroplast Ultrastructure in Pssu-ipt Tobacco During Plant Ontogeny

Changes in chloroplast ultrastructure and total content of endogenous cytokinins (CK) were studied during different phases of plant development in transgenic Pssu-ipt tobacco (Nicotiana tabacum L. cv. Petit Havana SR1). Permanent overproduction of CK was found in both rooted (SE) and grafted (G) Pssu-ipt plants in all phases of plant development with the peak in vegetative and flowering phase in the latter ones. No such a correlation was observed in SE on the contrary to control non-transgenic plants (SR1) and grafts (SRG), which showed also CK increase at juvenile and flowering phases. No significant differences in parameters of chloroplast ultrastructure, such as length of chloroplast, starch content, granum width, and number of thylakoids per granum, were proved between chloroplasts from young mature leaves of control and transgenic tobacco during plant ontogeny. Nevertheless, several anomalies in the ultrastructure of cell organelles were found in Pssu-ipt tobacco. Amoeboid shape of chloroplasts was often observed in connection with tubular clusters resembling peripheral reticulum. The distinct crystalline structures located in chloroplasts might be formed by LHC protein aggregates. Smaller crystals of unknown composition were found also in mitochondria. Numerous crystalline cores were present in peroxisomes. The alterations might be the result of imbalance of phytohormone content, degradation effect of CK overproduction, or the example of acclimation to permanent stress.     

High content of endogenous cytokinins stimulates activity of enzymes and proteins involved in stress response in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

Transgenic Pssu-ipt tobacco with elevated content of endogenous cytokinins grown under in vitro conditions exhibited elevated activities of antioxidant enzymes (i.e. catalase, ascorbate peroxidase, guaiacol and syringaldazine peroxidase, glutathione reductase) and some of enzymes involved in anaplerotic pathways such as glucose-6-phosphate dehydrogenase, glycolate oxidase, NADP-malic enzyme, NADP-isocitrate dehydrogenase, and glutamate dehydrogenase compared to control non-transgenic SR1 tobacco. Higher activities of peroxidases, NADP-malic enzyme, and glutamate dehydrogenase were maintained in transgenic grafts after several weeks of the growth under ex vitro conditions, while transgenic rooted plants showed only the increase in activity of glycolate oxidase compared to control non-transformed tobacco. Total activities of superoxide dismutase were lower in both types of Pssu-ipt tobacco contrary to controls under both growth conditions. The presence of PR-1 protein and proteins with elevated activities of chitinase was proved in the extracellular fluid in both transgenic types under both in vitro and ex vitro conditions.     

Cytokinin-induced activity of antioxidant enzymes in transgenic Pssu-ipt tobacco during plant ontogeny

Cytokinin (CK) content and activities of several antioxidant enzymes were examined during plant ontogeny with the aim to elucidate their role in delayed senescence of transgenic Pssu-ipt tobacco. Control Nicotiana tabacum L. (cv. Petit Havana SR1) and transgenic tobacco with the ipt gene under the control of the promoter of small subunit of Rubisco (Pssu-ipt) were both grown either as grafts on control rootstocks or as rooted plants. Both control plant types showed a decline in total content of CKs with proceeding plant senescence. Contrary to this both transgenic plant types exhibited at least ten times higher content of CKs than controls and a significant increase of CK contents throughout the ontogeny with maximal values in the later stages of plant development. Significantly higher portion of O-glucosides was found in both transgenic plant types compared to control ones. In transgenic plants, zeatin and zeatin riboside were predominant type of CKs. Generally, Pssu-ipt tobacco exhibited elevated activities of antioxidant enzymes compared to control tobacco particularly in the later stages of plant development. While in control tobacco activity of glutathione reductase (GR) and superoxide dismutase (SOD) showed increasing activity up to the onset of flowering and then gradually decreased, in both transgenic types GR increased and SOD activity showed only small change throughout the plant ontogeny. Ascorbate peroxidase (APOD) was stimulated in both transgenic types. The manifold enhancement of syringaldazine and guaiacol peroxidase activities was observed in transgenic grafts throughout plant ontogeny in contrast to control and transgenic rooted plants, where the increase was found only in the late stages. Electron microscopic examination showed higher number of crystallic cores in peroxisomes and abnormal interactions among organelles in transgenic tobacco in comparison with control plant. The overproduction of cytokinins resulted in the stimulation of activities of AOE throughout the plant ontogeny of transgenic Pssu-ipt tobacco.     

Effect of seed-specific expression of the <Emphasis Type="Italic">ipt</Emphasis> Gene on <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. seed composition

A transgenic approach to manipulation of endosperm development has been investigated. Nicotiana tabacum cv. Xanthi, an endosperm-containing dicotyledon, has been used as a model plant and the 2.6 kb wheat high molecular weight (HMW) glutenin subunit 12 promoter has been used fused either to the gus reporter gene (HMWgus construct)—to study promoter characteristics—or to the Agrobacterium ipt gene—to study the effect of cytokinin (CK) over-expression on assimilate accumulation in the seed. In transgenic tobacco the promoter:gus fusion showed that HMW is an endosperm-specific promoter with maximum expression 20 days after anthesis (DAA), corresponding to the mid to late stages of seed development. Transgenic plants containing the HMWipt construct showed no morphological abnormalities but they had an average increase in seed weight and total ethanol-insoluble carbohydrates and protein content of 8.1%, 7.0% and 8.3%, respectively. SDS PAGE analysis demonstrated that the effect on protein accumulation was non-specific. The highest values of the parameters analysed correlated with moderate increases in the levels of biologically active CKs. These results suggest that ectopic expression of small amounts of CKs can be used to increase storage assimilate accumulation without a detrimental effect on development.     

Constancy of organellar genome copy numbers during leaf development and senescence in higher plants

In higher plants, plastid and mitochondrial genomes occur at high copy numbers per cell. Several recent publications have suggested that, in higher plants like Arabidopsis and maize, chloroplast DNA is virtually absent in mature and old leaves. This conclusion was mainly based on DAPI staining of isolated chloroplasts. If correct, the finding that chloroplasts in mature leaves lack DNA would change dramatically our understanding of gene expression, mRNA stability and protein stability in chloroplasts. In view of the wide implications that the disposal of chloroplast DNA during leaf development would have, we have reinvestigated the age dependency of genome copy numbers in chloroplasts and, in addition, tested for possible changes in mitochondrial genome copy number during plant development. Analyzing chloroplast and mitochondrial DNA amounts in Arabidopsis and tobacco plants, we find that organellar genome copy numbers remain remarkably constant during leaf development and are present in essentially unchanged numbers even in the senescing leaves. We conclude that, during leaf development, organellar gene expression in higher plants is not significantly regulated at the level of genome copy number and we discuss possible explanations for the failure to detect DNA in isolated chloroplasts stained with DAPI.     

Über Plastidenstapel beiBotrydiopsis alpina sowie Anlage und Vermehrung der Stigmen bei dieser undHeterococcus (Xanthophyceae)

Under certain conditions inBotrydiopsis alpina stacks of chloroplasts are formed. They consist of up to 8 elements. In contrast to what is known from other algae in zoosporangia of this species and ofHeterococcus caespitosus, stigmata are formed in early developmental stages. They are reproduced together with the chloroplasts, in which they occupy a position at the edge and near the existing or future incision. At the side of the old stigma a new one is formed, and partitioning of the chloroplast between these two leads to their distribution to the daughter chloroplasts. Young daughter cells in the zoosporangia ofBotrydiopsis alpina contain one chloroplast which undergoes a last unequal division giving rise to one astigmate and usually somewhat smaller and to one stigmate chloroplast. In both species the capacity for locomotion may be suppressed, the presumptive zoospores thereby becoming aplanospores. Autospores in the proper sense were not observed. Their development quite generally is different from that of aplanospores (and zoospores), and both types of spores should be distinguished.

Herrn Professor Dr.Lothar Geitler zum 80. Geburtstag in Verehrung gewidmet.     

Endogenous cytokinins in shoots of Aloe polyphylla cultured in vitro in relation to hyperhydricity, exogenous cytokinins and gelling agents

The process of hyperhydricity in tissue cultured plants of Aloe polyphylla is affected by both applied cytokinins (CKs) and the type of gelling agent used to solidify the medium. Shoots were grown on media with agar or gelrite and supplemented with different concentrations of N⁶-benzyladenine (BA) or zeatin (0, 5 and 15 μM). Endogenous CKs were measured in in vitro regenerants after an 8-weeks cycle to examine whether the hyperhydricity-inducing effect of exogenous CKs and gelling agents is associated with changes in the endogenous CK content. On media with agar a reduction in hyperhydricity occurred, while the gelrite treatment produced both normal and hyperhydric shoots (HS). The content of endogenous CKs, determined by HPLC-mass spectrometry, in the shoots grown on CK-free media comprised isopentenyladenine-, trans-zeatin- and cis-zeatin-type CKs. The application of exogenous CKs resulted in an increase in the CK content of the shoots. Following application of zeatin, dihydrozeatin-type CKs were also detected in the newly-formed shoots. Application of BA to the media led to a transition from isoprenoid CKs to aromatic CKs in the shoots. Shoots grown on gelrite media contained higher levels of endogenous CKs compared to those on agar media. Total CK content of HS was higher than that of normal shoots grown on the same medium. We suggest that the ability of exogenous CKs and gelrite to induce hyperhydricity in shoots of Aloe polyphylla is at least partially due to up-regulation of endogenous CK levels. However, hyperhydricity is a multifactor process in which different factors intervene.     

巴西橡胶树HbWRKY1基因的克隆及转化烟草的初步研究

利用RACE结合RT-PCR技术,从巴西橡胶树(Hevea brasiliensis)总RNA中扩增得到长度为1234 bp的WRKY基因cDNA全长编码序列。通过氨基酸同源性比对,该序列推导的氨基酸序列与蓖麻、白杨的WRKY同源性分别为79%和73%,表明分离的cDNA序列为橡胶树WRKY基因,命名为HbWRKY1。通过构建pCAMBIA1304-HbWRKY1植物表达载体,经农杆菌GV3101介导,将HbWRKY1基因导入烟草(Nicotiana tabacum)中,对所获得的潮霉素抗性烟草株系进行PCR鉴定。结果表明,HbWRKY1基因已整合到65株转基因植株中。干旱胁迫试验表明,HbWRKY1的过量表达可以明显提高转基因烟草对干旱胁迫的耐受能力。这说明WRKY基因与橡胶树抗旱能力之间存在一定的关系。     

Growth retardation of plants transformed by overexpression of NtFtsZ1-2 in Tobacco

We transformed tobacco plants (Mcotiana tabacum L, Xanthi) by introducing a sense construct ofNtFtsZ1-2. This tobacco nuclear gene encodes a chloroplast-localized homologue of FtsZ, the bacterial cell-division protein. The overexpressing plants contained enlarged chloroplasts in their leaf mesophyll cells. In the T₁ progeny, we observed three different phenotypes: 1 ) plants with cells containing many small chloroplasts, which was the same as for wild-type plants; 2) plants in which the celts contained one to three enlarged chloroplasts (severe type); and 3) plants whose cells contained a combination of many small chloroplasts and one to three enlarged chloroplasts (intermediate type). The outward appearance of the severe and intermediate types of transgenic plants did not differ noticeably from the wild-types. However, the severe-type plants were most retarded in their growth under both high- and low-light conditions, followed by the intermediate-types. Under medium levels of light, the two types of transgenic plants exhibited growth rates comparable to that of the wild types. Based on the overall results, we suggest that many small chloroplasts, rather than a few large chloroplasts, are required for efficient use of light energy in the mesophyll cells.     

Chemistry and micromorphology of compound epicuticular wax crystalloids (Strelitzia type)

Certain non-liliiflorous taxa within the monocotyledons (e.g.,Strelitzia, Heliconia, Typha) are characterized by compound epicuticular wax rodlets (Strelitzia type). Similar rodlets are also encountered on the surface of the dicotyledonous plantBenincasa hispida (Thunb.)Cogn. Chemical analysis of the surface wax from both sources showed that the rodlets are chemically distinct. The rodlets of the monocotyledons consist exclusively of aliphatic wax lipids, mainly wax esters. In contrast, the rodlets ofBenincasa are cheifly composed of triterpenol acetates and triterpenols. Formation of rodlets is therefore interpreted as ultrastructural convergency. It is concluded that taxonomical studies on wax crystalloids can be misleading when interpreted in terms of micromorphology of crystalloids only.     

Random chloroplast segregation and frequent mtDNA rearrangements in fertile somatic hybrids between Nicotiana tabacum L. and N. glutinosa L.

Patterns of organelle inheritance were examined among fertile somatic hybrids between allotetraploid Nicotiana tabacum L. (2n=4x=48) and a diploid wild relative N. glutinosa L. (2n=2x=24). Seventy somatic hybrids resistant to methotrexate and kanamycin were recovered following fusion of leaf mesophyll protoplasts of transgenic methotrexate-resistant N. tabacum and kanamycin-resistant N. glutinosa. Evidence for hybridization of nuclear genomes was obtained by analysis of glutamate oxaloacetate transaminase and peroxidase isoenzymes and by restriction fragment length polymorphism (RFLP) analysis using a heterologous nuclear ribosomal DNA probe. Analysis of chloroplast genomes in a population of 41 hybrids revealed a random segregation of chloroplasts since 25 possessed N. glutinosa chloroplasts and 16 possessed N. tabacum chloroplasts. This contrasts with the markedly non-random segregation of plastids in N. tabacum (+)N. rustica and N. tabacum (+) N. debneyi somatic hybrids which we described previously and which were recovered using the same conditions for fusion and selection. The organization of the mitochondrial DNA (mtDNA) in 40 individuals was examined by RFLP analysis with a heterologous cytochrome B gene. Thirty-eight somatic hybrids possessed mitochondrial genomes which were rearranged with respect to the parental genomes, two carried mtDNA similar to N. tabacum, while none had mtDNA identical to N. glutinosa. The somatic hybrids were self-fertile and fertile in backcrosses with the tobacco parent.Contribution No. 1487 Plant Research Centre     

Changes in structure of isolated chloroplasts ofCodium fragile andCaulerpa filiformis in response to osmotic shock and detergent treatment

Summary The ultrastructure of chloroplasts from two genera of coenocytic green algae,Codium andCaulerpa, were examined after suspension in hypotonic solution and in detergent at various concentrations. The capacity of the suspensions to carry out CO₂-dependent and ferricyanide-dependent O₂ evolution was measured under the same conditions of osmotic strength and detergent concentration.The chloroplasts in the preparations were in the form of cytoplasts and gave rates of O₂ evolution comparable with those expected from undamaged chloroplasts. Suspension in hypotonic solution depressed the rate of CO₂-dependent O₂ evolution in both species, but this was partially restored in theCodium chloroplasts when these were re-suspended in iso-osmotic solutions. Major structural changes were observed only after suspension in buffer when theCodium chloroplasts lost their outer envelope, most of their stroma, and the thylakoids became swollen.Caulerpa chloroplasts were more variable in their response and, even when suspended in buffer only, the proportion of the plastids which had lost all of their stroma and thylakoid swelling was never as common as inCodium chloroplasts. However, once suspended in hyper-osmotic medium below 700 mosmolar,Caulerpa chloroplasts could not regain their capacity for CO₂-dependent O₂ evolution.Detergent treatment removed the cytoplast membrane but not the cytoplasmic material adhering to the chloroplast envelope. High concentrations of detergent were needed to cause loss of the chloroplast envelope, loss of stromal contents and unstacking of the thylakoids.Caulerpa chloroplasts were less sensitive to detergent than those ofCodium. There was no indication that specific structures such as the thylakoid organizing body were resistant to detergent action. The results show that exposure to hypotonic solutions and to detergent results in less damage to these chloroplasts than it would to those of higher plants. It is proposed that the basis of this unusual resistance is not due to the properties of the chloroplast membranes but to the presence of material which coats the organelles during isolation. This material is likely to be identical with the sulphated xylo-mannogalactan isolated from the vacuole contents of these algae and which has the visco-elastic properties essential to allow the organelles to resist disruption by osmotic forces and disintegration by detergents.     

Protein crystalloids in ribosome-deficient plastids of Aeonium domesticum cv. variegatum (Crassulaceae)

Protein crystalloids are typical constituents of Aeonium domesticum plastids. They are composed of hexagonally arranged tube-like elements situated in the stroma without a bordering membrane. The single tubule has an external diameter of about 20 nm and an internal one of about 10 nm. The green-white-green mesochimera Ae. domesticum cv. variegatum contains normal chloroplasts in the green tissue and colourless plastids in the pale tissue. The defective plastids have a double-layered envelope, scarce internal membrane structures and contain, in the mature stage, a large vacuole. Plastid ribosomes can be detected only rarely in proplastids. They lose their ribosome complement entirely in the course of development. Polyacrylamide gel electrophoresis of total nucleic acids extracted from white tissue revealed the absence of the 23S and 16S rRNA normally present in plastids. Despite the loss of ribosomes, the plastids contain large protein crystalloids, which are structurally identical with those of normal green chloroplasts. Consequences concerning problems of encoding and transport of crystalloid protein(s) are briefly discussed.Abbreviations CAM crassulacean acid metabolism - FIP fraction I protein - L I epidermis - L II subepidermal layer - L III leaf core - SPC succulent protein crystalloid This is the first part of a series on the crystalloid-forming succulent protein     

Isolation of a 6.2 kb genomic fragment carrying the Adh1 gene of tomato and its expression in transgenic tobacco

An 11 kb Eco RI genomic fragment containing the alcohol dehydrogenase (Adh1) gene was cloned. Cross-hybridization with three Adh2 cDNA clones suggested that the entire coding region of the Adh1 gene was contained on a 6.2 kb Xba I/Hind III subfragment. Using RFLP linkage analysis, the genomic clone was mapped on chromosome 4 between the markers TG 182 and TG 65 in a position corresponding to the Adh1 locus. To further confirm the Adh1 origin of the genomic clone, tobacco plants were transformed with the 6.2 kb Xba I/Hinb III genomic subfragment. Isozyme analysis demonstrated that in transgenic tobacco plants functional tomato specific ADH-1 homodimers were synthesized as well as heterodimers composed of tobacco and tomato subunits.     

The structure,composition and distribution of proteinaceous crystalloids in vegetative cells of the red algaWrangelia plumosa

Summary High concentrations of proteinaceous crystalloids accumulate in vegetative cells of the red algaWrangelia plumosa Harvey but disappear prior to sporogenesis. The distinctly structured crystalloids lack a bounding membrane and appear to autopolymerize within the cytoplasm. Chemical analysis of isolated crystalloids showed the presence of all amino acids except cysteine and cysteic acid. Carbohydrate accounted for 7.5% of the preparation. The crystalloids appear to have a storage function during growth and development.     

Expression of vde Gene Integrated Into Tobacco Genome in Antisense and Overexpressed Ways

Violaxanthin de-epoxidase (VDE) is localised in the thylakoid lumen of chloroplasts and catalyses de-epoxidation of violaxanthin into antheraxanthin and zeaxanthin. Tobacco vde gene was inserted into a binary vector pCAMBIA1301 with the hygromycin resistant gene for selection in antisense and overexpressed ways. Two constructs with antisense and overexpressed vde gene were introduced in tobacco (Nicotiana tabacum L.) using Agrobacterium tumefaciens strain LBA4404, PCR and Southern blot analyses demonstrated that the exogenous gene was integrated into genome of tobacco plants. VDE activity assay and HPLC analysis of pigments showed that the vde gene was expressed in the overexpressed transformants, whereas suppressed in the antisense ones. The chlorophyll fluorescence measurements proved that the contents of VDE in transgenic plants have a significant function in non-photochemical quenching.     

Gene activation in plastids by the CRE site-specific recombinase

We developed a novel system for gene activation in plastids that uses the CRE/loxP site-specific recombination system to create a translatable reading frame by excision of a blocking sequence. To test the system, we introduced an inactive gfp* gene into the tobacco plastid genome downstream of the selectable spectinomcyin resistance (aadA) marker gene. The aadA gene is the blocking sequence, and is flanked by directly oriented loxP sites for excision by the CRE. In the non-activated state, gfp* is transcribed from the aadA promoter, but the mRNA is not translated due to the lack of an AUG translation initiation codon. Green Fluorescent Protein (GFP) expression is activated by excision of the aadA coding segment to link up the gfp* coding region with the translation initiation codon of aadA. Tobacco plants that carry the inactive gfp* gene do not contain detectable levels of GFP. However, activation of gfp* resulted in GFP accumulation, proving the utility of CRE-induced protein expression in tobacco chloroplasts. The gene activation system described here will be useful to probe plastid gene function and for the production of recombinant proteins in chloroplasts.