Different modes of de novo telomere formation by plant telomerases.

The telomerase reverse transcriptase can recognize broken chromosome ends and add new telomeres de novo in a reaction termed "chromosome healing". Here we investigate new telomere formation in vitro by telomerases from a variety of flowering plant species. Comparing the electrophoretic mobilities and nucleotide sequences of the products, we uncovered three different modes of new telomere formation. The soybean telomerase, designated a Class I enzyme, only elongated DNA primers ending in telomeric nucleotides. Arabidopsis and maize telomerases, designated Class II enzymes, efficiently extended completely non-telomeric sequences by positioning the 3' terminus at a preferred site on the RNA template. Silene latifolia and sorghum telomerases constituted class III enzymes that elongated non-telomeric DNA primers by annealing them at alternative sites on the RNA template. For all enzymes, errors were prevalent during synthesis of the first two repeats, likely reflecting lateral instability of the primer 3' terminus on the template during the initial rounds of elongation. Class III telomerases, however, were five- to 13-fold more error prone than class II, generating more mistakes in distal repeats added to the primers. This remarkable variability in enzyme-DNA interactions among plant telomerases does not reflect phylogenetic relationships, and therefore implies that the telomerase active site can evolve rapidly.

Living with genome instability: plant responses to telomere dysfunction.

Loss of telomere function in metazoans results in catastrophic damage to the genome, cell cycle arrest, and apoptosis. Here we show that the mustard weed Arabidopsis thaliana can survive up to 10 generations without telomerase. The last five generations of telomerase-deficient plants endured increasing levels of cytogenetic damage, which was correlated with developmental anomalies in both vegetative and reproductive organs. Mutants ultimately arrested at a terminal vegetative state harboring shoot meristems that were grossly enlarged, disorganized, and in some cases, dedifferentiated into a callusoid mass. Unexpectedly, late-generation mutants had an extended life-span and remained metabolically active. The differences in plant and animal responses to dysfunctional telomeres may reflect the more plastic nature of plant development and genome organization.

Analysis of the G-overhang structures on plant telomeres: evidence for two distinct telomere architectures.

Telomeres are highly conserved structures essential for maintaining the integrity of eukaryotic genomes. In yeast, ciliates and mammals, the G-rich strand of the telomere forms a 3' overhang on the chromosome terminus. Here we investigate the architecture of telomeres in the dicot plants Silene latifolia and Arabidopsis thaliana using the PENT (primer extension/nick translation) assay. We show that both Arabidopsis and Silene telomeres carry G-overhangs longer than 20-30 nucleotides. However, in contrast to yeast and ciliate telomeres, only half of the telomeres in Silene seedlings possess detectable G-overhangs. PENT reactions using a variety of primers and reaction conditions revealed that the remaining fraction of Silene telomeres carries either no overhangs or overhangs less than 12 nucleotides in length. G-overhangs were observed in Silene seeds and leaves, tissues that lack telomerase activity. These findings suggest that incomplete DNA replication of the lagging strand, rather than synthesis by telomerase, is the primary mechanism for G-overhang synthesis in plants. Unexpectedly, we found that the fraction of telomeres with detectable G-overhangs decreased from 50% in seedlings to 35% in leaves. The difference may reflect increased susceptibility of the G-overhangs to nuclease attack in adult leaves, an event that could act as a precursor for the catabolic processes accompanying leaf senescence

Ty1-copia-retrotransposon behavior in a polyploid cotton.

Retrotransposons constitute a ubiquitous and dynamic component of plant genomes. Intragenomic and intergenomic comparisons of related genomes offer potential insights into retrotransposon behavior and genomic effects. Here, we have used fluorescent in-situ hybridization to determine the chromosomal distributions of a Ty1-copia-like retrotransposon in the cotton AD-genome tetraploid Gossypium hirsutum and closely related putative A- and D-genome diploid ancestors. Retrotransposon clone A108 hybridized to all G. hirsutum chromosomes, approximately equal in intensity in the A- and D-subgenomes. Similar results were obtained by hybridization of A108 to the A-genome diploid G. arboreum, whereas no signal was detected on chromosomes of the D-genome diploid G. raimondii. The significance and potential causes of these observations are discussed.

Disruption of the telomerase catalytic subunit gene from Arabidopsis inactivates telomerase and leads to a slow loss of telomeric DNA.

Telomerase is an essential enzyme that maintains telomeres on eukaryotic chromosomes. In mammals, telomerase is required for the lifelong proliferative capacity of normal regenerative and reproductive tissues and for sustained growth in a dedifferentiated state. Although the importance of telomeres was first elucidated in plants 60 years ago, little is known about the role of telomeres and telomerase in plant growth and development. Here we report the cloning and characterization of the Arabidopsis telomerase reverse transcriptase (TERT) gene, AtTERT. AtTERT is predicted to encode a highly basic protein of 131 kDa that harbors the reverse transcriptase and telomerase-specific motifs common to all known TERT proteins. AtTERT mRNA is 10-20 times more abundant in callus, which has high levels of telomerase activity, versus leaves, which contain no detectable telomerase. Plants homozygous for a transfer DNA insertion into the AtTERT gene lack telomerase activity, confirming the identity and function of this gene. Because telomeres in wild-type Arabidopsis are short, the discovery that telomerase-null plants are viable for at least two generations was unexpected. In the absence of telomerase, telomeres decline by approximately 500 bp per generation, a rate 10 times slower than seen in telomerase-deficient mice. This gradual loss of telomeric DNA may reflect a reduced rate of nucleotide depletion per round of DNA replication, or the requirement for fewer cell divisions per organismal generation. Nevertheless, progressive telomere shortening in the mutants, however slow, ultimately should be lethal.

New ribosomal RNA gene locations in Gossypium hirsutum mapped by meiotic FISH.

In this study we have mapped newly identified rDNA loci in Gossypium hirsutum. Four new minor 18S-26S rDNA loci, in addition to the sites previously identified, were mapped using fluorescence in situ hybridization (FISH) to heterozygous translocation (NT) quadrivalents (IVs). The newly detected 18S-26S rDNA loci were mapped to the right arms of chromosomes 8, 9, 15, 17, 19, 20, and 23 and the left arms of chromosomes 5, 11, 12, and 14. Using the rDNA loci as common reference points, we detected several erroneous arm assignments in the previously published map of NT breakpoints. The data are summarized in the form of an integrated map for all 17 known rDNA loci, relative to centromeres, telomeres, and NT breakpoints. This information will facilitate future locus-specific research on rRNA gene evolution and function.

Tissue-specific expression of the beta-subunit of tryptophan synthase in Camptotheca acuminata, an indole alkaloid-producing plant.

Camptothecin is an anticancer drug produced by the monoterpene indole alkaloid pathway in Camptotheca acuminata. As part of an investigation of the camptothecin biosynthetic pathway, we have cloned and characterized a gene from C. acuminata encoding the beta-subunit of tryptophan (Trp) synthase (TSB). In C. acuminata TSB provides Trp for both protein synthesis and indole alkaloid production and therefore represents a junction between primary and secondary metabolism. TSB mRNA and protein were detected in all C. acuminata organs examined, and their abundance paralleled that of camptothecin. Within each shoot organ, TSB was most abundant in vascular tissues. Within the root, however, TSB expression was most abundant in the outer cortex. TSB has been localized to chloroplasts in Arabidopsis, but there was little expression of TSB in C. acuminata tissues where the predominant plastids were photosynthetically competent chloroplasts. Expression of the promoter from the C. acuminata TSB gene in transgenic tobacco plants paralleled expression of the native gene in C. acuminata in all organs except roots. TSB is also highly expressed in C. acuminata during early seedling development at a stage corresponding to peak accumulation of camptothecin, consistent with the idea that Trp biosynthesis and the secondary indole alkaloid pathway are coordinately regulated.

Sustained harvest of camptothecin from the leaves of Camptotheca acuminata.

Over a 12-week period, new growth was collected at different intervals from Camptotheca acuminata trees to determine whether a leaf harvest strategy would be an efficient means for the production of the alkaloid camptothecin. Because camptothecin accumulates in young leaves and because the harvesting of young tissue stimulates axillary bud outgrowth, this strategy increased the harvestable amount of camptothecin from trees in a nondestructive manner.

Use of meiotic FISH for identification of a new monosome in Gossypium hirsutum L.

The extensive use of molecular cytogenetics in human genetics and clinical diagnostics indicates that analogous applications in plants are highly feasible. One sort of application would be the identification of new aneuploids, which traditionally involves either direct karyotypic identification, which is feasible in only a few plant species, or tests with markers (cytogenetic, genetic, or molecular), which require sexual hybridization and at least one subsequent seed or plant generation. We have used meiotic fluorescence in situ hybridization (FISH) to analyze a new monosome of cotton (Gossypium hirsutum L., 2n = 4x = 52, 2(AD)1) that had a phenotype which seemed to be distinct from monosomes in the Cotton Cytogenetic Collection. Painting with A2-genome DNA revealed the monosome's D-subgenome origin. DAPI-PI staining showed that the monosome carries a major NOR, delimiting it to the major NOR-bearing chromosomes of the D-subgenome, i.e., 16 or 23. Dual-color FISH with 5S and 18S-28S rDNAs indicated that the monosome contains separate major clusters of each of these two tandemly repeated rDNA elements, thus delimiting the monosome to chromosome 23, for which the Cotton Cytogenetic Collection has previously been devoid of any sort of deficiency. Of the 26 chromosomes in the cotton genome, the Collection now provides coverage for 16 (70%) in the form of monosomy, and 20 (77%) in the form of monosomy and (or) telosomy. Use of molecular cytogenetic methods to identify a new plant aneuploid in cotton exemplifies the fact that a physicochemical karyotypic chromosome identification system is not required a priori for application of new molecular cytogenetic methods, thus indicating their potential applicability to nearly all plant species.

A rapid procedure for the isolation of C0t-1 DNA from plants.

In situ hybridization (ISH) for the detection of single- or low-copy sequences, particularly large DNA fragments cloned into YAC or BAC vectors, generally requires the suppression or "blocking" of highly-repetitive DNAs. C0t-1 DNA is enriched for repetitive DNA elements, high or moderate in copy number, and can therefore be used more effectively than total genomic DNA to prehybridize and competitively hybridize repetitive elements that would otherwise cause nonspecific hybridization. C0t-1 DNAs from several mammalian species are commercially available, however, none is currently available for plants to the best of our knowledge. We have developed a simple 1-day procedure to generate C0t-1 DNA without the use of specialized equipment.

Plant telomeres and telomerases. A review.

Barbara McClintock began investigating plant telomeres during the 1930s, but little additional work was done in this area until a telomeric DNA sequence was isolated and characterized from Arabidopsis thaliana in 1988. This sequence, a simple repeat of the heptanucleotide 5'-TTTAGGG-3', has been found in telomeres of almost all plants analyzed. Telomere length in plants, which can be a long as 75 kb or as short as 2 kb, is controlled by both genetic and developmental factors. The major mechanism for synthesis of telomeres is telomerase, a ribonucleoprotein with reverse transcriptase activity. Telomerase expression is highly regulated in both plants and animals. For example, there is little or no detectable expression of telomerase in most vegetative tissues of plants nor in most somatic tissues of animals. In contrast to animals, plants do not specify a germ line until late in development, but telomerase is reactivated during flowering, possibly to ensure that gametes and embryos arising from them inherit fully functional chromosomes. Telomerase is also highly expressed in plant tissue culture cells, as might be expected for cells with an unlimited capacity for proliferation. Despite recent progress in investigating plant telomeres and telomerase at the molecular level, there is still much more to learn, especially concerning the developmental control of telomerase activity.

Characterization and developmental patterns of telomerase expression in plants.

Telomerase activity is developmentally regulated in mammals. Here we examine telomerase activity in plants, whose development differs in fundamental ways from that of animals. Using a modified version of the telomere repeat amplification protocol (TRAP) assay, we detected an activity in extracts from carrots, cauliflower, soybean, Arabidopsis, and rice with all the characteristics expected for a telomerase synthesizing the plant telomere repeat sequence TTTAGGG. The activity was dependent on RNA and protein components, required dGTP, dATP, and dTTP, but not dCTP, and generated products with a seven nucleotide periodicity. Telomerase activity was abundant in cauliflower meristematic tissue and undifferentiated cells from Arabidopsis, soybean, and carrot suspension cultures, but was low or not detectable in a sampling of differentiated tissues from mature plants. Telomerase from cauliflower meristematic tissues exhibited relaxed DNA sequence requirements, which might reflect the capacity to form telomeres on broken chromosomes in vivo. The dramatic differences in telomerase expression and their correlation with cellular proliferation capacity mirror changes in human telomerase levels during differentiation and immortalization. Hence, telomerase activation appears to be a conserved mechanism involved in conferring long-term proliferation capacity.

Distribution of 5S and 18S-28S rDNA loci in a tetraploid cotton (Gossypium hirsutum L.) and its putative diploid ancestors.

The most widely cultivated species of cotton, Gossypium hirsutum, is a disomic tetraploid (2n=4x=52). It has been proposed previously that extant A- and D-genome species are most closely related to the diploid progenitors of the tetraploid. We used fluorescent in situ hybridization (FISH) to determine the distribution of 5S and 18S-28S rDNA loci in the A-genome species G. herbaceum and G. arboreum, the D-genome species G. raimondii and G. thurberi, and the AD tetraploid G. hirsutum. High signal-to-noise, single-label FISH was used to enumerate rDNA loci, and simultaneous, dual-label FISH was used to determine the syntenic relationships of 5S rDNA loci relative to 18S-28S rDNA loci. These techniques provided greater sensitivity than our previous methods and permitted detection of six new G. hirsutum 18S-28S rDNA loci, bringing the total number of observed loci to 11. Differences in the intensity of the hybridization signal at these loci allowed us to designate them as major, intermediate, or minor 18S-28S loci. Using genomic painting with labeled A-genome DNA, five 18S-28S loci were localized to the G. hirsutum A-subgenome and six to the D-subgenome. Four of the 11 18S-28S rDNA loci in G. hirsutum could not be accounted for in its presumed diploid progenitors, as both A-genome species had three loci and both D-genome species had four. G. hirsutum has two 5S rDNA loci, both of which are syntenic to major 18S-28S rDNA loci. All four of the diploid genomes we examined contained a single 5S locus. In g. herbaceum (A1) and G. thurberi (D1), the 5S locus is syntenic to a major 18S-28S locus, but in G. arboreum (A2) and G. raimondii (D5), the proposed D-genome progenitor of G. hirsutum, the 5S loci are syntenic to minor and intermediate 18S-28S loci, respectively. The multiplicity, variation in size and site number, and lack of additivity between the tetraploid species and its putative diploid ancestors indicate that the behavior of rDNA loci in cotton is nondogmatic, and considerably more complex and dynamic than previously envisioned. The relative variability of 18S-28S rDNA loci versus 5S rDNA loci suggests that the behavior of tandem repeats can differ widely.

Fluorescent in situ hybridization of a bacterial artificial chromosome.

Fluorescent in situ hybridization (FISH) of a 130 kilobase cotton (Gossypium hirsuitum L.) bacterial artificial chromosome (BAC) clone containing a high proportion of single-copy DNA produced a large pair of FISH signals on the distal end of the long arm of a pair of chromosomes of the D-genome species G. raimondii Ulbr. and produced a fainter pair of signals on a small submetacentric pair of chromosomes of the A-genome species G. herbaceum L. The signals were synthetic with a nucleolar organizer region in G. raimondii and G. herbaceum. Signal pairs were easily recognized in interphase and metaphase cells either with or without suppression of repetitive sequences with unlabeled G. hirsutum C0t-1 DNA. High quality FISH results were consistently obtained and image analysis was not required for viewing or photography. Results indicate that FISH of BAC clones is an excellent tool for the establishment of new molecular cytogenetic markers in plants and will likely prove instrumental in the development of useful physical maps for many economically important crop species.

Sites of accumulation of the antitumor alkaloid camptothecin in Camptotheca acuminata.

Camptothecin is an anticancer and anti-viral alkaloid produced by the Chinese tree Camptotheca acuminata (Nyssaceae). Despite previous reports of low levels of anticancer activity in leaves of Camptotheca acuminata, we have discovered that camptothecin accumulates to approximately 0.4% of the dry weight of young leaves. This level is 1.5-fold higher than that of the seeds and 2.5-fold higher than that of the bark, the two currently used sources of the drug. As the leaves mature, the concentration and absolute amount of camptothecin decreases rapidly. The high levels of camptothecin in young leaves could provide an easily harvested, non-destructive source of this important drug.

Identification of a homeologous chromosome pair by in situ DNA hybridization to ribosomal RNA loci in meiotic chromosomes of cotton (Gossypium hirsutum).

In situ DNA hybridization with 18S-28S and 5S ribosomal DNA probes was used to map 18S-28S nucleolar organizers and tandem 5S repeats to meiotic chromosomes of cotton (Gossypium hirsutum L.). Mapping was performed by correlating hybridization sites to particular positions in translocation quadrivalents. Arm assignment required translocation quadrivalents with at least one interstitial chiasma and sufficient distance between the hybridization site and the centromere. We had previously localized a major 18S-28S site to the short arm of chromosome 9; here we mapped two additional major 18S-28S sites to the short arm of chromosome 16 and the left arm of chromosome 23. We also identified and mapped a minor 18S-28S site to the short arm of chromosome 7. Two 5S sites of unequal size were identified, the larger one near the centromere of chromosome 9 and the smaller one near the centromere of chromosome 23. Synteny of 5S and 18S-28S sites indicated homeology of chromosomes 9 and 23, while positions of the other two 18S-28S sites supplement genetic evidence that chromosomes 7 and 16 are homeologous.

Expression of a 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Camptotheca acuminata is differentially regulated by wounding and methyl jasmonate.

We have isolated a gene, hmg1, for 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) from Camptotheca acuminata, a Chinese tree that produces the anti-cancer monoterpenoid indole alkaloid camptothecin (CPT). HMGR supplies mevalonate for the synthesis of the terpenoid component of CPT as well as for the formation of many other primary and secondary metabolites. In Camptotheca, hmg1 transcripts were detected only in young seedlings and not in vegetative organs of older plants. Regulation of the hmg1 promoter was studied in transgenic tobacco using three translational fusions (-1678, -1107, -165) with the beta-glucuronidase (GUS) reporter gene. Histochemical analysis of plants containing each of the three promoter fusions showed similar developmental and spatial expression patterns. In vegetative tissues, GUS staining was localized to the epidermis of young leaves and stems, particularly in glandular trichomes. Roots showed intense staining in the cortical tissues in the elongation zone and light staining in the cortex of mature roots. hmg1::GUS expression was also observed in sepals, petals, pistils, and stamens of developing flowers, with darkest staining in the ovary wall, ovules, stigmas, and pollen. Leaf discs from plants containing each of the translational fusions showed a 15- to 20-fold wound induction of hmg1::GUS expression over 72 h; however, this increase in GUS activity was completely suppressed by treatment with methyl jasmonate. Taken together, these data show that a 165-bp fragment of Camptotheca hmg1 promoter is sufficient to confer developmental regulation as well as wound induction and methyl jasmonate suppression of GUS expression in transgenic tobacco.

Purification and characterization of a secreted purple phosphatase from soybean suspension cultures.

We purified and partially sequenced a purple (lambda(max) = 556 nanometers) acid phosphatase (APase; EC 3.1.3.2) secreted by soybean (Glycine max) suspension-culture cells. The enzyme is a metalloprotein with a Mn(2+) cofactor. This APase appears to be a glycoprotein with a monomer subunit molecular weight of 58,000 and an active dimer molecular weight of approximately 130,000. The protein has an isoelectric point of about 5.0 and a broad pH optimum centered near 5.5. The purified enzyme, assayed with p-nitrophenyl phosphate as the substrate, has a specific activity of 512 units per milligram protein and a K(m) of approximately 0.3 millimolar; phosphate is a competitive inhibitor with a K(i) of 0.7 millimolar. This APase is similar to one found in soybean seed meal but dissimilar to that found in soybean seedlings.

Expression of enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants.

Strictosidine, a precursor to over 1000 indole alkaloids including the anti-tumor drugs vinblastine, vincristine, and camptothecin, is produced by the condensation of tryptamine and secologanin. Strictosidine synthase, the enzyme responsible for this condensation, is the first committed step in the indole-alkaloid pathway. We have introduced a modified cDNA encoding Strictosidine synthase from Catharanthus roseus (L.) Don. (McKnight et al. 1990, Nucl. Acids Res. 18, 4939) driven by the CaMV 35S promoter into tobacco (Nicotiana tabacum L.). Transgenic tobacco plants expressing this construct had from 3 to 22 times greater strictosidinesynthase activity than C. roseus plants. Ultrastructural immunolocalization demonstrated that strictosidine synthase is a vacuolar protein in C. roseus and is correctly targeted to the vacuole in transgenic tobacco. Immunoblot analysis of strictosidine synthase showed that two distinct forms of the enzyme were produced in transgenic tobacco plants but that only a single form was made in C. roseus. This observation indicates that the second form of the protein is not simply a result of overexpression in tobacco, but may reflect differences in protein processing between tobacco and C. roseus.