Cloning and Characterization of an Agglutinin Gene from Arisaema lobatum

A novel agglutinin gene was cloned from Arisaema lobatum using SMART RACE-PCR technology. The full-length cDNA of Arisaema lobatum agglutinin (ala) was 1078 bp and contained a 774 bp open reading frame encoding a lectin precursor (proproprotein) of 258 amino acid residues with a 23 aa signal peptide. ALA contained three mannose-binding sites (QXDXNXVXY) with two-conserved domains of 45% identity, ALA-DOM1 and ALA-DOM2. The three-dimensional structure of ALA was very similar to that of GNA (Galanthus nivalis agglutinin). ALA shared varying identities, ranging from 40% to 85%, with mannose-binding lectins from other species of plant families, such as Araceae, Alliaceae, Iridaceae, Lillaceae, Amaryllidaceae and Bromeliaceae. Genomic sequence of ala was also cloned using genomic walker technology, and it was found to contain three putative TATA boxes and eight possible CAAT boxes in the 5′-flanking region. No intron was found within the region of genomic sequence. Southern blot analysis indicated that the ala belonged to a multi-copy gene family. Expression pattern analysis revealed that the ala preferentially expressed in the tissues with the higher expression being found in spadix, bud, leaf, spathe and tuber. The cloning of the ala gene not only provides a basis for further investigation of its structure, expression and regulation mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.     

Nuclear DNA variability withinPisum sativum (Leguminosae): Cytophotometric analyses

The cultivars or experimental lines ofPisum sativum were analyzed cytophotometrically for nuclear DNA content of early prophases after ethanol-acetic acid fixation. Wide variability was found (from 3.93 to 5.07pg per haploid nucleus). This result was confirmed by the cytophotometric analysis of interphase nuclei isolated from leaf tissues fixed in formalin. Analysis of interphase nuclei at different thresholds of optical density showed that certain chromatin fractions are involved in the variations.     

Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants

The propagation of plants through tissue culture can induce a variety of genetic and epigenetic changes. Variation in DNA methylation has been proposed as a mechanism that may explain at least a part of these changes. In the present study, the methylation of tomato callus DNA was compared with that of leaf DNA, from control or regenerated plants, at MspI/HpaII sites around five middle-repetitive sequences. Although the methylation of the internal cytosine in the recognition sequence CCGG varied from zero to nearly full methylation, depending on the probe used, no differences were found between callus and leaf DNA. For the external cytosine, small differences were revealed between leaf and callus DNA with two probes, but no polymorphisms were detected among DNA samples of calli or DNA samples of leaves of regenerated plants. When callus DNA cut with HindIII was studied with one of the probes, H9D9, most of the signal was found in high-molecular-weight DNA, as opposed to control leaf DNA where almost all the signal was in a fragment of 530 bp. Also, an extra fragment of 630 bp was found in the callus DNA that was not present in control leaf DNA. Among leaves of plants regenerated from tissue culture, the 630-bp fragment was found in 10 of 68 regenerated plants. This 630-bp fragment was present among progeny of only 4 of these 10 plants after selfing, i.e. it was partly inherited. In these cases, the fragment was not found in all progeny plants, indicating heterozygosity of the regenerated plants. The data are interpreted as indicating that a HindIII site becomes methylated in callus tissue, and that some of this methylation persists in regenerated plants and is partly transmitted to their progeny.     

Stability of RAPD fingerprints in potato: Effect of source tissue and primers

Variations in random amplified polymorphic DNA (RAPD) profiles from leaf, stem, root, and tuber tissues were observed in case of two glasshouse grown potato cultivars using 40 decamer primers suggesting possible danger of cultivar misidentification. Genomic DNA extracted from the above four tissues of four in vitro grown potato cultivars, however, produced more uniform RAPD fingerprints. A significant effect of random primers on fingerprint uniformity was observed in case of both glasshouse and in vitro grown samples. A new concept of stability index for random primers based on homogeneity of RAPD profiles obtained from different tissues of a single plant have been introduced. It is concluded that RAPD analysis of genomic DNA extracted from any tissue of in vitro grown potato plants using 14 selected decamer primers could be used to develop RAPD fingerprints for identification of Indian potato cultivars.     

A rapid and efficient method for the extraction of total DNA from mature leaves of the data palm (Phoenix dactylifera L.)

A method is presented for the rapid isolation of high-molecular-weight DNA from mature leaves of date palm (Phoenix dactylifera L.), using a CTAB-based buffer. The method yields up to 800 μg of DNA from 1 g of leaf tissues. The procedure was also suitable for DNA extraction from callus or buds from tissue culture. The DNA obtained through this method was a good substrate for at least seventeen restriction endonucleases. This method was also used to extract DNA from mature leaves of coconut and may be applicable to other species of palms.     

Nickel localization on tissues of hyperaccumulator species of Phyllanthus L. (euphorbiaceae) from Ultramafic Areas of Cuba

Two species of perennial Phyllanthus (Euphorbiaceae) (Phyllanthus orbicularis and Phyllanthus discolor, both endemic to ultramafic areas of Cuba, and their natural hybrid, Phyllanthus xpallidus) were selected for metal localization microanalysis. Different plant tissues were analyzed by X-ray fluorescence, inductively coupled plasma—atomic emission spectroscopy, and scanning electron microscopy coupled with an energy-dispersive X-ray probe. All of the studied taxa are nickel (Ni) hyperaccumulators and significant concentrations of this element were found in different leaf and stem tissues. The highest Ni content was found in the laticifer tubes, whereas leaf epidermis Ni content resulted to be much more relevant in terms of total metal storage. Calcium and magnesium were found more evenly distributed in leaf and stem tissues.     

Hydrolysis products of glucosinolates in Brassica napus tissues as inhibitors of seed germination

Enzymatic hydrolysis of glucosinolates, a class of compounds found in Brassica species, results in a number of products with potential to inhibit seed germination. To investigate the impact of both volatile and water-soluble allelochemicals, germination bioassays using Lactuca sativa seeds were conducted with root and combined leaf and stem tissues of Brassica napus. Tissues in which glucosinolates were hydrolyzed to remove volatile glucosinolate degradation products were compared with intact tissues and water controls. Only tissues containing glucosinolates produced volatiles that inhibited germination. Volatiles were trapped and identified using GC-MS. Volatiles produced in greater quanitity from intact tissues than from tissues without glucosinolates were almost exclusively glucosinolate hydrolysis products. Water-soluble components also inhibited germination. Chemical analysis of extracts confirmed the presence of glucosinolate hydrolysis products, but indicated the involvement of additional allelochemicals, especially in leaf and stem tissues. Results support the proposal that glucosinolate-containing plant tissues may contribute to reductions in synthetic pesticide use if weed seeds are targeted.Abbreviations ITC isothiocyanates - CN organic cyanides - OZT oxazolidinethione - iRoot intact root tissue - iL&S intact leaf and stem tissue - hRoot hydrolyzed root tissue - hL&S hydrolyzed leaf and stem tissue     

cDNA cloning and expression analysis of a mannose-binding lectin from <Emphasis Type="Italic">Pinellia pedatisecta</Emphasis>

Pinellia pedatisecta agglutinin (PPA) is a very basic protein that accumulates in the tuber of P. pedatisecta. PPA is a hetero-tetramer protein of 40 kDa, composed of two polypeptide chains A (about 12 kDa) and two polypeptides chains B (about 12 kDa). The full-length cDNA of PPA was cloned from P. pedatisecta using SMART RACE-PCR technology; it was 1146 bp and contained a 771 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acid residues with a 24 amino acid signal peptide. The PPA precursor contained 3 mannose-binding sites (QXDXNXVXY) and two conserved domains of 43% identity, PPA-DOM₁ (polypeptides A) and PPA-DOM₂ (polypeptides B). PPA shared varying identities, ranging from 40% to 85%, with mannose-binding lectins from other species of plant families such as Araceae, Alliaceae, Iridaceae, Liliaceae, Amaryllidaceae and Bromeliaceae. Southern blot analysis indicated that ppa belonged to a multi-copy gene family. Expression pattern analysis revealed that ppa expressed in most tested tissues, with high expression being found in spadix, spathe and tuber. Cloning of the ppa gene not only provides a basis for further investigation of its structure, expression and regulatory mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.     

Cell differentiation in a diploid state inLilium longiflorum tissues

Summary DNA content by cytophotometric analysis has been investigated in mature tissues ofLilium longiflorum plants.Cytophotometric measurements have revealed that many differentiated tissues of stem, root, leaf, scales and flower parts are in the diploid conditions.     

Nocturnal stachyose metabolism in leaf tissues of Xerosicyos danguyi H. Humb.

Leaf discs obtained from mature leaves of Xerosicyos danguyi were found to contain appreciable levels of stachyose throughout an 8-h nocturnal period during which this plant performs Crassulacean acid metabolism (CAM). In contrast, in mesophyll tissues obtained from paradermal sections of these same leaf discs and which were devoid of vascular tissues, stachyose pools were rapidly depleted during the nocturnal phase. The pattern of this depletion followed closely the depletion pattern observed for starch, indicating that mesophyll stachyose was possibly involved in nocturnal CAM processes and was not necessarily being used for export. Pulse-labelling of intact X. danguyi leaves prior to excision of leaf discs and mesophyll samples also indicated that, while labelled stachyose had turned-over completely in the mesophyll tissues by the end of the nocturnal period, substantial levels of labelled stachyose were always recovered from the leaf discs from which these mesophyll samples were derived. The data indicate the existence of two separate pools of stachyose in the X. danguyi leaf, one a mesophyll pool which turns over rapidly at night and which may be involved to a small extent in nocturnal CAM processes, and the other a pool associated with and possibly synthesized by the vascular tissues and which presumably represents the phloem-transport pool.Abbreviation CAM Crassulacean acid metabolism This work was supported by National Science Foundation Grant DCB 8901785.     

龙舌草果实和种子及其种苗发育形态学研究

采用形态观察和GMA切片相结合的方法,对龙舌草果实、种子及种苗发育的形态结构进行解剖学观察研究,探讨龙舌草个体发育中表现出的与水生环境相适应的结构特征.结果显示:(1)龙舌草由佛焰苞包被的果实通常由6心皮构成,心皮边缘不完全愈合,层片状胎座,水面形成的幼嫩果实被果柄拉入水中发育成熟;种子具毛,萌发时苗端先于根端发育,在胚根分化之前先出现单细胞的下胚轴毛,随后胚根发育为初生根,并在真叶的节处发育出不定根;果皮和叶肉内的维管束仅由少数细胞构成,且细胞分化不明显,有发达的通气道;叶表皮无角质层,细胞外凸,叶肉细胞数量少;表明龙舌草的结构特征与水生环境相适应.(2)研究还发现,龙舌草的种子无胚乳,下胚轴贮存种苗发育需要的大量淀粉粒,以及初生根和不定根均有根毛的结构,且与泽泻科的慈菇和泽泻的结构一致,证明水鳖科与泽泻科具有较近的亲缘关系.     

Transgenic rice plants with a synthetic cry1Ab gene from Bacillus thuringiensis were highly resistant to eight lepidopteran rice pest species

To fully explore the resistance potential of transgenic rice produced by Agrobacterium-mediated transformation, an elite line KMD1 was assessed for its resistance to eight lepidopteran rice pest species. KMD1 contained a synthetic cry1Ab gene from Bacillus thuringiensis under the control of a maize ubiquitin promoter. It was derived from a commercial japonica Chinese rice variety Xiushui 11, and bred true for both agronomic traits and a cry1Ab gene when the bioassays were done in 1998 in the R₅ generation. The eight lepidopteran pest species were: four Pyralidae species: Chilo suppressalis (striped stem borer, SSB), Scirpophaga incertulas (yellow stem borer, YSB), Cnaphalocrocis medinalis (leaf folder), Herpitogramma licarisalis; two Noctuidae: Sesamia inferens (pink stem borer, PSB) and Naranga anescens; one Stayridae: Mycalesis gotama; and one Hesperiidae, Parnara guttata. In laboratory bioassays, 100% mortality was observed in all insect species when their newly hatched or third-instar larvae were fed KMD1 leaf tissues, whereas only 9.65% of the neonates and none of the third-instar larvae died when fed the leaf tissues of non-transgenic control. Moreover, the leaf area of control tissues consumed in four days by stem borers was 20 to 40 times higher than that of KMD1 tissues, and the area of control tissues eaten by leaf-feeding species was 120 to 180 times greater than that of the transgenic tissues. Under natural infestation, no KMD1 plant was visibly damaged by the SSB, YSB and leaf folder in field evaluation. On the other hand, 80, 9.3 and 88.7% of control plants were injured by SSB, YSB, and leaf folder, respectively. These data disclosed that the transgenic line was highly resistant to a broad spectrum of lepidopteran insect species and could be useful in insect resistance breeding programs.     

Variation in potato microplant morphology in vitro and DNA methylation

Heterotrophic and autotrophic culture in agar and in polyurethane foam, the latter used as an alternative tissue support to agar, resulted in potato microplants with different in vitro morphologies. The microplants were visually characterised in terms of their relative developmental maturity, by comparing the respective leaf shapes in vitro with ontogenetic differences in leaf shape in glasshouse-grown potato plants. Cytosine methylation in the DNA of microplants of the different morphologies was determined using a method based on the AFLP technique but employing methylation-sensitive restriction enzymes (MSAP analysis) to test the hypothesis that DNA methylation could be used to characterise differences in microplant development in vitro. In three of the four treatments there was a good correlation between the visual assessment of relative morphological maturity and DNA base methylation levels. In these microplants there was increased DNA methylation in the leaves with mature leaf morphology represented by a decreased number of restriction fragments. The fourth in vitro morphology had the most juvenile leaf shape but did not have the predicted level of DNA methylation, having a relatively low number of restriction fragments. Subtraction analysis was used to discriminate the fragments that were unique to the juvenile and mature in vivo leaf morphologies. Comparison of the fragment patterns from the microplants with the latter reference profiles, confirmed the relationship with the total DNA methylation as detected by MSAP analysis, that is, the number of common fragments with the juvenile or mature in vivo leaf profiles, respectively. However, none of the fragment profiles, while sharing some common bands at random, was identical to any other; or to that of either the juvenile or mature in vivo leaf. The anomalous relationship of the microplants with most juvenile leaf shape and highest DNA methylation was confirmed. The measurement of DNA methylation in in vitro plants is discussed in the context of the development of a method to assess the quality of microplants produced by different in vitro protocols.     

A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris

A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)₄ repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.     

Alternative approach for consistent yields of total genomic DNA from cotton (Gossypium hirsutum L.)

A persistent limitation to molecular biological research on cotton (Gossypium spp.) has been the difficulty in isolation of total genomic DNA from the plant tissue. This report describes a reliable strategy for isolation of genomic DNA from cotton. The mini-preparation procedure involves use of lyophilized, etiolated cotyledons and an anion exchange column kit. The isolated DNA had a molecular weight in excess of 50 kb with minimal degradation or shearing. Routine yields ranged from 5 to 7 μg DNA per etiolated cotyledon pair (corresponding to 100 ng/mg dry weight), in contrast to little or no DNA from equivalent amounts of either green cotyledons or mature leaf tissue. The decreased yields from the latter tissues appeared to be correlated with increased afmounts of flavonoid. The DNA was amenable to routine molecular applications as demonstrated by: digestibility with a number of restriction enzymes (Eco RI,HindIII,Sau 3A), and hybridization of a tomato genomic clone containing the gene for S-adenosylmethionine synthetase to a 13.3-kbEco RI fragment of cotton. Using DNA from an isoline immune to root-knot nematodes, we observed no impediment to genomic cloning.     

Faithful inheritance of cytosine methylation patterns in repeated sequences of the allotetraploid tobacco correlates with the expression of DNA methyltransferase gene families from both parental genomes

The widespread occurrence of epigenetic alterations in allopolyploid species deserves scrutiny that DNA methylation systems may be perturbed by interspecies hybridization and polyploidization. Here we studied the genes involved in DNA methylation in Nicotiana tabacum (tobacco) allotetraploid containing S and T genomes inherited from Nicotiana sylvestris and Nicotiana tomentosiformis progenitors. To determine the inheritance of DNA methyltransferase genes and their expression patterns we examined three major DNA methyltransferase families (MET1, CMT3 and DRM) from tobacco and the progenitor species. Using Southern blot hybridization and PCR-based methods (genomic CAPS), we found that the parental loci of these gene families are retained in tobacco. Homoeologous expression was found in all tissues examined (leaf, root, flower) suggesting that DNA methyltransferase genes were probably not themselves targets of uniparental epigenetic silencing for over thousands of generations of allotetraploid evolution. The level of CG and CHG methylation of selected high-copy repeated sequences was similar and high in tobacco and its diploid progenitors. We speculate that natural selection might favor additive expression of parental DNA methyltransferase genes maintaining high levels of DNA methylation in tobacco, which has a repeat-rich heterochromatic genome. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AM946602–AM946620 and FM872474–FM872476.     

Comparative analysis of telomeric restriction fragment lengths in different tissues of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> trees of different age

Ginkgo trees of four different ages were selected as experimental material. Telomeric restriction fragment (TRF) lengths, as an indicator of telomere length, were determined for different tissues by Southern hybridization analysis. Statistical analysis was performed to compare two aspects of TRF length. By determining TRF lengths for different tissues for each age, a latent tendency was found. TRF length varied from short to long in these tissues in the order microspore < embryonal callus < leaf < branchlet. TRF lengths for leaf tissue and branchlet tissue were dissimilar for female and male mature trees, although this difference between TRF lengths for the two sexes was not statistically significant. Evaluation of TRF lengths for each tissue for trees of all four ages revealed TRF lengths increased with age to some extent. Different rates of change were found for leaf tissue and for branchlet tissue, although tendencies to increase were not linear for either. Finally, a simple mathematical model was formulated to describe the relationship between telomere length and age for Ginkgo biloba L.     

Integrated small RNA profiling and degradome analysis of Anthurium andraeanum cultivars with different-colored spathes

MicroRNAs (miRNAs) are known to play vital roles in coloration of leaves, flowers, and fruits in plants. However, their functions in spathe coloration are poorly known. Anthurium andraeanum is a popular ornamental plant with various spathe colors. In this study, small RNA and degradome libraries from three A. andraeanum cultivars with different-colored spathes were constructed and sequenced. Illumina sequencing resulted in 94 conserved miRNAs, and 34 novel miRNAs in total were then identified based on precursor sequences and hairpin structures. Differential expression analysis showed that 52, 51, and 49 miRNAs were differentially expressed in comparisons of orange- versus white-colored spathe, purple- versus white-colored spathe, and purple- versus orange-colored spathe, respectively. The expression patterns of miRNAs and their corresponding targets involved in spathe coloration were further analyzed, and displayed that miR156b and miR529 were highly abundant in the spathes with higher anthocyanin content. These two miRNAs co-targeted a gene encoding SPL17, which may function as a negative regulator in anthocyanin accumulation. In addition, miR408 was also abundantly expressed in purple- and orange-colored spathes, and its typical targets were also identified. This comprehensive integrated analysis provides insight into the miRNA-mediated genetic regulation in spathe coloration of A. andraeanum.

     

Agrobacterium tumefaciens-mediated expression ofgusA in maize tissues

To develop a system forAgrobacterium-mediated transformation of maize (Zea mays L.), we have investigated histochemically the transient expression of -glucuronidase (GUS) activity in maize seedling tissue segments using binary vectors that allow minimal (pKIWI105 and pCNL1) or undetectable (p35S-GUS-INT and pCNL56) levels of GUS activity inA. tumefaciens. Tissue segments from three- to five-day-old sterile seedlings of maize genotype A188 were inoculated withA. tumefaciens. Four days after inoculation, transient expression of GUS activity was found in mesocotyl segments originating from the intercalary meristem region. This GUS activity was specific to the vascular cylinder and was not found in the internal cortical or epidermal layers, nor was it found in mature mesocotyl tissue (segments 5 mm below the coleoptilar node). Transient GUS activity was also detected in leaf and coleoptile tissues of shoot segments, but not in the shoot apexper se or in leaves younger than the first leaf. Maize tissues inoculated withA. tumefaciens strains that harbourgusA-containing binary vectors but no Ti-plasmid did not show GUS activity, supporting evidence from previous work thatvir gene activity was essential for the observed GUS activity.A. tumefaciens strains containing different types of Ti-plasmids were also tested. A strain harbouring an agropine-type Ti-plasmid was the most effective for expressing GUS activity in mesocotyl segments, whereas a strain harboring a nopaline-type Ti-plasmid was most effective for expression of GUS activity in the apical meristem-containing segment. These results indicate that different interactions occurred between the differentA. tumefaciens strains and the susceptible plant tissues. Maize genotype specificity for GUS activity in mesocotyl tissues was observed; variations in the cocultivation medium had a profound effect on the frequency of expression of GUS activity.     

DNA probes for the Y-chromosome ofSilene latifolia,a dioecious angiosperm

Summary In order to obtain markers for the Y chromosome ofSilene latifolia, we pooled equal weights of leaf tissue from 18 female siblings into one sample and repeated the process with 18 male siblings. Pooling was intended to provide a common genetic background for each sample, leaving the absence or presence of the Y chromosome as the primary difference between the two samples. DNA was extracted from each sample and subjected to polymerase chain reaction (PCR) amplification with arbitrary 10 bp primers. Four of 60 primers used gave an amplification with the male DNA not found among those from the female DNA. Each of these was subsequently shown to provide a reliable marker for the Y chromosome.