We isolated PaAP3, a homolog of the class B MADS-box transcription factor gene APETALA3 (AP3), from the monoecious plant London plane tree (Platanus acerifolia Willd.). PaAP3 encodes a protein that shares good levels of identity with class B genes from Arabidopsis thaliana (35 and 51 % identity with PISTILLATA (PI) and AP3, respectively), and also with class B genes of other woody species (59 % identity with PTD from Populus trichocarpa and 66 % with TraAP3 from Trochodendron aralioides). Reverse transcription polymerase chain reaction showed that PaAP3 was expressed in both the female and male flowers of P. acerifolia, but almost no signal was detected in the vegetative tissues or mature embryos. The PaAP3 expression in male flowers showed a relationship with developmental stage. There was a small transient increase during differentiation of the flower primordia in June, but maximal levels occurred during December when flower development appeared arrested. Increased PaAP3 expression was also detected in March of the following year, corresponding to meiotic divisions of the microspore mother cells, but this was lost by April when the pollen was mature.

Iron is one of the essential micronutrients required by all living organisms. In this study, we isolated a gene encoding putative citrate synthase (CS) from Malus xiaojinensis, designated as MxCS1. The MxCS1 gene encodes a protein of 473 amino acid residues with a predicted molecular mass of 52.5 kDa and a theoretical isoelectric point of 8.67. The expression of MxCS1 was enriched in the leaf rather than in phloem and root, however, its expression was hardly detected in the xylem. The gene expression was strongly induced by Fe stress treatment in the M. xiaojinensis seedlings. Over-expression of MxCS1 improved Fe deficiency tolerance in transgenic Arabidopsis. We argued that MxCS1 is a new member of the CS genes, and it may function as a regulator in response to iron stress in plants.

Plant C2 domain proteins play important roles in diverse cellular processes including growth, development, and membrane targeting, as well as in abiotic and biotic stress adaptations by sensing intracellular Ca²⁺ signals. In this study, we isolated a novel C2 domain protein gene, TaERG3, from wheat infected by Puccinia striiformis f. sp. tritici. TaERG3 was predicted to encode a 144 amino acid protein with molecular mass of 15.68 kD and isoelectric point of 3.93. Analysis of the deduced amino acid sequence of TaERG3 using InterProScan revealed the presence of an N-terminal calciumdependent phospholipid-binding module (C2 domain, 5 to 103). Transient expression analysis showed that the TaERG3 protein was predominately and uniformly localized in the plasmalemma and nucleus of onion epidermal cells. Quantitative real-time PCR analyses indicated that TaERG3 transcript was differentially induced in both incompatible and compatible interactions, as well as by applied abscisic acid (ABA) and CaCl₂. However, the significant transcript changes induced by methyl jasmonate, ethylene, and salicylic acid treatments were not as dramatic as those induced by ABA. TaERG3 was also up-regulated by environmental stimuli including low temperature and high salinity. These results imply that TaERG3 might be involved in wheat defence responses against stripe rust and abiotic stresses in an ABA-dependent signalling pathway.

The ABC1 protein family (ABC1P), a new family of putative kinases, widely existed in procaryote and eucaryote. A comprehensive genome-wide analysis was carried out in this study to find all ABC1Ps in rice (Oryza sativa subsp. japonica). We identified 15 ABC1P genes in rice. All the ABC1Ps contained an ABC1 domain of about 120 conserved amino acid residues and conserved kinase motifs-VAIK (VAVK, VAMK) and DFG (DEG). The phylogenetic analysis showed that all the ABC1Ps were grouped into three subgroups, and formed a total of 12 sister pairs. Conserved motifs analysed by MEME program indicated that almost all ABC1Ps contains motifs 1, 3, 7, 8 and 9. Predictably, the ABC1Ps were localized in mitochondria or chloroplasts, which implied that the ABC1Ps might be involved in energy metabolism in plants. RT-PCR assays demonstrated that all 15 ABC1P genes were active, and some of them were affected by abiotic stresses (NaCl, high temperature, methyl viologen, abscisic acid and cadmium).

In this study, bulked segregant analysis (BSA) was used on Larix leptolepis × Larix olgensis hybrids to identify a random amplified polymorphic DNA (RAPD) marker associated with high rooting ability in larch. Two DNA bulks: H (high rooting ability) bulk and L (low rooting ability) bulk were constructed according to the rooting percentages of the stock plants. Among the 328 primers, only S356 could amplify a specific band, named S356₄₄₅, which only existed in the H bulk and was further confirmed following selective genotyping of individual hybrids. Grounded on the border sequences, S356₄₄₅ was converted to a sequence characterized amplified region (SCAR) marker, HRL445, which can be useful in marker-assisted selection (MAS) to screen for larch with high rooting ability. All the results strongly indicated that S356₄₄₅ and HRL445 were closely associated with high rooting ability in larch.

In order to produce human lactoferrin (Lf) in alfalfa (Medicago sativa L.), a construct containing human Lf cDNA under the control of cauliflower mosaic virus 35S promoter was engineered. As selectable marker bar gene, whose expression in plant cells confers tolerance to L-phosphinothricin (ppt) was used. Plants from a highly embryogenic alfalfa clone from the Bulgarian cultivar Obnova 10 were transformed using Agrobacterium tumefaciens mediated leaf disc method. Transgenic alfalfa plants were established from ppt-resistant calli via indirect somatic embryogenesis. The presence of human Lf cDNA in the genome of the selected regenerants was confirmed by polymerase chain reaction (PCR). Reverse transcriptase (RT)-PCR and Western blot showed expression of human Lf in leaf tissue. Studies on antibacterial effect of the recombinant glycoprotein were conducted and resistance of the transgenic alfalfa plants to two phytopathogens, Pseudomonas syringae pv. syringae and Clavibacter michiganensis, was demonstrated. The obtained results suggest that the expression of human Lf in alfalfa could be beneficial not only for producing recombinant protein for clinical application but also for crop quality improvement.

Acyl carrier protein (ACP), as an essential protein cofactor, plays an important role in de novo synthesis of fatty acids in plastids. In this study, the expression profile of peanut (Arachis hypogaea) AhACP1-1 and AhACP1-2 was analyzed in different tissues. The expression level of AhACP1-1 was highest in the seed, whereas expression was barely detected in the shoot, and AhACP1-2 was expressed in every tissue analyzed with the highest expression level detected in the leaf and seed. Overexpression (OE) and antisense-inhibition (AT) of AhACP1 in transgenic tobacco modified the transcript level of endogenous NtACPs, and the content of total lipids and composition of fatty acid in leaves were altered compared with the wild-type control. Transgenic OE-AhACP1 or AT-AhACP1 tobacco exhibited a significant increase or decrease in polyunsaturated C18:2 and C18:3 fatty acid content, and were more tolerant or sensitive to cold stress, respectively. It is suggested that AhACP1 bound with C18:1 might be the specific substrate of oleoyl-ACP thioesterase or glycerol-3-phosphate acyltransferase, and participates in membrane lipid synthesis.

A segregating population from the cross between drought sensitive (Variant-2) and drought tolerant (Cham-6) genotypes was made to identify molecular markers linked to wheat (Triticum aestivum L.) flag leaf senescence under water-stress. From 38 random amplified polymorphic DNA (RAPD) primers, 25 inter-simple sequence repeat (ISSR) primers and 46 simple sequence repeat (SRR) primers, tested for polymorphism among parental genotypes and F2 population. Quantitative trait locus (QTL) for flag leaf senescence was associated with 1 RAPD marker (Pr9), 4 ISSR markers (Pr8, AD5, AD2 and AD3), and 1 SSR marker (Xgwm382) and explained 44, 50, 35, 31, 22 and 73 % phenotypic variation, respectively. The genetic distance between flag leaf senescence gene and Pr9 was 10.0 cM (LOD score 22.9). The markers Pr8, AD5, AD2 and AD3 had genetic distances of 10.5, 14.6, 15.6 and 18.1 cM, respectively (LOD scores 22.6, 17.8, 17.5 and 14.6). The genetic distance between Xgwm382 was 3.9 cM (LOD score 33.8). Therefore, the RAPD, ISSR and SSR markers linked to the QTL for the drought-induced flag leaf senescence can be further used in breeding for drought tolerance in wheat.

Three wheat and two barley populations were studied in order to find loci responsible for dormancy and pre-harvest sprouting. A classical quantitative trait loci analysis was combined with an association mapping approach. Many quantitative trait loci and marker trait associations could be detected on all seven chromosome groups of wheat and on the chromosomes 2H, 3H, 5H, 6H, and 7H of barley. Especially, the known regions on chromosomes 3A and 4A for wheat and 5H for barley were confirmed. Putative functions could be found via a candidate homologues search and via expressed sequence tag annotation. On chromosome 3A, the viviparous1 gene is located which is associated to preharvest sprouting and dormancy. On chromosome 4A, a protein is detected which belongs to the aquaporin family. In barley, an association with the aleurain gene on chromosome 5H was found. The expression of aleurain is regulated by abscisic acid and gibberelic acid. An influence of both hormones on dormancy and pre-harvest sprouting is known. It can be concluded that dormancy and pre-harvest sprouting are very complex traits regulated by multigenes and/or quantitative trait loci.

The protein elicitor cerato-platanin (CP) is known to induce defence-related responses in various plants. Some of these responses occur very quickly. In the present work, transcriptional changes caused by CP in leaves from Platanus × acerifolia (Aiton) Willd. were studied. With a cDNA microarray, 131 differentially regulated transcripts were identified as responsive to CP after 24 h of treatment. Eighty-six of these were cold-or ozone-modulated transcripts, thus revealing a significant overlap between genes responsive to CP and to cold/ozone stress. The transcriptional changes caused by CP were compared with the CP-orthologous protein Pop1 in a time-course analysis performed after 3, 6, 12, and 24 h of treatment by real-time RT-PCR on five defence-related genes. Despite some differences, CP and Pop1 were both able to induce early transcriptional changes (WRKY was overexpressed after only 3 h) confirming that pathogenassociated molecular patterns (PAMPs) act very quickly on gene transcription.

The objective of this study was to examine the role of nitrate reductase, nitric oxide and non-symbiotic hemoglobin in imparting waterlogging tolerance in mung bean genotypes. Experiment was conducted with two cultivated mung bean [Vigna radiata (L.) Wilczek] genotypes T 44 (tolerant) and Pusa Baisakhi (susceptible) and a highly tolerant wild species Vigna luteola (Jacq.) Benth. The content of nitric oxide increased up to 6 d of waterlogging in Vigna luteola and T 44, and up to 4 d of treatment in Pusa Baisakhi. Increase in nitrate reductase (NR) activity was observed only up to 4 d of waterlogging in Vigna luteola and T 44, and up to 2 d of treatment in Pusa Baisakhi, and thereafter the activity decreased in all the genotypes. The increase in NO content and NR activity was greater in Vigna luteola and T 44 than in Pusa Baisakhi. Non-symbiotic hemoglobin (NSHb) and cNR mRNA expressions were observed only in waterlogging treated roots of Vigna luteola and T 44, while very little expression was observed in control plants of Vigna luteola and T 44, and in control and waterlogged plants of Pusa Baisakhi. PCR bands of Hb and cNR were cloned, and nucleotide and deduced amino acid sequences were obtained and conserved regions and domains were identified using database.

Leaf senescence constituted the final stage of leaf development and it is always accompanied by the leaf yellowing. The non-yellowing gene (NYE1), initially identified from Arabidopsis in our laboratory, is a key regulatory gene responsible for chlorophyll degradation during senescence. In this study, an orthologue of AtNYE1 was isolated from the bamboo (Bambusa emeiensis cv. Viridiflavus) and tentatively named BeNYE1. The full length sequence of 1 386 bp contains an open reading frame of 801 bp. The protein encoded by BeNYE1 consists of 266 amino acids. Sequence analysis revealed that BeNYE1 had high similarity with other NYE/SGR proteins from various monocotyledon species. BeNYE1 was strongly induced by natural senescence and dark-induced senescence in bamboo. Driven by a 1.5 kb upstream fragment of AtNYE1, BeNYE1 could rescue the stay-green phenotype of nye1-1. The constitutive over-expression of BeNYE1 could accelerate the chlorophyll degradation. These results indicated that BeNYE1 might play an important role in the regulation of chlorophyll degradation during leaf senescence in bamboo.

Treatment with 0.5 mM salicylic acid (SA) significantly alleviated growth inhibition induced by drought in wheat seedlings, manifested by less decreassed fresh mass, dry mass, plant height, root length, and less increased lipid peroxidation. Under drought stress, SA significantly increased the content of ascorbate (ASA) and glutathione (GSH). We determined the full-length cDNA sequences of genes encoding the glutathione-S-transferase 1 (GST1) and 2 (GST2) and we also measured the transcription of eight genes related to ASA-GSH cycle. The results indicated that exogenous SA significantly enhanced the transcription of GST1, GST2, glutathione reductase (GR), and monodehydroascorbate reductase (MDHAR) genes during almost the entire drought period, but only increased those of dehydroascorbate reductase (DHAR) at 12 h, glutathione peroxidase (GPX1) at 48 h, phospholipid hydroperoxide glutathione peroxidase (GPX2) at 12 and 24 h, and glutathione synthetase (GSHS) at 12, 24, and 48 h. This implies that SA alleviates the detrimental effects of drought stress on wheat seedling growth by influencing the ASA-GSH cycle.

Soil salinity is a major abiotic stress and salt overly sensitive (SOS) pathway plays an important role in imparting tolerance to salinity by reinstating cellular ionic equilibrium. Salt overly sensitive 1 (SOS1) gene of SOS pathway has been implicated in increasing salt tolerance in plants. In this study, a 734 bp fragment of SOS1 promoter (SbUSOS1) was isolated from a halophyte Salicornia brachiata Roxb. In silico analysis of SbUSOS1 predicted several cis-acting regulatory elements such as DOF motif, GT elements, ABRE-like sequence, and root specific motifs. Functional validation of SbUSOS1 into tobacco stems and leaves using the GUS reporter gene showed that this promoter is induced by salt stress (250 mM NaCl) but not by ABA (500 μM) and cold (4 °C) stresses. This study indicated that SbUSOS1 was functional with predicted cis-acting elements that could be responsible for its salt-inducible nature. It can be used for the development of salt stress tolerant transgenic plants.

Glutathione-S-transferases (GSTs) are ubiquitous enzymes that play a key role in stress tolerance and cellular detoxification. The GST gene GsGST14 selected from the gene expression profiles of Glycine soja under alkaline stress was transformed into alfalfa (Medicago sativa L.). Transgenic alfalfa plants showed 1.73-1.99 times higher GST activity than wild-type plants. Transgenic alfalfa grew well in the presence of 100 mM NaHCO₃, while wild-type plants exhibited chlorosis and stunted growth, even death. There were marked changes in malondialdehyde content and relative membrane permeability caused by alkaline stress in non-transgenic lines compared to transgenic lines. The results indicate that the gene GsGST14 could enhance alkaline resistance in transgenic alfalfa.

After identifying regions of cDNA conserved between the symbiotic gene DMI1 of the model species Medicago truncatula and the homologous genomic region of Arabidopsis thaliana, universal primers were designed from 8 of 12 exons to allow the routine amplification of plant homologs. As an example, the complete homologous sequence from the pea (Pisum sativum L.) was amplified and sequenced, although the poorly conserved 5'-end and 5'-flanking region of the gene had to be amplified using a modified TAIL-PCR strategy. The identity of this amplified homolog with the SYM8 gene was independently confirmed by the presence of a single nucleotide change in the coding sequence of the mutant line Risnod27 (sym8) that cosegregated with the asymbiotic phenotype. Five insertions in pea introns responsible for increasing the total length of SYM8 by 1443 bp, compared to the M. truncatula homolog DMI1, belong to known transposon and retrotransposon families of pea and legumes in general. In view of the predicted function of SYM8 as an ion channel, the Risnod27 mutation (His309Tyr) appears to be localized in the selectivity filter domain. This finding confirms the essential role of histidine 309 in the symbiotic function of SYM8 and provides a guide to its ionic specificity. In view of the Risnod27 symbiotic phenotype, we hypothesize that SYM8 does not have identical functions in the transduction of rhizobial and mycorrhizal signals. The variability of the N-proximal region of the known legume homologs of DMI1 suggests an interaction with a variable ligand.

A near isogenic line (NIL) of Brassica oleracea var. botrytis with resistant and susceptible lines C712 and C731, was used in this study. More than 100 differentially expressed cDNA fragments were obtained from black rot resistant cauliflower plants obtained using cDNA-amplified fragment length polymorphism (AFLP) after infection with the pathogen. Thirteen of these fragments were cloned and subjected to reverse Northern blot analysis using both infected and control cDNA pools. Two positive clones, M2 and M6, were isolated. Northern dot blot and Northern blot analyses showed that M2 was constitutively expressed, whereas M6 contained a gene that was differentially expressed during pathogen infection. Moreover, M6 cDNA fragment was also highly expressed 16-24 h after H₂O₂ treatment. Southern blots showed that M6 is a single copy gene in the cauliflower genome, and encodes a protein with 84 % homology to gene on Arabidopsis chromosome 1. The deduced M6 protein has 91 % positive homology with the Arabidopsis 2A6 protein, which regulates ethylene synthesis; 76 % homology with a 1-aminocyclopropane-1-carboxylate oxidase (ACO), the last enzyme in ethylene synthesis; and 70 % homology with an ethylene induced DNA binding factor. These results suggest that M6 gene fragment is a new H₂O₂ downstream defense related gene fragment and can be induced by Xanthomonas campestris pv. campestris and H₂O₂.

This study aims to understand the changes in the transcriptome of durum wheat (Tricitum durum cv. Balcali-85) upon exposure to varying Cd concentrations using mRNA differential display (mRNA DD) technique. Sequence analyses of the two heavily induced genes upon exposure to Cd showed high homology to NADH dehydrogenase subunit 1 (EC907725) and PsaC gene encoding a photosystem 1 (PS 1) 9 kDa subunit protein (EC907731). Additionally, three differentially expressed genes (EC907726, EC907729 and EC907730) were identified. Their sequence analyses revealed no significant homologies to known genes. The expressions of NADH dehydrogenase subunit 1 and PsaC genes were confirmed by Northern blot analysis and quantified by real time PCR. This is the first report for the induction of NADH dehydrogenase subunit 1 gene during Cd stress in wheat.

An unknown brownish protein was purified by ammonium sulfate precipitation and DEAE-cellulose column and hydroxyapatite column chromatographies from pumpkin callus treated with a high concentration of 2,4-D. The apparent molecular mass and isoelectric point of the purified protein were estimated to be 38 kD and 4.6, respectively. The absorption spectra of the protein showed a shoulder at around 280 nm and a sharp peak at 405 nm. In order to determine what the purified protein is, a cDNA library of the callus treated with a high concentration of 2,4-D was immunoscreened with antiserum raised against the purified protein. The obtained positive cDNA clone encoded a thioredoxin h having a predicted molecular mass of 13 123 D and a predicted isoelectric point of 5.24, suggesting that the purified protein might be a trimer that was formed by oxidative polymerization of the thioredoxin h.

DNA cassette consisting of an Arabidopsis dehydration-responsive element binding factor 1 (DREB1B) cDNA, driven by a cauliflower mosaic virus 35S promoter, was introduced into potato plants (Solanum tuberosum L.) through Agrobacterium tumefaciens-mediated gene transfer. The presence and expression of the gene in transgenic plants were confirmed by the PCR and RT-PCR techniques, respectively. Northern hybridization using a DREB1B cDNA probe revealed high levels of DREB1B expression among the most transgenic lines. Overexpression of DREB1B imparted a significant freezing and drought tolerance gain in the transgenic potato lines. In comparison with the wild-type plants, the transgenic potatoes contained higher proline content under drought and freezing conditions, and maintained their relative water content higher under water stress. The enhancement of tolerance in transgenic potato highlights the presence of genes responding to the transcription factor DREB1B in this plant.

To induce virus resistance in tobacco and rice we constructed hairpin RNA expression system harbouring inverted repeat fragments of coat protein cDNA of Potato virus Y (PVY) or Rice stripe virus (RSV). These structures were driven by three promoters [cauliflower mosaic virus 35S (CaMV 35S), polyubiqutin gene of maize (Ubi), and Pharbitis nil leucine zipper gene (PNZIP)] which have different tissue-specific activity. PVY resistance ratios were 65.18, 24.33 and 83.54 % in transgenic tobacco plants harboring p35S-PVY, pUbi-PVY and pPNZIP-PVY. RSV resistance was 16.21, 28.61 and 29.33 % in transgenic rice plants harboring p35S-RSV, pUbi-RSV and pPNZIP-RSV. Northern blotting and GUS assay demonstrated that virus resistance levels were related to promoter activity. Therefore, choice of the more effective and tissue-specific promoter to reinforce transcription of hpRNAs will favour the cultivation of highly virusresistant transgenic plants.

Mantisia spathulata Schult. and M. wengeri Fischer, two critically-endangered, endemic and rare species of the genus Mantisia (Zingiberaceae), have been rediscovered from Lunglei province of Mizoram, India, after two decades. For sustainable conservation and utilization of the Mantisia species, in vitro seed and clonal propagation methods have been developed earlier by our research group and plantlets have been reintroduced to their natural habitat for species recovery. To comprehend the plausible reasons for endemism and endangeredness of both the species at DNA level, they were analyzed to assess natural genetic variation using three different polymerase chain reaction (PCR) based DNA markers viz. random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and directed amplification of minisatellite DNA regions (DAMD), both individually and cumulatively, which are popularly regarded as single primer amplification reaction (SPAR) methods. A total of 107 primers belonging to three SPARs are used which collectively endow low genetic variation (15 and 20 %, respectively) in both M. spathulata and M. wengeri. The use and efficacy of SPAR methods to reveal the natural genetic variation in Mantisia species at intra-specific level has been recorded for the first time. To impede the extinction risk of these two species of genus Mantisia, large scale conservation strategies including in situ and ex situ conservation are recommended.

Germination of Lepidium apetalum Wild. seeds is invariably arrested by cold stress. cDNA-amplified fragment length polymorphism (AFLP) technique was used to isolate genes relevant to chilling stress (4 °C) during seedling emergence. 43 transcript-derived fragments (TDFs) were found to be up-regulated and 17 down-regulated during chilling stress. Eighteen TDF of up-regulated genes were cloned and sequenced. Some of these genes are involved in the stress response, some play important roles in energy and substrate metabolism, and some encode unknown proteins such as TDF119. Two sequences, designated TDF217 and TDF223, may correspond to novel genes. The expression profiles of 6 from 18 TDFs were analyzed by quantitative real-time PCR under chilling and abscisic acid (ABA) stress. It was demonstrated that all 6 genes were significantly induced by chilling and their expression was decreased when the temperature was shifted from 4 to 25 °C. The transcriptional levels of 5 TDFs were strongly enhanced also in response to exogenous ABA. Based on the characteristics of genes isolated from seedlings exposed to cold stress, we conclude that Lepidium adapts to cold stress by regulating many signal transduction pathways, including both ABA-dependent and ABA-independent signaling pathways.

The Potato virus X (PVX)-based vector was used for the construction of N- and C-terminally modified PVX coat protein (XCP) chimeras. N-terminal XCP modifications do not influence the viral life cycle, whereas the simple XCP C-terminal fusion impedes the viral replication. We designed several C-terminally modified XCP chimeras and tested their viabilities in various Nicotiana benthamiana genotypes. Our results showed the negative impact of 3'-terminal modification of XCP on the chimera's life cycle. To ensure chimeric constructs stability, the second copy of the last 60 nucleotides of XCP followed by the 3'-untranslated region (UTR) was added downstream of the recombinant sequence. Simultaneously, the first copy of the last 60 nucleotides of XCP was mutated in order to prevent recombination between the two identical sequences. The movement protein of Tobacco mosaic virus expressed in transgenic N. benthamiana plants positively affected the cell-to-cell spread of C-terminally modified XCP chimeras.

Identification of gene expression patterns in mangroves grown under salinity will help to reveal the molecular mechanisms of salt tolerance. Here, 10 cDNAs of genes were isolated from Kandelia candel and identified by representational difference analysis of cDNA (cDNA RDA) under different NaCl concentrations. Of five genes expressed preferentially under salt condition, two were unknown, three were two kinds of low molecular mass heat-shock proteins (sHSPs) and ADP-ribosylation factor, respectively. The expressions of other five genes were repressed under NaCl stress, two encoded cyclophilins, three were tonoplast intrinsic protein, early light-induced protein and 60S ribosomal protein, respectively.

A Na⁺/H⁺ antiporter catalyzes the transport of Na⁺ and H⁺ across the tonoplast membrane. We isolated a vacuolar Na⁺/H⁺ antiporter cDNA (SsNHX1) clone from a euhalophyte, Suaeda salsa. The nuclear sequence contains 2262 bp with an open reading frame of 1665 bp. The deduced amino acid sequence is similar to that of AtNHX1 and OsNHX1 in rice, with the highest similarities within the predicted transmembrane segments and an amiloride-binding domain. Northern blot analysis shows that the expression of the S. salsa gene was increased by salt stress. The results suggest that the SsNHX1 product is likely a Na⁺/H⁺ antiporter and may play important roles in the salt tolerance of S. salsa.

Randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers were applied to assess the genetic stability of micropropagated olive (Olea europaea L. cv. Maurino) plants regenerated by axillary buds. Initial olive explants, isolated from one donor tree, were multiplied on Murashige and Skoog medium for 12 repeated subcultures. A total of 40 RAPD and 10 ISSR markers resulted in 301 distinct and reproducible band classes showing homogeneous RAPD and ISSR patterns. The amplification products revealed genetic stability among the micropropagated plants and between them and the donor plant. The results demonstrate the genetic stability of nine year old mature micropropagated olive plants cultured in field, and corroborated the fact that axillary multiplication is the safest mode for multiplication of true to type plants.

Although most plastid transformation studies have focused on chloroplast expression, plastid transformation can also be used to express genes in plastids of a wide variety of plant tissues by using appropriate plastid promoters. Based on the sequence of the Gossypium hirsutum chloroplast genome, we developed primers and amplified segments of 20 different plastid genes. The PCR products were labeled and used in filter dot blot hybridization studies to characterize their expression levels and patterns in total RNA isolated from light- and dark-grown cotton tissues at different developmental stages. A subset of 6 genes among these was further characterized by real time PCR. Highest expression levels were observed for rrn16 and psbA. Four genes were expressed in all samples at relatively constant levels: accD, atpA, matK and rrn16. Expression in root tissue was generally low. The results of our study can be used to predict which operons and promoters are most likely to be preferentially expressed in the plastids of tissues of interest at levels that would result in the desired phenotype, facilitating the development of plastid transformation vectors.

Chalcone isomerase (CHI, EC 5.5.1.6) is an entrance enzyme in the flavonoid biosynthesis, which catalyzes the conversion of chalcones to flavanones. In this study, the full-length CHI cDNA from grape vine (Vitis vinifera L.) was cloned, the recombinant protein was purified and the polyclonal antibody was prepared. Using these tools, the expression and tissue localization of CHI in developing grape berry was analyzed by RT-PCR, gel blot hybridization and immunohistochemical techniques. The expression of CHI was dependent on developmental stage, and CHI protein was mainly distributed in vascular bundles throughout all the stages of berry development, which suggested that flavonoids in the berry might have been partially synthesized in situ.

The coat protein (CP) gene of Tobacco streak virus (TSV) from sunflower (Helianthus annuus L.) was amplified, cloned and sequenced. A 421 bp fragment of the TSV coat protein gene was amplified and a gene construct encoding the hairpin RNA (hpRNA) of the TSV-CP sequence was made in the plasmid pHANNIBAL. The construct contains sense and antisense CP sequences flanking a 742 bp spacer sequence (Pdk intron) under the control of the constitutive CaMV35S promoter. A 3.6 kb Not I fragment containing the hpRNA cassette (TSV-CP) was isolated from pHANNIBAL and sub-cloned into the binary vector pART27. This chimeric gene construct was then mobilized into Agrobacterium tumefaciens strain LBA4404 via triparental mating using pRK2013 as a helper. Sunflower (cv. Co 4) and tobacco (cv. Petit Havana) plants were transformed with A. tumefaciens strain LBA4404 harbouring the hpRNA cassette and in vitro selection was performed with kanamycin. The integration of the transgene into the genome of the transgenic lines was confirmed by PCR analysis. Infectivity assays with TSV by mechanical sap inoculation demonstrated that both the sunflower and tobacco transgenic lines exhibited resistance to TSV infection and accumulated lower levels of TSV compared with non-transformed controls.