A full-length cDNA encoding phosphoenolpyruvate carboxylase (PEPC) was isolated from tropical epiphytic CAM orchid Mokara Yellow. The cDNA designated as Mpepc1 is 3 450 bp in length with an open reading frame of 2 862 bp encoding 954 amino acids. The deduced amino acid sequence of Mpepc1 shows 83 % identity with pepc2 of sorghum, 82 % with pepc1 and pepc2 of maize and 81 % with pepc of Arabidopsis thaliana. RT-PCR analysis showed that Mpepc1 was expressed in mature leaves, immature leaves, and aerial roots of M. Yellow. No expression was detected in the flower.

Histone deacetylases (HDACs) catalyze histone deacetylation and play an important role in suppression of gene transcription in multiple cellular processes. HDACs are widely distributed in eukaryotes, however, detailed characterrization of HDACs in woody plants is not available. In this study, the sequences of reduced potassium dependency 3/histone deacetylase 1 (RPD3/HDA1) family proteins in black cottonwood (Populus trichocarpa Torr. & A. Gray) were characterized and their expression patterns in response to cold and salt stresses were determined. The RPD3/HDA1 proteins had conserved HDAC domains and can be divided into three classes based on sequence similarity and phylogenetic analysis. The transcripts of the HDAC genes were detected in different amounts in leaves, stems, and roots. The expressions of HDAC genes HDA902, HDA903, HDA904, HDA909, and HDA912 were up-regulated in a cold stress. Interestingly, in a salt stress, most of the HDAC genes were down-regulated. These results indicate that the poplar HDAC genes were regulated by the cold and salt stresses, and the members of the RPD3/HDA1 family play a role in stress responses.

Suppressive subtraction hybridization (SSH) was used to identify differentially expressed genes caused by a chlorophyll-reduced mutation in B. napus. The cDNA fragments, derived from SSH positive subtractive library (tester: normal wild type, driver: mutant) were cloned into pMD18-T vector. Two hundred SSH cDNA clones were screened by dot blot array, and 151 clones were identified as differentially expressed cDNA fragments in Cr3529 line. Thirty-six positive clones which showed marked expression differences were selected and sequenced. After redundant cDNAs were removed, 33 differentially expressed unique cDNA section clones were obtained. Among the 33 clones, two clones possess different parts of the cDNA sequence of the same gene coding geranylgeranyl reductase, four clones belong to unknown proteins, and the rest share homology to genes of diverse class. Sequence analysis showed that at least 12 genes were discovered to be related to the photosynthesis, seven of them coded the proteins which belong to the subunit of photosystem 2. RNA gel blot analysis showed that compared with 3529, the gene expression of the chlorophyll a/b-binding protein Lhcb2 in photosystem 2 declined markedly in the cotyledons and seedling leaves of Cr3529, indicating that the reduced light-harvesting complex 2 accumulation in thylakoid membrane of Cr3529 was due to the decrease of the related gene mRNA level for translation.

This study aimed to investigate sucrose-metabolizing enzymes and their genes in fruits of two longan (Dimocarpus longan Lour) cultivars Cihezhong (CHZ) and Lidongben (LDB). Content of sucrose, glucose, and fructose were measured by high-performance liquid chromatography. The genes of sucrose-metabolizing enzymes were cloned by combining reverse transcription polymerase chain reaction and rapid amplification of cDNA ends, and enzyme activities were analyzed at various points in the fruiting cycle. The total soluble solid (TSS) content of longan arils rose and was positively correlated with sucrose content during maturation and then declined as the fruit senesced. Cihezhong showed a more rapid decrease in sucrose content than LDB. The activities of both sucrose phosphate synthase (SPS) and sucrose synthase (SS) were lower in CHZ, whereas the activities of soluble acid invertase (SAI) and neutral invertase (NI) were higher. The full-length cDNA of the genes of the sucrose-metabolizing enzymes were cloned successfully. The patterns of changes of sucrose synthase-2 (DlSS-2), sucrose synthase-3 (DlSS-3), and neutral invertase-3 (DlNI-3) gene expressions corresponded to those of SS and NI activities. The rate of sucrose decline in the longan fruits was related to sugar receding, sucrose metabolizing enzyme activities, and corresponding gene expressions.

C-repeat/dehydration-responsive element binding factor (CBF) plays important roles in cold response network in plants. Here, one member of CBF coding gene family in trifoliate orange (Poncirus trifoliata), designated as PtCBF, was isolated. Semi-quantitative reverse transcription-polymerase chain reactions showed up-regulation of PtCBF not only under low temperature but also induced by abscisic acid. Additionally, the CBF genomic fragments in four citrus species including trifoliate orange, sweet orange (Citrus sinensis), pummel (Citrus grandis) and rough lemon (Citrus jambhiri) were isolated with complete open reading frames. According to the results of alignment analysis between full length cDNA and genomic DNA sequences in trifoliate orange, there were no introns in PtCBF. Moreover, the results of multiple sequence alignment analysis and phylogenetic analysis on putative protein sequences suggested that the AP2 DNA binding domains and CBF signature sequences were highly conserved in four citrus CBF proteins. Finally, the CBF promoters in above citrus species were isolated, which provides some information concerning promoter function.

Histone H3 lysine 4 methylations catalyzed by histone lysine methyltransferases (HKMTs), like the Arabidopsis thaliana ATX1 and ATX2, are important epigenetic modifications related to chromatin decondensation and gene activation. In order to study this epigenetic mechanism in monocot cereal plants, we performed homology searches of ATX1 and ATX2 against the Brachypodium distachyon L. Beauv and rice (Oryza sativa L. spp. japonica) genomes, discovering single homologues for each cereal crop representing both Arabidopsis sequences. Using this information, we employed the rolling circle amplification - rapid amplification of cDNA ends (RCA-RACE) method to isolate, clone and characterize HvTX1 from RNA extracted from barley (Hordeum vulgare L.) tissues and studied its expression during seed development and under drought stress. The cloned cDNA sequence contained a 3 093 bp ORF homologous to ATX1 and ATX2. Characterization of the translated HvTX1 transcript sequence revealed the multi-domain nature of the putative protein, including all conserved regions characteristic for ATX1 and ATX2. By comparative genomic analysis and homology searches in EST databases we located, with high probability, the gene coding for HvTX1 on the barley chromosome 5H. Constant elevation of HvTX1 expression was observed during seed development. Expression of HvTX1 after drought stress was analyzed by quantitative real-time polymerase chain reaction (qPCR) in two different barley cultivars with varying drought stress tolerance, revealing HvTX1 drought-induction in a tolerance-specific manner.

The gene of Zinnia elegans L. coding for S-like extracellular ribonuclease (ZRNase II) was used to produce transgenic tobacco plants with an increased ribonuclease activity. The protein-coding part of ZRNase II included the signal peptide sequence so the transgenic protein was located extracellularly. The cDNA of ZRNase II was cloned under the control of 2'-promoter of the mannopine synthase (MAS 2') gene from Ti-plasmid of Agrobacterium tumefaciens. It was shown that the resultant transgenic plants had an increased ribonuclease activity of the crude extracts and the induction of MAS 2' promoter by wounding additionally increased the activity. The plants of two transforming lines characterized by different ribonuclease activities were used to analyze the transgene influence on plant resistance to tobacco mosaic virus. The plants demonstrated either absence of disease symptoms or a significant delay in their appearance, depending on the virus content in the inoculum and ribonuclease activity.

The present study describes the assessment of genetic diversity and relationships among 79 Job's tears (Coix lacrymajobi L.) accessions collected from China and Korea using 17 microsatellite markers. A total of 57 alleles were detected with an average of 3.4 alleles per locus. A high frequency of rare alleles (36.3 %) was observed within the collection. Values for observed (H_O), expected heterozygosity (H_E) and Shannon's information index (I) within the analysis ranged from 0.00 (GBssrJT183) to 0.81 (GBssrJT130), from 0.01 (GBssrJT170) to 0.65 (GBssrJT130) and from 0.034 (GBssrJt170) to 1.13 (GBssrJT130), respectively. The locus GBJT130 was the most informative marker with the highest values for observed and effective alleles as well as for H_O, H_E and I. Based on the UPGMA algorithm, the majority of the Chinese accessions grouped in one cluster, whereas all the Korean accessions grouped together in a separate cluster, indicating that Chinese accessions are genetically quite distinct from Korean accessions. No relation between genetic relatedness among Job's tears accessions and their place of collection was observed. Chinese accessions exhibited greater within population polymorphism (P = 95 %, H_E = 0.30, I = 0.52) than the accessions from Korea (P = 68 %, H_E = 0.13, I =0.24), indicating their potentiality as a reservoir of novel alleles for crop improvement. However, in general the low diversity within each population indicates a narrow genetic base within our collection.

Analysis of structural changes of octoploid triticale genomes was conducted in F₂ and F₃ generations. The plants were derived from crosses of five cultivars and breeding lines of hexaploid wheat (Triticum aestivum L.) with one cultivar of rye (Secale cereale L). The study used four marker systems: inter-simple sequence repeat (ISSR), inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP), and a technique named inter-transposon amplified polymorphism (ITAP) developed by the authors. Most frequently, elimination of specific bands was observed, especially of rye bands. Depending on the cross combination, the percentage of eliminated rye bands ranged from 73.6 to 80.6 %. A lower percentage of wheat bands was eliminated, i.e., from 57.6 to 76.48 %, depending on the combination of crosses. The emergence of new types of bands in hybrids absent in the parental forms was the rarest phenomenon (14.5-17.9 %). The results indicate the ongoing process of genome rearrangements at the molecular level in the early generations of plant crosses that also involve repeated nucleotide sequences of DNA.

C2H2-type zinc finger proteins belong to a major family of transcription factors that play a crucial role in plant stress responses. In this study, seven C2H2-type zinc finger genes (EgrZFP1-7) in Eucalyptus grandis were cloned using the RACE PCR method. EgrZFP1-7 proteins included a QALGGH motif, two zinc finger domains, and an EAR motif. They belong to a Q-type C2H2 zinc finger protein family and are classified into the subset C1. EgrZFP4 and EgrZFP6 had a higher transcription in roots than in leaves and stems, whereas the expression of the other genes did not differ in various plant tissues. The expression of EgrZFP genes in seedlings was induced by low temperatures. Time course experiments at temperatures lower than 4 °C revealed oscillating expression of EgrZFP1-6 during the cold treatment. However, EgrZFP7 showed a phasic expression pattern at the same conditions. The expression of EgrZFP1-6 was found to be enhanced by 200 mM NaCl, whereas the expression of EgrZFP7 was inhibited.

Pepper (Capsicum annum L.) fruit colours vary from green, ivory, or yellow at the juvenile stage to red, orange, and yellow at the mature stage. Carotenoid accumulation causes fruit colour formation in ripe Capsicum fruits, and capsanthin is a main carotenoid in ripe red pepper fruits. In this study, pepper cultivars with red, yellow, and orange fruits were employed and four key genes named phytoene synthase (Psy), lycopene-β-cyclase (Lcyb), β-carotene hydroxylase (Crtz), and capsanthin/capsorubin synthase (Ccs) in the capsanthin biosynthetic pathway were analyzed. Results show that a clear expression of the Ccs gene in cv. CK7 (yellow) was detected, but the expressions of the Psy and Lcyb genes were low comparing with the other tested cultivars. In addition, the expression of the Ccs gene was not observed in cv. R37-1 (orange). Furthermore, though the four key genes (Psy, Lcyb, Crtz, and Ccs) were detected in red cultivar R15, the expressions of them were low. Sequence analysis further indicated that the Ccs genes in cv. CK7 and cv. R37-1 had a high sequence identity (97.36 %). Interestingly, amino acid sequences coded by the Ccs genes from cv. CK7, cv. R37-1, and cv. R15 were the same. The above results suggest that the genes Psy, Lcyb, Crtz, and Ccs were required for capsanthin synthesis in pepper, and their varied expression patterns resulted in the variety of pepper fruit colours.

Previously, the ascorbate peroxidase (APX1) activity and gene expression in Chinese cabbage (Brassica campestris, Bc) heat-tolerant cv. ASVEG2 were found to be significantly higher than in heat-sensitive cv. RN720 under a heat stress. Furthermore, BcAPX2 and BcAPX3, isoforms of BcAPX1, were cloned in this study. Our objective was to transfer BcAPX cDNA under the control of the ubiquitin promoter to Arabidopsis via Agrobacterium tumefaciens strain GV3101. We found that BcAPX genes were overexpressed in transgenic Arabidopsis, and the expression of APX, and the APX activity in transgenic lines were higher than in non-transgenic (NT) plants under high temperatures. The chlorophyll content and the germination rate were significantly higher, and the malondialdehyde content was lower in BcAPX1-3, 2-1, and 3-5 lines subjected to the heat-stress treatment than those in the NT plants. Compared to the NT plants, a lower heat-induced H₂O₂ accumulation was detected by diaminobenzidine staining in leaves of the transgenic lines with a high APX activity indicating that the overexpression of BcAPX in Arabidopsis could enhance heat tolerance by eliminating H₂O₂.

Tospoviruses are devastating plant viruses causing severe economic losses in a diverse range of crops worldwide. Here, we describe the development and evaluation of an RNA interference (RNAi) broad-spectrum virus resistance strategy based on a unique and short hairpin-RNA-generating construct (pNhpRNA). This construct was designed from a region of the nucleocapsid gene (N) of Tomato spotted wilt virus (TSWV) that showed a high sequence identity to the corresponding region in the related species Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV). To test the effectiveness of the pNhpRNA construct, we developed a silencing reporter assay based on three fusion proteins in which the complete viral N gene sequence from each of the three tospoviruses was fused in frame to the green fluorescent protein (GFP) sequence. Co-agroinoculation of these constructs with pNhpRNA into leaves of Nicotiana benthamiana resulted in a strong silencing phenotype determined by GFP decay and suppression of the three N genes at the RNA and protein levels. To test the potential of the pNhpRNA construct to generate virus-resistant plants, we infiltrated the whole shoots of N. benthamiana with pNhpRNA. When these infiltrated plants were mechanically inoculated with the mentioned viruses 100, 70, and 60 % resistance phenotypes to TSWV, GRSV, and TCSV, respectively, were observed. The induction of a broad tospovirus resistance with a simple construct and a minimized off-target effect are the main contributions of pNhpRNA.

Pogostemon cablin (Blanco) Benth is a medicinal plant rich in terpenoids and flavonoids. Currently, the molecular mechanism of flavonoid biosynthesis in P. cablin remains unclear. In this study, three copies of the phenylalanine ammonia lyase (PAL) gene designated as PcPAL1 - PcPAL3, one copy of the chalcone synthase (CHS) gene referred to as PcCHS, and four copies of the CHS-like gene referred to as PcCHSL1 - PcCHSL4 were isolated from P. cablin. PcPAL1, PcPAL2, and PcPAL3 comprised 2 136, 2 136, and 2 148 bp full-length open reading frames (ORFs) encoding 711, 711, and 715 amino acids, respectively. PcCHS, PcCHSL1, PcCHSL2, PcCHSL3, and PcCHSL4 contained 1 173, 1 176, 1 179, 1 173, and 1 170 bp ORFs encoding 390, 391, 392, 390, and 389 amino acids, respectively. A phylogenetic analysis indicates that PcPAL2 was closer to PcPAL3 than PcPAL1, and that PcCHS was clustered with other plant CHS genes but independently to the PcCHSLs subgroup. A sequence analysis shows that PcCHSLs diverged in functional residues when compared to plant CHSs including PcCHS, suggesting that PcCHSLs may be divergent in tertiary structures. A quantitative reverse-transcription polymerase chain reaction analysis indicates that the expression profiles of PcPALs, PcCHS, and PcCHSLs in leaves and stems differed among four cultivars. Our results suggest that divergence in sequence and expression profiles of the tested genes may contribute to the diversity in flavonoids among the four cultivars.

Understanding the basis of the genetic variations responsible for the complex traits found in Eucalyptus cladocalyx under arid environmental conditions is crucial for designing genetic architecture studies. Forty-five half-sib families from Australia were used to identify inter-simple sequence repeat (ISSR) markers that are associated with growth (height, diameter at breast height, and stem straightness), flowering traits (flowering intensity, flowering precocity, reproductive capacity, and late flowering) and tree survival under arid conditions in southern Atacama Desert, Chile. Each DNA pellet consisted of a pool of five trees from each family. ISSR markers were associated with all the traits studied and accounted for 9.8 to 23.4 % of the phenotypic variation. Several loci were associated with more than one trait. For example, UBC_{810(450-500 bp)}, ISO_{1(600-610 bp)}, and TGT_{9(780-800 bp)} were associated with three of the traits studied. These identified genomic regions may contribute to the increase of the efficiency of the conventional tree breeding program for E. cladocalyx.

The JNK motif is repeated 4 000 times in an additional heterochromatin band. Flanking sequences of a JNK region, which form extra heterochromatin in 2R rye (Secale vavilovii Grossh.) chromosomes, were studied using a genome walking technique. The results clearly indicate that there were blocks of JNK sequences adjacent to the R173 family of repeated sequences. Moreover, it appears that the R173 are sequences flanking in both directions, i.e., upstream and downstream. Downstream, the R173 is adjacent to the JNKs in an anti-parallel orientation, whereas upstream it is adjacent in a parallel orientation. In order to confirm the presence of the R173 sequence, fluorescence in situ hybridization was carried out. Using both JNK and R173 molecular probes, overlapping hybridization signals in the 2RL pair of chromosomes were observed, indicating an identical location of the two sequence elements.

A segregating F4 population from the cross between drought sensitive (Yecora Rojo) and drought tolerant (Pavon 76) genotypes was made to identify molecular markers linked to a wheat (Triticum aestivum L.) abscisic acid (ABA) content at two water regimes. The parents and 150 F₄ lines were evaluated phenotypically for drought tolerance using two irrigation treatments [0.25 and 0.75 m³(H₂O) m^-2(soil)]. Forty different target region amplification polymorphism (TRAP) primer combinations, 98 different sequence-related amplified polymorphism (SRAP) primer combinations, and 400 simple sequence repeat (SSR) primers were tested for polymorphism among the parental genotypes and the F₄ lines. Seven loci in the F₄ lines treated with the drought stress were identified. Single quantitative trait loci (QTLs) were located on chromosomes 1B, 2A, 3A, 5D, and 7B and each of them explained from 15 to 31 % of phenotypic variance with a LOD value of 7.2 to 15.7. Five QTLs were located on chromosome 4A and six QTLs on chromosome 5A. In control (well-watered) F₄ lines, two QTLs were mapped on chromosome 3B and one QTL on each chromosome 5B and 5D. Statistically the most significant groups of QTLs for the ABA content were identified in the regions of chromosomes 3B, 4A, and 5A mostly near to Barc164, Wmc96, and Trap9 markers. Therefore, these markers linked to QTLs for the drought-induced ABA content can be further used in breeding for drought tolerance in wheat.

Based on information from the Arabidopsis model system, a putative transcriptional activator of cuticle formation (TaSHN1) was selected among the expressed sequence tags in wheat (Triticum aestivum L.). RT-PCR indicated the preferential expression of this gene in the basal, but not in the middle parts of wheat leaves. This leaf region is a likely site of cuticle formation in cereals. TaSHN1 was cloned and expressed in Arabidopsis, resulting in shiny leaf surfaces and the overproliferation of cuticular material as observed by electron microscopy. Unlike the Arabidopsis WAX INDUCER/SHINE1 (WIN/SHN1) gene, TaSHN1 triggered disorganized cuticular ultrastructure in the transgenic leaves, with the continuous layers replaced by large electrodense bodies embedded in amorphous lipid material. Toluidine blue staining and dark-adapted water release indicated increased cuticular permeability in TaSHN1-expressing Arabidopsis leaves. The expression of TaSHN1 resulted in a moderate decrease of the total number of stomata per unit leaf area in comparison with the wild type. Drought tolerance of Arabidopsis was unaffected by the transgene. The data indicate that this putative wheat orthologue of WIN/SHN transcription factors (TaSHN1) elicited both overlapping and new, distinctive phenotypes compared to other WIN/SHN-overexpressing plants. TaSHN1 transgenic Arabidopsis lines should provide a rich source of material for further comparative biochemical, physiological, and genetic studies.

The halophyte Thellungiella salsuginea is a new model plants due to its small genome size, short life cycle, and copious seed production. Although T. salsuginea shares a high sequence identity with its close relative Arabidopsis thaliana, it shows a greater tolerance to salinity, drought, freezing, heat, and cold. To elucidate the mechanism of abiotic stress resistance in T. salsuginea, we characterized its cytosolic Apx1 gene (TsApx1) and established A. thaliana transgenic lines overexpressing TsApx1. Under 300 mM NaCl, the content of H₂O₂, malondialdehyde, and proline were lower and the activities of superoxide dismutase, catalase, glutathione peroxidase, and ascorbate peroxidase were all higher in the transgenic plants overexpressing TsApx1 (35S:TsApx1-GFP) than in the wild-type plants. The atapx1 mutant plants of A. thaliana had a NaCl/mannitol-sensitive phenotype. The ectopic expression of TsApx1 in the atapx1 mutant effectively remedied the phenotype. These results suggest that TsApx1 plays an important role in scavenging reactive oxygen species in the cytoplasm under salinity or drought. Although TsApx1 in T. salsuginea was constantly expressed at a high level, this gene was clearly inducible. In summary, the high constitutive expression and rapid induction of TsApx1 may contribute to the tolerance to abiotic stresses in T. salsuginea.

The δ-aminolevulinic acid synthase (ALA-S) is an enzyme which catalyzes the synthesis of δ-aminolevulinic acid (ALA). The Bradyrhizobium japonicum ALA-S coding sequence lacking plastidal transit sequence was introduced into the rice genome (C line). The transgenic lines, C4 and C5, were compared with the transgenic lines expressing TALA-S gene with plastidal transit sequence (P line) to investigate whether the plastidal sequence affects the targeting capacity of B. japonicum ALA-S gene and the ALA-synthesizing capacity in rice plants. The B. japonicum ALA-S mRNA was expressed efficiently in C lines and the protein was localized in the stroma of chloroplasts regardless of the transit sequence as in P lines. The resulting transgenic plants, C line, had similar levels of ALA-S activity, ALA, protoporphyrin IX and chlorophylls, compared to those of P lines. In response to irradiance of 350 μmol m^-2 s^-1, transgenic lines C4 and C5 displayed the characteristic phenotypes of photodynamic damage, i.e., decreases in photosynthetic parameter F_v/F_m, as in P5 and P14 lines, whereas wild type did not. These results indicate that the lack of the plastidal transit sequence influences neither chloroplast translocation of B. japonicum ALA-S nor ALA-synthesizing capacity in the transgenic rice.

Aluminium toxicity in acid soils is the main limitation to crop production worldwide. In wheat (Triticum aestivum L.), the Al-activated malate transporter (TaALMT1) gene located on chromosome 4DL is associated with malate efflux and Al-tolerance. To introgress Al-tolerance from the breeding line CAR3911 into the high yielding Al-sensitive cultivar Kumpa-INIA, phenotypic and molecular characterizations of gene/QTL underlying Al-tolerance in CAR3911 followed by marker-assisted backcrossing (MAS-BC) were undertaken. Al-tolerant backcross (BC) lines were selected using the functional marker ALMT1-4 designed immediately upstream of the TaALMT1 coding region. Foreground and background selections using ALMT1-4 and microsatellite markers were conducted. Linkage and sequence analyses suggest that the TaALMT1 gene could underly the Al-tolerance in CAR3911, possessing the same promoter type (V) as the Al-tolerant genotypes Carazinho and ET8. The MAS-BC strategy allowed the selection of Al-tolerant lines with the smallest introgressed region (6 cM) on 4D and the highest recurrent parent genome (RPG) (98 %) covering 2 194 cM of the wheat genome. The homozygous BC3F2 line named Kumpa-INIA-TaALMT1 expressed a 3-fold higher Al-tolerance than its isogenic line Kumpa-INIA at 40 μM Al in the hydroponic solution, and similarly to CAR3911 and Carazinho. The MAS-BC strategy was successful for the introgression of the TaALMT1 gene into Kumpa-INIA in only three BC generations, shortening the breeding cycle to 24 months, which promises to increase wheat production and a greater yield stability in the acid soils of Southern Chile.

C-repeat binding factor (CBF), also called the dehydration-responsive element binding factor 1 (DREB1), can be induced by low-temperature (LT), and plays an important role in abiotic stress tolerance in higher plants. In present study, two new homologous genes of CBF from Prunus mume (PmCBFb and PmCBFc) have been identified and characterized. The complete coding sequences of PmCBFb and PmCBFc were 714 and 723 bp, respectively. They encoded putative proteins of 237 and 240 amino acids. Neither of them had introns. Genome PCR sequencing showed that PmCBFb was arranged in tandem with PmCBFa (another CBF/DREB1 homolog in P. mume) within a region of nearly 4 kb. Promoter prediction analyses indicated that multiple types of cis-elements related to abiotic stress and irradiance existed in the putative promoter region of PmCBFb. LT treatment of seedlings showed that the expression of PmCBF genes were induced by 2 °C within 30 min, and their expression reached a peak after 8-12 h. In addition, PmCBFa and PmCBFb appeared more sensitive to LT than PmCBFc. However, the exact roles of PmCBF genes in plant cold tolerance need to be further investigated.

The Wx gene, which encodes waxy proteins, is the sole gene responsible for amylose synthesis in the wheat seed endosperm. In this study, we characterized, at the molecular level, several Wx alleles in durum wheat (Triticum turgidum L. ssp. durum Desf.) that had previously been catalogued at the protein level. Our data show a misclassification of the alleles in both Wx genes: Wx-A1 alleles from Blanqueta (Wx-A1a) and Astrodur (Wx-A1b) have been reclassified as Wx-A1a and Wx-A1h, respectively. A sequence comparison of the Wx-B1e allele shows that there were up to five different alleles under this denomination, which confirms that the protein analysis by SDS-PAGE separation needs to be combined with the PCR amplification-sequencing analysis in order to obtain a correct classification of the alleles, which will facilitate their use in wheat quality improvement.

A full-length cDNA clone encoding cytosolic glutamine synthetase (GS1; EC 6.3.1.2) was isolated from melon (Cucumis melo L.) for the first time by RT-PCR and RACE approach. The clone, designated as M-GS1 (accession No. DQ851867), contains 1494 nucleotides with an open reading frame (ORF) of 1068 nucleotides. The deduced 356 amino acid sequence showed high similarity with previously reported GS1s from various plant species. Sequence analysis revealed that the predicted protein contains a GS β-Grasp domain, a GS catalytic domain, and the main conserved motifs characteristic of a plant GS1. The phylogenetic analysis displayed that M-GS1 is related most closely to the GS1 from Datisca glomerata. Southern blot analysis indicated that M-GS1 belongs to a small gene family of 2 or 3 members. M-GS1 was expressed in all plant tissues without evident tissue specificity, but with different patterns when the melon plants were fed in hydroponic culture with different forms and concentration of nitrogen. Ammonium dramatically enhanced the contents of M-GS1 transcripts in all tested tissues, while nitrate stimulated M-GS1 transcription only in the roots and leaves, but not in the stems; glutamate, however, depressed M-GS1 transcripts in the roots, but resulted in no significant change to the levels of M-GS1 transcripts in the stems and leaves. Moreover, the same effects were observed at the GS enzyme activity level. These results indicated that melons respond to changes of N nutrition by regulating M-GS1 expression.

Leucoanthocyanidin reductase (LAR) converts leucoanthocyanidin to (+)-catechin, a precursor of proanthocyanidins abundant in Japanese persimmon (Diospyros kaki Thunb.) fruits. A putative LAR gene (DkLAR) was isolated by rapid amplification of cDNA ends from young fruits. The full-length cDNA of DkLAR gene was 1 356 bp long and encoded an open reading frame of 349 residues. The deduced DkLAR protein was closely related to the homolog in other plant species. The expression of the DkLAR gene in Chinese pollination-constant non-astringent (PCNA) genotype was coincident with the tannin cell development, but was not in Japanese PCNA and Chinese pollination-variant astringent (PCA) genotypes.

The effect of plant growth regulators (PGRs), gelling agents, sucrose and heat shock on shoot multiplication, shoot growth, rooting and subsequent survival of Chlorophytum borivilianum Sant. et Fernand was evaluated. Benzyladenine (BA) was found to be better cytokinin over kinetin (KIN) for shoot multiplication. Sucrose concentrations from 116-290 mM in the basal medium (BM) promoted shoot multiplication. Heat shock (50 °C, 1 h) also promoted shoot multiplication at these sucrose concentrations on both BM medium and BM supplemented with 5.0 μM BA. Beneficial effect of sucrose was also observed on rooting of shoots on BM as well as BM supplemented with 5.0 μM indole-3-butyric acid (IBA). Phytagel as a gelling agent was found to be more effective for shoot proliferation and growth compared to agar. Amongst various soil mixtures tested, higher survival of plants was observed in soil containing Vermicompost. It was interesting to note that a maximum plant survival (> 95 %) was observed when plants were directly transferred to net-house (irradiance reduced to 50 % with green net, without humidity and temperature control) than poly-house (with humidity and temperature control). Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis of regenerated plants showed genetic similarity to mother plant.

Four different markers [random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), and selective amplified microsatellite polymorphism length (SAMPL)] were applied for evaluating somaclonal variation of micropropagated genotypes of stone pine (Pinus pinea L.). The total number of primers tested was 130, with 223 combinations assayed. A high number of them amplified successfully (178), representing 79.82 % of the total, and the average number of amplified fragments ranged from 2.47 (ISSR) to 65.76 (SAMPL). Based on internal controls, no problem of reproducibility was detected. Almost no somaclonal variation was detected within the clones. Of the tested markers, ISSR, AFLP, and SAMPL showed monomorphic amplification profiles, with only RAPD markers showing some interclonal variation.

Metallothioneins are involved in detoxification of heavy metals. A cDNA encoding type 3 metallothionein (PcMT3) was isolated from the salt stressed leaf cDNA library of Porteresia coarctata (Roxb.) Tateoka (wild rice) that grows well in the heavy metal laden estuarine soils. The PcMT3 cDNA (581 bp) encodes a protein of 64 amino acids. PcMT3 is highly homologous (82 %) to OsMT-I-3a of rice, but is unique from other type 3 plant MTs due to the presence of an additional glycine residue in the C-terminal domain. Analysis of the 5' upstream region of PcMT3 showed the presence of cis-acting elements like the CG box and STRE previously reported to be involved in gene expression under heavy metal stress. Southern analysis suggested the presence of more than one copy of PcMT3-like sequences in the P. coarctata genome. Analysis of genomic clone of PcMT3 revealed the presence of two introns. A comparison of the genomic sequence of PcMT3 with closely similar type 3 MTs from rice and mangrove species revealed conservation in the number and position of introns. Transcript profiling for PcMT3 in P. coarctata leaves in the presence of Cd, Cu and Zn showed an increase in transcript accumulation.

Among the 400 soybean (Glycine max) landraces, we selected 3 tolerant (KAS150-9, KAS160-15, and KAS170-9) and 3 susceptible lines (KAS160-14, KAS160-20, and KAS201-6-1) by the survival percentage and injury scores. Susceptible lines showed decrease in chlorophyll content and increase in glucose and malondialdehyde (MDA) contents under waterlogging stress, while tolerant lines did not change significantly. For AFLP analysis, 8 EcoRI (+3) and 8 MseI (+3) primers used in 32 primer combinations generated a total of 2 566 bands with a mean of 80 bands per primer combination, of which 1 117 (43.5 %) were clearly polymorphic between the tolerant and susceptible lines. A genetic similarity coefficient, based on cluster analysis using an unweighted pair grouping method of average (UPGMA), was 0.79 for the tolerant group, while the susceptible landraces were genetically less related, with a genetic similarity coefficient of 0.17. The 10 reproducible polymorphic PCR products present in the 3 tolerant or susceptible lines were sequenced and converted into sequence tagged site (STS) markers. These STS primer sets were designated GmWT01-GmWT06 and GmWS01-GmWS04. Two STS primer sets, GmWT06 and GmWS02, generated a single monomorphic PCR product identical in size to the original AFLP fragments. For the broad application of these STS markers in marker-assisted selection (MAS) for soybean genotypes tolerant to waterlogging stress, two developed STS markers are being evaluated with putative waterlogging tolerant mutant lines induced by γ-radiation in soybean mutation breeding programs.

The full-length cDNA of LeSGR1 was cloned from tomato by RT-PCR and RACE. The cDNA encoded a protein of 272 amino acid residues and was deposited in GenBank (accession No. DQ100158). Northern analysis suggests that LeSGR1 gene specifically expresses in senescent leaves and mature fruits of tomatoes. Desiccation and flooding induce the expression of LeSGR1 in tomato leaves and stems. Both in ethylene-insensitive mutants (Nr) and ripening inhibitor mutants (rin), the expression of LeSGR1 is markedly decreased compared with that in the wild type. Alignment of the nucleotide sequence of SGR1 cloned from the tomato green flesh (gf) mutant with that from the wild type tomato shows a single nucleotide change leading to an amino acid substitution in gf mutant. Furthermore, LeSGR1 gene silencing by RNA interference results in inhibited chlorophyll degradation similar to the phenotype in gf mutant. Thus, we conclude that LeSGR1 is crucial to chlorophyll degradation and the mutation of SGR1 protein might be responsible for gf tomato properties.