Marker-free transgenic cucumber (Cucumis sativus L.) cv. Poinsett 76 SR plants were produced by Agrobacterium mediated transformation. A transformation efficiency of 1.62 was observed on using Agrobacterium tumefaciens strain LBA4404 harbouring Arabidopsis cbf1 gene driven by the inducible promoter RD29A in a binary vector system pCAMBIA. Transgene integration and single copy insert in transgenic cucumber was confirmed by polymerase chain reaction (PCR) and Southern blot analysis in T₀ lines and also confirmed marker-free status in T₁ generation. Transgene expression was confirmed by reverse transcription (RT)-PCR in T₁ generation transgenic cucumber and advanced to T₂ generation. Upon exposure to chilling stress (4 °C), the T₂ generation transgenic plants survived up to 36 h; however, wild-type plants could not survive and gradually died. A significant decrease in membrane injury index (MII), increase in activities of antioxidant enzymes (SOD and CAT), free proline content and relative water content (RWC) in the leaves were observed in transgenic cucumber as compared to wild-type under chilling stress. Thus, the transgenic cucumber plants expressing Arabidopsis cbf1 gene conferred protection against chilling stress.

The influence of abscisic acid (ABA) on plastidial and cytosolic terpenoids and on two key enzymes for terpenoid biosynthesis was determined in vegetative stage of Cannabis sativa L. Low concentration of ABA (1 μM) increased 1-deoxy-D-xylulose 5-phosphate synthase (DXS) activity in treated plants in comparison to control plants. The amounts of chlorophyll a and carotenoids increased in response to ABA treatment but chlorophyll b content declined. The accumulation of α-tocopherol was stimulated only by 10 μM ABA. The ABA-treated plants showed a decline in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity which was followed by a decrease in squalene and phytosterol content. ABA also decreased tetrahydrocannabinol (THC) and cannabidiol (CBD) contents. The essential oil had higher ratios of monoterpenes to sesquiterpenes as ABA-treated plants had less numbers of sesquiterpenes in comparison with control plants. Influence of ABA on the amounts of sesquiterpenes was different, some of them showed decrease of content and others increase of content.

In Pinus peuce zygotic embryo culture grown on Gresshoff and Doy (1972; GD) basal medium, 2.22 μM benzyladenine (BA) was superior in promoting adventitious bud induction during 4 weeks comparing to kinetin or BA + kinetin. Shoot elongation was achieved on half-strength GD medium devoid of plant growth regulators and containing activated charcoal. Pulse treatment with 1 mM indole-3-butyric acid (IBA) for 2 h, followed by transfer to half-strength GD medium, produced the most efficient rooting. Rooted shoots were transplanted to the greenhouse and plantlets continued to grow and developed into phenotypically normal plants. Up to 10 plants per explant can be obtained within 36 weeks from culture initiation.

The transcript levels and activities of the superoxide dismutase isoenzyme (Cu/ZnSOD) were assessed in winter (Tarm-92) and spring (Zafer-160) barley cultivars during cold acclimation, freezing stress and after rewarming. Leaf Cu/ZnSOD activity and Cu/ZnSOD expression level were not significantly changed during cold acclimation. The Cu/ZnSOD expression increased evidently at mild freezing stress (-3 °C; F1), while Cu/ZnSOD1 activity did not show any response and Cu/ZnSOD2 activity decreased continuously during F1 and F2 (-7 °C) in Tarm-92. On the other hand, root Cu/ZnSOD2 activity was in accordance with Cu/ZnSOD expression in Zafer-160 after F2 treatment. Rewarming periods did not cause any significant changes in the Cu/ZnSOD activity and expression of Cu/ZnSOD in both cultivars when compared to freezing stresses. These results showed that freezing stress can regulate differently Cu/ZnSOD transcription and enzyme activity.

During the period of senescence of desert plant Alhagi sparsifolia Shap. the maximum photochemical quantum yield measured as variable to maximum fluorescence ratio (F_v/F_m) remained relatively high, although the number of active reaction centres per cross section (RCs) decreased significantly. The efficiency of electron acceptors beyond the primary quinone acceptor (Q_A) decreased. The effect of temperature and irradiance on photosystem activity was maximum after 6 d. Our results suggest that: 1) the down-regulation of photosystem activity was due to the decline of both RCs and electron acceptance between plastoquinone (PQ) and cytochrome (cyt) b6/f; 2) photosystem activity presented negative correlation with daily mean temperature, and 3) reduction of daily sunshine period and increase of temperature at noon can stimulate the speed of senescence.

Differences among three clones of Gentiana punctata L. hairy root shoot regenerants were investigated in relation to their growth patterns, production of secondary metabolites and 2D protein profiles. Prominent differences in growth parameters were stable thus qualifying regenerant clones as true somaclones. Marked differences in protein spots were registered among the regenerant clones but not in comparison with the non-transformed control. Southern blot hybridization of regenerants showed the absence of rolA, B and C genes, initially present in the main hairy root lines. Orf13 and rolD were present and orf8 was missing in all three regenerant clones whereas orf3 was missing only in clone 2. Although lacking the three major rol genes, plants of regenerant clones retained characteristics of the hairy root phenotype.

The effect of exogenously applied nitric oxide on the heat tolerance of Chrysanthemum morifolium was investigated by applying the NO donor sodium nitroprusside (SNP). We found that the SNP partially alleviated the heat stress by slowing down the reduction of photosynthetic pigment content and net photosynthetic rate. SNP treatment also lowered the increase in the non-photochemical quenching of fluorescence and malondialdehyde content and maintained higher activities of superoxide dismutase, peroxidase, catalase and ascorbate peroxidase.

The anatomic and functional leaf characteristics related to photosynthetic performance of Castanea sativa growing in vitro and in nursery were compared. The irradiance saturated photosynthesis in in vitro grown plantlets was significantly lower compared to nursery plants (65 vs. 722 μmol m^-2 s^-1). The maximum photosynthetic rate (P_Nmax) was 4.0 and 10.0 μmol(CO₂) m^-2 s^-1 in in vitro microshoots and nursery plant leaves, respectively. Carboxylation efficiency (C_E) and electron transport rate (ETR) were three-folds higher in nursery plants than in microshoots. The nonphotochemical quenching (NPQ) was saturated at 80 μmol m^-2 s^-1 in microshoots suggesting limited photoprotection by thermal dissipation. The microshoots had wide open, spherical stomata and higher stomatal density than nursery plants and they had almost no epicuticular wax. Consequently, the microshoots had high stomatal conductance and high transpiration rate. These anatomic and functional leaf characteristics are likely major causes of the low survival rates of plantlets after ex vitro transfer.

Branching is an important step in higher plant development, which not only determines the configuration of the plant directly, but also affects its adaptability to the environment. The interactions between hormones, genes, environmental and other factors subtly regulate the process of branching. Strigolactone is a newly recognized phytohormone and its content and distribution might be a key factor affecting branching. This review is focused on the genes related to synthesis and transduction pathway of strigolactone, and summarizes the inhibitory role of strigolactone in plant branching. Discussions about the issues to be clarified and prospects of the future research were also proposed.

Antioxidative responses and proline accumulation induced by exogenous H₂O₂ were investigated in the callus from halophyte Nitraria tangutorum Bobr. H₂O₂-treated callus exhibited higher H₂O₂ content than untreated callus. The activities of catalase (CAT) and peroxidase (POD) significantly increased in the callus treated with H₂O₂, while ascorbate peroxidase (APX) activity decreased. In addition, significantly enhanced proline content was observed in the callus treated by H₂O₂, which could be alleviated by H₂O₂ scavenger dimethylthiourea and calcium (Ca) chelator ethylene glycol bis-(β-aminoethyl ether)-N,N,N',N'-tetra-acetic acid (EGTA). Moreover, γ-glutamyl kinase (GK) activity increased in H₂O₂-treated callus, but proline dehydrogenase (PDH) activity decreased significantly, and the reduction was partly abolished by EGTA or Ca channel blocker verapamil. Assays using a scanning electron microscope showed significantly enhanced Ca content in H₂O₂-treated callus.

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in supplying reduced nicotine amide cofactors for biochemical reactions and in modulating the redox state of cells. In this study, the role of G6PDH in thermotolerance of the calli from Przewalskia tangutica and tobacco (Nicotiana tabacum L.) was investigated. Results showed that Przewalskia tangutica callus was more sensitive to heat stress than tobacco callus. The activity of G6PDH and antioxidant enzymes (ascorbate peroxidase, catalase, peroxidase and superoxide dismutase) in calli from Przewalskia tangutica and tobacco increased after 40 °C treatment, although two calli exhibited a difference in the degree and timing of response to heat stress. When G6PDH was partially inhibited by glucosamine pretreatment, the antioxidant enzyme activities and thermotolerance in both calli significantly decreased. Simultaneously, the heat-induced H₂O₂ content and the plasma membrane NADPH oxidase activity were also reduced. Application of H₂O₂ increased the activity of G6PDH and antioxidant enzymes in both calli. Diphenylene iodonium, a NADPH oxidase inhibitor, counteracted heatinduced H₂O₂ accumulation and reduced the heat-induced activity of G6PDH and antioxidant enzymes. Moreover, exogenous H₂O₂ was effective in restoring the activity of G6PDH and antioxidant enzymes after glucosamine pretreatment. Western blot analysis showed that G6PDH gene expression in both calli was also stimulated by heat and H₂O₂, and blocked by DPI and glucosamine under heat stress. Taken together, under heat stress G6PDH promoted H₂O₂ accumulation via NADPH oxidase and the elevated H₂O₂ was involved in regulating the activity of antioxidant enzymes, which in turn facilitate to maintain the steady-state H₂O₂ level and protect plants from the oxidative damage.

The leaf apoplast is a dynamic compartment in contact with plant pathogenic bacteria after infection. Among the very first interaction events is the receptor-mediated perception of bacterial surface molecules such as flagellin or other conserved microbe-associated molecular patterns (MAMPs). Apoplast proteins likely play a role in basal resistance (BR) or pattern-triggered immunity (PTI). Here, a proteomic approach was carried out on water soluble - potentially the most mobile - apoplast proteins from flagellin-treated tobacco (Nicotiana tabacum) leaves. As the quickness of BR/PTI seems crucial for its efficacy, samples were taken as early as 2.5 and 7 h post inoculation. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Forty-nine different proteins from 28 protein spots changed in their density compared to the water-inoculated control. Eleven protein spots appeared de novo in response to EBR induction. There are glycohydrolases and redox-active proteins besides pathogenesis-related proteins among them, predicting plant cell wall structural modifications and more direct antimicrobial effectors as earliest changes related to BR/PTI.

Grapevine rootstocks belonging to different species of Vitis differ in their response to stress. Vines of rootstocks 110R and 1613C were subjected to different salt and water stresses individually and in combination. Expression of VvNHX1, a Na⁺/H⁺ antiporter gene, was analyzed at 7 and 21 d of stress treatment. In 110R, the expression of VvNHX1 gene increased in response to both salinity and water stress already after 7 d. Under salinity and combined stress enhanced expression of this gene was observed also after 21 d whereas expression decreased under water stress. In 1613C, expression of this gene did not increase under salinity stress. There was delayed response to water and combined stress and expression increased several fold after 21 d of stress. The stressed vines of 110R maintained lower sodium content and higher K⁺/Na⁺ ratio as compared to rootstock 1613C.

The removal of Mg²⁺ is an important step in the chlorophyll degradation pathway and extracts from senescent and presenescent Arabidopsis thaliana leaves were analyzed for Mg-dechelatase activity, using chlorophyllin, an artificial derivative of the natural substrate, chlorophyllide. The optimum temperature and pH for this reaction were determined to be at approximately 50 °C and 7.2, respectively. Mg-dechelatase activity was enhanced by addition of EDTA and inhibited by MgCl₂, HgCl₂ and reduced glutathione, indicating phenomenons such as retroinhibition by reaction products and dependence on the redox state of the mixture. Size exclusion chromatography was performed on Arabidopsis leaf extracts, and Mg-dechelatase activity was found in the fraction corresponding to molecular mass of about 42 kDa, which indicates that the Mg-dechelating compound in Arabidopsis is considerably larger than in other systems. During dark-induced senescence, the activity increased over time until reaching a maximum at day 4, and then decreased. The addition of plant growth regulators indicated that the accumulation of Mg-dechelatase was activated by ethylene and delayed by 6-benzylaminopurine.

Dark-grown seedlings of Picea abies (L) Karst. are able to accumulate the highest amounts of chlorophyll (Chl) and its precursor protochlorophyllide (Pchlide) in all Pinaceae, but calli derived from 14-d-old green cotyledons of P. abies are completely white during the cultivation in the dark. Pchlide reduction is catalysed in the dark by light-independent protochlorophyllide oxidoreductase (DPOR). This enzyme complex consists of three protein subunits ChlL, ChlN and ChlB, encoded by three plastid genes chlL, chlN and chlB. Using semiquantitative RT-PCR, we observed very low expression of chlLNB genes in dark-grown calli. It seems, that chlLNB expression and thus Chl accumulation could be modulated by light in P. abies calli cultures. This hypothesis is supported by the fact, that we observed low contents of glutamyl-tRNA reductase and Flu-like protein, which probably affected Chl biosynthetic pathway at the step of 5-aminolevulinic acid formation. ChlB subunit was not detected in dark-grown P. abies calli cultures. Our results indicated limited ability to synthesize Chl in callus during cultivation in the dark.

Methylglyoxal (MG) is a highly reactive α-oxoaldehyde, demonstrating anticancer effect on plant neoplastic tumours. In in vivo studies it was observed that MG destroyed crown gall tumours in Nicotiana tabacum produced by Agrobacterium tumefaciens, without any adverse effect on the host. The efficacy of MG in comparison to other anticancer drugs viz. cisplatin and ellagic acid in the treatment of crown gall was investigated. A slight degeneration of galls was noted in plants treated with cisplatin and ellagic acid but the plants died subsequently. With MG however, crown galls were completely cured and the plants completed their usual life cycle by flowering and producing seeds. MG inhibited the respiration of crown gall calluses suggesting that energy depletion resulted in tumour destruction.

Mature pollen grain represents a highly desiccated structure with an extremely tough cell wall. Thanks to it, it resists common proteomic protocols. Instead, a robust homogenization has to be performed since proteins are needed to burst out of the cell to be included in the extracted proteome fraction. Here, a novel way of pollen homogenization employing Roche MagNA Lyser Instrument is presented, sparing time and laborious work. However, plant proteomics does not rely solely on perfect homogenization; also the choice of the extraction protocol is of key importance. The composition of the extraction buffer has a decisive influence on which proteome fraction will be extracted. Therefore the second part of our study is dedicated to the comparison of different extraction protocols with respect to subsequent proteomic analyses.

Lipoxygenase (LOX) from opium poppy (Papaver somniferum L.) chloroplasts was isolated and 126.1-fold purified to electrophoretic homogeneity by combination of ion-exchange chromatography on HA-Ultragel column and affinity chromatography on a linoleyl-aminopropyl agarose column. The relative molecular mass of the LOX determined by SDS-PAGE was 92 kDa. Kinetic properties of purified LOX were determined in spectrophotometric assay by using of linoleic acid (K_M = 1.78 mM and V_max = 11.4 μmol mg^-1 min^-1) and linolenic acid (K_M = 1.27 mM and V_max = 10.2 μmol mg^-1 min^-1). The optimum pH was 6.0 for both linoleic and linolenic acid dioxygenation catalyzed by LOX. HPLC analysis of the products revealed a dual positional specificity of linoleic acid dioxygenation at pH 6.0 with ratio of 9- and 13-hydroperoxide products being about 1:1. The activity of purified LOX was stimulated by Mg²⁺ and Ca²⁺.

Photosynthetic pigments, gas exchange, chlorophyll (Chl) a fluorescence kinetics, antioxidant enzymes and chloroplast ultrastructure were investigated in ginkgo (Ginkgo biloba L.) leaves from emergence to full size. Under natural conditions, the net photosynthetic rate (P_N), contents of Chl a, Chl b and total soluble proteins and fresh and dry leaf mass gradually increased during leaf expansion. The maximum photochemical efficiency of photosystem (PS) 2 (variable to maximum fluorescence ratio, F_v/F_m) was considerably higher at the early stages of leaf development than in fully expanded leaves. During daily course, only reversible decrease in F_v/F_m was distinguished at various stages, implying that no photo-damage occurred. Absorption flux per cross section (CS) and trapped energy flux per CS were significantly lower in newly expanding leaves compared with fully expanded ones, however, dissipated energy flux per CS was only slightly lower in expanding leaves. The ratio of carotenoids (Car)/Chl decreased gradually during leaf expansion due to increasing Chl content. Moreover, activities of the antioxidant enzymes, such as superoxide dismutase, ascorbate peroxidase, catalase and peroxidase, increased at the early stages of leaf expansion. The appearance of osmiophilic granules in fully expanded leaves further proves that photo-protection is significantly strengthened at the early stages of leaf expansion.

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.

The relationship between somatic embryogenesis (SE) and the expression of the BABY BOOM (BBM) gene was studied in cultured immature zygotic embryos (IZEs) using a transgenic line of Arabidopsis thaliana containing a BBMPro::GUS construct. Results showed spatio-temporal differences in BBM expression in explants during culture. BBM promoter activity was observed in freshly isolated IZEs except distal parts of cotyledons. At the beginning of culture, considerable increase of GUS staining intensity was observed in all parts of explants, which maintained at high level over next few days and coincide with cell divisions. Gradual decrease of GUS distribution in explants was observed at about the 5th day of culture. BBM promoter activity became largely restricted to dividing cells, then to developing somatic embryos, shoot-like structures and callus. In parts of explants not involved in morphogenesis BBM promoter activity was absent or hardly seen. Thus the in vitro expression of BBM coincides with cell proliferation and morphogenesis.

Mature embryonic axes were used for chickpea (Cicer arietinum L.) regeneration via somatic embryogenesis. Qualitative and quantitative estimation of protein profile during somatic embryogenesis by SDS-PAGE and densitometric analysis showed differential expression of various storage proteins at different stages of somatic embryo development, which was compared with the profile of developing seeds. Total protein content in somatic embryos of chickpea increased from globular stage [2.9 μg mg^-1(f.m.)] to cotyledonary stage [4.8 μg mg^-1(f.m.)] and then started decreasing during onset of maturation and germination [up to 1.5 μg mg^-1(f.m.)]. Differential expression of seed storage proteins, late embryogenesis abundant (LEA) proteins and proteins related with stress response were documented at different stages of somatic embryogenesis. Germinating somatic embryos showed degradation products of several seed storage proteins and the appearance of new polypeptides (76.8, 67.6, 49.9 and 34.2 kDa), which were absent during differentiation of somatic embryos. A low molecular mass (17.7 kDa) polypeptide was uniformly present during all stages of somatic embryogenesis and it may belong to a group of stress-related proteins. This study describes the expression of true seed storage proteins like legumin, vicilin, convicilin and their subunits at different stages of somatic embryogenesis, which may serve as excellent markers for embryogenic pathway of regeneration in chickpea.

Differences in Cd accumulation and Cd tolerance between Thlaspi arvense ecotype Aigues Vives (AV) from a commercial grower in South France and ecotype Jena collected in the polluted urban area of Jena (Germany) were reported here. Ecotype Jena exhibited considerable Cd-tolerance. Shoot and root masses were unaffected and root elongation was even enhanced by exposure to 50 μM Cd. In contrast, growth of ecotype AV was severely affected by this Cd treatment. Ecotype Jena was much more efficient in excluding Cd from both roots and shoots than ecotype AV. Despite the efficient restriction of Cd transport from roots to shoots in Jena, this ecotype maintained high root to shoot transport of Zn and Fe under Cd exposure. Cd supply strongly decreased the activities of antioxidant enzymes in AV, while in the Cd resistant Jena these activities either remained unaffected (SOD, APX) or were increased (CAT) by Cd supply. In conclusion, naturally selected Cd-tolerance in Thlaspi arvense is due to efficient Cd exclusion. The mechanisms underlying exclusion of Cd from the shoots seem Cd-specific yet they did not affect the homeostasis of Fe and Zn in the shoots.

Arabidopsis thaliana plants (wild type accessions Col and N1438) were grown in nutrient solution for 34 d with or without 50 mM NaCl. Salt stress inhibited plant growth rate more in Col than in N1438 and a decrease in K⁺, Ca²⁺ and nitrogen contents was observed in both accessions. NaCl diminished accumulation of malate, fumarate and citrate only in Col accession. To measure the effect of NaCl on transcript level of superoxide dismutase (SOD) isoforms and copper chaperone for SOD genes, a semi-quantitative polymerase chain reaction (RT-PCR) method was developed using cDNA normalized against the EF1a gene in parallel with quantitative real time RT-PCR (Q-PCR) technique. Both methods gave the same results. The abundance of transcripts of the three genes coding for Cu/Zn-SOD responded similarly to NaCl in both accessions: CSD1 gene was overexpressed, and CSD2 and CSD3 genes were repressed. However, the genes coding for Fe-SOD (FSD1), Mn-SOD (MSD1) and Cu-chaperone for SOD (CCS) responded to NaCl differently in Col and N1438: the former gene was overexpressed in Col and repressed in N1438, and the opposite behaviour was observed for the latter two genes.

The accumulation of cold-induced dehydrin and proline was related to the frost tolerance (FT) in several Brassica species or cultivars. A dehydrin of molecular mass 47 kDa was detected in the leaves of an Ethiopian mustard (B. carinata) and a pair of dehydrins of similar molecular mass in the three (two winter, one spring) oilseed rape (B. napus) cultivars, when plants were maintained at 4 °C for one-month under two different irradiances. More dehydrin was accumulated in oilseed rape than in Ethiopian mustard under the high irradiance. A significant correlation was observed between leaf dehydrin content and FT, and no relationship between proline content and FT or between the proline and dehydrin contents. Protoplast-derived callus cells behaved differently from leaves sampled from intact plants, as they did not accumulate dehydrin and proline in response to cold stress.

Thinopyrum intermedium is an important species with potential utilization value in breeding of wheat. In this study, the non-coding intergenic region of trnH-psbA was investigated to assess the genetic diversity and infer the maternal origin within T. intermedium accessions. Eleven haplotypes were distinguished among the thirty-five accessions of T. intermedium. They showed a relatively low nucleotide diversity (π) of 0.00473 ± 0.00037 and a moderately high haplotype diversity (H_d) of 0.733 ± 0.061. In the phylogenetic analysis, all accessions of T. intermedium were positioned into two clades, which corresponded to the different diploid donors. These results suggested that there were two phylogenetically divergent maternal donors in T. intermedium.

The effects of inhibition of mitochondrial alternative oxidase (AOX) respiratory pathway on photosynthetic apparatus in Rumex K-1 leaves were studied. Under high irradiance, the inhibition of AOX pathway caused over-reduction of photosystem (PS) 2 acceptor side, a decrease in the energy transfer in the PS 2 units, damage of donor side of PS 2 and decrease in pool size of electron acceptors. The inhibition of AOX pathway also decreased photosynthetic performance index (PI_ABS), actual photochemical efficiency (Φ_PS2), photochemical quenching (qP) and photosynthetic O₂ evolution rate. The results demonstrate that mitochondrial AOX pathway plays a vital role in photoprotection of photosynthetic apparatus.

In order to characterize the response of selected grasses to water stress, relative water content (RWC) in leaves and quantum efficiency of photosystem 2 (F_v/F_m) were measured in Phleum pratense L., P. bertolonii DC. and P. phleoides H. Karst. during 6 d of water stress. The results indicated differential responses to water stress among the three Phleum species with higher water deficit sensitivity of P. pratense and P. bertolonii than that of P. phleoides. The cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique was applied to identify differentially expressed genes responding to water stress in P. pratense. Cloned and sequenced differentially expressed fragments (DEFs) were used for primer design in order to identify orthologous genes in Lolium perenne L. Twelve genes orthologous to P. pratense DEFs were mapped in the L. perenne mapping population VrnA based on a high resolution melting curve analysis (HRM). This study provides genomic information about 29 differentially expressed genes after water stress in P. pratense and reports on the identification and mapping of twelve orthologs in L. perenne.

Seventeen polymorphic microsatellite markers were isolated from enriched genomic libraries for Theobroma cacao, providing additional tools for studying the genetic diversity and map saturation of this species. These markers were characterized in 32 accessions of the T. cacao germplasm collection from the Centro de Pesquisas do Cacau. The number of alleles at each locus varied from 2 to 8, with an average of 4.41 alleles per locus. The polymorphism information content varied from 0.060 to 0.695, with an average of 0.333. The markers characterized in this study will be employed in map saturation studies and diversity assessments of cacao genotypes.

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.