Search

Main Conclusion: CsERF2, an ethylene response factor, plays a role in leaf variegation. Leaf variegation is a main ornamental characteristic in Cymbidium sinense (C. sinense). However, the mechanisms of leaf color variegation remain largely unclear. In the present study, we analyzed the cytological and physiological features, as well as molecular analyses of leaves from wild-type (WT) and leaf variegation mutants of Cymbidium sinense 'Dharma'. Chloroplasts with typical and functional structures were discovered in WT and green sectors of the mutants leaves (MG), but not in yellow sectors of the mutant leaves (MY). The activities of key enzymes involved in chlorophyll (Chl) degradation and their substrate contents were significantly increased in MY. Genes related to Chl degradation also showed a significant up-regulation in MY. Transcriptomic analysis showed that the expression of all identified ethylene response factors (ERFs) was significantly up-regulated, and the 1-aminocyclopropane-1-carboxylic acid (ACC) content in MY was significantly higher compared with MG. QRT-PCR analysis validated that the expression levels of genes related to Chl degradation could be positively affected by ethylene (ETH) treatment. Stable overexpression of CsERF2 in Nicotiana tabacum (N. tabacum) led to a decrease in Chl content and abnormal chloroplast. Transcriptomic analysis and qRT-PCR results showed that the KEGG pathway related to chloroplast development and function showed significant change in transgenic N. tabacum. Therefore, the leaf color formation of C. sinense was greatly affected by chloroplast development and Chl metabolism. CsERF2 played an important role in leaf variegation of C. sinense.

Main Conclusion: The changes in the expression of key sugar metabolism enzymes (SPS and SUS), sucrose content and arrangement of chloroplast starch may play a significant role in the cold response in M. giganteus and maize plants. To understand the mechanism of the chilling-response of two closely-related C 4 plants, we investigated the changes in the expression of sucrose phosphate synthase (SPS) and sucrose synthase (SUS) as well as changes in their potential products: sucrose, cellulose and starch in the leaves of Miscanthus × giganteus and Zea mays. Low temperature (12-14 °C) increased SPS content in Miscanthus (MG) and chilling-sensitive maize line (Zm-S), but not in chilling-tolerant one (Zm-T). In Zm-S line, chilling also caused the higher intensity of labelling of SPS in the cytoplasm of mesophyll cells, as demonstrated by electron microscopy. SUS labelling was also increased by cold stress only in MG plants what was observed in the secondary wall between mesophyll and bundle sheath cells, as well as in the vacuoles of companion cells. Cold led to a marked increase in total starch grain area in the chloroplasts of Zm-S line. In turn, Fourier transform infrared spectroscopy (FTIR) showed a slight shift in the cellulose band position, which may indicate the formation of more compact cellulose arrangement in Zm-T maize line. In conclusion, this work presents new findings supporting diversified cold-response, not only between two C 4 plant species but also within one species of maize.

Main Conclusion: The accumulation of NiCo following the termination of the accumulation of iron in chloroplast suggests that NiCo is not solely involved in iron uptake processes of chloroplasts. Chloroplast iron (Fe) uptake is thought to be operated by a complex containing permease in chloroplast 1 (PIC1) and nickel-cobalt transporter (NiCo) proteins, whereas the role of other Fe homeostasis-related transporters such as multiple antibiotic resistance protein 1 (MAR1) is less characterized. Although pieces of information exist on the regulation of chloroplast Fe uptake, including the effect of plant Fe homeostasis, the whole system has not been revealed in detail yet. Thus, we aimed to follow leaf development-scale changes in the chloroplast Fe uptake components PIC1, NiCo and MAR1 under deficient, optimal and supraoptimal Fe nutrition using Brassica napus as model. Fe deficiency decreased both the photosynthetic activity and the Fe content of plastids. Supraoptimal Fe nutrition caused neither Fe accumulation in chloroplasts nor any toxic effects, thus only fully saturated the need for Fe in the leaves. In parallel with the increasing Fe supply of plants and ageing of the leaves, the expression of BnPIC1 was tendentiously repressed. Though transcript and protein amount of BnNiCo tendentiously increased during leaf development, it was even markedly upregulated in ageing leaves. The relative transcript amount of BnMAR1 increased mainly in ageing leaves facing Fe deficiency. Taken together chloroplast physiology, Fe content and transcript amount data, the exclusive participation of NiCo in the chloroplast Fe uptake is not supported. Saturation of the Fe requirement of chloroplasts seems to be linked to the delay of decomposing the photosynthetic apparatus and keeping chloroplast Fe homeostasis in a rather constant status together with a supressed Fe uptake machinery.

Main Conclusion: In alfalfa, the B form of Sucrose phosphate synthase synthesizes sucrose in the leaves while the A form participates in regulatory cycles of synthesis/breakdown of sucrose/starch in the root nodules. Sucrose (Suc) is the major stable product of photosynthesis that is transported to all heterotrophic organs as a source of energy and carbon. The enzyme sucrose phosphate synthase (SPS) catalyzes the synthesis of Suc. Besides the leaves, SPS is also found in heterotrophic organs. There are two isoforms of SPS in alfalfa (Medicago sativa): SPSA and SPSB. While SPSA is expressed in the vasculature of all the organs and in the N 2 -fixing zone in the nodules, SPSB is exclusively expressed in the photosynthetic cells. Two classes of alfalfa transformants were produced, one with a gene construct consisting of the alfalfa SPSA promoter and the other with the SPSB promoter-both driving the maize SPS coding region-referred to as SPSA-ZmSPS and SPSB-ZmSPS, respectively. Both classes of transformants showed increased growth compared to control plants. The SPSB-ZmSPS transformants showed increased SPS protein levels and activity along with a significant increase in the Suc levels in the leaves. The SPSA-ZmSPS transformants showed an increase in the SPS protein level and enzyme activity both in the leaves and the nodules with no increase in Suc content in the leaves but a substantial increase in the nodules. Both SPSA and SPSB have unique roles in the nodules (sink) and leaves (source). SPSB is responsible for the synthesis of Suc in the photosynthetic cells and SPSA participates in a regulatory cycle in which Suc is simultaneously degraded and re-synthesized; both these functions contribute to plant growth in rhizobia nodulated alfalfa plants.

Main Conclusion: Ethylene receptor is crucial for PCD and aerenchyma formation in Typha angustifolia leaves. Not only does it receive and deliver the ethylene signal, but it probably can determine the cell fate during aerenchyma morphogenesis, which is due to the receptor expression quantity. Aquatic plant oxygen delivery relies on aerenchyma, which is formed by a programmed cell death (PCD) procedure. However, cells in the outer edge of the aerenchyma (palisade cells and septum cells) remain intact, and the mechanism is unclear. Here, we offer a hypothesis: cells that have a higher content of ethylene receptors do not undergo PCD. In this study, we investigated the leaf aerenchyma of the aquatic plant Typha angustifolia. Ethephon and pyrazinamide (PZA, an inhibitor of ACC oxidase) were used to confirm that ethylene is an essential hormone for PCD of leaf aerenchyma cells in T. angustifolia. That the ethylene receptor was an indispensable factor in this PCD was confirmed by 1-MCP (an inhibitor of the ethylene receptor) treatment. Although PCD can be avoided by blocking the ethylene receptor, excessive ethylene receptors also protect cells from PCD. TaETR1, TaETR2 and TaEIN4 in the T. angustifolia leaf were detected by immunofluorescence (IF) using polyclonal antibodies. The result showed that the content of ethylene receptors in PCD-unsusceptible cells was 4-14 times higher than that one in PCD-susceptible cells, suggesting that PCD-susceptible cells undergo the PCD programme, while PCD-unsusceptible cells do not due to the content difference in the ethylene receptor in different cells. A higher level of ethylene receptor content makes the cells insensitive to ethylene, thereby avoiding cell death and degradation.

Main Conclusion: Plastid genome engineering is an effective method to generate drought-resistant potato plants accumulating glycine betaine in plastids. Glycine betaine (GB) plays an important role under abiotic stress, and its accumulation in chloroplasts is more effective on stress tolerance than that in cytosol of transgenic plants. Here, we report that the codA gene from Arthrobacter globiformis, which encoded choline oxidase to catalyze the conversion of choline to GB, was successfully introduced into potato (Solanum tuberosum) plastid genome by plastid genetic engineering. Two independent plastid-transformed lines were isolated and confirmed as homoplasmic via Southern-blot analysis, in which the mRNA level of codA was much higher in leaves than in tubers. GB accumulated in similar levels in both leaves and tubers of codA-transplastomic potato plants (referred to as PC plants). The GB content was moderately increased in PC plants, and compartmentation of GB in plastids conferred considerably higher tolerance to drought stress compared to wild-type (WT) plants. Higher levels of relative water content and chlorophyll content under drought stress were detected in the leaves of PC plants compared to WT plants. Moreover, PC plants presented a significantly higher photosynthetic performance as well as antioxidant enzyme activities during drought stress. These results suggested that biosynthesis of GB by chloroplast engineering was an effective method to increase drought tolerance.

Main Conclusion: The LysM receptor-like kinase K1 is involved in regulation of pea-rhizobial symbiosis development. The ability of the crop legume Pisum sativum L. to perceive the Nod factor rhizobial signals may depend on several receptors that differ in ligand structure specificity. Identification of pea mutants defective in two types of LysM receptor-like kinases (LysM-RLKs), SYM10 and SYM37, featuring different phenotypic manifestations and impaired at various stages of symbiosis development, corresponds well to this assumption. There is evidence that one of the receptor proteins involved in symbiosis initiation, SYM10, has an inactive kinase domain. This implies the presence of an additional component in the receptor complex, together with SYM10, that remains unknown. Here, we describe a new LysM-RLK, K1, which may serve as an additional component of the receptor complex in pea. To verify the function of K1 in symbiosis, several P. sativum non-nodulating mutants in the k1 gene were identified using the TILLING approach. Phenotyping revealed the blocking of symbiosis development at an appropriately early stage, strongly suggesting the importance of LysM-RLK K1 for symbiosis initiation. Moreover, the analysis of pea mutants with weaker phenotypes provides evidence for the additional role of K1 in infection thread distribution in the cortex and rhizobia penetration. The interaction between K1 and SYM10 was detected using transient leaf expression in Nicotiana benthamiana and in the yeast two-hybrid system. Since the possibility of SYM10/SYM37 complex formation was also shown, we tested whether the SYM37 and K1 receptors are functionally interchangeable using a complementation test. The interaction between K1 and other receptors is discussed.

Main Conclusion: Although the PAO/phyllobilin pathway of chlorophyll breakdown is active in grass leaf senescence, the abundance of phyllobilins is far below the amount of degraded chlorophyll. The yellowing of fully developed leaves is the most prominent visual symptom of plant senescence. Thereby, chlorophyll is degraded via the so-called pheophorbide a oxygenase (PAO)/phyllobilin pathway to a species-specific set of phyllobilins, linear tetrapyrrolic products of chlorophyll breakdown. Here, we investigated the diversity and abundance of phyllobilins in cereal and forage crops, i.e. barley, rice, ryegrass, sorghum and wheat, using liquid chromatography-mass spectrometry. A total of thirteen phyllobilins were identified, among them four novel, not yet described ones, pointing to a rather high diversity of phyllobilin-modifying activities present in the Gramineae. Along with these phyllobilins, barley orthologs of known Arabidopsis thaliana chlorophyll catabolic enzymes were demonstrated to localize in the chloroplast, and two of them, i.e. PAO and pheophytin pheophorbide hydrolase, complemented respective Arabidopsis mutants. These data confirm functionality of the PAO/phyllobilin pathway in grasses. Interestingly, when comparing phyllobilin abundance with amounts of degraded chlorophyll in senescent leaves, in most analyzed grass species only minor fractions of chlorophyll were recovered as phyllobilins, opposite to A. thaliana where phyllobilin quantities match degraded chlorophyll rather well. These data show that, despite the presence and activity of the PAO/phyllobilin pathway in barley (and other cereals), phyllobilins do not accumulate stoichiometrically, implying possible degradation of chlorophyll beyond the phyllobilin level.

Main Conclusion: Stilbene synthase (STS) and its metabolic products are accumulated in senescing grapevine leaves. Ectopic expression of VpSTS29 in Arabidopsis shows the presence of VpSTS29 in oil bodies and increases trans-piceid in developing leaves. Stilbenes are the natural antimicrobial phytoalexins that are synthesised via the phenylpropanoid pathway. STS is the key enzyme catalysing the production of stilbenes. We have previously reported that the VpSTS29 gene plays an important role in powdery mildew resistance in Vitis pseudoreticulata. However, the synthesis and accumulation of these stilbene products in plant cells remain unclear. Here, we demonstrate that VpSTS29 is present in cytosolic oil bodies and can be transported into the vacuole at particular plant-developmental stages. Western blot and high-performance liquid chromatography showed that STS and trans-piceid accumulated in senescent grape leaves and in pVpSTS29::VpSTS29-expressing Arabidopsis during age-dependent leaf senescence. Subcellular localisation analyses indicated VpSTS29-GFP was present in the cytoplasm and in STS-containing bodies in Arabidopsis. Nile red staining, co-localisation and immunohistochemistry analyses of leaves confirmed that the STS-containing bodies were oil bodies and that these moved randomly in the cytoplasm and vacuole. Detection of protein profiles revealed that no free GFP was detected in the pVpSTS29::VpSTS29-GFP-expressing protoplasts or in Arabidopsis during the dark-light cycle, demonstrating that GFP fluorescence distributed in the STS-containing bodies and vacuole was the VpSTS29-GFP fusion protein. Intriguingly, in comparison to the controls, over-expression of VpSTS29 in Arabidopsis resulted in relatively high levels of trans-piceid, chlorophyll content and of photochemical efficiency accompanied by delayed leaf senescence. These results provide exciting new insights into the subcellular localisation of STS in plant cells and information about stilbene synthesis and storage.

Main Conclusion: This study showed that a galactosyltransferase, AgUCGalT1, is involved in anthocyanin galactosylation in purple celery. Celery is a well-known vegetable because of its rich nutrients, low calories, and medicinal values. Its petioles and leaf blades are the main organs acting as nutrient sources. UDP-galactose: cyanidin 3-O-galactosyltransferase can transfer the galactosyl moiety from UDP-galactose to the 3-O-position of cyanidin through glycosylation. This process enhances the stability and water solubility of anthocyanins. In the present study, LC-MS data indicated that abundant cyanidin-based anthocyanins accumulated in the petioles of purple celery ('Nanxuan liuhe purple celery'). A gene encoding UDP-galactose: cyanidin 3-O-galactosyltransferase, namely AgUCGalT1, was isolated from purple celery and expressed in Escherichia coli BL21 (DE3). Sequence alignments revealed that the AgUCGalT1 protein contained a highly conserved putative secondary plant glycosyltransferase (PSPG) motif. The glycosylation product catalyzed by AgUCGalT1 was detected using UPLC equipment. The recombinant AgUCGalT1 had an optimal enzyme activity at 35 °C and pH 8.0, and showed highest enzyme activity toward cyanidin among the enzyme activities involving other substances, namely, peonidin, quercetin, and kaempferol. The expression levels of AgUCGalT1 were positively correlated with the total anthocyanin contents in purple and non-purple celery varieties. Crude enzymes extracted from purple celery exhibited glycosylation ability, whereas crude enzymes obtained from non-purple celery did not have this ability. This work provided evidence as a basis for investigations on the function of AgUCGalT1 in anthocyanin glycosylation in purple celery.

Main Conclusion: Different levels of salt stress affected the OAS-TL expression levels in Pancratium maritimum organs (bulb, leaf and root). A detailed method has been described for the identification of the conserved domain of the OAS-TL cDNA in sea daffodil given the scarce data available for the Amaryllidaceae family. Pancratium maritimum or sea daffodil (Amaryllidaceae) is a bulbous geophyte growing on coastal sands. In this study, we investigated the involvement of cysteine synthesis for salt tolerance through the expression of the enzyme O-acetylserine(thio)lyase (OAS-TL) during the stress response to NaCl treatments in P. maritimum. Quantitative real-time PCR was used in different organs (bulb, leaf and root).

Main Conclusion: Over-expression of AtFDH controlled by the promoter of Rubisco small subunit in chloroplasts increases formaldehyde uptake and metabolism in tobacco leaves. Our previous study showed that formaldehyde (HCHO) uptake and resistance in tobacco are weaker than in Arabidopsis. Formate dehydrogenase in Arabidopsis (AtFDH) is a key enzyme in HCHO metabolism by oxidation of HCOOH to CO 2 , which enters the Calvin cycle to be assimilated into glucose. HCHO metabolic mechanism in tobacco differs from that in Arabidopsis. In this study, AtFDH was over-expressed in the chloroplasts of transgenic tobacco using a light inducible promoter. 13 C-NMR analysis showed that the carbon flux from H 13 CHO metabolism was not introduced into the Calvin cycle to produce glucose in transgenic tobacco leaves. However, the over-expression of AtFDH significantly enhanced the HCHO metabolism in transgenic leaves. Consequently, the productions of [4- 13 C]Asn, [3- 13 C]Gln, [U- 13 C]oxalate, and H 13 COOH were notably greater in transgenic leaves than in non-transformed leaves after treatment with H 13 CHO. The increased stomatal conductance and aperture in transgenic leaves might be ascribed to the increased yield of oxalate in the guard cells with over-expressed AtFDH in chloroplasts. Accordingly, the transgenic plants exhibited a stronger capacity to absorb gaseous HCHO. Furthermore, the higher proline content in transgenic leaves compared with non-transformed leaves under HCHO stress might be attributable to the excess formate accumulation and Gln production. Consequently, the HCHO-induced oxidative stress was reduced in transgenic leaves.

Main Conclusion: PpORS knockout mutants produced abnormal leaves with increased dye permeability and were more susceptible to dehydration, consistent with PpORS products being constituents of a cuticular structure in the moss. Type III polyketide synthases (PKSs) have co-evolved with terrestrial plants such that each taxon can generate a characteristic collection of polyketides, fine-tuned to its needs. 2'-Oxoalkylresorcinol synthase from Physcomitrella patens (PpORS) is basal to all plant type III PKSs in phylogenetic trees and may closely resemble their most recent common ancestor. To gain insight into the roles that ancestral plant type III PKSs might have played during early land plant evolution, we constructed and phenotypically characterized targeted knockouts of PpORS. Ors gametophores, unless submerged in water while they were developing, displayed various leaf malformations that included grossly misshapen leaves, missing or abnormal midribs, multicellular protuberances and localized necrosis. Ors leaves, particularly abnormal ones, showed increased permeability to the hydrophilic dye, toluidine blue. Ors gametophores lost water faster and were more susceptible to dehydration than those of the control strain. Our findings are consistent with ors leaves possessing a partially defective cuticle and implicate PpORS in synthesis of the intact cuticle. PpORS orthologs are present in a few moss species but have not been found in other plants. However, conceivably an ancestral ORS in early land plants may have contributed to their protection from dehydration.

Main Conclusion: Post-transcriptional gene silencing (PTGS) of a chalcone synthase ( DvCHS2 ) occurred in the white part of bicolor petals and flavonoid-poor leaves; however, it did not in red petals and flavonoid-rich leaves. Petal color lability is a prominent feature of bicolor dahlia cultivars, and causes plants to produce not only original bicolor petals with colored bases and pure white tips, but also frequently single-colored petals without white tips. In this study, we analysed the molecular mechanisms that are associated with petal color lability using the red-white bicolor cultivar 'Yuino'. Red single-colored petals lose their white tips as a result of recover of flavonoid biosynthesis. Among flavonoid biosynthetic genes including four chalcone synthase (CHS)-like genes (DvCHS1, DvCHS2, DvCHS3, and DvCHS4), DvCHS1 and DvCHS2 had significantly lower expression levels in the white part of bicolor petals than in red petals, while DvCHS3, DvCHS4, and other flavonoid biosynthetic genes had almost the same expression levels. Small RNAs from the white part of a bicolor petal were mapped onto DvCHS1 and DvCHS2, while small RNAs from a red single-colored petal were not mapped onto any of the four CHS genes. A relationship between petal color and leaf flavonoid accumulation has previously been demonstrated, whereby red petal-producing plants accumulate flavonoids in their leaves, while bicolor petal-producing plants tend not to. The expression level of DvCHS2 was down-regulated in flavonoid-poor leaves and small RNAs from flavonoid-poor leaves were mapped onto DvCHS2, suggesting that the down-regulation of DvCHS2 in flavonoid-poor leaves occurs post-transcriptionally. Genomic analysis also suggested that DvCHS2 is the key gene involved in bicolor formation. Together, these results suggest that post-transcriptional gene silencing of DvCHS2 plays a key role in phenotypic lability in this bicolor dahlia.

Main Conclusion: Monoterpenoid indole alkaloids (MIAs) have remarkable biological properties that have led to their medical uses for a variety of human diseases. Mutagenesis has been used to generate plants with new alkaloid profiles and a useful screen for rapid comparison of MIA profiles is described. The MIA mutants identified are useful for investigating MIA biosynthesis and for targeted production of these specialised metabolites. The Madagascar periwinkle (Catharanthus roseus) is the sole source of the dimeric anticancer monoterpenoid indole alkaloids (MIAs), 3',4'-anhydrovinblastine and derivatives, which are formed via the coupling of the MIAs, catharanthine and vindoline. While intense efforts to identify parts of the complex pathways involved in the assembly of these dimers have been successful, our understanding of MIA biochemistry in C. roseus remains limited. A simple thin layer chromatography screen of 4000 ethyl methanesulfonate-metagenized M2 plants is described to identify mutant lines with altered MIA profiles. One mutant (M2-1865) accumulated reduced levels of vindoline inside the leaves in favour of high levels of tabersonine-2,3-epoxide and 16-methoxytabersonine-2,3-epoxide on the leaf surface. This MIA profile suggested that changes in tabersonine 3-reductase (T3R) activity might be responsible for the observed phenotype. Molecular cloning of mutant and wild type T3R revealed two nucleotide substitutions at cytosine residues 565 (CAT to TAT) and 903 (ACC to ACA) in the mutant corresponding to substitution (H189Y) and silent (T305T) amino acid mutations, respectively, in the protein. The single amino acid substitution in the mutant T3R protein diminished the biochemical activity of T3R by 95% that explained the reason for the low vindoline phenotype of the mutant. This phenotype was recessive and exhibited standard Mendelian single-gene inheritance. The stable formation and accumulation of epoxides in the M2-1865 mutant provides a dependable biological source of these two MIAs.

Main Conclusion: Carbonylation inactivates sorghum C 4 PEPCase while nitrosylation has little impact on its activity but holds back carbonylation. This interplay could be important to preserve photosynthetic C 4 PEPCase activity in salinity. Previous work had shown that nitric acid (NO) increased phosphoenolpyruvate carboxylase kinase (PEPCase-k) activity, promoting the phosphorylation of phosphoenolpyruvate carboxylase (PEPCase) in sorghum leaves (Monreal et al. in Planta 238:859-869, 2013b). The present work investigates the effect of NO on C 4 PEPCase in sorghum leaves and its interplay with carbonylation, an oxidative modification frequently observed under salt stress. The PEPCase of sorghum leaves could be carbonylated in vitro and in vivo, and this post-translational modification (PTM) was accompanied by a loss of its activity. Similarly, PEPCase could be S-nitrosylated in vitro and in vivo, and this PTM had little impact on its activity. The S-nitrosylated PEPCase showed increased resistance towards subsequent carbonylation, both in vitro and in vivo. Under salt shock, carbonylation of PEPCase increased in parallel with decreased S-nitrosylation of the enzyme. Subsequent increase of S-nitrosylation was accompanied by decreased carbonylation. Taken together, the results suggest that S-nitrosylation could contribute to maintain C 4 PEPCase activity in stressed sorghum plants. Thus, salt-induced NO synthesis would be protecting photosynthetic PEPCase activity from oxidative inactivation while promoting its phosphorylation, which will guarantee its optimal functioning in suboptimal conditions.

Main Conclusion: PP2A catalytic subunit C2 is of special importance for light/dark regulation of nitrate reductase activity. The level of unmethylated PP2A catalytic subunits decreases in darkness. Protein phosphatase 2A (PP2A) dephosphorylates and activates nitrate reductase (NR) in photosynthetically active tissue when plants are transferred from darkness to light. In the present work, investigation of Arabidopsis thaliana PP2A mutant lines revealed that one of the five PP2A catalytic subunit genes, e.g., C2, was of special importance for NR activation. Impairment of NR activation was, especially pronounced in the c2c4 double mutant. Though weaker, NR activation was also impaired in the c2 single mutant, and c1c2 and c2c5 double mutants. On the other hand, NR activation in the c4c5 double mutant was as efficient as in WT. The c4 single mutant had low PP2A activity, whereas the c2 single mutant possessed WT levels of extractable PP2A activity. PP2A activity was low in both c2c4 and c4c5. Differences in extracted PP2A activity among mutants did not strictly correlate with differences in NR activation, but underpinned that C2 has a special function in NR activation in vivo. The terminal leucine in PP2A catalytic subunits is generally methylated to a high degree, but regulation and impact of methylation/demethylation is barely studied. In WT and PP2A mutants, the level of unmethylated PP2A catalytic subunits decreased during 45 min of darkness, but did not change much when light was switched on. In leucine carboxyl methyl transferase1 (LCMT1) knockout plants, which possess mainly unmethylated PP2A, NR was still activated, although not fully as efficient as in WT.

Main Conclusion: This study provides new insights into the biosynthesis regulation and in planta function of the lignan yatein in flax leaves. Pinoresinol-lariciresinol reductases (PLR) catalyze the conversion of pinoresinol into secoisolariciresinol (SECO) in lignan biosynthesis. Several lignans are accumulated in high concentrations, such as SECO accumulated as secoisolariciresinol diglucoside (SDG) in seeds and yatein in aerial parts, in the flax plant (Linum usitatissimum L.) from which two PLR enzymes of opposite enantioselectivity have been isolated. While LuPLR1 catalyzes the biosynthesis of (+)-SECO leading to (+)-SDG in seeds, the role(s) of the second PLR (LuPLR2) is not completely elucidated. This study provides new insights into the in planta regulation and function of the lignan yatein in flax leaves: its biosynthesis relies on a different PLR with opposite stereospecificity but also on a distinct expression regulation. RNAi technology provided evidence for the in vivo involvement of the LuPLR2 gene in the biosynthesis of (-)-yatein accumulated in flax leaves. LuPLR2 expression in different tissues and in response to stress was studied by RT-qPCR and promoter-reporter transgenesis showing that the spatio-temporal expression of the LuPLR2 gene in leaves perfectly matches the (-)-yatein accumulation and that LuPLR2 expression and yatein production are increased by methyl jasmonate and wounding. A promoter deletion approach yielded putative regulatory elements. This expression pattern in relation to a possible role for this lignan in flax defense is discussed.

Main Conclusion: Regulation of a gene encoding coniferaldehyde 5-hydroxylase leads to substantial alterations in lignin structure in rice cell walls, identifying a promising genetic engineering target for improving grass biomass utilization. The aromatic composition of lignin greatly affects utilization characteristics of lignocellulosic biomass and, therefore, has been one of the primary targets of cell wall engineering studies. Limited information is, however, available regarding lignin modifications in monocotyledonous grasses, despite the fact that grass lignocelluloses have a great potential for feedstocks of biofuel production and various biorefinery applications. Here, we report that manipulation of a gene encoding coniferaldehyde 5-hydroxylase (CAld5H, or ferulate 5-hydroxylase, F5H) leads to substantial alterations in syringyl (S)/guaiacyl (G) lignin aromatic composition in rice (Oryza sativa), a major model grass and commercially important crop. Among three CAld5H genes identified in rice, OsCAld5H1 (CYP84A5) appeared to be predominantly expressed in lignin-producing rice vegetative tissues. Down-regulation of OsCAld5H1 produced altered lignins largely enriched in G units, whereas up-regulation of OsCAld5H1 resulted in lignins enriched in S units, as revealed by a series of wet-chemical and NMR structural analyses. Our data collectively demonstrate that OsCAld5H1 expression is a major factor controlling S/G lignin composition in rice cell walls. Given that S/G lignin composition affects various biomass properties, we contemplate that manipulation of CAld5H gene expression represents a promising strategy to upgrade grass biomass for biorefinery applications.

Main Conclusion: Fackel (FK) is involved in the flowering of Arabidopsis mainly via the gibberellin pathway and vernalization pathway. This new function of FK is partially dependent on the FLOWERING LOCUS C ( FLC ). A common transitional process from vegetative stage to reproductive stage exists in higher plants during their life cycle. The initiation of flower bud differentiation, which plays a key role in the reproductive phase, is affected by both external environmental and internal regulatory factors. In this study, we showed that the Arabidopsis weak mutant allele fk-J3158, impaired in the FACKEL (FK) gene, which encodes a C-14 reductase involved in sterol biosynthesis, had a long life cycle and delayed flowering time in different photoperiods. In addition, FK overexpression lines displayed an earlier flowering phenotype than that of the wild type. These processes might be independent of the downstream brassinosteroid (BR) pathway and the autonomous pathway. However, the fk-J3158 plants were more sensitive than wild type in reducing the bolting days and total leaf number under gibberellic acid (GA) treatment. Further studies suggested that FK mutation led to an absence of endogenous GAs in fk-J3158 and FK gene expression was also affected under GA and paclobutrazol (PAC) treatment. Moreover, the delayed flowering time of fk-J3158 could be rescued by a 3-week vernalization treatment, and the expression of FLOWERING LOCUS C (FLC) was accordingly down-regulated in fk-J3158. We also demonstrated that flowering time of fk-J3158 flc double mutant was significantly earlier than that of fk-J3158 under the long-day (LD) conditions. All these results indicated that FK may affect the flowering in Arabidopsis mainly via GA pathway and vernalization pathway. And these effects are partially dependent on the FLOWERING LOCUS C (FLC).

Main Conclusion: Acrolein is a lipid-derived highly reactive aldehyde, mediating oxidative signal and damage in plants. We found acrolein-scavenging glutathione transferase activity in plants and purified a low K M isozyme from spinach. Various environmental stressors on plants cause the generation of acrolein, a highly toxic aldehyde produced from lipid peroxides, via the promotion of the formation of reactive oxygen species, which oxidize membrane lipids. In mammals, acrolein is scavenged by glutathione transferase (GST; EC 2.5.1.18) isozymes of Alpha, Pi, and Mu classes, but plants lack these GST classes. We detected the acrolein-scavenging GST activity in four species of plants, and purified an isozyme showing this activity from spinach (Spinacia oleracea L.) leaves. The isozyme (GST-Acr), obtained after an affinity chromatography and two ion exchange chromatography steps, showed the K M value for acrolein 93 μM, the smallest value known for acrolein-detoxifying enzymes in plants. Peptide sequence homology search revealed that GST-Acr belongs to the GST Tau, a plant-specific class. The Arabidopsis thaliana GST Tau19, which has the closest sequence similar to spinach GST-Acr, also showed a high catalytic efficiency for acrolein. These results suggest that GST plays as a scavenger for acrolein in plants.

Main Conclusion: Class II and III chitinases belonging to different glycoside hydrolase families were major nectarins in Rhododendron irroratum floral nectar which showed significant chitinolytic activity. Previous studies have demonstrated antimicrobial activity in plant floral nectar, but the molecular basis for the mechanism is still poorly understood. Two chitinases, class II (Rhchi2) and III (Rhchi3), were characterized from alkaline Rhododendron irroratum nectar by both SDS-PAGE and mass spectrometry. Rhchi2 (27 kDa) and Rhchi3 (29 kDa) are glycoside hydrolases (family 19 and 18) with theoretical pI of 8.19 and 7.04. The expression patterns of Rhchi2 and Rhchi3 were analyzed by semi-quantitative RT-PCR. Rhchi2 is expressed in flowers (corolla nectar pouches) and leaves while Rhchi3 is expressed in flowers. Chitinase in concentrated protein and fresh nectar samples was visualised by SDS-PAGE and chitinolytic activity in fresh nectar was determined spectrophotometrically via chitin-azure. Full length gene sequences were cloned with Tail-PCR and RACE. The amino acid sequence deduced from the coding region for these proteins showed high identity with known chitinases and predicted to be located in extracellular space. Fresh R. irroratum floral nectar showed significant chitinolytic activity. Our results demonstrate that class III chitinase (GH 18 family) also exists in floral nectar. The functional relationship between class II and III chitinases and the role of these pathogenesis-related proteins in antimicrobial activity in nectar is suggested.

Main Conclusion: p -Methoxybenzoic acid carboxyl methyltransferase (MBMT) was isolated from loquat flowers. MBMT displayed high similarity to jasmonic acid carboxyl methyltransferases, but exhibited high catalytic activity to form methyl p -methoxybenzoate from p -methoxybenzoic acid. Volatile benzenoids impart the characteristic fragrance of loquat (Eriobotrya japonica) flowers. Here, we report that loquat produces methyl p-methoxybenzoate, along with other benzenoids, as the flowers bloom. Although the adaxial side of flower petals is covered with hairy trichomes, the trichomes are not the site of volatile benzenoid formation. Here we identified four carboxyl methyltransferase (EjMT1 to EjMT4) genes from loquat and functionally characterized EjMT1 which we found to encode a p-methoxybenzoic acid carboxyl methyltransferase (MBMT); an enzyme capable of converting p-methoxybenzoic acid to methyl p-methoxybenzoate via methylation of the carboxyl group. We found that transcript levels of MBMT continually increased throughout the flower development with peak expression occurring in fully opened flowers. Recombinant MBMT protein expressed in Escherichia coli showed the highest substrate preference toward p-methoxybenzoic acid with an apparent K m value of 137.3 µM. In contrast to benzoic acid carboxyl methyltransferase (BAMT) and benzoic acid/salicylic acid carboxyl methyltransferase, MBMT also displayed activity towards both benzoic acid and jasmonic acid. Phylogenetic analysis revealed that loquat MBMT forms a monophyletic group with jasmonic acid carboxyl methyltransferases (JMTs) from other plant species. Our results suggest that plant enzymes with same BAMT activity have evolved independently.

Main Conclusion: We show that changing the expression of a putative feruloyl transferase gene belonging to the BAHD acyl-transferase family alters the levels of cell wall esterified ferulates and diferulates in Brachypodium distachyon cell walls. While the potential of grass cell walls for biofuel production has been realized, the technology for lignocellulosic biomass conversion for the production of ethanol is still inefficient because of structural mechanisms that plants have evolved to make the cell wall recalcitrant to enzymatic attack. One of these mechanisms in grasses involves the esterification of arabinoxylans in the cell wall with ferulic acid via an ester linkage to arabinose side chains on xylans. These ferulates undergo oxidative coupling reactions to form ferulate dimers, thus crosslinking polysaccharides. Arabinoxylan feruloylation is an important factor that determines cell wall recalcitrance because it directly cross-links xylans and because ferulates act as nucleating sites for the formation of lignin and for the linkage of lignin to the xylan/cellulose network. Here we report on the effects of changing the expression of Bradi2g43520 (BdAT1), a homologue of the rice feruloyl transferase gene Os01g42880 belonging to the Pfam PF02458 family, in Brachypodium distachyon. Down regulation in several independent RNAi::BdAT1 lines, resulted in up to a 35 % reduction of ferulate levels in both leaves and stems compared to control plants, over 2-3 generations of selfing. In contrast, overexpression of putative BdAT1 resulted in an increase of up to 58 and 47 % of ferulate levels in leaves and stems, respectively, compared to control plants and analyzed over 2-3 generations of selfing. These findings suggest that Bradi2g43520 may be a good candidate for feruloylation of AX in Brachypodium.

Main Conclusion: Green tissue-specific simultaneous overexpression of two defense-related genes ( OsCHI11 & OsOXO4 ) in rice leads to significant resistance against sheath blight pathogen ( R. solani ) without distressing any agronomically important traits. Overexpressing two defense-related genes (OsOXO4 and OsCHI11) cloned from rice is effective at enhancing resistance against sheath blight caused by Rhizoctonia solani. These genes were expressed under the control of two different green tissue-specific promoters, viz. maize phosphoenolpyruvate carboxylase gene promoter, PEPC, and rice cis-acting 544-bp DNA element, immediately upstream of the D54O translational start site, P D54O-544 . Putative T0 transgenic rice plants were screened by PCR and integration of genes was confirmed by Southern hybridization of progeny (T1) rice plants. Successful expression of OsOXO4 and OsCHI11 in all tested plants was confirmed. Expression of PR genes increased significantly following pathogen infection in overexpressing transgenic plants. Following infection, transgenic plants exhibited elevated hydrogen peroxide levels, significant changes in activity of ROS scavenging enzymes and reduced membrane damage when compared to their wild-type counterpart. In a Rhizoctonia solani toxin assay, a detached leaf inoculation test and an in vivo plant bioassay, transgenic plants showed a significant reduction in disease symptoms in comparison to non-transgenic control plants. This is the first report of overexpression of two different PR genes driven by two green tissue-specific promoters providing enhanced sheath blight resistance in transgenic rice.

Main Conclusion: The total capacity of the GS-mediated ligation of free ammonium and glutamate to form glutamine in the leaves of maize plants is not impaired upon severe magnesium starvation. Magnesium deficiency does not obligatorily lead to the decreased total protein concentrations in the leaves. Magnesium (Mg) is an integral component of the enzyme glutamine synthetase (GS), having both a structural and a catalytic role. Moreover, Mg is relevant for the post-translational regulation of the GS. Glutamine synthetase is one of the key enzymes in nitrogen assimilation, ligating-free ammonium (NH4 (+)) to glutamate to form glutamine and it is therefore crucial for plant growth and productivity. This study was conducted in order to test whether a severe Mg-deficiency impairs the total capacity of the GS-catalyzed synthesis of glutamine in maize leaves. Maize was grown hydroponically and the GS activity was analyzed dependent on different leaf developmental stages. Glutamine synthetase activity in vitro assays in combination with immune-dot blot analysis revealed that both the total activity and the abundance of glutamine synthetase was not impaired in the leaves of maize plants upon 54 days of severe Mg starvation. Additionally, it was shown that Mg deficiency does not obligatorily lead to decreased total protein concentrations in the leaves, as assayed by Bradford protein quantification. Moreover, Mg resupply to the roots or the leaves of Mg-deficient plants reversed the Mg-deficiency-induced accumulation of free amino acids in older leaves, which indicates impaired phloem loading. The results of our study reveal that the total GS-mediated primary or secondary assimilation of free NH4 (+) is not a limiting enzymatic reaction under Mg deficiency and thus cannot be accountable for the observed restriction of plant growth and productivity in Mg-deficient maize.

Main Conclusion: Of the three functional FPPS identified in maize, fpps3 is induced by herbivory to produce FDP important for the formation of the volatile sesquiterpenes of plant defense. Sesquiterpenes are not only crucial for the growth and development of a plant but also for its interaction with the environment. The biosynthesis of sesquiterpenes proceeds over farnesyl diphosphate (FDP), which is either used as a substrate for protein prenylation, converted to squalene, or to volatile sesquiterpenes. To elucidate the regulation of sesquiterpene biosynthesis in maize, we identified and characterized the farnesyl diphosphate synthase (FPPS) gene family which consists of three genes. Synteny analysis indicates that fpps2 and fpps3 originate from a genome duplication in an ancient tetraploid ancestor. The three FPPSs encode active enzymes that produce predominantly FDP from the isopentenyl diphosphate and dimethylallyl diphosphate substrates. Only fpps1 and fpps3 are induced by elicitor treatment, but induced fpps1 levels are much lower and only increased to the amounts of fpps3 levels in intact leaves. Elicitor-induced fpps3 levels in leaves increase to more than 15-fold of background levels. In undamaged roots, transcript levels of fpps1 are higher than those of fpps3, but only fpps3 transcripts are induced in response to herbivory by Diabrotica virgifera virgifera. A kinetic of transcript abundance in response to herbivory in leaves provided further evidence that the regulation of fpps3 corresponds to that of tps23, a terpene synthase, that converts FDP to the volatile (E)-ß-caryophyllene. Our study indicates that the differential expression of fpps1 and fpps3 provides maize with FDP for both primary metabolism and terpene-based defenses. The expression of fpps3 seems to coincide with the herbivore-induced emission of volatile sesquiterpenes that were demonstrated to be important defense signals.

Main Conclusion: The AOX pathway in C. protothecoides plays an important role in the photoprotection of PSII by alleviating the inhibition of the repair of the photodamaged PSII during H2 photoproduction. We had demonstrated that nitrogen limitation (LN) substantially enhanced H2 photoproduction in Chlorella protothecoides. In the present study, the mitochondrial alternative oxidase (AOX) pathway capacity was found to increase significantly during H2 photoproduction under LN or under LN simultaneously with sulfur deprivation (LNS) conditions. The purpose of this study was to clarify the role of the AOX pathway during H2 photoproduction in C. protothecoides. The AOX pathway can affect H2 photoproduction in the following ways: (1) consuming O2, which is favorable for the establishment of anaerobiosis; (2) consuming NADPH and competing with hydrogenase for photosynthetic electrons, which would decrease the H2 photoproduction; (3) protecting photosystem (PS) II, which is a direct electron source for H2 photoproduction, from photoinhibition. In LN and LNS cultures, the inhibition of the AOX pathway reduced the H2 photoproduction significantly, and did not increase the amount of O2. But, the inhibition of the AOX pathway decreased the maximal photochemical efficiency of PSII (F v/F m) and the actual photochemical efficiency of PSII (Φ PSII) significantly, leading to photoinhibition, which would decrease the photosynthetic electrons transferred to hydrogenase. And, the inhibition of the AOX pathway did not change the level of photoinhibition in the presence of D1 protein synthesis inhibitor chloramphenicol, indicating that the inhibition of the AOX pathway did not accelerate the photodamage to PSII directly but inhibited the repair of the photodamaged PSII. Therefore, the mitochondrial AOX pathway in C. protothecoides plays an important role in the photoprotection of PSII by alleviating the inhibition of the repair of the photodamaged PSII during H2 photoproduction, which is thus able to supply more electrons to hydrogenase under LN and LNS conditions.

Main Conclusion: Rice heme oxygenase 2 (OsHO2) mutants are chlorophyll deficient with distinct tetrapyrrole metabolite and transcript profiles, suggesting a potential regulatory role of the stromal-localized OsHO2 in tetrapyrrole biosynthesis. In plants, heme oxygenases (HOs) are classified into the subfamilies HO1 and HO2. HO1 are highly conserved plastid enzymes required for synthesizing the chromophore in phytochromes which mediate a number of light-regulated responses. However, the physiological and biochemical functions of HO2, which are distantly related to HO1, are not well understood, especially in crop plants. From a population of (60)Coγ-irradiated rice mutants, we identified the ylc2 (young leaf chlorosis 2) mutant which displays a chlorosis phenotype in seedlings with substantially reduced chlorophyll content. Normal leaf pigmentation is gradually restored in older plants while newly emerged leaves remain yellow. Transmission electron microscopy further revealed defective chloroplast structures in the ylc2 seedlings. Map-based cloning located the OsYLC2 gene on chromosome 3 and it encodes the OsHO2 protein. The gene identification was confirmed by complementation and T-DNA mutant analyses. Subcellular localization and chloroplast fractionation experiments indicated that OsHO2 resides in the stroma. However, recombinant enzyme assay demonstrated that OsHO2 is not a functional HO enzyme. Analysis of tetrapyrrole metabolites revealed the reduced levels of most chlorophyll and phytochromobilin precursors in the ylc2 mutant. On the other hand, elevated accumulation of 5-aminolevulinic acid and Mg-protoporphyrin IX was observed. These unique metabolite changes are accompanied by consistent changes in the expression levels of the corresponding tetrapyrrole biosynthesis genes. Taken together, our work suggests that OsHO2 has a potential regulatory role for tetrapyrrole biosynthesis in rice.

Main Conclusion: The present study demonstrates the first direct evidence of the novel role of OsACA6 in providing Cd (2+) stress tolerance in transgenic tobacco by maintaining cellular ion homeostasis and modulating ROS-scavenging pathway. Cadmium, a non-essential toxic heavy metal, interferes with the plant growth and development. It reaches the leaves through xylem and may become part of the food chain, thus causing detrimental effects to human health. Therefore, there is an urgent need to develop strategies for engineering plants for Cd(2+) tolerance and less accumulation. The members of P-type ATPases family transport metal ions including Cd(2+), and thus play important role an ion homeostasis. The present study elucidates the role of P-type 2B Ca(2+) ATPase (OsACA6) in Cd(2+) stress tolerance. The transcript levels of OsACA6 were up-regulated upon Cd(2+), Zn(2+) and Mn(2+) exposure. Transgenic tobacco expressing OsACA6 showed tolerance towards Cd(2+) stress as demonstrated by several physiological indices including root length, biomass, chlorophyll, malondialdehyde and hydrogen peroxide content. The roots of the transgenic lines accumulated more Cd(2+) as compared to shoot. Further, confocal laser scanning microscopy showed that Cd(2+) exposure altered Ca(2+) uptake in OsACA6 transgenic plants. OsACA6 expression in tobacco also protected the transgenic plants from oxidative stress by enhancing the activity of enzymatic (SOD, CAT, APX, GR) and non-enzymatic (GSH and AsA) antioxidant machinery. Transgenic lines also tolerated Zn(2+) and Mn(2+) stress; however, tolerance for these ions was not as significant as observed for Cd(2+) exposure. Thus, overexpression of OsACA6 confers Cd(2+) stress tolerance in transgenic lines by maintaining cellular ion homeostasis and modulating reactive oxygen species (ROS)-scavenging pathway. The results of the present study will help to develop strategies for engineering Cd(2+) stress tolerance in economically important crop plants.

Phospholipase D (PLD) is crucial for plant responses to stress and signal transduction, however, the regulatory mechanism of PLD in abiotic stress is not completely understood; especially, in crops. In this study, we isolated a gene, TaPLDα, from common wheat (Triticum aestivum L.). Analysis of the amino acid sequence of TaPLDα revealed a highly conserved C2 domain and two characteristic HKD motifs, which is similar to other known PLD family genes. Further characterization revealed that TaPLDα expressed differentially in various organs, such as roots, stems, leaves and spikelets of wheat. After treatment with abscisic acid (ABA), methyl jasmonate, dehydration, polyethylene glycol and NaCl, the expression of TaPLDα was up-regulated in shoots. Subsequently, we generated TaPLDα-overexpressing transgenic Arabidopsis lines under the control of the dexamethasone-inducible 35S promoter. The overexpression of TaPLDα in Arabidopsis resulted in significantly enhanced tolerance to drought, as shown by reduced chlorosis and leaf water loss, higher relative water content and lower relative electrolyte leakage than the wild type. Moreover, the TaPLDα-overexpressing plants exhibited longer roots in response to mannitol treatment. In addition, the seeds of TaPLDα-overexpressing plants showed hypersensitivity to ABA and osmotic stress. Under dehydration, the expression of several stress-related genes, RD29A, RD29B, KIN1 and RAB18, was up-regulated to a higher level in TaPLDα-overexpressing plants than in wild type. Taken together, our results indicated that TaPLDα can enhance tolerance to drought and osmotic stress in Arabidopsis and represents a potential candidate gene to enhance stress tolerance in crops.

Many plants accumulate hydroxycinnamoyl esters to protect against abiotic and biotic stresses. Caffeoyl esters in particular can be substrates for endogenous polyphenol oxidases (PPOs). Recently, we showed that perennial peanut (Arachis glabrata Benth.) leaves contain PPO and identified one PPO substrate, caftaric acid (trans-caffeoyl-tartaric acid). Additional compounds were believed to be cis- and trans-p-coumaroyl tartaric acid and cis- and trans-feruloyl-tartaric acid, but lack of standards prevented definitive identifications. Here we characterize enzymatic activities in peanut leaves to understand how caftaric acid and related hydroxycinnamoyl esters are made in this species. We show that peanut leaves contain a hydroxycinnamoyl-CoA:tartaric acid hydroxycinnamoyl transferase (HTT) activity capable of transferring p-coumaroyl, caffeoyl, and feruloyl moieties from CoA to tartaric acid (specific activities of 11 ± 2.8, 8 ± 1.8, 4 ± 0.8 pkat mg(-1) crude protein, respectively). The HTT activity was used to make cis- and trans-p-coumaroyl- and -feruloyl-tartaric acid in vitro. These products allowed definitive identification of the corresponding cis- and trans-hydroxycinnamoyl esters extracted from leaves. We tentatively identified sinapoyl-tartaric acid as another major phenolic compound in peanut leaves that likely participates in secondary reactions with PPO-generated quinones. These results suggest hydroxycinnamoyl-tartaric acid esters are made by an acyltransferase, possibly a BAHD family member, in perennial peanut. Identification of a gene encoding HTT and further characterization of the enzyme will aid in identifying determinants of donor and acceptor substrate specificity for this important class of biosynthetic enzymes. An HTT gene could also provide a means by genetic engineering for producing caffeoyl- and other hydroxycinnamoyl-tartaric acid esters in forage crops that lack them.

Nitric oxide (NO) is a signaling molecule that mediates many plant responses to biotic and abiotic stresses, including salt stress. Interestingly, salinity increases NO production selectively in mesophyll cells of sorghum leaves, where photosynthetic C₄ phosphoenolpyruvate carboxylase (C₄ PEPCase) is located. PEPCase is regulated by a phosphoenolpyruvate carboxylase-kinase (PEPCase-k), which levels are greatly enhanced by salinity in sorghum. This work investigated whether NO is involved in this effect. NO donors (SNP, SNAP), the inhibitor of NO synthesis NNA, and the NO scavenger cPTIO were used for long- and short-term treatments. Long-term treatments had multifaceted consequences on both PPCK gene expression and PEPCase-k activity, and they also decreased photosynthetic gas-exchange parameters and plant growth. Nonetheless, it could be observed that SNP increased PEPCase-k activity, resembling salinity effect. Short-term treatments with NO donors, which did not change photosynthetic gas-exchange parameters and PPCK gene expression, increased PEPCase-k activity both in illuminated leaves and in leaves kept at dark. At least in part, these effects were independent on protein synthesis. PEPCase-k activity was not decreased by short-term treatment with cycloheximide in NaCl-treated plants; on the contrary, it was decreased by cPTIO. In summary, NO donors mimicked salt effect on PEPCase-k activity, and scavenging of NO abolished it. Collectively, these results indicate that NO is involved in the complex control of PEPCase-k activity, and it may mediate some of the plant responses to salinity.

Carnivory in plants evolved as an adaptation strategy to nutrient-poor environments. Thanks to specialized traps, carnivorous plants can gain nutrients from various heterotrophic sources such as small insects. Digestion in traps requires a coordinated action of several hydrolytic enzymes that break down complex substances into simple absorbable nutrients. Among these, several pathogenesis-related proteins including β-1,3-glucanases have previously been identified in digestive fluid of some carnivorous species. Here we show that a single acidic endo-β-1,3-glucanase of ~50 kDa is present in the digestive fluid of the flypaper-trapped sundew (Drosera rotundifolia L.). The enzyme is inducible with a complex plant β-glucan laminarin from which it releases simple saccharides when supplied to leaves as a substrate. Moreover, thin-layer chromatography of digestive exudates showed that the simplest degradation products (especially glucose) are taken up by the leaves. These results for the first time point on involvement of β-1,3-glucanases in digestion of carnivorous plants and demonstrate the uptake of saccharide-based compounds by traps. Such a strategy could enable the plant to utilize other types of nutritional sources e.g., pollen grains, fungal spores or detritus from environment. Possible multiple roles of β-1,3-glucanases in the digestive fluid of carnivorous sundew are also discussed.

The plant hormone ethylene is known to affect various developmental processes including dormancy and growth. Yet, little information is available about the role of ethylene during paradormancy release in underground adventitious buds of leafy spurge. In this study, we examined changes in ethylene evolution and the ethylene biosynthetic enzyme ACC oxidase following paradormancy release (growth induction). Our results did not show an obvious increase in ethylene during bud growth. However, when buds were incubated with 1 mM ACC, ethylene levels were higher in growing than non-growing buds, suggesting that the levels of ACC oxidase increased in growing buds. Real-time qPCR indicated that the transcript of a Euphorbia esula ACC oxidase (Ee-ACO) increased up to threefold following growth induction. In addition, a 2.5- to 4-fold increase in ACO activity was observed 4 days after decapitation, and the Ee-ACO accounted for 40 % of the total ACO activity. Immunoblot analyses identified a 36-kD Ee-ACO protein that increased in expression during bud growth. This protein was highly expressed in leaves, moderately expressed in crown buds, stems and meristems, and weakly expressed in roots and flowers. Immunolocalization of Ee-ACO on growing bud sections revealed strong labeling of the nucleus and cytoplasm in cells at the shoot apical meristem and leaf primordia. An exception to this pattern occurred in cells undergoing mitosis, where labeling of Ee-ACO was negligible. Taken together, our results indicated an increase in the levels of Ee-ACO during paradormancy release of leafy spurge that was not correlated with an increase in ethylene synthesis.

Salinity increases phosphoenolpyruvate carboxylase kinase (PEPCase-k) activity in sorghum leaves. This work has been focused on the mechanisms responsible for this phenomenon. The light-triggered expression of SbPPCK1 gene, accountable for the photosynthetic C4-PEPCase-k, is controlled by a complex signal transduction chain involving phospholipases C and D (PLC and PLD). These two phospholipase-derived signalling pathways were functional in salinized plants. Pharmacological agents that act on PLC (U-73122, neomycin) or PLD (n-butanol) derived signals, blocked the expression of SbPPCK1, but had little effect on PEPCase-k activity. This discrepancy was further noticed when SbPPCK1-3 gene expression and PEPCase-k activity were studied in parallel. At 172 mM, the main effect of NaCl was to decrease the rate of PEPCase-k protein turnover. Meanwhile, 258 mM NaCl significantly increased both SbPPCK1 and SbPPCK2 gene expression and/or mRNA stability. The combination of these factors contributed to maintain a high PEPCase-k activity in salinity. LiCl increased calcium-dependent protein kinase (CDPK) activity in illuminated sorghum leaves while it decreased the rate of PEPCase-k degradation. The latter effect was restrained by W7, an inhibitor of CDPK activity. Recombinant PEPCase-k protein was phosphorylated in vitro by PKA. A conserved phosphorylation motif, which can be recognized by PKA and by plant CDPKs, is present in the three PEPCase-ks proteins. Thus, it is possible that a phosphorylation event could be controlling (increasing) the stability of PEPCase-k in salinity. These results propose a new mechanism of regulation of PEPCase-k levels, and highlight the relevance of the preservation of key metabolic elements during the bulk degradation of proteins, which is commonly associated to stress.

Calcium-dependent protein kinases (CDPKs) are key components of calcium regulated signaling cascades in plants. In this work, isoform StCDPK3 from Solanum tuberosum was studied and fully described. StCDPK3 encodes a 63 kDa protein with an N-terminal variable domain (NTV), rich in prolines and glutamines, which presents myristoylation and palmitoylation consensus sites and a PEST sequence indicative of rapid protein degradation. StCDPK3 gene (circa 11 kb) is localized in chromosome 3, shares the eight exons and seven introns structure with other isoforms from subgroup IIa and contains an additional intron in the 5'UTR region. StCDPK3 expression is ubiquitous being transcripts more abundant in early elongating stolons (ES), leaves and roots, however isoform specific antibodies only detected the protein in leaf particulate extracts. The recombinant 6xHis-StCDPK3 is an active kinase that differs in its kinetic parameters and calcium requirements from StCDPK1 and 2 isoforms. In vitro, StCDPK3 undergoes autophosphorylation regardless of the addition of calcium. The StCDPK3 promoter region (circa 1,800 bp) was subcloned by genome walking and fused to GUS. Light and ABRE responsive elements were identified in the promoter region as well as elements associated to expression in roots. StCDPK3 expression was enhanced by ABA while GA decreased it. Potato transgenic lines harboring StCDPK3 promoter∷GUS construct were generated by Agrobacterium tumefaciens mediated plant transformation. Promoter activity was detected in leaves, root tips and branching points, early ES, tuber eyes and developing sprouts indicating that StCDPK3 is expressed in actively growing organs.

In order to obtain a tuberous root-specific promoter to be used in the transformation of cassava, a 1,728 bp sequence containing the cassava granule-bound starch synthase (GBSSI) promoter was isolated. The sequence proved to contain light- and sugar-responsive cis elements. Part of this sequence (1,167 bp) was cloned into binary vectors to drive expression of the firefly luciferase gene. Cassava cultivar Adira 4 was transformed with this construct or a control construct in which the luciferase gene was cloned behind the 35S promoter. Luciferase activity was measured in leaves, stems, roots and tuberous roots. As expected, the 35S promoter induced luciferase activity in all organs at similar levels, whereas the GBSSI promoter showed very low expression in leaves, stems and roots, but very high expression in tuberous roots. These results show that the cassava GBSSI promoter is an excellent candidate to achieve tuberous root-specific expression in cassava.

An endo-xylanase from Trichoderma reesei (xyn2) has been expressed in tall fescue targeted to the vacuole, apoplast or Golgi, constitutively under the control of the rice actin promoter, and to the apoplast under the control of a senescence enhanced gene promoter. Constitutive xylanase expression in the vacuole, apoplast, and golgi, resulted in only a small number of plants with low enzyme activities and in reduced plant growth in apoplast, and golgi targeted plants. Constitutive expression in the apoplast also resulted in increased levels of cell wall bound hydroxycinnamic acid monomers and dimers, but no significant effect on cell wall xylose or arabinose content. In situ constitutive xylanase expression in the Golgi also resulted in increased ferulate dimers. However, senescence induced xylanase expression in the apoplast was considerably higher and did not affect plant growth or the level of monomeric hydroxycinnamic acids or lignin in the cell walls. These plants also showed increased levels of ferulate dimers, and decreased levels of xylose with increased levels of arabinose in their cell walls. While the release of cell wall hydroxycinnamic acids on self digestion was enhanced in these plants in the presence of exogenously applied ferulic acid esterase, changes in cell wall composition resulted in decreases in both tissue digestibility and cellulase mediated sugar release. In situ detection of H(2)O(2) production mediated by ethylene release in leaves of plants expressing apoplast xylanase could be leading to increased dimerisation. High-level xylanase expression in the apoplast also resulted in necrotic lesions on the leaves. Together these results indicate that xylanase expression in tall fescue may be triggering plant defence responses analogous to foliar pathogen attack mediated by ethylene and H(2)O(2).

β-Carotene ketolase (BKT) catalyzes the rate-limiting steps for the biosynthesis of astaxanthin. Several bkt genes have been isolated and explored to modify plant carotenoids to astaxanthin with limited success. In this study, five algal BKT cDNAs were isolated and characterized for the engineering of high-yield astaxanthin in plants. The products of the cDNAs showed high similarity in sequence and enzymatic activity of converting β-carotene into canthaxanthin. However, the enzymes exhibited extremely different activities in converting zeaxanthin into astaxanthin. Chlamydomonas reinhardtii BKT showed the highest conversion rate (ca 85%), whereas, Neochloris wimmeri BKT exhibited very poor activity of ketolating zeaxanthin. Expression of C. reinhardtii BKT in tobacco led to a twofold increase of total carotenoids in the leaves with astaxanthin being the predominant. The bkt genes described here provide a valuable resource for metabolic engineering of plants as cell factories for astaxanthin production.

During the last decade, there has been growing interest in the role of trehalose metabolism in tolerance to abiotic stress in higher plants, especially cold stress. So far, this metabolism has not yet been studied in Vitis vinifera L., despite the economic importance of this crop. The goal of this paper was to investigate the involvement of trehalose metabolism in the response of grapevine to chilling stress, and to compare the response in plants bacterised with Burkholderia phytofirmans strain PsJN, a plant growth-promoting rhizobacterium that confers grapevine chilling tolerance, with mock-inoculated plants. In silico analysis revealed that the V. vinifera L. genome contains genes encoding the enzymes responsible for trehalose synthesis and degradation. Transcript analysis showed that these genes were differentially expressed in various plant organs, and we also characterised their response to chilling. Both trehalose and trehalose 6-phosphate (T6P) were present in grapevine tissues and showed a distinct pattern of accumulation upon chilling. Our results suggest a role for T6P as the main active molecule in the metabolism upon chilling, with a possible link with sucrose metabolism. Furthermore, plants colonised by B. phytofirmans and cultivated at 26°C accumulated T6P and trehalose in stems and leaves at concentrations similar to non-bacterised plants exposed to chilling temperatures for 1 day. Overall, our data suggest that T6P and trehalose accumulate upon chilling stress in grapevine and might participate in the resistance to chilling stress conferred by B. phytofirmans.

Potato steroidal glycoalkaloids (SGAs) are toxic secondary metabolites whose total content in tubers must be regulated. SGAs are biosynthesized by the sterol branch of the mevalonic acid/isoprenoid pathway. In a previous study, we showed a correlation between SGA levels and the abundance of transcript coding for HMG-CoA reductase 1 (HMG1) and squalene synthase 1 (SQS1) in potato tissues and potato genotypes varying in SGA content. Here, Solanum tuberosum cv. Desirée (low SGA producer) was transformed with a gene construct containing the coding region of either HMG1 or SQS1 of Solanum chacoense Bitt. clone 8380-1, a high SGA producer. SGA levels in transgenic HMG-plants were either greater than (in eight of 14 plants) or no different from untransformed controls, whereas only four of 12 SQS-transgenics had greater SGA levels than control, as determined by HPLC. Quantitative real-time PCR was used to estimate relative steady-state transcript levels of isoprenoid-, steroid-, and SGA-related genes in leaves of the transgenic plants compared to nontransgenic controls. HMG-transgenic plants exhibited increased transcript accumulation of SQS1, sterol C24-methyltransferase type1 (SMT1), and solanidine glycosyltransferase 2 (SGT2), whereas SQS-transgenic plants, had consistently lower transcript levels of HMG1 and variable SMT1 and SGT2 transcript abundance among different transgenics. HMG-transgenic plants exhibited changes in transcript accumulation for some sterol biosynthetic genes as well. Taken together, the data suggest coordinated regulation of isoprenoid metabolism and SGA secondary metabolism.

Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris.

Biosynthesis of the p-menthane monoterpenes in peppermint occurs in the secretory cells of the peltate glandular trichomes and results in the accumulation of primarily menthone and menthol. cDNAs and recombinant enzymes are well characterized for eight of the nine enzymatic steps leading from the 5-carbon precursors to menthol, and subcellular localization of several key enzymes suggests a complex network of substrate and product movement is required during oil biosynthesis. In addition, studies concerning the regulation of oil biosynthesis have demonstrated a temporal partition of the pathway into an early, biosynthetic program that results in the accumulation of menthone and a later, oil maturation program that leads to menthone reduction and concomitant menthol accumulation. The menthone reductase responsible for the ultimate pathway reduction step, menthone-menthol reductase (MMR), has been characterized and found to share significant sequence similarity with its counterpart reductase, a menthone-neomenthol reductase, which catalyzes a minor enzymatic reaction associated with oil maturation. Further, the menthone reductases share significant sequence similarity with the temporally separate and mechanistically different isopiperitenone reductase (IPR). Here we present immunocytochemical localizations for these reductases using a polyclonal antibody raised against menthone-menthol reductase. The polyclonal antibody used for this study showed little specificity between these three reductases, but by using it for immunostaining of tissues of different ages we were able to provisionally separate staining of an early biosynthetic enzyme, IPR, found in young, immature leaves from that of the oil maturation enzyme, MMR, found in older, mature leaves. Both reductases were localized to the cytoplasm and nucleoplasm of the secretory cells of peltate glandular trichomes, and were absent from all other cell types examined.

Plastid genome transformation offers an attractive methodology for transgene expression in plants, but for potato, only expression of gfp transgene (besides the selective gene aadA) has been published. We report here successful expression of β-glucuronidase in transplastomic Solanum tuberosum (var. Desiree) plants, with accumulation levels for the recombinant protein of up to 41% of total soluble protein in mature leaves. To our knowledge, this is the highest expression level reported for a heterologous protein in S. tuberosum. Accumulation of the recombinant protein in soil-grown minitubers was very low, as described in previous reports. Interestingly, microtubers developed in vitro showed higher accumulation of β-glucuronidase. As light exposure during their development could be the trigger for this high accumulation, we analyzed the effect of light on β-glucuronidase accumulation in transplastomic tubers. Exposure to light for 8 days increased β-glucuronidase accumulation in soil-grown tubers, acting as a light-inducible expression system for recombinant protein accumulation in tuber plastids. In this paper we show that plastid transformation in potato allows the highest recombinant protein accumulation in foliar tissue described so far for this food crop. We also demonstrate that in tubers high accumulation is possible and depends on light exposure. Because tubers have many advantages as protein storage organs, these results could lead to new recombinant protein production schemes based on potato.

Despite extensive research over the past years, regeneration from protoplasts has been observed in only a limited number of plant species. Protoplasts undergo complex metabolic modification during their isolation. The isolation of protoplasts induces reactive oxygen species (ROS) generation in Brassica napus leaf protoplasts. The present study was conducted to provide new insight into the mechanism of ROS generation in B. napus leaf protoplasts. In vivo localization of H(2)O(2) and enzymes involved in H(2)O(2) generation and detoxification, molecular antioxidant-ascorbate and its redox state and lipid peroxidation were investigated in the leaf and isolated protoplasts. Incubating leaf strips in the macerating enzyme (ME) for different duration (3, 6, and 12 h) induced accumulation of H(2)O(2) and malondialdehyde (lipid peroxidation, an index of membrane damage) in protoplasts. The level of H(2)O(2) was highest just after protoplast isolation and subsequently decreased during culture. Superoxide generating NADPH oxidase (NOX)-like activity was enhanced, whereas superoxide dismutase (SOD) and ascorbate peroxidase (APX) decreased in the protoplasts compared to leaves. Diaminobenzidine peroxidase (DAB-POD) activity was also lower in the protoplasts compared to leaves. Total ascorbate content, ascorbate to dehydroascorbate ratio (redox state), were enhanced in the protoplasts compared to leaves. Higher activity of NOX-like enzyme and weakening in the activity of antioxidant enzymes (SOD, APX, and DAB-POD) in protoplasts resulted in excessive accumulation of H(2)O(2) in chloroplasts of protoplasts. Chloroplastic NADPH oxidase-like activity mediated perpetual H(2)O(2) generation probably induced apoptotic-like cell death of B. napus leaf protoplasts as indicated by parallel DNA laddering and decreased mitochondrial membrane potential.

Grapes berries produce and accumulate many reactive secondary metabolites, and encounter a wide range of pathogen- and human-derived xenobiotic compounds. The enzymatic glucosylation of these metabolites changes their reactivity, stability and subcellular location. Two ESTs with more than 90% nucleotide sequence identity to three full-length glucosyltransferases are expressed in several grape tissues. The full-length clones have more than 60% amino acid sequence similarity to previously characterized flavonoid 7-O-glucosyltransferases, catechin O-glucosyltransferases and anthocyanin 5-O-glucosyltransferases. In vitro, these enzymes glucosylate flavonols and the xenobiotic 2,4,5-trichlorophenol (TCP). Kinetic analysis indicates that TCP is the preferred substrate for these enzymes, while expression analysis reveals variable transcription of these genes in grape leaves, flowers and berry tissues. The in vivo role of these Vitis labrusca glucosyltransferases is discussed.

Glycine betaine (GB) is a compatible solute accumulated by many plants under various abiotic stresses. GB is synthesized in two steps, choline → betaine aldehyde → GB, where a functional choline-oxidizing enzyme has only been reported in Amaranthaceae (a chloroplastic ferredoxin-dependent choline monooxygenase) thus far. Here, we have cloned a cDNA encoding a choline monooxygenase (CMO) from barley (Hordeum vulgare) plants, HvCMO. In barley plants under non-stress condition, GB had accumulated in all the determined organs (leaves, internodes, awn and floret proper), mostly in the leaves. The expression of HvCMO protein was abundant in the leaves, whereas the expression of betaine aldehyde dehydrogenase (BADH) protein was abundant in the awn, floret proper and the youngest internode than in the leaves. The accumulation of HvCMO mRNA was increased by high osmotic and low-temperature environments. Also, the expression of HvCMO protein was increased by the presence of high NaCl. Immunofluorescent labeling of HvCMO protein and subcellular fractionation analysis showed that HvCMO protein was localized to peroxisomes. [(14)C]choline was oxidized to betaine aldehyde and GB in spinach (Spinacia oleracea) chloroplasts but not in barley, which indicates that the subcellular localization of choline-oxidizing enzyme is different between two plant species. We investigated the choline-oxidizing reaction using recombinant HvCMO protein expressed in yeast (Saccharomyces cerevisiae). The crude extract of HvCMO-expressing yeast coupled with recombinant BBD2 protein converted [(14)C]choline to GB when NADPH was added as a cofactor. These results suggest that choline oxidation in GB synthesis is mediated by a peroxisomal NADPH-dependent choline monooxygenase in barley plants.

Artemisia annua L. is the only natural resource that produces artemisinin (Qinghaosu), an endoperoxide sesquiterpene lactone used in the artemisinin-combination therapy of malaria. The cross-hybridization properties of A. annua do not favor studying artemisinin biosynthesis. To overcome this problem, in this study, we report on selection of self-pollinated A. annua plants and characterize their development and artemisinin biosynthesis. Self-pollinated F2 plants selected were grown under optimized growth conditions, consisting of long day (16 h of light) and short day (9 h of light) exposures in a phytotron. The life cycles of these plants were approximately 3 months long, and final heights of 30-35 cm were achieved. The leaves on the main stems exhibited obvious morphological changes, from indented single leaves to odd, pinnately compound leaves. Leaves and flowers formed glandular and T-shaped trichomes on their surfaces. The glandular trichome densities increased from the bottom to the top leaves. High performance liquid chromatography-mass spectrometry-based metabolic profiling analyses showed that leaves, flowers, and young seedlings of F2 plants produced artemisinin. In leaves, the levels of artemisinin increased from the bottom to the top of the plants, showing a positive correlation to the density increase of glandular trichomes. RT-PCR analysis showed that progeny of self-pollinated plants expressed the amorpha-4, 11-diene synthase (ADS) and cytochrome P450 monooxygenase 71 AV1 (CYP71AV1) genes, which are involved in artemisinin biosynthesis in leaves and flowers. The use of self-pollinated A. annua plants will be a valuable approach to the study of artemisinin biosynthesis.

The effects of cadmium (Cd) on aminopeptidase (AP) activities and Leucine-AP (LAP) expression were investigated in the roots of tomato (Solanum lycopersicum L., var Ibiza) plants. Three-week-old plants were grown for 10 days in the presence of 0.3-300 μM Cd and compared to control plants grown in the absence of Cd. AP activities were measured using six different p-nitroanilide (p-NA) substrates. Leu, Met, Arg, Pro and Lys hydrolyzing activities increased in roots of Cd-treated plants, while Phe-pNA cleavage was not enhanced after Cd treatments. The use of peptidase inhibitors showed that most of the Leu-pNA hydrolyzing activity was related to one or several metallo-APs. Changes in Lap transcripts, protein and activities were measured in the roots of 0 and 30-μM Cd-treated plants. LapA transcript levels increased in Cd-treated roots, whereas LapN RNAs levels were not modified. To assess amount of Leu-pNA hydrolyzing activity associated with the hexameric LAPs, LAP activity was measured following immunoprecipitation with a LAP polyclonal antiserum. LAP activity increased in Cd-treated roots. There was a corresponding increase in LAP-A protein levels detected in 2D-immunoblots. The role of LAP-A in the proteolytic response to Cd stress is discussed.