Mutation of AtPME2, a pH-Dependent Pectin Methylesterase, Affects Cell Wall Structure and Hypocotyl Elongation.

Pectin methylesterases (PMEs) modify homogalacturonan's chemistry and play a key role in regulating primary cell wall mechanical properties. Here, we report on Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We showed that dark-grown hypocotyl elongation was reduced in knock-out mutant lines as compared to the control. The latter was related to the decreased total PME activity as well as increased stiffness of the cell wall in the apical part of the hypocotyl. To relate phenotypic analyses to the biochemical specificity of the enzyme, we produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum. AtPME2 is more active at neutral compared to acidic pH, on pectins with a degree of 55-70% methylesterification. We further showed that the mode of action of AtPME2 can vary according to pH, from high processivity (at pH8) to low processivity (at pH5), and relate these observations to the differences in electrostatic potential of the protein. Our study brings insights into how the pH-dependent regulation by PME activity could affect the pectin structure and associated cell wall mechanical properties.

Cloning and expression analysis of HY5 transcription factor gene of safflower in response to light signal.

The flower of the safflower (Carthamus tinctorius L.) is a traditional Chinese medicine that can improve cerebral blood flow due to its enrichment in flavonoids. Light is one of the main environmental factors that affects safflower growth and flavonoid synthesis. Elongated hypocotyl 5 (HY5) plays an important role in plants' light signal transduction. However, no study of HY5 in safflower has been conducted. In this study, a 462-bp sequence of CtHY5 was successfully cloned. The expression pattern of CtHY5 in different safflower tissues and the expression patterns of CtHY5 and CtCHS1 in full-blooming flowers that were treated under different light intensities were studied. The subcellular localization and the overexpression of CtHY5 were carried out as well. CtHY5 has a DNA-binding region belonging to the basic leucine zipper transcription factor family. CtHY5 was specifically expressed in flowers. The expression level of CtHY5 first increased and then decreased with increasing light intensity, which was similar to the expression pattern of CtCHS1. The subcellular localization study was implemented in safflower protoplasts and the YFP fluorescence was observed in nucleus. The overexpression analysis initially verified the promotion effect of CtHY5 to the expression of CtCHS1 and the content of flavonoids.

Scarlet Flax Linum grandiflorum (L.) In Vitro Cultures as a New Source of Antioxidant and Anti-Inflammatory Lignans.

In vitro cultures of scarlet flax (Linum grandiflorum L.), an important ornamental flax, have been established as a new possible valuable resource of lignans and neolignans for antioxidant and anti-inflammatory applications. The callogenic potential at different concentrations of α-naphthalene acetic acid (NAA) and thidiazuron (TDZ), alone or in combinations, was evaluated using both L. grandiflorum hypocotyl and cotyledon explants. A higher callus induction frequency was observed on NAA than TDZ, especially for hypocotyl explants, with a maximum frequency (i.e., 95.2%) on 1.0 mg/L of NAA. The presence of NAA (1.0 mg/L) in conjunction with TDZ tended to increase the frequency of callogenesis relative to TDZ alone, but never reached the values observed with NAA alone, thereby indicating the lack of synergy between these two plant growth regulators (PGRs). Similarly, in terms of biomass, NAA was more effective than TDZ, with a maximum accumulation of biomass registered for medium supplemented with 1.0 mg/L of NAA using hypocotyls as initial explants (DW: 13.1 g). However, for biomass, a synergy between the two PGRs was observed, particularly for cotyledon-derived explants and for the lowest concentrations of TDZ. The influence of these two PGRs on callogenesis and biomass is discussed. The HPLC analysis confirmed the presence of lignans (secoisolariciresinol (SECO) and lariciresinol (LARI) and neolignan (dehydrodiconiferyl alcohol [DCA]) naturally accumulated in their glycoside forms. Furthermore, the antioxidant activities performed for both hypocotyl- and cotyledon-derived cultures were also found maximal (DPPH: 89.5%, FRAP 866: µM TEAC, ABTS: 456 µM TEAC) in hypocotyl-derived callus cultures as compared with callus obtained from cotyledon explants. Moreover, the anti-inflammatory activities revealed high inhibition (COX-1: 47.4% and COX-2: 51.1%) for extract of hypocotyl-derived callus cultures at 2.5 mg/L TDZ. The anti-inflammatory action against COX-1 and COX-2 was supported by the IC50 values. This report provides a viable approach for enhanced biomass accumulation and efficient production of (neo)lignans in L. grandiflorum callus cultures.

Locally restricted glucose availability in the embryonic hypocotyl determines seed germination under abscisic acid treatment.

Abiotic stresses affect plant growth and development by causing cellular damage and/or restricting resources. Plants often respond to stresses through abscisic acid (ABA) signaling. Exogenous ABA application can therefore be used to mimic stress responses, which can be overridden by glucose (Glc) addition during seed germination. It remains unclear whether ABA-mediated germination inhibition is due to regional or global suppression of Glc availability in germinating Arabidopsis seeds. We used a genetically engineered Förster resonance energy transfer (FRET) sensor to ascertain whether ABA affects the spatiotemporal distribution of Glc, 14 C-Glc uptake assays to track potential effects of ABA on sugar import, and transcriptome and mutant analyses to identify genes associated with Glc availability that are involved in ABA-inhibited seed germination. Abscisic acid limits Glc in the hypocotyl largely by suppressing sugar allocation as well as altering sugar metabolism. Mutant plants carrying loss-of-function ABA-inducible sucrose-phosphate synthase (SPS) genes accumulated more Glc, leading to ABA-insensitive germination. We reveal that Glc antagonizes ABA by globally counteracting the ABA influence at the transcript level, including expansin (EXP) family genes suppressed by ABA. This study presents a new perspective on how ABA affects Glc distribution, which likely reflects what occurs when seeds are subjected to abiotic stresses such as drought and salt stress.

Mechanochemical feedback mediates tissue bending required for seedling emergence.

Tissue bending is vital to plant development, as exemplified by apical hook formation during seedling emergence by bending of the hypocotyl. How tissue bending is coordinated during development remains poorly understood, especially in plants where cells are attached via rigid cell walls. Asymmetric distribution of the plant hormone auxin underlies differential cell elongation during apical hook formation. Yet the underlying mechanism remains unclear. Here, we demonstrate spatial correlation between asymmetric auxin distribution, methylesterified homogalacturonan (HG) pectin, and mechanical properties of the epidermal layer of the hypocotyl in Arabidopsis. Genetic and cell biological approaches show that this mechanochemical asymmetry is essential for differential cell elongation. We show that asymmetric auxin distribution underlies differential HG methylesterification, and conversely changes in HG methylesterification impact the auxin response domain. Our results suggest that a positive feedback loop between auxin distribution and HG methylesterification underpins asymmetric cell wall mechanochemical properties to promote tissue bending and seedling emergence.

A cotton (Gossypium hirsutum) WRKY transcription factor (GhWRKY22) participates in regulating anther/pollen development.

Anther/pollen development is a highly programmed process in flowering plants. However, the molecular mechanism of regulating anther/pollen development is still largely unclear so far. Here, we report a cotton WRKY transcription factor (GhWRKY22) that functions in anther/pollen development. Quantitative RT-PCR and GUS activity analyses revealed that GhWRKY22 is predominantly expressed in the late developing anther/pollen of cotton. The transgenic Arabidopsis plants expressing GhWRKY22 displayed the male fertility defect with the fewer viable pollen grains. Expression of the genes involved in jasmonate (JA) biosynthesis was up-regulated, whereas expression of the JA-repressors (JAZ1 and JAZ8) was down-regulated in the transgenic Arabidopsis plants expressing GhWRKY22, compared with those in wild type. Yeast one-hybrid and ChIP-qPCR assays demonstrated that GhWRKY22 modulated the expression of JAZ genes by directly binding to their promoters for regulating anther/pollen development. Yeast two-hybrid assay indicated that GhMYB24 could interact with GhJAZ8-A and GhJAZ13-A. Furthermore, expression of AtMYB24, AtPAL2 and AtANS2 was enhanced in the transgenic Arabidopsis plants, owing to GhWRKY22 overexpression. Taking the data together, our results suggest that GhWRKY22 acts as a transcriptional repressor to regulate anther/pollen development possibly by modulating the expression of the JAZ genes.

LOT regulates TGN biogenesis and Golgi structure in plants.

Trans-Golgi Network (TGN) is an essential organelle in eukaryotic cells. It acts not only as the sorting station of trafficking cargoes, but also as a signaling hub. In plant cells, TGN simultaneously takes the role of early endosome (EE) and contributes to the endocytic recycling. We recently characterized the first Golgi-localized protein Loss of TGNs (LOT) that is critical for TGN biogenesis and demonstrated its role during pollen tube growth in Arabidopsis. We also showed that the homozygous lot plant is dwarf and smaller than the wild type plant. As LOT is a single-copy gene and shows ubiquitous expression pattern, knowledge of its role in vegetative tissues, besides the pollen, is important for understanding the regulation of TGN/EE dynamics and signaling in plant development. Here, in this short communication, we present data to show that LOT also regulates TGN formation and Golgi structure in root meristem cells, and is critical for the elongation of hypocotyl and stamen filament.

Dynamic changes in polyphenol compounds, antioxidant activity, and PAL gene expression in different tissues of buckwheat during germination.

BACKGROUND: There is a growing interest in buckwheat germination regarding the improvement of its health benefits. The aims of this study were to evaluate the effects of germination on polyphenol compounds, antioxidant activity, and phenylalanine ammonia-lyase (PAL) gene expression in different tissues (cotyledon, hypocotyl, and radicle) of buckwheat sprouts during germination for 12 days, as well as to investigate their interactions. RESULTS: Total polyphenol and total flavonoid contents, antioxidant activity, main polyphenol components, and PAL gene expression significantly increased during germination. On day 12, the rutin content in cotyledons was elevated to 88.6 g kg-1 , which was 7.7-times and 39.4-times compared to those in buckwheat seeds and radicles, respectively. Meanwhile, chlorogenic acid in hypocotyls reached 7.84 g kg-1 , which was 36.3-fold higher than those in radicles. However, the PAL gene showed the highest expression in radicles. CONCLUSION: Present results showed that polyphenol compounds mainly accumulated in cotyledons and hypocotyls. There was a negative correlation between polyphenol compounds and PAL gene expression. The discrepancy suggested that polyphenol compounds might experience transportation within buckwheat sprouts. The study could provide useful information for further application of buckwheat in functional foods, and revelation of the correlation between bioactive components and related gene expressions. © 2018 Society of Chemical Industry.

Activation tagging of Arabidopsis POLYGALACTURONASE INVOLVED IN EXPANSION2 promotes hypocotyl elongation, leaf expansion, stem lignification, mechanical stiffening, and lodging.

Pectin is the most abundant component of primary cell walls in eudicot plants. The modification and degradation of pectin affects multiple processes during plant development, including cell expansion, organ initiation, and cell separation. However, the extent to which pectin degradation by polygalacturonases affects stem development and secondary wall formation remains unclear. Using an activation tag screen, we identified a transgenic Arabidopsis thaliana line with longer etiolated hypocotyls, which overexpresses a gene encoding a polygalacturonase. We designated this gene as POLYGALACTURONASE INVOLVED IN EXPANSION2 (PGX2), and the corresponding activation tagged line as PGX2AT . PGX2 is widely expressed in young seedlings and in roots, stems, leaves, flowers, and siliques of adult plants. PGX2-GFP localizes to the cell wall, and PGX2AT plants show higher total polygalacturonase activity and smaller pectin molecular masses than wild-type controls, supporting a function for this protein in apoplastic pectin degradation. A heterologously expressed, truncated version of PGX2 also displays polygalacturonase activity in vitro. Like previously identified PGX1AT plants, PGX2AT plants have longer hypocotyls and larger rosette leaves, but they also uniquely display early flowering, earlier stem lignification, and lodging stems with enhanced mechanical stiffness that is possibly due to decreased stem thickness. Together, these results indicate that PGX2 both functions in cell expansion and influences secondary wall formation, providing a possible link between these two developmental processes.

Characteristic fingerprinting based on macamides for discrimination of maca (Lepidium meyenii) by LC/MS/MS and multivariate statistical analysis.

BACKGROUND: Macamides with a benzylalkylamide nucleus are characteristic and major bioactive compounds in the functional food maca (Lepidium meyenii Walp). The aim of this study was to explore variations in macamide content among maca from China and Peru. Twenty-seven batches of maca hypocotyls with different phenotypes, sampled from different geographical origins, were extracted and profiled by liquid chromatography with ultraviolet detection/tandem mass spectrometry (LC-UV/MS/MS). RESULTS: Twelve macamides were identified by MS operated in multiple scanning modes. Similarity analysis showed that maca samples differed significantly in their macamide fingerprinting. Partial least squares discriminant analysis (PLS-DA) was used to differentiate samples according to their geographical origin and to identify the most relevant variables in the classification model. The prediction accuracy for raw maca was 91% and five macamides were selected and considered as chemical markers for sample classification. CONCLUSION: When combined with a PLS-DA model, characteristic fingerprinting based on macamides could be recommended for labelling for the authentication of maca from different geographical origins. The results provided potential evidence for the relationships between environmental or other factors and distribution of macamides. © 2016 Society of Chemical Industry.

Tuning of Pectin Methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER.

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.

RESPONSE OF PHENOLIC METABOLISM INDUCED BY ALUMINIUM TOXICITY IN FAGOPYRUM ESCULENTUM MOENCH. PLANTS.

Buckwheat genus (Fagopyrum Mill.) is one of the aluminium tolerant taxonomic units of plants. The aim of the study was an evaluation of the aluminium (50 µM effect on phenolic accumulation in various parts of buckwheat plants (Fagopyrum esculentum Moench). Detection of increasing of total phenolic content, changes in flavonoid and anthocyanin content and phenylalanine ammonia-lyase activity (PAL) were revealed over a period of 10 days of exposure to aluminium. The most significant effects of aluminium treatment on phenolic compounds accumulation were total phenolic content increasing (by 27.2%) and PAL activity rising by 2.5 times observed in leaves tissues. Received data could be helpful to understand the aluminium tolerance principles and relationships of phenolic compounds to aluminium phytotoxicity.

Characterization of transmembrane auxin transport in Arabidopsis suspension-cultured cells.

Polar auxin transport is a crucial process for control and coordination of plant development. Studies of auxin transport through plant tissues and organs showed that auxin is transported by a combination of phloem flow and the active, carrier-mediated cell-to-cell transport. Since plant organs and even tissues are too complex for determination of the kinetics of carrier-mediated auxin uptake and efflux on the cellular level, simplified models of cell suspension cultures are often used, and several tobacco cell lines have been established for auxin transport assays. However, there are very few data available on the specificity and kinetics of auxin transport across the plasma membrane for Arabidopsis thaliana suspension-cultured cells. In this report, the characteristics of carrier-mediated uptake (influx) and efflux for the native auxin indole-3-acetic acid and synthetic auxins, naphthalene-1-acetic and 2,4-dichlorophenoxyacetic acids (NAA and 2,4-D, respectively) in A. thaliana ecotype Landsberg erecta suspension-cultured cells (LE line) are provided. By auxin competition assays and inhibitor treatments, we show that, similarly to tobacco cells, uptake carriers have high affinity towards 2,4-D and that NAA is a good tool for studies of auxin efflux in LE cells. In contrast to tobacco cells, metabolic profiling showed that only a small proportion of NAA is metabolized in LE cells. These results show that the LE cell line is a useful experimental system for measurements of kinetics of auxin carriers on the cellular level that is complementary to tobacco cells.

Proline partially overcomes excess molybdenum toxicity in cabbage seedlings grown in vitro.

In vitro grown cabbage (Brassica oleracea var. capitata) seedlings exposed to excess molybdenum (Mo) ions exhibited severely reduced plant growth at the cotyledonary stage. Adding 80 mM proline (Pro) to the Mo-treated medium could help 50% seedlings to overcome the toxicity and grow true leaves. Under excess Mo stress, seedlings accumulated blue/purple anthocyanin in their cotyledons and hypocotyls. The anthocyanin content under Mo with 40 mM Pro was 4-fold higher than the control medium, MS with 40 mM Pro. The presence of Pro in the excess-Mo condition reduced chlorophyll a, whereas the chlorophyll b content was much higher than the control media of MS with and without Pro. Moreover, supplementing various concentrations of Pro into the Mo-stressed condition promoted the seedlings with higher antioxidant enzyme activities of superoxide dismutase, ascorbate peroxidate, and catalase. In addition, genes in the anthocyanin biosynthesis and accumulation pathways, phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), flavonone 3-hydroxylase (F3H), leucoanthocyanidin dioxygenase (LDOX), and glutathione-S-transferase (GST), were all upregulated. Our study indicated that, under excess Mo stress, the antioxidant activity of cabbage seedlings was induced in an attempt to protect plants from the Mo-induced toxicity and exacerbated growth. Pro, on the other hand, functioned in producing higher antioxidant enzyme activity to partially help recover plant growth.

Production of oleanolic acid glycosides by hairy root established cultures of Calendula officinalis L.

In order to initiate hairy root culture initiation cotyledons and hypocotyls of Calendula officinalis L. were infected with Agrobacterium rhizogenes strain ATCC 15834 or the same strain containing pCAMBIA 1381Z vector with ß-glucuronidase reporter gene under control of promoter of NIK (Nematode Induced Kinase) gene. The efficiency of induction of hairy roots reached 33.8% for cotyledons and 66.6% for hypocotyls together for both transformation experiments. Finally, eight control and nine modified lines were established as a long-term culture. The hairy root cultures showed the ability to synthesize oleanolic acid mainly (97%) as glycosides; control lines contained it at the average 8.42 mg · g(-1) dry weight in tissue and 0.23 mg · dm(-3) in medium; modified lines: 4.59 mg · g(-1) for the tissue, and 0.48 mg · dm(-3) for the medium. Additionally lines showed high positive correlation between dry/fresh weight and oleanolic acid concentration in tissue. Using the Killiani mixture in acidic hydrolysis of oleanolic acid glycosides released free aglycones that were partially acetylated in such conditions.

Basal root whorl number: a modulator of phosphorus acquisition in common bean (Phaseolus vulgaris).

BACKGROUND AND AIMS: Root architectural phenes enhancing topsoil foraging are important for phosphorus acquisition. In this study, the utility of a novel phene is described, basal root whorl number (BRWN), that has significant effects on topsoil foraging in common bean (Phaseolus vulgaris). METHODS: Whorls are defined as distinct tiers of basal roots that emerge in a tetrarch fashion along the base of the hypocotyl. Wild and domesticated bean taxa as well as two recombinant inbred line (RIL) populations were screened for BRWN and basal root number (BRN). A set of six RILs contrasting for BRWN was evaluated for performance under low phosphorus availability in the greenhouse and in the field. In the greenhouse, plants were grown in a sand-soil media with low or high phosphorus availability. In the field, plants were grown in an Oxisol in Mozambique under low and moderate phosphorus availability. KEY RESULTS: Wild bean accessions tended to have a BRWN of one or two, whereas cultivated accessions had BRWN reaching four and sometimes five. BRWN and BRN did not vary with phosphorus availability, i.e. BRWN was not a plastic trait in these genotypes. Greater BRWN was beneficial for phosphorus acquisition in low phosphorus soil. Genotypes with three whorls had almost twice the shoot biomass, greater root length and greater leaf area than related genotypes with two whorls. In low phosphorus soil, shoot phosphorus content was strongly correlated with BRWN (R(2) = 0.64 in the greenhouse and R(2) = 0.88 in the field). Genotypes with three whorls had shallower root systems with a greater range of basal root growth angles (from 10 to 45 ° from horizontal) than genotypes with two whorls (angles ranged from 60 to 85 ° from horizontal). CONCLUSIONS: The results indicate that BRWN is associated with increased phosphorus acquisition and that this trait may have value for selection of genotypes with better performance in low phosphorus soils.

MIDGET connects COP1-dependent development with endoreduplication in Arabidopsis thaliana.

In Arabidopsis thaliana, loss of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) function leads to constitutive photomorphogenesis in the dark associated with inhibition of endoreduplication in the hypocotyl, and a post-germination growth arrest. MIDGET (MID), a component of the TOPOISOMERASE VI (TOPOVI) complex, is essential for endoreduplication and genome integrity in A. thaliana. Here we show that MID and COP1 interact in vitro and in vivo through the amino terminus of COP1. We further demonstrate that MID supports sub-nuclear accumulation of COP1. The MID protein is not degraded in a COP1-dependent fashion in darkness, and the phenotypes of single and double mutants prove that MID is not a target of COP1 but rather a necessary factor for proper COP1 activity with respect to both, control of COP1-dependent morphogenesis and regulation of endoreduplication. Our data provide evidence for a functional connection between COP1 and the TOPOVI in plants linking COP1-dependent development with the regulation of endoreduplication.

Group III-A XTH genes of Arabidopsis encode predominant xyloglucan endohydrolases that are dispensable for normal growth.

The molecular basis of primary wall extension endures as one of the central enigmas in plant cell morphogenesis. Classical cell wall models suggest that xyloglucan endo-transglycosylase activity is the primary catalyst (together with expansins) of controlled cell wall loosening through the transient cleavage and religation of xyloglucan-cellulose cross links. The genome of Arabidopsis (Arabidopsis thaliana) contains 33 phylogenetically diverse XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) gene products, two of which were predicted to be predominant xyloglucan endohydrolases due to clustering into group III-A. Enzyme kinetic analysis of recombinant AtXTH31 confirmed this prediction and indicated that this enzyme had similar catalytic properties to the nasturtium (Tropaeolum majus) xyloglucanase1 responsible for storage xyloglucan hydrolysis during germination. Global analysis of Genevestigator data indicated that AtXTH31 and the paralogous AtXTH32 were abundantly expressed in expanding tissues. Microscopy analysis, utilizing the resorufin ß-glycoside of the xyloglucan oligosaccharide XXXG as an in situ probe, indicated significant xyloglucan endohydrolase activity in specific regions of both roots and hypocotyls, in good correlation with transcriptomic data. Moreover, this hydrolytic activity was essentially completely eliminated in AtXTH31/AtXTH32 double knockout lines. However, single and double knockout lines, as well as individual overexpressing lines, of AtXTH31 and AtXTH32 did not demonstrate significant growth or developmental phenotypes. These results suggest that although xyloglucan polysaccharide hydrolysis occurs in parallel with primary wall expansion, morphological effects are subtle or may be compensated by other mechanisms. We hypothesize that there is likely to be an interplay between these xyloglucan endohydrolases and recently discovered apoplastic exo-glycosidases in the hydrolytic modification of matrix xyloglucans.

Development of efficient Catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants.

BACKGROUND: As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus) produces many terpenoid indole alkaloids (TIAs), such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus, the establishment of an efficient genetic transformation method is required. RESULTS: To develop a genetic transformation method for C. roseus, Agrobacterium tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report ß-glucuronidase (GUS) gene and a selectable marker neomycin phosphotransferase II gene (NTPII). The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 µM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC) showed that overexpression of DAT increased the yield of vindoline in transgenic plants. CONCLUSIONS: In the present study, we report an efficient Agrobacterium-mediated transformation system for C. roseus plants with 11% of transformation frequency. To our knowledge, this is the first report on the establishment of A. tumefaciens mediated transformation and regeneration of C. roseus. More importantly, the C. roseus transformation system developed in this work was confirmed in the successful transformation of C. roseus using a key gene DAT involved in TIAs biosynthetic pathway resulting in the higher accumulation of vindoline in transgenic plants.

Mutations in multiple XXT genes of Arabidopsis reveal the complexity of xyloglucan biosynthesis.

Xyloglucan is an important hemicellulosic polysaccharide in dicot primary cell walls. Most of the enzymes involved in xyloglucan synthesis have been identified. However, many important details of its synthesis in vivo remain unknown. The roles of three genes encoding xylosyltransferases participating in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana) were further investigated using reverse genetic, biochemical, and immunological approaches. New double mutants (xxt1 xxt5 and xxt2 xxt5) and a triple mutant (xxt1 xxt2 xxt5) were generated, characterized, and compared with three single mutants and the xxt1 xxt2 double mutant that had been isolated previously. Antibody-based glycome profiling was applied in combination with chemical and immunohistochemical analyses for these characterizations. From the combined data, we conclude that XXT1 and XXT2 are responsible for the bulk of the xylosylation of the glucan backbone, and at least one of these proteins must be present and active for xyloglucan to be made. XXT5 plays a significant but as yet uncharacterized role in this process. The glycome profiling data demonstrate that the lack of detectable xyloglucan does not cause significant compensatory changes in other polysaccharides, although changes in nonxyloglucan polysaccharide amounts cannot be ruled out. Structural rearrangements of the polysaccharide network appear responsible for maintaining wall integrity in the absence of xyloglucan, thereby allowing nearly normal plant growth in plants lacking xyloglucan. Finally, results from immunohistochemical studies, combined with known information about expression patterns of the three genes, suggest that different combinations of xylosyltransferases contribute differently to xyloglucan biosynthesis in the various cell types found in stems, roots, and hypocotyls.