Composition and structural characterization of pectin in micropropagated and conventional plants of Premma puberula Pamp.

The metabolites produced by plants can be enhanced by plant tissue culture. In Premma puberula Pamp., the pectin content in leaves is 30 %-40 %, and it is widely used in the food industry and medicine. However, inefficient propagation has seriously restricted the utilization of pectin resources. Therefore, we established an efficient micropropagation technology for P. puberula through comparative analysis in mature leaves of regenerated and conventionally propagated plants. The results showed that the pectin composition of their leaves was similar in terms of galacturonic acid, monosaccharide composition, degree of esterification, functional groups, nuclear magnetic resonance spectrum and morphological characteristics. Furthermore, micropropagated plants had better hardness, gumminess and chewiness characteristics than conventionally propagated plants and were similar in emulsion stability, adhesiveness, springiness, cohesiveness and viscoelasticity. Therefore, micropropagation technology will provide an important guarantee for the industrial production of pectin from P. puberula. The technical essentials include callus induction, embryoid formation, and root induction, followed by acclimatization and transplanting.

Structural Alteration of Rice Pectin Affects Cell Wall Mechanical Strength and Pathogenicity of the Rice Blast Fungus Under Weak Light Conditions.

Pectin, a component of the plant cell wall, is involved in cell adhesion and environmental adaptations. We generated OsPG-FOX rice lines with little pectin due to overexpression of the gene encoding a pectin-degrading enzyme [polygalacturonase (PG)]. Overexpression of OsPG2 in rice under weak light conditions increased the activity of PG, which increased the degradation of pectin in the cell wall, thereby reducing adhesion. Under weak light conditions, the overexpression of OsPG decreased the pectin content and cell adhesion, resulting in abnormally large intercellular gaps and facilitating invasion by the rice blast fungus. OsPG2-FOX plants had weaker mechanical properties and greater sensitivity to biotic stresses than wild-type (WT) plants. However, the expression levels of disease resistance genes in non-infected leaves of OsPG2-FOX were more than twice as high as those of the WT and the intensity of disease symptoms was reduced, compared with the WT. Under normal light conditions, overexpression of OsPG2 decreased the pectin content, but did not affect cell adhesion and sensitivity to biotic stresses. Therefore, PG plays a role in regulating intercellular adhesion and the response to biotic stresses in rice.

Ethnopharmacological investigations of the leaves of Cecropia pachystachya Trécul (Urticaceae): A native Brazilian tree species.

ETHNOPHARMACOLOGICAL RELEVANCE: Cecropia pachystachya Trécul (Urticaceae) is a medicinal plant popularly known as 'embaúba'. In Brazil, the leaves of this species are used for the treatment of various kidney and cardiovascular diseases. However, there are no detailed studies on the renal and cardiovascular activities of this species. No studies on the anatomy or the quality control of this herbal drug is available thus far. AIM: This study was aimed to investigate the ethnopharmacological properties of the leaves of C. pachystachya. MATERIAL AND METHODS: The leaves of C. pachystachya were analyzed by light and scanning electron microscopy for pharmacobotanical and anatomical characterization. The ethanol-soluble fraction of C. pachystachya leaf extract (ESCP) was characterized by high-performance liquid chromatograph equipped with diode array detector and mass spectrometry (HPLC-DAD-MS). The acute oral toxicity of ESCP on female Wistar rats was assessed. The acute and prolonged diuresis and antioxidant effects of ESCP (30, 100, and 300 mg/kg) were evaluated in male Wistar rats. In addition, the hypotensive effects of the ESCP as well as the vasodilatory activity in isolated and perfused mesenteric vascular beds were investigated. RESULTS: The anatomical markers obtained in this study can help in the identification of C. pachystachya, as well as to distinguish it from the other 'embaúbas'. The metabolites found in the ESCP were phenolic compounds, mainly C- and O-glycosylated flavonoids. The ESCP did not exhibit any toxic effects at a dose of 2000 mg/kg. Significant diuretic activities were observed at the doses of 30, 100, and 300 mg/kg. In addition, a significant modulating activity of the tissue redox state was observed after prolonged treatment. On the other hand, no hypotensive or vasodilator activity was observed. CONCLUSION: The key findings of the present study can contribute to the taxonomy, species identification and quality control of C. pachystachya. Chemical studies have shown the presence of glycosylated flavonoids, phenylpropanoid derivative and proanthocyanidins. The pharmacological studies showed significant diuretic and antioxidant effects of C. pachystachya leaf extract, indicating a possible validation of its popular medicinal use.

Identification of lupeol produced by Vernonanthura patens (Kunth) H. Rob. leaf callus culture.

The lupeol detection in callus of Vernonanthura patens (Kunth) H. Rob. leaves is discussed. Leaf segments previously treated with sodium hypochlorite, ethanol, and distilled water were placed in MS basal medium (Murashige and Skoog) for 7 days. Next, callus induction were done in two complemented MS medium for 6 weeks. Then, callus propagation were performed in MS medium supplemented with 1.0 mg/L of benzylaminopurine (BAP) and 0.5 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) for 50 days. Fresh callus were extracted every 10 days in an ultrasonic bath using ethyl acetate (1.0 g/10 mL). The identification was carried out by Gas Chromatography-Mass Spectrometry (GC-MS) using selected ion monitoring (SIM) acquisition mode with characteristic ions of lupeol. The results obtained indicate the occurrence of lupeol in callus extract after twenty days of proliferation. These findings could be use in subsequent scale-up studies for biomass production containing this active compound in order to replace conventional methods.

Differential Distribution of Flavonoids and Phenolic Acids in Leaves of Kalanchoe delagoensis Ecklon & Zeyher (Crassulaceae).

Kalanchoe delagoensis is adapted to intense solar irradiation, drought, and heat, partially due to the presence of phenols, important photo-protective compounds and antioxidants. This study aimed to evaluate the distribution of flavonoids and phenolic acid derivatives throughout the erect-tubular leaves of K. delagoensis. Specimens grown under sunny conditions were used for histochemical and high-performance liquid chromatography coupled with diode array detection (liquid HPLC-DAD) analysis. The NP (2-aminoethyl diphenylborinate) test suggested the presence of phenolic acids throughout the leaf blade below the epidermis and in chloroplasts, mainly in the leaf base. Flavonoids were detected specifically in chloroplasts, on the adaxial side of the middle third and at the leaf apex, near the meristematic cells. There was a tendency of flavonoid accumulation from the middle third to the apex, especially surrounding the gem, while phenolic acids were observed mainly in the base. This can be explained by the more exposed leaf apex and to the presence of apical buds (high production and regulation sites of ROS). The HPLC-DAD analysis showed different classes of flavonoids and phenolic acid derivatives in the leaf extracts, agreeing with the NP test results. This is the first time that the substitution of phenolic acids by flavonoids from the leaf base to the apex has been described.

Comparative foliar anatomical and pollen morphological studies of Acanthaceae using light microscope and scanning electron microscope for effective microteaching in community.

In this study, foliar anatomy and pollen morphology of 10 species of Acanthaceae has been investigated using light and scanning electron microscopy. The study was aimed to highlight the role of microscopy in microteaching at community for proper characterization of plants using palyno-anatomical characters including pollen type, exine sculpturing, shape of epidermal cells, pattern of anticlinal wall, type and size of stomata, and trichome. Most of the species have polygonal cell shapes but some species have irregular, tetragonal, and pentagonal shape of epidermal cells. The largest epidermal cell length on adaxial and abaxial surface were observed in Asystasia gangetica 66.95 and 87.40 µm whereas least was observed on adaxial surface in Justicia adhatoda 36.9 µm and on abaxial surface in Barleria cristata 35.65 µm. In anatomy, species have diacytic type of stomata, whereas stomata of paracytic type observed in two species, while in A. gangetica cyclocytic type of stomata are present. Quantitively on abaxial surface, largest stomata length 29.9 µm and width 24.30 µm was noted in B. cristata. While shortest stomata length was observed in Ruellia prostrata 25.95 µm whereas minimum width of stomata was examined in Barleria acanthoides 2.05 µm. The diversity of trichomes are present in all species except in Ruellia brittoniana. Acanthaceae can be characterized by exhibiting different pollen morphology having five types of pollen shapes, prolate, spheroidal, perprolate, subprolate, and oblate spheroidal. Exine peculiarities showing variations such as reticulate, granulate, coarsely reticulate, lophoreticulate, perforate tectate, and granulate surface were examined.

Blast resistance gene Pi54 over-expressed in rice to understand its cellular and sub-cellular localization and response to different pathogens.

Rice blast resistance gene, Pi54 provides broad-spectrum resistance against different strains of Magnaporthe oryzae. Understanding the cellular localization of Pi54 protein is an essential step towards deciphering its place of interaction with the cognate Avr-gene. In this study, we investigated the sub-cellular localization of Pi54 with Green Fluorescent Protein (GFP) as a molecular tag through transient and stable expression in onion epidermal cells (Allium cepa) and susceptible japonica cultivar rice Taipei 309 (TP309), respectively. Confocal microscopy based observations of the onion epidermal cells revealed nucleus and cytoplasm specific GFP signals. In the stable transformed rice plants, GFP signal was recorded in the stomata, upper epidermal cells, mesophyll cells, vascular bundle, and walls of bundle sheath and bulliform cells of leaf tissues. These observations were further confirmed by Immunocytochemical studies. Using GFP specific antibodies, it was found that there was sufficient aggregation of GFP::Pi54protein in the cytoplasm of the leaf mesophyll cells and periphery of the epidermal cells. Interestingly, the transgenic lines developed in this study could show a moderate level of resistance to Xanthomonas oryzae and Rhizoctonia solani, the causal agents of the rice bacterial blight and sheath blight diseases, respectively. This study is a first detailed report, which emphasizes the cellular and subcellular distribution of the broad spectrum blast resistance gene Pi54 in rice and the impact of its constitutive expression towards resistance against other fungal and bacterial pathogens of rice.

Comparative pollen and foliar micromorphological studies using light microscopy and scanning electron microscopy of some selected species of Lamiaceae from Alpine Zone of Deosai Plateau, Western Himalayas.

The study was conducted to highlight a detailed account of morphology of pollen chosen species of Lamiaceae through scanning electron microscopy, and the anatomical characteristics of leaf epidermis of seven species using simple light microscopy. In results, Anisomeles indica and Otostegia aucheri belong to subfamily Lamioideae because it has tricolpate pollen while the rest eight species belong to subfamily Nepetoideae (hexacolpate pollen). The exine sculpturing of pollen of studied species was found to be reticulate. In the family Lamiaceae, four kinds of stomata were found anomocytic, anisocytic, diacytic, and actinocytic, respectively. The cell wall patterns of epidermal cells were irregular or polygonal with straight or undulate walls. It was noted that the variety of the epidermal trichomes seems of taxonomically important for the identification of species of Lamiaceae. Both nonglandular and glandular trichomes were analyzed. The nonglandular trichomes were characterized with long, thin, and pointed apical unicellular cells. The nonglandular trichomes were A-shaped in Thymus linearis. In Perovskia abrotanoides, stellate glandular trichomes were observed whereas in A. indica and Mentha royleana both glandular and nonglandular trichomes were found. In A. indica, the nonglandular trichomes were sessile and peltate in M. royleana. For the first time in this study, pollen and foliar micromorphological features of selected species of this area are carried out. These taxonomic characters were found to be important in discrimination of species from each other. In future, the detailed study with comprehensive morphology coupled with other important characters is required for delimitation of taxa at various levels.

Spectral quality as an elicitor of bioactive compound production in Solanum aculeatissimum JACQ cell suspension.

Solanum aculeatissimum Jacq. is a common plant in much of Brazil. Despite containing metabolites with a wide range of pharmacological applications, there are few tissue culture reports for this plant. The possibility of large-scale in vitro production of this material has significant biotechnological potential. Therefore, the objective of this study was to investigate the effect of light conditions on the growth of cells in suspension, observing the production and yield of biomass and bioactive compounds and the enzymatic behavior. Calli obtained from leaf segments were cultured in solid medium supplemented with 1 mg L-1 of 2,4-D, 2.5 mg L-1 kinetin, pH 5.7, in the dark. After 110 days of subculture, the calli were transferred to liquid medium. Cells were kept in the dark under agitation at 110 rpm and 25 °C and subcultured every 30 days. After 90 days of culture, 20 mL aliquots of cell suspension were added to flasks containing approximately 20 mL of medium (1:1) and cultured at different wavelengths (white, green, blue, red, and blue/red) under a photoperiod of 16 h with irradiance of 50 µmol m-2 s-1) and in the absence of light. The experiment was performed in a 6 × 6 factorial design (light condition × culture time). The cell cultures showed viability throughout the entire cycle, and chlorogenic and ferulic acids, orientin, quercitrin and, in higher amounts, quercetin, were detected in the first 7 days of culture. There was an increase in superoxide dismutase and catalase and a decrease in ascorbate peroxidase after exposure to different light conditions; for phenylalanine ammonia lyase, no differences were observed. The different light conditions were not sufficient to trigger responses in the concentrations of bioactive compounds, despite the detection of increased levels of the enzymes involved in cellular homeostasis.

Transcriptomic and metabolomic analysis reveals the role of CoA in the salt tolerance of Zygophyllum spp.

BACKGROUND: Zygophyllum is an important medicinal plant, with notable properties such as resistance to salt, alkali, and drought, as well as tolerance of poor soils and shifting sand. However, the response mechanism of Zygophyllum spp. to abiotic stess were rarely studied. RESULTS: Here, we aimed to explore the salt-tolerance genes of Zygophyllum plants by transcriptomic and metabolic approaches. We chose Z. brachypterum, Z. obliquum and Z. fabago to screen for salt tolerant and sensitive species. Cytological observation showed that both the stem and leaf of Z. brachypterum were significantly thicker than those of Z. fabago. Then, we treated these three species with different concentrations of NaCl, and found that Z. brachypterum exhibited the highest salt tolerance (ST), while Z. fabago was the most sensitive to salt (SS). With the increase of salt concentration, the CAT, SOD and POD activity, as well as proline and chlorophyll content in SS decreased significantly more than in ST. After salt treatment, the proportion of open stomata in ST decreased significantly more than in SS, although there was no significant difference in stomatal number between the two species. Transcriptomic analysis identified a total of 11 overlapping differentially expressed genes (DEGs) in the leaves and roots of the ST and SS species after salt stress. Two branched-chain-amino-acid aminotransferase (BCAT) genes among the 11 DEGs, which were significantly enriched in pantothenate and CoA biosynthesis, as well as the valine, leucine and isoleucine biosynthesis pathways, were confirmed to be significantly induced by salt stress through qRT-PCR. Furthermore, overlapping differentially abundant metabolites showed that the pantothenate and CoA biosynthesis pathways were significantly enriched after salt stress, which was consistent with the KEGG pathways enriched according to transcriptomics. CONCLUSIONS: In our study, transcriptomic and metabolomic analysis revealed that BCAT genes may affect the pantothenate and CoA biosynthesis pathway to regulate the salt tolerance of Zygophyllum species, which may constitute a newly identified signaling pathway through which plants respond to salt stress.

Eliciting callus culture for production of hepatoprotective flavonoids and phenolics from Sequoia sempervirens (D. Don Endl).

The aim of our study is to estimate the hepatoprotective effects of the ethanolic extract of the leaves of Sequoia sempervirens by determination of liver biomarkers (ALT, AST, total bilirubin and albumin in serum) and by histopathological examinations using thioacetamide-induced (TAA) liver injury model. Concurrent administration of ethanolic extracts of S. sempervirens leaves improved the alterations in liver morphology where it was a potent protector of the liver. The potential of L-phenylalanine and silver nitrate as chemical elicitors as well as UV radiation as a physical elicitor on flavonoid production in callus culture of S. sempervirens were emphasized. Murashige and Skoog's medium fortified with phenylalanine and silver nitrate enhanced the production of flavonoids and phenolics. HPLC analysis was performed for qualitative and quantitative estimation of some flavonoid compounds in the produced calli in comparison with the mother plant. This finding highlights the potential use of S. sempervirens in the treatment of liver dysfunction.

Apparent penetration depth in attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy of Allium cepa L. epidermis and cuticle.

Over the past decades, ATR-FTIR has emerged as promising tool for the identification of plants at the genus and (sub-) species level through surface measurements of intact leaves. Theoretical considerations regarding the penetration depth of the evanescent wave into the sample and the thickness of plant leaf cuticles suggest that the structure and composition of the cuticle represent universal taxonomic markers. However, experimental evidence for this hypothesis is scarce. In the current contribution, we present results of a series of simple experiments on epidermal monolayers derived from the bulbs of Allium cepa L. (Amaryllidaceae) as a model system to study the effect of an IR active probe located beyond the theoretical penetration depth of the evanescent wave. We found that this probe had a significant influence on the ATR-FTIR spectra for up to 4 epidermal layers stacked on top of each other corresponding to a total thickness of around 60 µm, exceeding the theoretical penetration depth of the evanescent wave by a factor of around 20. Altogether, our data indicate a major discrepancy between theory and practice in ATR-FTIR spectroscopy in general and provide strong evidence that in general plant leaf spectra cannot be fully explained by the structure and composition of the cuticle alone.

Regulation of color transition in purple tea (Camellia sinensis).

MAIN CONCLUSION: Comparative proteomics and metabolomics study of juvenile green, light purple and dark purple leaf to identify key proteins and metabolites that putatively govern color transition in Camellia sinensis. Color transition from juvenile green to dark purple leaf in Camellia sinensis is a complex process and thought to be regulated by an intricate balance of genes, proteins and metabolites expression. A molecular-level understanding of proteins and metabolites expression is needed to define metabolic process underpinning color transition in C. sinensis. Here, purple leaf growth of C. sinensis cultivar was divided into three developmental stages viz. juvenile green (JG), light purple (LP) and dark purple (DP) leaf. Scanning electron microscope (SEM) analysis revealed a clear morphological variation such as cell size, shape and texture as tea leaf undergoing color transition. Proteomic and metabolomic analyses displayed the temporal changes in proteins and metabolites that occur in color transition process. In total, 211 differentially expressed proteins (DEPs) were identified presumably involved in secondary metabolic processes particularly, flavonoids/anthocyanin biosynthesis, phytohormone regulation, carbon and nitrogen assimilation and photosynthesis, among others. Subcellular localization of three candidate proteins was further evaluated by their transient expression in planta. Interactome study revealed that proteins involved in primary metabolism, precursor metabolite, photosynthesis, phytohormones, transcription factor and anthocyanin biosynthesis were found to be interact directly or indirectly and thus, regulate color transition from JG to DP leaf. The present study not only corroborated earlier findings but also identified novel proteins and metabolites that putatively govern color transition in C. sinensis. These findings provide a platform for future studies that may be utilized for metabolic engineering/molecular breeding in an effort to develop more desirable traits.

The genuine localization of indole alkaloids in Vinca minor and Catharanthus roseus.

Based on the occurrence of indole alkaloids in so-called "chloroform leaf surface extracts", it was previously deduced that these alkaloids are present in the cuticle at the leaf surface of Catharanthus roseus and Vinca minor. As no symplastic markers were found in these extracts this deduction seemed to be sound. However, since chloroform is known to destroy biomembranes very rapidly, these data have to be judged with scepticism. We reanalyzed the alleged apoplastic localization of indole alkaloids by employing slightly acidic aqueous surface extracts and comparing the corresponding alkaloid patterns with those of aqueous total leaf extracts. Whereas in the "chloroform leaf surface extracts" all alkaloids are present in the same manner as in the total leaf extracts, no alkaloids occur in the aqueous leaf surface extracts. These results clearly show that chloroform had rapidly destroyed cell integrity, and the related extracts also contain the alkaloids genuinely accumulated within the protoplasm. The related decompartmentation was verified by the massively enhanced concentration of amino acids in aqueous surface extracts of chloroform treated leaves. Furthermore, the chloroform-induced cell disintegration was vividly visualized by confocal laser scanning microscopical analyses, which clearly displayed a strong decrease in the chlorophyll fluorescence in chloroform treated leaves. These findings unequivocally display that the indole alkaloids are not located in the apoplastic space, but exclusively are present symplastically within the cells of V. minor and C. roseus leaves. Accordingly, we have to presume that also other leaf surface extracts employing organic solvents have to be re-investigated.

ABCC Transporters Mediate the Vacuolar Accumulation of Crocins in Saffron Stigmas.

Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast (Saccharomyces cerevisiae), and in vitro transportomic assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify Crocus sativus transporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug and toxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the gene encoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana leaves.plantcell;31/11/2789/FX1F1fx1.

The complexity of intercellular localisation of alkaloids revealed by single-cell metabolomics.

Catharanthus roseus is a medicinal plant well known for producing bioactive compounds such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). Although the leaves of this plant are the main source of these antitumour drugs, much remains unknown on how TIAs are biosynthesised from a central precursor, strictosidine, to various TIAs in planta. Here, we have succeeded in showing, for the first time in leaf tissue of C. roseus, cell-specific TIAs localisation and accumulation with 10 µm spatial resolution Imaging mass spectrometry (Imaging MS) and live single-cell mass spectrometry (single-cell MS). These metabolomic studies revealed that most TIA precursors (iridoids) are localised in the epidermal cells, but major TIAs including serpentine and vindoline are localised instead in idioblast cells. Interestingly, the central TIA intermediate strictosidine also accumulates in both epidermal and idioblast cells of C. roseus. Moreover, we also found that vindoline accumulation increases in laticifer cells as the leaf expands. These discoveries highlight the complexity of intercellular localisation in plant specialised metabolism.

Report: Pharmacognostic and physicochemical screening of Euphorbia nivulia Buch.-Ham.

Euphorbia nivulia Buch.-Ham. (Euphorbiaceae) is commonly known as Indian Spurge Tree in English, and "Saj Thor" or "Jhanami booti" in local language. The plant is used traditionally in the treatment of various diseases like inflammation, fever, worm infection, asthma, cough, wounds and diabetes. In current study fresh as well as dried aerial parts of the plant and cut sections were examined, both macroscopically and microscopically. The study also deals with fluorescence analysis and phytochemical characteristics and other WHO recommended methods for standardization. WHO guidelines on quality control for medicinal plants materials were used for pharmacognostical evaluation of E. nivulia, phytochemical screening helps in determining the predominant classes of active constituents responsible for the activity. The present work will be helpful in identification of the fresh and dried samples of aerial parts pharmacognostically and anatomically. These studies will serve as a reference for correct identification and may be helpful in checking any type of adulteration. These observations will also help in differentiating this species from closely related species of the same genus and family.

Craterostigma plantagineum cell wall composition is remodelled during desiccation and the glycine-rich protein CpGRP1 interacts with pectins through clustered arginines.

Craterostigma plantagineum belongs to the desiccation-tolerant angiosperm plants. Upon dehydration, leaves fold and the cells shrink which is reversed during rehydration. To understand this process changes in cell wall pectin composition, and the role of the apoplastic glycine-rich protein 1 (CpGRP1) were analysed. Cellular microstructural changes in hydrated, desiccated and rehydrated leaf sections were analysed using scanning electron microscopy. Pectin composition in different cell wall fractions was analysed with monoclonal antibodies against homogalacturonan, rhamnogalacturonan I, rhamnogalacturonan II and hemicellulose epitopes. Our data demonstrate changes in pectin composition during dehydration/rehydration which is suggested to affect cell wall properties. Homogalacturonan was less methylesterified upon desiccation and changes were also demonstrated in the detection of rhamnogalacturonan I, rhamnogalacturonan II and hemicelluloses. CpGRP1 seems to have a central role in cell adaptations to water deficit, as it interacts with pectin through a cluster of arginine residues and de-methylesterified pectin presents more binding sites for the protein-pectin interaction than to pectin from hydrated leaves. CpGRP1 can also bind phosphatidic acid (PA) and cardiolipin. The binding of CpGRP1 to pectin appears to be dependent on the pectin methylesterification status and it has a higher affinity to pectin than its binding partner CpWAK1. It is hypothesised that changes in pectin composition are sensed by the CpGRP1-CpWAK1 complex therefore leading to the activation of dehydration-related responses and leaf folding. PA might participate in the modulation of CpGRP1 activity.

[Study on tissue culture system of Polygonatum cyrtonema].

In order to accelerate the breeding of the excellent seedlings of Polygonatum cyrtonema,tissue culture system of P. cyrtonema was established through the comprehensive regulation of key factors( leaf age,leaf location,basic media and plant growth regulators) and cytological basis of callus formation and differentiation was analyzed through paraffin section. The results showed that the 30-day-old leaf base explanton medium MS+6-BA 1. 50 mg·L~(-1)+2,4-D 0. 20 mg·L~(-1) had the highest induction rate( 80. 00%). The callus was initiated from cells on leaf base epidermis and near cortex,formed by the differentiation of middle vascular bundle cells. The optimal medium for adventitious bud differentiation was MS+ 6-BA 4. 00 mg·L~(-1)+ 2,4-D 0. 20 mg·L~(-1) with the differentiation rate of90. 33%,and the average number of buds was 5. 16. The adventitious buds had two origin types: exogenous and endogenous origin,formed by callus proximal cells and callus internal meristemoid. The adventitious bud proliferation medium was screened by orthogonal design,which determined the optimum combination was MS+ 6-BA 2. 00 mg·L~(-1)+NAA 0. 10 mg·L~(-1) and MS+ 6-BA 2. 00 mg·L~(-1)+NAA 0. 20 mg·L~(-1). The tubers with three leaves were cut and inoculated in the medium 1/2 MS+IBA 2. 00 mg·L~(-1),showing the highest rooting rate of 94. 00%. The rooting seedlings transplanted into the peat-vermiculite( 1 ∶ 1) matrix grew healthy and the survival rate was over 85. 00%. This research provided a novel solution for large-scale cultivation of P. cyrtonema seedling.

Integrated analysis of miRNAs and their targets reveals that miR319c/TCP2 regulates apical bud burst in tea plant (Camellia sinensis).

MAIN CONCLUSION: The roles of microRNA-mediated epigenetic regulation were highlighted in the bud dormancy-activity cycle, implying that certain differentially expressed miRNAs play crucial roles in apical bud burst, such as csn-miR319c/TCP2. microRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by targeting mRNA transcripts for cleavage or directing translational inhibition. To investigate whether miRNAs regulate bud dormancy-activation transition in tea plant, which largely affects the yield and price of tea products and adaptability of tea trees, we constructed small RNA libraries from three different periods of bud dormancy-burst transition. Through sequencing analysis, 262 conserved and 83 novel miRNAs were identified, including 118 differentially expressed miRNAs. Quantitative RT-PCR results for randomly selected miRNAs exhibited that our comprehensive analysis is highly reliable and accurate. The content of caffeine increased continuously from the endodormancy bud to flushing bud, and differentially expressed miRNAs coupling with their targets associated with bud burst were identified. Remarkably, csn-miR319c was downregulated significantly from the quiescent bud to burst bud, while its target gene CsnTCP2 (TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR 2) displayed opposite expression patterns. Co-transformation experiment in tobacco demonstrated that csn-miR319c can significantly suppress the functions of CsnTCP2. This study on miRNAs and the recognition of target genes could provide new insights into the molecular mechanism of the bud dormancy-activation transition in tea plant.