Micropropagation of Rosaceous Species SAM Grown in Temperate Climate.

The benefits of in vitro plant cultivation are mainly due to very high multiplication rate. Cultivation of plant material in vitro can be carried out during the whole year regardless of the time of the year or weather conditions. We create artificial conditions in the lab (heat, light, humidity), and we can regulate these conditions at any time. For the preservation of cultivar identity, we recommend establishing in vitro cultures from shoot tips usually larger than 0.2 mm. In practice, in vitro cultivation of plants uses these growth regulators to achieve organogenesis, for example, root formation, prolonged growth, or multiplication. During each subculture, these cultures are then transferred on a solid agar medium in the form of actively growing multiple shoots with a well-differentiated shoot tip containing meristematic area. Cytokinins are important for cell division and causes branching of plants. Auxins, both endogenous and exogenous, act at as a trigger for the differentiation and formation of root primordia. Morphological characteristics (formation of leaves or callus) and shoot development should be observed during in vitro multiplication and after transfer to ex vitro conditions.

Physiological and de novo transcriptome analysis of the fermentation mechanism of Cerasus sachalinensis roots in response to short-term waterlogging.

BACKGROUND: Cerasus sachalinensis is widely used in cool regions as a sweet cherry rootstock and is known for its sensitivity to soil waterlogging and waterlogging stress. However, the limited availability of Cerasus genomic resources has considerably restricted the exploration of its waterlogging response mechanism. To understand its reaction to short-term waterlogging, we analyzed the physiology and transcriptomes of C. sachalinensis roots in response to different waterlogging durations. RESULTS: In this study, 12,487 differentially expressed genes (DEGs) were identified from Cerasus sachalinensis roots under different waterlogging durations. Carbon metabolism and energy maintenance formed the first coping mechanism stage of C. sachalinensis in response to low oxygen conditions. Root energy processes, including root respiration and activities of the fermentation enzymes alcohol dehydrogenase, pyruvate decarboxylase, and lactate dehydrogenase, showed unique changes after 0 h, 3 h, 6 h, and 24 h of waterlogging exposure. Ribonucleic acid sequencing was used to analyze transcriptome changes in C. sachalinensis roots treated with 3 h, 6 h, and 24 h of waterlogging stress. After de novo assembly, 597,474 unigenes were recognized, of which 355,350 (59.47%) were annotated. To identify the most important pathways represented by DEGs, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were used to compare these genes. The first stage of root reaction to waterlogging stress was activation of carbohydrate metabolism to produce more glucose and maintain energy levels. At 3 h, the glycolytic and fermentation pathways were activated to maintain adenosine triphosphate production. At 24 h, pathways involved in the translation of proteins were activated to further assist the plant in tolerating waterlogging stress. These findings will facilitate a further understanding of the potential mechanisms of plant responses to waterlogging at physiological and transcriptome levels. CONCLUSIONS: Carbon metabolism and energy maintenance formed the first coping mechanism C. sachalinensis in response to low oxygen conditions, and they may be responsible for its short-term waterlogging response. Our study not only provides the assessment of genomic resources of Cerasus but also paves the way for probing the metabolic and molecular mechanisms underlying the short-term waterlogging response in C. sachalinensis.

Different responses of alpine plants to nitrogen addition: effects on plant-plant interactions.

The different responses of plant species to resource stress are keys to understand the dynamics of plant community in a changing environment. To test the hypothesis that nitrogen (N) increase would benefit N competitive species, rather than N stress-tolerant species, to compete with neighbours, we conducted an experiment with neighbour removal, N addition and soil moisture as treatments in an alpine grassland on the southeastern Tibetan Plateau. Both growths and competitive-response abilities (CRA, the ability to tolerate the inhibitory effects of neighbors) of Kobresia macrantha, Polygonum viviparum and Potentilla anserine in wet site were facilitated by N addition, conversely, both growths and CRA of Taraxacum mongolicum and Ligularia virgaurea were suppressed by N addition, indicating that the responses of CRA of target species under N addition were consistent with the N utilization strategies of them. Moreover, the facilitative effects of N addition on competitive-response abilities of Kobresia macrantha and Polygonum viviparum were not found at the dry site, illustrating that soil moisture can alter the changes of neighbour effects caused by N addition. Life strategy of dominant species in plant community on the undisturbed southeastern Tibetan Plateau may shift from N stress-tolerant to N competitive, if the N increases continuously.

Low induction of non-photochemical quenching and high photochemical efficiency in the annual desert plant Anastatica hierochuntica.

Non-photochemical quenching (NPQ) plays a major role in photoprotection. Anastatica hierochuntica is an annual desert plant found in hot deserts. We compared A. hierochuntica to three other different species: Arabidopsis thaliana, Eutrema salsugineum and Helianthus annuus, which have different NPQ and photosynthetic capacities. Anastatica hierochuntica plants had very different induction kinetics of NPQ and, to a lesser extent, of photosystem II electron transport rate (PSII ETR), in comparison to all other plants species in the experiments. The major components of the unusual photosynthetic and photoprotective response in A. hierochuntica were: (1) Low NPQ at the beginning of the light period, at various light intensities and CO2 concentrations. The described low NPQ cannot be explained by low leaf absorbance or by low energy distribution to PSII, but was related to the de-epoxidation state of xanthophylls. (2) Relatively high PSII ETR at various CO2 concentrations in correlation with low NPQ. PSII ETR responded positively to the increase of CO2 concentrations. At low CO2 concentrations PSII ETR was mostly O2 dependent. At moderate and high CO2 concentrations the high PSII ETR in A. hierochuntica was accompanied by relatively high CO2 assimilation rates. We suggest that A. hierochuntica have an uncommon NPQ and PSII ETR response. These responses in A. hierochuntica might represent an adaptation to the short growing season of an annual desert plant.

Isolation of the four methyl jasmonate stereoisomers and their effects on selected chiral volatile compounds in red raspberries.

The four stereoisomers present in a commercial sample of methyl jasmonate (MJ) were isolated at semi-preparative scale by HPLC, using a permethylated ß-cyclodextrin column. This allowed the baseline resolution and collection of both major (methyl jasmonates) and minor (epi-methyl jasmonates) stereoisomers. When 1.5 mL of a 5mg per mL MJ solution were injected, isolated amounts were 3.56 mg for (-) and (+)-methyl jasmonates, with respective purities of 96.1% and 99.9%, and 0.18 mg for (-)- and (+)-epi-methyl jasmonates, with 98.6% and 91.6% respective purities. The post-harvest treatment of red raspberry fruits with the pure methyl jasmonate stereoisomers isolated proved that (-)-epi-MJ either promotes the bioformation of (+)-limonene or inhibits that of (-)-limonene to a greater extent than the other three MJ stereoisomers, while the biosynthesis of the (+)-enantiomer of α-ionone is favoured equally, whichever MJ stereoisomer used. The results obtained in the present study might be used to obtain food products with improved sensory characteristics.

Exogenous melatonin improves Malus resistance to Marssonina apple blotch.

We examined whether exogenously applied melatonin could improve resistance to Marssonina apple blotch (Diplocarpon mali) by apple [Malus prunifolia (Willd.) Borkh. cv. Donghongguo]. This serious disease leads to premature defoliation in the main regions of apple production. When plants were pretreated with melatonin, resistance was increased in the leaves. We investigated the potential roles for melatonin in modulating levels of hydrogen peroxide (H2O2), as well the activities of antioxidant enzymes and pathogenesis-related proteins during these plant-pathogen interactions. Pretreatment enabled plants to maintain intracellular H2O2 concentrations at steady-state levels and enhance the activities of plant defence-related enzymes, possibly improving disease resistance. Because melatonin is safe and beneficial to animals and humans, exogenous pretreatment might represent a promising cultivation strategy to protect plants against this pathogen infection.

Oxidative stress responses and root lignification induced by Fe deficiency conditions in pear and quince genotypes.

We analysed Pyrus communis cv. Conference and Cydonia oblonga BA29, differently tolerant to lime-induced chlorosis, to identify the key mechanisms involved in their different performance under Fe deficiency induced by the absence of Fe (-Fe) or by the presence of bicarbonate (+FeBic). Under our experimental conditions, a decrease in root elongation was observed in BA29 under bicarbonate supply. Superoxide dismutase (SOD) and peroxidase (POD) activities were analysed and the relative isoforms were detected by native electrophoresis. The data obtained for both genotypes under -Fe and for BA29 +FeBic suggest the occurrence of overproduction of reactive oxygen species (ROS) and, at the same time, of a scarce capacity to detoxify them. The detection of ROS (O(2)(-) and H(2)O(2)) through histochemical localization supports these results and suggests that they could account for the modifications of mechanical properties of the cell wall during stress adaptation. On the other hand, in the cv. Conference +FeBic, an increase in non-specific POD activity was detected, confirming its higher level of protection in particular against H(2)O(2) accumulation. Peroxidases involved in lignification were assayed and histochemical analysis was performed. The results suggest that only in BA29 under bicarbonate supply can the presence of ROS in root apoplast be correlated with lignin deposits in external layers and in endodermis as a consequence of the shift of PODs towards a lignification role. We suggest that in BA29 the decrease in root growth could impair mineral nutrition, generating susceptibility to calcareous soils. In the cv. Conference, the allocation of new biomass to the root system could improve soil exploration and consequently Fe uptake.

(+)-methyl jasmonate-induced bioformation of myricetin, quercetin and kaempferol in red raspberries.

The effect of postharvest treatment with enantiomers of methyl jasmonate (MJ) in conjunction with ethanol on bioformation of myricetin, quercetin and kaempferol in red raspberry was studied. For comparison, postharvest treatment with the commercial stereoisomeric mixture of MJ in conjunction with ethanol was simultaneously accomplished. The levels obtained were contrasted with those determined in untreated (control) samples. Exogenous (+)-MJ induced an enhancement in the levels of myricetin, quercetin and, particularly, kaempferol whereas the exposition to (-)-MJ exhibited the opposite effect. Enzymatic assays were carried out in presence and absence of (-)-MJ and (+)-MJ to evaluate possible changes in the activity of the enzymes regulating the bioformation of flavonols in red raspberries as a consequence of the treatments. From the results of the assays both (-)-MJ and (+)-MJ inhibited the activity of flavanone 3ß-hydroxylase (FHT) and flavonol synthase (FLS), which are directly involved in the formation of flavonols from (-/+)-naringenin. From these results, it is speculated that the activity of phenylalanine ammonia lyase (PAL) regulating the formation of (-/+)-naringenin from l-phenylalanine by (+)-MJ in conjunction with ethanol is promoted. Postharvest treatment of red raspberry with (+)-MJ in ethanol is proposed as a mean to increase flavonol content in red raspberries.

Vitamins C and E improve regrowth and reduce lipid peroxidation of blackberry shoot tips following cryopreservation.

Oxidative processes involved in cryopreservation protocols may be responsible for the reduced viability of tissues after liquid nitrogen exposure. Antioxidants that counteract these reactions should improve recovery. This study focused on oxidative lipid injury and the effects of exogenous vitamin E (tocopherol, Vit E) and vitamin C (ascorbic acid, Vit C) treatments on regrowth at four critical steps of the plant vitrification solution number 2 (PVS2) vitrification cryopreservation technique; pretreatment, loading, rinsing, and regrowth. Initial experiments showed that Vit E at 11-15 mM significantly increased regrowth (P < 0.001) when added at any of the four steps. There was significantly more malondialdehyde (MDA), a lipid peroxidation product, at each of the steps than in fresh untreated shoot tips. Vit E uptake was assayed at each step and showed significantly more alpha- and gamma-tocopherols in treated shoots than those without Vit E. Vit E added at each step significantly reduced MDA formation and improved shoot regrowth. Vit C (0.14-0.58 mM) also significantly improved regrowth of shoot tips at each step compared to the controls. Regrowth medium with high iron concentrations and Vit C decreased recovery. However, in iron-free medium, Vit C significantly improved recovery. Treatments with Vit E (11 mM) and Vit C (0.14 mM) combined were not significantly better than Vit C alone. We recommend adding Vit C (0.28 mM) to the pretreatment medium, the loading solution or the rinse solution in the PVS2 vitrification protocol. This is the first report of the application of vitamins for improving cryopreservation of plant tissues by minimizing oxidative damage.

Differential responses in pear and quince genotypes induced by Fe deficiency and bicarbonate.

Most of the studies carried out on Fe deficiency condition in arboreous plants have been performed, with the exception of those carried out on plants grown in the field, in hydroponic culture utilizing a total iron depletion growth condition. This can cause great stress to plants. By introducing Fe deficiency induced by the presence of bicarbonate, we found significant differences between Pyrus communis L. cv. Conference and Cydonia oblonga Mill. BA29 and MA clones, characterized by different levels of tolerance to chlorosis. Pigment content and the main protein-pigment complexes were investigated by HPLC and protein gel blot analysis, respectively. While similar changes in the structural organization of photosystems (PSs) were observed in both species under Fe deficiency, a different reorganization of the photosynthetic apparatus was found in the presence of bicarbonate between tolerant and susceptible genotypes, in agreement with the photosynthetic electron transport rate measured in isolated thylakoids. In order to characterize the intrinsic factors determining the efficiency of iron uptake in a tolerant genotype, the main mechanisms induced by Fe deficiency in Strategy I species, such as Fe3+-chelate reductase (EC 1.16.1.7) and H+-ATPase (EC 3.6.3.6) activities, were also investigated. We demonstrate that physiological and biochemical root responses in quince and pear are differentially affected by iron starvation and bicarbonate supply, and we show a high correlation between tolerance and Strategy I activation.

Benzothiadiazole affects the leaf proteome in arctic bramble (Rubus arcticus).

Benzothiadiazole (BTH) induces resistance to the downy mildew pathogen, Peronospora sparsa, in arctic bramble, but the basis for the BTH-induced resistance is unknown. Arctic bramble cv. Mespi was treated with BTH to study the changes in leaf proteome and to identify proteins with a putative role in disease resistance. First, BTH induced strong expression of one PR-1 protein isoform, which was also induced by salicylic acid (SA). The PR-1 was responsive to BTH and exogenous SA despite a high endogenous SA content (20-25 microg/g fresh weight), which increased to an even higher level after treatment with BTH. Secondly, a total of 792 protein spots were detected in two-dimensional gel electrophoresis, eight proteins being detected solely in the BTH-treated plants. BTH caused up- or down-regulation of 72 and 31 proteins, respectively, of which 18 were tentatively identified by mass spectrometry. The up-regulation of flavanone-3-hydroxylase, alanine aminotransferase, 1-aminocyclopropane-1-carboxylate oxidase, PR-1 and PR-10 proteins may partly explain the BTH-induced resistance against P. sparsa. Other proteins with changes in intensity appear to be involved in, for example, energy metabolism and protein processing. The decline in ATP synthase, triosephosphate isomerase, fructose bisphosphate aldolase and glutamine synthetase suggests that BTH causes significant changes in primary metabolism, which provides one possible explanation for the decreased vegetative growth of foliage and rhizome observed in BTH-treated plants.

Impact of agrochemicals on Peronospora sparsa and phenolic profiles in three Rubus arcticus cultivars.

The main arctic bramble ( Rubus arcticus) cultivars are susceptible to downy mildew ( Peronospora sparsa), which seriously threatens the cultivation. The efficiency of Aliette, Euparen M, phosphite-containing Phosfik, Phostrol, Farm-Fos-44, and Kaliumfosfiet, as well as Bion was evaluated in the greenhouse. Fewer symptoms and less Peronospora DNA were found in plants treated with Euparen M and Bion, whereas Aliette, Phosfik, and Phostrol gave moderate protection. Three arctic bramble cultivars showed varying susceptibility to P. sparsa. An inexpensive and fast in vitro plate test gave results parallel with those obtained in the greenhouse. Quantitative differences were found in the phenolic profiles of the leaves of different cultivars and in different treatments. Several phenolic compounds were tentatively identified in arctic bramble for the first time, for example, monomeric and oligomeric ellagitannins and galloylglucoses. Negative correlation was found between the amount of P. sparsa DNA and flavonol glycosides and some ellagitannins in the leaves 8 days after inoculation, suggesting a possible role for these phenolics in the defense.

Physiological behaviour of loquat and anger rootstocks in relation to salinity and calcium addition.

The salinity tolerance of two commercial rootstocks used for loquat plants (Eribotrya japonica Lindl.), loquat and anger, was studied in a pot experiment. The plants were irrigated using solutions containing 5 and 50mM NaCl and 5 and 25mM calcium acetate for 4 months. The growth, tissue mineral content, water status, and leaf gas exchange responses to salt treatment with and without additional calcium were examined. Plant growth was not modified by salinity in anger (50mM), but was reduced in loquat; leaf biomass and stem diameter were particularly affected. However, Cl(-) levels leaf increased with salinity to a greater extent in anger, while the Na(+) content increased to the same extent in both species, indicating that ion transport from root to leaves was not inhibited in either species. Additional calcium (25mM) reduced Na(+) and Cl(-) concentrations in both species, but did not minimise the effects of salinity on the growth of salt-treated loquat plants. The decrease in K(+) concentrations had no effect on growth, as anger was the most tolerant rootstock and had lowest leaf K(+) content. Salinity reduced the Ca(2+) concentration in the roots of both species. However, when calcium was added, the concentration of Ca(2+) increased in the roots of salinised plants. Leaf water potential at pre-dawn decreased significantly in both species under saline conditions. Leaf gas exchange, stomatal conductance and, in particular, net CO(2) assimilation, decreased with salinity only in loquat, indicating that photosynthesis could be the growth-limiting factor in this species.

Do xylem fibers affect vessel cavitation resistance?

Possible mechanical and hydraulic costs to increased cavitation resistance were examined among six co-occurring species of chaparral shrubs in southern California. We measured cavitation resistance (xylem pressure at 50% loss of hydraulic conductivity), seasonal low pressure potential (P(min)), xylem conductive efficiency (specific conductivity), mechanical strength of stems (modulus of elasticity and modulus of rupture), and xylem density. At the cellular level, we measured vessel and fiber wall thickness and lumen diameter, transverse fiber wall and total lumen area, and estimated vessel implosion resistance using (t/b)(h)(2), where t is the thickness of adjoining vessel walls and b is the vessel lumen diameter. Increased cavitation resistance was correlated with increased mechanical strength (r(2) = 0.74 and 0.76 for modulus of elasticity and modulus of rupture, respectively), xylem density (r(2) = 0.88), and P(min) (r(2) = 0.96). In contrast, cavitation resistance and P(min) were not correlated with decreased specific conductivity, suggesting no tradeoff between these traits. At the cellular level, increased cavitation resistance was correlated with increased (t/b)(h)(2) (r(2) = 0.95), increased transverse fiber wall area (r(2) = 0.89), and decreased fiber lumen area (r(2) = 0.76). To our knowledge, the correlation between cavitation resistance and fiber wall area has not been shown previously and suggests a mechanical role for fibers in cavitation resistance. Fiber efficacy in prevention of vessel implosion, defined as inward bending or collapse of vessels, is discussed.

In vitro regeneration of Hagenia abyssinica (Bruce) J.F. Gmel. (Rosaceae) from leaf explants.

A protocol for shoot regeneration of Hagenia abyssinica (Bruce) J.F. Gmel. has been developed using leaf explants originating from in vitro seedlings and mature material. The explants were cultured on Murashige and Skoog medium containing various concentrations of alpha-naphthaleneacetic acid and thidiazuron (TDZ). Concentrations of TDZ lower than 1.0 microM promoted direct shoot regeneration, but higher concentrations promoted callus induction. Around 96-100% regeneration was obtained between 1.0 and 10 microM TDZ. The average number of shoots per explant at 1.0 microM TDZ was 8.4+/-4.8. Among the different explants used, the highest percentage of regeneration and shoots per explant was obtained from complete leaf explants. A significant (P< or =0.05) difference in regeneration capacity was observed among the five genotypes examined. The resulting shoots were multiplied on multiplication medium, rooted and acclimatised in a greenhouse.

Novel oligosaccharides isolated from Fusarium oxysporum L. rapidly induce PAL activity in Rubus cells.

Activation of the phenolic pathway is known to be part of a defense response against cell wall-derived elicitors from pathogens. Many examples of a defense response by increasing the synthesis of phenolic compound against the elicitor were demonstrated in the past, but the elicitor structure has so far been poorly characterized. Our results indicate that a disaccharide fraction containing the following structure: alpha-D-mannopyranosyl (1-->2)alpha/beta-D-glucopyranosyl and alpha-D-mannopyranosyl (1-->x) inositol, isolated from Fusarium oxysporum L., promotes rapid and transient phenylalanine ammonia lyase activity in Rubus fructicosus cells at nanomolar concentration. The disaccharides were isolated by size-exclusion chromatography directly from extracts obtained by alkaline treatment of F. oxysporum mycelium. Their structure was determined by 500-MHz-1H-NMR spectroscopy combined with methylation analysis and fast atom bombardment mass spectrometry.

Novel insight into vascular, stress, and auxin-dependent and -independent gene expression programs in strawberry, a non-climacteric fruit.

Using cDNA microarrays, a comprehensive investigation of gene expression was carried out in strawberry (Fragaria x ananassa) fruit to understand the flow of events associated with its maturation and non-climacteric ripening. We detected key processes and novel genes not previously associated with fruit development and ripening, related to vascular development, oxidative stress, and auxin response. Microarray analysis during fruit development and in receptacle and seed (achene) tissues established an interesting parallelism in gene expression between the transdifferentiation of tracheary elements in Zinnia elegans and strawberry. One of the genes, CAD, common to both systems and encoding the lignin-related protein cinnamyl alcohol dehydrogenase, was immunolocalized to immature xylem cells of the vascular bundles in the strawberry receptacle. To examine the importance of oxidative stress in ripening, gene expression was compared between fruit treated on-vine with a free radical generator and non-treated fruit. Of 46 genes induced, 20 were also ripening regulated. This might suggest that active gene expression is induced to cope with oxidative stress conditions during ripening or that the strawberry ripening transcriptional program is an oxidative stress-induced process. To gain insight into the hormonal control of non-climacteric fruit ripening, an additional microarray experiment was conducted comparing gene expression in fruit treated exogenously with auxin and control fruit. Novel auxin-dependent genes and processes were identified in addition to transcriptional programs acting independent of auxin mainly related to cell wall metabolism and stress response.

High performance liquid chromatography and photodiode array detection of ferulic acid in Rubus protoplasts elicited by O-glycans from Fusarium sp. M7-1.

So far only little data have been available concerning the eliciting capacity of well defined glycan molecules isolated from plant pathogens. This study brings new information about changes in plant cells caused by fungal pathogens. Sugar fractions derived from glycoproteins isolated from the fungus Fusarium sp. M7-1 have been tested here as signaling molecules. The ability of three O-glycan fractions (named in this work inducer I, II, III) to trigger responses in Rubus protoplasts has been examined. It was found that inducer III was the most efficient as it elicited changes in the levels of phenylpropanoid pathway intermediates in relation to phenylalanine-ammonia lyase (PAL) activation.