Shoot Tip Cryopreservation of Lamprocapnos spectabilis (L.) Fukuhara Using Different Approaches and Evaluation of Stability on the Molecular, Biochemical, and Plant Architecture Levels.

The aim of this study is to optimize and evaluate the effectiveness of vitrification, droplet-vitrification, and encapsulation-vitrification techniques in the cryopreservation of Lamprocapnos spectabilis (L.) Fukuhara 'Gold Heart', a popular medicinal and ornamental plant species. In vitro-derived shoot tips were used in the experiments. All three techniques were based on explant dehydration with plant vitrification solution 3 (PVS3; 50% glycerol and 50% sucrose) for 0, 30, 60, 90, 120, 150, or 180 min. The recovered microshoots were subjected to morphometric, biochemical, and molecular analyses (RAPD, ISSR, SCoT). The highest recovery level was reported with the encapsulation-vitrification protocol based on 150 min dehydration (73.1%), while the vitrification technique was the least effective (maximum 25.8% recovery). Explants cryopreserved with the encapsulation-vitrification technique produced the highest mean number of shoots (4.9); moreover, this technique was optimal in terms of rooting efficiency. The highest fresh weight of shoots, on the other hand, was found with the vitrification protocol based on a 30-min PVS3 treatment. The concentrations of chlorophyll a and b were lower in all cryopreservation-derived plants, compared to the untreated control. On the other hand, short dehydration and cryopreservation of non-encapsulated explants stimulated the synthesis of anthocyanins. A small genetic variation in 5% of all samples analyzed was detected by RAPD and ISSR marker systems. Only plants recovered from the encapsulation-vitrification protocol had no DNA sequence alternations.

Factors affecting the dormancy and germination of bleeding heart [Lamprocapnos spectabilis (L.) Fukuhara] seeds.

Information on the optimal conditions to promote the germination of Lamprocapnos spectabilis (L.) Fukuhara seeds is limited; consequently, this study was conducted to establish the requirements to break seed dormancy and promote germination. The selected seeds had morphophysiological dormancy and had not begun embryo development. To study the dormancy breaking and embryo development processes, seeds were subjected to constant or changing temperature treatments during moist stratification. High temperature and humidity resulted in vigorous embryo growth, with the longest embryos occurring after 1 month of incubation at 20 °C. At 4 °C, the seeds required incubation period of at least 3 months to germinate. Embryo growth and germination were higher with changing high and low temperatures than under a constant temperature, and changing temperatures also considerably changed the endogenous hormone levels, embryo development and germination. Bioactive gibberellin (GA) content was higher in seeds incubated at 20 °C for 1 month, then at 4 °C for 2 months. The content of endogenous abscisic acid in seeds subjected to the same treatment decreased by 97.6% compared with that of the untreated seeds. Embryo growth and seed germination require changing high and low temperatures; however, exogenous GA3 could substitute for high temperatures, as it also causes accelerated germination. In this study, the seeds of L. spectabilis were identified as an intermediate simple type, a sub-level of morphophysiologically dormant seeds.

Integrative study of subcellular distribution, chemical forms, and physiological responses for understanding manganese tolerance in the herb Macleaya cordata (papaveraceae).

Macleaya cordata is a perennial herb, a candidate phytoremediation plant with high biomass and manganese (Mn) tolerance. To study the mechanism underlying its Mn adaptability, Mn2+ at various concentrations (0, 1000, 5000, 10000, 15000, and 20000 µM) were applied to M. cordata to investigate the subcellular distribution and chemical forms of Mn, as well as the resulting physiological and biochemical changes by pot culture experiment under natural light in a greenhouse. According to our results, Mn level in M. cordata increased with exogenous Mn concentrations; and Mn contents in different tissues exhibited a leaf > root > stem pattern. Meanwhile, biomass and the level of photosynthetic pigments increased at lower Mn concentrations but declined as Mn concentration further ascended. Subcellular distribution analysis revealed that Mn was sequestered in cell wall and vacuole; in addition, it was incorporated into pectates and protein, phosphates, and oxalates. These findings revealed a possible effective strategy for M. cordata to reduce Mn mobility and toxicity. Moreover, a continuous boost in the level of malondialdehyde was observed with Mn gradient; whereas contents of soluble proteins and proline, and the activities of superoxide dismutase and peroxidase were initially increased and then dropped. Altogether, these results indicated that most Mn was trapped in the cell wall and soluble fractions in low toxicity forms such as pectates and protein, phosphates, and oxalates. These strategies, that is functioning cooperatively with the well-coordinated antioxidant defense systems and the non-enzymatic metabolites, confer strong resistance to Mn in M. cordata.

Enhancement of repeated applications of chelates on phytoremediation of uranium contaminated soil by Macleaya cordata.

A greenhouse pot experiment was performed to investigate the enhancement of repeated applications of citric acid (CA), ethylenediamine disuccinic acid (EDDS), and Oxalic acid (OA) on phytoremediation of uranium (U) contaminated soil by Macleaya Cordata. The chelates followed the order CA > EDDS > OA in terms of the enhancement on uranium uptake by M. cordata. The repeated applications of the chelates were found to be more effective than the one time application at the equal dose as the U concentration of soil solution increased significantly from the 8th to 14th day. The repeated applications of 10 mmol kg-1 CA promoted the solubilization of U in the U-contaminated soil by significantly decreasing the pH of soil solution, achieved the maximum U concentration of soil solution (1463.6 µg L-1), bioconcentration factors (BCFs, 11.4), bioaccumulation factors (BAFs, 21.4) and transfer factors (TFs, 1.9), which were 215.2, 5.7, 30.6 and 16.3 times as compared with the control group, respectively. The three applied chelates significantly affected the activities of the antioxidant enzymes in the leaves. Repeated applications of CA further enhanced the activities of the antioxidant enzymes in the leaves of M. cordata as compared with the control, EDDS and OA, mitigated the oxidative stress induced by uranium and chelates, and maximized the enhancement on the uranium uptake, which will be beneficial for the enhancement on the phytoremediation of uranium contaminated soil by U hyperaccumulating plants. These results indicated that the phytoavailability of uranium in soil solution as well as the accumulation of U by M. cordata were both significantly increased after repeated applications of CA, and that the repeated applications of 10 mmol kg-1 CA increased the activities of the antioxidant enzymes and promoted U accumulation by M. cordata. The study provided an environmentally friendly alternative for the enhancement on the phytoremediation of uranium contaminated soil using M. cordata.

Effects of climate warming and prolonged snow cover on phenology of the early life history stages of four alpine herbs on the southeastern Tibetan Plateau.

PREMISE OF THE STUDY: Much research has focused on plant responses to ongoing climate change, but there is relatively little information about how climate change will affect the early plant life history stages. Understanding how global warming and changes in winter snow pattern will affect seed germination and seedling establishment is crucial for predicting future alpine population and vegetation dynamics. METHODS: In a 2-year study, we tested how warming and alteration in the snowmelt regime, both in isolation and combination, influence seedling emergence phenology, first-year growth, biomass allocation, and survival of four native alpine perennial herbs on the southeastern Tibetan Plateau. KEY RESULTS: Warming promoted seedling emergence phenology of all four species and biomass per plant of two species but reduced seedling survival of three species. Prolonged snow cover partly mediated the affects of warming on Primula alpicola (survival and biomass), Pedicularis fletcheri (phenology, biomass, and root:shoot ratio) and Meconopsis integrifolia (survival). For the narrowly distributed species M. racemosa, seedling growth was additively decreased by warming and prolonged snow cover. CONCLUSIONS: Both warming and alteration of the snow cover regime can influence plant recruitment by affecting seedling phenology, growth, and survival, and the effects are largely species-specific. Thus, climate change is likely to affect population dynamics and community structure of the alpine ecosystem. This is the first experimental demonstration of the phenological advancement of seedling emergence in the field by simulated climate warming.

Virus-induced gene silencing (VIGS) in Cysticapnos vesicaria, a zygomorphic-flowered Papaveraceae (Ranunculales, basal eudicots).

BACKGROUND AND AIMS: Studies of evolutionary diversification in the basal eudicot family Papaveraceae, such as the transition from actinomorphy to zygomorphy, are hampered by the lack of comparative functional studies. So far, gene silencing methods are only available in the actinomorphic species Eschscholzia californica and Papaver somniferum. This study addresses the amenability of Cysticapnos vesicaria, a derived fumitory with zygomorphic flowers, to virus-induced gene silencing (VIGS), and describes vegetative and reproductive traits in this species. METHODS: VIGS-mediated downregulation of the C. vesicaria PHYTOENE DESATURASE gene (CvPDS) and of the FLORICAULA gene CvFLO was carried out using Agrobacterium tumefaciens transfer of Tobacco rattle virus (TRV)-based vectors. Wild-type and vector-treated plants were characterized using reverse transcription-PCR (RT-PCR), in situ hybridization, and macroscopic and scanning electron microscopic imaging. KEY RESULTS: Cysticapnos vesicaria germinates rapidly, can be grown at high density, has a short life cycle and is self-compatible. Inoculation of C. vesicaria with a CvPDS-VIGS vector resulted in strong photobleaching of green parts and reduction of endogenous CvPDS transcript levels. Gene silencing persisted during inflorescence development until fruit set. Inoculation of plants with CvFLO-VIGS affected floral phyllotaxis, symmetry and floral organ identities. CONCLUSIONS: The high penetrance, severity and stability of pTRV-mediated silencing, including the induction of meristem-related phenotypes, make C. vesicaria a very promising new focus species for evolutionary-developmental (evo-devo) studies in the Papaveraceae. This now enables comparative studies of flower symmetry, inflorescence determinacy and other traits that diversified in the Papaveraceae.

The ant, Aphaenogaster picea, benefits from plant elaiosomes when insect prey is scarce.

Myrmecochory is a facultative, mutualistic interaction in which ants receive a protein-rich food reward (elaiosome) in return for dispersing plant seeds. In North American northeastern hardwood forests, Aphaenogaster ants are the primary genus dispersing myrmecochorous plants. In these forests, myrmecochores occur in plant guilds of understory spring ephemerals or seasonal greens. This mutualism has been demonstrated for Aphaenogaster rudis (Emery) and individual plant species, but it has not been demonstrated for other Aphaenogaster species or guilds of myrmecochores as they naturally occur. Aphaenogaster picea (Wheeler) colonies were fed three treatments over 5 mo: 1) a mixture of only elaiosomes from an entire plant guild, 2) a diet of only insect protein and 3) a combination diet of both elaiosomes and insect protein. This experiment investigated two potential hypotheses through which elaiosomes can benefit ants: 1) elaiosome proteins can substitute for protein nutritional requirements when ants are prey-limited, and 2) elaiosome nutrition can supplement insect protein when prey is ample. First, a mixture of elaiosomes from four myrmecochorous plant species provided to A. picea colonies was sufficient to maintain worker production, larval growth, and fat stores when no other food was available. A. picea colonies consuming elaiosomes as their only protein source could be sustained for a growing season (5 mo). Second, colonies fed both elaiosomes and protein did not yield more productive colonies than a control diet of just insect protein. These results support the hypothesis that myrmecochory is indeed a facultative mutualism in which ants take advantage of the protein content of elaiosomes when it is favorable, but when they are not limited by insect prey they do not gain any additional benefit from elaiosomes.

Evolutionary genetics of an S-like polymorphism in Papaveraceae with putative function in self-incompatibility.

BACKGROUND: Papaver rhoeas possesses a gametophytic self-incompatibility (SI) system not homologous to any other SI mechanism characterized at the molecular level. Four previously published full length stigmatic S-alleles from the genus Papaver exhibited remarkable sequence divergence, but these studies failed to amplify additional S-alleles despite crossing evidence for more than 60 S-alleles in Papaver rhoeas alone. METHODOLOGY/PRINCIPAL FINDINGS: Using RT-PCR we identified 87 unique putative stigmatic S-allele sequences from the Papaveraceae Argemone munita, Papaver mcconnellii, P. nudicuale, Platystemon californicus and Romneya coulteri. Hand pollinations among two full-sib families of both A. munita and P. californicus indicate a strong correlation between the putative S-genotype and observed incompatibility phenotype. However, we also found more than two S-like sequences in some individuals of A. munita and P. californicus, with two products co-segregating in both full-sib families of P. californicus. Pairwise sequence divergence estimates within and among taxa show Papaver stigmatic S-alleles to be the most variable with lower divergence among putative S-alleles from other Papaveraceae. Genealogical analysis indicates little shared ancestral polymorphism among S-like sequences from different genera. Lack of shared ancestral polymorphism could be due to long divergence times among genera studied, reduced levels of balancing selection if some or all S-like sequences do not function in incompatibility, population bottlenecks, or different levels of recombination among taxa. Preliminary estimates of positive selection find many sites under selective constraint with a few undergoing positive selection, suggesting that self-recognition may depend on amino acid substitutions at only a few sites. CONCLUSIONS/SIGNIFICANCE: Because of the strong correlation between genotype and SI phenotype, sequences reported here represent either functional stylar S-alleles, tightly linked paralogs of the S-locus or a combination of both. The considerable complexity revealed in this study shows we have much to learn about the evolutionary dynamics of self-incompatibility systems.

Impact of cytomixis on meiosis, pollen viability and pollen size in wild populations of Himalayan poppy (Meconopsis aculeata Royle).

We report the occurrence of cytomixis in wild populations of Himalayan poppy (Meconopsis aculeata Royle),which is considered to be an important and threatened medicinal plant growing in the high hills of the Himalayas. The impact of cytomixis on meiotic behaviour, reduced pollen viability and heterogeneous-sized pollen grains was also studied. Cytological studies in the seven wild populations from the high hills of Himachal Pradesh revealed that all the Himalayan populations exist uniformly at the tetraploid level (2n=56) on x=14. The phenomenon of chromatin transfer among the proximate pollen mother cells (PMCs) in six populations caused various meiotic abnormalities. Chromatin transfer also resulted in the formation of coenocytes, aneuploid, polyploid and anucleated PMCs. Among individuals that showed chromatin transfer, chromosome stickiness and interbivalent connections were frequently observed in some PMCs. The phenomenon of cytomixis in the species seems to be directly under genetic control; it affects the meiotic course considerably and results in reduced pollen viability.

Self-incompatibility in plants.

Sexual reproduction in many flowering plants involves self-incompatibility (SI), which is one of the most important systems to prevent inbreeding. In many species, the self-/nonself-recognition of SI is controlled by a single polymorphic locus, the S-locus. Molecular dissection of the S-locus revealed that SI represents not one system, but a collection of divergent mechanisms. Here, we discuss recent advances in the understanding of three distinct SI mechanisms, each controlled by two separate determinant genes at the S-locus. In the Brassicaceae, the determinant genes encode a pollen ligand and its stigmatic receptor kinase; their interaction induces incompatible signaling(s) within the stigma papilla cells. In the Solanaceae-type SI, the determinants are a ribonuclease and an F-box protein, suggesting the involvement of RNA and protein degradation in the system. In the Papaveraceae, the only identified female determinant induces a Ca2+-dependent signaling network that ultimately results in the death of incompatible pollen.

Anti-ulcerogenic activity of some plants used as folk remedy in Turkey.

Five herbal remedies used as gastroprotective crude drugs in Turkey were assessed for anti-ulcerogenic activity using the EtOH-induced ulcerogenesis model in rat. The crude drugs investigated comprises fruits of Elaeagnus angustifolia L. (Elaeagnaceae), fresh fruits of Hibiscus esculentus L. (Malvaceae), fresh roots of Papaver rhoeas L. (Papaveraceae), leaves of Phlomis grandiflora H.S. Thomson (Lamiaceae) and fresh fruits of Rosa canina L. (Rosaceae). Extracts were prepared according to the traditional indications of use. Under our experimental conditions, all extracts exhibited statistically significant gastroprotective effect with better results for Phlomis grandiflora and Rosa canina (100%). At the concentration under study, both crude drugs were more effective than the reference compound misoprostol at 0.4 mg/kg. Even the weakest anti-ulcerogenic effect observed for Papaver rhoeas roots was found statistically potent (95.6%). Histopathological studies confirmed the results of the in vivo test.

Elevational trends in defense chemistry, vegetation, and reproduction in Sanguinaria canadensis.

Evaluation of biotic interactions along geographic gradients reveals that pressure on plant populations by herbivores and pathogens increases as latitude decreases, and is accompanied by a parallel increase in the number and toxicity of alkaloid-bearing plants. We compared rhizome alkaloid content with plant reproductive and vegetative characters in Sanguinaria canadensis (Papaveraceae) along an elevational gradient over two growing seasons to ascertain 1) if alkaloid production in bloodroot varies among populations and systematically with elevation, and 2) if there exists a correlation between isoquinoline alkaloid, vegetative and reproductive production. In general, alkaloid content in bloodroot rhizomes declines with elevation, increases with rhizome water content, varies by site, and fluctuates seasonally with plant growth and reproduction. Alkaloid content was positively correlated with vegetative and reproductive effort with few exceptions. Analysis of total protopine and benzophenanthridine alkaloid concentrations revealed generally similar patterns as those of individual alkaloid concentrations, although significant differences did appear between individual alkaloid concentrations.

Mechanisms of self-incompatibility in flowering plants.

Self-incompatibility is a widespread mechanism in flowering plants that prevents inbreeding and promotes outcrossing. The self-incompatibility response is genetically controlled by one or more multi-allelic loci, and relies on a series of complex cellular interactions between the self-incompatible pollen and pistil. Although self-incompatibility functions ultimately to prevent self-fertilization, flowering plants have evolved several unique mechanisms for rejecting the self-incompatible pollen. The self-incompatibility system in the Solanaceae makes use of a multi-allelic RNase in the pistil to block incompatible pollen tube growth. In contrast, the Papaveraceae system appears to have complex cellular responses such as calcium fluxes, actin rearrangements, and programmed cell death occurring in the incompatible pollen tube. Finally, the Brassicaceae system has a receptor kinase signalling pathway activated in the pistil leading to pollen rejection. This review highlights the recent advances made towards understanding the cellular mechanisms involved in these self-incompatibility systems and discusses the striking differences between these systems.