De novo transcriptome analysis provides insights into formation of in vitro adventitious root from leaf explants of Arnebia euchroma.

BACKGROUND: Adventitious root formation is considered a major developmental step during the propagation of difficult to root plants, especially in horticultural crops. Recently, adventitious roots induced through plant tissue culture methods have also been used for production of phytochemicals such as flavonoids, anthocyanins and anthraquinones. It is rather well understood which horticultural species will easily form adventitious roots, but the factors affecting this process at molecular level or regulating the induction process in in vitro conditions are far less known. The present study was conducted to identify transcripts involved in in vitro induction and formation of adventitious roots using Arnebia euchroma leaves at different time points (intact leaf (control), 3 h, 12 h, 24 h, 3 d, 7 d, 10 d and 15 d). A. euchroma is an endangered medicinal Himalayan herb whose root contains red naphthoquinone pigments. These phytoconstituents are widely used as an herbal ingredient in Asian traditional medicine as well as natural colouring agent in food and cosmetics. RESULTS: A total of 137.93 to 293.76 million raw reads were generated and assembled to 54,587 transcripts with average length of 1512.27 bps and N50 of 2193 bps, respectively. In addition, 50,107 differentially expressed genes were identified and found to be involved in plant hormone signal transduction, cell wall modification and wound induced mitogen activated protein kinase signalling. The data exhibited dominance of auxin responsive (AUXIN RESPONSE FACTOR8, IAA13, GRETCHEN HAGEN3.1) and sucrose translocation (BETA-31 FRUCTOFURANOSIDASE and MONOSACCHARIDE-SENSING protein1) genes during induction phase. In the initiation phase, the expression of LATERAL ORGAN BOUNDARIES DOMAIN16, EXPANSIN-B15, ENDOGLUCANASE25 and LEUCINE-rich repeat EXTENSION-like proteins was increased. During the expression phase, the same transcripts, with exception of LATERAL ORGAN BOUNDARIES DOMAIN16 were identified. Overall, the transcriptomic analysis revealed a similar patterns of genes, however, their expression level varied in subsequent phases of in vitro adventitious root formation in A. euchroma. CONCLUSION: The results presented here will be helpful in understanding key regulators of in vitro adventitious root development in Arnebia species, which may be deployed in the future for phytochemical production at a commercial scale.

Flow cytometric seed screen data are consistent with models of chromosome inheritance in asymmetrically compensating allopolyploids.

Angiosperms have evolved a mechanism of double fertilization, which results in the production of a separate embryo (new individual) and endosperm (nutritive tissue). The flow cytometric seed screen (FCSS) was developed to infer plant reproduction modes based on endosperm-to-embryo DNA content ratio (Pind ). A ratio of 1.5 indicates sexual reproduction, whereas higher values of ≥2.0 are consistent with apomixis. Although FCSS has been successfully applied to the study of sexual and asexual plants, the limits of FCSS and particularly its potential for determination of reproduction modes in hemisexual plants have not been explored. Here, we evaluated the application of FCSS to the study of reproduction modes in two asymmetrically compensating allopolyploids (ACAs), Onosma arenaria and Rosa canina. These two species are characterized by the presence of asexually inherited univalent-forming and sexually inherited bivalent-forming chromosome sets. They both use asymmetric meiosis, which eliminates univalent-forming chromosome sets from the male gamete and retains them in the female gamete. Different chromosomal behavior in male and female meiosis in these plants is reflected in different theoretically derived Pind values, which deviate from a sexual 1.5 value. Here, we determined Pind FCSS-based values in seeds of ACAs, and compared the results to sexual species. As expected, we determined that the mean Pind is 1.51, 1.52, and 1.52 in the sexual plants, that is, Capsella bursa-pastoris, Crataegus monogyna, and O. pseudoarenaria, respectively. In the ACAs, different mean Pind values were determined for O. arenaria (1.61) and R. canina (1.82). These values are consistent with the theoretical Pind values determined based on models of chromosome inheritance. This study highlights the precision of flow cytometry in determining DNA content and it's utility in screening reproduction modes. Additionally, it advocates for more in-depth investigations into rapid screening of accessions where the Pind ratio has deviated from the 1.5 value typical of sexual species, which may indicate meiotic irregularities.

Gender specialisation and stigma height dimorphism in Mediterranean Lithodora fruticosa (Boraginaceae).

Dimorphism in style height has evolved repeatedly in flowering plants, with some individuals having short and others long styles; in the case of distylous species, stigma position varies reciprocally with that of the anthers. Distyly can be associated with divergence in the functional gender between long- and short-styled individuals, but gender divergence has rarely been investigated in species with a simple stigma height polymorphism in the absence of reciprocal dimorphism in anther position. To evaluate the relation between stigma height polymorphism and gender, I measured the dimensions of floral morphology and seed production for the two morphs of a large population of the Iberian species Lithodora fruticosa (Boraginaceae). Results confirm the existence of a stigma height polymorphism in L. fruticosa, with long- and short-styled individuals at a 1:1 ratio in the studied population. Long-styled individuals produced substantially more seeds than did short-styled individuals, pointing to strong divergence in functional gender between the two morphs. The results of this study are puzzling in light of recent work that suggests that L. fruticosa has a multi-allelic self-incompatibility system. I discuss the significance of gender divergence in L. fruticosa and evaluate hypotheses that might explain it.

[Local population of Eritrichium caucasicum as an object of mathematical modelling. I. Life cycle graph and a nonautonomous matrix model].

For the plant species, which is considered a short-lived perennial, we have composed a scale of ontogenetic stages and the life cycle graph (LCG) according to annual observations on permanent sample plots in an Alpine lichen heath during the 2009-2014 period. The LCG that reflects seed reproduction has been reduced to the one that avoids the stage of soil seed bank, yet preserves the arcs of annual recruitment. The corresponding matrix model of stage-structured population dynamics has four stages: juvenile plants (including seedlings), virginal, generative, and 'terminally generative' (the plants die after seed production). Model calibration reduces to directly calculating the rates of transition between stages and those of delays within stages from the data of only one time step, while keeping the two reproduction rates uncertain, yet confined to the quantitative bounds of observed recruitment. This has enabled us to determine a feasible range for the dominant eigenvalue of the model matrix, i.e., the quantitative bounds for the measure of how the local population adapts to its environment, at each of the five time steps, resulting in aformally nonautonomous model. To obtain 'age-specific parameters' from a stage-classified model, we have applied the technique that constructs a virtual absorbing Markov chain and calculates its fundamental matrix. In a nonautonomous model, the estimates of life expectancy also depend on the time of observation (that fixes certain environmental conditions), and vary from two to nearly seven years. The estimates reveal how specifically short lives the short-lived perennial, while their range motivates the task to average the model matrices over the whole period of observation. The model indicates that Eritrichium caucasicum plants spend the most part of their life span in the virginal stage under each of the environment conditions observed, thus revealing the place retention strategy by C. K6rner (2003), or the delayed-development strategy by L.A. Zhukova (1995). We discuss the prospects of model experiments with a logically nonautonomous model to forecast the long-term dynamics of E. caucasicum under a scenario of climate changes.

[Response of vegetative growth and biomass allocation of Lappula semiglabra seedlings to dew gradient.] / ç‹­æžœé¹¤è™±å¹¼è‹—è¥å »ç”Ÿé•¿å’Œç”Ÿç‰©é‡åˆ†é å¯¹å‡ç»“æ°´æ¢¯åº¦çš„å“åº”.

Three dew amount gradients,i.e., few amount of dew (W0), natural amount of dew (W1) and the doubled amount of natural dew (W2) were set to study the response strategies of the morphology, physiology and the dry matter allocation of Lappula semiglabra seedlings on dew amount in Ebinur Lake Wetland National Nature Reserve in Xinjiang, China. The results showed that plant traits in connection with absorbing water by plant leaves, involving in relative content of chlorophyll, leaf water potential, plant height, crown breadth, stem mass and leaf mass ratio increased significantly with the amount of dew. On the contrary, the main stem diameter, root length and root diameter which associated with water absorption and transportation by root had no significant change with the amount of dew. Plant height and the relative content of chlorophyll were the most obvious traits in response to dew amount among all leaf absorption water traits. The proportion of stem biomass had no significant difference along the dew amount gradient. However, the root mass ratio declined gradually along the growth of seedlings, and seedlings in W0 had the smallest decline rather than W1 and W2. In conclusion, L. semiglabra seedlings changed aboveground traits in response to the change of dew amount. Specifically, it increased its photosynthetic capacity and ratio of leaf dry matter to adapt to the variation on dew amount.

Positive effects of non-native grasses on the growth of a native annual in a southern california ecosystem.

Fire disturbance is considered a major factor in the promotion of non-native plant species. Non-native grasses are adapted to fire and can alter environmental conditions and reduce resource availability in native coastal sage scrub and chaparral communities of southern California. In these communities persistence of non-native grasses following fire can inhibit establishment and growth of woody species. This may allow certain native herbaceous species to colonize and persist beneath gaps in the canopy. A field manipulative experiment with control, litter, and bare ground treatments was used to examine the impact of non-native grasses on growth and establishment of a native herbaceous species, Cryptantha muricata. C. muricata seedling survival, growth, and reproduction were greatest in the control treatment where non-native grasses were present. C. muricata plants growing in the presence of non-native grasses produced more than twice the number of flowers and more than twice the reproductive biomass of plants growing in the treatments where non-native grasses were removed. Total biomass and number of fruits were also greater in the plants growing in the presence of non-native grasses. Total biomass and reproductive biomass was also greater in late germinants than early germinants growing in the presence of non-native grasses. This study suggests a potential positive effect of non-native grasses on the performance of a particular native annual in a southern California ecosystem.

Differential host-finding abilities by a weed biocontrol insect create within-patch spatial refuges for nontarget plants.

Many modern weed biocontrol insects exhibit transient "spillover" nontarget herbivory when and where insects are in high density, such as following biocontrol releases, or around dense target weed infestations. Understanding spatial patterns of herbivory is important for predicting efficacy and safety of biocontrol, as refuges from herbivory can buffer plants from population-level impacts. Here, we demonstrate that differential host-finding and arrestment behaviors by an oligophagous biocontrol insect lead to spatial refuges from nontarget herbivory around insect release points within mixed patches of target and nontarget plants. We created transient insect outbreaks by releasing large numbers of Mogulones crucifer Pallas (Coleoptera: Curculionidae) into naturally occurring rangeland patches of the nontarget plant Hackelia micrantha (Eastwood) J.L. Gentry with varying densities of its target weed Cynoglossum officinale L., and monitored spatial patterns of herbivory around release points after 4-7 wk. In complement, we conducted a mark-release-recapture (MRR) experiment to compare M. crucifer's target and nontarget host-finding and arrestment behaviors. For rangeland releases, 95% of nontarget herbivory occurred within 4.25 m of release points, independent of target plant density. Target herbivory occurred throughout our evaluation radii (up to 14 m), where maximum density of diffusing M. crucifer was 1/10 of that in the nontarget herbivory radius. In the MRR experiment, more weevils were recaptured on C. officinale (but not H. micrantha) than expected by chance. M. crucifer's lack of specialized nontarget host-finding and arrestment behaviors means that spatial refuges from herbivory are created for H. micrantha just meters away from sources of high weevil density.

Effects of exogenous methyl jasmonate on the biosynthesis of shikonin derivatives in callus tissues of Arnebia euchroma.

The shikonin derivatives, accumulated in the roots of Arnebia euchroma (Boraginaceae), showed antibacterial, anti-inflammatory, and anti-tumor activities. To explore their possible biosynthesis regulation mechanism, this paper investigated the effects of exogenous methyl jasmonate (MJ) on the biosynthesis of shikonin derivatives in callus cultures of A. euchroma. The main results include: Under MJ treatment, the growth of A. euchroma callus cultures was not inhibited, but the expression level of both the genes involved in the biosynthesis of shikonin derivatives and their precursors and the genes responsible for intracellular localization of shikonin derivatives increased significantly in the Red Strain (shikonin derivatives high-producing strain). The quantitative analysis showed that six out of the seven naphthoquinone compounds under investigation increased their contents in the MJ-treated Red Strain, and in particular, the bioactive component acetylshikonin nearly doubled its content in the MJ-treated Red Strain. In addition, it was also observed that the metabolic profiling of naphthoquinone compounds changed significantly after MJ treatment, and the MJ-treated and MJ-untreated strains clearly formed distinct clusters in the score plot of PLS-DA. Our results provide some new insights into the regulation mechanism of the biosynthesis of shikonin derivatives and a possible way to increase the production of naphthoquinone compounds in A. euchroma callus cultures in the future.

Effect of salicylic acid on the activity of PAL and PHB geranyltransferase and shikonin derivatives production in cell suspension cultures of Arnebia euchroma (Royle) Johnst--a medicinally important plant species.

Cell suspension cultures of Arnebia euchroma were established from the friable callus on liquid Murashige and Skoog medium supplemented with 6-benzylaminopurine (10.0 µM) and indole-3-butyric acid (5.0 µM). Salicylic acid was used to study its effect on the enzymes which participate in shikonin biosynthesis with respect to metabolite (shikonin) content in the cell suspension culture of A. euchroma. In our study, phenylalanine ammonia lyase and PHB geranyltransferase were selected from the entire biosynthetic pathway. Results showed that phenylalanine ammonia lyase is responsible for growth and PHB geranyltransferase for metabolite production. Salicylic acid exhibited an inverse relationship with the metabolite content (shikonin); salicylic acid (100 µM) completely inhibited shikonin biosynthesis. The results presented in the current study can be successfully employed for the metabolic engineering of its biosynthetic pathway for the enhancement of shikonin, which will not only help in meeting its industrial demand but also lead to the conservation of species in its natural habitat.

An improved micropropagation of Arnebia hispidissima (Lehm.) DC. and assessment of genetic fidelity of micropropagated plants using DNA-based molecular markers.

An efficient and improved in vitro propagation method has been developed for Arnebia hispidissima, a medicinally and pharmaceutically important plant species of arid and semiarid regions. Nodal segments (3-4 cm) with two to three nodes obtained from field grown plants were used as explants for shoot proliferation. Murashige and Skoog's (MS) medium supplemented with cytokinins with or without indole-3-acetic acid (IAA) or naphthalene acetic acid was used for shoot multiplication. Out of different PGRs combinations, MS medium containing 0.5 mg l(-1) 6-benzylaminopurine and 0.1 mg l(-1) IAA was optimal for shoot multiplication. On this medium, explants produced the highest number of shoots (47.50 ± 0.38). About 90 % of shoots rooted ex vitro on sterile soilrite under the greenhouse condition when the base (2-4 mm) of shoots was treated with 300 mg l(-1) of indole-3-butyric acid for 5 min. The plantlets were hardened successfully in the greenhouse with 85-90 % survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro-regenerated plants of A. hispidissima. Out of 40 (25 RAPD and 15 ISSR) primers screened, 15 RAPD and 7 ISSR primers produced a total number of 111 (77 RAPD and 34 ISSR) reproducible amplicons. The amplified products were monomorphic across all the micropropagated plants and were similar to the mother plant. To the best of our knowledge, it is the first report on the assessment of the genetic fidelity in micropropagated plants of A. hispidissima.

Mechanisms for success after long-term nutrient enrichment in a boreal forest understory.

Global levels of reactive nitrogen are predicted to rise in the coming decades as a result of increased deposition from the burning of fossil fuels and the large-scale conversion of nitrogen into a useable form for agriculture. Many plant communities respond strongly to increases in soil nitrogen, particularly in northern ecosystems where nitrogen levels are naturally very low. An experiment in northern Canada that was initiated in 1990 has been investigating the effects of long-term nutrient enrichment (fertilizer added annually) on a boreal forest understory community. We used this experiment to investigate why some species increase in abundance under nutrient enrichment whereas others decline. We focused on four species that differed in their responses to fertilization: Mertensia paniculata and Epilobium angustifolium increased in abundance, Achillea millefolium remained relatively constant and Festuca altaica declined. We hypothesized that the two species that were successful in the new high-nutrient, light-limited environment would be taller, have higher specific leaf area, change phenology by growing earlier in the season and be more morphologically plastic than their less successful counterparts. We compared plant height, specific leaf area, growth spurt date and allocation to leaves in plants grown in control and fertilized plots. We demonstrated that each of the two species that came to dominate fertilized plots has a different combination of traits and responses that likely gave them a competitive advantage; M. paniculata has the highest specific leaf area of the four species whereas E. angustifolium is tallest and exhibits morphological plasticity when fertilized by increasing biomass allocation to leaves. These results indicate that rather than one strategy determining success when nutrients become available, a variety of traits and responses may contribute to a species' ability to persist in a nutrient-enriched boreal forest understory.

Nutrient enrichment affects the mechanical resistance of aquatic plants.

For many plant species, nutrient availability induces important anatomical responses, particularly the production of low-density tissues to the detriment of supporting tissues. Due to the contrasting biomechanical properties of plant tissues, these anatomical responses may induce important modifications in the biomechanical properties of plant organs. The aim of this study was to determine the effects of nutrient enrichment on the anatomical traits of two freshwater plant species and its consequences on plant biomechanical performance. Two plant species were grown under controlled conditions in low versus high nutrient levels. The anatomical and biomechanical traits of the plant stems were measured. Both species produced tissues with lower densities under nutrient-rich conditions, accompanied by modifications in the structure of the aerenchyma for one species. As expected, nutrient enrichment also led to important modifications in the biomechanical properties of the stem for both species. In particular, mechanical resistance (breaking force and strength) and stiffness of stems were significantly reduced under nutrient rich conditions. The production of weaker stem tissues as a result of nutrient enrichment may increase the risk of plants to mechanical failure, thus challenging plant maintenance in mechanically stressful or disturbed habitats.

Detailed investigations on the solid cell culture and antimicrobial activities of the Iranian Arnebia euchroma.

In pursuit of strong shikalkin-producing cell lines, seeds of the Iranian Arnebia euchroma were collected from Dena altitudes in the central Zagross. Chemical analysis showed that the dried root of the plant contained about 8.5% (w/w) shikalkin pigment. The root explants of the young plantlets, obtained from the germinated seeds, were used for establishing callus. Then, parameters effective on proliferation and pigment production of the resulting calli were studied in detail. Accordingly, two modified media called mLS and mM9 were optimized for propagation and pigment production, respectively. Using these media, the biomass of the A. euchroma calli was increased to 600%, and the pigment production reached to a maximum of 16.3 mg per gram of the wet biomass in a period of a subculture (21 days). Parallel to these experiments, the antimicrobial activity of shikalkin pigment was examined on some fungi and gram-positive and gram-negative bacteria. Results indicated that the pigment was almost ineffective on fungi and gram-negative bacteria, but it was meaningfully effective against Micrococcus luteus.

Differential responses to guano fertilization among tropical tree species with varying functional traits.

PREMISE OF THE STUDY: Seabirds often cause significant changes to soil properties, and seabird-dominated systems often host unique plant communities. This study experimentally (1) examined species-specific responses to seabird guano gradients, (2) considered the role that differential functional traits among species play in altering plant response to guano, and (3) investigated the implications of seabird guano on range-expanding species. METHODS: Using a greenhouse fertilization experiment, we examined how guano fertilization affects the growth and functional traits of four tree species dominant in the Pacific Islands: Cocos nucifera, Pisonia grandis, Scaevola sericea, and Tournefortia argentea. In these systems, seabirds are frequently found in association with three of these four species; the remaining species, C. nucifera, is a recently proliferating species commonly found in the region but rarely associated with seabirds. KEY RESULTS: We determined that responses to guano addition differed significantly between species in ways that were consistent with predictions based on differing functional traits among species. Notably, we demonstrated that C. nucifera showed no growth responses to guano additions, whereas all seabird-associated plants showed strong responses. CONCLUSIONS: These results provide experimental evidence of differential species response to guano additions, suggesting that differences in species functional traits may contribute to changes in plant communities in seabird-dominated areas, with seabird-associated species garnering performance advantages in these high-nutrient environments. Among these species, results also suggest that C. nucifera may have a competitive advantage in low-nutrient environments, providing an unusual example of how a range-expanding plant species can profit from low-nutrient environments.

Introducing short roots in a desert perennial: anatomy and spatiotemporal foraging responses to increased precipitation.

• The desert flora possesses diverse root architectures that result in fast growth in response to precipitation. We introduce the short root, a previously undescribed second-order root in the aridland chamaephyte Cryptantha flava, and explore fine root production. • We describe the short root anatomy and associated fine roots, correlate standing fine root crop with soil moisture, and explore the architectural level - the short root, third-order lateral roots, or the whole root system - at which fine roots are induced by watering and the amount of water required. • We show that short roots are borne at intervals on lateral roots and produce fine roots at their tips; new fine roots are white and have root hairs, while brown and black fine roots are apparently dead; and fine root production is triggered at the level of lateral roots and with relatively low precipitation (≤ 2 cm). • Short roots are suberized and thus are probably not capable of water uptake themselves, but serve as initiation sites for fine roots that grow rapidly in response to rainfall. Thus, C. flava should be a beneficiary of projected precipitation increases in habitats where rainfall is pulsed.

Flowering life-history strategies differ between the native and introduced ranges of a monocarpic perennial.

Life-history theory makes several key predictions about reproductive strategies on the basis of demographic vital rates, particularly the relationship between juvenile and adult survival. Two such predictions concern the optimal time to begin reproducing and whether semelparity or iteroparity is favored. I tested these life-history predictions and explored how they might differ between the native and introduced ranges of the monocarpic perennial Cynoglossum officinale. I first compared vital rates between ranges. I then used these vital rates to parameterize integral projection models to calculate the population growth rate (lambda) and net reproductive rate (R(0)) as surrogates for fitness to compare strategies within and between ranges. I found that both survival and growth were higher in the introduced range, where size at flowering was larger and iteroparity was much more common than in the native range. The observed and predicted strategies for size at flowering were similar in the native range. In the introduced range, however, even though plants flowered at a larger size, the observed size was not as large as the optimum predicted by lambda or the higher optimum predicted by R(0). Iteroparity conferred higher fitness in both ranges, as measured by both fitness metrics, suggesting that severe constraints, potentially specialist herbivores, prevent this strategy from becoming more common in the native range.

[Characteristics of natural radionuclide distribution in different organs of plants growing on territory with increased radiation background].

We investigated wild-growing grassy plants such as Alhagi pseudalhagi, Zygophyllum, Juncus acutus and Argusia sibirica with the purpose of studying the mechanism of carry of radionuclides from ground in plants and an establishment most the common laws of distribution of radionuclides between different organs of plants. The results of the research show that the researched plants are mainly accumulated 40K. On a degree of accumulation, 226Ra occupies the second place, but 232Th doesn't participate almost in processes of carry from ground in plants. It is established that root systems of plants possesses unequal ability to absorb same radionuclides from the same ground. For example, the root system of Argusia sibirica is characterized by smaller ability to absorb 226Ra, but 40K is more accessible. It is shown that biological availability radionuclides in the given ground depends both on a kind of plants and on radionuclides. For example, Argusia sibirica, Zygophyllum show higher accumulating ability to 40K and in relation to 226Ra, Alhagi pseudalhagi and Juncus acutus are more sensitive. It is established that accumulating ability of stalks of different plants in relation to 40K are appreciably different. Distribution of radionuclides in seeds of plants has other character. Thus Alhagi pseudalhagi in the seeds had also certain amount of 232Th, and leaves Argusia sibirica in comparison with Zygophyllum have a high degree of accumulation as for 40K, so for 226Ra.

Cell growth and shikonin production of Arnebia euchroma in a periodically submerged airlift bioreactor.

Arnebia euchroma was grown in a 2-l periodically submerged, airlift bioreactor (PSAB) in which the non-submerged (immobilization culture) and submerged (suspension culture) operations were controlled automatically. PSAB had advantages in improving cell growth, shikonin content, shikonin production and cell aggregation compared with suspension culture. Under the optimal submerged/non-submerged period of 10 min/15 h, the shikonin content (4.6%, w/w) and, cell dry mass (16.8 g/l) were 229 and 26% higher than those in suspension culture.

[Biosynthesis of naphthoquinoine pigments in plants from Boraginaceae family in nature and in vitro culture].

Literature data on the production of naphthoquinoine pigment, shikonin, by the intact plants and cell cultures derived from the members of Boraginaceae family have been reviewed. The results of our own studies on generation of highly productive Arnebia euchroma cell lines able to accumulate up to 20% of shikonin in dry biomass are presented. Data on localization, application of naphthoquinoine pigments, peculiarities and enzyme control of their biosynthesis and transportation within plant cells and in culture in vitro are summarized.