The influence of different light spectra and intensities was evaluated in an in vitro culture of Achillea millefolium L. (yarrow). The treatments were: use of light emitting diode (LED) lamps in the blue, red, green and white wavelengths, and the intensities of 13; 27; 35; 47 and 69 µmol m−2 s−1, obtained with a cool fluorescent lamp. At 45 days of culture in hormone-free MS medium, the production of dry matter, survival, rooting, length of shoots and roots, numbers of roots, pigments, as well as volatile constituents, were evaluated. The quality and intensity of light significantly influenced the in vitro growth of yarrow. In the experiment with LEDs, the blue spectrum provided the highest dry matter accumulation, number of roots, percentage of rooting and survival. In different light intensities, 27 µmol m−2 s−1 showed the highest values for the variables analyzed. Thus, blue LED spectrum or cool fluorescent lamp with 27 µmol m−2 s−1 benefits the in vitro growth of yarrow. A variation in number, content and profile of volatile constituents under the influence of quality and light intensity was also observed. The major constituents identified were sabinene, 1,8 cineole, borneol, β-caryophyllene and β-cubebene, independent of the light treatments. The amount and composition of the volatile compounds ranged with the intensity and quality of light. Thus, it is possible to adjust the ambient light in order to yield the compounds of interest.

In vitro culture of Achillea millefolium L.: quality and intensity of light on growth and production of volatiles

Gerbera jamesonii cv. Dura is a well-known and economically important ornamental crop that is produced using micropropagation. This study aimed to evaluate the effect of various light wavelengths in six different light emitting diode (LED) treatments on biometric attributes and photosynthetic pigment content in gerbera plants grown in vitro. The LED treatments were B (100% blue), RB1 (50% red and 50% blue), RB2 (70% red and 30% blue), RBW (40% red, 40% blue, and 20% white), RBfR (49% red, 49% blue, and 2% far red), and R (100% red). Light quality was seen to significantly affect plant growth and development. The highest shoot multiplication rate was observed in plants grown under both RB1 and RB2. Plantlets grown under R displayed the greatest shoot elongation, and their petioles were three times longer than those grown under B. Both B and R resulted in reduced leaf blade area. Rooting of shoots was observed in plants grown under all light treatments; however, R stimulated adventitious root formation. The average number of roots produced by plants under all light treatments was calculated and found to match that produced under RB2. The RBfR treatment caused a reduction in leaf dry weight compared to that produced under B, which represented the highest leaf dry weight. Control fluorescent lighting, supplied by Philips TL-D 36W/54 lamps, had a positive effect on root dry weight. Photosynthetic pigment content was higher in the leaves of rooted plants compared to that in plants at the multiplication stage. RB2 resulted in higher concentrations of chlorophyll a and b and carotenoids, whereas lower accumulation of photosynthetic pigments was observed under R. These results demonstrate that light wavelength manipulation through LEDs can be strategically used for the rapid and large-scale propagation of gerbera. The outcomes of this study offer potential to improve micropropagation efficiency and reduce the costs of in vitro plant production.

Impact of LED light sources on morphogenesis and levels of photosynthetic pigments in Gerbera jamesonii grown in vitro

Dysphania ambrosioides L. is a medicinal plant with anti-helmintic potential. The aim of this study was to evaluate separately the effect of light spectra and elicitors on Dysphania ambrosioides growth and volatile constituents in vitro. Thus, plantlets were first cultured under blue (B), red (R), white, combinations of B:R (1:1, 2:1, 1:2) from LEDs and fluorescent lamps. Secondly, nodal segments were inoculated in the medium supplemented with chitosan (0, 50, 100, 150, and 200 mg L− 1) and salicylic acid (0, 3, 6, 9, and 12 mg L− 1). After 40 days of cultivation, the growth parameters and chemical composition of volatile constituents were evaluated. The light spectra significantly influenced in vitro growth of D. ambrosioides. The best growth occured using white LED or a blue:red combination of 2:1. It was also observed that the blue LEDs inhibited the synthesis of Z-ascaridole, while fluorescent light promoted a greater conversion of α-terpinene into ascaridole. The elicitors, chitosan and salicylic acid had a negative effect on the growth of nodal segments. However, the highest Z-ascaridole content was obtained at 50 to 100 mg L− 1 of chitosan and with 6 to 9 mg L− 1 of salicylic acid. The present study demonstrates that shoots regenerated from nodal segments exposed to different light spectra or on MS medium containing chitosan and salicylic acid can exhibit an altered growth and increased volatile constituents of interest. Elicitors and different wavelengths provided by LEDs and fluorescent lamp were developed for Dysphania ambrosioides, altering growth and increase volatile constituents of interest.

Influence of light spectra and elicitors on growth and ascaridole content using in vitro cultures of Dysphania ambrosioides L.

Phomopsis asparagi is one of the most serious fungal pathogens, which causes stem blight disease in Asparagus officinalis (AO), adversely affecting its production worldwide. Recently, the development of novel asparagus varieties using wild Asparagus genetic resources with natural P. asparagi resistance has become a priority in Japan due to the lack of resistant commercial AO cultivars. In this study, comparative metabolome and transcriptome analyses of susceptible AO and resistant wild Asparagus kiusianus (AK) 24 and 48 h postinoculated (AOI_24 hpi, AOI_48 hpi, AKI_24 hpi and AKI_48 hpi, respectively) with P. asparagi were conducted to gain insights into metabolic and expression changes associated with AK species. Following infection, the resistant wild AK showed rapid metabolic changes with increased levels of flavonoids and steroidal saponins and decreased asparagusic acid glucose ester content, compared with the susceptible AO plants. Transcriptome data revealed a total of 21  differentially expressed genes (DEGs) as the core gene set that displayed upregulation in the resistant AK versus susceptible AO after infection with P. asparagi. Kyoto Encyclopedia of Genes and Genomes pathway analysis of these DEGs identified 11 significantly enriched pathways, including flavonoid biosynthesis and primary metabolite metabolism, in addition to plant signaling and defense-related pathways. In addition, comparative single-nucleotide polymorphism and Indel distributions in susceptible AO and resistant AK plants were evaluated using the latest AO reference genome Aspof.V1. The data generated in this study are important resources for advancing Asparagus breeding programs and for investigations of genetic linkage mapping, phylogenetic diversity and plant defense-related genes.

Comparative Metabolome and Transcriptome Analyses of Susceptible Asparagus officinalis and Resistant Wild A. kiusianus Reveal Insights into Stem Blight Disease Resistance.

The most relevant wavelengths in photobiology are the ultraviolet, the visible light, and the infrared. Wavelengths between 300 and 900 nm can affect plants growth and development. Nevertheless, not only light quality can influence plant growth processes. Light properties such as its intensity and duration, as well as climatic factors, are also involved. The development of studies using a single wavelength is very useful to identify the most responsive spectrum in the target application. However, detecting the influences of adjacent spectrum segments in certain applications is difficult because the spectrum is continuous, with no apparent margins between neighboring colors. The agricultural technique of colored covering, although not new, can still be explored in order to clarify aspects concerning its use as light stimulus and the response of plants to it, and thus offer to farmers alternatives of crop management, which will also contribute significantly to the study of plant physiology. Research focused on the influence of light on plant growth is wide; however, it is necessary to discuss its findings so as to offer solid evidence if the aim is to introduce modifications in the light environment as a strategy for crop management. © 2015. Acad. Colomb. Cienc. Ex. Fis. Nat.

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Modificaciones fotomorfogénicas inducidas por la calidad de la luz en plantas cultivadas

Gentians are medicinal plants whose roots and rhizomes have been used as natural medicines for ga strointestinal problems. �e active components are bitter compounds known as secoiridoid glycosides such as gentiopicrin and swertiamarin. Previously, procedures for the in vitro clonal culture of gentian plantlets were established. In this study, the eect of dierent wavelengths of light emitted by LED on plantlet growth and the concentrations of active components w as investigated. We found that plantlet growth was promoted aer irradiation with red light. Because a similar trend was observed in all gentian cultivars tested, the improved eect of red light on plantlet growth seemed to be a common characteristic of Gentiana species. We also found that both leaves and roots of the plantlets contained gentiopicrin and swertiamarin. Gentiana triora 'M aciry' had the highest concentration of both compounds compared with several gentian lines and cultivars. Furthermore, the concentrations of roots were increased by far-red and blue light irradiation. �ese results suggest that modication of light quality by LED is an eective strategy for improving the growth and concentration of active components in gentian plantlets that can be used as a novel ingredient in natural medicine.

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Modification of light quality improves the growth and medicinal quality of clonal plantlets derived from the herbal plant Gentiana

Gentiobiose, a β-1,6-linked glycosyl-disaccharide, accumulates abundantly in Gentianaceae and is involved in aspects of plant development, such as fruits ripening and release of bud dormancy. However, the mechanisms regulating the amount of gentio-oligosaccharide accumulation in plants remain obscure. The present study aimed to identify an enzyme that modulates gentio-oligosaccharide amount in gentian (Gentiana triflora). A protein responsible for gentiobiose hydrolysis, GtGen3A, was identified by partial purification and its peptide sequence analysis. The enzyme had a molecular mass of ∼67 kDa without a secretory signal peptide sequence. Sequence analysis revealed that GtGen3A could be a β-glucosidase member belonging to glycoside hydrolase family 3 (GH3). GtGen3A showed a homology to GH3 β-glucan exohydrolases, ExoI of Hordeum vulgare, and ExgI from Zea mays, which preferentially hydrolyzed β-1,3- and β-1,4-linked oligosaccharides. The purified recombinant GtGen3A (rGtGen3A) produced in Escherichia coli showed optimal reaction at pH 6.5 and 20°C. The rGtGen3A liberated glucose from β-1,2-, β-1,3-, β-1,4-, and β-1,6-linked oligosaccharides, and showed the highest activity toward gentiotriose among the substrates tested. Kinetic analysis also revealed that rGtGen3A preferentially hydrolyzed gentiotriose. Virus-induced gene silencing of Gtgen3A in gentian plantlets resulted in predominant accumulation of gentiotriose rather than gentiobiose. Furthermore, the expression level of Gtgen3A was almost similar to the amount of gentiobiose in field-grown gentians. These findings suggest that the main function of GtGen3A is the hydrolysis of gentiotriose to gentiobiose, and that GtGen3A plays a role in modulating gentiobiose amounts in gentian.

Gtgen3A, a novel plant GH3 β-glucosidase, modulates gentio-oligosaccharide metabolism in Gentiana .

The elongation of flower longevity increases the commercial value of ornamental plants, and various genes have been identified as influencing flower senescence. Recently, EPHEMERAL1 (EPH1), encoding a NAC-type transcription factor, was identified in Japanese morning glory as a gene that promotes flower senescence. Here we attempted to identify an EPH1 homolog gene from cultivated Japanese gentians and characterized the same with regard to its flower senescence. Two EPH1-LIKE genes (EPH1La and EPH1Lb), considered as alleles, were isolated from a gentian cultivar (Gentiana scabra × G. triflora). Phylogenetic analyses revealed that EPH1L belongs to the NAM subfamily. The transcript levels of EPH1L increased along with its senescence in the field-grown flowers. Under dark-induced senescence conditions, the gentian-detached flowers showed the peak transcription level of EPH1L earlier than that of SAG12, a senescence marker gene, suggesting the involvement of EPH1L in flower senescence. To reveal the EPH1L function, we produced eph1l-knockout mutant lines using the CRISPR/Cas9 system. When the flower longevity was evaluated using the detached flowers as described above, improved longevity was recorded in all genome-edited lines, with delayed induction of SAG12 transcription. The degradation analysis of genomic DNA matched the elongation of flower longevity, cumulatively indicating the involvement of EPH1L in the regulation of flower senescence in gentians.

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Isolation and Functional Analysis of EPHEMERAL1-LIKE (EPH1L) Genes Involved in Flower Senescence in Cultivated Japanese Gentians

Glycoside hydrolases require carboxyl groups as catalysts for their activity. A retaining xylanase from Streptomyces olivaceoviridis E-86 belonging to glycoside hydrolase family 10 possesses Glu128 and Glu236 that respectively function as acid/base and nucleophile. We previously developed a unique mutant of the retaining xylanase, N127S/E128H, whose deglycosylation is triggered by azide. A crystallographic study reported that the transient formation of a Ser-His catalytic dyad in the reaction cycle possibly reduced the azidolysis reaction. In the present study, we engineered a catalytic dyad with enhanced stability by site-directed mutagenesis and crystallographic study of N127S/E128H. Comparison of the Michaelis complexes of N127S/E128H with pNP-X2 and with xylopentaose showed that Ser127 could form an alternative hydrogen bond with Thr82, which disrupts the formation of the Ser-His catalytic dyad. The introduction of T82A mutation in N127S/E128H produces an enhanced first-order rate constant (6 times that of N127S/E128H). We confirmed the presence of a stable Ser-His hydrogen bond in the Michaelis complex of the triple mutant, which forms the productive tautomer of His128 that acts as an acid catalyst. Because the glycosyl azide is applicable in the bioconjugation of glycans by using click chemistry, the enzyme-assisted production of the glycosyl azide may contribute to the field of glycobiology.

Sugar Composition in Asparagus Spears and Its Relationship to Soil Chemical Properties.

The lacquer tree, Toxicodendron vernicifluum, is a common industrial crop in East Asia. However, T. vernicifluum seeds are extremely difficult to germinate, which poses a major obstacle to establishing seedlings for sap production. In this study, we examined the germination properties of T. vernicifluum seeds in order to establish an inexpensive and effective method to promote seed germination. The seeds are covered with a hard endocarp, which we degrade using conventional sulfuric acid-based methods. Although sulfuric acid was effective in promoting seed germination, the germination rate was less than 5%. In addition to treatment with sulfuric acid, co-treatment with cold temperatures or the phytohormone gibberellic acid increased the germination rate to 22–35%. Seed viability analysis combined with specific gravity-based seed selection revealed that more than half of the seeds housed embryos that were incapable of germination. In additions, specific gravity-based seed selection aided in the selection of seeds capable of germination and improved the germination rate to approximately 47%. Taken together, our results suggest that the low germination rate of T. vernicifluum seeds is due to deep seed dormancy—which is controlled by physical and physiological mechanisms—and low embryo viability. To improve the germination rate of T. vernicifluum seeds, we propose an effective method whereby seeds with good germination capacity are selected based on specific gravity, following which their physiological dormancy is inactivated through cold pretreatment.

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Chiharu Yoshida

Papers

Modification of light quality improves the growth and medicinal quality of clonal plantlets derived from the herbal plant Gentiana

Gtgen3A, a novel plant GH3 β-glucosidase, modulates gentio-oligosaccharide metabolism in Gentiana .

Isolation and Functional Analysis of EPHEMERAL1-LIKE (EPH1L) Genes Involved in Flower Senescence in Cultivated Japanese Gentians

Sugar Composition in Asparagus Spears and Its Relationship to Soil Chemical Properties.

Methods to promote seed germination in the lacquer tree, Toxicodendron vernicifluum (Stokes) F.A. Barkley