Photoperiod modulates growth and pigments and 20-hydroxyecdysone accumulation in Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen] grown in vitro

Plant Cell, Tissue and Organ Culture (PCTOC) - Photoperiod regulates different morphophysiological processes in plants, directly impacting photosynthetic performance and, consequently, primary and...     

A CO2-enriched atmosphere improves in vitro growth of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]

The aim of the present study was to evaluate the effects of forced ventilation and CO₂ enrichment (360 or 720 μmol mol^?1 CO₂) on the in vitro growth and development of Pfaffia glomerata, an endangered medicinal species, under photomixotrophic or photoautotrophic conditions. P. glomerata nodal segments showed substantial differences in growth, relative water content and water loss from leaves, photosynthetic pigments, stomatal density, and leaf anatomical characteristics under these different treatments. CO₂ enrichment led to increased photosynthetic pigments and reduced stomatal density of in vitro cultivated P. glomerata. A lack of sucrose in the culture medium increased 20-hydroxyecdysone levels, but the increase in CO₂ levels did not further elevate the accumulation of 20-hydroxyecdysone. All growth increased in a CO₂-enriched atmosphere. In addition, CO₂ enrichment, with or without sucrose, gave a lower relative water loss from leaves. This finding indicates that either a photoautotrophic or photomixotrophic system in a CO₂-enriched atmosphere may be suitable for large-scale propagation of this species.     

Morphophysiological in vitro performance of Brazilian ginseng (Pfaffia glomerata (Spreng.) Pedersen) based on culture medium formulations

Pfaffia glomerata has potential pharmacological and medicinal properties due to the production of a secondary metabolite known as the phytoecdysteroid 20-hydroxyecdysone (20E). There have been increasing efforts for massive in vitro propagation of Pfaffia plants due to high extractivism and overharvesting of this species. Research on the species has shown that photoautotrophic cultivation can improve the production of 20E. In addition, other abiotic factors such as the formulations of culture media can influence the morphophysiological behavior of the plants in vitro. Therefore, the objective of this study was to analyze the morphological and physiological performances of P. glomerata plants in different formulations of culture media, under photoautotrophic and photomixotrophic propagation conditions. Six medium formulations, the Driver and Kuniyuki medium (DKW), Correia et al. medium (JADS), Murashige and Skoog medium (MS), Quoirin and Lepoivre medium (QL), Rugini medium (OM), and Woody Plant medium (WPM), all supplemented with DKW vitamins, 100 mg L⁻¹ myo-inositol, 6.5 g L⁻¹ agar, and with or without 3% (w/v) sucrose, were evaluated. Cultures were maintained at 25 ± 2°C, with a 16 h-photoperiod under 60 μmol m⁻² s⁻¹ of irradiance under a fluorescent lamp for 50 d. Results showed that the presence or absence of sucrose, and the different nutritional formulations influenced growth, photosynthetic pigment content, endogenous levels of sugars, leaf morphology, levels of 20E, and transport of water and minerals in P. glomerata. Notably, OM, DKW, QL, and WPM media promoted higher production of 20E under photomixotrophic growth conditions.

     

CO2 enrichment and supporting material impact the primary metabolism and 20-hydroxyecdysone levels in Brazilian ginseng grown under photoautotrophy

Plant cell and organ cultures via the implementation of effective elicitation strategies can offer attractive biotechnological platforms for the enhanced production of phytochemicals of pharmaceutical interest. For the first time, the elicitation of exogenous signal molecules was conducted to enhance the production of pharmacologically active alkaloids and flavonoids in Isatis tinctoria L. hairy root cultures (ITHRCs). ITHRCs III and V correspondingly possessing high alkaloid and flavonoid productivity were adopted for elicitation treatments. The maximum accumulation of alkaloids in ITHRCs III elicited by 142.61 µM salicylic acid for 28.18 h and flavonoids in ITHRCs V elicited by 179.54 µM methyl jasmonate for 41.87 h increased 5.89- and 11.21-folds as compared with controls, respectively. Moreover, expressions of 11 genes involved in alkaloid and flavonoid biosynthetic pathways were significantly up-regulated following elicitation, among which YUCCA, CHI and F3′H genes might play a crucial role in the target phytochemical augmentation. Overall, two effective elicitation protocols were provided here to improve the yields of bioactive alkaloids and flavonoids in ITHRCs, which was useful for the scale-up production of these valuable compounds to meet the demands for natural bioactive ingredients by pharmaceutical industries.

     

A low-cost alternative membrane system that promotes growth in nodal cultures of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]

In vitro propagated plants under conditions of low gas exchange generally show morphological and physiological anomalies that lead to high mortality rates during ex vitro acclimatization. The use of gas-permeable membranes increases natural ventilation in culture vessels, photosynthesis and growth rates. However, commercial membranes are expensive, which limits their application. In this study, low-cost, simple to manufacture, alternative membranes were developed to promote gas exchange in jars used for in vitro plant tissue culture. The membranes were developed using polytetrafluoroethylene film and two or three layers of microporous tape (Missner & Missner^?), and were designed to increase the growth of nodal cultures of Pfaffia glomerata (Brazilian ginseng). Conditions that provided higher gas exchange led to an increase in plant growth and content of photosynthetic pigments compared to a closed system without a gas-permeable membrane. The alternative membranes showed similar results for water vapor loss rate and photosynthetic pigments when compared to a commercial membrane. The alternative membranes were also an efficient barrier against contamination and remained intact after being autoclaved multiple times. Among the membranes tested, the traits of the P. glomerata in vitro-derived plants were similar when propagated using the alternative membrane with three layers of microporous tape or the commercial membrane. However, the alternative membrane has a unit cost that is ten times lower than the commercial membrane.     

Physiological,epigenetic, and proteomic responses in Pfaffia glomerata growth in vitro under salt stress and 5-azacytidine

Protoplasma - Plants adjust their complex molecular, biochemical, and metabolic processes to overcome salt stress. Here, we investigated the proteomic and epigenetic alterations involved in the...     

Response of oxidative stress defense systems in rice (Oryza sativa) leaves with supplemental UV-B radiation

The impact of elevated ultraviolet-B radiation (UV-B, 280–320 nm) on membrane systems and lipid peroxidation, and possible involvement of active oxygen radicals was investigated in leaves of two UV-B susceptible rice cultivars (Oryza sativa L. cvs IR74 and Dular). Rice seedlings were grown in a greenhouse for 10 days and then treated with biologically effective UV-B (UV-B_BE) radiation for 28 days. Oxidative stress effects were evaluated by measuring superoxide anion (O₂) generation rate, hydrogen peroxide (H₂O₂) content, malondialdehyde (MDA) concentration and relative electrolyte conductivity (EC) for IR74 and Dular at 0 (control), 6 or 13 kJ m^?2 day^?1 UV-B_BE. Significant increases in these parameters were found in rice plants grown at 13 vs 0 kJ m^?2 day^?1 UV-B_BE after 28 days; indicating that disruption of membrane systems may be an eventual reason for UV-B-induced injury in rice plants. There was a positive correlation between O₂^? generation and increases in EC or MDA in leaves. Activities of enzymatic and nonenzymatic free radical scavengers were measured for IR74 after 7, 14, 21 and 28 days of exposure to 13 or 0 UV-B_BE to evaluate dynamics of these responses over time. Activities of catalase and superoxide dismutase (but not ascorbate peroxidase) and concentrations of ascorbic acid and glutathione were enhanced by 13 vs 0 UV-B_BE after 14 days of UV-B exposure. Further exposure to 28 days of UV-B was associated with a decline in enzyme activities and ascorbic acid, but not glutathione. It is suggested that UV-B-induced injury may be associated with disturbance of active oxygen metabolism through the destruction and alteration of both enzymatic and nonenzymatic defense systems in rice.     

Exogenous 7,8-dihydro-8α-20-hydroxyecdysone application improves antioxidative enzyme system,photosynthesis, and yield in rice under high-temperature condition

7,8-Dihydro-8α-20-hydroxyecdysone (DHECD) has biological activity similar to brassinosteroids. In this study, we investigated the role of DHECD on the antioxidative enzyme activity and photosynthetic performance of rice subjected to high-temperature conditions. DHECD at either 1 or 10 µM was used as a foliar application to rice leaves at 23 days after sowing, and then, at 31 days after sowing, rice plants were moved to a heat chamber at 40/30 °C day/night for 9 days. Application of DHECD significantly increased the activities of the antioxidative enzymes: superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase. The results of this study provide the first direct evident for DHECD-elevated antioxidative enzyme activity and decreased lipid peroxidation, which possibly induced thermotolerance in rice plants. Furthermore, DHECD was effective in increasing the net photosynthetic rate, stomatal conductance, and water use efficiency, as well as increasing the maximal quantum efficiency of photosystem II and decreasing non-photochemical quenching. Thus, enhancement of photosynthesis by DHECD application resulted in a high grain yield for rice plants subjected to high-temperature conditions.     

Effects of environmentally relevant concentrations of nonylphenol on growth and 20-hydroxyecdysone levels in mysid crustacean, Americamysis bahia

We investigated the effects of environmentally relevant concentrations of nonylphenol (NP) on survival, growth, sexual development and molting of the mysid Americamysis bahia (Crustacea: Mysidacea), including assay of 20-hydroxyecdysone (20E) levels. Seven-day-old mysids were exposed to NP concentrations of 0.3, 1, 3, 10 and 30 microg/L for 14 days. Among growth-related traits we assayed, an effect on body length was the most prominent, with the effects observed at concentrations as low as 1 microg/L NP. In addition, the total number of molting exposed to NP for 14 days was significantly decreased in the treatment groups at 10 or 30 microg/L relative to the controls. These results clearly indicate that subchronic exposure to environmentally relevant concentrations of NP affects growth of mysids. Furthermore, we investigated the effect of NP on production of 20E in female mysids during the molting period. Production of 20E in pre-molting mysids exposed to 30 microg/L of NP was lower than those in control groups, and exposure of female mysids to NP disrupted molting cycles. Taken together, the results suggest that environmentally relevant concentrations of NP can disrupt growth of A. bahia via inhibition of 20E levels and an associated delay in molting.     

Enhanced UV-B radiation under field conditions increases anthocyanin and reduces the risk of photoinhibition but does not affect growth in the carnivorous plant Pinguicula vulgaris

The effects of enhanced UV-B radiation were investigated in the carnivorous plant Pinguicula vulgaris in a field experiment performed in Abisko, North Sweden (68° 21' N, 18° 49' E, 380 m above sea level). Potted plants were exposed to either ambient or ambient plus supplemental UV-B radiation, simulating a 15% ozone depletion. No effect was observed on either the epicuticular (external) or cellular (internal) UV absorbing capacity of the leaves. However, the anthocyanin content was more than doubled by supplemental UV-B radiation. In laboratory experiments, the anthocyanin rich, UV-B treated leaves were less susceptible to a low temperature/high light photoinhibitory treatment, as judged by in vivo chlorophyll fluorescence measurements. Yet, this potential benefit did not considerably affect the growth of the plant in the field (leaf area and dry mass, reproductive dry mass, flowering frequency, senescence rates, dry mass of winter buds). However, there was a marginally significant increase in root dry mass and in the root to shoot ratio, which may underlie the significant increase in the nitrogen content of the leaves. We suggest that P. vulgaris is resistant against UV-B radiation damage and that the possible negative effects of additional UV-B radiation on the growth of these plants may have been effectively counterbalanced by the lower risk of photoinhibition, due to the concomitant increase in anthocyanins.     

Seed germination and seedling growth of two Pseudobombax species (Malvaceae) with contrasting habitats from Brazilian Cerrado

Pseudobombax tomentosum and P. longiflorum are common trees in the Cerrado region, but the former species is more common in forest edges while the later is present in open cerrado areas. This work aimed to investigate differences in seed germination and seedling growth in these species, from seed collected from Cerrado areas in Central Brazil. For this, a seed germination experiment was designed and included four replicates with 25 seeds per species; seeds were randomly distributed in the germination chamber. To evaluate initial seedling growth, seedlings height was measured up to 67 days after seedling emergence; besides, some of these seedlings were grown for biomass evaluation during nine months. Results showed that seeds of the two species had the same germinability (near 100%) and mean germination time (ca. 12 days). However, P. longiflorum showed a more spread seed germination through time, with higher values of coefficient of variation in germination time and uncertainty index; and lower values of synchronization than P. tomentosum. The two species showed basically the same growth pattern, but lower values for height of apical meristem, diameter of underground structures (mostly roots), dry mass of shoots, underground structure and total mass of seedlings in P. tomentosum were obtained, compared to P. longiflorum. Both species allocated more dry mass to underground structures in detriment of shoot. This probably allows resprouting behavior which prevents hydric stress and detrimental fire action typical of the open Cerrado areas.     

Influence of UV-B radiation and Cd2+ on chlorophyll fluorescence, growth and nutrient content in Brassica napus

The possible interaction of two stresses, UV-B radiation and cadmium, applied simultaneously, was investigated in Brassica napus L. cv. Paroll with respect of chlorophyll fluorescence, growth and uptake of selected elements. Plants were grown in nutrient solution containing CdCl2, (0, 0.5, 2 or 5 M) and irradiated with photosynthetically active radiation (PAR, 400-700 nm, 800 mol m^-2 s^-1) with or without supplemental ultraviolet-B radiation (UV-B, 280-320 nm, 15 kJ m^-2 d^-1, weighted irradiance). After 14 d of treatment, the most pronounced effects were found at 2 and 5 M CdCl2 with and without supplemental UV-B radiation. Exposure to cadmium significantly increased the amount of Cd in both roots and shoots. In addition, increases occurred in the concentrations of Fe, Zn, Cu, and P in roots, while K was reduced. In shoots the S content rose significantly both in the presence and absence of UV-B radiation, while significant increases in Mg, Ca, P, Cu, and K occurred only in plants exposed to Cd and UV-B radiation. Manganese decreased significantly under the combined exposure treatment. The rise in S content may have been due to stimulated glutathione and phytochelatin synthesis. Cadmium exposure significantly decreased root dry weight, leaf area, total chlorophyll content, carotenoid content, and the photochemical quantum yield of photosynthesis. As an estimation of energy dissipation processes in photosynthesis, non-photochemical quenching (qNPQ) was measured using a pulse amplitude modulated fluorometer. The qNPQ increased with increasing Cd, while the combination of cadmium and UV-B reduced the qNPQ compared to that in plants exposed only to cadmium or UV-B radiation. The chlorophyll a:b ratio showed a reduction with UV-B at no or low Cd concentrations (0 M, 0.5 M CdCl2), but not at the higher Cd concentrations used (2 M, 5 M CdCl2). Thus in some instances there appeared to be a UV-B and Cd interaction, while in other plants response could be attributed to either treatment alone.Keywords: Brassica napus, cadmium, ultraviolet-B radiation.      

Changes in growth and pigment concentrations with leaf age in pea under modulated UV-B radiation field treatments

We assessed whether growth of garden pea (Pisum sativum mutant Argenteum) was reduced under ecologically relevant enhancements of ultraviolet-B radiation (UV-B, 280–320 nm) by employing modulated field lampbanks which simulated 0, 16 or 24% ozone depiction. In addition, we determined whether enhanced UV-B altered the concentration and distribution of chlorophyll and UV-B-absorbing compounds in leaves, and whether this was dependent on leaf age. There were no significant UV-B effects on the four whole-plant parameters we examined (height, above-ground biomass, total leaflet area or average leaflet area). Of the 12 leaf-level parameters we examined, UV-B had a significant effect (P < 0.05) on only one parameter: the ratio of UV-B-absorbing compounds to chlorophyll, which was greatest at the highest UV-B level. Total chlorophyll concentrations tended to be lower under enhanced UV-B (P= 0.11), while the proportion of UV-B-absorbing compounds in the adaxial epidermis tended to be higher (P= 0.11). Total leaf concentrations of UV-B-absorbing compounds were unaffected by UV-B level. Cooler, suboptimal growing conditions during this late summer/early autumn experiment may have masked some potential UV-B effects. In contrast to the UV-B effects, we found strong leaf-age effects on nearly all parameters that we assessed. On an area basis, concentrations of total chlorophyll and UV-B-absorbing compounds increased with leaf age, while Chlorophyll a/b) ratios decreased. One of the few parameters unaffected by leaf age was the ratio of UV-B-absorbing compounds to total chlorophyll, which remained constant within a given UV-B treatment. Pea was much less sensitive to enhanced UV-B than in previous growth-chamber and greenhouse studies, and in nearly all cases UV-B treatment effects were overshadowed by leaf-age effects. In view of the large effect leaf age had on concentrations of UV-B-absorbing compounds, as well as their distribution within leaves, researchers may need to consider leaf age in UV-B experimental designs.     

Effects of UV-B radiation on growth rates,pigment content and ultrastructure of red (wild type), greenish-brown and green strains of Gracilaria birdiae (Gracilariales,Rhodophyta)

Based on physiological characteristics, we hypothesized that different strains of Gracilaria birdiae from two distinct geographical areas of the Brazilian coast (2500 km apart) would have different responses to long-term exposure to UV-B radiation (UV-B). The locations differ in their environmental conditions: one is a warmer area, Ceará State (CE), closer to the equator; the other is a colder area, Espirito Santo State (ES), closer to the Tropic of Capricorn. To test the hypothesis that the CE population is more resistant to UV-B than the ES population, apical segments of the red (RD_CE, RD_ES), green (GR_CE) and greenish-brown (GB_CE) strains were cultivated in the laboratory under two treatments: control (PAR) and artificial UV-B (PAR + UV-B). Algal performance was evaluated by considering growth rates, pigment content and ultrastructural analysis. Compared with the control, all strains showed a decrease in growth rates after exposure to UV-B. Of all strains, RD_ES showed the greatest sensitivity to UV-B. However, a decrease in growth rate and morphological changes were observed to a lesser extent in the RD_CE strain. Moreover, exposure to UV-B resulted in a decrease in the concentrations of phycobiliproteins in the RD_CE strain. The GB_CE strain showed an increase in phycoerythrin (PE)/allophycocyanin (APC) and phycocyanin (PC)/allophycocyanin (APC) ratios after exposure to UV-B, suggesting this strain had a higher tolerance to the radiation. No differences in the chlorophyll a and carotenoid content were found between the control and UV-B treated samples for all strains. Ultrastructural changes, such as damage to chloroplasts and mitochondria, were present in all strains after exposure to UV-B. In summary, our findings support the hypothesis that the population from Ceará State has adapted to the higher irradiation and is thus more resistant to increased UV-B. Additionally, of the strains tested, the GB_CE and RD_CE strains appear to be more resistant to this radiation.     

Influence of ultraviolet-B (UV-B) radiation on photosynthetic and growth characteristics in field-grown cassava (Manihot esculentum Crantz)

The effects of ultraviolet-B (UV-B between 290 and 320 nm) on photosynthesis and growth characteristics were investigated in field grown cassava (Manihot esculentum Crantz). Plants were grown at ambient and ambient plus a 5.5kJ m^?2 d^?1 supplementation of UV-B radiation for 95 d. The supplemental UV-B fluence used in this experiment simulated a 15% depletion in stratospheric ozone at the equator (0°N). Carbon dioxide exchange, oxygen evolution, and the ratio of variable to maximum fluorescence (F_v/F_m) were determined for fully expanded leaves after 64–76 d of UV-B exposure. AH plants were harvested after 95 d of UV-B exposure, assayed for chlorophyll and UV-B absorbing compounds, and separated into leaves, petioles, stems and roots. Exposure to UV-B radiation had no effect on in situ rates of photosynthesis or dark respiration. No difference in the concentration of UV-B absorbing compounds was observed between treatments. A 2-d daytime diurnal comparison of F_v to F_m ratios indicated a significant decline in F_v/F_m ratios and a subsequent increase in photoinhibition under enhanced UV-B radiation if temperature or PPF exceeded 35°C or 1800μmol m^?2 s^?1, respectively. However, UV-B effects on fluorescence kinetics appeared to be temporal since maximal photosynthetic rates as determined by oxygen evolution at saturated CO₂ and PPF remained unchanged. Although total biomass was unaltered with UV-B exposure, alterations in the growth characteristics of cassava grown with supplemental UV-B radiation are consistent with auxin destruction and reduced apical dominance. Changes in growth included an alteration of biomass partitioning with a significant increase in shoot/root ratio noted for plants receiving supplemental UV-B radiation. The increase in shoot/root ratio was due primarily to a significant decrease in root weight (–32%) with UV-B exposure. Because root production determines the harvest-able portion of cassava, UV-B radiation may still influence the yield of an important tropical agronomic species, even though photosynthesis and total dry biomass may not be directly affected.     

CO2-enriched atmosphere and supporting material impact the growth,morphophysiology and ultrastructure of in vitro Brazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen] plantlets

This study aimed to evaluate under photoautotrophic conditions the effect of CO₂-enriched atmosphere (360 or 1,000 μmol CO₂ mol^?1 air) combined with two substrate types (agar or Florialite^®) in vitro on plants of Pfaffia glomerata, an endangered medicinal species with promising applications in phytotherapy and phytomedicine. The effects of the treatments on the growth, stomatal density, Rubisco activity, carbon isotopic discrimination, metabolite accumulation, photosynthetic pigments and ultrastructural characteristics were investigated. After a 35-day cultivation period, the in vitro-growth of P. glomerata nodal segments under the different treatments resulted in plants with substantial differences in relation to their growth, photosynthetic pigments, stomatal density and leaf ultrastructural characteristics. The enrichment with CO₂ coupled with a porous substrate increased the growth of P. glomerata. The stomatal density in the abaxial epidermis more than doubled in response to the high CO₂ supply in both supporting types, whereas the Rubisco activity and activation state were both unresponsive to the treatments. Regardless of the CO₂ supply, the plants grown in agar displayed higher carbon isotope discrimination than their counterparts grown in Florialite^®. We propose that the long-term photosynthetic performance was improved using Florialite^® as a growth support in combination with a high CO₂ supply. No apparent signs of photosynthetic down-regulation could be found under elevated CO₂ conditions. The enrichment of in vitro atmospheres with CO₂ coupled with a porous substrate offers new possibilities for improving the growth and production on a commercial scale of high morphological and physiological quality Pfaffia plants.     

Solar radiation interception by leafless, semi-leafless and leafed peas (Pisum sativum) under contrasting field conditions

Foliage composition, photosynthetic area index (PAI) and radiation interception were measured for crop canopies of leafless (var. Filby), semi-leafless (var. BS3) and leafed (var. Birte) peas (Pisum sativum). Tendrils and petioles contributed more than 60% of total leaf area for leafless peas but less than 30% for semileafless and leafed pea canopies. PAI was related to radiation interception by calculating attenuation coefficients which indicated that leafless peas intercepted more radiation per unit PAI than either semi-leafless or leafed peas. Data interpretation, however, was complicated because of difficulties in estimating the tendril and petiole surface area contribution to PAI. Radiation interception was related to dry matter accumulation by calculating photosynthetic efficiencies. Leafless and semi-leafless peas converted intercepted radiation into dry matter as efficiently as leafed peas. Under conditions of moisture stress, leafed and leafless peas both intercepted radiation more effectively but converted it into dry matter with reduced photosynthetic efficiency.     

Temporal changes in the growth, saponin content and antioxidant defense in the adventitious roots of Panax ginseng subjected to nitric oxide elicitation

Nitric oxide (NO) is a diffusible, gaseous signaling molecule. In plants, NO influences growth and development, and it can also affect plant responses to various stresses. Because NO induces root differentiation and interacts with reactive oxygen species, we examined the temporal effect of NO elicitation on root growth, saponin accumulation and antioxidant defense responses in the adventitious roots of mountain ginseng (Panax ginseng). The observations revealed that NO is involved in root growth and saponin production. Elicitation with sodium nitroprusside (SNP) activated O₂ ⁻-generating NADPH oxidase (NOX) activity, which most probably subsequently enhanced growth of adventitious roots of mountain ginseng. A severe inhibition of NOX activity and decline in dry weight of SNP elicited adventitious roots in the presence of NOX inhibitor (diphenyl iodonium, DPI), which further supports involvement of NOX in root growth. Enhanced activities of antioxidant enzymes by SNP appear to be responsible for low H₂O₂, less lipid peroxidation, and modulation of ascorbate and non-protein thiol statuses in the adventitious roots of mountain ginseng. Dry mass, saponin content and NOX activity was related with NO content present in adventitious roots of mountain ginseng.