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Boletín de la Sociedad Chilena de Química

Print version ISSN 0366-1644

Bol. Soc. Chil. Quím. vol.47 no.4 Concepción Dec. 2002

http://dx.doi.org/10.4067/S0366-16442002000400022 

Evidence of UVB differential response in
Sophora microphylla from shady and sunny places

Susan Hess*, José Luis Alvarez***, Griselda Iturra** and Magdalena Romero**,

*Instituto de Química, **Instituto de Botánica, Facultad de Ciencias,
Universidad Austral de Chile, Independencia 641, Campus Isla Teja, Valdivia, Chile
***Centro Ciencias Básicas, Universidad Austral de Chile,
Los Pinos SN, Campus Pelluco, Puerto Montt, Chile

(Received: August 27, 2001 - Accepted: August 13, 2002)

ABSTRACT

The effect of the supplemental UVB radiation in forest species was studied in Sophora microphylla, a native tree that grow at low latitudes in the Central-South of Chile and important by their association with soil bacteria and mycorhizal fungi. Seeds of sun and shade variety growing at different light intensities were collected. Using indoor supplemental UVB radiation germination and seedling grow were examined. As response to UVB radiation, seeds and young emerging seedling from high irradiance habitat (PPF ± 2200 m molm-2s-1), sun variety plants show more tolerance than plants from habitat with less irradiance (PPF ± 1400 m molm-2s-1), shade variety plants. Germination and growing of sunny plants was faster than shady plants. Photosynthetic pigments (chlorophyll a and chlorophyll b) content increased in shady plants and was almost invariable for sunny plants. Total protein content decreased in both cases. Biosynthesis of flavonoids and epicuticular waxes was induced by UVB, especially in sun variety seedling. UVB screening total flavonoids were determined with UV spectroscopy. Epicuticular wax analysis was performed with FT IR technique. All this results suggest that an increment of UVB radiation is more harmful for plants accustomed to low radiation intensity, than for those growing at high light intensity.

KEYWORDS: UVB stress, germination, seedling growth, pigments, Sophora microphylla

RESUMEN

Se estudio en laboratorio el efecto de radiación UBV adicional en Sophora microphylla, una especie forestal nativa de bajas latitudes en el centro-sur de Chile importante por su asociación con bacterias y hongos micorricicos del suelo. Semillas de Sophora en variedad sol y sombra (± 2200 y ± 1400 m molm-2s-1 respectivamente) y las plantulas emergidas fueron irradiadas durante dos meses con un exceso de UVB (9.6 m W cm-2 x 5 min.) y 120 m molm-2s-1 PPF diarios, con lamparas que simulan el espectro solar con un incremento de la radiacion UVB. Semillas de variedad sol cultivadas en exceso de UVB germinaron mas rápidamente que su control y que las de variedad sombra, siendo su crecimiento y biomasa también mayor. La radiación UVB indujo reducción en el crecimiento y biomasa en ambas variedades, aumento en la síntesis de pigmentos fotosinteticos principalmente en variedad sol y aumento en la síntesis de flavonoides y ceras epicuticulares también en variedad sol. Esta mayor resistencia a eventos esporádicos de UVB en exceso sugieren que un incremento de la radiación UVB podría afectar mayormente a plantas acostumbradas a intensidades de radiación bajas (variedad sombra) que en aquellas que naturalmente crecen bajo altas intensidades de luz (variedad sol).

PALABRAS CLAVES: Estrés UVB, germinación, crecimiento, pigmento, Sophora microphyla

INTRODUCTION

Since the starting of the Ozone depletion in the stratosphere at the seventies, more ultraviolet (UV) radiation rice earth, causing impacts in biological organism (1). Terrestrial plants are especially vulnerable to this radiation due to their requirement of sunlight for photosynthesis (2, 3). The UVB radiation dilemma has conducted to many scientific experiments (4-7). The research has been focused mainly in agricultural species (8-13). Most of the research has concerning destructive and inhibiting effects to this radiation (14), however in the last years even regulatory effects have been considered (15-18).

Plants respond in different ways to UV- radiation, including growth and physiological changes. A wide intraspecific variability in terms of reduction in total dry weight by effect of UVB radiation has been informed (10-12). However, little data evaluate the range of variability present in native tree species (19). Many plants from habitats with high UVB dose have more pronounced adaptive mechanisms than plants from ambient with less UVB (20, 21). Additionally, shade-grown plants seem to be more susceptible to high light stress than sun-grown (22). The observed adaptive mechanisms are among others: decreased biomass, altered photosynthetic pigment production and increased biosynthesis of molecules that avoid penetration of UVB radiation on the photosynthetic system. These molecules are mainly leaf surface (epicuticular) waxes and UVB screening pigments (flavonoids) (23-26). However, in general the ability of plants to acclimate to UVB radiation depends on their capacity to detect elevates levels of damaging radiation. As the UVB to visible light ratio increases, this ability diminishes and the amount of physiological damages rises (27).

The present study evaluate the germination dynamic, grow response and vigor (expressed in dry weight variation) of young emerging seedlings of S. microphylla from sunny and shady places, as response to UVB radiation. This growth stage is very critical and affects directly survival (28). We postulate that plants acclimated or adapted to high light intensities (sunny plants) are less sensitive to the effects of UVB than plants naturally growing in lower light intensities (shady plants). To test this hypothesis, we choose a woody species from South Central Chile, Sophora microphylla Ait. This species grow at low altitudes and under different light conditions. It is colonist thanks to his root symbiotic association with nitrogen fixing soil bacteria and mycorrhizal fungi. These associations increase the available pool of nutrients for the plant, improving soil recuperation and restoration of degraded area (29). As response to enhanced UVB radiation we performed physiological (germination, growth and biomass production), and chemical analysis (total proteins, epicuticular waxes and photosynthetic and UVB screening pigments, as well).

MATERIAL AND METHODS

Seeds of S. microphylla from a shady (noon photosynthetic photon flux (PPF): @ 1400 m molm-2s-1) (P1) and a sunny (noon PPF: @ 2200 m molm-2s-1) (P2) places were collected in August 2000 at Botanic Garden of Universidad Austral de Chile, Valdivia (39° 48’ S 73° 14’ W at 9 m o.s.l.). Measurements were made in December with a LI/COR Quantum Photometer, mod. Li 189.

Simulated ambient solar radiation with enhanced UV-B radiation was provided with Q-PANEL UV 313 Philips lamp (Hanna Instruments Neurtek). The lamps were suspended 30 cm over the plants after been pre-burnt for 50 h to reach stable output. The intensity of the radiation was regulated with glass filters. Wavelengths below 280 nm were removed with a 0.13 mm cellulose diacetate filter (transmission down to 280 nm). Filters were changed weekly to avoid aging effects on their spectral transmission of UV-B light. Photosynthetically active radiation (PAR, 400-700 nm) was provided by metal halide lamps (Osram HQI-T 400 W). The spectral irradiance was measured with a spectroradiometer Cole Parmer Instrument Company, serie 9811.

Seeds were mechanically scarificated and sterilized with Natrium Hypochlorite at 0.1% for 10 seconds and washed with sterile distilled water (30). One hundred seeds were imbibed with distilled water in Petri dishes on two layers of Whatman filter paper. The dishes were constantly checked to maintain appropriate humidity. For UV-B treatment 4 replicates of 25 seeds each were used. The dishes were maintained at ambient conditions (20°C day and 15oC night) with a PPF of 100 m mol m-2s-1 and a supplemental UV-B dose of 9.6 m W cm-2 (1.5 m mol m-2s-1) under four minutes daily, until full germination. Non treated seeds (control) were kept at the same temperature without supplemental UV-B application.

Five germinated seeds (with a 2-4 mm length radicle) were sawn in plastic pots containing organic soil. Young seedlings were watered every second day and kept under ambient conditions as mentioned above. When the first shoot was emerged, the supplemental UV-B application was reinitiated. Pots with young emerging seedlings were distributed in two groups: One group (control), was exposed to ambient UV-B radiation (UV-B: PAR 1:100) and the other ones was exposed daily, around 12.00 noon, to 4 minutes pulses of supplemental UV-B radiation (9.6 m W cm-2) under 48 days. At the end of the UV-B treatment, three-month-old seedlings were harvested for chemical and physiological studies. To evaluate the effects of the enhanced UV-B, plant growth parameters (plant height, number of leaves, number of leaflets per leaf and biomass) were measured directly at the end of the treatment. The recovery capacity (without enhanced UV-B) was determined after 60 days in four-month-old seedlings. Seedlings coming from shady and sunny seeds will be also designed as P1 and P2 respectively.

Relative growth rate (RGR) was expressed as dry matter increment per time unit respect to initial weight of the plant (28). RGR was calculated according the expression RGR= DW/ (DT x W). Where DW= final weight minus the initial weight (in this case considered zero, since we started the experiment with seeds), DT= seedlings time irradiation and W= dry weight. Water content (WC) was calculated according the expression: WC = (FW-W) 100/ FW. Where FW= fresh weight, W= dry weight after drying by 48 hours until constant weight.

Proteins, pigments and epicuticular wax were measured directly at the end of UV-B treatment. To analyze total protein content, leaf samples (0.15 g) from different stem positions were extracted and proteins measured according to Bradford et al. (31). Total chlorophylls (a and b) and UV-B screening pigments (flavonoids), were extracted from 100 mg of fresh leaf material and incubated in 10 mL methanol 80% for 24 h at 4°C in the dark. Thereafter, the extracts were filtered through 0.2 m m pore filter paper and absorption spectra was immediately measured. The spectral absorbance of all the pigments was determined by scanning the extracts from 200 to 750 nm with a UV-VISIBLE Spectrometer (Unicam Instruments) resulting an integrated spectra with the absorption of photosynthetic and UV-B screening pigments. Total flavonoids absorbed strongly from 240 to 350 nm and total chlorophylls present absorption bands around 420 and 650 nm (32). To identify and quantify different components in the integrated spectra two methods were used: 1) comparison with the absorption spectra of standards of chlorophyll a, chlorophyll b and two flavonoids, which has been identified early in plants as UV-B screening pigments (kaempferol and quercitin). 2) generation of the individual spectra using software "octave" for Linux and the algorithm Nelder-Mead simplex (direct search method) (33 and 34). As control method to the Nelder algorithm, the determination of photosynthetic pigments (chlorophyll and carotenoids) was performed using Lichtenhaler and Welburn method (35).

Epicuticular waxes were extracted on adaxial and abaxial leaf surfaces at the end of the experiments. In 0.25 g fresh weight, of randomly selected leaves, surface waxes were removed by dipping them individually, with agitation (three times for up to 10 sec.) in 5 mL petroleum ether (Merck PA) at room temperature, according to Salatino (36). The waxy material, accurately weighed and finely powered was mixed with potassium bromide powder to make a disk at high pressure. The disk was placed in instrument beam to measure the IR spectra in a fourier-transform spectrometer (Nicolet Instruments). Infra-red analysis of the extracts a main peak at 3600-3000 cm-1 corresponding to hydroxyl group and a smaller peak 1750-1700 indicating the presence of esters. Quantitative analysis of the IR spectra was made on the hydroxyl group based on the base-line method for determination of the absorbance of the sample (37).

To analyze the combined effects of enhanced UV-B treatment and plant variety on the studied parameters, a two way ANOVA analysis was performed.

RESULT AND DISCUSSION

Germination dynamic and growth response of young emerging Sophora seedlings from shady and sunny seeds showed some differences when growing under UV-B irradiation. With supplemental UV-B, sunny seed (P2), in comparison to shady seed (P1) and controls, showed the shortest germination time and the highest percentage of germination (Fig. 1). This response may be due to the presence or increased contents of flavonoids in the coat of P2 seeds, which can provide the young seedlings a preliminary UV screen and facilitate the germination under UV-B stress as reported for other plants (38). Similar results in seeds from species that presented an improved germination as response to enhanced UV-B radiation have been reported by Musil et al. (39) and Gitz et al (40).


Fig. 1. Effect of ehanced UVB irradiation in the dynamics of radicle emergence (germination) of Sophora microphylla seeds. Values are means of four replicates. Vertical bars denote ± SD. Seeds were collected from trees growing in habitats with different levels of irradiation. The PPF dose in P1 was 30% lower than in P2. These irradiation measurements were made at noon in December.
P1 = Shady; P2 = Sunny places (P<0.05)

Initially, non UV-B treated seedlings (control), from sunny places, showed higher fresh and dry biomass, total height, water content and leaves number, respect to shady places (Table 1 and Fig. 2) evidencing a major vigor. After the UV-B treatment, both groups of seedlings presented a statistically significant reduction (P>0.05) in fresh and dry weight, water content and total height respect to their controls. The dry weight reduction was higher in the P2 (24.3 %) than in the P1 (17%) seedlings. In addition, roots, stems and leaves from P2 seedlings showed a higher biomass reduction (35, 27 and 17% respectively) than in P1 (18, 13.5 and 18.7 % respectively) after UV-B treatment (Fig. 3). However, the number of leaves was increase (Table 1).

Table 1: Effect of enhanced UVB radiation in total high, leaves number and water content of three months old Sophora microphylla seedlings shade (P1) and sun (P2) variety. The results were evaluated by a two-way ANOVA test, values are means of 20 replicates ± SD, different letters indicate statistical significantly differences within a file (P<0.05).


  P1 P2
  Control +UVB Control +UVB

Total high (cm) 21.5±3.48 a 18.7±2.97 b 23.5±2.07 c 20.1±1.94 b
Leaves number 5.29±1.30 a 4.52±1.18 b 6.10±0.84 c 6.90±1.42 d
Water content (g) 0.20±0.06 a 0.18±0.05 a 0.27±0.05 a 0.22±0.05 c


Fig. 2. Total fresh and dry weight of 3 month old seedling that grow under UVB ambient (control) and supplemental UVB irradiation (+UVB). P1 = Shade variety; P2 = Sun variety. Values are means of 20 replicates ± DS. Different letters indicate statistically significantly differences within of parameter (P>0.05)


Fig. 3. Integrated spectra contributio of mathanol soluble pigments in ophora microphylla sun variety. Chlorophyll a present a strong Sorét bant at 430nm, a weack Qy band at 670 nm and a shoulder corresponding to Qx band at 600-630 nm. Total flavonoids present a broad ban between 270 and 330 nm and a small between 220 and 240 nm. Signal before 220 nm correspond to the solvent.

Relative growth rates (RGR) and shoot/root ratios of UV-B irradiated P1 and P2 seedlings, decreased respect to their controls (Table 2). RGR decrement was slightly higher (23%) in P2 with respect to P1 (21%) seedlings. Suggesting that growth of both groups of plants are similarly adapted to UV-B radiation despite the different germination capacity (Fig. 1). Sullivan and Rozema (41) reported that sun plants are better adapted to higher photon fluxes densities than shade plants, although sun plants were sensitive to UVB changes. Shoot/root ratio reduction in UV-B treated P1 P2 S. microphylla seedlings showed non-significant differences when compared with their controls. A well-balanced shoot/root ratio is necessary to ensuring mineral supply and a favorable water economy, and thus an adequate biomass allocation (12 and 42). Early works have reported that enhanced UV-B may affect more the biomass partitioning and photomorphogenesis than biomass "per se". This effect may be important in determining ecosystem structures in terms of seedling establishment and competition (42 and 43).

Table 2: Relative growth rate (RGR) and shoot/root ratio content of three months old Sophora microphylla seedlings shade (P1) and sun (P2) variety. In each variety, the RGR of UVB radiated seedlings and their controls were evaluated by a two way ANOVA test. Values are means of 20 replicates ± SD, different letters indicate statistical significantly differences within a file (P<0.05).


  P1 P2
  Control +UVB Control +UVB

RGR (mg g-1 d-1) 0.63±0.20 a 0.50±0.12 b 0.74±0.11 c 0.57±0.13 db
Shoot/root radio 4.54±0.77 a 4.87±1.75 a 5.50±2.65 a 5.75±2.36 a

Protein contents of non UV-B treated S. microphylla P1 seedlings were higher (36%) than those of P2. This initial difference may be related with adaptations to the different environmental conditions of the collection places (28). The UV-B radiation induce a decreament of protein concentration in both groups of plants, being this decrement slightly lower in P2 (13%) compared to P1 (17%). This reduction could be associated to flavonoid production, since UV-B radiation stimulated the synthesis of enzymes linked to flavonoid pathway (44). In addition these results can also be due to the degradation of D1 and D2 proteins in PSII, that occur even at very low UV-B radiation flow (45).

The UV-B treatment increased the total photosynthetic pigments (Table 4) in P1 seedlings (45% chlorophylls and 48% carotens), while in P2 seedlings a slightly decrease (8% chlorophylls and 9% carotens) with respect their controls, was found. These responses are in agreement with results obtained by Hunt et al. (46). They founded that under enhanced UV-B, in shade tolerant tree photoinhibition was diminished in about 50% with respect to shade intolerant species. The increment in carotenoid contents in association with UV-B treatment is in agreement with early reports. Carotenoids may have important functions in plant (47): as natural UV-B filter, avoiding chlorophyll photobleaching and as antioxidant, inactivating possible free radicals generated by non filtered UV-B (27).

Table 3: Effect of enhanced UVB on basal proteins concentration in shade and sun variety of Sophora microphylla (n=5, SD=0.04).


  P1 P2
  Control +UVB Control +UVB

Total Proteins (mg/g DW) 37.4 31.1 24.1 21.0
Decrement (%) 17 13

Table 4: Effect of enhanced UVB on total photosynthetic pigments concentration in shade and sun variety of Sophora microphylla, according Lichtenhaler and Welburn method (n=5, SD=0.04).


  P1 P2
  Control +UVB Control +UVB

Total Chlorophylls (mg/g FW) 1.70 2.48 1.76  
Variation (%) 45 -8
Total Carotenoids (mg/g FW) 0.31 0.46 0.35 0.32
Variation (%) 48 -9

The absorption spectra of methanol soluble pigments (Table 5 and Fig. 4), calculated from absorption integrated spectra showed that P1 and P2 seedlings evidenced small increments in chlorophylls a and b production (18% and 10% respectively) with respect to the controls. The possible disagreement on chlorophyll increments when compared with the contents in Table 4, depends on the measurement wavelengths. Spectroscopic studies in natural photosynthetic system have shown that chlorophyll absorption bands shifts (48). Normally chlorophyll presents tree absorption bands. The strong Sorét band at ~ 430 nm for Chl a and ~ 470 nm for Chl b, which correspond to several unresolved transitions in the spectrum. The lowest band corresponding to the Qy transition at ~670 nm for Chl a and ~650 nm for Chl b, and next lowest corresponding to the Qx transition at ~600-630 nm for both Chl a and Chl b, in methanol. These bands vary somewhat, specially Qy band shift to lower wavelengths depending on chlorophyll self-aggregation, due to solvent polarity and concentration. Therefore comparing measurements of chlorophyll absorbance at some wavelengths and at some region of the electromagnetic spectra (scanning) can induce to disagreement (48 and 49).

Table 5: Absorbance (expressed absorption units) of total Chlorophyll and Flavonoids as response to UVB radiation, calculated from the integrated absorption spectra of pigment extract (100 mg fresh material in 10 mL methanol 90%) of S. microphylla from different natural habitats (n=3, S=0.05).


  P1 P2
  Control +UVB Control +UVB

Total Chlorophylls (AU) 0.25 0.30 0.20 0.22
Increment (%) 18 10
Total Flavonoids (AU) 0.51 0.55 0.69 1.0
Increment (%) 8 43


Fig. 4. Dry weight decrement (% respect to control) in root, stem and leaves of shadt (P1) and sunny (P2) plants. Seedings were 3 month old and the measurements were made 30 days after the UVB treatment was finished. Values are means of 20 replicates.

Early observation about the relation between chlorophylls content and UV-B radiation are contradictory. Results showed in Tables 4 and 5 are in agreement with different studies that informed that chlorophyll content is one way to asses UV-B resistance capacity of plants (27, 50 and 51). However, in UV-B resistant crop species, chlorophyll concentration was unaffected after treatment with enhanced UV-B, while UV-B sensitive species increase their chlorophyll content (4). On the other hand, unvarying levels of chlorophyll indicate tolerance or adaptation of plants to higher UV-B levels (52)

Flavonoids production increased on both varieties as response to UV-B, being the increment with respect to the controls more significant on the sunny seedlings (43%) than in the shady (8%) (Table 5). These results suggest that P2 seedlings are apparently better prepared to respond efficiently to enhanced UV-B than the P1 seedlings, whose normally receive less radiation. Similar UV-B adaptive capacity of plants with higher contents of flavonoids, have been reported for conifers from sunny habitats (53 and 54). In addition, Meijkamp et al. (55) have suggest that flavonoids levels increase until a certain UV-B doses and above this, flavonoid content decrease, probably as a consequence of damage of general cell metabolism.

Since the major component of the leaf surface cuticular lipids (epicuticular waxes) are primary alcohols, we calculated from the IR spectra, the absorbance of the main peak at 3600-3000 cm-1 corresponding to hydroxyl group. In both cases (P1 and P2 seedlings) an increment in the epicuticular wax production as response to UVB stress (Table 6) were found. P1 and P2 seedlings produced more epicuticular waxes (15 % and 67 % respectively) than their controls. The absorption of the waxy extracts of both varieties and their controls was calculated at 3600 cm-1 (Table 6). These results corresponds well with early works in Pisum sativum, where a wax increase from 20 to 40 % as response to UVB stress was found (26).

Table 6: Effect of enhanced UVB on epicuticular waxes absorbance (1g/5 mL petroleum ether) of the hydroxyl band measured at 3500 cm-1 in S. microphylla growth at different natural habitats, P1 grow at low light intensity and P2 grow at high light intensity (n=3, S=0.06).


  P1 P2
  Control +UVB Control +UVB

Absorbance (mUA) 0.51 0.62 0.46 0.77
Increment (%) 21 67

In order to establish if the observed changes were reversible or not, morphological parameters were controlled 60 days after the UV-B treatment was finished (Fig. 5). According to the dry weight of the different organs the P2 seedlings showed a much better recovery capacity than the P1 seedlings. While the P2 seedlings recovered a 116 % of their biomass with respect to the controls, P1 only recovered only a 2 %. The higher recovery capacity of biomass of P2 seedlings is probably due to their genetically determined vigor as observed in the parameters determined before start of the UV-B treatment.


Fig. 5. Dry weight recovery capacity (% respect to control) in root, stem and leaves of shady (P1) and sunny (P2) plants. Seedlings were 4 month old and the measurements were made 60 days after the UVB treatment was finished. Values are means of 20 replicates.

Despite, that in P2 seedlings total flavonoids and epicuticular waxes concentrations increased considerably by UV-B effect, compared to P1 seedlings (Table 5 and 6), dry weight of organs decreased in both varieties after the UV-B treatment (Fig. 3). However, P2 dry weights were strongly increased after 60 days without UV-B, showing a higher recovery capacity with respect to P1 seedlings (Fig. 5). These results may imply that the enhanced flavonoids and epicuticular wax production can trigger resistance mechanisms not only to UV-B stress. But also for other environmental conditions as reported in other plants (56, 57), suggesting that sporadic enhanced UV-B events could have a stimulatory effect in plants adapted to high solar radiation (sunny habitats).

ACKNOWLEDGEMENTS

This work was supported by DID-UACH, grant S-1999-2 We would thanks Dr. Miren Alberdi for critics reading and English correction

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