Analysis of the relationship between blue-light photon flux density and the photosynthetic properties of spinach (Spinacia oleracea L.) leaves with regard to the acclimation of photosynthesis to growth irradiance

Abstract

Blue light has been suggested to participate in the acclimation of photosynthesis to growth irradiance. We analyzed the effects of blue light intensity on the photosynthetic properties of leaves with regard to acclimation to irradiance. Spinach (Spinacia oleracea L.) plants were grown under mixtures of blue and red light with blue-light photon flux densities (PFDs) of 0, 30, 100 and 150 µmol m⁻² s⁻¹ at a total photosynthetic PFD of 300 µmol m⁻² s⁻¹. The light-saturated rate of photosynthesis under white light, leaf N content per unit leaf area, leaf dry weight per unit leaf area and the ratio of cytochrome (Cyt) f content to light-harvesting chlorophyll-binding protein of photosystem II (LHCII) content were evaluated. The photosynthetic rate tended to increase with increasing blue-light PFD up to 100 µmol m⁻² s⁻¹, and this was associated with an increase in leaf N content per unit leaf area. However, the increase in leaf N content per unit leaf area did not necessarily result from an increase in leaf dry weight per unit leaf area. The Cyt f to LHCII content ratio increased linearly with increasing blue-light PFD up to 100 µmol m⁻² s⁻¹, indicating that plants grown under higher blue-light PFD up to this value resembled plants grown under higher irradiance in terms of N partitioning between electron-transport components and light-harvesting components. This result suggests that the level of blue light is involved only in the acclimation to relatively low growth irradiances at the chloroplast level.

Key words:

• blue light
• light acclimation
• nitrogen partitioning
• photosynthesis
• spinach
• Spinacia oleracea L.

INTRODUCTION

Blue light strongly affects the growth and development of higher plants. There have been many studies on blue-light responses, including photomorphogenesis, mediated by blue-light photoreceptors (Briggs and Huala 1999; Lin 2000). In addition, blue light influences leaf photosynthetic properties. Leong and Anderson (1984a) found that Atriplex triangularis plants grown under blue light showed a higher rate of leaf photosynthesis under light-saturating conditions than those plants grown under red light. Plants grown under blue light have a higher chlorophyll (Chl) a/b ratio (Leong and Anderson 1984a; Lichtenthaler et al. 1980; López-Juez and Hughes 1995), a greater cytochrome (Cyt) f content (Leong and Anderson 1984a; López-Juez & Hughes 1995) and a greater ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content (Eskins et al. 1991; López-Juez and Hughes 1995) than plants grown under red light. The photosynthetic properties of leaves developing under blue light are similar to those acclimating to high irradiance. In addition, Walters and Horton (1995) found that neither the light-saturated rate of photosynthetic O₂ evolution nor the Chl a/b ratio in Arabidopsis thaliana leaves were affected by growth irradiance of red light completely deficient in blue light. Considering these results, several authors suggested that blue light is involved in the acclimation of leaf photosynthesis to growth irradiance (Anderson et al. 1995; Senger and Bauer 1987; Walters 2005).

In most previous studies, the effects of blue-enriched light have been compared with those of red-enriched light at an equal photosynthetic photon flux density (PPFD) using colored fluorescent lamps. It is not known whether plants perceive only the presence of weak blue light or use the absolute level of blue light as a signal to induce acclimation responses to growth irradiance. Thus, it is unclear how the level of blue light in irradiated light is related to the changes in photosynthetic properties during the acclimation to growth irradiance. An analysis of this relationship would contribute to clarifying the mechanism of the acclimation of plants to growth irradiance.

In the present study, we grew spinach plants under mixtures of blue and red light at an equal total PPFD (300 µmol m⁻² s⁻¹) with different blue-light photon flux densities (PFDs) (0, 30, 100 and 150 µmol m⁻² s⁻¹). Blue and red light-emitting diodes (LED) were used as the light source so that the blue-light PFD could be controlled independently of total PPFD (Matsuda et al. 2004; Ohashi-Kaneko et al. 2006). The acclimation of leaf photosynthesis to growth irradiance has been well characterized in terms of changes in the light-saturated rate of photosynthesis, leaf N content and N partitioning among photosynthetic components within chloroplasts (Anderson 1986; Björkman 1981; Boardman 1977). We determined the photosynthetic rate under saturating white light and the amounts of N and some photosynthetic components in young, fully expanded leaves developing under various blue-light PFDs. Then, we evaluated quantitatively how blue-light intensity was related to changes in the photosynthetic properties of leaves and we discuss the relationships with regard to the acclimation of leaf photosynthesis to growth irradiance.

MATERIALS AND METHODS

Plant material and growth conditions

Seeds of spinach (Spinacia oleracea L., cv. Megaton) plants were sown in vermiculite and grown under white fluorescent lamps in an environmentally controlled chamber for 7 days. The chamber was first operated with 150 µmol m⁻² s⁻¹ PPFD at the tops of the plants during a 12-h photoperiod and 25 ± 1/20 ± 1°C day/night temperatures. Seven days after germination, each seedling was transplanted to a 0.5-L plastic bottle containing a nutrient solution. The seedlings were then grown hydroponically in environmentally controlled chambers equipped with an LED array (Iwasaki Electric, Tokyo, Japan). The LED array was composed of 1296 red and 1296 blue LED chips. From 7 days after germination, the plants were grown under mixtures of blue and red light with four levels of blue-light PFD. The blue-light and red-light PFDs were 0 and 300, 30 and 270, 100 and 200 and 150 and 150 µmol m⁻² s⁻¹, respectively. Total PPFD was 300 µmol m⁻² s⁻¹ for any lighting treatment. Nine seedlings were subjected to each lighting treatment. The chambers were maintained with 12 h photoperiod and 20 ± 1/15 ± 1°C day/night temperatures. The basal nutrient solution used was prepared according to Makino et al. (1988) except that a N concentration of 8.0 mmol L⁻¹ (6.0 mmol L⁻¹ NaNO₃ plus 1.0 mmol L⁻¹ NH₄NO₃) was used. The nutrient solution was renewed every 5 days and was continuously aerated. The strength of the nutrient solution was varied depending on plant growth (days after germination): half strength at 7 days and full strength from 12 days. Gas-exchange measurements and biochemical assays were carried out on young, fully expanded leaves of 32-day-old to 47-day-old plants.

Light environment measurements

The PPFD was measured using a quantum sensor (LI-190SA with LI-250, LI-COR, Lincoln, NE, USA). Spectral PFD distributions for the lighting treatments (Fig. 1) were calculated from spectral energy distributions, which were measured using a spectroradiometer (HR2000, Ocean Optics, Dunedin, FL, USA). The spectral energy distributions were measured every 0.4–0.5 nm. We calculated phytochrome photoequilibrium (P_fr/P_total) from the spectral PFD distribution for each lighting treatment with the photoconversion cross section of phytochrome according to Sager et al. (1988). The P_fr/P_total represents the fraction of the active form of phytochrome in the total phytochrome possessing red/far-red reversibility. The P_fr/P_total ratio for all lighting treatments was almost identical (0.87–0.88). This means that differences in the reversible action of phytochrome were negligible under the conditions of this study.

Gas-exchange measurements

Gas-exchange rates were measured using a portable gas-exchange measurement system (LI-6400, LI-COR). Light was provided from a 35 W white halogen lamp. Measurements were made at a PPFD of 1,800 µmol m⁻² s⁻¹, an atmospheric CO₂ partial pressure of 36 Pa, a leaf temperature of 25°C and a leaf-to-air vapor pressure deficit of 1.1 ± 0.1 kPa.

Biochemical assays

A single leaf was homogenized with a chilled mortar and pestle in 50 mmol L⁻¹ Na-phosphate buffer (pH 7.0) containing 2 mmol L⁻¹ Na-iodoacetate, 0.8% (v/v) 2-mercaptoethanol and 5% (v/v) glycerol. The leaf N,

Figure 1  Spectral photon flux density (PFD) distributions for the lighting treatments. Blue-light PFDs were (a) 0, (b) 30, (c) 100 and (d) 150 µmol m−2 s−1. Total photosynthetic photon flux density was 300 µmol m−2 s−1 in any lighting treatment. The PFD was measured every 0.4–0.5 nm.

Rubisco, Cyt f, ligsht-harvesting chlorophyll-binding protein of photosystem II (LHCII) and Chl contents were determined from parts of this homogenate. Leaf N, Rubisco and Chl contents were determined according to the methods of Makino et al. (1994). Cyt f content was estimated from the difference between the hydroquinone-reduced and ferricyanide-oxidized spectra of the thylakoid membrane according to Ohashi et al. (1998). The millimolar extinction coefficient used was 20 mmol L⁻¹ cm⁻¹ (Bendall et al. 1971). The LHCII content was determined spectrophotometrically by formamide extraction of the Coomassie Brilliant Blue R-250 stained bands separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis, as previously described (Matsuda et al. 2004). Calibration curves were made with bovine serum albumin.

RESULTS AND DISCUSSION

Plants grown under high irradiance exhibit a higher rate of light-saturated photosynthesis at a normal CO₂ level associated with a greater leaf N content per unit leaf area (LA) than those grown under low irradiance (Hirose and Werger 1987; Makino et al. 1997; Sims and Pearcy 1989). The light-saturated rate of photosynthesis per unit LA at an ambient partial pressure of 36 Pa CO₂ under white light tended to increase as the blue-light PFD increased, up to 100 µmol m⁻² s⁻¹, while the rate at 150 µmol m⁻² s⁻¹ was nearly identical to that at 100 µmol m⁻² s⁻¹ (Fig. 2A). This increase in photosynthetic rate did not result from an increase in stomatal conductance because intercellular CO₂ partial pressure was independent of blue-light PFD during growth (data not shown). Leaf N content per unit LA also increased with increasing blue-light PFD except at 150 µmol m⁻² s⁻¹, where it tended to decrease slightly from that at 100 µmol m⁻² s⁻¹ (Fig. 2B). Thus, in the range between 0 and 100 µmol m⁻² s⁻¹, plants grown under higher blue-light PFD resembled those grown under higher irradiance in terms of the capacity for photosynthesis as well as N content on an LA basis. The dependency of Rubisco content per unit LA, a determinant for the light-saturated rate of photosynthesis at normal CO₂ levels (Makino et al. 1985), on blue-light PFD was similar to that of leaf N content per unit LA (data not shown).

In general, the greater leaf N content per unit LA in plants grown under high irradiance is almost accounted for by a greater leaf dry weight (DW) per unit LA, rather than by a greater leaf N content per unit DW (Evans 1996; Evans and Poorter 2001; Sims and Pearcy 1994). This means that, in acclimation to growth irradiance, the change in leaf N content on an LA basis mainly results from morphological alteration. However, the change in leaf N content per unit LA depending on the blue-light PFD was not correlated with changes in leaf DW per unit LA (Fig. 3A). Both leaf DW per unit LA

Figure 2  The relationship between (a) the light-saturated rate of photosynthesis per unit leaf area (LA) and (b) leaf N content per unit LA as functions of blue-light photon flux density in spinach leaves. Gas-exchange measurements were made at a photosynthetic photon flux density of 1,800 µmol m−2 s−1, an ambient CO2 partial pressure of 36 Pa, a leaf temperature of 25°C and a leaf-to-air vapor pressure deficit of 1.1 ± 0.1 kPa. Light for measurements was provided from a white halogen lamp. Error bars represent standard error (n ≥ 4). Means with different letters are significantly different using Tukey's honestly significant difference test (P < 0.05).

and leaf N content per unit DW were greater at 30 µmol m⁻² s⁻¹ blue light than at 0 µmol m⁻² s⁻¹, and they almost equally contributed to the greater leaf N content per unit LA. The greater leaf N content per unit LA at 100 and 150 µmol m⁻² s⁻¹ blue light than that at 0 µmol m⁻² s⁻¹ was primarily brought about by greater leaf N content per unit DW (Fig. 3B). Thus, although the change in leaf DW per unit LA depending on blue-light PFD contributed only slightly to the change in leaf N per unit LA, the extent of the contribution appeared to be small relative to that generally observed in plants acclimated to different growth irradiances. This means that the changes in the photosynthetic rate and leaf N content per unit LA under 0 to 100 µmol m⁻² s⁻¹ blue light (Fig. 2A,B) cannot be considered as acclimation phenomena to growth irradiance.

When plants acclimate to growth irradiance, changes are observed not only in the leaf N content on an LA

Figure 3  The relationship between (a) leaf dry weight (DW) per unit leaf area (LA) and (b) leaf N content per unit DW as functions of blue-light photon flux density in spinach leaves. Error bars represent standard error (n ≥ 6). Means with different letters are significantly different using Tukey's honestly significant difference test (P < 0.05).

basis, but also in N partitioning among photosynthetic components within chloroplasts. Plants grown under high irradiance allocate more N to electron-transport and ATP-synthesis components and less N to Chl-binding protein complexes than plants grown under low irradiance (De la Torre and Burkey 1990b; Evans 1987; Leong and Anderson 1984c; Terashima and Evans 1988). In addition, within Chl-binding protein complexes, N allocation to reaction–center complexes increases whereas that to light-harvesting components decreases in response to high irradiance (De la Torre & Burkey 1990a; Evans 1987; Leong and Anderson 1984b). The ratio of N allocated to Rubisco hardly changes with growth irradiance (Hikosaka 2005; Makino et al. 1997; Miyake et al. 2005). Therefore, changes in N partitioning among thylakoid components should be evaluated as an acclimation response to irradiance at the chloroplast level. We measured the amounts of Cyt f as one of the rate-limiting factors for electron transport and LHCII and Chl as representative light-harvesting components (see Hidema et al. 1992; Makino et al. 1994). The Cyt f content was significantly greater at 100 and 150 µmol m⁻² s⁻¹ blue light than at 0 µmol m⁻² s⁻¹ (Fig. 4A). The LHCII content

Figure 4  The relationships between (a) cytochrome (Cyt) f, (b) light-harvesting chlorophyll-binding protein of photosystem II (LHCII) and (c) chlorophyll (Chl) content per unit leaf area (LA) and (d) Chl a/b ratio as functions of blue-light photon flux density in spinach leaves. Error bars represent standard error (n ≥ 3). Means with different letters are significantly different using Tukey's honestly significant difference test (P < 0.05).

was significantly greater at 30 and 100 µmol m⁻² s⁻¹ blue light than at 0 µmol m⁻² s⁻¹ (Fig. 4B). The Chl content and its a/b ratio were significantly greater above 30 µmol m⁻² s⁻¹ blue light than at 0 µmol m⁻² s⁻¹ (Fig. 4C,D). As N allocations to electron-transport components and light-harvesting components are especially variable, depending on growth irradiance, we considered the ratio of Cyt f content to LHCII content to be a trait reflecting the N partitioning among photosynthetic components during the acclimation to irradiance. The ratio of Cyt f content to LHCII content increased almost linearly with increasing blue-light PFD up to 100 µmol m⁻² s⁻¹, at which time the ratio was approximately 1.2-fold as high as that at 0 µmol m⁻² s⁻¹ (Fig. 5). The Cyt f to LHCII content ratio at 150 µmol m⁻² s⁻¹ blue light was not significantly different from that at 100 µmol m⁻² s⁻¹. Variation in the Cyt f to LHCII content ratio below 100 µmol m⁻² s⁻¹ blue light was not attributable to a change in the N status of their leaves, because a similar response to blue-light PFD was observed when the plants were grown at different N concentrations (0.5 and 2.0 mmol L⁻¹ N, data not shown). Thus, below 100 µmol m⁻² s⁻¹ blue light, plants tend to alter N allocation to Cyt f and LHCII in response to blue-light PFD as they respond to growth irradiance, suggesting a

Figure 5  The relationship between the ratio of cytochrome (Cyt) f content to light-harvesting chlorophyll-binding protein of photosystem II (LHCII) content as a function of blue-light photon flux density in spinach leaves. Eror bars represent standard error (n = 3). Means with different letters are significantly different using Tukey's honestly significant difference test (P < 0.05).

role for blue-light PFD in the acclimation of chloroplasts to growth irradiance.

In conclusion, the level of blue light in a low-PFD range (below 100 µmol m⁻² s⁻¹ under the conditions of this study) appears to be related to the alteration in N partitioning among thylakoid components to growth irradiance. In contrast, blue light took little part in the change in leaf DW per unit LA, even though the change in leaf DW per unit LA is important in changing leaf N content and the light-saturated rate of photosynthesis per unit LA during the acclimation to irradiance. Several authors suggested that responses to growth irradiance at the chloroplast level and at the leaf morphological level could be regulated by different mechanisms (Murchie and Horton, 1998; Weston et al. 2000; Yano and Terashima 2001). The level of blue light may be involved only in the chloroplast-level acclimation to relatively low growth irradiances.