: A. MAKINO, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan. Email: firstname.lastname@example.org
The effects of temperature on photosynthesis, ribulose-bisphosphate carboxylase (Rubisco) content and whole plant growth were investigated in the assimilation shoots of a rose (Rosa hybrida L.). Assimilation shoots were grown at two different day/night temperature regimes of 20/15°C (LT) and 30/25°C (HT) for 42 days after 1-month growth. Although LT initially suppressed the photosynthetic rate during the first 7 days, prolonged growth at LT enhanced potential photosynthesis. This was associated with increases in Rubisco and N contents at the level of a single leaf. Rubisco content and the photosynthetic rate at 25°C were 2.8-fold and 1.6-fold higher in the LT plants than in the HT plants at day 42, respectively. The relative growth rate at the level of the whole plant was lower in the LT plants during the first 28 days and the leaf area ratio was smaller in the LT plants throughout the experiment. However, enhanced photosynthesis during growth at LT led to increases in the net assimilation rate at the level of the whole plant, and final biomass at day 42 did not differ between the two temperature treatments. To enhance the photosynthetic capacity in assimilation shoots of a rose, cultivation at 20/15°C is better than cultivation at 30/25°C.
Roses are one of the most popular plants in greenhouse cultivation. In rose cultivation, the basal shoots emerging in the early stage of growth are artificially bent down as assimilation shoots to effectively catch sunlight, and then new shoots emerging successively are harvested as cut flowers for rose products (Okawa and Suematsu 1999). This “shoot-bending” (or “arching”) cultivation technique leads to a higher yield and high quality of the flowering shoot (Okawa and Suematsu 1999). This technique makes it possible that higher amounts of carbohydrate are transported to flowering shoots from the leaves of bent shoots.
To attain high yields of cut flowers using the shoot-bending technique, a few important approaches are proposed. One is to optimize the leaf area index (LAI) of the bent-shoot canopies, and the others are to enhance the photosynthetic capacity of the assimilation shoots before shoot bending and to maintain a high photosynthetic capacity in the shoots after bending. For example, it is proposed that an LAI of 3.0 should be optimal for the production of the shoot-bending technique (Shimomura et al. 2003). However, it is not known how photosynthesis can be enhanced in the assimilation shoots before bending and how it can be maintained at a high capacity after bending. Many cultural environments, such as nutrition, temperature and sunlight, may affect the photosynthetic capacity of rose leaves. As roses are generally grown in soil-less culture under sufficient nutritional conditions, nutrition does not limit plant growth and the photosynthetic capacity of the leaves. In conventional greenhouse cultivation, temperature largely changes throughout the year, and the difference in average temperature in a greenhouse between summer and winter is approximately 10°C. However, it is unknown how temperature affects photosynthesis and rose growth.
In the present study, we examined the effects of growth temperature on the photosynthetic capacity at a single leaf level in the assimilation shoots of rose before shoot bending. The uppermost, young leaf was used throughout the experimental period. As roses are generally cultivated over a range of 20–30°C for day temperature and 18–20°C for night temperature (Beeson 1990; Bredmose 1998; Gonzalez-Real and Baille 2000; Jiao and Grodzinski 1998; Kim and Lieth 2003; Kool et al. 1996), we grew the assimilation shoots at two different day/night temperature regimes of 20/15°C and 30/25°C. We analyzed the difference in the growth rate of the assimilation shoots under these different temperatures.
MATERIALS AND METHODS
Rosa hybrida L. cv. Asami Red (Roterose) plants were used. Three rooted single-node cuttings with a five-leaflet leaf grown on rockwool blocks (5 cm × 5 cm × 5 cm) were planted on a rockwool plate (30 cm length × 20 cm width × 7.5 cm height). The rooted cutting plants had a 5–6 cm new shoot. Two plates were placed in a plastic container. The plants were cultivated in a greenhouse at Tsukuba, Japan. The plants were fertilized with a nutrient solution containing 0.3 mmol L−1 KH2PO4, 0.5 mmol L−1 MgSO4, 1 mmol L−1 CaCl2, 1 mmol L−1 KCl, 0.5 mmol L−1 NH4NO3, 1 mmol L−1 KNO3, 50 µmol L−1 Fe-ethylenediaminetetraacetic acid, 50 µmol L−1 H3BO3, 9 µmol L−1 MnSO4, 0.3 µmol L−1 CuSO4, 0.8 µmol L−1 ZnSO4 and 0.1 µmol L−1 Na2MoO4. The nutrient solution (2 L per plastic container) was renewed once (0–2 weeks after plantation) or twice (2–4 weeks after plantation) per week. When the solution was renewed the pH was adjusted to 5.3 with HCl.
After 1 month, the plants (approximately 25 cm shoot length and 5–8 five-leaflet leaves) were transferred to temperature-controlled growth chambers operating at two different day/night temperature regimes of 20/15°C (LT) and 30/25°C (HT) under natural sunlight conditions. The plants were fertilized with the same nutrient solution. The nutrient solution (5 L per plastic container) was renewed twice per week, and the pH was adjusted to 5.3 with HCl. The plants were grown for 42 days and all rose buds were removed throughout the experimental period.
Plants were harvested every 2 weeks between day 0 and day 42 after transfer to different growth temperatures. The harvested plants were divided into leaves and stems. The leaf area was measured. The leaves and stems were oven-dried at 80°C for more than 3 days. The rockwool plates were oven-dried at 80°C for more than 7 days, and then the roots were carefully picked out with tweezers. The leaves, stems and roots were weighed and milled. Relative growth rate (RGR), net assimilation rate (NAR), leaf area ratio (LAR), leaf weight ratio (LWR) and specific leaf area (SLA) were calculated from total dry weight and leaf area.
Determination of chlorophyll, Rubisco and total leaf N in a leaf
The uppermost, young leaf was cut at day 0, 14 and 42 after transfer to the different growth temperatures and stored at −80°C. The chlorophyll (Chl) and total leaf N contents were determined according to the method of Makino and Osmond (1991), except that a buffer of 100 mmol L−1 Na-phosphate, pH 7.5, 0.8% (v/v) 2-mercaptoethanol, 4 mmol L−1 iodoacetic acid and 20% (v/v) glycerol was used.
Rubisco contents were also determined according to the method of Makino and Osmond (1991) with some modifications. One leaf was powdered in liquid N2 in a mortar with a pestle and sea sand, and then homogenized in 100 mmol L−1 Na-phosphate buffer, pH 7.5, containing 0.8% (v/v) 2-mercaptoethanol, 4 mmol L−1 iodoacetic acid, 20% (v/v) glycerol and 2% (w/v) polyvinylpyroridone. The homogenate was treated with a lithium dodecylsulfate solution (4%[w/v] final concentration) and 2-mercaptoethanol (2%[v/v] final concentration) at 100°C for 90 s. After centrifugation at 10,000 g for 8 min the supernatant fluid was stored at −30°C until analysis by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Gas exchange measurements
Gas exchange rates were measured at the level of a single leaf with a LI-6400 portable photosynthesis system (Li-Cor, Lincoln, NE, USA). All measurements were made at a photosynthetical photon flux density (PPFD) of 1,000 µmol quanta m−2 s−1 (10% blue light-emitting diodes [LEDs] in red LEDs), a leaf-to-air vapor pressure difference of 1.0–1.2 kPa and a partial pressure of CO2 of 37 Pa between 09:00 and 12:00 hours. To measure the rates at growth temperature, the rates were measured at a leaf temperature of 20°C for the LT plants and at 30°C for the HT plants, respectively. A young, expanding leaf was used for the measurements between day 0 and day 10 after transfer to the different growth temperatures, and then a young, fully expanded leaf was used between day 14 and day 42. In addition, the rates at a leaf temperature of 25°C were also measured for both treatments.
Photosynthesis, total N and Rubisco contents in a leaf
Changes in the rates of photosynthesis measured at growth temperature are shown in Fig. 1. The photosynthetic rate was higher in the HT plants than in the LT plants during the first 7 days, and then the rates were not significantly different between day 10 and day 18 after transfer to the different growth temperatures. However, the photosynthetic rate at day 42 was significantly higher in the LT plants than in the HT plants (P < 0.01). Table 1 shows the rates of CO2 assimilation measured at the same leaf temperature of 25°C. The rates at 25°C were slightly higher at day 14 in the LT plants, and the difference became greater at day 42. Stomatal conductance tended to show a similar response to that of photosynthesis. No difference in the intercellular CO2 partial pressure was found at day 14, but it was higher in the HT plants at day 42.
Table 1. Photosynthetic rate, stomatal conductance and intercellular CO2 partial pressure measured at 25°C in a young rose leaf after transfer to two different temperatures of 20/15°C and 30/25°C (day/night)
Photosynthesis (CO2µmol m−2 s−1)
Stomatal conductance (mol m−2 s−1)
Intercellular CO2 (Pa)
Data are mean ± standard deviation (n = 5–14). *P < 0.05; **P < 0.01.
Table 2 shows the changes in total N, Chl and Rubisco contents. Total leaf N and Rubisco contents were slightly greater in LT plants at day 14. At day 42, all parameters were markedly higher in the LT plants than in the HT plants. Among them, the Rubisco content was 2.8-fold greater in the LT plants. Thus, all parameters increased in the LT plants over the experimental period, and decreased in the HT plants after day 14.
Table 2. Total leaf N, chlorophyll and Rubisco contents in a young rose leaf after transfer to two different temperatures of 20/15°C and 30/25°C (day/night)
Total N (mmol m−2)
Chl (mmol m−2)
Rubisco (g m−2)
Data are mean ± standard deviation (n = 3–4). *P < 0.05; **P < 0.01. Chl, chlorophyll.
Table 3 shows the changes in plant mass at the whole plant level, the dry weight of each organ, and the total leaf area. The total plant mass was greater in the HT plants than in the LT plants between day 0 and day 28, but the final biomass at day 42 was not significantly different. The total leaf area per plant was greater in the HT plants than in the LT plants throughout the experimental period. The root biomass was not different between the LT and HT plants.
Table 3. Total plant mass, dry weight of each organ and total leaf area of rose after transfer to two different temperatures of 20/15°C and 30/25°C (day/night)
Total dry weight (g)
Leaf dry weight (g)
Stem dry weight (g)
Root dry weight (g)
Leaf area (m2 plant−1)
Data are mean ± standard deviation (n = 17 at day 0, n = 11–12 between day 14 and day 28 and n = 5 at day 42). *P < 0.05; **P < 0.01.
The growth rate was analyzed at the level of the whole plant (Fig. 2). The RGR, which is defined as the dry weight increment per dry weight per day, was higher in the HT plants than in the LT plants during the first 14 days, but this difference became smaller between day 14 and day 28, and was reversed between day 28 and day 42 (i.e. was higher in the LT plants). The change in NAR was similar to that of RGR, and NAR between day 28 and day 42 was also higher in the LT plants. The LAR was greater in the HT plants throughout the period. This was caused by greater LWR and SLA in the HT plants, although the difference between day 14 and day 28 was not significant.
Our results with rose assimilation shoots indicate that low growth temperature (20/15°C) initially suppressed the photosynthetic rate during the first 7 days, but prolonged growth at low temperature enhanced potential photosynthesis (Fig. 1, Tables 1,2). Similar trends have been reported for several cold-habitat plants, such as spinach (Holaday et al. 1992), Arabidopsis (Strand et al. 1999), winter rye (Hurry et al. 1994), winter wheat and winter rape (Hurry et al. 1995). In these plants, low temperature increases the activities of several photosynthetic enzymes, such as Rubisco, stromal fructose-1,6-bisphosphatase and sucrose-phosphate synthase. Yamori et al. (2005) also reported that the amount of Rubisco increased in spinach leaves grown under low temperature. In addition, Sage and Kubien (2007) have recently pointed out that plants acclimated to cooler temperatures often exhibit enhanced Rubisco content. As shown in Table 2, as low temperature enhanced total leaf-N content, such increases in the photosynthetic components may have been associated with an increase in leaf-N content at low temperature. In our case, however, the photosynthetic rate at growth temperature was lower in the LT plants just after transfer to the low temperature. According to Kim and Lieth (2003), the photosynthetic rate in rose does not differ between 20 and 30°C. Therefore, an increase in the amount of Rubisco as well as in the photosynthetic capacity during growth at low temperatures can be one of acclimation phenomena to low temperatures in rose. Actually, in rice, which belongs to a typical summer crop, the photosynthetic rate continuously decreased during growth at 20/17°C and was never restored (Hirotsu et al. 2004).
Rubisco is a limiting factor for light-saturated photosynthesis under atmospheric CO2 levels (Evans 1986; Makino et al. 1985). However, although Rubisco content at day 42 was 2.8-fold greater in the LT plants (Table 2), the photosynthetic rate at 25°C was only 1.6-fold higher (Table 1). Similarly, total leaf N and Chl contents were 1.8-fold and 1.7-fold greater in the LT plants than in the HT plants. Thus, the increase in Rubisco content during the growth at low temperature did not quantitatively lead to an increase in potential photosynthesis. The reason for this discrepancy is not known, but one possible explanation is an increase in the resistance to CO2 diffusion from the intercellular airspace to the chloroplasts in the low-temperature-grown rose. Makino et al. (1994) suggested the possibility that the conductance to CO2 diffusion between the intercellular airspace and the chloroplasts decreases when rice is grown under low temperature. However, although Makino et al. (1994) observed a large decrease in stomatal conductance during growth at low temperature, stomatal conductance in rose increased (Table 1). Thus, an increase in the stomatal conductance at low growth temperatures may also be one of acclimation phenomena to low temperatures in rose. Another possibility is that Pi regeneration limitation occurred in the LT plants. A selective enhancement of Rubisco content often leads to a photosynthetic limitation in Pi regeneration (Makino and Sage 2007).
The RGR was higher in the HT plants during the first 28 days and LAR was higher in the HT plants throughout the experimental period (Fig. 2). Total leaf area was also always greater in the HT plants (Table 3). These results indicate that it can take less time to obtain appropriate leaf area of the bent shoots in 30/25°C cultivation than in 20/15°C cultivation (Fig. 2, Table 3). In fact, the expansion rate of the leaf was faster in the HT plants (data not shown). Thus, HT led to a rapid leaf expansion and resulted in a higher initial growth rate. However, because potential photosynthesis was not enhanced, we conclude that HT does not suit the growth of the assimilation shoots of rose before shoot bending. In sweet pepper (Nilwik 1981) and Secale cerea (Huner 1985), low temperature also led to decreases in the LAR and the SLA. In these plants, narrower and thicker leaf development was observed. Such morphological characteristics were similar to those found for rose (Fig. 2).
Loveys (2002) reported that there is a species-dependent difference in temperature response of NAR and it determines a difference in RGR under different temperatures. In rose, NAR increased during growth at low temperatures (Fig. 2), and this increase led to a large increase in RGR during the late growth. In addition, the increase in NAR compensated for a decrease in LAR over the whole growth period. This increase in NAR may have been caused by an increase in the amount of Rubisco during growth at the low temperature (Table 2).
Our results clearly indicate that the photosynthetic capacity in rose strongly depends on the growth temperature even if the nutrition conditions are the same. Photosynthetic capacity is initially suppressed under growth at 20/15°C, but prolonged growth at a low temperature enhances potential photosynthesis. This is associated with increases in Rubisco and N contents in a leaf. In addition, enhanced photosynthesis leads to increases in NAR and RGR at the level of the whole plant. Thus, to enhance the photosynthetic capacity in rose assimilation shoots, cultivation at 20/15°C is better than cultivation at 30/25°C.
We thank Mr Hideo Shimaji for his valuable comments and support over the period of this research.