Leaf Photosynthetic Reduction at High Temperatures in Various Genotypes of Passion Fruit (Passiflora spp.)

Abstract

Passion fruit (Passiflora spp.) vines, mostly indigenous to tropical highlands, although some species can be cultivated in tropical lowlands, are susceptible to high temperatures. To increase the resilience of passion fruit to the warming climate, there is an urgent need to evaluate existing genetic resources for traits suited to high temperatures and to efficiently select the superior genotypes. We investigated the genotypic variation in leaf photosynthetic reduction at high temperatures (> 30°C) as a base target trait for warming climates. Leaf photosynthesis and dark respiration were measured at 30–45°C leaf temperatures for 13 genotypes from various Passiflora spp. using a portable gas exchange system. Temperature-net and -gross photosynthetic rate curves were plotted, and the relationships between the photosynthetic rate and the transpiration rate and stomatal conductance were analyzed. The net photosynthetic rate decreased with reduced stomatal conductance and transpiration rate as the leaf temperature increased from 30°C to 40°C. Up to 45°C, the net photosynthetic rate continued decreasing with increasing dark respiration rate, whereas the gross photosynthetic rate tended to stop decreasing as the stomatal conductance and transpiration rate stopped decreasing. The respiration load of photosynthesis varied among genotypes: Alata seedling #1 (P. alata) and ‘MaQuatro’ (P. edulis f. flavicarpa) showed the lowest load, together with the smallest reduction in photosynthetic rate at high temperatures, while the Iriomote strain (P. laurifolia) showed the largest load despite the smallest reduction in photosynthetic rate. Although the trend in the physiological response to high temperatures was similar among genotypes, the rate of leaf photosynthetic reduction at high temperatures varied among passion fruit genotypes both inter- and intra-specifically. Reduction of the photosynthetic rate at high temperatures was significantly correlated with stomatal activity under non-stress conditions at 30°C leaf temperature. Stomatal length was closely correlated with photosynthetic and transpiration rates at higher leaf temperatures in the P. edulis group, whereas no correlation was detected when including the lowland relatives. We concluded that genotypes showing higher stomatal conductance and transpiration rates at 30°C leaf temperature maintained a higher leaf photosynthetic rate at temperatures > 40°C, providing potential indicators for screening.

Introduction

Global warming due to climate change is increasing the average annual temperature (IPCC, 2014). Production of existing crops in temperate and subtropical regions is thus threatened by unexpected high-temperature events and damage (Ferrise et al., 2011; Peng et al., 2004; Sato et al., 2006; Sugiura, 2019); crops indigenous to the tropics are also susceptible to high-temperature damage because of climate change-related increases in extreme temperatures (Chauhan et al., 2014; Dinesh and Reddy, 2012). Crop species indigenous to tropical highlands, where the climate is cool year-round, are highly sensitive to high temperatures (Leon-Garcia and Lasso, 2019). For example, when grown in temperate and subtropical regions, as well as tropical lowlands, growth inhibition due to high temperatures during summer is often observed in potatoes (Ewing, 1981), tomatoes (Abdul-Baki, 1991), cherimoya (Higuchi et al., 1998), and passion fruit (Shimada et al., 2017). Under greenhouse conditions, air temperatures sometimes exceed 40°C. To increase resilience to climate change, there is an urgent need to evaluate existing genetic resources for traits that are superior for growth in high-temperature environments, and to efficiently select the genotypes with these traits (Chauhan et al., 2014; Driedonks et al., 2016).

Passion fruit species are indigenous to the tropical highlands of South America, stretching across Brazil, Paraguay, and Argentina (Ulmer and MacDougal, 2004). Purple passion fruit (Passiflora edulis Sims), one of the main cultivated species, is known to be sensitive to high temperatures and is not suitable for cultivation in tropical lowlands (Akamine and Girolami, 1959; Morton, 1987); however, it has been widely grown mainly in temperate and subtropical areas for fresh consumption. In contrast, yellow passion fruit (P. edulis f. flavicarpa Deg.), a cultivated mutant of purple passion fruit, is relatively tolerant to high temperatures and has been cultivated in tropical lowlands (Lim, 2012) mainly for processing as juice. In recent years, hybrids (P. edulis × P. edulis f. flavicarpa) with intermediate traits have been bred in various temperate and subtropical regions, including Europe (Nave et al., 2010), Asia (Chang and Cheng, 1992; Iwai and Omatsu, 2002), and Australia (Menzel et al., 1987). The production of fruits for fresh consumption is increasing along with demand in these regions (Kondo et al., 2020). Therefore, these regions are expected to be new potential areas for growing tropical crops such as passion fruit under increasing temperatures due to climate change; however, production is often unstable because of high temperatures in mid-summer and in greenhouses (Chang and Cheng, 1992; Matsuda and Higuchi, 2020). Water lemon (P. laurifolia L.) and sweet passion fruit (P. alata Curtis), relatives of purple and yellow passion fruit, respectively, are expected to have excellent heat tolerance in terms of growth because they are also cultivated in tropical lowlands. As these species can be hybridized with purple and yellow passion fruit (Payán and Martin, 1975), they are also considered breeding materials. Although passion fruit species vary widely in the climates of suitable cultivation areas and are particularly influenced by temperature, we found no quantitative studies regarding genotypic differences in their responses to high temperature.

To quantify the physiological responses and growth under environmental stresses, leaf photosynthesis measurements have been previously used in passion fruit (Kondo and Higuchi, 2013; Niwayama and Higuchi, 2019; Rodríguez et al., 2019) and various other species (Hikosaka et al., 2007; Khan et al., 2021; Leakey et al., 2003). In passion fruit, the high-temperature response of individual leaf photosynthesis has begun to be investigated using only a few hybrids with high commercial value. Shimada et al. (2017) measured and compared individual leaf photosynthesis in two cultivars under air temperature conditions of 20–40°C. They reported a clear difference in the reduction rate between the two cultivars at temperatures higher than 30°C. Regarding the photosynthetic response to high temperatures in passion fruit, higher temperature ranges exceeding 40°C and various genetic resources have not yet been examined.

In this study, to clarify the genotypic variations in leaf photosynthetic reduction at high temperatures in passion fruit (Passiflora spp.) and to examine a simple indicator for evaluating the thermal responses, we investigated the gas exchange characteristics of individual leaves under high temperature conditions (30–45°C) using various genotypes from various species.

Materials and Methods

Plant materials

Two one-year-old passion fruit plants, including four Passiflora and one hybrid species, were used for each of the 13 genotypes (Table 1): Kagoshima strain (PK) for purple passion fruit (P. edulis); two cultivars ‘MaDois (MD)’ and ‘MaQuatro (MQ)’ for yellow passion fruit (P. edulis f. flavicarpa); six cultivars ‘King Ruby (KR)’, ‘Minami-jujisei (MJ)’, ‘Ruby Star (RS)’, ‘Suico (SC)’, ‘Summer Queen (SQ)’, and ‘Sunny Shine (SS)’ for the hybrid (P. edulis × P. edulis f. flavicarpa); two strains including Alata seedling #1 (A1) and Amami-B (AB) for sweet passion fruit (P. alata); and two strains including Laurifolia seedling #2 (L2) and Iriomote strain (LI) for water lemon (P. laurifolia). The plants were propagated by cutting in December 2020. They were then transplanted to 18-L pots in March 2021 and grown under greenhouse conditions without heating at the Japan International Research Center for Agricultural Sciences on Ishigaki Island (24.4°N, 124.2°E), Japan. The roof windows of the greenhouse were automatically opened when the indoor air temperature exceeded 30°C. Figure 1 shows the plant training method. Each plant was trimmed to a single stem, which was first trained vertically to a height of 1.5 m and then trained horizontally. After allowing the horizontally trained vine to elongate along a horizontal circle with the same diameter as that of the pot, the terminal buds were pinched. Three newly emerged young axillary vines spiraled downward along the cylindrical outer circumference. When these axillary vines reached the pot, the terminal buds were pinched. In mid-September, these vines were cut back, leaving two to three leaves per vine, and three newly emerged young axillary vines per plant were trained as described above. Plants were irrigated daily and fertilized three times a week with liquid fertilizer containing 19.4 mM nitrogen, 1.1 mM phosphorus, 3.2 mM potassium, 1.0 mM magnesium, 91 μM calcium, 18 μM manganese, 29 μM boron, and 36 μM iron. Of the total nitrogen in the fertilizer, approximately 25% (w/w) was nitrate nitrogen, which is preferred for passion fruit cultivation (Kondo and Higuchi, 2013). The air temperature conditions in the greenhouse are shown in Figure S1.

Table 1

Passion fruit plant materials (Passiflora spp.) and genotypes used in the study.

Fig. 1

Training method for passion fruit plants. The thin green line represents a single stem trained vertically to a height of 1.5 m and then horizontally along a horizontal circle with the same diameter as that of the pot. Green solid and dashed lines represent newly emerged young axillary vines (AV), which were trained spirally downward along the cylindrical outer circumference. Arrowheads represent the mature leaves used for measuring photosynthesis and respiration.

Photosynthetic measurements

In June–July, two mature leaves (i.e., leaf age > one month) were randomly selected from each plant. These leaves were considered well acclimated to temperatures > 30°C in the greenhouse (Fig. S1). SPAD values (relative chlorophyll content) were recorded for each leaf using a chlorophyll meter (SPAD-502; KONICA MINOLTA JAPAN Inc., Tokyo, Japan). Plants were transferred from the greenhouse to a growth chamber (Growth Cabinet TGH-9H-S; ESPEC MIC Corp., Osaka, Japan) at noon, and photosynthetic measurements were then conducted using a portable gas exchange system (LI-6400; LI-COR Inc., Lincoln, NE, USA) inside the growth chamber until late afternoon. In the leaf chamber, the flow rate and reference CO₂ concentration were set as 300 μmol·s⁻¹ and 380 ppm, respectively. The photosynthetic photon flux density (PPFD) was 2,000 μmol·m⁻²·s⁻¹ according to photosynthesis-light curves (Fig. S2). To examine the temperature-photosynthesis curves, measurements were conducted at six levels of leaf temperature (T_leaf) ranging from 30 to 45°C for each leaf in a day. The temperature of each leaf was first set at around 30°C as a control under the growth chamber air temperature (T_gc) conditions of 28–29°C, as the photosynthetic rate in passion fruit was reported to peak at an air temperature around 30°C in orchard conditions (Shimada et al., 2017). T_leaf was gradually increased to ≈44°C by changing the LI-6400 block temperature, along with the T_gc up to 40°C as follows: 28–29°C T_gc was for ≈30°C T_leaf, 30°C T_gc for ≈34°C T_leaf, 35°C T_gc for ≈38°C and ≈40°C T_leaf, and 40°C T_gc for ≈42°C and ≈44°C T_leaf, considering that the T_leaf control capacity of LI-6400 is ± 5–7°C outside the leaf chamber and that whole plant warming together with T_leaf are effective to obtain stable measurements. PPFD inside the growth chamber was 1,000–1,200 μmol·m⁻²·s⁻¹. For every interval between the different levels of T_leaf measurements, the leaf chamber was opened and removed from the measured portion, and the leaf was exposed to the ambient air inside the growth chamber for more than 30 min. Photosynthetic rate (A), stomatal conductance (g_s), leaf–air vapor pressure deficit (VPD), transpiration rate (E), intercellular CO₂ concentration (C_i), and T_leaf were recorded for each leaf after these values stablized.

Passion fruit is a hypostomatous species. After photosynthetic measurements, the stomatal apertures on the abaxial surface of each measured leaf were obtained using Suzuki’s universal micro-printing (SUMP) method with polyvinyl acetate resin. The copied stomatal apertures were then observed under an optical microscope, and the stomatal pore length (Fig. S3) and density per square millimeter were recorded (Table S1). The mean values of stomatal length were calculated for each genotype using the data from nine stomata per leaf.

Respiration measurements

From early to mid-December, two mature leaves per plant were selected, and measurements of dark respiration were conducted in a growth chamber using a portable gas exchange system. In the leaf chamber, the flow rate and CO₂ concentration were the same as those used for the photosynthesis measurements. After measuring the A of each leaf at 30°C T_leaf under high PPFD (2,000 μmol·m⁻²·s⁻¹) conditions to ensure a high photosynthetic ability similar to that of the leaves measured in June–July, the PPFD inside the growth chamber was darkened to 600 μmol·m⁻²·s⁻¹, the light in the chamber was turned off, and the dark respiration rate (DR) was measured at approximately 30°C T_leaf. For genotypes PK, MQ, and SQ, measurements were conducted at five levels of T_leaf ranging from 30 to 42°C, followed by confirmation of the linear relationship between T_leaf and DR. For other genotypes, the measurements were conducted at two levels, around 30°C and 40°C.

Data analysis

To examine the temperature-A curves for each genotype, a decreasing sigmoid curvilinear regression was performed for each genotype using the following equation:

  

$$A=\frac{a-d}{1+{{\displaystyle \left(\frac{T_{\mathit{\text{leaf}}}}{c}\right)}}^b}+d$$

where a, b, c, and d are the coefficients and constants calculated using the Excel solver add-in. To examine the temperature-DR relations in each genotype, linear regression was performed using data from two levels of T_leaf, around 30°C and 40°C. The measured values of A were used in the regression to evaluate the gross photosynthetic rate, whereas relative A values (A at 30°C = 1 in each measured leaf) were used for the regression to examine the correlation between the photosynthetic rate reduction at 35°C, 40°C, or 45°C and the photosynthetic measurements (A, E, g_s, C_i, and VPD).

Results

The relationships of leaf A with g_s and E in Passiflora spp. (all 13 genotypes) are shown in Figure 2. The A at 30–45°C T_leaf was fitted as a logarithmic function of g_s, and the coefficient of determination (R²) was higher than 0.8 (Fig. 2a). The A decreased even with equal values of g_s when T_leaf increased. Both A and g_s values tended to decrease at higher temperatures. The A at each level of T_leaf was also fitted separately as a logarithmic function of g_s (R² > 0.5), and the R² was higher than 0.8 for T_leaf at 34–42°C (Fig. 2b). Meanwhile, A was fitted as a linear function of E (R² > 0.4) (Fig. 2c, d). R² was higher than 0.8 for T_leaf ≥ 34°C. In addition to the relationship between A and g_s, A decreased with equal values of E at higher T_leaf. The E tended to decrease as T_leaf increased up to ≈40°C, but started to increase at higher T_leaf. The mean E values were approximately 5 mmol·m⁻²·s⁻¹ at 30°C T_leaf, decreasing to approximately 2 mmol·m⁻²·s⁻¹ at 40°C T_leaf, and slightly increasing to approximately 3 mmol·m⁻²·s⁻¹ at 45°C T_leaf.

Fig. 2

Relationship between photosynthetic rate and stomatal conductance (left) and transpiration rate (right) of measured leaves in passion fruit (Passiflora spp.). Leaf temperature conditions are shown as mean ± SD. Logarithmic regression curves and linear regression lines are fitted (P < 0.01) for stomatal conductance (a, b) and transpiration rate (c, d) in each measurement, leaf temperature condition (solid) and total (dashed). The values indicated for each regression curve and line represent the coefficients of determination (R²).

The temperature-A and temperature-DR relations in P. edulis, P. edulis f. flavicarpa, P. alata and P. laurifolia are shown in Figure 3. The temperature-A relationship was fitted as a sigmoidal curve for all genotypes, and R² values were higher than 0.7, except for LI (0.58). For MQ, A was higher than 15 μmol·m⁻²·s⁻¹ at 30°C and greatly decreased to approximately 10 μmol·m⁻²·s⁻¹ at 34°C. A then gradually decreased > 35°C and reached approximately 5 μmol·m⁻²·s⁻¹ at 45°C. The DR at 30°C was approximately 3 μmol·m⁻²·s⁻¹ and increased linearly (R² > 0.6) to > 5 μmol·m⁻²·s⁻¹ at > 40°C. The difference in the reduction percentage from values under 30°C between net and gross A was less than 30% at 45°C in MQ. For PK, A at 30°C was lower than 10 μmol·m⁻²·s⁻¹, and A decreased at higher temperatures the same as MQ. A at > 40°C was almost 0 μmol·m⁻²·s⁻¹ in PK. The DR values of PK were similar to those of MQ within 30–45°C and increased linearly (R² = 0.76) at higher temperatures. The difference in the percentage reduction between net and gross A at 45°C was larger (> 45%) in PK than that in MQ. MD shows the intermediate characteristics of the temperature-A reduction curve. The temperature-DR line in MD was similar to those of MQ and PK. For sweet passion fruit A1, A at 30°C was higher than 15 μmol·m⁻²·s⁻¹, and decreased to approximately 5 μmol·m⁻²·s⁻¹ at 45°C, similar to that in the yellow passion fruit MQ (Fig. 3). For AB, A was low (around 10 μmol·m⁻²·s⁻¹) at 30°C, largely decreased toward 35°C, and almost reached 0 μmol·m⁻²·s⁻¹ at 45°C, similar to values observed in purple passion fruit PK (Fig. 3). For P. laurifolia, A at 30°C was within 10–15 μmol·m⁻²·s⁻¹ and decreased to below 5 μmol·m⁻²·s⁻¹ at 45°C in both genotypes. The decrease in A from 30 to 40°C was larger in L2. Temperature-DR lines of P. alata and P. laurifolia were similar to those observed in purple and yellow passion fruits. The difference in the reduction percentage between net and gross A at 45°C varied largely with the genotype in P. alata and P. laurifolia, with the smallest (23.5%) in A1 and the largest (50.6%) in LI.

Fig. 3

Leaf photosynthetic and respiration rate under high temperature (≥ 30°C) conditions in purple passion fruit (Passiflora edulis), yellow passion fruit (P. edulis f. flavicarpa), sweet passion fruit (P. alata) and water lemon (P. laurifolia). Sygmoid curvilinear regression (R² > 0.58, P < 0.01, n = 24) was conducted for the net photosynthetic rate using the data obtained at 30–45°C, and linear regression (R² > 0.62, P < 0.01, n = 8) was performed for the respiration rate using the data at 30°C and 40°C. The gross photosynthetic rate (dotted curve) was calculated using the regression values of net photosynthetic and respiration rates. Percentages shown in the upper figures indicate the difference in reduction percentage (30°C = 100%) between gross and net photosynthesis at 45°C.

The temperature-A and temperature-DR relationships in hybrid (P. edulis × P. edulis f. flavicarpa) cultivars are shown in Figure 4. The temperature-A relation was fitted as a sigmoidal curve (R² > 0.7) in all six cultivars, as well as in purple and yellow passion fruit (Fig. 3). For MJ, RS, SC, and SQ, A at 30°C was approximately 15 μmol·m⁻²·s⁻¹, and decreased to below 5 μmol·m⁻²·s⁻¹ at 45°C, but the decrease in A from 30–35°C was larger in SC and SQ than that in MJ and RS. For KR and SS, A at 30°C was slightly lower (10 < A < 15 μmol·m⁻²·s⁻¹) than that of the other cultivars. DR at 30°C was approximately 3 μmol·m⁻²·s⁻¹ and increased linearly to > 5 μmol·m⁻²·s⁻¹ at > 40°C for all six cultivars, which was consistent with the observations in the purple and yellow passion fruits. The difference in the reduction percentage between net and gross A at 45°C was lower than 35% in MJ, RS, SC, and SQ and higher than 40% in KR and SS. Temperature-A curves in the hybrid cultivars showed intermediate characteristics between MQ and PK (Fig. 3).

Fig. 4

Leaf photosynthetic and respiration rate under high temperature (≥ 30°C) conditions in six hybrid passion fruit (Passiflora edulis × P. edulis f. flavicarpa) cultivars. Sygmoid curvilinear regression was performed for net photosynthetic rate using the data at 30–45°C, and linear regression was performed for respiration rate using the data at 30°C and 40°C. Gross photosynthetic rate (dotted curve) was calculated using the regression values of the net photosynthetic and respiration rates. Percentages shown in the upper figures indicate the differences in reduction percentage (30°C = 100%) between gross and net photosynthesis at 45°C.

The results of the correlation analysis between the reduction ratio of A at 35–45°C to A at 30°C and the photosynthetic measurements in Passiflora spp. are shown in Table 2. For the measured values of A, E, and g_s, significant positive correlations were generally detected with the reduction ratio of A at 35–45°C to A at 30°C, with some exceptions, indicating that genotypes with higher A, E, and g_s ability tended to show smaller A reduction under high temperature conditions. For C_i values, significant correlations were detected less than those for A, E, and g_s, but significant positive and negative correlations were found at 34°C and 44°C, respectively. For VPD, significant negative correlations were found at temperatures lower than 40°C, whereas no significant correlation was detected at higher temperatures.

Table 2

Correlation coefficients between the reduction ratios of the photosynthetic rate at 35°C, 40°C, or 45°C to 30°C and photosynthetic measurements at each leaf temperature condition within 30–45°C of measured leaves in 13 genotypes of Passiflora spp.

In the P. edulis group (P. edulis, P. edulis f. flavicarpa, and their hybrids), significant positive correlations were generally detected in the reduction ratio of A at 35–45°C to A at 30°C for A, E, and g_s (Table 3), but tended to be lower than those in Passiflora spp. Overall (Table 2). For C_i and VPD, even fewer significant correlations were found in the P. edulis group.

Table 3

Correlation coefficients between the reduction ratios of the photosynthetic rate at 35°C, 40°C, or 45°C to 30°C and photosynthetic measurements at each leaf temperature condition within 30–45°C of measured leaves in nine genotypes of Passiflora edulis, P. edulis f. flavicarpa and their hybrids.

The results of the correlation analysis between the reduction ratio of A at 35–45°C to A at 30°C and leaf chlorophyll content and stomatal characteristics are shown in Table 4. For the SPAD value, a significant positive correlation was detected only for the reduction at 35°C in the P. edulis group, whereas no significant correlation was found in Passiflora spp. For stomatal density, a significant negative correlation was detected only with the ratio of A at 35°C in both Passiflora spp. and P. edulis groups, indicating that genotypes with higher stomatal density tended to show larger A reduction at 35°C. In contrast, for stomatal length, significant positive correlations were detected with the ratio of A at temperatures higher than 40°C in the P. edulis group. Genotypes in the P. edulis group with larger stomata tended to show smaller A reduction at higher temperatures. In Passiflora spp., no significant correlations were found with stomatal length.

Table 4

Correlation coefficients between reduction ratios of photosynthetic rate at 35°C, 40°C, or 45°C to 30°C and chlorophyll content, stomatal density, and size of measured leaves in Passiflora spp.

Based on the results showing significant correlations between A reduction under high temperature conditions and E and g_s (Tables 2 and 3), correlations between these two measurements, leaf chlorophyll content, and stomatal characteristics were also analyzed (Table 5). For SPAD values, significant positive correlations were detected only with E and g_s at 34°C in Passiflora spp., whereas in the P. edulis group, stronger correlations were detected at temperatures lower than 35°C. For stomatal density, significant negative correlations were detected at temperatures < 35°C and > 44°C, indicating that genotypes with higher stomatal density tended to show less vigorous stomatal activity. For stomatal length, significant positive correlations were generally detected at 30–42°C in the P. edulis group, except at 34°C. No correlation was found at temperatures > 44°C. In Passiflora spp., significant positive correlations were found only at temperatures < 35°C, and these were weaker than those in the P. edulis group.

Table 5

Correlation coefficients between transpiration rate (E) and stomatal conductance (g_s) at six leaf temperature conditions ranging from 30°C to 45°C and leaf chlorophyll content and stomatal characteristics in Passiflora spp.

Discussion

Factors inhibiting photosynthesis at high temperatures

In the present study, the stomatal conductance and transpiration rate of passion fruit decreased as the leaf temperature increased to 40°C (Fig. 2). In contrast, for leaf temperature conditions > 40°C, stomatal conductance barely decreased, and the transpiration rate showed a slight increase as the leaf temperature increased. In each genotype, the net photosynthetic rate continued to decrease up to 45°C, while the gross photosynthetic rate tended to stop decreasing at temperatures higher than 40°C with an increase in the dark respiration rate (Figs. 3 and 4). These results indicate that stomatal limitation, as well as an increase in dark respiration, caused photosynthetic rate reduction from 30°C to 40°C, while the increase in dark respiration largely reduced the net photosynthetic rate at temperatures > 40°C. Such stomata-dependent characteristics of passion fruit were also reflected in the strong correlations of photosynthetic rate reduction at high temperatures with transpiration and stomatal conductance (Tables 2 and 3).

Under low VPD conditions, higher leaf temperatures can generally increase stomatal conductance and transpiration rates (Jones, 1998; Jumrani et al., 2017; Leegood and Edwards, 1996). However, in our study, it was difficult to maintain a low VPD at higher temperatures because the VPD tends to largely increase under high-temperature conditions in the growth chamber. Under humid air conditions in orchards, the leaf photosynthetic rates of passion fruit at high temperatures may be higher compared to the values obtained in the present study. Under orchard conditions, Shimada et al. (2017) reported a smaller decrease in photosynthetic rate at approximately 40°C than that observed in our study.

The small decrease in gross photosynthetic rate observed at > 40°C (Figs. 3 and 4) indicates that increased dark respiration was the major cause of the decreased net photosynthetic rate in passion fruit, although the photosynthetic rate decrease at higher temperatures can be attributed to stomatal limitations and increased dark respiration, as well as to increased photorespiration and damage to the leaf photosynthetic apparatus (Schrader et al., 2004). High temperatures and strong light conditions accelerate photoinhibition (Demmig-Adams and Adams, 1992; Powles, 1984). Damage to the photosynthetic apparatus, mainly photosystem II, has been reported at temperatures > 40°C in potatoes (Havaux, 1993) and tomatoes (Camejo et al., 2005). In the present study, passion fruit leaves were exposed to extremely high temperatures for less than an hour during each measurement. Exposure to extremely high temperatures for a longer time may also damage the photosynthetic apparatus in passion fruit.

Genotypic variation in high temperature response

Our results clearly demonstrate genotypic differences in the thermal response of leaf photosynthesis in the P. edulis group. Two yellow passion fruit cultivars showed a smaller reduction in photosynthetic rate under the high-temperature regime than that in purple passion fruit (Fig. 3), in accordance with the known species’ characteristics; yellow passion fruit is more suitable for cultivation in warmer areas, as it shows superior tolerance to high temperatures than purple passion fruit (Akamine and Girolami, 1959; Morton, 1987; Ulmer and MacDougal, 2004). For hybrid cultivars, RS was reported to show a smaller decrease in the photosynthetic rate under high-temperature conditions (35–40°C) compared to SQ (Shimada et al., 2017). Ogata et al. (2016) reported that SS showed a lower incidence of immature fruit dropping and superior fruit quality in a high temperature (above 30°C) season than SQ. These observations are consistent with the temperature-A curves of the cultivars used in the present study (Fig. 4). In species cultivated on tropical lowlands (i.e., P. alata and P. laurifolia), A1 and LI showed marked tolerance to high temperatures, similar to the results in yellow passion fruit MQ; however, the response to high temperatures varied among genotypes, and AB was susceptible to high temperatures similar to purple passion fruit PK (Fig. 3). A1 is a genotype selected on Ishigaki Island, whereas AB is a genotype selected on Amami Island (Table 1), where the climate is cooler. Among the hybrid cultivars, RS and SS, which show superior tolerance to high temperatures (Fig. 4), were bred in Taiwan and Ishigaki Island, respectively, with warmer climates than that on Tanegashima Island, where SQ was bred (Table 1). Matsuda and Ogata (2020) reported that the thermal response of pollen germination in hybrid passion fruit cultivars reflects the climate of the cultivar selection region. The thermal photosynthetic response is also likely to reflect the climate of the genotypic origin.

Although the dark respiration rate and its increase at high temperatures barely differed among genotypes, the reduction in the gross and net photosynthetic rates at high temperatures for each genotype was significantly changed by dark respiration (Figs. 3 and 4). MQ and A1 showed the lowest respiration load on CO₂ assimilation, together with the smallest reduction in net photosynthetic rate at high temperatures, whereas LI showed the largest respiration load despite the smallest reduction in net photosynthetic rate. Thus, MQ and A1 can be regarded as highly efficient genotypes for CO₂ absorption, i.e., a lower ratio of dark respiration rate to gross photosynthetic rate. Among the hybrid cultivars, MJ, RS, SC, and SQ were relatively efficient, whereas KR and SS were less efficient. Plant respiration is a large component of carbon balance (Amthor, 1991), and half of photosynthetic CO₂ assimilation is estimated to be lost to respiration in plants (Amthor, 1989; Kira, 1975). The efficiency of CO₂ absorption in photosynthesis can thus be one of the target characteristics for breeding tolerance to high temperatures in passion fruits.

High temperature tolerance evaluation

Measured values of photosyntehtic rate vary widely from leaf to leaf even for the same genotype and same leaf age. Accordingly, evaluation using relative values is considered preferable to fairly compare the degree of photosynthetic reduction among genotypes. Leaf photosynthetic reduction at high temperatures was highly correlated with stomatal activity in passion fruit; genotypes showing higher transpiration rates and stomatal conductance at 30°C leaf temperatures tended to maintain higher photosynthetic rates at higher temperatures (Tables 2 and 3). Moreover, transpiration at 30°C tended to be vigorous in genotypes with longer stomata at lower densities (Table 5). In the P. edulis group, genotypes with longer stomata tended to maintain higher photosynthetic rates at higher temperatures (Table 4). Using these characteristics, superior passion fruit genotypes with high temperature tolerance in terms of leaf photosynthesis can be screened.

High stomatal density can theoretically be correlated with high maximum stomatal conductance (Franks and Beerling, 2009). Higher stomatal density has been correlated with higher photosynthetic and transpiration rates, and stomatal conductance in Leymus chinensis (Xu and Zhou, 2008) and Arabidopsis thaliana (Tanaka et al., 2013). Jumrani et al. (2017) observed that soybeans grown at higher temperatures showed a higher transpiration rate with higher stomatal densities, even though the photosynthetic rate decreased. In contrast, Crawford et al. (2012) reported that A. thaliana plants grown under higher temperatures had lower stomatal densities, despite increased transpiration. Zhao et al. (2015) also reported a negative correlation between stomatal density and transpiration rate in maize subjected to water stress. Although stomatal density may be a target trait for improving the photosynthetic capacity of plants (Tanaka et al., 2013), stomatal characteristics (density and length) can vary widely among species, as well as with growing environments (Jones, 1977); therefore, they may not always be good target traits for breeding (Wang et al., 2013). In the present study, stomatal length was highly correlated with photosynthetic and transpiration rates at higher leaf temperatures in the P. edulis group, whereas no correlation was detected when including the lowland relatives (Tables 4 and 5). Therefore, we recommend transpiration rate and stomatal conductance at 30°C as practical target traits in passion fruit.

Conclusion

Our measurements demonstrate that the genotypic differences in thermal response characteristics of leaf photosynthesis in passion fruit reflected the temperature characteristics of the climate where each genotype originated and was cultivated. At temperatures higher than 30°C, leaf photosynthesis in passion fruit was found to be highly dependent on stomatal activity and dark respiration. The leaf photosynthetic rate was maintained at a higher level even at approximately 40°C in genotypes that showed higher stomatal activity under non-stressed conditions (i.e., high transpiration rate and stomatal conductance at 30°C), which can be considered potential indicators for screening genotypes that are highly tolerant to high temperatures in passion fruit breeding.

Acknowledgements

We are grateful to Ms. Matsuura and Ms. Nemoto for providing support with daily cultivation management.

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