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Pepper Photosynthesis, Stomatal Conductance, Transpiration, and Water Use Efficiency Differ with Variety, Indigenous Habitat, and Species of Origin

The instantaneous photosynthetic rate (Pn), transpiration rate (E), and stomatal conductance (gS) were measured for 33 outdoor-grown Capsicum varieties (varying in species of origin and indigenous habitat) between 29 July and 22 Aug. 2017 using a portable gas exchange meter. Cuvette leaf temperature (Tleaf) and relative humidity (RH) were recorded at that same time. Pn differed from 3.6 to 3.7 for ‘Malawi Piquante’ and ‘Korean Long Green’ peppers to 16.3 μmol CO2/m2/s (fixed) for ‘Thai Hot’ peppers. The gS differed from 0.01 to 0.05 among 13 varieties to 0.28 mmol H2O/m2/s for ‘Thai Hot’ peppers. E differed from 0.43 to 0.59 among three varieties to 4.14 to 4.20 mmol H2O/m2/s for ‘CGN 22091’ and ‘Peruvian Purple’ peppers. Water use efficiency (WUE; Pn/E) varied from 2.92 to 3.43 among three varieties to 5.10 to 7.20 for 16 other varieties. C. annuum derived varieties had higher Pn (9.4 μmol CO2/m2/s fixed) than varieties derived from other species (4.5–8.6 μmol CO2/m2/s fixed). Varieties originating from dry climates had higher Pn (12.5 μmol CO2/m2/s fixed) than those originating from temperate or tropical climates (8.0–8.8 μmol CO2/m2/s fixed). Tleaf (27 to 33 °C) and RH (38% to 39% and 57% to 59%) differed among varieties. Pn was positively correlated with gS, E, and RH and was negatively correlated with WUE. We found that Capsicum Pn, E, and gS varied more than has been previously reported, and our data suggested that Pn, gS, and E data of outdoor-grown peppers should be used only when selecting parents for a breeding program (unless progeny is intended for greenhouse production).

Peppers (Capsicum sp.) were one of the first crops domesticated in the Western Hemisphere using several independent, geographically distinct, and regional domestication efforts (Bosland and Votava, 2012). Cultivated peppers are derived from five species (C. annuum L., C. baccatum L., C. chinense Jacq., C. frutescens L., and C. pubescens) among the 32 documented species in the Capsicum genus (Solanaceae family) (Qin et al., 2014). These domestication efforts resulted in peppers becoming a globally important fresh vegetable (fruit) and spice crop that is now consumed by nearly one-quarter of the world's population, with fresh and dry pepper production reaching 32.2 and 3.8 million tons annually, respectively, in 2014 (FAO, 2014). In the United States, 44,800 acres of bell peppers and 19,400 acres of chili pepper were produced in 2015, with a wholesale value of $806 million and $135 million, respectively (USDA NASS Report, 2016). Peppers are consumed as a fresh food and contain carbohydrates, fiber, and a variety of nutrients, including antioxidants such as vitamin C, vitamin E, b-carotene, and carotenoids (Carvalho et al., 2011;Howard et al., 2000;Howard and Wildman, 2007;Kantar et al., 2016;Palevitch and Craker, 1995;Russo and Howard, 2002;Topuz and Ozdemir, 2007;Wahyuni et al., 2011). Other pepper products are consumed as spices, including dried pepper flakes, processed ''hot'' sauces, and infused/pressed oils, among other products (Bosland, 1996;Zewdie and Bosland, 2001).
Abiotic factors such as low temperatures, high temperatures, salt stress, drought (Serrano et al., 2017), and waterlogging can limit pepper yield (Ou and Zou, 2012;Zhai et al., 2016). For instance, optimal temperatures for pepper photosynthesis range from 25 to 35°C, and temperatures outside this range can limit the yield; for example, C. chinense flower abortion increased 2-fold and fruit set decreased 3-fold when greenhouse temperatures were increased from 30 to 40°C (Garruna-Hernandez et al., 2014). Erickson and Markhart (2002) showed that Capcisum flower abortion after pollination was particularly sensitive to high temperatures (33°C).
We determined whether photosynthetic and water use attributes differed among outdoor-grown pepper varieties that differed in parental species and indigenous climates. Previous studies of Capsicum focused on differences in photosynthetic and water use attributes among species only or varieties within a species. Past Capsicum research often used greenhouse-grown or controlled environment-grown plants. Our research objectives were to: 1) determine whether instantaneous P n , g S , E, WUE meter cuvette T leaf , and RH of 33 outdoor-grown pepper varieties differed; 2) to determine whether variety photosynthetic and water use attributes differed based on indigenous parents or habitat; and 3) to determine whether pepper photosynthetic and/or water use attributes were correlated with each other.

Materials and Methods
Thirty-three pepper varieties derived from five species (C. annuum L., C. baccatum L., C. chinense Jacq., C. frutescens L., and C. pubescens) from three indigenous habitats (dry, temperate, and tropical) were selected (Table 1). Seeds were sourced from different seed producers/suppliers, and the reported species of origin were confirmed by phenotyping of flowers, leaves, and fruits during the experiment (Table 1). Seeds were sown 0.6 cm deep in Master Garden Premium media (Premier Tech Horticulture, Ltd., Ontario, Canada) in 32-cell trays (one seed per cell; individual cell volume = 150 cm 3 ) in January and February 2017. Trays were then covered with a clear plastic lid (10 cm above media) and were placed in a greenhouse (23 ± 2°C air temperature). After seeds germinated and cotyledons unfolded, the lid was removed and plants were grown for 9 to 14 additional weeks under natural daylight plus 25 mmol · m -2 · s -1 supplemental irradiance (0800-0200 HR; Sunblaze T5 fluorescent lights; Sunlight Supply, Inc., WA; +1.62 mol · m -2 · d -1 daily light integral) in the same greenhouse. After five leaves unfolded, plants were transplanted into 18.9-L plastic pots with the same media. Potted plants were placed outdoors (St. Paul, MN) on 1 June and were watered with a drip irrigation system as needed. Nutrients were provided by incorporating granular Sustane Natural Fertilizer (4-6-4; Cannon Falls, MN) with media at a rate of 12 g/L to provide 100 ppm of N (3.2N-6P 2 O 5 -4K 2 O). P n , g S , and E data were collected from the third unfolded mature leaf (expansion completed) below the shoot tip using a LI-COR LI6400XT portable gas exchange meter (LI-COR, Inc., Lincoln, NE) with an external cuvette with a built-in LED light source (cuvette dimensions = 3 · 2 cm 2 ) on 29 July, 3 Aug., 5 Aug., 10 Aug., 17 Aug., and 22 Aug. 2017 (similar weather conditions). Because data were collected at different times, a different leaf was used each time as the plant grew. Data were recorded 5 min after placing the cuvette around a leaf (P n had stabilized). Cuvette T leaf and RH were also recorded at that time. Cuvette irradiance, carbon dioxide concentration, and atmospheric flow rate were set to 1000 mmol · m -2 · s -1 , 400 ppm (approximately outdoor ambient), and 500 mL · min -1 , respectively. The outdoor air tem-perature, RH, and photoperiod on collection dates varied from 22.4 to 27.9°C, 40% to 70%, and 13.25 to 15.5 h, respectively. Garruna-Hernandez et al. (2014) reported that C. chinense g S varied during the day and was highest between 1100 and 1300 HR; therefore, we only collected data between 1100 and 1300 HR. WUE was calculated by dividing P n by E (Ou and Zou, 2012).
The experiment was organized in a completely randomized statistical design. P n , E, g S , and WUE were dependent variables, and variety (33 levels), species of origin (5 levels), and indigenous habitat (3 levels) Table 1. Variation in the instantaneous photosynthetic rate (P n ; mmol·m -2 ·s -1 ), stomatal conductance (g S ; mmol H 2 O/m 2 /s), transpiration rate (E; mmol H 2 O/m 2 /s), cuvette leaf temperature (T leaf ;°C), and relative humidity after 5 min (RH; %) and water use efficiency (WUE; WUE = P n /E) among 33 outdoor-grown pepper varieties that vary in indigenous habitat and species. Indigenous habitats used for analysis are shown as superscript numbers after the variety name ( 1 desert; 2 temperature; 3 tropical; ? unknown).  (Table 1).

Discussion
Capsicum P n values varied more in our experiment (3.6-3.7 to 16.3 mmol CO 2 /m 2 /s fixed) than they did in other experiments reported by others; for example, Ou and Zou (2012) reported that the P n of five Capsicum species varied from 15.8 to 21.8 mmol CO 2 /m 2 /s (fixed). Among pepper varieties within a species, P n also varied more in our experiment than it did in others (Ghasemi et al., 2016;Rosado-Souza et al., 2015). Borisev et al. (2012) reported that the P n of 10 C. annuum varieties differed from 14.5 to 16.6 mmol CO 2 /m 2 /s (fixed); the C. annuum variety P n (22 varieties) in our work ranged from 3.7 to 16.3 mmol CO 2 /m 2 /s (fixed) ( Table 1). Ridzuan et al. (2018) reported that the C. annuum variety/accession P n differed from 11.5 to 19.1 mmol CO 2 /m 2 /s (fixed). In contrast, Hassan et al. (2014) reported that the C. annuum variety P n ranged from 2.5 to 5.3 mmol CO 2 /m 2 /s (fixed; irradiance not reported). P erez-Grajales et al. (2004) reported similar P n rates (at a lower irradiance of 500 mmol/m 2 /s) for C. pubescens. Rosado-Souza et al. (2015) reported that the C. chinense accession P n varied from 17 to 25 mmol CO 2 /m 2 /s (fixed).
Differences between our P n values and others may have been related to the irradiance levels when P n was measured, where plants were grown, or the number of varieties studied. For instance, irradiance in our work was at saturating photosynthetic levels (1000 mmol · m -2 · s -1 ), whereas irradiance in other studies was undocumented or at 300 to 500 mmol · m -2 · s -1 . Because peppers are typically grown in the field, where they are routinely exposed to saturating irradiance levels, we believe that our data more accurately reflected actual field P n . Furthermore, P n can vary more for field-grown than for greenhouse-grown/controlled environmentgrown Capsicum. Borisev et al. (2012) reported that the P n of nine Capsicum varieties differed from 14.5 to 16.6 mmol CO 2 /m 2 /s (fixed), but that P n differed from 12.8 to 18.7 mmol CO 2 /m 2 /s (fixed) when those same varieties were grown in the field. Furthermore, the greater variation in P n in this study compared with that in some other studies may have occurred because we evaluated a greater number of varieties that were intentionally selected to represent a broad array of genetic and indigenous backgrounds.
Our data regarding the varieties derived from C. annuum included collectively higher P n than the data regarding the varieties derived from other species. However, broad conclusions regarding which species have higher P n should be made cautiously because abiotic factors can interact with species and affect P n . For instance, Ou and Zou (2012) reported that the P n of C. frutescens was higher than that of four other Capsicum species (21.8 vs. 15.8-17.7 mmol CO 2 /m 2 /s fixed) when plants were grown at 35°C, but the P n of C. pubescens was highest when plants were grown at 15°C. In the same study, the P n of C. baccatum and the P n of C. pubescens were higher than that of three other Capsicum species when plants were drought-stressed. In contrast, Okunlola et al. (2017) reported that the C. chinense P n was more drought-tolerant than the P n of C. annuum or C. frutescens. The environment where a plant is grown or the water status of a plant when P n data are collected can lead to different conclusions about which Capsicum species has higher P n . Although varieties are reportedly derived from a species, many are interspecific hybrids (often unreported) (Petkova et al., 2014) and genetically related (Amarul J'unior et al., 2005). That hybridization alone may impact the photosynthetic or water use data. Petkova et al. (2014) showed that the P n of four F 1 pepper hybrids was more tolerant to high temperatures (33 to 35°C) than that of parental species.
The range and g S values reported in our study (0.01-0.28 mmol H 2 O/m 2 /s) were less than those reported by others. Ridzuan et al. (2018) reported that the g S of the C. annuum variety (14) varied from 0.25 to 0.68 mmol H 2 O/m 2 /s. Hassan et al. (2014) reported that the g S of the C. annuum variety varied from 0.50 to 0.60 mmol H 2 O/m 2 /s. Differences between our g S data and that of others may have been related to the environments where plants were grown. For instance, plants grown at temperatures below 15°C or above the optimal temperature for pepper P n can decrease the Capsicum g S (Jaimez and Rada, 2016). Because the T leaf data were collected on some days when the temperature was above the optimal temperature reported for Table 2. Pearson correlation between instantaneous photosynthetic rate (P n ), stomatal conductance (g S ), transpiration rate (E), cuvette leaf temperature (T leaf ), and humidity after 5 min (RH) and water use efficiency (WUE; WUE = P n /T). Capsicum P n (25 to 30°C), the g S may have been impacted (Table 3). Our data indicated that g S does not vary among domesticated Capsicum species (Table 1). Milla et al. (2013) also found that g S did not vary between wild-type and domesticated C. annuum or C. baccatum. Interestingly, we observed that g S differed among varieties derived from those species. Rosado-Souza et al. (2015) reported that the C. annuum variety g S differed. Furthermore, Kang et al. (2001) reported that the C. chinense variety g S differed. Yet, in both of those studies, the species g S did not differ. Similarly, Percy et al. (1996) showed that the g S of interspecific selections of cotton (Gossypium barbadense L) was higher than that of the parents or ancestral lines. Differences in the g S of varieties observed by us and others may have been due to the integration of a mutation into breeding programs over time and/or interspecific hybridization resulting in greater variations in g S than that observed for genetic parents. Furthermore, a recent study has shown a transgenerational effect on watercress (Lepidium sativum) g S ; the maternal light environment effects on stomatal density and g S were expressed in seedpropagated progeny, suggesting that differences in g S in this experiment and in past experiments, to some degree, may be impacted by the environment in which seeds were produced (Vrablova et al., 2018). A similar relationship between the maternal water status and progeny WUE was also reported for Eucalyptus (Vivas et al., 2019).
The E values reported here also varied more than those reported by others, although the maximum E values were similar (3.21-4.05 mmol H 2 O/m 2 /s) (Ou and Zou, 2012). Ridzuan et al. (2018) reported that the E of the C. annuum variety differed from 4.61 to 7.11 mmol H 2 O/m 2 /s (indoor-grown). Furthermore, differences in E values in our work and that of others may have been related to where plants were grown. Borisev et al. (2012) reported that the E of Capsicum variety (nine) differed more for outdoorgrown than indoor-grown peppers (4.4-6.1 vs. 4.7-5.0 mmol H 2 O/m 2 /s, respectively).
In contrast to the P n , E, and g S data presented here, the WUE values we observed (3.1-7.2) were similar to those reported by others for peppers grown in a controlled environment (3.9-6.8) (Ou and Zou, 2012). However, our values were generally higher than those reported by Borisev et al. (2012) who also reported that the WUE of indoorgrown C. annuum variety (nine) varied less than that of field-grown plants (3.2-3.5 and 2.3-3.9, respectively). The WUE of Capsicum is reportedly impacted by irradiance; WUE was higher when plants were grown under high irradiance (400 mmol · m -2 · s -1 ; 2.2) compared to low irradiance (200 mmol · m -2 · s -1 ; 1.8) (Fu et al., 2010). However, irradiance in our study was 1000 mmol · m -2 · s -1 ; therefore, it would be considered high. Moreover, the negative correlation between Capsicum WUE and P n that we observed agreed with the observations of Antony and Singandhupe (2004). Ridzuan et al. (2018) reported that the C. annuum variety E and g S were correlated with P n , as we observed (0.55 and 0.87, respectively). However, P n is not always correlated with g S . Yun and Ahn (2009) reported that increasing temperature and atmospheric CO 2 concentrations increased the pepper P n , but not the pepper g S . The strong correlation between pepper g S and P n reported here (0.95) (Table 2) supports the assertion that g S limitations may reduce pepper P n (Serrano et al., 2017). However, the observations of Yun and Ahn (2009) suggested that g S limitations on P n may occur less if CO 2 concentrations are increased, as is common for greenhouse-produced bell peppers (Dorais, 2003;Erwin and Gesick, 2017).
A negative correlation between g S and T leaf was previously reported (r 2 = 0.76) for cotton (G. barbadense L.-derived) (Radin et al., 1994), although that correlation was greater than what we observed here for pepper (Table 2). Interestingly, some have suggested that g S measurements obtained later in the afternoon (after 1300 HR) may be important when evaluating plants for higher yield based on g S ; for instance, Rebetzke et al., (2003) recommended that wheat breeding projects should involve selected progeny with higher g S collected later in the day because g S collected later in the day correlated more with yield than g S collected earlier. Similarly, Radin et al. (1994) found that the cotton yield (related to P n ) was associated more with g S collected in the afternoon than in the morning, and that varieties with high yield under hot temperatures had higher g S in the afternoon, but not in the morning (Lu et al., 1997). We collected data when P n and g S were highest during the day; whether Capsicum yield is associated more with g S collected at certain times of the day is not known.
Differences in outdoor environmental conditions for data collection dates during our experiment may have reduced the resolution of our experiment. T leaf was higher on collection dates 3 and 4 (33.6 to 34°C; overreported P n optima) than on other dates (27.7 to 29.6°C) (Table 3). Furthermore, cuvette RH was higher on collection dates 2, 3, 4, and 5 (48% to 58%) than on collection dates 1 and 6 (40% to 43%) ( Table 3). Because T leaf and RH are correlated with P n , g S , and E (Table 2), the environmental differences among collection dates may have impacted values.
Care should be taken when determining conclusions about plant photosynthesis or yield based on instantaneous P n or water use data based on the leaf area per unit. Collecting data based on the leaf area per unit does not account for differences in the individual leaf area, plant leaf number, whole-plant leaf area, or whole-plant photosynthesis. Furthermore, instantaneous P n data do not account for plant acclimation to changing environmental conditions (Hikosaka et al., 2006). Therefore, our study is of value because it compared photosynthetic and water use attributes of a larger number of pepper varieties at the same time of day and under the same environmental conditions simultaneously. Our study also demonstrated that outdoor-grown pepper variety photosynthetic and water use attributes vary more than previously reported, and that the reported parental species and indigenous habitat are associated with some photosynthetic and water use attributes.  Table 3. Variation in instantaneous photosynthetic rate (P n ; mmol CO 2 /m 2 /s fixed), stomatal conductance (g S ; mmol H 2 O/m 2 /s), transpiration rate (E; mmol H 2 O/m 2 /s), cuvette leaf temperature (T leaf ;°C), and relative humidity (RH; %) and water use efficiency (WUE; WUE = P n /E) among 33 outdoor-grown pepper varieties. NS, *, **, ***Nonsignificant or significant at P # 0.05, 0.01, or 0.001, respectively.