INTRODUCTION
The lulo plant (Solanum quitoense Lam.; Solanaceae) is a tropical shrub from the inter-Andean areas (Huertas et al. 2011), whose fruit is desired for its organoleptic characteristics, such as soft and exotic aroma, taste, color and bright appearance in the pulp (Ardila et al., 2015). In addition, it has antioxidant properties and a high nutritional potential with its important source of vitamins and minerals (Gancel et al., 2008). It is widely consumed in Colombia, desired mainly for its high potential in agroindustry with uses for its pulp, nectars and juices (González et al., 2014). It belongs to the group of exotic Solanaceae that enjoys demand in the international market, especially in Europe (Álvarez-Herrera et al., 2015; Cruz et al., 2007; Orjuela-Castro et al., 2017).
In Colombia, two botanical varieties predominate: a) septentrionale, with thorns on the stem and leaves and higher acidity in the fruits; and b) quitoense, without thorns and with sweeter fruits (Bonnet and Cárdenas, 2012). The lulo exhibits its best development in sites with temperatures of 15 to 24°C, with an optimum of 20°C, requires slightly acidic soils, with pH between 5.5 and 6.0, with good moisture retention capacity and deep and good drainage (Gómez et al., 2014). It is a plant native to humid forests, adapted to fresh and shaded zones, but, in the course of its domestication, commercial crops have been adapted to areas free from light exposure (Ardila et al., 2015; Huertas et al., 2011).
Fischer et al. (2018) stated that "the environmental conditions of the site (climate and soil) are crucial for the process of crop quality formation" when plants are exposed to multiple factors of abiotic and biotic stress (Visser et al., 2015). The Intergovernmental Panel on Climate Change (IPCC) reported that the increase in rainfall as a result of climate change might have a greater impact on tropical regions as a result of the increased world hydrological cycle (Bailey-Serres and Voeseneck, 2008), generating waterlogging conditions as a result of heavy rains and poor drainage and gloomy weather because of the greater presence of clouds. This situation is aggravated on mixed plantations and can negatively affect photosynthesis and the performance of crops (Soleh et al., 2018). The lulo was classified as a species susceptible to waterlogging (Flórez-Velazco et al., 2015) and moderately susceptible to shaded conditions (Cardona et al., 2016).
In general, plants are often limited by a number of stressful factors that occur simultaneously, which makes it difficult to predict their geographical distribution based on physiological responses of an individual factor (Mittler, 2006; Fischer et al., 2016; Fischer and Melgarejo, 2020); thus, the effect of a combined stress on plant physiology is likely to be key to understanding the mechanisms of susceptibility to stress under natural field conditions (Casierra-Posada and Cutler, 2017). However, the potential effects of the stress combination may vary depending on the level of each of the individual stresses combined (e.g. severe vs. low) and the type of plant or pathogen involved (Mittler, 2006; Villarreal-Navarette et al., 2017; Jiménez et al., 2015).
Some physiological responses reported about the effects of stress from waterlogging and cloudy conditions suggest that both factors interact in such a way that the adverse effect of the shade is amplified in flooded soils (Laan et al., 1990). Waterlogging and shading can affect the growth of plants independently or interact in such a way that a factor reduces or increases the impact of additional factors (Urbas and Zobel, 2000).
The effects of abiotic stress caused by waterlogging and shading, mainly on growth and development factors, have been studied in Solanaceae species, such as the cape gooseberry (Physalis peruviana L.) by Aldana et al. (2014), tomatoes (Solanum lycopersicum L.) by Ezin et al. (2010) and Baracaldo et al. (2014) and lulos (Solanum quitoense) by Flórez-Velasco et al. (2015).
It has been shown that the decrease in chlorophyll content under stressful conditions can be considered a typical symptom of oxidative stress as a result of photooxidation of pigments and degradation of photosynthetic pigments (Anjum et al., 2011). Chlorophyll fluorescence, which indicates the photosynthetic efficiency of photosystem II (PSII) (Bansal et al., 2019), is a highly informative parameter of plant traits under adverse environmental conditions. Nowadays, chlorophyll fluorescence measurements are nearly universally used in ecophysiological plant studies, where, especially under natural and managed growth conditions, plant reactions can be easily understood when factors are changing (Hanelt, 2018).
Generally, environmental stress can be detected early through the quantification of chlorophyll fluorescence since this methodology uses information on the photochemical activity of plants (Marques et al., 2017). This is possible because the chlorophyll molecule is fluorescent and, through the dissipation of photons, changes in electron transfer at the level of chloroplast membranes can be detected (Do Nascimento and Marques, 2018). However, studies on the effects of the interaction of two abiotic stresses on the fluorescence in lulo plants are scarce.
Therefore, the objective was to evaluate the effects of two abiotic stresses (shading and waterlogging) and their interaction on physiological parameters such as fluorescence of chlorophyll, relative water content and electrolyte leakage in seedlings of lulo (Solanum quitoense) var. septentrionale.
MATERIALS AND METHODS
Plant material and experiment conditions
This study was conducted in a plastic greenhouse at the Faculty of Agricultural Sciences of the Universidad Nacional de Colombia, Bogota, located at 4°35'56'' N and 74°04'51'' W and at 2,556 m a.s.l.
The plant material was lulo seedlings (Solanum quitoense var. septentrionale) obtained from seeds, with an age of 10 weeks. The seedlings were transplanted into 5 L pots, which contained a substrate composed of quartzite sand and sieved soil at a ratio of 1:1 v/v. The substrate had the following physical and chemical properties: pH 5.2; organic carbon 6.33%; N 0.55%; Ca 6.21 meq/100 g; K 1.7 meq/100 g; Mg1.42 meq/100 g; Na 0.29 meq/100 g; P 37.2 mg kg-1; Cu 0.20 mg kg-1; Fe 47.0 mg kg-1; Mn 4.57 mg kg-1; Zn 1.45 mg kg-1; B 0.34 mg kg-1; AI 0.23 meq/100 g; CEC 9.94 meq/100 g; and a sandy loam texture (sand 72%, lime 17% and clay 11%).
Experiment design and treatments
A completely randomized design was carried out with a factorial arrangement, where the first factor consisted of the two levels of light conditions: a) without shading (WS); with an average noon radiation of 66,694±10,981 lux; b) shading (SH); with an average shading percentage of 65±4% (radiation of 29,935±3,428 lux), which was done through the installation of a black polyshade, 1.5 m high, covering the seedlings on all sides. The second factor corresponded to the four levels of waterlogging (WA) period (0, 3, 6 and 9 d). To do this, the drainage holes of the materials were closed hermetically, and, through irrigation, the water level was established at 3 cm above the substrate during the experiment. At the end of each period of waterlogging, the drainage holes of the pots were reopened to begin the recovery period. The experiment had a total of 8 treatments with three replicates for each one. Additionally, the response variables were recorded at 6, 12 and 18 d after the application of the waterlogging treatments began.
The mean air temperature for the two light conditions, with and without shading, was 25.3 and 25.6°C, respectively, while the mean relative humidity in the polyshade chamber was higher than that of without shading, with 61.2 and 25.7%, respectively.
Relative water content of leaves
The relative water content (RWC) was determined by extracting 1 cm diameter discs, taken from the middle area of the leaf blade of the second fully expanded leaf. The following equation was used to calculate the RWC
where, FW is the fresh weight, WT is the weight at turgidity, measured after 24 h of saturation in distilled water at 4°C in darkness and DW is the dry weight determined after 48 h of drying in an oven at 80°C.
Electrolyte leakage
The methodology described by Jiang and Zhang (2001) was followed to determine the electrolyte leakage. Ten discs, with a 0.5 mm diameter, were extracted from the middle area of the leaf blade from the second fully expanded leaf (counted from the apical part of the plant) in each treatment replicate. The discs were lightly washed with deionized water and placed in a test tube with 30 mL of deionized water. The tubes were subsequently incubated in a water bath (model B-480; Büchi Waterbath, Flawil, Switzerland) at 30°C for 2 h. The initial electrical conductivity (EQ) was measured with a conductimeter (Bench Meter, model P700; Oakton Instruments, Vernon Hills, IL). Subsequently, they were incubated again in the water bath, for 15 min at a temperature of 100°C, in order to extract all released electrolytes, and, with a conductivity meter, the final electrical conductivity (EC 2 ) was measured. The percentage of electrolytes was calculated with the following equation (2)
Chlorophyll content
The chlorophyll content was estimated using a chlorophyllometer (atLEAF, FT Green, Wilmington, DE). The readings were taken on the second fully expanded leaf, taking three data per replicate.
Fluorescence of chlorophyll a
The maximum quantum efficiency of photosystem II (PSII) (Fv/Fm), the effective photochemical quantum yield of PSII (YII), the photochemical quenching (qP) and the non-photochemical quenching (NPQ) were determined at 6, 12 and 18 DAOW, using a Modulated fluorescence chlorophyll meter (MINI-PAM, Walz, Effeltrich, Germany). The leaves were adapted to darkness with clips for 15 min. The maximal fluorescence (Fm) was estimated with a 0.8 s long saturating light pulse (2,600 ¿¿mol m-2 s-1), with 20,000 Hz frequency. The variable fluorescence (Fv) was estimated with the difference between F0 and Fm. The potential maximal PSII quantum yield (Fv/Fm ratio) was calculated from the Fv and Fm. The photochemical and non-photochemical quenching were assessed as qP = (Fm' - F)/(Fm' - F0), and NPQ = (Fm - Fm')/Fm' according to Schreiber et al. (1994).
RESULTS AND DISCUSSION
Relative water content (RWC) of leaves
The RWC expresses the percentage of water content and is relative to the turgidity or total saturation in a given tissue (Melgarejo, 2010). Significant differences were found in the waterlogging factor at the three evaluation moments (Tab. 1). The waterlogging period of 6 d and longer negatively affected the RWC (Tab. 2). Although at 12 and 18 d after the onset of waterlogging (DAOW) the plants in the 6 and 9 d waterlogging treatments had a recovery time (6 and 3 d at the first evaluation point; 12 and 9 d at the second evaluation point, respectively), the values were close to 55 and 65% of the RWC at the two evaluation points, respectively, and were significantly lower than the control treatment (about 80% RWC).
NS: non-significant; *significance level P≤0.05, **significance level P≤0.01, ***significance level P≤0.001.
Means with different letters indicate a significant statistical differences according to Tukey test (P≤0.05) (n=3 ± standard error); CV, coefficient of variation.
The reduction of the RWC after 6 d of waterlogging was possibly caused by the low absorption and transfer of water to the leaves, negatively affecting cellular turgor (Moreno and Fischer, 2014) as caused by anoxia conditions in the root system (Moreno et al., 2019), a situation that can damage numerous metabolism processes in plants (Yan et al., 2018). Similarly, six tree species in Bogota constantly decreased their RWC with an increase in waterlogged time between 0 and 28 d (Moreno et al., 2019).
The plants with up to 3 d of waterlogging (DWA) without any influence on the RWC or recovery time may have activated mechanisms that diminished the water potential in the plant cells through the synthesis of compatible osmolytes, such as proline, soluble sugars, or glycine betaine in order to maintain a water potential gradient that favored water intake (Oh and Komatsu, 2015). On the other hand, and according to Cardona et al. (2016), lulo plants waterlogged for 3 d do not suffer damage to their root system, so the absorption and transport of water in this organ is much greater and is related to the high RWC values compared to the 6 and 9 d waterlogged plants, which presented more serious damage, especially through the reduced diameter of the root neck.
Electrolyte leakage
For the loss of electrolytes, significant differences were found at the level of the shadingXwaterlogging interaction at the three sampling points (Fig. 1).
In general, it was observed that the treatments that did not have the additional shade (SH) presented the lowest values for electrolyte leakage. At 6 DAOW, the loss of electrolytes from the seedlings with shading maintained values close to 40%, while the seedlings without shading had values that were on average 35%. At 12 DAOW, the leakage of electrolytes from the seedlings with shadowy conditions was higher than 90%. Also, for the seedlings without shading with 0 d of waterlogging, the electrolyte leakage values were close to 60%, probably because of a mild dry air stress during this evaluation period (Sánchez-Reinoso et al., 2019). For the plants with some period of waterlogging stress, these values were close to 80%. At 18 DAOW the percentage of electrolyte leakage from the plants of the different SH treatments was close to 85%. However, the highest values found in the seedlings of the different WS treatments were observed in those that had 9 d of waterlogging, with about 60% electrolyte leakage, and the lowest values were evidenced in the seedlings with 0 d of waterlogging (about 40% electrolyte leakage). However, the treatment of the lulo seedlings with 9 d of waterlogging reached values of approximately 60% loss of electrolytes.
Obviously, the flooding stress was increased by the conditions of shading, increasing the leakage of electrolytes, which is related to oxidative stress (Moreno et al., 2019). Similar results were found by Bansal et al. (2019) in black beans, in which the membrane stability decreased as a result of the greater loss of electrolytes generated by the conditions of the lack of oxygen in the radical system (waterlogging for 10 d starting at 30 d after sowing), which was also manifested by cell membrane damage by ROS-induced peroxidation of membrane lipids (Andrade et al., 2018).
Chlorophyll content
For the chlorophyll content, significant differences were obtained in the shadingXwaterlogging interaction at the three sampling points (Fig. 2; Tab. 1). Particularly, the plants in the different shading treatments had lower chlorophyll contents than the shaded plants. Additionally, the chlorophyll values at 6 DAOW were, on average, ~ 50 at-LEAF units. In general, it was evident that, with the increase in the period of waterlogging, the reduction of chlorophyll content was greater. This reduction was more pronounced in the treatments with 6 and 9 d in the last two evaluation points when the plants were unshaded, which reached values close to 30 at-LEAF units. However, the SH plants with 9 d of waterlogging reached the lowest values at 18 DAOW (about ~35 at-LEAF units), followed by SH plants with 6 d of waterlogging (~ 40 at-LEAF units) at the same evaluation point.
A decrease in chlorophyll content with the duration of waterlogging stress has been observed in many species, such as the cape gooseberry (Aldana et al., 2014), tomatoes (Ezin et al., 2010; Baracaldo et al., 2014), black beans (Bansal et al., 2019), and cabbage (Casierra-Posada and Cutler, 2017), among others, which is manifested by foliar yellowing, followed by wilting, affecting photosynthesis (Wu et al., 2015) and consequently reducing the biomass of the lulo plants, such as with a decrease in leaf area and root volume (Cardona et al., 2016).
The lower reduction of chlorophyll content in the lulo plants under additional shade as waterlogging continued (Fig. 2) coincided with that observed by Mielke and Schaffer (2009) in the Solanaceae pitanga, taking into account that shaded leaves generally have a higher concentration of chlorophyll and more pigment molecules per electron transport chain than leaves in full light (Kadereit et al., 2014). However, Visser et al. (2015) found the opposite in Solanum dulcamara, which means that the flooded and shaded plants synthesized a smaller amount of chlorophyll than those waterlogged and in full light.
Chlorophyll fluorescence parameters
The results of the maximum quantum efficiency of PSII (Fv/Fm), effective photochemical quantum yield of PSII (YII), photochemical quenching (qP), and non-photochemical quenching (NPQ) were affected by the waterlogging (Fig. 3). In general, regardless of the duration of waterlogging or the shading condition, there was a tendency towards a reduction of the parameters Fv/Fm, Y(II), and qP. Additionally, the reduction was higher in the seedlings that were not shaded (NS), being lower in those that had 9 d of waterlogging (Fig. 3A, B and C). However, the NPQ tended to increase its values up to 40% with respect to the waterlogging condition, being greater when the lulo plants were exposed to a greater number of days (especially 6 and 9 d of waterlogging). However, at 6 DAOW, the highest values were found in the plants in shading conditions, but, as time progressed, the shaded plants had higher values (Fig. 3D).
For the reduction of the NPQ values at 12 and 18 DAOW, it is supposed that the lulo plants under shady conditions did not have stress conditions, with no need to dissipate energy in the form of heat since lulo plants are native to undergrowth areas (Bonnet and Cárdenas, 2012). This may be related mainly to the fact that the light compensation point may be lower than in other species grown with free exposure (Taiz et al., 2018).
A reduction in the maximum quantum efficiency of PSII, when the condition of waterlogging stress is increased, is common in many different plant species (e.g. black beans, Bansal et al., 2019; pitanga, Mielke and Schaffer, 2010; and several tree species, Moreno et al., 2019). Most likely, the decrease in Fv/Fm in the flooded lulo indicated that it adversely affected the photochemistry of photosynthesis in these stressed plants (Mielke and Schaffer, 2010), while Janowiak et al. (2002) observed tomato damage in the light-harvesting complex of PSII in waterlogged plants.
The strong decrease in the Fv/Fm ratio was especially evident in the plants with 9 d of waterlogging and 12 of recovery, with values below 0.7, which indicated severe stress that generated serious damage in PSII (Moreno et al., 2019) and showed, according to Ashraf (2012), the inability of the plants to regenerate Rubisco under these stressful conditions. In the other treatments, with levels that exceeded 0.7, the functioning of PSII was not impaired (Bansal et al., 2019); rather, there was a dynamic photoinhibition, without presenting real damage to the photosystems (Moreno et al., 2019).
The waterlogged and shaded lulo showed a lower reduction in the maximum efficiency of PSII than the plants at full light, a result that Mielke and Schaffer (2010) also observed in pitanga. Supposedly, a higher concentration of chlorophyll, larger grana and a higher proportion of PSII compared to PSI in shaded leaves (Kadereit et al., 2014) led to the increase in Fv/Fm in the lulo.
The effective photochemical quantum yield of PSII (YII) indicates the part of the energy absorbed by chlorophyll, which is associated with PSII and is used in the photochemical activity, evidencing the sum of transported electrons and, thus, becomes an indicator for photosynthesis (Lichtenthaler et al., 2005; Jiménez-Suancha et al., 2015). Overall, the Yn of the waterlogged lulo plants decreased with the increased stress, as Else et al. (2009) observed in tomatoes. With the increase in recovery time (at 12 and 18 DAOW), this stress was reduced.
Photochemical quenching (qP) quantifies the photochemical capacity of PSII and refers to the proportion of excitation energy trapped by open PSII reaction centers (Lichtenthaler et al., 2005; Hanelt, 2018). While non-photochemical quenching (NPQ) indicates the activation of non-photochemical processes that lead to the dissipation of non-radiant energy, such as changes in the transthylacoidal pH gradient, photoinhibition, interruption of light collection complexes, and formation of zeaxanthin, among others (Jiménez-Suancha et al., 2015; Rohacek, 2002).
The reduction of the qP value in all treatments, especially between 0 and 3 d of waterlogging and also in the comparison between the 6 and 9 d waterlogged plants, indicated that the plants were under stress. Casierra-Posada and Cutler (2017) and Wu et al. (2015) observed a reduction of photochemical dissipation in Triticum aestivum as the result of a reduction in the efficiency of the excited energy collected in the open photosystem II centers. In the lulo, it was observed that the plants tried to tolerate waterlogging in a specific way when they were stressed for 6 d, as seen in the increases in the qP and YII values, mainly in the shading treatments (Fig. 3B and C), but, in the end, they could not resist, as the results showed at 9 DWA, at recovery times of 12 and 18 DAOW The increase in the non-photochemical dissipation with the increase in the stress duration was observed in the lulo only when the recovery of the plants occurred for 6 d (Fig. 3D), an effect that occurred in tomatoes (Else et al., 2009) and manifested mainly in plants under full light (Waldhoff et al., 2002); this situation did not occur in the plants with recovery periods of 12 and 18 d.
Interestingly, the qP values were near 0.8 at 0 DWA on 12DAOW; presumedly, the low radiation accompanied by a mild dry air stress condition that occurred in the evaluation period resulted in this value (Sánchez-Reinoso et al., 2019).
CONCLUSIONS
In the lulo seedlings, the effect of waterlogging and shading was especially observed in the treatments evaluated at 12 and 18 d after the onset of the treatments, in which a lower light intensity (shade) favored the chlorophyll content and the maximum and real efficiency of photosystem II, but also increased the electrolyte leakage.
The lack of water absorption and translocation that resulted from the waterlogging of the root system reduced the relative water content in the leaves, especially in the plants with a longer waterlogging time (6 and 9 d) and observation (12 and 18 d).
For the chlorophyll a fluorescence, the measurements of the maximum quantum efficiency of PSII (Fv/Fm), the effective photochemical quantum yield of PSII (YII), and the photochemical quenching (qP) proved to be a good indicator of the effects of this double stress on the lulo seedlings.