Screening Genipa americana Progenies for Their Ability to Maintain Leaf Vitality Under Severe Dehydration Using Chlorophyll Fluorescence ^†

Abstract

Damage to the photosynthetic apparatus during leaf dehydration is an indicator of the maintenance of leaf vitality and the resilience of tree seedlings to severe drought. Genipa americana is a tree widely distributed in the neotropical region but with great ecological and sociocultural importance in the south of the state of Bahia, Brazil, where its fruits are harvested from subspontaneous trees. This study aimed to compare the feasibility of the maximum quantum efficiency of photosystem II (Fv/Fm) and performance indexes derived from the JIP test, i.e., performance index on absorption basis (PI_abs) and total performance index (PI_total), for screening G. americana seedlings from different mother plants for leaf damage caused by dehydration. From leaf dehydration curves, we calculated the values of relative water content (RWC) in which Fv/Fm, PI_abs, and PI_total reach a loss of 10% and 50% in relation to the values of fully hydrated leaves. PI_total was the only parameter that revealed consistent significant differences between progenies for RWC at 50% of percentage loss. Significant differences were observed among progenies for leaf traits; however, no correlation was detected between these traits and chlorophyll fluorescence parameters. Monitoring the PI_total values during leaf dehydration is a useful tool for screening G. americana progenies in relation to their capacity to maintain leaf vitality under occasional severe droughts.

1. Introduction

Tropical trees have been impacted by sudden changes in temperature and water constraints, induced by global climate change [1], with negative impacts on biodiversity and forest ecosystem services worldwide [2]. Rising average surface temperatures on Earth have contributed to an increase in the frequency and intensity of extreme weather events. One key driver of these changes is the El Niño–Southern Oscillation (ENSO), a climate phenomenon involving periodic variations in ocean temperatures in the central and eastern tropical Pacific. These temperature fluctuations significantly influence global weather patterns and can lead to severe droughts and floods, particularly in tropical regions [3,4]. With the increased frequency and intensity of extreme events, many tropical rainforests are expected to be impacted by the increased occurrence of droughts [5]. For example, studying eight tropical forests in central Panama, [6] reported that the 2014/16 ENSO increased seedling mortality by an average of 11%, with a range of about 2% to 21%. Also, during the last extreme 2014/15 ENSO, the cocoa agroforests in southern Bahia, Brazil, were extremely impacted by the drought, causing an average cocoa tree mortality of 15% and a decrease in cocoa production of 89% [7].

Tree planting has been suggested as a way to mitigate global climate change [8]. However, the increased occurrence of extreme climatic events, such as occasional severe droughts, is an imminent challenge, as it may affect the survival and growth of seedlings after planting. The initial establishment of tree seedlings is a critical phase, especially in the first post-planting year [9]. In recent decades, studies have shown that many tree seedlings are already exposed to upper limits of climatic extremes and resource limitations in planting environments, representing a great challenge for ecological restoration and plantations of tree crops in agroecosystems [10,11]. Compared to adult trees, seedlings have a smaller volume of roots and a smaller stock of carbon reserves. Thus, the seedling phase, which corresponds to the period of full establishment of the new plant, is the phase of greatest vulnerability due to the effects of stress factors, such as unexpected severe droughts [12,13]. Because tree seedlings typically have few leaves, maintaining leaf vitality under low relative water content is important for their survival during extreme drought, as well as for recovery after periods of drought stress. Thus, knowing the limits of maintaining leaf vitality in conditions of low relative water content is important for the selection of tree species [14] or progenies of different mother trees in order to increase the success of plantings in sites prone to occasional severe droughts.

The analysis of chlorophyll fluorescence emission by portable equipment is a non-destructive and fast technique, considered very useful for analyzing the photochemical efficiency of plants under abiotic stress [15]. The maximum quantum yield of photosystem II, or the ratio between variable and maximum fluorescence emission (Fv/Fm), is one of the most applied parameters in plant ecophysiological studies to assess environmental stress in plants, being considered an excellent indicator of photochemical efficiency and leaf vitality [16,17], including water deficit [18,19]. Recent studies have demonstrated that the chlorophyll fluorescence emission technique can be a useful tool to assess the vitality of leaves under continuous dehydration and, consequently, to screen tree species in relation to their capacity to survive periods of unexpected severe droughts [14,20]. Although [20] reported that the relative water content (RWC) value at which the Fv/Fm ratio reaches 50% of its initial value is lower than the limiting leaf rehydration capacity for plant species with different tolerances to water deficit, [14] demonstrated that drops in the Fv/Fm ratio in relation to leaf dehydration were effective in separating tree seedlings from nine tropical species due to their susceptibility to severe leaf dehydration.

The fast phase of chlorophyll fluorescence emission is called OJIP and reflects the photochemical processes associated with photosystem II (PSII) and information about the photosynthetic electron transport chain integrity of the photosynthetic electron transport chain [21,22,23]. The OJIP curve is analyzed by the JIP test, which quantifies the characteristics of the structure and vitality of photosystems II (PSII) and I (PSI) [21,24]. Different parameters can be derived from the JIP test, such as performance indices based on absorption and energy conservation (PI_abs and PI_total), respectively [21]. These indices have been used to effectively quantify photosynthetic sensitivity to various abiotic stresses [17,21,25]. This is because PI_abs and PI_total integrate the energy conservation for electron transport rate outside the PSII, being potentially more sensitive than other fluorescence parameters used in most studies, such as Fv/Fm [21]. The PI_abs and PI_total performance indices have been used as indicators of drought stress in plants [26,27] and for screening tolerant genotypes [18,28,29]. Despite this, we are unaware of published studies on the impacts of continuous leaf dehydration on these JIP test parameters in seedlings of tropical trees, especially in progenies originating from native and sub-spontaneous mother trees.

Genipa americana (Rubiaceae) is a neotropical fruit tree with a wide territorial distribution, being found in the tropical forests of South and Central America [15]. The fruit of the species is popularly known as “Jenipapo” and has a high iron content. Furthermore, in its initial state of maturation, the fruit is an excellent natural source of genipin [30]. The species is recognized for its valuable wood and for its ecological role in tropical ecosystems, being used as food for many animals, and mainly for its tolerance to soil flooding [31]. It has great sociocultural importance in the south of the state of Bahia, Brazil, a tropical region with rainfall well distributed throughout the year, where its fruits are harvested from sub-spontaneous trees [32]. In addition, G. americana is a rustic species, presenting fast growth and high acclimatization to different environmental conditions [32,33]. These characteristics contribute to the tree being considered an essential tree crop in tropical agroecosystems and indicated for silvicultural management in the Brazilian Atlantic Forest biome [34]. However, although G. americana is a species widely recognized for its tolerance to flooding, information about its tolerance to extreme severe drought is still scarce, and it is an interesting model species to test the use of different chlorophyll fluorescence variables to screen progenies in relation to their capacity for leaf vitality maintenance under unexpected severe droughts.

This study aimed to compare the feasibility of the maximum quantum efficiency of photosystem II (Fv/Fm) and variables from the JIP test (PI_abs and PI_total) for screening G. americana seedlings originating from different mother plants to their capacity for leaf vitality maintenance under leaf dehydration. As PIabs and PItotal integrate more steps of the electron transport chain and are potentially more sensitive than the Fv/Fm ratio in detecting environmental stress in plants, we tested the hypothesis that these derived JIP test parameters are more efficient than the Fv/Fm ratio for screening seedlings of G. americana progenies in relation to their leaf vitality maintenance under unexpected severe droughts. We believe that this is a simple methodology that can contribute to advances in the process of selecting mother trees, aiming at the production of seedlings with greater resilience to extreme climate events in a context of tree planting under climate threats.

2. Materials and Methods

2.1. Plant Material

The seeds of G. americana used to produce the seedlings were obtained from the fruits of twelve mother plants growing spontaneously in the southern region of Bahia, Brazil. To ensure genetic diversity, the fruits were collected from mother trees located at a minimum distance of 30 m from each sampling individual. According to the Köppen classification [33], the climate in the region is humid tropical with an average monthly temperature of 24–26 °C and annual precipitation greater than 1.500 mm. Fruits were collected from farms within an altitude gradient of 29–400 masl, and only healthy, fully ripened fruits were used. The seedlings were cultivated in containers measuring 21.0 cm in height × 17 cm in diameter with a volume of 5.0 L, in the nursery of the State University of Santa Cruz (UESC), located in Ilhéus, BA, Brazil (39°13′59″ W; 14°45′15″ S). After a growth period of 198 days, analyses of leaf dehydration essays were carried out.

2.2. Leaf Dehydration Essays and Fluorescence Parameters

The methodology used during the leaf dehydration essays was adapted from the methodologies proposed by [14,20]. The leaf dehydration curves were assessed through two independent essays. Initially, leaf dehydration curves were assessed in progenies from six mother plants, always considering nine seedlings per mother plant, totaling 54 seedlings. Before starting the leaf dehydration curves, always at the end of the day, before each test, nine well-irrigated plants of G. americana were taken to the laboratory. In the laboratory, the seedlings were sprayed with water and wrapped in black plastic bags. The environmental conditions of the laboratory for the foliar dehydration experiments were rigorously controlled, maintaining the ambient temperature between 23 and 25 °C and the relative humidity between 65% and 70%. Chlorophyll fluorescence was quantified using a Pocket PEA portable fluorometer (Hansatech Instruments, King’s Lynn, UK). To ensure complete oxidation of the photosystem II (PSII) electron transport chain, one leaf per plant was selected and kept in the dark for a minimum period of 30 min using a specific clip. After this period, the leaves were exposed to a saturating light pulse (3500 μmol photons m⁻² s⁻¹, wavelength of 650 nm, duration of 1 s). Subsequently, the recorded transient fluorescence data were analyzed through the JIP test, allowing the calculation of maximum quantum efficiency of PSII (Fv/Fm) and the potential performance indexes for energy conservation from exciton to the reduction of intersystem electron acceptors (PI_abs) and for energy conservation from exciton to the reduction of PSI end acceptors (PI_total). The Fv/Fm ratio was calculated by the formula Fv/Fm = (Fm − F₀)/Fm, whereas PI_abs and PI_total were calculated according to the formulas proposed by [25]: PI_abs = (RC/ABS)·(TR₀/DI₀)·[ET₀/(TR₀ − ET₀)] and PI_total = PIabs·[(RE₀/ET₀)/(1 − RE₀/ET₀)]; where RC/ABS is the density of active reaction centers, TR₀/DI₀ is the ratio between the energy capture and dissipation flux, and (ET₀)/(TR₀ − ET₀) is the efficiency of electron transport after the primary acceptor QA⁻.

After the initial measurement of chlorophyll fluorescence, the leaves were detached and digitized using a flatbed scanner. In addition, measurements of leaf thickness (LT) were carried out, and the leaves were weighed to obtain the saturated fresh mass (LMs). From the digitalized images, the individual leaf area (LA) was measured using the ImageJ software version 1.54g [35]. The LT was measured with an analog micrometer (Zaas Precision, São Paulo, Brazil). After these procedures, the leaves were placed under the laboratory bench and weighed at intervals of 0, 3, 6, 10, 24, 32, 48, 52, 58, 72, 76, 82, and 96 h to obtain the fresh mass during dehydration (LMde). These time intervals were established from previous tests. Right after the leaves were weighed, the chlorophyll fluorescence emission was measured. At the end of 96 h of leaf dehydration, the leaves were placed separately in a forced air circulation oven at 60 °C to obtain the dry mass of individual leaves (LMd). The leaf mass per area (LMA) was calculated using the formula LMA = LMd/LA. To determine the relative water content (RWC), the values of LMs, LMde, and LMd were used for each measurement time, through the formula RWC = (LMde − LMd)/(LMs − LMd). The percentage loss for photosystem II maximum quantum yield on dehydration (PL[Fv/Fm]) were calculated using the formula PL[Fv/Fm] = 100 × (1 − [Fv/Fm]de/[Fv/Fm]s); where [Fv/Fm] is the value of the Fv/Fm ratio at each LMde value and [Fv/Fm]s is the value of the Fv/Fm ratio of the leaf in water saturation, which corresponds to the fresh saturated mass of the individual leaf before dehydration (LMs). Likewise, this same formula was used for the performance index on an absorption basis (PL[PI_abs]), in this case PL[PI_abs] = 100 × (1 − [PI_abs]de/[PI_abs]s), and for the performance index for energy conservation (PL[PI_total]), in this case PL[PI_total]= 100 × (1 − [PI_total]de/[PI_total]s).

Subsequently, a second essay was conducted using the best chlorophyll fluorescence parameter and the time at which this parameter reaches 50% in relation to time zero (0 h). For this, we used the same six progenies used for the first essay, in addition to six more independent progenies (twelve mother plants and 108 seedlings in total, one leaf per plant).

2.3. Data Analysis

For data analysis in this study, initially, six progenies were named from A to E. The relative water content (RWC) for percentage loss of 10% and 50% of Fv/Fm, PI_abs, and PI_total for each seedling was determined by fitting the relationships between percentage loss (PL) of the parameters over decreasing RWC and using the best fit of three differently shaped functions, as reported by [14]: linear, $PL=b·(100-RWC)$; exponential, $PL=a·e^{\left(-b·RWC\right)}$; and sigmoidal, $PL=a/\left\{1+e^{\left[-\left(RWC-X_0\right)/b\right]}\right\}$ (Table S1, Supplementary Materials). All functions were compared using maximum likelihood, and the best fit was selected according to the lowest Akaike information criterion corrected for small n [36]. The chlorophyll fluorescence parameters derived from the leaf dehydration curves and time course (

Table 1. Abbreviations, definitions, and units of all variables measured.

Abbreviature	Definition	Units
Chlorophyll fluorescence’s parameters derived
Fv/Fm	Maximum quantum yield of photochemical energy conversion in photosystem II	Unitless
PIabs	Performance index (potential) for energy conservation from photons absorbed by PSII to the reduction of intersystem electron acceptors	Unitless
PItotal	Performance index (potential) for energy conservation from photons absorbed by PSII to the reduction of PSI end electron acceptors	Unitless
PL[Fv/Fm]	Percentage loss of Fv/Fm	%
PL[PIabs]	Percentage loss of PIabs	%
PL[PItotal]	Percentage loss of PItotal	%
PL[Fv/Fm]10	PL[Fv/Fm] at 10%	%
PL[Fv/Fm]50	PL[Fv/Fm] at 50%	%
PL[PIabs]10	PL[PIabs] at 10%	%
PL[PIabs]50	PL[PIabs] at 50%	%
PL[PItotal]10	PL[PItotal] at 10%	%
PL[PItotal]50	PL[PItotal] at 50%	%
Parameters derived from the response of chlorophyll fluorescence to loss of relative water content (RWC) and time-course
RWCPL[Fv/Fm]10	RWC at percentage loss of Fv/Fm at 10%	%
RWCPL[Fv/Fm]50	RWC at percentage loss of Fv/Fm at 50%	%
RWCPL[PIabs]10	RWC at percentage loss of PIabs at 10%	%
RWCPL[PIabs]50	RWC at percentage loss of PIabs at 50%	%
RWCPL[PItotal]10	RWC at percentage loss of PItotal at 10%	%
RWCPL[PItotal]50	RWC at percentage loss of PItotal at 50%	%
Time_RWCPL[Fv/Fm]10	Time at RWCPL[Fv/Fm]10	hour
Time_RWCPL[Fv/Fm]50	Time at RWCPL[Fv/Fm]50	hour
Time_RWCPL[PIabs]10	Time at RWCPL[PIabs]10	hour
Time_RWCPL[PIabs]50	Time at RWCPL[PIabs]50	hour
Time_RWCPL[PItotal]10	Time at RWCPL[PItotal]10	hour
Time_RWCPL[PItotal]50	Time at RWCPL[PItotal]50	hour
Leaf traits
LT	Leaf thickness	μm
LS	Leaf succulence	g m−2
LMA	Leaf mass per area	g m−2
ILA	Individual leaf area	cm2

) were compared with Fisher’s one-way ANOVA or Kruskal–Wallis, and post-hoc Tukey’s Honest Significant Difference test (Tukey HSDs test for ANOVA) or Least Significant Difference test (LSDs test for Kruskal–Wallis). After this, the data of leaf traits and the best chlorophyll fluorescence parameter(s) for screening leaf dehydration tolerance of twelve progenies, named from A to L, were submitted to analysis of variance (one-way ANOVA), followed by the Scott–Knott test (p < 0.05). Spearman’s correlation coefficients of bivariate cross-correlations of leaf trait variables with RWC and the best chlorophyll fluorescence parameter in the first essay were also calculated. All statistical analyses were performed with the programming language R, version 4.1.0 [37]. with a level of significance of α = 0.05.

3. Results

The chlorophyll fluorescence parameter most sensitive to leaf dehydration was PL_[PItotal] (90.0–91.0% and 50.1–55.2% RWC for PL_[PItotal] at 10% and 50%, respectively; Figure 1,

Table 2. Model of best fit between the parameters of percentage loss of chlorophyll fluorescence (F_v/F_m, PI_abs, and PI_total) and relative water content (RWC) for each of the G. americana progenies.

Progenies	Model	R2	AICc	Pm
Percentage loss of Fv/Fm
A	87.4 · e (−0.093 RWC)	0.82	866.1	***
B	96.4 · e (−0.065 RWC)	0.78	947.9	***
C	157.9 · e (−0.125 RWC)	0.80	959.3	***
D	180.0 · e (−0.109 RWC)	0.88	730.1	***
E	105.6 · e (−0.068 RWC)	0.78	974.5	***
F	137.8 · e (−0.097 RWC)	0.82	777.1	***
Percentage loss of PIabs
A	0.893 · (100 − RWC)	0.84	794.2	***
B	0.942 · (100 − RWC)	0.73	986.1	***
C	0.938 · (100 − RWC)	0.87	646.4	***
D	0.997 · (100 − RWC)	0.85	665.7	***
E	1.005 · (100 − RWC)	0.82	569.2	***
F	1.018 · (100 − RWC)	0.88	653.9	***
Percentage loss of PItotal
A	1.002 · (100 − RWC)	0.90	988.1	***
B	1.029 · (100 − RWC)	0.84	1033.3	***
C	1.061 · (100 − RWC)	0.89	825.7	***
D	1.091 · (100 − RWC)	0.84	777.9	***
E	1.110 · (100 − RWC)	0.87	860.7	***
F	1.102 · (100 − RWC)	0.89	944.7	***

R²: coefficient of determination; AIC_c: Akaike Information Criterion corrected; P_m: model probability; ***: p < 0.001. See abbreviation in Table 1.

), followed by PL_[PIabs] (88.7–90.1% and 43.7–50.6% RWC), and finally PL_[Fv/Fm] (18.9–27.5% and 7.1–13.8% RWC). As for the parameters of chlorophyll fluorescence response to RWC loss and time course (

Table 3. Summary of the ANOVA of the comparison among G. americana progenies for parameters derived from the response of chlorophyll fluorescence to loss of relative water content (RWC) and time-course and leaf trait variables.

Variables (Abbreviature)	F or KW β	R2	Pm
Parameters from the response of chlorophyll fluorescence to loss of RWC and time-course γ
RWCPL[Fv/Fm]10	8.53 β	-	n.s.
RWCPL[Fv/Fm]50	10.87 β	-	n.s.
RWCPL[PIabs]10	9.77 β	-	n.s.
RWCPL[PIabs]50	9.75 β	-	n.s.
RWCPL[PItotal]10	9.21 β	-	n.s.
RWCPL[PItotal]50	15.31 β	-	**
Time_RWCPL[Fv/Fm]10	14.05 β	-	*
Time_RWCPL[Fv/Fm]50	4.71	0.29	**
Time_RWCPL[PIabs]10	9.37 β	-	n.s.
Time_RWCPL[PIabs]50	8.61 β	-	n.s.
Time_RWCPL[PItotal]10	9.71 β	-	n.s.
Time_RWCPL[PItotal]50	10.50 β	-	n.s.
Leaf traits
Individual leaf area (ILA)	4.41	0.24	**
Leaf thickness (LT)	6.07	0.32	***
Leaf mass per area (LMA)	8.9 β	-	n.s.
Leaf succulence (LS)	2.73	0.14	*

F: Fisher coefficient’s value; KW: Kruskal–Wallis’s test (^β); R²: coefficient of determination; P_m: model probability; n.s.: not significant; *: p < 0.05; **: p < 0.01; and ***: p < 0.001. ^γ: See abbreviation in Table 1.

), overall RWC_PL[Fv/Fm] and RWC_PL[PIabs] at 10% and 50% showed no significant differences between progenies (Figure 2A–C), but RWC_PL[PItotal] at 50% presented significant differences between progenies (Figure 2E), where progeny A showed the highest resistance to dehydration and the lowest progeny E; Time_RWC_PL[Fv/Fm] at 10% and 50% showed significant differences between progenies (Figure 2B), but Time_RWC_PL[PIabs] and Time_RWC_PL[PItotal] at 10% and 50% did not exhibit differences (Figure 2D–F).

Although the progenies came from seeds collected from mother plants of the same region, significant differences among progenies for the leaf traits were observed (

Table 4. Mean values comparing the variables of leaf characters for RWC and PL_[PItotal] in the percentage loss of 24 h for twelve progenies of G. americana.

		Variables
Progenies	LA	LT	LS	LMA	RWC24h	PL[PItotal]24h
A	420.77 a	20.44 b	3.88 a	58.91 a	53.65 a	57.22 a
B	428.88 a	23.00 a	3.77 a	44.44 a	56.10 a	57.24 a
C	358.33 a	22.11 a	3.11 a	57.83 a	50.21 b	64.39 a
D	383.00 a	24.33 a	3.82 a	49.51 a	58.78 a	57.55 a
E	373.44 a	22.11 a	3.68 a	51.59 a	55.88 a	54.87 a
F	390.66 a	24.00 a	3.50 a	48.16 b	53.50 a	54.18 a
G	350.88 a	21.55 b	2.74 b	44.79 b	53.87 a	48.51 b
H	346.55 a	20.22 b	3.21 a	52.17 a	52.32 a	51.21 b
I	412.88 a	21.55 b	3.40 a	51.20 a	55.10 a	50.69 b
J	385.55 a	19.44 b	2.46 b	40.18 b	50.70 b	57.14 a
K	343.33 a	19.88 b	2.65 b	45.75 b	45.87 b	57.86 a
L	323.88 a	20.66 b	2.48 b	41.76 b	52.97 a	48.87 b

Equal letters indicate no statistical difference between the progenies of G. americana (p > 0.05, Scott–Knott test). (n = 108). See abbreviation in Table 1.

). All leaf traits showed significant differences (LT, LS, and LMA), except for ILA Progeny A stood out with the highest LMA and highest LS. In addition, ten progenies had an average value of PL[PI_total]_24h above 50% as a function of RWC, except for progenies G and L, PL[PI_total]_24h, around 49% (Table 4). In addition, no correlation was observed between the chlorophyll fluorescence parameters and leaf trait variables (

Table 5. Correlations between all measured variables of G. americana progenies, leaf trait variables, and parameters derived from RWC and PL[_PItotal] at 24 h percentage loss.

	Leaf Traits Variables
Parameters	LA	LT	LMA	LS
RWC24h	0.04	0.07	−0.00	0.21 *
PL[PItotal]24h	0.06	−0.05	0.03	−0.04

Spearman’s correlation coefficients of bivariate cross-correlations (n = 108). Bold values indicate significant correlations (*: p < 0.05). See abbreviation in Table 1.

). Except for RWC_24h, the only parameter correlated with leaf succulence (LS; r_s = 0.21) (Table 5).

4. Discussion

Global climate change has already led to an increase in extreme weather events, such as heat waves and extreme occasional droughts in tropical regions [4]. Although still under debate, tree death caused by severe drought may occur due to two main mechanisms, that is, by hydraulic failure (when the loss of water through transpiration exceeds the absorption of water by the roots) and carbon starvation (when the stomata close and leaves fall to prevent excessive water loss, resulting in the depletion of carbon reserves necessary to maintain vital physiological processes) [1]. In this study, we compared the capacity of seedlings of a neotropical tree from different mother plants to maintain leaf vitality during dehydration. As observed, the decline in the RWC had a strong influence on the performance indexes, in which the percentages of loss for 10% and 50% of PI_abs and PI_total were obtained at RWC values much higher than those of Fv/Fm. The tree seedlings are more vulnerable to unexpected severe droughts than adult individuals, as their roots have less capacity to exploit soil resources and have less capacity to store carbon reserves. Under such conditions, maintaining leaf vitality under low water content is important for the resumption of vital functions after periods of unexpected severe droughts.

Chlorophyll fluorescence emission is a physicochemical process that occurs in all photosynthetic organisms in an extremely fast time step [17]. So, the analysis of chlorophyll fluorescence parameters allows very rapid and highly accurate inferences to be made about the conversion of physical light energy into chemical energy, as well as the integrity of the photosynthetic electron transport chain [24]. The Fv/Fm ratio is a classic chlorophyll fluorescence parameter related to the transformation of the physical energy of light into chemical energy, indicating the probability of the energy of an absorbed photon reaching the reaction center and triggering a photochemical event [38]. Although it has been well established that the Fv/Fm ratio is an important indicator of environmental stress in plants for a long time [39,40]. It has been reported that this quantitative parameter is not sensitive enough to detect physiological changes to mild water stress [22]. Furthermore, with technological advances in the manufacture of portable fluorescence meters, the JIP test, which corresponds to a series of time steps on the curve between the initial (F₀) and maximum (Fm) fluorescence emissions, has allowed a more detailed and efficient assessment of how environmental stress affects the performance of fundamental processes related to the photochemistry of photosynthesis [38]. Thus, other chlorophyll fluorescence variables, originating from the JIP test, have been considered more efficient in detecting the response of plants to water stress, such as performance indices (PI_abs and PI_total) used in this study [16,22,23,41]. The two performance indices used in this study integrate a series of events related to the conservation of the energy absorbed in the antenna pigments of PSII until the reduction of electron acceptors after PSII (PI_abs) and after PSI (PI_total) [22]. Because the JIP curve performance indexes integrate several stages of the chloroplast electron transport chain, several studies have considered PI_abs and PI_total as the best predictors to measure photosynthetic efficiency [21,22]. Thus, any alteration in terms of the PI_abs and PI_total components can be easily observed, mainly in the most sensitive performance index. In some cases, PI_total is the most prevalent and most sensitive parameter, as it reflects changes in photosynthetic electron transport activity outside the PSII [42].

Our results indicated that PI_total was the most responsive parameter related to fluorescence emission in dehydrated leaves of different G. americana progenies. Our results suggest a possible limitation of the electron transport chain at the PSI level [43], indicating that leaf dehydration negatively affected the photochemical efficiency of the progenies. Previous studies have also shown similar trends regarding leaf dehydration tolerance for different genotypes [36]. Although we observed that there were no significant differences between progenies for RWC at PL_[PItotal]10, it was shown that there were consistent significant differences between progenies for RWC at PL_[PItotal]50. An explanation for this pattern is related to the linear regression model, since it considers the intercept at zero (at the origin). The values of all progenies are very close in 10%; that is, in 10% it was still not possible to observe changes. On the other hand, we observed that statistical differences between progenies increased as RWC decreased. This finding is consistent with previous studies on tolerance to leaf dehydration, which showed important declines in RWC and loss of photochemical efficiency in tree species [14,20,44].

In the context of global climate change, the increase in average temperature with consequent changes in the water regime brings with it extreme ecological filters that can limit the survival and initial growth of many forest species. Water stress reduces the ability of newly planted seedlings to fix their roots in the soil, with serious impacts on nutrient and water absorption, being even more marked in the first months after planting [45]. In this study, we used G. americana as a model species to compare the tolerance to leaf vitality during dehydration of seedlings from different mother trees. Even considering a single species, our results were accurate in indicating that the PI_total parameter was the best variable related to the emission of chlorophyll fluorescence capable of separating the progenies. The methodology proposed was effective for the fast screening of native forest trees in relation to leaf dehydration using chlorophyll fluorescence (PI_total). We suggest that it can be applied to other tropical tree species as an alternative to reduce seedling mortality after planting in the field. In addition, considering that the increased occurrence of heat waves is one of the main effects of global climate change on plant physiological performance [12,46], we suggest the development of new studies aiming to simultaneously test the effects of high temperatures and leaf dehydration on chlorophyll fluorescence parameters.

5. Conclusions

In summary, the results of Fv/Fm and PI_abs showed no consistent significant differences between progenies, while PI_total was the only parameter that revealed consistent significant differences between progenies for RWC at 50% of percentage loss. Among the chlorophyll fluorescence parameters tested, the PI_total was the best for screening G. americana genotypes in relation to photosynthetic damage caused by leaf dehydration in the seedlings’ ontogenetic stage. Although other approaches at leaf and whole plant scales are highly related to drought tolerance, monitoring the PI_total values during leaf dehydration is a useful tool for screening G. americana progenies in relation to their capacity to maintain leaf vitality under occasional severe droughts. From a practical point of view, considering ecosystems more prone to the occurrence of severe droughts, or even areas with soils with low water retention capacity, studies comparing the leaf dehydration tolerance of seedlings of tree species in the initial stage of growth can be useful to assist in the selection of mother trees for the production of high-quality seedlings for planting in tropical agroecosystems or forest restoration.