*2.4. Statistical Analysis*

Data were subjected to analysis of variance using Genstat 20th edition (VSN International, Hempstead, UK). The mean data for the two seasons were combined for analysis. Means were separated using Fisher's protected least significant difference (LSD) test at the 5% significance level. Pearson's correlation coefficients were calculated using IBM SPSS Statistics 25.0 (SPSS Inc., Chicago, IL, USA) to determine the magnitude of the relationship among physiological traits. Principal component analysis (PCA) based on a correlation matrix was used to identify influential traits under NS and DS conditions using R Studio version 4.0, ggplot2 (R Core Team, 2018). Biplots were built using XLSTAT to determine relationships among the accessions and response variables (physiological traits). Principal component biplot diagrams were used to identify drought-tolerant and drought-susceptible okra accessions using XLSTAT. ClustVis (https://biit.cs.ut.ee/clustvis\_large (accessed on 23 November 2022)) was used to visualise the heatmap analysis of physiological traits.

#### **3. Results**

#### *3.1. Leaf Gas Exchange and Chlorophyll Fluorescence Parameters in Response to Drought*

The effects of genotype, water regime and interaction of genotype × water regime were significantly different for most evaluated traits of leaf gas exchange and chlorophyll fluorescence (Table 2). Drought stress significantly reduced gs, A and A/C*i* among the evaluated accessions (Tables 3 and 4). Accessions LS02, LS09, LS10, LS17, LS19 and LS26 recorded *gs* values of >0.3 mmol m−<sup>2</sup> s−<sup>1</sup> under NS conditions. Under DS, accessions LS04, LS11, LS13 and LS20 recorded *gs* values <0.1 μmol m−<sup>2</sup> s<sup>−</sup>1. Regarding T, accessions LS03, LS13, LS15, LS19, LS23 and LS24 recorded values ≥ 7.01 mmol H2O m−<sup>2</sup> <sup>s</sup>−<sup>1</sup> under NS conditions, while, under DS conditions, genotypes LS01, LS03, LS04, LS08, LS09, LS11, LS12, LS14, LS19 and LS22 recorded T values ≤ 1.00 mmol H2O m−<sup>1</sup> <sup>s</sup>−1. Under NS conditions, A values of ≥ <sup>30</sup> <sup>μ</sup>mol CO2 <sup>m</sup>−<sup>2</sup> <sup>s</sup>−<sup>1</sup> were observed from accessions LS08, LS10 and LS21, while values ≤ <sup>20</sup> <sup>μ</sup>mol CO2 <sup>m</sup>−<sup>2</sup> <sup>s</sup>−<sup>1</sup> were recorded for accessions LS03 and LS06.


**Table 2.** Analysis of variance indicating mean squares and significant tests of leaf gas exchange and chlorophyll fluorescence parameters of 26 okra genotypes evaluated under non-stress and drought-stress conditions averaged across two seasons.

d.f.: degree of freedom, *gs*: stomatal conductance, *T*: transpiration rate, *A*: net CO2 assimilation, *Ci*: intercellular CO2 concentration, *A*/*Ci*: CO2 assimilation rate/intercellular CO2 concentration, *Ci*/*Ca*: ratio of intercellular and atmospheric CO2, WUE*i*: intrinsic water use efficiency, WUE*ins*: instantaneous water use efficiency, *F*<sup>0</sup> - : minimum fluorescence, *Fm*- : maximum fluorescence, *Fv*- /*Fm*- : maximum quantum efficiency of photosystem II photochemistry, *φPSII*: the effective quantum efficiency of PSII photochemistry, *qP*: photochemical quenching, *qN*: non-photochemical quenching, ETR: electron transport rate, ETR/A: relative measure of electron transport to oxygen molecules, AES: alternative electron sinks, YPP: yield per plant, \* and \*\* denote significance at 5 and 1% probability levels, respectively, ns: non-significant.

**Table 3.** Means of leaf gas exchange and chlorophyll fluorescence parameters of okra accessions under non-stressed conditions.



**Table 3.** *Cont.*

*gs*: stomatal conductance (mmol m <sup>−</sup><sup>2</sup> s−1), T: transpiration rate (mmol H20 m <sup>−</sup><sup>1</sup> s−1), A: net CO2 assimilation (μmol CO2 m−<sup>1</sup> s<sup>−</sup>1), A/Ci: CO2 assimilation rate/intercellular CO2 concentration (μmol.mol <sup>−</sup>1), Ci: intercellular CO2 concentration (μmol.mol <sup>−</sup>1), Ci/Ca: ratio of intercellular and atmospheric CO2, WUEi: intrinsic water use efficiency ((μmol (CO2)m−2), WUEins: instantaneous water use efficiency (μmol.mol−1), F0 - : minimum fluorescence, Fm- : maximum fluorescence, *Fv*- */Fm*- : maximum quantum efficiency of photosystem II photochemistry (ratio), *φPSII*: the effective quantum efficiency of *PSII* photochemistry, *qP*: photochemical quenching, *qN*: non-photochemical quenching, ETR: electron transport rate (μmol e−<sup>1</sup> m−<sup>2</sup> s−1), ETR/A: relative measure of electron transport to oxygen molecules (μmol e μmol−<sup>1</sup> CO2), AES: alternative electron sinks, SED: standard deviation, YPP: yield per plant (g/plant), LSD: least significant difference, CV: coefficient of variation, \* and \*\* denote significance at 5 and 1% probability levels, respectively, ns: non-significant.

**Table 4.** Means of leaf gas exchange and chlorophyll fluorescence parameters of okra accessions under drought-stressed conditions.


**Table 4.** *Cont.*


*gs*: stomatal conductance (mmol m <sup>−</sup><sup>2</sup> s−1), T: transpiration rate (mmol H20 m <sup>−</sup><sup>1</sup> s−1), A: net CO2 assimilation (μmol CO2 <sup>m</sup>−<sup>1</sup> <sup>s</sup>−1), A/*Ci*: CO2 assimilation rate/intercellular CO2 concentration (μmol·mol <sup>−</sup>1), *Ci*: intercellular CO2 concentration (μmol·mol <sup>−</sup>1), *Ci*/*Ca*: ratio of intercellular and atmospheric CO2, WUE*i*: intrinsic water use efficiency (μmol (CO2)m<sup>−</sup>2), WUE*ins*: instantaneous water use efficiency (μmol·mol<sup>−</sup>1), *<sup>F</sup>*<sup>0</sup> - : minimum fluorescence, *Fm*- : maximum fluorescence, *Fv*- */Fm*- : maximum quantum efficiency of photosystem II photochemistry (ratio), *φPSII*: the effective quantum efficiency of *PSII* photochemistry, *qP*: photochemical quenching, *qN*: non-photochemical quenching, ETR: electron transport rate (μmol e−<sup>1</sup> m−<sup>2</sup> s<sup>−</sup>1), ETR/A: relative measure of electron transport to oxygen molecules (μmol e μmol−<sup>1</sup> CO2), AES: alternative electron sinks, YPP: yield per plant (g/plant), SED: standard deviation, LSD: least significant difference, CV: coefficient of variation, \* and \*\* denote significance at 5 and 1% probability levels, respectively, ns: non-significant.

Non-significant (*p* > 0.05) differences were observed among accessions under NS and DS conditions for *Ci*. Okra genotypes LS02 and LS21 exhibited high *A/Ci* values of 0.23 and 0.28 μmol. mol <sup>−</sup>1, respectively, under DS conditions compared to other accessions. Significant (*p* < 0.05) differences were observed in Ci/Ca values among accessions under both NS and DS conditions. Intrinsic water use efficiency and instantaneous water use efficiency were increased by drought stress (Table 4). Accessions LS13 and LS20 had the highest *WUEi* under drought-stress conditions, with 1438.80 and 1256.10 μmol CO2 m−2, respectively. The highest WUE*ins* values under drought stress were recorded for accessions LS04 (2164.70 <sup>μ</sup>mol·mol<sup>−</sup>1) and LS22 (2161.00 <sup>μ</sup>molmol<sup>−</sup>1).

The effect of drought stress on chlorophyll fluorescence parameters among the tested okra accessions are highlighted in Table 2. Chlorophyll fluorescence parameters indicated significant differences for genotype, water regime and genotype x water regime interaction, showing that the evaluated genotypes responded differently under non-stress and droughtstress conditions. Non-significant differences were observed for *Fo* under non-stress, while significant (*p* < 0.001) differences were recorded under drought-stress conditions (Tables 3 and 4). Genotypic variability (*p* < 0.001) with respect to *Fm* was observed under non-stress and droughtstress conditions. Drought stress decreased *Fv*- */Fm*- , from 0.51 under non-stressed to 0.35 under drought-stressed conditions. The *φPSII* varied significantly among the tested genotypes under non-stress and drought-stress conditions. LS07, LS12 and LS19 revealed considerably higher values for *φPSII* ≥ 0.40 compared to other genotypes under non-stress conditions.

Photochemical quenching was significantly reduced from 0.32 to 0.13 by drought stress among the evaluated genotypes, of which LS04, LS12 and LS13 had the highest values of *qP* > 0.40. A variable genotypic response was observed with respect to *qN* under non-stress and drought-stress conditions. The mean for *qN* was higher under drought-stress (1.96) than non-stress conditions (1.39). The *qN* values ranged from 0.68 to 2.80 under non-stress (Table 3) and from 0.66 to 3.75 under drought-stress conditions (Table 4). LS02, LS03 and LS11 revealed *qN* values ≥ 2 under non-stress conditions. Genotypes LS01, LS02, LS10, LS11 and LS18 showed *qN* values ≥ 3 under drought-stress conditions. Non-significant differences were observed for ETR under non-stress conditions, while genotypic variation was observed for ETR under drought-stress conditions. LS08, LS09 and LS17 revealed the highest ETR value of ≥34,541 <sup>μ</sup>mol e−<sup>1</sup> <sup>m</sup>−<sup>1</sup> <sup>s</sup>−1, whereas LS16, LS22 and LS26 showed the lowest ETR ≤ 8071 under DS conditions. Drought stress significantly increased ETR/A (Table 4). The highest ETR/A (≥1542 μmol e μmol-1 CO2) was recorded from LS03, LS08, LS09 and LS17 under drought-stress conditions. Drought stress significantly increased AES (154.72) compared to NS (26.98). AES ranged from 12.77 to 61.12 under non-stress and from 64.90 to 562.80 under drought-stress conditions. Yield per plant was significantly reduced, from 7.20 g/plant to 4.31 g/plant, by drought stress among the evaluated genotypes. Accessions LS11, LS19, LS21, LS22 and LS24 had the highest yield (>9 g/plant) under NS conditions, whereas LS05, LS06, LS07, LS08, LS10, LS11, LS18 and LS23 exhibited the highest yield (>5 g/plant) under DS conditions.

#### *3.2. Correlation between Leaf Gas Exchange and Chlorophyll Fluorescence Parameters under Non-Stressed and Drought-Stressed Conditions*

Pearson correlation coefficients showing relationships among leaf gas exchange and chlorophyll fluorescence parameters among the tested okra accessions under NS and DS conditions are presented in Table 5. Under NS conditions, Ci/Ca was highly and significantly correlated with Ci (*r* = 1, *p* < 0.001), WUE*i* with gs (*r* = −0.75, *p* < 0.001), WUE*ins* with T (r = −0.75, *p* < 0.001) and *φPSII* with A/Ci (*r* = 0.61, *p* < 0.001). In addition, *qP* was positively and significantly correlated with A (*r* = 0.55, *p* < 0.05), C*i* (*r* = 0.48, *p* < 0.05) and C*i*/C*a* (*r* = 0.48, *p* < 0.05). ETR was positively and highly significantly correlated with A (*r* = 0.71, *p* < 0.001) and *qP* (*r* = 0.52, *p* < 0.001). Positive and high significant correlation was observed between ERT/A and ETR (*r* = 0.86, *p* < 0.001) and AES and q*P* (*r* = 0.52, *p* < 0.001), while a negative and highly significant association was observed between YPP and A (*r* = −0.69, *p* < 0.001). A significant positive correlation was observed between YPP and ETR/A (r = 0.49, *p* < 0.05), YPP and Ci (*r* = 0.34, *p* < 0.05) and YPP and C*i*/C*a* (r = 0.45, *p* < 0.05), while a negative significant correlation was observed between YPP and qN (*r* = −0.45, *p* < 0.05) under NS conditions.

**Table 5.** Correlation coefficients for gas exchange and chlorophyll fluorescence parameters under non-stressed (bottom diagonal) and drought-stressed (top diagonal) conditions.


*gs*: stomatal conductance, T: transpiration rate, A: net CO2 assimilation, A/*Ci*: CO2 assimilation rate/intercellular CO2 concentration, *Ci*: intercellular CO2 concentration, *Ci*/*Ca*: ratio of intercellular and atmospheric CO2, WUE*i*: intrinsic water use efficiency, WUE*ins*: instantaneous water use efficiency, F0 - : minimum fluorescence, *Fm*- : maximum fluorescence, *Fv*- */Fm*- : maximum quantum efficiency of photosystem II photochemistry (ratio), *φPSII*: the effective quantum efficiency of *PSII* photochemistry, *qP*: photochemical quenching, *qN*: nonphotochemical quenching, ETR: electron transport rate, ETR/A: relative measure of electron transport to oxygen molecules, AES: alternative electron sinks, YPP: pod yield per plant. \* and \*\* denote significance at 5 and 1% probability levels, respectively, ns: non-significant.

Under DS conditions, a significant positive correlation was detected between A and gs (*r* = 0.57, *p* < 0.05), while A/C*i* was negatively and highly significantly correlated with C*i* (*r* = −0.61, *p* < 0.001). A highly significant negative association was observed between

C*i*/C*a* and A/Ci (*r* = −0.57, *p* < 0.001). WUE*i* was positively and significantly correlated with A (*r* = 0.48, *p* < 0.05), while WUE*ins* was negatively and highly significantly correlated with T (*r* = −0.55, *p* < 0.001). *Fv*- */Fm* was positively correlated with gs (*r* = 0.47, *p* < 0.05). *φPSII* was positively and highly significantly correlated with gs (*r* = 0.54, *p* < 0.001), while significantly associated with A (*r* = 0.42, *p* < 0.05) and *Fv*- */Fm*- (*r* = 0.46, *p* < 0.05). *qP* was positively correlated with WUEins (*r* = 0.39, *p* < 0.05) and highly significantly correlated with *Fm*- (r = 0.55, *p* < 0.001). Positive correlations were observed between *qN* and A (*r* = 0.48, *p* < 0.05) and C*i* (*r* = 0.48, *p* < 0.05) and WUE*i* (*r* = 0.43, *p* < 0.05). ETR was positively correlated with gs (*r* = 0.45, *p* < 0.05), A (*r* = 0.45, *p* < 0.05), *Fv*- */Fm*- (*r* = 0.53, *p* < 0.001) and *φPSII* (r = 0.82, *p* < 0.001). Relative measure of electron transport to oxygen molecules was positively and significantly correlated with WUE*i* (*r* = 0.68, *p* < 0.001) and ETR (*r* = 0.82, *p* < 0.001), while AES was positively correlated with T (*r* = 0.45, *p* < 0.05) and *qP* (*r* = 0.48, *p* < 0.05). YPP was highly positively correlated with C*i* (*r* = 0.66, *p* < 0.001), *Fo*- (*r* = 0.83, *p* < 0.001) and Ci/Ca (*r* = 0.67, *p* < 0.001), while significantly associated with WUE*i* (*r* = 0.48, *p* < 0.05) and Fm- (*r* = 0.40, *p* < 0.05) and negatively correlated with ETR/A (*r* = −0.60, *p* < 0.001) under DS conditions.
