Sorghum Drought Tolerance is Associated with Deeper Root System and Decreased Transpiration Rate

Drought decreases grain yield of sorghum [ Sorghum bicolor (L.) Moench], and understanding the mechanism(s) related to drought tolerance is critical for sustaining sorghum production. Variation in root and shoot traits associated with drought tolerance were analyzed to provide an integrated view of factors that underlie the drought tolerance of sorghum. The plants were grown in the root column up to the five-leaf stage, then exposed to either 0.9 fraction of transpirable soil water (FTSW) or 0.4 FTSW for five days. In another experiment, at the five-leaf stage, stress was imposed for 14 days. Various root and shoot traits associated with drought tolerance were recorded. The seminal root angle of IS13540 was lower (24.4  ) than IS23143 (42.6  ). Drought stress increased the maximum root length (40%) and total root length (58%) of IS13540 than its irrigated control. In contrast, the maximum root length and total root length were decreased in IS23143. Similarly, across the lines, drought stress decreased stomatal conductance (37%), transpiration rate (42%), photosynthetic rate (40%), photosystem II quantum yield (20%), photochemical quenching (44%), and total dry matter production (34%) than irrigated control. An increased transpiration rate was observed in IS23143 than IS13540 under irrigated and drought stress. In IS23143, the reduction in photosynthetic rate under drought may be a combination of stomatal and non-stomatal factors. However, in IS13540, the reduction is especially by the stomatal factors. It is evident that IS13540 is a drought-tolerant line, and tolerance is related to a deep prolific root system and reduced transpiration rate.

relationship between the root angle and drought tolerance of sorghum. Genotypes with narrow root angles had a higher root length density at deeper soil depths at the seedling stage, enhancing drought tolerance [7]. Similarly, studies on winter wheat [Triticum aestivum (L.)] [8], upland rice [Oryza sativa (L.)] [9], and maize [Zea mays (L.)] [10] indicated that a deep and prolific root system is advantageous under drought stress. Besides deeper roots, higher root length density (RLD) at depth and low RLD at the surface are also considered as desired traits for drought tolerance [11]. In contrast, studies on rice [12], peanut (Arachis hypogaea L.) [13], and chickpea (Cicer arietinum L.) [14] showed that deep and profuse root system is not associated with drought tolerance. Also, it was demonstrated that a shallow, well-branched root system was associated with drought tolerance in spring wheat [8] and rice [15]. Hence, it can be hypothesized that an ideotype that increases the water uptake from deeper soil may not always yield higher under drought stress conditions. Apart from root traits, stomatal closure is one of the earliest responses of the crop to drought stress [16][17]. Stomatal conductance and vapor pressure deficit (VPD) are the major drivers influencing transpiration rates [18]. A substantial amount of water can be lost from the leaves even when the stomata are at minimum aperture [19]. Furthermore, decreased stomatal conductance can affect the photosynthetic rate through reduced availability of CO2 to the enzyme Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCo), consequently lowering biomass [20]. Researches showed that the primary effect of drought was decreased photosynthetic rate through stomatal and non-stomatal limitations like damaging ultrastructure of chloroplast, RuBisCo regeneration, and carboxylation efficiency, inactivation of photosystem (PS) II reaction center, the maximum quantum yield of PS II (Fv/Fm ratio), electron transport rate (ETR), photochemical quenching (qP), leaf water potential, and osmotic adjustment [21][22][23][24][25][26][27].
In sorghum, the shoot and root traits associated with drought tolerance are studied independently. In this study, the various root traits like maximum root length, total root length, root diameter, and root: shoot ratio and shoot traits like transpiration rate, stomatal conductance, photosynthetic rate, and leaf photochemistry that can change under drought stress was quantified to understand the mechanism associated with drought tolerance of sorghum. This study fills a knowledge gap by recording the root architectural changes caused by drought stress and linking these with shoot traits related to transpiration and photosynthesis.

Materials
The sorghum lines IS13540 and IS23143 were selected based on our previous experiment [28]. The study indicated that under progressive soil drying, the line IS13540 had a lower transpiration rate than IS23143.

Measurement of root angle 2.2.1. Nodal root angle
The seeds of IS13540 and IS23143 were surface sterilized using sodium hypochlorite (10% v/v) for 5 min, followed by washing in deionized water for five times and sown in a plastic pot (25.7 x 25.5 x 22.1 cm; L x B X H) containing 9 kg of sandy clay loam soil. The pots had holes at the bottom for drainage. There were five replications for each line. In each pot, three seeds were sown at a depth of 3 cm. After seedling emergence, the seedlings were thinned to one per pot. To each pot, 2 grams of urea, 1 gram of diammonium phosphate, and 1 gram of potash were added after thinning. The plants were irrigated on alternate days from emergence to 21 days after emergence (DAE). On the 22 nd day, the soil was washed from the pot, and the roots were carefully removed with hand. The roots were washed with deionized water with hand to remove the debris and stone. The roots of the upper node are tough and thicker than the lower nodes, and the root angle remained in the original shape after removing soil and washing with water. The root angle of the first nodal root from the top of the plant was determined by measuring the root angle between the horizontal (i.e., soil surface) and the line along the slope of the nodal root at 2 cm from the root base using a protractor [29].

Seminal root angle
The seeds of IS13540 and IS23143 were surface-sterilized as described earlier, then germinated in a Petri plate (100 mm x 15 mm; diameter x thickness) using Whatman number 42 filter paper, moistened with 5 mL of deionized water for two days. In each Petri plate, ten seeds were placed. A 2% (w/v) agar (Type A; Sigma Chemicals, Bangalore, India) solution was prepared and autoclaved at standard temperature (121  C) and pressure (15 lbs sq inch -1 ) for 20 min. Next, the sterilized agar was poured into the Petri plate (12 x 12 x 1.7 cm; L x W x H) up to the rim under aseptic condition and allowed to solidify. Followed by this, all the sides of the Petri plates were sealed using clear cellophane tape (Amazon Basics Tape 1.9 cm x 1605 cm, Amazon, India). On the second day, a uniformly sized seedling (radicle length of 0.2-0.3 cm) was selected and placed one per Petri plate containing agar in a vertical position with the radicle facing downwards through the cuts of the Petri plates. The Petri plates were positioned vertically in an incubation room maintained at 24.5  1.5  C for 14 days. There were five replications per line. On the 15 th day, the angle of the first pair of seminal roots was measured at a 3 cm distance from the seed relative to a vertical line passing through the stem base [30].

Effects of vegetative stage drought stress: root column experiment 2.3.1. Plant materials and crop husbandry
An outdoor experiment in a completely randomized block with a split plot treatment structure was conducted at the Department of Crop Physiology, Tamil Nadu Agricultural University, Coimbatore, (11°N; 77°E; 426.7 m MSL), India. Before starting the experiment, the clay-loamy soil was sieved through a 2 mm sieve, and the soil fraction that did not pass through the sieve was discarded. Next, the soil fraction (<2 mm) was sieved using a 1 mm sieve, and the fraction which passed through the sieve was collected. Finally, 5 kg of vermicompost was added to 200 kg of sieved soil and thoroughly mixed and filled in the column.
The soil was sampled to quantify the available soil moisture using a pressure plate apparatus [31]. Analysis indicated that the available soil moisture of the soil was 18  0.5%. The soil pH and electrical conductivity were 7.7 and 0.32 dS m -1 , respectively. The plant was grown in polyvinyl chloride (PVC) column (100 x 7.5 cm; length x inner diameter), and the bottom of the column had a plastic cap with a central hole of 1 cm diameter for drainage. Before sowing, each PVC column was filled with 6.5 kg of soil, and 2 g of urea, 2 g of diammonium phosphate, and 1 g of muriate of potash were added on top of the soil and mixed well. Then, the column was irrigated to 100% field capacity (FC). Three seeds of sorghum line (IS23143 or IS13540) were sown at 3 cm depth in each PVC column. After seedling emergence, the seedling was thinned to one and maintained till the completion of the experiment. The plants were irrigated daily in the evening up to FC. Plants were grown under the natural sunlit condition from sowing to the five-leaf stage (40 days after emergence; DAE). Afterward, the plants were divided into two groups; one served as irrigated control, and the other was drought stress.

Imposition of drought stress
The total transpirable soil water (TTSW) content was determined as the difference between the soil water content at FC and wilting point (WP). The FC and WP were determined through a pressure plate experiment, and, using FC and WP values, the fraction of transpirable soil water (FTSW) was calculated [FTSW = (actual water content -water content at the wilting point) / TTSW] [32]. Drought stress was imposed by maintaining the plants at 0.4 FTSW, and the control plants were maintained at 0.9 FTSW.
At the five-leaf stage, all the plants were irrigated to FC, then the top of the column was completely covered with a PVC sheet by making a small slit. The slit was covered with white packing adhesive tape around plant collars such that there was no space between the plant and slit to minimize evaporative losses from the soil surface [33]. Then, the column weight was recorded in the morning (06:00 -07:00 hours) to arrive at the weight corresponding to FC. The irrigated plants were weighed daily at 07:00 hours, and the quantity of water required to bring 0.9 FTSW was worked out and added. In the drought-stressed plants, the irrigation was withheld until the soil moisture dropped to 0.4 FTSW. At that point, the required quantity of water to maintain 0.4 FTSW was added. The plants were maintained at 0.9 or 0.4 FTSW for five days. Our previous study indicated that the FTSW reached a value of 0.3 on the 4 th day of progressive soil drying. At 0.3 FTSW, the photosynthetic rate and stomatal conductance were decreased by 70 and 81%, respectively, from the initial value [28]. Apart from this, Gano et al. [5] reported that in sorghum, at 0.3 FTSW, the plants experienced drought stress as revealed by leaf rolling. Similarly, in this study, the plants at 0.4 FTSW on the 2 nd day showed leaf rolling symptoms. Hence, the drought stress was imposed for five days.
The environmental condition during the drought stress period was presented in supplementary figure 1. During the drought period, the daytime maximum temperature ranged between 37 and 38  C and the nighttime minimum temperature ranged from 22 to 23  C. Similarly, the daytime relative humidity ranged between 45 and 50% and nighttime humidity from 90 to 95%. The plants were harvested after five days of drought stress.

Traits recorded 2.3.3.1. Growth traits
Plant height was measured as the distance from the soil surface to the tip of the fully expanded leaf and expressed as cm. At harvest, the PVC column was gently tilted at about 80  , and water was flushed slowly to the bottom of the column. The wet loose soil was removed by hand so that the soil particle alone slipped out of the column. The entire root system was recovered and washed thrice in water by hand to remove any attached soil. The root system was laid on a flat surface and straightened to measure the maximum root length (rooting depth; measured from the base of the stem to the tip of the root) and expressed as cm. Washed roots were stored in a plastic container containing 30% ethanol until further analysis [34]. The whole root system was cut into 30 cm long portions, and each part was carefully spread in a transparent tray (20 x 15 x 2 cm; L x W x H) containing water to minimize root overlap and scanned using an Epson photo scanner (Epson Perfection V800 with 100 dpi resolution, Epson, Long Beach, CA, USA). Images of scanned roots were analyzed using the WinRHIZO Pro image system (Regent Instruments, Inc., Quebec City, QC, Canada) to estimate total root length and root diameter as explained by McPhee [35] and Singh et al. [36]. The total root length and root diameter were expressed in m and mm, respectively. Root length: shoot length ratio was calculated as the ratio of maximum root length to plant height [8,[37][38]].

Physiological measurements
Plant transpiration rate was estimated on an hourly basis (from 08:00 to 15:00 hours) on all days of observation. The pigments, leaf area, canopy temperature, chlorophyll a fluorescence kinetics, and gas exchange traits were recorded on the 5 th day of drought stress.
2.3.3.2.1. Leaf area, chlorophyll index, and canopy temperature The chlorophyll index and canopy temperature were recorded on the attached tagged fully expanded top leaf from each replication between 10:00 and 14:00 hours in three replications. After harvest, the leaves were removed, and the leaf area was measured using a benchtop leaf area meter (LI-COR 3000; Lincoln, NE, USA) and expressed as cm 2 plant -1 . Chlorophyll index was measured using a chlorophyll meter [Soil Plant Analysis Development (SPAD); Model 502, Spectrum Technologies, Plainfield, IL, USA] and expressed as SPAD units. Canopy temperature was measured using an infrared camera (FLIR Systems, Inc., Wilsonville, OR, USA) and expressed as  C.

Chlorophyll a fluorescence measurement
The chlorophyll a fluorescence traits were measured on attached tagged fully expanded top leaves on the 5 th day of drought stress using a modulated fluorometer (OS1p, Optisciences, Hudson, NH, USA). The chlorophyll a fluorescence kinetics was measured in dark-adapted leaves using a dark adaptation clip. The leaves were continuously irradiated with white actinic light to measure the initial fluorescence in leaves acclimated to irradiation (Fo), steady-state fluorescence yield (Fs), and maximum fluorescence yield (Fms) of irradiated leaves [39]. By using the above parameters, the following chlorophyll a fluorescence parameters were calculated: The leaf-level gas exchange measurements (photosynthesis, stomatal conductance, and transpiration rate) were measured on the tagged fully expanded top leaf using an LI-COR 6400XT portable photosynthesis system (LI-COR, Lincoln, NE, USA). The gas exchange measurements were recorded at daytime growth temperature and ambient CO2 conditions (410 ppm). The internal light-emitting diode light source was set at 1600 µmol m -2 s -1 to ensure a constant, uniform light across all measurements [41]. The total dry matter production was dried at 60  C for ten days and weighed and expressed as g plant -1 .

Effects of vegetative stage drought stress under field condition
A field experiment was conducted in a split plot design with three replications. The main plot and sub-plots were irrigation regimes and lines, respectively. The main plot had two levels (1. Irrigated control: plants are irrigated at five-day intervals and 2. drought stress: withholding water for 15 d from 35 DAE). The sub plot had two levels (lines: 1. IS13540 and 2. IS23143).
Sorghum lines were sown in a paired row method of planting (60/30 x 30 cm) at a depth of 3 cm. The fertilizer dose of 90:45:45 kg N:P2O5:K2O ha -1 was followed. The nitrogenous fertilizer was applied in two doses, one at sowing and another at 30 DAE. In addition, phosphorus and potassium fertilizers were added during sowing. The crop was irrigated once in five days until the five-leaf stage (35 DAE). After that, three irrigations were skipped for drought stress treatment from the five-leaf stage, and the plants were irrigated on 50 DAE. At the 5 th leaf stage, the primary stalk and the top fully expanded leaf were tagged to record the physiological traits. The weather parameters that prevailed during the drought stress period are presented in supplementary figure 2.

Traits recorded
Physiological traits were recorded on the attached fully expanded top leaf from each replication between 10:00 and 14:00 hours on the 14 th day of withholding irrigation. The Fv/Fm ratio and the performance index (PIABS) were measured through fast chlorophyll fluorescence induction kinetics as suggested by Strasser et al. [42]. Leaf-level gas exchange measurements (photosynthesis, stomatal conductance, and transpiration rate) were recorded on three replications using an LI-COR 6400XT portable photosynthesis system as described earlier. The total dry matter production was estimated by sampling five plants per replication after completion of drought stress, and the samples were dried at 60  C for ten days and weighed, and the average was expressed as g plant -1 .

Statistical Analyses
The seminal and nodal root angles were analyzed using the PROC TTEST procedure of the SAS 9.3 version [43]. A P value of  0.05 was considered statistically significant. The root column experiment was designed in a completely randomized block with a split plot treatment structure with three replications (n=3). The irrigation regime was the main plot factor, and the line was the sub plot factor. The PROC MIXED procedure of SAS was used for data analysis. The hourly transpiration data was analyzed by considering irrigation regime, line, days of observation, and time of observation (hourly transpiration rates) as fixed factors and block as a random factor. Results revealed that the trends of transpiration rate in both lines were similar for all days of observation. Therefore, the data were analyzed by combining days of observation by considering the irrigation regime, line, and time of observation as fixed factors and days of observation and block as random factors to get the overall effects of the irrigation regime and line. The data on leaf area, chlorophyll index, canopy temperature, Fv/Fm ratio, chlorophyll a kinetics, the photosynthetic rate, stomatal conductance, transpiration rate, and total dry matter production were analyzed by considering irrigation regime and lines as fixed factors and block as a random factor. The Tukey-Kramer adjustment was used to separate the treatment means. The field experiment was conducted in a split plot design with three replications. The main plot was the irrigation regime, and the sub plot was the line. The PROC MIXED procedure of SAS was used for data analysis as described earlier.

Nodal and seminal root angle (  )
The lines varied significantly (P0.01) for nodal (Fig. 1a) and seminal root angle (Fig.  1b). The nodal root angle was substantially higher in IS13540 (59.4  ) than IS23143 (41.4  ). In contrast, the seminal root angle was significantly lower in IS13540 (24.4  ) than IS23143 (42.6  ) (Fig. 1a-b). The effect of the line was significant for maximum root length (P0.05) and total root length (P0.01) (Fig. 2a, 2c), and the effect of the irrigation regime was significant (P0.01) for maximum root length (Fig. 2a) and root length: shoot length ratio (Fig. 2b). However, there were significant (P0.001) effects of the interaction of lines and irrigation regime for maximum root length, root length: shoot length ratio, average root diameter, and total root length (Fig. 2a-d).
Differential response for maximum root length and root length: shoot length ratio was observed between the lines (Fig. 2a-b). The line IS23143 had a higher maximum root length and root length: shoot length ratio under irrigated conditions than drought stress. However, the line IS13540 had a higher maximum root length and root length: shoot length ratio under drought than irrigated control. Between the lines, IS13540 had an increased total root length (58%) under drought than irrigated control. However, there was no difference between drought and irrigated conditions for total root length in IS23143 (Fig. 2c). The average root diameter of IS13540 was higher under drought than other treatment combinations (Fig. 2d).

Hourly transpiration rate (g h -1 plant -1 )
The rate of transpiration varied significantly (P0.05) for line, irrigation regime, and their interactions from 8:00 to 15:00 hours (Fig. 3). Under the irrigated condition, the highest transpiration rate was observed around 12:00 to 13:00 hours (Fig. 3). However, under drought stress, the highest transpiration rate was observed from 10:00 to 10:30 hours. Irrespective of the time of the observation, IS23143 had an increased transpiration rate than IS13540. Within each line, the irrigated plants had a higher transpiration rate than drought-stressed plants (Fig. 3).  There were significant differences (P0.05) between lines, irrigation regime, and their interaction on leaf area, canopy temperature, and Fv/Fm ratio (Fig. 4b-d). However, the chlorophyll index varied significantly (P0.05) for drought stress alone (Fig. 4a). In both lines, drought stress decreased the chlorophyll index (Fig. 4a). The leaf area of IS23143 decreased significantly (P0.01) under drought stress compared to irrigated control; however, there was no significant difference between irrigated and drought stress for IS13540 (Fig. 4b). Between the lines, drought stress increased the canopy temperature (Fig. 4c) and decreased Fv/Fm ratio (Fig. 4d). The highest decrease in Fv/Fm ratio was observed in IS23143, along with the lowest canopy temperature increase under drought (Fig. 4c-d).
Between the irrigation regime, drought stress significantly (P0.05) decreased the PSII, qP, and ETR. In contrast, the NPQ was increased by drought stress (Fig. 4e-h). Between the lines, IS13540 had a higher PSII, qP, and ETR than IS23143 under drought stress. However, there were no differences between the lines on chlorophyll a fluorescence traits under the irrigated condition (Fig. 4e, f, h). In addition, the NPQ level was significantly lower in IS13540 than IS23143 under both irrigated and drought stress conditions (Fig. 4g).

4.2.4.
Photosynthetic rate (mol m -2 s -1 ), stomatal conductance (mol m -2 s -1 ), transpiration rate (mmol m -2 s -1 ), and total dry matter production (g plant -1 ) Line, irrigation regime, and interaction of line and irrigation regime varied significantly (P0.05) for photosynthetic rate, stomatal conductance, transpiration rate, and total dry matter production (Fig. 5a-d). Drought stress significantly (P0.05) decreased photosynthetic rate than irrigated control, and between the lines, IS13540 had a higher photosynthetic rate than IS23143 under drought stress (Fig. 5a). The stomatal conductance and transpiration rate was decreased under drought stress than irrigated control (Fig. 5b, c), and the line IS13540 had lower stomatal conductance and transpiration rate than IS23143. In contrast, the total dry matter production was the highest in IS13540 under drought stress compared to IS23143 (Fig. 5d). The chlorophyll index and leaf area varied significantly (P0.01) for lines and irrigation regimes (Table 1). Between the irrigation regime, drought stress decreased chlorophyll index (9.5%) and leaf area plant -1 (21%) compared to irrigated control (Table 1). Similarly, between the lines, IS23143 had a higher leaf area (12%) than IS13540. In contrast, the chlorophyll index was lower (7%) in IS23143 than in IS13540 (Table 1). , photosynthetic rate (mol m -2 s -1 ), stomatal conductance (mol m -2 s -1 ), transpiration rate (mmol m -2 s -1 ), and total dry matter production (g plant -1 )] There were significant (P0.01) differences in Fv/Fm ratio and PIABS for lines, drought stress, and the interactions of lines and drought stress (Fig. 6a-b). In both the lines, drought stress significantly (P0.01) decreased Fv/Fm ratio and PIABS compared to irrigated condition; however, the decrease was higher in IS23143 than IS13540 (Fig. 6a-b).
The photosynthetic rate, stomatal conductance, transpiration rate, and total dry matter production were significant (P0.01) for lines, moisture regimes, and their interactions (Fig. 6c-f). A higher decrease in photosynthetic rate and total dry matter production was observed in IS23143 under drought stress than IS13540 (Fig. 6c, 6e, 6f). In contrast, the line IS23143 had a higher transpiration rate and stomatal conductance under drought stress than IS13540 (Fig. 6d, e). , and (f) total dry matter production (g plant -1 ) in sorghum lines. Each datum is the mean ± SEM of three plants (n = 3). Means with different letters are significantly different at P ≤ 0.05.

Discussion
Drought is important abiotic stress, limiting sorghum yield in arid and semi-arid regions [44]. The root architecture and stomatal conductance are the essential traits associated with drought tolerance under limited soil moisture because roots maintain water uptake [45], and stomatal conductance regulates transpiration rate [46]. Hence, targeting root architecture and stomatal conductance can be a better approach for improving the drought tolerance of sorghum [47][48][49].
The profuse and deep root systems are generally considered drought-tolerant traits. Deep rooting is a complex trait governed by a narrow root angle and higher maximum root length [9,29,50]. The present study confirmed the above findings, evidenced by a higher maximum root length, root length: shoot length ratio under drought stress, and a narrow seminal root angle (~25  ) in the line IS13540 compared to IS23143. Apart from maximum root length, the line IS13540 had a higher total root length than IS23143, showing that this line can extract more water from deep soil layers, provided moisture was present in deeper soil horizons [38,51]. The deep rooting of IS13540 could be associated with narrow root angle [9], greater root penetration rate ability [52], achieved by large root diameter [53]. This has been confirmed in rice, wheat, and maize, as deep rooting with profuse branching was associated with drought tolerance [9,30,[54][55].
Previous studies have reported strong coordination between below-ground and above-ground morphological changes under drought stress [56][57]. In general, drought avoidance in plants is achieved by maintaining turgor through increased water absorption from deeper soils through increased rooting depth and/or reduction in water loss through decreased leaf area and stomatal conductance under drought stress [48][49][58][59]. These findings indicate that both root and shoot system traits are involved in the drought tolerance of sorghum. In line with these, the genotype IS13540 had a deeper and more extensive root system, decreased transpiration rate under drought stress (Fig. 2a, 2c, 4b, and 5c).
Genotypes with deep and prolific root systems under drought stress would be advantageous if the size of the shoot were smaller that consumed less water [60]. Under drought stress, compared to IS23143, the line IS13540 had a lower transpiration rate due to decreased stomatal conductance. The relationship between root length and stomatal conductance was explained by Fraccasso et al. [61], namely, the sorghum lines with higher root: shoot ratio also reduced stomatal conductance and transpiration rate under drought, as observed in the present study. The above changes explain the water saver mechanism of drought tolerance.
The decrease in photosynthetic rate under drought stress may be due to stomatal and/or non-stomatal factors. Chlorophyll pigment is involved in capturing light energy, and a reduction in chlorophyll content under drought could be associated with damage to the chloroplast structure [62][63]. Between the lines, IS13540 had a higher chlorophyll content than IS23143, indicating a comparatively higher drought tolerance ability of IS13540 than IS23143. Between the lines, IS23143 had a lower increase in canopy temperature than IS13540 under drought stress, indicating that this line had a higher transpiration rate. This was validated in the hourly transpiration rate from 8:00 hours to 15:00 hours. In the IS13540 line, the reduction in photosynthetic rate under drought stress was associated with stomatal factor alone, as evidenced by a higher decrease in stomatal conductance (48%) than PSII (11%) and ETR (8%) compared to its irrigated control [64]. In contrast, in IS23143, the decrease in photosynthetic rate is a combination of both stomatal and non-stomatal factors [65], as demonstrated by an equal reduction in stomatal conductance (25%), PSII (24%), and ETR (25%) compared to its irrigated control. The above findings indicate that the line IS13540 had better tolerance to drought stress than IS23143.
The decrease in Fv/Fm ratio under drought stress may be associated with an increase in Fo value. The rise in Fo value under drought stress indicates the inactivation of reaction centers [66], photoinhibition, and damage of PS II [67][68]. The decrease in qP (Fig. 4f) and PSII (Fig. 4e) and increase in NPQ values (Fig. 4g) under drought than irrigated control suggest that the photosynthetic electron transport activity decreases under drought (Fig.  4h). Therefore, more light energy has to be dissipated as heat through non-photochemical pathways [69]. The performance index (PI) represents the energy flow efficiency of photosynthetic electron transport beyond PSII [70]. A higher value of PI under drought stress was recorded in IS13540 than IS23143, indicating its efficient electron transport rate (Fig.  6b). The above trait might have contributed to IS13540 per se performance under drought conditions.

Conclusions
The major conclusions from this study are (i) drought stress increased maximum root length, root length: shoot length ratio, total root length, and decreased the photosynthetic rate, stomatal conductance, transpiration rate,  PSII, photochemical quenching, and electron transport rate and total dry matter production of sorghum, (ii) under both irrigated and drought stress, a decreased transpiration rate, and increased photosynthetic rate was observed in IS13540 than IS23143, (iii) the decrease in the photosynthetic rate under drought stress in IS13540, was mainly associated with stomatal factors, and in IS23143 it was associated with both stomatal and non-stomatal factors, and (iv) the drought tolerance ability of IS13540 is associated with a deep root system and decreased transpiration rate.
Author Contributions: Conducted laboratory, glasshouse, and field experiment, G.S.; Conceptualization, monitored the experiments data curation, written, editing and review M.D. and G.M.; edited the manuscript and followed up with field experiment T.N.; edited the manuscript and followed up with experiments J.P; and editing the manuscript and review G.M. Funding: