Stress Tolerance in Sugar Beet under Various Drought Stress Cycles

Plants under field cultivation face one or more unfavourable environmental conditions.  Around 90% of the world’s arable lands are affected by several stressors (Rojas, 2020), leading in yield reductions up to 70% loss in major crops (Mantri et al., 2012). Drought, a common abiotic stress, affects approximately 45% of the global cultivated area (Ashraf and Foolad, 2007) and around 35% of Indian agricultural land (Pal et al., 2022). Drought stress alters plant distribution (Ghaffari and Tadayon, 2019), limits crop productivity and stability and affects plant growth, development, physiological and biochemical processes. Limited water supply causes leaf wilting, early maturation reduces leaf area and weight, that alters root length (Jaleel et al., 2009).  Furthermore, the reduction in relative water content is a common consequence of water stress in many crop species (Deka et al., 2018). Plants respond to water stress primarily by maintaining water status through increased water uptake and decreased water loss, employing various strategies to overcome drought stress and adaptation. Under limited water availability, crop plants close their stomata, reduce their height, leaf number, leaf area and accumulate more osmolytes. Sugar beet, a root crop accounting for approximately one-quarter of global sugar production (Singh et al., 2018), is also used as fodder and a source of bioethanol. Although it is a temperate crop primarily grown in cold and mediterranean climates, gradually many heat-resistant varieties have been developed for cultivation in tropical and subtropical areas. Sugar beet requires less water than other sugar crops and exhibits moderate drought tolerance, however, limited water supply can hinder its growth, development and quality (Moosavi et al., 2017). This is the part of master research work presented with the objectives to evaluate the sugar beet tolerance ability to various stress durations under water deficit condition during summer.

Plant material and experimental details
 
Tropical sugar beet LS-6 variety seeds were supplied by the Indian Institute of Sugarcane Research (IISR), Lucknow, India and used as planting material for this research. The pot experiment was conducted at the ICAR-CTCRI and FAS, SOA for lab work, Bhubaneswar, Odisha, India, during January to May 2021. Sugar beet seeds were sown in the poly-pots containing 25 kg of soil each in three replicates. Adequate number of pots per replication was prepared as per the experiment in a completely randomized design. After 7 weeks of normal growth, the plants were subjected to drought stress by withholding water. The treatments were exposed to single stress (1^st cycle) of 5, 10, 15 and 20 days durations as in T1, T2, T3 and T4 respectively. After first regime of stress T1, T2, T3 and T4 were re-watered continuously till harvest, while double stress (1^st cycle + 2^nd cycle) was imposed in T5, T6, T7 and T8 treatments, initially subjected to 5, 10, 15  and 20 days of 1^st cycle stress respectively, which were then re-watered for 15 days after completing 1^st cycle and then subjected to 2^nd cycle of 3 days stress by withholding water supply in the pots where, control plants were watered daily.
 
Growth parameters
 
The number of fully expanded healthy green leaves was counted in the field for each treatment. Total plant leaf area was measured using a leaf area meter and the leaf area index was calculated by following formula.
 
 

 
 

 
Root to shoot ratio
 
The fresh weight of the root and shoot of each sample plant was measured separately to calculate the root to shoot ratio.
 
Physiological parameters
 
Relative water content (RWC%)
 
To measure the relative water content in percentage, the fresh weight of five fully expanded healthy leaves from each plant was recorded. These leaves were then submerged in water for 30 minutes to determine their turgid weight before being placed in a hot air oven at 80^oC for drying to measure the dry weight. Relative water content percentage (RWC %) was calculated using the formula:
 

 

 

 Chlorophyll stability index (CSI)%
 
Chlorophyll stability index was calculated by taking fresh leaf samples. Two sets of test tubes containing 100 mg of leaf sample in water were prepared. Half of each set was placed in an 85^oC water bath and the other half was kept at normal conditions for 30 minutes. Both were then macerated with 80% ethanol and the mixture was centrifuged. The optical density (OD) of the supernatant was measured at 652 nm using a spectrophotometer for both treated and untreated samples. The Chlorophyll Stability Index (%) was calculated using the formula:
 
 

 
 

 
Biochemical analysis
 
Proline
 
Proline determination was obtained by using ninhydrin method (Bates et al., 1973). About 100 mg of fresh, fully expanded leaves were macerated with 3% sulfo-salicylic acid. Then it was centrifuged for 15 minutes. A mixture of 2 ml each of the supernatant, ninhydrin and glacial acetic acid was placed in a boiling water bath for 1 hour.  Then, it was separated with 4 ml toluene to measure the optical density at 520 nm. The amount of proline in the sample was calculated in μg/g fresh weight and then the percentage increase or decrease over the control was also calculated for each treatment using the formula:
 
 

 
 

 Total soluble carbohydrate
 
Total carbohydrate percentage in leaves was determined by using anthrone method (Scott and Melvin,1953). About 100 mg of fresh sample were hydrolysed in a boiling water bath for three hours with 2.5 N HCl and neutralized with solid sodium carbonate after cooling. The volume was then adjusted to 100 ml. Then a mixture of sample extract, distilled water and anthrone reagent prepared and incubated in boiling water bath before cooling it to measure the optical density of the green solution at 630 nm. The percentage of total soluble carbohydrate in the sample was calculated to express TSC% increase or decrease over control using the formula below.
 

 

 
Starch
 
Starch content was determined from dry leaves using the anthrone method (Hedge and Hofreiter, 1962). Oven-dried residue of previously extracted sugar was used for starch extraction. A mixture of 2.5 ml starch extract and 5 ml of anthrone reagent was taken and incubated in boiling water bath, then cooled in an ice bath to measure the absorbance at 630 nm and the amount of starch was calculated from glucose as standard and the percentage increase or decrease in starch was calculated for each treatment by following formula:
 

  

Yield parameters
 
Biological yield was determined by measuring the weight of the whole harvested plants and economic yield by measuring the tuber weight. The harvest index (HI) was calculated for each treatment using the formula.
 
 

 

 
 Statistical analysis
 
One-way analysis of variance (CRD) was performed using SPSS v 26. Statistically significant differences between treatments were indicated at P<0.05. During stressful periods, each treatment was analysed and compared with its respective controls. Fisher’s t-test (P<0.05) was applied to study their significance level.

Growth parameters
 
Reduction in plant growth was observed at p<0.05 as the number of leaves per plant was reduced to half compared to the control after 20 days of withholding irrigation, 7.33 leaves/plant in T4 to 14.67 leaves/plant in control (Fig 1). Moreover, a significant reduction in the photosynthetic area in terms of leaf area per plant was observed 1235.11cm² after 5 days of stress and 433.59cm² exposed to 20 days stress against the respective control plants, which were 1971.42 cm² and 2650.01 cm², respectively (Table 1). This decline in leaf area resulted due to decrease in plant growth under severe stress. Leaf area index also reduced which was reached minimum value of 0.61 at 20 days long stress duration (Table 1). With gradual increase of drought duration in sugar beet plants, 15 days and 20 days of water withholding caused a significant reduction in leaf number, leaf area and LAI under first cycle of stress against respective controls. Unlike other growth parameters, the root: shoot ratio increased by increasing drought duration showed more biomass accumulation and reaching 0.37 in 20 days stress treated plants by the end of 1^st duration of drought stress (Fig 2). Drought impact was somehow managed and recovered in 10 days of water stressed plants than other treatments, the result was more pronounced in double stress of 3 days after 15 days re-watering, same trend of results observed for all growth parameters, where the plants were able to obtain more growth and the results showed recovery in the previous parameters. The number of leaves per plant increased compared to the first cycle of stress. The differences between treatments to that of control were less 13.67 at T5 and 14 at T8 where as in control 16.00 and 17.00 leaves per plant, respectively. Similarly, plant leaf area and leaf area index were observed to recover during the second stress period in plants under double drought stress cycles, where leaf area was 1045.97cm² at T8 with LAI 1.480 in contrast to its control 1981.43 cm²and 2.80 respectively. On the other hand, the increment in foliar growth resulted a decrease in the root-to-shoot ratio in the plants after 15 days of watering with 3 days of 2^nd cycle of stress, reaching its maximum at T6 was 0.33. The reduction in sugar beet growth under drought stress has been reported previously (Hamed and Emara, 2019), which was explained a significant reduction in leaf area index as a result of increasing water scarcity also match with current results of this investigation, indicated a reduction in leaf area and leaf area index under moderate to severe drought under 14-days of irrigation intervals (Mehanna et al., 2020, Majeed et al., 2016).When water availability is limited due to drought, root: shoot ratio increases because low water potentials cause more reduction in shoots than roots growth, presented the same in the sugar beet (Wu and Cosgrove, 2000, Khorshid et al., 2018).






 
Physiological parameters
 
In terms of chlorophyll stability index (CSI) %, the observation showed a gradual decrease with increasing drought duration, up to 71.58% at 20 days of stress in T4. This somehow recovered after 15 days of re-watering and reached 77.16% in T8. On the other hand, sugar beet plants showed a significant reduction in relative water content under water deficit conditions, from 81.18% at 5 days of stress to 51.29% at 20 days of stress, against the respective controls 85.58% and 86.73%. Imposing 2nd stress of 3 days water withholding did not cause further reduction in RWC, but the effect of the recovery period was clear in managing the water balance in the plants, as the results showed a slight increment in leaf RWC% as 79.25% for T8 compared to the control, 82.86% (Table 1). Under water deficit condition green pigments were reduced and photosynthetic activity also declined in terms of chlorophyll total (Loho et al., 2022), which was expressed in CSI%. Previous researches indicated a decrease in RWC after 7 to 9 days of stress (Wedeking et al., 2018). In the present study, RWC% gradually declined as the drought period increased but maximum decrease in RWC% in leaf was visible at longer drought duration. Reports explained that higher RWC% in leaves as an important characteristics of stress tolerance in plants (Shaw et al., 2002, Kebede et al., 2020).
 
Biochemical parameters
 
The results showed a significant accumulation of proline over the control in sugar beet leaves during the drought period, which increased with the duration of stress from 63.14% at 5 days to 276.36% at 20 days of single stress (Fig 3). Proline regulated ROS (hydroxyl radicles) to evoke resistance in plants under prolonged drought stress (Rana et al., 2017). After the recovery period of rewatering, the crop tolerated the second stress duration showing less proline accumulation compared to first stress cycle (Leufen et al., 2016). In this study it was observed that proline accumulation in sugar beet leaves was higher under long duration stress of 20 days, has been reported by many researchers (Loho et al., 2022).This increase in proline, combined with a gradual increase in total soluble carbohydrates in leaves upto 15 days of stress (92.78%) over respective control, then declined with prolonged stress exposure, reaching 24.55% at 20 days stress, explaining its utilization to overcome severe stress. The same trend was observed after the second stress cycle with rewatering (Fig 4). Plants can overcome the adverse effect of water stress by accumulating more TSC in their leaves (Rana et al., 2017). The current results are matched with a strong correlation with previous researches explained under drought stress (Ghaffari et al., 2019). More decrease in leaf starch% was resulted at 20 days of stress, also reflected in second round of stress (-1.61%) even after re-irrigation. The stress effect was recovered after irrigation in T5, T6 and T7, where short period of 3 days water withholding showing least effect (Fig 5). Exhibiting the tolerance activity, primary starch accumulation and its mobilization in the leaves are the most valuable factors within plant responses (Thalmann and Santelia, 2017). When plants growing older, starch accumulation is gradually declined in the leaves under long stress periods and the evidence showing its utilization towards metabolic and cellular adjustments under stress (Wedeking et al., 2018), which was observed in the present experiment during T7  and T8.






       
In case of sugar beet roots an increase in total soluble carbohydrates deposit also resulted after 15 days of re-watering. In root at harvest, reduction in TSC% over control was observed under long exposure (15 and 20 days) to drought stress along with summer climatic temperature. More accumulation of TSC % was observed in T6, T7 and T8 as 126.98%, 106.36% and 21.53% respectively, showing its growth maturity and stress recovery over control (Fig 6). The starch% in root tubers reduced against control in all types of stress durations. Percentage increased in starch was less decreased in roots of 10 days (T2) stressed plants (-3.30%) and the same also resulted in 2^nd phase of stressed plants in T6 (-1.32%) than other treatments exhibiting their tolerance against drought durations (Fig 7). Sugar beet has no such specific self-regulatory mechanism to enhance sugar accumulation in their roots which was observed in the total sugar accumulation and starch content reduction under long duration of drought stress as 15 and 20 days of dryness, even observed at harvest, but is affected by external stimuli based on climatic factors to a great extent (Petkeviciene, 2009).
 




Yield
 
More than 50% of yield reduction was observed in both biological and economic yield of 20 days stressed plants (18.65 t/ha and 13.92 t/ha) compared to control (37.23 t/ha and 27.65 t/ha) respectively. After 15 days of re-watering with 3-days stress, the 10days stressed plants were managed their economic yield to 26.304 t/ha compared to the control 27.65 t/ha. Harvest index from treatments was mostly coincide with normal watering plants and non-significantly increased after re-watering (Fig 8). The depletion in sugar beet yield under drought conditions has been noted in previous findings (Mehanna et al., 2020). The results implies that sugar beet plants had the ability to manage their biomass accumulation under drought stress. Economic Yield and quality of the product is the complex set of interactions, expressed during crop growth and development due to genetic, agronomic and environmental factors which influence sugar beet yield (Brar et al., 2015). Temperature more than 30^oC retards yield and quality of sugar beet root (Singh et al., 2019), which was observed throughout the experimental period and reflected in yield results at harvest with respective duration of drought regimes.

Imposing drought stress on sugar beet plants restricted their growth, affecting plant water relations, pigment stability and yield. However, the plants coped with water scarcity by accumulating more osmolytes, such as proline and carbohydrates and using the accumulated starch as an energy source to withstand the condition. Long duration stress affected sugar beet performances and these harmful effects reduced after 15-days recovery period of normal watering, leading to less significant effects from the shorter period of second drought duration, where the plants largely recovered and returned to normal conditions in the studied parameters.  The tropical sugar beet LS-6 variety can be considered as moderately tolerant to water deficit, even in summer condition, showing good performance under drought up to 10 days of withheld irrigation and exhibiting the ability to recover after rewatering.
 

The authors would like to extend their gratitude to the Indian Institute of sugar cane research (IISR) Lucknow, India for providing the seeds for this research and the Centre of Tuber Crops Research Institute (ICAR- CTCRI) Bhubaneswar, India for their support in this research.
 
Disclaimers
 
All views or claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.