Impact of Drought on Chlorophyll, Soluble Protein, Abscisic Acid, Yield and Quality Characters of Contrasting Genotypes of Tomato ( Solanum lycopersicum )

Impact of drought stress on chlorophyll, chlorophyll fluorescence (Fv/Fm), chlorophyll stability index (CSI), soluble protein, abscisic acid (ABA), yield and quality of tomato ( Solanum lycopersicum ) genotypes was investigated for the assessment of drought tolerance under field conditions in rainout shelter. The drought condition was created first day from transplanting based on Irrigation water (IW):Cumulative Pan Evaporation (CPE) of soil. Experiment was laid out with 10 genotypes by adopting FRBD with three replications and two treatments of 1 IW:CPE and 0.5 IW:CPE. The result revealed that the reductions in chlorophyll content, Fv/Fm, chlorophyll stability index (CSI), soluble protein and yield were noticed at drought condition (0.5 IW/CPE). The genotypes LE 114, LE 57, and LE 118 which showed significantly less reduction in the above parameters during drought were considered as drought tolerant. ABA content and quality characters such as total soluble solids (TSS), lycopene content were increased under drought condition. Genotypes LE 1 and LE 125 which recorded the lowest chlorophyll content, Fv/Fm, CSI, soluble protein and higher ABA content ultimately poor yield were considered as drought susceptible.


INTRODUCTION
Drought is the major inevitable and recurring feature of semi-arid tropics and despite our improved ability to predict their onset, duration and impact, crop scientists are still concerned about it as it remains the single most important factor affecting the yield potentials of crop species. It is one of the serious environmental factor affecting plant growth, yield, and quality. It induces various physiological and biochemical adaptations in plants. Drought is one of the most important factors for yield reduction in the majority of the cultivated areas, affected 40 to 60% of the world's agriculture lands [1].
Water deficit leads to the perturbation of most of the physiological and biochemical processes and consequently reduces plant growth and yield [2]. Gladden et al. [3] showed that water deficit earlier in the growth of tomato caused a significant reduction in leaf chlorophyll content. Abdellah et al. [4] recorded the highest reduction in the chlorophyll content in susceptible wheat cultivar under water stress of 30% FC. Water stress reduced the total chlorophyll content significantly in different genotypes of moth bean and reduction was more pronounced in late flowering genotypes [5]. Sanadhya et al. [6] reported that the water stress reduced the chlorophyll content and hill activity with increased levels of stress in mung bean.
There was a reduction of only 1.3% and 2.2% in Fv/Fm under moderate and severe stress compared to control in Withania somnifera [7]. Chlorophyll fluorescence emission well on the level of water stress and, thus, can be used to identify elevated drought tolerance in tomato for selection of resistant genotypes [8]. Decreased chlorophyll content and chlorophyll stability index under both moisture stress and temperature stress were found by Sairam et al. [9] in wheat.
Daniel and Triboi [10] showed that heat stress decreased the duration of soluble protein accumulation in terms of days after anthesis but not in terms of thermal time. Few studies have investigated the combined influence of drought and heat stress on nitrogen metabolism. Abdellah et al. [4] reported that the increased ABA content was observed in wheat cultivar by water stress (30% FC) over control. Under intense water stress, the concentrations of ABA in plants increases, which trigger a number of processes starting from decrease in turgor pressure, decline in cellular expansion and stomatal closure to reduce water loss in leaves [11].
[12] studied the physiological parameter governing drought tolerance in maize and recorded more than 80 per cent reduction in yield in highly susceptible lines while in relatively tolerant genotypes reduction was up to 50 per cent. Manojkumar et al. [13] reported that water stressed tomato plants showed significant difference in the TSS level at different irrigation levels. As the irrigation frequency increased TSS level decreased. Maximum per-cent TSS was observed under IW/CPE ratio of 0.60 (6.10%) and the minimum was recorded at the IW/CPE ratio of 1.20 (4.80%). The fruit quality improvement was observed under water deficit condition in tomato as a result of the synthesis of ascorbic acid, citric acid and malic acid [14].
Tomato (Solanum lycopersicum) is one of the most popular and widely grown vegetables in the world. Considering the potentiality of this crop, there is plenty of scope for its improvement, especially under the drought situation. Some of the adaptive mechanisms of plants to drought stress, which do not decrease plant yield to a greater extent, assume greater importance. There are several physiological and biochemical traits contributing to the drought tolerance of horticultural crops. However, a large number of tomato genotypes have not been screened for drought tolerance or exploited for their cultivation under drought situation and field condition.
To breed drought tolerant genotypes, it is necessary to identify physiological traits of plants, which contributes to drought tolerance. Therefore, the present investigation was carried out to study the chlorophyll characters, soluble protein and ABA to facilitate the screening and selection of tomato genotypes for drought tolerance.

MATERIALS AND METHODS
The study was undertaken to find out effect of drought on chlorophyll characters, soluble protein, ABA, yield and quality in tomato in the field experiment at Rainout Shelter of Crop Physiology Department, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu. The experiment was conducted with ten tomato genotypes viz., LE 1, LE 27, LE 57, LE 114, LE 118, LE 125, CO 3, PKM 1, TH CO 2 and TNAU TH CO 3 and two treatments viz., 1.0 IW/CPE and 0.5 IW/CPE with three replications. Seeds of selected genotypes were sown in trays filled with vermicompost for nursery. Twenty five days old seedlings were transplanted and drought was imposed at first day after transplanting onwards based on IW/CPE, 0.5 IW/CPE for drought stress and 1.0 IW/CPE for control were maintained by irrigation the field at regular interval based cumulative pan evaporation. Crop was supplied with fertilizers and other cultivation operations including plant protection measures as per recommended package of practices of Tamil Nadu Agricultural University, Coimbatore. All the observations were recorded on third leaf from top at 60 DAT. The experiment was laid out in factorial randomized block design with three replications.

Chlorophyll Characters
Total chlorophyll content was estimated following the method suggested by Arnon [15] and expressed as mg g -1 . 250 mg of fresh leaf sample was weighed and transferred to a pestle and mortar. The sample was macerated with 10 ml of 80% Acetone. The content was centrifuged at 3000 rpm for 10 minutes. After centrifuge, the supernatant was collected and made up the volume to 25 ml by using 80% acetone. The optical density was measured at 652 nm in a spectrophotometer.
Chlorophyll fluorescence measurements were recorded using Plant Efficiency Analyzer (Hansatech, UK) following the method advocated by Lu and Zhang [16]. Measurements were made on intact leaves, which were dark adapted for 30 min prior to measurement. The minimal fluorescence level (F 0 ) with all PS II reaction centers open was assessed by measuring the modulated light, which was sufficiently low (< 0.1 µmol m -2 s -1 ) not to induce any significant variable fluorescence. The maximal fluorescence level (Fm) with all PS II reaction centers closed were determined by a 0.8 s saturating pulse at 8000 µmol m -2 s -1 in dark adapted leaves [17]. Using light and dark fluorescence parameters, the maximal efficiency of PS II photochemistry in the dark adapted state, Fv/Fm = (Fm-Fo) / Fm [18] was calculated.
Estimation of CSI was carried out based on the protocol of Koleyoras [19]. Two clean test tubes (Control and treatment) were taken. Two 250 mg of leaf samples were weighed and cut into 8 to 10 leaf bits and transferred to test tubes. 20 ml of distilled water to control tube and 20 ml of hot water (55°C) to treatment test tube were added. The treatment tube was kept in a hot water bath for exactly 30 minutes control tube in the lab condition. After the completion of the reaction time, the leaf bits were taken out from the test tube and macerated with 10 ml of 80% acetone. The contents were centrifuged at 3000 rpm for 10 minutes. The supernatant was collected and made up the volume to 25 ml by using 80% acetone. OD was measured at 652 nm in a spectrophotometer and total chlorophyll content of control and treated samples were calculated. CSI expressed in terms of per cent by using following formula. Chlorophyll stability index (CSI) = Total chlorophyll content (Treated)/Total chlorophyll content (Control) X 100.

Estimation of Protein and ABA Content
Soluble protein content of leaf was estimated as per the method of Lowry et al. [20]. 250 mg of leaf sample was weighed and macerated with 10 ml of phosphate buffer solution. The content was centrifuged at 3000 rpm for 10 minutes and the supernatant was collected and made up to 25 ml. 1 ml of the supernatant was pipette out to a test tube and 5 ml of alkaline copper tartarate reagent and 0.5 ml of folin reagent were added. The colour intensity was measured at 660 nm in spectrophotometer and the amount of soluble protein present in the sample was calculated by using bovine serum albumin as standard and expressed as mg g -1 fresh weight.
Quantification of abscisic acid was done by using the instrument HPLC cyber lab with the column of RP 18 (4.6 mm ID x 250 mm) and mobile phase of acetonitrile (60) and water (40) by adopting the protocol of Krochko et al. [21]. Leaf samples were powdered and representative sample (10 g) in triplicate was extracted by homogenizing with extracted using 40 ml of 80 per cent chilled methanol for 30 min at 4°C. The mixture was filtered in a separate conical flask using Whatman filter paper No. A volume of 20 µL of sample was injected into HPLC. The elution was carried out by a binary gradient of 60 per cent HPLC grade acetonitrile for 20 minute at the flow rate of 1 mL min -1 . The column elutes were passed through an UV detector set at 254 nm and the ABA were estimated measuring the peak area and comparing with standard curve of hormones. The peak areas were measured and ABA concentration quantified using the standard curve obtained from ABA.

Yield and Quality Characters
The total weight of fruits harvested from each plant of all picking was added and average yield per plant was worked out and expressed in gram per plant. Later the yield per hectare was calculated and expressed as tonnes per hectare.
Drop of juice extracted from cut fruit was used to determine TSS with the help of Hand Refractometer (0 to 32°Brix) at room temperature and the value was noted in °Brix. Lycopene content of fruit was extracted by using petroleum ether and OD of the extract was measured at 503 nm in UV-VISspectrophotometer using petroleum ether as a blank [22].
Lycopene content of the sample was calculated by using the following formula and expressed in mg 100 g The data on various parameters were analyzed statistically as per the procedure suggested by Gomez and Gomez [23]. Wherever the treatment differences are found significant, critical differences were worked out at five per cent probability level and the values were furnished and discussed.

Impact of Drought on Chlorophyll Characters
The intensity of the greenness in terms of chlorophyll content of the plant had influenced the photosynthetic rate and thereby the efficiency of the plant for increased biomass production. Chlorophyll content in terms of SPAD values can be used for evaluation for the response of plant species to the drought and heat stresses in the field [24]. Ma et al. [25] reported a highly significant correlation of chlorophyll in terms of SPAD value with photosynthetic rate in soybean and Kapotis et al. [26] in weed species (Amaranthus viltus L.). In the present study, the adverse effect of drought on greenness of the leaf could be observed through about 23.48% reduction in mean total chlorophyll content. The reduction of chlorophyll content under drought might be due to the fact that drought stress blemishes the chlorophyll content through causing internal modification in the thylakoid membrane.
Among the genotypes, highest reduction of total chlorophyll content was recorded in the genotype LE 1 (34.76%) followed by LE 125 (33.10%) and CO TH 2 (31.65%) under drought ( Table 1). The present study also indicated the ability of the genotypes LE 57 (18.79%), LE114 (19.65%) and LE 118 (21.37%) in maintaining total chlorophyll content under drought (0.5 IW/CPE) by showing less reduction. Therefore, these genotypes were able to endure drought injury better than the sensitive lines. Similar to this finding, Ghaffari et al. [27] stated that the tolerant sunflower line had higher chlorophyll than the susceptible line under drought. These findings are in agreement with the earlier findings of Petcu et al. [28] in sunflower.
A considerable reduction in chlorophyll fluorescence (Fv/Fm) was observed due to the drought treatment. A possible reason for this effect is that the drought stressed plants have lower capacity for the use of transported electrons and their electron transport chain is more reduced at any light condition [29].
For the treatments, smaller mean fluorescence value (0.63) was registered by 0.5 IW/CPE with the reduction of 25.88 per cent than 1.0 IW/CPE (0.85). Relating to the genotypes, LE 57 was significantly superior chlorophyll fluorescence value (0.74) followed by LE 118 and LE 27 while the lowest was recorded by LE 125 (0.47). The genotype, LE 57 proved its supremacy with less reduction (20.69%) of Fv/Fm followed by LE 118 (20.69%) ( Table 1). The high Fv/Fm ratio indicates that genotype has more efficient in protecting their photosynthetic apparatus under drought. This result is in agreement with Mishraa et al. [8] in tomato. Lower values of Fv/Fm ratio under drought, indicated an injury to electron transfer system in photo system II, causing an imbalance between generation and utilization of electrons, resulting changes of quantum yield efficiency [30].
Chlorophyll Stability Index (CSI) is an indicator of the stress tolerance capacity of the plants and is a measure of integrity of membrane [31]. A higher CSI helps the plants to withstand stress through better availability of chlorophyll, leading to increased photosynthetic rate, more dry matter production and higher productivity. Kilen and Andrew [32] observed a high correlation between CSI and drought tolerance in corn.

Drought
condition aggravates chlorophyll degradation in later part of growth due to loss of membrane compartmentation. Membrane stability index decreased significantly under water stress condition over control in wheat varieties [33].
In the present study also corroborates the earlier findings with 18.49% reduction of CSI in drought (0.5 IW/CPE) compared to 1.0 IW/CPE. The primary effect of drought at the cellular level is to affect the integrity of membrane which in turn leads to disruption of cellular compartment ultimately destruction chlorophyll contents. The earlier findings of Fariduddin et al. [34] confirm the present study.
The lowest reduction of CSI was observed in the genotypes LE 114 (14.68%) followed by LE 118 (15.46%) while the highest reduction was showed by LE 125 (24.73%) and CO TH 2 (24.29%) under drought condition ( Table 2). The ability of the genotype maintained the higher CSI under drought is a desirable character for tolerance. Maintenance of CSI at drought condition by the genotype might be due to high membrane stability. Beena et al. [35] reported that high membrane stability index and chlorophyll stability index were recorded in tolerant inbred lines of rice than in susceptible lines under water stress condition.

Impact of Drought on Soluble Protein
The soluble protein content of the leaf, being a measure of Rubisco activity was considered as an index for photosynthetic efficiency due to the important enzyme involved in photosynthesis. Rubisco enzyme forms nearly 80 per cent of the soluble proteins in leaves of many plants [36]. Diethelm and Shibles [37] opined that the Rubisco content per unit leaf area was positively correlated with that of soluble protein content of the leaf. The amount of Rubisco in leaves is controlled by the rate of synthesis and degradation. Even under drought stress the Rubisco holo enzyme is relatively stable with a half-life of several days [38].  However, drought stress in tomato [39], arabidopsis [40] and rice [41] leads to a rapid decrease in the abundance of Rubisco small subunit (rbcS) transcripts, which may indicate decreased synthesis. In the present study also confirms the earlier findings with 32.28% reduction of soluble protein content under drought. The reduction of soluble protein content might be due to the degradation of available soluble protein in plant and reduction of synthesis of new protein.
Among the genotypes, CO TH 2 (15.63) and TH CO 3 (15.18) registered highest soluble protein content at under 1.0 IW/CPE ratio level. During drought (0.5 IW/CPE), LE 57 recorded significantly superior soluble protein content (11.99), however the genotype LE 118 proved its endurance to water deficit with less reduction (19.48%) and LE 125 showed highest reduction of 52.66%. Biochemical limitations of photosynthetic carbon fixation by the inhibition of Rubisco activity play an important role mostly under conditions of prolonged or more severe drought [42,43]. Maintenance of soluble protein content by the genotypes could be attributed to higher rubisco activity leads to more carbon fixation and ultimately to higher photosynthetic efficiency under drought is one of the important traits for drought tolerance.

Impact of Drought on ABA Content
It was found a significant per cent increment of ABA content in leaf under drought condition (39.45%) over control. The increment of ABA content under drought condition was reported by several workers [4,11,44]. Accumulation of ABA under drought condition is a favourable mechanism for drought tolerance through reducing transpiration rate by closing of stomata. However, complete closure of stomata leads to increment of leaf temperature which produces reactive oxygen species ultimately death of the plant.
Among the genotypes, the elevation in ABA was less in LE 114 (24%) under drought, whereas nearly double fold increment of ABA content was observed in LE 125 and LE 1 (Fig. 1). ABA synthesized in response to drought stress, is known to induce stomatal closure which leads to reduced transpirational water loss [45]. In the present study, LE 1 and LE 125 showed higher ABA content which ultimately recorded less transpiration rate by closing of stomata. However, the genotype LE 114 showed a moderate increment of leaf ABA content leads to partial closure of stomata with maintains the photosynthetic rate and leaf temperature. Hence, both the physiological characters are important for drought tolerance. The present study is in agreement with earlier findings of Wang and Huang [46], who reported that highly significant negative correlation between ABA content and leaf water potential, stomatal conductance, transpiration rate and net photosynthetic rate.

Impact of Drought on Yield Characters
Comparing two treatments, plants received 1 A significantly lesser reduction of 32.49% was exhibited by LE 118 followed by LE 57 (33.13%) and LE 114 (38.55%) showing their tolerance nature to drought stress. Therefore, it could be clearly revealed that water deficit as the result of drying soil caused a major adverse effect on yield and yield components even in tolerant genotypes. The reduction in fruit yield and related parameters under drought probably due to reduction of water content in plant which disrupting leaf gas exchange properties which   [48]. Izzeldin et al. [49] also explained that the impact of drought before the time of flowering affects the reproductive system with the increasing sterility of flowers, so that flowering and fruiting will fail if the water shortage is prolonged.

Impact of Drought on Quality Characters
Plants imposed with 0.5 IW/CPE ratio recorded higher Total Soluble Solids (TSS: o Brix) brix value (3.01) than 1.0 IW/CPE ratio (2.89). Among the genotypes, TH CO 3 recorded higher average brix value of 4.00 than the rest of the genotypes. At 0.5 IW/CPE ratio condition, the highest TSS value was recorded by TH CO 3 (4.1) followed by CO TH 2 (3.9), PKM 1 (3.6) and CO 3 (3.4) while the lowest was registered by LE 125 (2.2). Regarding treatments, plants imposed with 0.5 IW/CPE ratio recorded higher lycopene content (3.23) than 1.0 IW/CPE ratio (3.02). With respect to the genotypes, CO 3 recorded significantly higher average lycopene content (4.69). Hence, the present study indicated that the quality parameters like TSS and lycopene increased slightly under drought compared to control.
Present study corroborates with early findings of Ali et al. [50] in tomato. Nahar et al. [51] also explained that the fruit quality improvement under water deficit condition in tomato might be due to the synthesis of ascorbic acid, citric acid and malic acid. In the present study, LE 118, LE 57 and LE 27 showed their primacy with highest increment of TSS and lycopene content. This finding was strongly supported by Tambussi et al. [52] and it was also explained that the increase in lycopene and TSS might be an effective strategy to protect membranes from oxidative damage in water stressed condition.

CONCLUSION
Water stress causes detrimental effects on plant activities, which are likely to alter the yielding potential of the crops. Hence, to identify the physiological parameters, which get altered under drought conditions is pre-requisite to evaluate drought tolerant varieties. It is concluded that the tomato genotypes LE 118, LE 57 and LE 114 were identified as the most tolerant lines to drought stress imposed provided with Rainout shelter. As the genotypes LE 125 and LE 1 recorded significantly lesser yield under the same condition, these two genotypes were considered as susceptible to water deficit.