Atlas Journal of Biology 2016, pp. 267–273 doi: 10.5147/ajb.2016.0140 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Influence of Drought Stress on Several Root Traits and their Correlation with Seed Protein and Oil Contents in Soybean Ambrocio Zenis1, Stella Kantartzi2, Khalid Meksem2, and My Abdelmajid Kassem1* 1 Plant Genetics, Genomics, and Biotechnology Lab, Department of Biological Sciences, Fayetteville State Uni- versity, Fayetteville, NC 28301, USA; 2 Department of Plant, Soil, and Agricultural Systems, Southern Illinois University, Carbondale, IL 62901, USA. Received: May 2, 2016 / Accepted: June 3, 2016 __________________________________________________ * Corresponding author: mkvzant@siu.edu 267 Abstract The important crop soybean [Glycine max (L.) Merr.] is cul- tivated worldwide and the US is its number one exporter. However, farmers face many challenges in cultivating soy- beans, including drought and diseases that reduce yields drastically. The root system is very important for plants, in- cluding crops, because it receives water and minerals from the soil so that the plant/crop can photosynthesize, grow, and increase its yield. The objective of this study was to grow the ‘MD 96-5722’ by ‘Spencer’ recombinant inbred line (RIL) population (n=86) in the greenhouse under normal (Group I) and drought stress (Group II) conditions, and compare the root length (RL), root surface area (RSA), average root diam- eter (ARD), and average root volume (ARV) in the two groups of plants. WhinRhizo software was used to measure the root traits and SPSSTM was used to evaluate population perfor- mance under normal and drought conditions. JMPTM was used to compare the root traits under normal and drought conditions, and to analyze the correlation between root traits, protein and oil contents. The results showed that there is a huge variation in these traits among the parents ‘MD 96-5722’ and ‘Spencer’, and among their RILs. For Group I plants, the RL of parents and RILs ranged from 20.67 cm to 2,327.88 cm; the RSA ranged from 4.57 cm2 to 1,176.79 cm2; the ARD ranged from 0.38 mm to 4.04 mm; and the ARV ranged from 0.08 cm3 to 47.34 cm3. For Group II plants, the RL of parents and RILs ranged from 15.70 cm to 3,562.42 cm; the RSA ranged from 4.15 cm2 to 829.72 cm2; the ARD ranged from 0.24 mm to 5.74 mm; and the ARV ranged from 0.03 cm3 to 23.67 cm3. It is clear from the results that Group Introduction Soybean [Glycine max (L.) Merr.] is a Leguminosae crop grown for its oil, proteins, isoflavones, and other bioactive compounds. It also widely used in the food industry and as a biofuel (Rosenthal et al., 2001; Kinney and Clemente, 2005). According to USDA, North Carolina planted soybeans in 1.82 Million acres and produced 57.3 Million Bushels for a total of $487 Million in 2015 (USDA, 2015). In North Carolina, soybean production amount was 31 million bushels from 1.4 million acres and a total value of $174 million (NCSUCE, 2015). Soybeans are usually planted in early soybean production system (ESPS) This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecom- mons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the origi- nal work is properly cited. I plants have higher means of RL [572.58 cm vs. 537.33 cm], RSA [201.20 cm2 vs. 165.50 cm2], ARD [3.96 mm vs. 1.45 mm], and ARV [6.31 cm3 vs. 5.61 cm3] compared to Group II plants which demonstrates that drought-stressed plants have reduced overall plant growth and development. However, statistically, these differences were not significant; therefore, further studies with several replicates should be conducted both in the greenhouse and the field in order to determine the effects of drought stress on the ‘MD 96-5722’ by ‘Spencer’ RILs. Moreover, studies of quantitative trait loci (QTL) map- ping of the root traits studied here are underway to genetically map QTL for these root traits in this soybean RIL population. Keywords: Root length (RL), Root surface area (RSA), Average root diameter (ARD), Average root volume (ARV), MD 96-5722, Spencer. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 268 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) (April) or conventional soybean production system (CSPS, May- June) across the US (Bowers, 1995; Taylor et al., 2005; Ouertani et al., 2011). Soybean plants grown in ESPS have increased yield and are drought-tolerant compared to plants grown in CSPS (Taylor et al., 2005; Ouertani et al., 2011). Plant breeders and researchers are trying to develop culti- vars that are resistant to diseases and drought stress. Drought stress reduces the ability of roots to absorb water and nutrients from the soil and it has been demonstrated that plants with vig- orous and extensive root systems are able to cope with drought and become water deficit-tolerant (WDT) (Pandey et al., 1984; Benjamin and Nielsen, 2006; Reubens et al., 2006). During drought stress seasons, plants usually change the distribution of their roots and grow them deeper to absorb water and miner- als as a mechanism of drought tolerance (Pandey et al., 1984; Benjamin and Nielsen, 2006). In another study, drought-stressed soybean plants have a sharp decrease in their photosynthetic rates, leaf water potentials, starch concentration, and leaf su- crose contents, and pod growth (Liu et al., 2004). Defending themselves against drought stress, soybean plants close their stomata to escape dehydration but the overall pho- tosynthetic rate is reduced by limiting CO2 intake (via stomata), causing a reduced metabolism that might be reversible with enough supply or availability (Kramer et al., 1995; Specht et al., 2001; Flexas and Medrano, 2002; Liu et al., 2004; Greg- ory, 2006; Mastrodomenico et al., 2013). The reduced photo- synthetic rate has a drastic effect on reducing grain yield. The physiological effects of drought can be estimated by measuring the leaf relative water contents (RWC), root architecture, and reactive oxygen species (ROS) (Hunt et al., 1987; Reddy et al., 2004; Chiante et al., 2006; Reubens et al., 2006; Miller et al., 2010; Comas et al., 2013). During seed fill and pod develop- ment stages, soybean plants, like other crops, need an adequate supply of water and any drought stress will decrease devel- opmental and physiological functions that will lead to high de- creased seed yields (Kadhem et al., 1985; Kramer et al., 1995; Chiante et al., 2006; Comas et al., 2013). To withstand drought stress, soybean plants and other legumes use several important mechanisms. Among these are growing extensive root systems, water use efficiency, and high efficient nitrogen fixation (Nunberg et al., 2006; Mastrodomenico et al., 2013; Hussain et al., 2014). It is known that drought-stressed plants tend to grow extensive root systems with continuous elon- gation at low root water potentials (Westgate and Boyer, 1985; Kramer et al., 1995; Bing et al., 2005; Nunberg et al., 2006; Mastrodomenico et al., 2013; Hussain et al., 2014). The objectives of this study are to (1) grow the ‘MD 96-5722’ by ‘Spencer’ recombinant inbred line population (n=86) of soy- bean under drought stress in the greenhouse, study the influence of drought stress on several root traits such as root length (RL), root surface area (RSA), average root diameter (ARD), and av- erage root volume (ARV), and (2) investigate the correlation be- tween root trait measurements, protein and oil contents of ‘MD 96-5722’ by ‘Spencer’. Materials and Methods Plant Material In this study, the ‘MD 96-5722’ by ‘Spencer’ recombinant in- bred line (RIL) population (n=86) of soybean was used (Akond et al., 2013). These seeds were obtained at the National Soy- bean Research Laboratory (NSRL) in 2006 from Dr. Kantart- zi. The parents of ‘MD 96-5722’ were from a cross between KS4644 and ‘Corsica’ KS4694, whose parents were ‘Shermon and Toano’. ‘Spencer’ came from the crossings between A75- 305022 by ‘Century’ (Akond et al., 2013). Experimental Conditions The experiment was performed during 2013 (June 2013 to September 2013) in a Greenhouse at Fayetteville State Uni- versity, Fayetteville, North Carolina. Four seeds of parents, ‘MD 96-5722’, ‘Spencer’, and each RIL (n=86; n is the population size) were sown at 1.5–2.0 inches deep in 2-gallon pots (20.32 x 17.78 cm) filled with potting soil of organic material (Miracle Grow potting mix) under daylight temperatures of 24–34o C. The pots were set on a brown sheet, avoiding water capillar- ity from the bottom. The pots were divided into two groups, a control group (n=86) for normal growth and a treatment group (n=86) for drought conditions. The treatment group would un- dergo drought stress during the entire growth development. First week, normal watering was given to each seedling, 3.76 liters of water. Control group would receive normal watering throughout the whole study or when needed. Treatment group was submit- ted through intense drought stress for two weeks after normal watering. Afterwards, watering the plants at least once a week and at 1.5 liters of water. Root Extraction Method After eight weeks of planting, roots were carefully extracted from the pots one by one, placed inside a tray filled with wa- ter and washed by hand. Later, the roots were left to dry and have just enough moisture in order to separate the lateral roots from the primary root. Roots were separated by using a plastic transparent wrap, the wraps texture allowed for lateral roots to separate easily without damaging the root and minimizing loss of fine lateral roots. Afterwards, to preserve the roots, they were placed inside Ziploc bags and placed in a refrigerator for later analysis. Several root traits such as root length (RL), root surface area (RSA), average root diameter (ARD), and average root volume (ARV) were measured. Protein and Oil Contents Quantification Seed protein and oil contents have been measured by Nacer Bellaloui at the USDA-ARS, Stoneville, MS according to Bellaloui et al. (2009). Briefly, 25 g of seed from each plant were grind- ed using a Lab Mill 3600 (Perten, Springfield, IL). Near infrared reflectance was used for analyses using a diode array feed an- alyzer AD 7200 (Perten, Springfield, IL) (Bellaloui et al., 2009). A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Root Trait Analysis The root traits root length (RL), root surface area (RSA), aver- age root diameter (ARD), and average root volume (ARV) were measured in both groups (Control and Experimental Groups) using an Epson Scanner and the WhinRhizo software Package 3.10 version (Regent Instruments Inc. Quebec, Canada). The im- ages were visualized and root data analyzed using the Epson Scanner and the WhinRhizo software. Root traits analyzed were root length (RL), root surface area (RSA), average root diameter (ARD), and average root volume (ARV). Statistical Analysis A distribution chart was constructed based on the differ- ences in root trait measurements in (RIL) population. SPSSTM statistical software package was used to evaluate population performance under normal and drought conditions. The evalu- ation was based on mean differences, estimation of variance and descriptive comparisons. JMPTM was used to compare the root length (RL), root surface area (RSA), and average root di- ameter (ARD) and average root volume (ARV) under normal and drought conditions. Also, JMPTM statistical software package was used to analyze the correlation between root traits, protein and oil content. Recombinant inbred lines of the parents were also compared along with the RILs evaluation. T-Test and Pear- son correlations were performed to compare the root traits in plants grown in the two conditions and between the root traits and protein and oil contents. Results and Discussion Comparison of Root Traits Data Examples of scanned images of the root system of RIL 2 un- der normal water regime and drought stress are shown in Figure 1. Measurements of root length (RL), root surface area (RSA), average root diameter (ARD), and average root volume (ARV) of ‘MD 96-5722 by ‘Spencer’ recombinant inbred lines root sys- tems under normal (Group I) and drought (Group II) growth con- ditions have been recorded. For control plants (Group I), the root length (RL) of parents and RILs ranged from 20.67 cm (RIL 63) to 2,327.88 cm (RIL 2); the root surface area (RSA) parents and RILs ranged from 4.57 cm2 (RIL 41) to 1,176.79 cm2 (RIL 2); the average root diameter of parents and RILs ranged from 0.38 mm (RIL 80) to 4.04 mm (RIL 43); and the average root volume (ARV) of parents and RILs ranged from 0.08 cm3 (RIL 41) to 47.34 cm3 (RIL 2). For drought-stressed plants (Group II), the root length (RL) of parents and RILs ranged from 15.70 cm (RIL 80) to 3,562.42 cm (MD, 2,497.9 cm for RIL 87); the root surface area (RSA) par- ents and RILs ranged from 4.15 cm2 (RIL 46) to 829.72 cm2 (MD, 669.62 cm2 for RIL 87); the average root diameter of parents and RILs ranged from 0.24 mm (RIL 7) to 5.74 mm (RIL 80); and the average root volume (ARV) of parents and RILs ranged from 0.03 cm3 (RIL 46) to 23.67 cm3 (RIL 32). Our results showed that both RL, RSA, ARD, and ARD have been significantly reduced by drought stress which is in agree- ment with several studies reporting the influence of drought on root traits (Xiong et al., 2006, Pandey and Shukla 2015). For example, Xiong et al. (2006) reported that drought stress re- duced significantly lateral root growth in Arabidopsis thaliana as a mechanism of drought adaptation (Xiong et al., 2006). Ga- ray and Wilhelm (1982) studies the effect of drought stress on root traits of two soybean isolines of ‘Harosoy’ (Harosoy normal (HN) and Harosoy dense (HD)) and found that the root systems have been concentrated in the upper 0.15 m of the soil. How- ever, after one month of drought stress, the root systems were denser at 0.90-1.2 m layer of the soil which means that the root system grew deeper into the soil to pump more water and mineral (Garay and Wilhelm, 1982). However, another study reported effect of drought stress on root distribution (Benjamin and Nielsen, 2006). Statistical Data Analysis Root traits data for the two groups (Control plants – Group I and Drought Stressed plants – Group II) were analyzed for the mean values, standard deviation, range, and coefficient of variation and the results are shown in Table 1. In Group I plants (normal water conditions), the mean of the average of root length (RL) was 572.58 cm with a coefficient of variance of (CV) of 0.95; the mean of the average of root sur- face area (RSA) was 201.20 cm2 with a coefficient of variance of (CV) of 1.13; the mean of the average root diameter (ARD) was 3.96 cm with a coefficient of variance of (CV) of 6.01; and the mean of the average root volume (ARV) was 6.3 cm3 with a coefficient of variance of (CV) of 8.02 (Table 1). In Group II plants (water stress conditions), the mean of the average of root length (RL) was 537.33 cm with a coefficient of variance of (CV) of 1.25; the mean of the average of root surface area (RSA) was 165.5 cm2 with a coefficient of variance of (CV) of 1.01; the mean of the average root diameter (ARD) was 1.45 cm with a coefficient of variance of (CV) of 7.89; and the mean of the average root volume (ARV) was 5.6 cm3 with a coefficient of variance of (CV) of 1.03 (Table 1). It is clear from the results that Group I plants have higher means of root length (RL) [572.58 cm vs. 537.33 cm], root sur- face area (RSA) [201.20 cm2 vs. 165.50 cm2], average root di- 269 (A) (B) Figure 1. Scanned images of the root system of RIL 2 under normal water regime (A) and drought stress (B). A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) ameter (ARD) [3.96 mm vs. 1.45 mm], and average root volume (ARV) [6.31 cm3 vs. 5.61 cm3] compared to Group II plants (Table 1) which demonstrate that drought-stressed plants have reduced overall plant growth and development. A paired-samples t-Test shown in Table 2 was used to dem- onstrate whether there was a significant mean difference of root measurements (RL, RSA, ARD, ARV) between normal and drought growth in ‘MD 96-5722’ by ‘Spencer’ population planted under greenhouse conditions. Levels of root trait measurements for root length elicited a difference of 58.60821 (95% Confidence Inter- val (CI), -167.15727 to 284.37370) cm when comparing normal and drought growth conditions, t (61) = .519, p > .005. The comparison for root surface area (cm2) displayed a difference of 42.32496 (95% CI, -34.88817 to 119.53809) and a t (61) = 1.096, p > .005. The average root diameter in comparison showed a difference of -.30523 (95% CI, -.63563 to .02518) mm and t (61) = -1.847, p < .005. Last, when comparing aver- age root volume, the measurements demonstrated a difference of 1.06339 (95% CI, -1.56043 to 3.68721) cm3 with a t (61) = .810, p > .005. There is no significant difference between root length, root surface area, and average root volume. However, there was statistical difference between normal and drought growth conditions for average root diameter. A paired statistic test was used to determine the different effects between two experimental conditions for each of the four root traits. In (Table 2), results indicate a decrease for root measurements of length (cm) (529.14 -77.299), surface area (cm2) (167.59- 20.069), root volume (cm3) (5.66- 0.719), and an increase in average diameter (1.42-0.140). In normal growth conditions there was a slight increase in measurements in length (587.75-72.141), 270 Root Traits N Range Minimum Maximum Mean SD CV% Normal Growth Length (cm) 69 2307.201 20.679 2327.881 572.580 548.901 0.958 Surface Area (cm2) 69 1172.221 4.572 1176.793 201.208 227.754 1.131 Average Diameter (mm) 69 3.653 0.388 4.042 1.090 0.537 4.927 Average Root Volume (cm3) 69 47.260 0.080 47.340 6.311 8.028 1.272 Drought Conditions Length (cm) 75 2482.200 15.700 2497.901 497.001 582.135 1.171 Surface Area (cm2) 75 665.496 4.156 669.653 156.646 150.044 0.957 Average Diameter (mm) 75 5.499 0.245 5.745 1.449 1.154 7.961 Average Root Volume (cm3) 75 23.638 0.039 23.677 5.610 5.796 1.033 Table 1. Comparison of root trait measurements of ‘MD 96-5722 by ‘Spencer’ RILs under normal growth conditions and drought. N: Population size (69 plants in group I and 75 plants in groups II survived among 86 planted plants in total). Root Traits Mean N SD Pair 1 Length-Normal 587.753 62 568.043 Length-Drought 529.145 62 608.654 Pair 2 Surface Area-Normal 209.915 62 235.000 Surface Area-Drought 167.590 62 158.028 Pair 3 Average Diameter-Normal 1.112 62 .556 Average Diameter-Drought 1.417 62 1.105 Pair 4 Volume-Normal 6.726 62 8.351 Volume-Drought 5.663 62 5.660 Table 2. T-Test showing summary of paired statistics for the two ex- perimental conditions for each of the four root traits listed. For each condition we display the mean, number of participants (N), the stan- dard deviation and standard error. N: Population size. Root Traits N Correlation Significance Level Pair 1 Length (Normal)-Length (Drought) 62 -.141 .276 Pair 2 Surface Area (Normal) & Surface Area (Drought) 62 -.165 .200 Pair 3 Average Diameter (Normal) & Average Diameter (Drought) 62 -.131 .310 Pair 4 Volume (Normal) & Volume (Drought) 62 -.053 .685 Table 3. The Pearson correlation between the two conditions, whether the difference between the means of the two conditions was large enough not to result, and last, 95% confidence interval was calculated. N: Population size. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) surface area (209.91-29.845), volume (6.73-1.060) and a decrease in average diameter (1.11-.071). A paired sample correlation test was used to determine the significance between the two conditions and the root traits demonstrated no correla- tion between RL, RSA, ARD, and ARV under drought and normal conditions. There is no correlation when compared to parents of RIL (Tables 3 and 4). In a recent study, Fenta et al. (2014) studied three soybean cultivars: A drought-sensitive (A5409RG), a drought-tolerant (‘Jackson’), and an intermediate drought-tolerant cultivar (‘Prima 2000’). They found that Prima 2000 outperformed the other cultivars under drought stress with the highest grain yield, shoot biomass, and nodules. They also found a positive correlation be- tween nodule size and seed yield and shoot biomass in both con- trol and water stressed plants (Fenta et al., 2014). In general, drought stress is a negative environmental condition affecting soybean production, growth and development (Garay and Wil- helm, 1982; Benjamin and Nielsen, 2006; Du et al., 2009; Fenta et al., 2014). This demonstrates that the carefully chosen root traits can be used to screen for drought tolerance in soybean. Increasing water deficit tolerance (WDT) increases agricultural plant productivity. Since the roots are the main organs for water and minerals uptake, they play a vital role in agricultural plant productivity. Water stress or water deficit (WD) also decreases shoot biomass, leaf cuticular waxes, seed yield, nitrogen fixa- tion, and seed development (Purcell and King, 1996; Serraj et al., 1999; Kim et al., 2007). Root systems are reported to be critical to cope with WD and increase yield. For example, deep and dense roots with high abilities of penetration and branching into the soil help rice plants to be drought tolerant (Pandey and Shukla, 2015). Correlation Between Root Traits and Seed Protein and Oil Contents A Pearson correlations test was done between roots traits of Group I (normal) and Group II (drought), protein and oil content as shown in Table 5 and Table 6. The frequency distribution charts for all root traits showed different measurement distri- butions. In Group I plants, a positive correlation was observed between all root traits studied (RL, RSA, ARD, and ARV) and seed protein content while a negative correlation was observed between all these root traits and seed oil content (Table 5). In Group II plants, a negative correlation was observed between RL, RSA, and ARV and seed protein content while a positive correlation was observed between ARD and protein content. A negative correlation was observed between RL, RSA, and ARV and seed oil content while a positive correlation was observed between ARD and seed oil content (Table 6). Several studies reported correlations between shoot traits and protein and oil contents in soybean (Johnson et al., 1955; Mansur et al., 1995; SoyBase, 2016); however, studies report- ing correlations between root traits and protein or oil contents are non-existent our knowledge to date. In conclusion, we have demonstrated that water deficit de- creased RL, RSA, and ARV but not ARD in soybean. It is very im- portant to understand the root architecture and root traits under WD to be able to increase seed yield, seed quality, and crop productivity which will enhance the development of WDT variet- 271 Root Traits Mean SD Std. Error Mean 95% Confidence Interval of the Difference t df Sig. Lower Upper Pair 1 Length (Normal) – Length (Drought) 58.608 889.006 112.903 -167.157 284.373 .519 61 .606 Pair 2 Surface Area (Normal) – Surface Area (Drought) 42.324 304.045 38.613 -34.888 119.538 1.096 61 .277 Pair 3 Average Diameter (Normal) – Average Diameter (Drought) -.305 1.301 .165 -.635 .025 -1.847 61 .070 Pair 4 Volume (Normal) – Volume (Drought) 1.063 10.331 1.312 -1.560 3.687 .810 61 .421 Table 4. Paired Samples Test. The Pearson correlation between the two conditions, whether the difference between the means of the two conditions was large enough not to result, and last, 95% confidence interval was calculated. Sig.: significance level. N Correlation Sig. Pair 1 Length & Protein 69 0.086 0.480 Pair 2 Length & Oil 69 -0.240 0.047 Pair 3 Surface Area & Protein 69 0.113 0.354 Pair 4 Surface Area & Oil 69 -0.301 0.012 Pair 5 Average Diameter & Protein 69 0.084 0.492 Pair 6 Average Diameter & Oil 69 -0.293 0.015 Pair 7 Volume & Protein 69 0.077 0.531 Pair 8 Volume & Oil 69 -0.294 0.014 Table 5. The Pearson correlation between root traits from Group I (nor- mal growth), protein and oil content. Sig.: significance level. N Correlation Sig. Pair 1 Length & Protein 75 -0.225 0.052 Pair 2 Length & Oil 75 0.221 0.057 Pair 3 Surface Area & Protein 75 -0.304 0.008 Pair 4 Surface Area & Oil 75 0.246 0.034 Pair 5 Average Diameter & Protein 75 0.029 0.804 Pair 6 Average Diameter & Oil 75 -0.026 0.827 Pair 7 Volume & Protein 75 -0.233 0.044 Pair 8 Volume & Oil 75 0.148 0.204 Table 6. The Pearson correlation between root traits from Group II (drought growth), protein and oil content. Sig.: significance level. 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