The Role of Deep Roots in Sorghum Yield Production under Drought Conditions

Abstract

Root function plays a vital role in maintaining crop production. However, the role of deep roots in yield production and their effects on photosynthetic performance in sorghum remain unclear. This study aimed to provide theoretical supports for establishing highly efficient root systems of sorghum to achieve more yield under certain conditions. In this study, two sorghum (Sorghum bicolor L. Moench) cultivars, Jiza127 and Jiza305, were cultivated in soil columns as experimental materials. Three treatments (no roots removed, CK; roots removed at 30 cm underground, R30; roots removed at 60 cm underground, R60) were carried out under drought conditions during the filling stage. The root bleeding intensity, endogenous substances in the root bleeding sap, photosynthetic characteristics, dry matter accumulation, and yield were measured. The results showed that R30 and R60 significantly reduced yield in both sorghum cultivars, and the effect of R30 on yield was greater than that of R60. The contributions of roots below 30 cm to the yield of both sorghum hybrids were notably higher than those below 60 cm. R30 significantly reduced the dry matter weights (DMWs) of leaves, stems, sheaths, and panicles. R60 significantly reduced the DMW of panicles but had no significant effect on the DMWs of leaves and stems. R30 significantly reduced the photosynthetic level and PSII reaction center activity; however, the effect of R60 was not significant. Although both R30 and R60 significantly reduced root activity and the soluble sugar, amino acid, gibberellin (GA₃), and abscisic acid (ABA) contents of the root bleeding sap, some of the above indicators in R60 were significantly higher than those in R30 during the filling stage, indicating that the deeper roots (below 30 cm) had a critical regulatory effect on the physiological processes of the aerial parts in sorghum, which resulted in a stronger effect on yield, especially under drought conditions. In brief, the deep roots of sorghum played a key role in yield production, but the roots in different soil depths regulated yield production in different ways. Our results indicate that deep roots of sorghum deserve consideration as a potential trait for yield improvement especially under drought conditions.

1. Introduction

Plant roots are important organs that absorb water and nutrients from the soil and are a center for the biosynthesis and transport of plant hormones such as abscisic acid (ABA) [1,2,3,4]. The morphological and physiological characteristics of roots affect the growth of plant aerial parts [1]. The roots and aerial parts of crops are interactive and interdependent [5]. Therefore, maintaining high root vitality is essential [6], and changing root structure is more likely to promote crop growth than changing the stems and leaves [7,8]. Alteration of root traits to adapt to the surrounding environment and optimize resource utilization plays a vital role in the adaptation of wheat to different environments [9]. The study of Sebastian et al. [10] showed that stronger suppression of crown roots actually may benefit crop productivity under a water deficit. Hammer et al. [11] reported that root structure optimization and water utilization may be the basis for historic advances in yield breeding in the U.S. maize belt. Hence, to understand what kind of root system is beneficial to crop growth, it is necessary to explore the role of the root system on crop growth and yield production, especially the role of deep roots under adverse conditions such as drought stress.

Gewin revealed that deep roots play a vital role in mitigating water stress in many crops [12]. Deep roots have a greater ability to absorb water and nutrients than shallow roots and act as the central hub of the water and nutrient cycle [3,13,14]. In addition, deep roots play a key role in crop adaptation to circumstances. Manschadi et al. [15] reported that the roots of drought-tolerant wheat are tighter, more uniform, and longer than those of drought sensitive wheat, and the plants have higher water use efficiency. The deep roots of crops can not only increase yield by improving water and nitrogen uptake but also reduce environmental nitrogen leaching [16,17,18]. Therefore, breeders strongly emphasize the role of deep roots in absorbing water and nutrients when developing new varieties [19,20,21,22]. Chaves et al. [23] revealed that since deeper soil layers have higher moisture contents in arid environments, deep-rooted plants are more likely to survive under these conditions. Deep roots can absorb more substances, including water and nutrients, in arid environments and maintain the function of the shallow roots through material transport [24,25,26]. Although some functions of the root system are well known, some of the physiological functions of deep roots and their effects on aboveground crop production have rarely been reported.

Sorghum is mainly planted in arid and semiarid regions of Asia and Africa, and the planting area in these regions accounts for 85% of the worldwide planting area according to 2018 data [27]. However, filling stage is the most important period for yield production, at which drought can lead to severe decline of yield in sorghum [28]. Sorghum can feed at least 5 million people in these areas due to its unique drought adaptability [29,30,31], and sorghum roots play an important role in its drought tolerance [32]. Wang et al. reported that a higher root activity has been found in the stay green sorghum B35 as compared to non-green-stayed sorghum Sanchisan and is considered as one of the drought-resistant mechanisms under drought conditions [33]. Photosynthetic parameters and osmotic adjustment ability are the principal factors associated with sorghum yield under drought conditions [28]. However, research on root systems is time consuming, expensive, and difficult because plant roots are hidden under the ground. Therefore, compared with studies of the aboveground characteristics of plants, there are few studies on the physiological functions of underground roots. In recent decades, although the number of root system studies has significantly increased, these studies have mainly been limited to shallow root systems [34,35] or the effects of different root types on plant productivity [1]. Specifically, little is known about the effects of deep root systems on the aboveground physiology and yield production of sorghum plants [4]. Therefore, the objective of this study was to determine (1) the contribution of roots in different soil depths to yield production in sorghum and (2) the manner in which deep roots affect photosynthesis and yield. Overall, we hypothesize that the deep roots of sorghum play a crucial role in yield production, and that the roots in different soil depth regulate yield production differently.

2. Materials and Methods

2.1. Plant Materials and Experimental Design

This experiment was conducted at the experimental base of Shenyang Agricultural University from 2015 to 2016. Average temperature and sunshine duration are similar in both experimental years (Figure 1). The sorghum hybrids Jiza127 (J127) and Jiza305 (J305) were selected as experimental materials which are mainly cultivated in northeastern China. Before the test, PVC tubes (tube length 1 m, inner diameter 30 cm) were cut into two sections of the same size longitudinally, fixed tightly with pipe hoops (Figure 2), and placed in a 1 m deep soil pit. The original soil layer was placed into the PVC tubes to ensure the spatial distribution of the original soil layer. After that, the soil was compacted by watering. The tubes were arranged in two rows with a large ridge (ridge spacing 66 cm, row spacing 33 cm), and the planting density was 6 plants·m⁻². Five uniformly sized seeds were sown in each soil column on May 8. One seedling was left at the five-leaf stage. Surface soil samples were taken at 5 cm soil depth. Soil was silt loam with a pH of 7.0, organic matter content of 30.82 g kg⁻¹, alkali hydrolysable N of 114.52 mg kg⁻¹, available P of 78.33 mg kg⁻¹, available K of 102.92 mg kg⁻¹. At 45 cm soil depth, soil was loam with organic matter content of 14.82 g kg⁻¹, alkali hydrolysable N of 59.21 mg kg⁻¹, available P of 12.61 mg kg⁻¹, available K of 51.46 mg kg⁻¹. At 80 cm soil depth, there were organic matter content of 13.19 g kg⁻¹, alkali hydrolysable N of 45.35 mg kg⁻¹, available P of 12.53 mg kg⁻¹, available K of 75.85 mg kg⁻¹ in the loam soil. Diammonium phosphate (2.46 g) was used as seed fertilizer, and urea (3.33 g) was applied to each column at the jointing stage based on local sorghum cultivation. Three treatments (no roots removed, CK; roots removed at 30 cm underground, R30; roots removed at 60 cm underground, R60) were carried out during the filling stage. There were 60 columns per treatment. When the roots were removed, the pipe hoop was opened, and the roots were cut with a saw at the desired depth. Meanwhile, the soil was not moved and the pipes were closed again. A 30-d drought period was started at the same time as the root removal, and the soil moisture was controlled to produce moderate drought conditions (the soil water content was 45%–50% of the maximum water holding capacity in the field). The soil columns were protected by a mobile rain shelter from the rain during the drought-stressed period. The soil water content was measured daily at soil depth of 10 cm using the soil water sensor ML2x (DELTA-T, United Kingdom). To ensure that the soil water content remained constant, an automatic equipment of drip irrigation was used to supplement the soil water to maintain the aimed soil water content. After root removal (from the beginning of the filling stage, August 17), plant aerial part and root bleeding sap from three plants in each treatment were collected every 10 d, and photosynthetic parameters and fluorescence parameters from ten plants per treatment, biomass and substances in root bleeding sap from three plants in each treatment were measured. The plants in all treatments were rewatered from 30 d until maturity and harvested on September 28 in the two years. All the parameters in the experiment except yield were collected in 2015.

2.2. Determination of Photosynthetic Parameters, Fluorescence Parameters and SPAD Values.

The net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) of the second leaf from the top were measured using an LI-6400 portable photosynthetic system analyzer (LI-COR Inc., Lincoln, NE, USA) following the method of Chang et al. [36]. Ten plants were randomly selected for each treatment. Chlorophyll fluorescence parameters, including the initial fluorescence (Fo), maximum photosynthetic efficiency (Fv/Fm), photochemical quenching coefficient (qL) and electron transfer efficiency (ETR), were determined on the same leaf selected for determination of photosynthetic parameters by using a Junior-PAM fluorometer (Walz, Effeltrich, Germany) following the method described by Khoshbakht et al. [37]. The SPAD value (relative chlorophyll content) of the same leaf was determined with a SPAD chlorophyll meter; the upper, middle and lower parts of the leaf were measured, and the average value was calculated.

2.3. Collection of Root Bleeding Sap

Three uniform plants were selected in each treatment, and the plants were quickly cut with branch shears at the middle of the second stem node above the ground. After the incision was washed with distilled water and covered with preweighed absorbent cotton, the incision was placed in a valve bag and sealed tightly with waterproof tape. The root bleeding sap was collected from 6:00 am. to 18:00 pm., and the root bleeding intensity was determined by weighing (g·plant⁻¹·12 h⁻¹).

2.4. Determination of Osmotic Adjustment Substance and Hormone Content in Root Bleeding Sap

The soluble sugar content of the root bleeding sap was determined with the anthrone-sulfuric acid method described by Quan et al. [38]. The soluble protein content was measured with the Coomassie Brilliant Blue G-250 method described by Guzel and Terzi [39]. The free amino acid content was determined using the ninhydrin colorimetry method according to Sun et al. [40]. The ABA and gibberellin (GA₃) contents were determined using enzyme-linked immunoassay (ELISA). The kit was provided by the Crop Chemical Control Research Center of China Agricultural University.

2.5. Determination of Dry Matter Weight and Yield

Three plants were sampled from each treatment on the 10th, 20th, and 30th days from the initiation of root removal, and the dry matter weights (DMWs) of different organs (leaves, stems, sheaths, and panicles) were measured. The plant samples were killed at 105 °C for 30 min and dried at 80 °C to a constant weight. At harvest, 142 days after seeding, 10 plants were taken from each treatment, and the biological yield and grain yield were determined after air-drying.

2.6. Contribution of Roots of Different Depths to Yield

$\begin{array}{c}\mathrm{Contribution} \mathrm{of} \mathrm{roots} \mathrm{below} 30 \mathrm{cm} \mathrm{to} \mathrm{yield}=\frac{\mathrm{yield} \mathrm{of} \mathrm{CK}-\mathrm{yield} \mathrm{of} \mathrm{R}30}{\mathrm{yield} \mathrm{of} \mathrm{CK}}\times 100\%\\ \mathrm{Contribution} \mathrm{of} \mathrm{roots} \mathrm{below} 60 \mathrm{cm} \mathrm{to} \mathrm{yield}=\frac{\mathrm{yield} \mathrm{of} \mathrm{CK}-\mathrm{yield} \mathrm{of} \mathrm{R}60}{\mathrm{yield} \mathrm{of} \mathrm{CK}}\times 100\%\end{array}$

2.7. Statistical Analysis

This experiment was conducted as a complete randomized design. Significance of main effects of root removal was determined using one way analysis of variance in software SPSS 18.0. Means were separated by Duncan’s multiple range test at p < 0.05. SPSS 18.0 software was also used for regression analysis. The data are presented as the means ± standard deviation from all replications. Different characters indicate significant differences.

3. Results

3.1. Effects of Root Removal on Photosynthetic Performance

The photosynthetic parameters and SPAD values of plants in the different treatments were ranked as follows: CK > R60 > R30 (

Table 1. Effects of root removal in different soil depths on photosynthetic parameters and SPAD values.

Cultivar	Days after Root Removal	Treatment	Pn (μmol·m−2·s−1)	Gs (mmol·m−2·s−1)	Tr (mmol·m−2·s−1)	SPAD Value
J127	10 d	R30	20.29 ± 2.51b	0.20 ± 0.024b	3.77 ± 0.79b	45.07 ± 2.02b
		R60	27.16 ± 0.20a	0.22 ± 0.014ab	5.90 ± 0.41a	49.40 ± 3.58a
		CK	30.00 ± 1.85a	0.25 ± 0.015a	6.73 ± 0.48a	55.65 ± 4.26a
	20 d	R30	17.97 ± 0.31b	0.11 ± 0.004b	3.05 ± 0.49c	35.13 ± 2.86b
		R60	23.18 ± 2.55a	0.13 ± 0.027a	4.10 ± 0.20b	37.93 ± 1.16b
		CK	25.37 ± 2.40a	0.24 ± 0.025a	4.96 ± 0.37a	47.30 ± 2.61a
	30 d	R30	9.16 ± 2.40b	0.06 ± 0.016b	1.35 ± 0.37b	29.03 ± 2.78b
		R60	11.39 ± 2.13ab	0.09 ± 0.014ab	1.64 ± 0.28a	31.60 ± 4.92ab
		CK	13.50 ± 2.92a	0.13 ± 0.014a	1.65 ± 0.28a	39.00 ± 4.40a
J305	10 d	R30	22.26 ± 1.81b	0.20 ± 0.031b	4.51 ± 0.51b	48.63 ± 1.70b
		R60	32.25 ± 2.79a	0.25 ± 0.016ab	5.38 ± 0.25ab	54.50 ± 3.95ab
		CK	32.81 ± 2.44a	0.30 ± 0.050a	6.12 ± 0.99a	57.66 ± 6.39a
	20 d	R30	21.24 ± 1.98b	0.18 ± 0.017b	3.52 ± 0.85a	43.33 ± 6.64b
		R60	24.45 ± 2.25ab	0.23 ± 0.045a	4.35 ± 0.83a	49.37 ± 1.30ab
		CK	27.88 ± 3.98a	0.27 ± 0.011a	5.09 ± 0.72a	54.36 ± 6.21a
	30 d	R30	10.54 ± 2.52b	0.10 ± 0.012b	1.59 ± 0.25b	32.00 ± 2.98b
		R60	12.47 ± 1.77a	0.16 ± 0.008a	1.76 ± 0.15a	38.03 ± 3.07ab
		CK	13.17 ± 1.39a	0.17 ± 0.010a	1.81 ± 0.20a	42.18 ± 3.76a

No roots removed, CK; roots removed at 30 cm underground, R30; roots removed at 60 cm underground, R60; the net photosynthetic rate, Pn; stomatal conductance, Gs; transpiration rate, Tr; chlorophyll relative content, SPAD value. Different characters within the same column indicate significant differences at p < 0.05. The data are represented as the means ± standard deviation (n = 10).

). In most measurement periods, R60 showed no significant difference as compared to CK, while R30 showed significantly reduced photosynthetic parameters and SPAD values. The greater reduction of the photosynthetic parameters as compared to CK was measured in variety J127. At 30 d after root removal, the Pn, Gs, Tr, and SPAD values of R30 in J127 were reduced by 32.15%, 53.85%, 18.18%, and 25.56% compared with those of CK in J127, respectively. The relative decreases at 10 or 20 d after root removal were similar, but slightly less pronounced decreases as compared to those at 30 days.

Fo in different treatments was highest in R30, followed by R60 and then CK, and R30 showed a significantly increased Fo compared with CK at all measurement periods (

Table 2. Effects of root removal in different soil depths on fluorescence parameters.

Cultivar	Days after Root Removal	Treatment	Fo	Fv/Fm	qL	ETR
J127	10 d	R30	255.36 ± 5.66a	0.75 ± 0.050a	0.22 ± 0.009a	24.49 ± 0.90b
		R60	230.67 ± 6.66b	0.77 ± 0.003a	0.24 ± 0.019a	28.48 ± 0.89a
		CK	219.66 ± 5.64b	0.79 ± 0.002a	0.25 ± 0.029a	31.05 ± 2.32a
	20 d	R30	278.35±9.10a	0.69 ± 0.006b	0.18 ± 0.029a	21.53 ± 1.24c
		R60	250.41±4.87b	0.75 ± 0.026a	0.20 ± 0.034a	25.61 ± 0.40b
		CK	236.31±5.00c	0.77 ± 0.001a	0.22±0.022a	28.24 ± 0.94a
	30 d	R30	319.00±11.37a	0.63 ± 0.039b	0.17±0.015b	16.05 ± 2.42b
		R60	293.463±16.57ab	0.65 ± 0.019ab	0.20 ± 0.034a	21.80 ± 1.48a
		CK	258.33±15.51b	0.70 ± 29a	0.21 ± 25a	24.24 ± 1.95a
J305	10 d	R30	236.14 ± 16.97a	0.77 ± 0.004a	0.22 ± 0.033b	28.32 ± 1.00b
		R60	227.20 ± 7.14ab	0.78 ± 0.001a	0.31 ± 0.054a	31.14 ± 2.92ab
		CK	216.46 ± 6.85b	0.79 ± 0.028a	0.32 ± 0.025a	34.66 ± 2.08a
	20 d	R30	261.96 ± 4.88a	0.71 ± 0.022b	0.17 ± 0.005c	24.49 ± 0.53b
		R60	238.42 ± 7.23b	0.75 ± 0.020ab	0.23 ± 0.016b	27.48 ± 0.39ab
		CK	228.67 ± 6.03c	0.76 ± 0.025a	0.27 ± 0.020a	30.03 ± 2.80a
	30 d	R30	292.00 ± 19.67a	0.62 ± 0.079b	0.11 ± 0.100b	20.06 ± 0.79b
		R60	262.67 ± 11.50ab	0.67 ± 0.023ab	0.14 ± 0.149a	23.56 ± 2.02ab
		CK	246.67 ± 11.59b	0.70 ± 0.012a	0.16 ± 0.156a	24.42 ± 0.74a

No roots removed, CK; roots removed at 30 cm underground, R30; roots removed at 60 cm underground, R60; initial fluorescence, Fo; maximum photosynthetic efficiency, Fv/Fm; photochemical quenching coefficient, qL; electron transfer efficiency, ETR. Different characters within the same column indicate significant differences at p < 0.05. The data are represented as the means ± standard deviation (n = 10).

). In contrast, Fv/Fm, qL, and ETR were ranked as follows: CK > R60 > R30. At 30 d after root removal, both varieties in the R30 treatment showed significantly reduced Fv/Fm, qL, and ETR, which were decreased by 10.00%, 19.05%, 33.79% in J127 and 11.43%, 31.25%, and 17.85% in J305, respectively. R60 showed no significant difference from CK. The relative decreases at 10 or 20 d after root removal were similar, but slightly less pronounced decreases as compared to those at 30 days.

3.2. Effects of Root Removal on Root Bleeding

Root bleeding intensity was significantly affected by root excision (Figure 3). R30 and R60 significantly reduced root bleeding intensity in J127 at 10 and 20 d after root excision. The root bleeding intensity was decreased by 42.69% and 35.30% in R30 and R60 at 10 d after root removal and 41.22% and 33.64% at 20 d after root removal compared with that in CK, respectively. In J305, R30 and R60 showed significantly reduced root bleeding intensity at 20 and 30 d after root removal. Compared with that in CK, the root bleeding intensity was decreased by 46.52% and 24.46% in R30 and R60 at 20 d after root removal and 52.60% and 33.99% at 30 d after root removal, respectively.

The content of osmotic adjustment substances in the root bleeding sap was significantly affected by root removal (Figure 4). R30 led to a significantly reduced soluble sugar content. R60 led to a significantly reduced soluble sugar content in J127 compared with CK at 10 and 20 d after root removal; the decreases were 25.71% and 12.98%, respectively. In J305, R60 also led to significantly decreased soluble sugar contents; the decreases were 20.94%, 17.33%, and 11.78% at 10, 20, and 30 d after root excision, respectively. Furthermore, R30 significantly reduced the soluble protein content of the root bleeding sap of J305 by 26.71%, 20.09%, and 31.55% at 10, 20, and 30 d after root removal, respectively. In contrast, R60 had no significant effect on the soluble protein content of the root bleeding sap at 10 and 30 d after root removal. R30 significantly reduced the amino acid content of the root bleeding sap by 47.41%, 34.63%, and 32.33% in J127 at 10, 20, and 30 d after root removal, respectively, and by 40.59%, 29.82%, and 30.23% in J305 at 10, 20, and 30 d after root removal, respectively. R60 significantly reduced the amino acid content in J127 by 30.94%, 20.65%, and 25.95% at 10, 20, and 30 d after root excision, respectively, and by 28.42% and 18.76% in J305 at 10 and 30 d after root excision, respectively.

The GA₃ content of the root bleeding sap was significantly affected by root excision. R30 significantly reduced the GA₃ content by 36.81%, 39.21%, and 49.04% in J127 at 10, 20, and 30 d after root removal and by 25.11%, 35.02%, and 37.87% in J305, respectively, compared with that in CK. R60 significantly reduced the GA₃ content in J127 by 22.25%, 25.67%, and 36.66% at 10, 20, and 30 d after root removal, respectively, and by 20.44% and 30.13% in J305 at 20 and 30 d after root removal (Figure 5).

At 10 d after root excision, compared with that of CK, the ABA content in the root bleeding sap of R30 was significantly increased, while that of R60 showed no significant difference from CK. R30 significantly reduced the ABA content in the root bleeding sap of J127 at 20 and 30 d after root removal. R60 significantly reduced the ABA content in the root bleeding sap of J127 at 20 and 30 d after root removal and that of J305 at 30 d after root removal (Figure 5).

3.3. Effects of Root Removal on Dry Matter Accumulation and Yield

Compared with CK, the dry weight of leaves, stems, sheaths, and panicles in J127 decreased by 33.10%, 46.88%, 43.08%, and 24.63% under R30 at 30 d after root removal, respectively (

Table 3. Effects of root removal in different soil depths on dry matter accumulation.

Cultivar	Days after Root Removal	Treatment	Leaf (g·plant−1)	Stem (g·plant−1)	Sheath (g·plant−1)	Panicle (g·plant−1)
J127	10 d	R30	21.67 ± 3.94a	40.18 ± 4.76b	14.95 ± 1.72a	75.25 ± 3.53b
		R60	24.41 ± 2.22a	57.73 ± 9.56a	16.92 ± 2.22a	87.37 ± 6.85a
		CK	26.22 ± 1.94a	61.14 ± 5.57a	19.08 ± 3.65a	98.31 ± 6.30a
	20 d	R30	18.53 ± 0.71b	35.35 ± 1.32b	12.05 ± 0.78c	70.39 ± 5.39b
		R60	22.59 ± 1.85a	48.85 ± 7.88a	14.87 ± 0.70b	78.71 ± 9.31ab
		CK	24.32 ± 1.54a	56.94 ± 6.93a	17.51 ± 0.66a	87.72 ± 8.78a
	30 d	R30	14.15 ± 1.97b	27.31 ± 13.34b	8.43 ± 0.34c	63.98 ± 2.61c
		R60	21.07 ± 0.74a	40.87 ± 4.19ab	10.35 ± 0.67b	73.73 ± 3.13b
		CK	21.15 ± 1.41a	51.41 ± 3.79a	14.81 ± 0.77a	84.89 ± 2.88a
J305	10 d	R30	25.70 ± 2.98a	63.77 ± 7.72b	19.12 ± 6.13a	89.17 ± 8.81b
		R60	28.76 ± 1.24a	83.02 ± 4.04a	19.73 ± 2.50a	106.48 ± 1.73a
		CK	29.23 ± 1.68a	85.83 ± 10.71a	21.61 ± 3.31a	115.24 ± 11.30a
	20 d	R30	21.98 ± 2.05b	53.44 ± 10.77b	14.52 ± 2.67a	84.18 ± 2.93b
		R60	26.46 ± 2.51a	79.43 ± 2.91a	17.44 ± 2.53a	102.95 ± 7.12a
		CK	28.27 ± 1.27a	80.87 ± 6.90a	20.59 ± 1.13a	109.80 ± 5.87a
	30 d	R30	19.76 ± 1.84b	50.41 ± 5.23b	12.81 ± 2.45b	70.06 ± 1.58c
		R60	25.22 ± 2.13a	67.64 ± 8.78a	16.77 ± 3.87a	81.56 ± 0.23b
		CK	25.49 ± 2.95a	73.15 ± 5.33a	19.60 ± 1.60a	95.14 ± 2.82a

No roots removed, CK; roots removed at 30 cm underground, R30; roots removed at 60 cm underground, R60. Different characters within the same column indicate significant differences at p < 0.05. The data are represented as the means ± standard deviation (n = 3).

). R60 had no significant effect on the dry weights of leaves and stems in J127 but significantly reduced those of sheaths and panicles by 30.11% and 13.15% compared with CK, respectively. In J305, R60 had no significant effects on the dry weights of the leaves, stems, and sheaths but significantly decreased the dry weight of the panicles, which was reduced by 14.27% compared to that of CK. The relative decreases at 10 or 20 d after root removal were similar, but slightly less pronounced decreases as compared to those at 30 days.

Both R30 and R60 significantly reduced grain yield (

Table 4. Effects of root removal in different soil depths on grain and biological yield in 2015 and 2016.

Year	Cultivar	Treatment	Grain Yield (g/plant)	Biological Yield (g/plant)
2015	J127	R30	60.34 ± 1.00c	161.18 ± 8.30c
		R60	72.99 ± 3.64b	175.39 ± 0.71b
		CK	102.43 ± 9.24a	197.71 ± 11.47a
	J305	R30	83.34 ± 4.82b	193.44 ± 3.83b
		R60	94.77 ± 4.21b	213.13 ± 5.56b
		CK	125.37 ± 7.72a	231.10 ± 6.36a
2016	J127	R30	54.48 ± 0.45c	184.39 ± 25.07c
		R60	84.82 ± 2.41b	243.04 ± 9.08b
		CK	144.30 ± 2.28a	295.32 ± 36.83a
	J305	R30	42.72 ± 10.88b	164.21 ± 9.34b
		R60	66.37 ± 6.97b	204.78 ± 20.35b
		CK	117.82 ± 13.33a	290.40 ± 49.96a

No roots removed, CK; roots removed at 30 cm underground, R30; roots removed at 60 cm underground, R60. Different characters within the same column indicate significant differences at p < 0.05. The data are represented as the means ± standard deviation (n = 10).

). The two-year average grain yield of R30 and R60 decreased by 53.46% and 36.04% compared with that of CK in J127 and by 48.16% and 33.74% in J305. The biological yield was also significantly affected by root removal. The average biological yield of R30 and R60 in J127 decreased by 19.41% and 9.40% compared with that of CK and by 31.42% and 19.86% in J305, respectively. The yield trend of the two-year experiment was consistent.

3.4. Correlation of Root Bleeding with Grain Yield and Pn

A significant positive relationship (R² = 0.6072, p < 0.01) was found between the Pn and root bleeding intensity, indicating that the increasing root bleeding intensity had a positive influence on Pn. Similarly, there was a significant positive relationship (R² = 0.5862, p < 0.01) between grain yield and root bleeding intensity, indicating that the increasing root bleeding intensity had a positive influence on grain yield (Figure 6).

4. Discussion

Root activity reflects the strength of root metabolic capacity and has an important role in promoting the formation of yield [41,42]. Munns et al. [43] reported that a decline in osmotic adjustment substances increased the osmotic potential of plant root cells, resulting in a decrease in the water absorption capacity of the root system. In this study, R30 and R60 significantly reduced root bleeding intensity and osmotic adjustment substance content, indicating that roots below 30 cm still had strong water absorption capacity under drought conditions during the filling stage. Meanwhile, some indicators in the root bleeding sap of R60, such as soluble protein and soluble sugar, were significantly higher than those of R30 during the filling process, indicating that deeper root systems (below 30 cm soil depth) hid high physiological activity and strong regulatory effect on the aboveground parts. With respect to signaling substances, root excision significantly reduced the GA₃ content in the root bleeding sap. This result indicates that the reduction of GA₃ synthesis in the root system should lead to a decrease of the available GA₃ in the aerial part. GA₃ is an important hormone in plants that can promote plant growth and material accumulation [44]. These results suggested that the decrease in synthesis and output of GA₃ may be one of the reasons for the reduction in aboveground dry matter, and this effect was more obvious in R30. The changes in ABA were more complicated due to root excision and plant senescence. On the one hand, the change trend in ABA content in the root bleeding sap of CK reflects a response to drought conditions and senescence during the filling stage. A similar change trend of ABA was also found in the results of Abid et al. [45]. The changes in ABA content in R30 and R60 may indicate a stress response in the root system in the early stage due to root excision. This damage and loss in the root system decreased the ability of the roots to synthesize ABA, which significantly reduced the ABA content exported from the roots to the shoots. Lower ABA content in the leaves could reduce plant drought resistance [46], ultimately leading to a reduction in yield. The correlation of the root bleeding intensity with Pn and grain yield also implied that higher root activity could better support the development of photosynthetic potential that ultimately resulted in higher yield.

Song et al. [47] and Qi et al. [48] reported that deep roots play a critical role to improve root vitality, maintain root nutrition and water supply to the aerial part, promote the production of photosynthetic materials and grain filling, and increase yield. In the present study, regardless of R30 or R60, the yield of sorghum was significantly reduced by root removal, reflecting the important role of deep roots in yield production. Root excision at 30 cm underground significantly reduced leaf chlorophyll content (SPAD value), photosynthetic level, Fv/Fm, qL, and ETR and increased Fo. An increased Fo and a decreased Fv/Fm indicate that the PSII reaction center is damaged [49]. These results revealed that root excision directly reduced the function of the PSII reaction center, and photosynthetic activity and electron transfer were affected. As a result, the photosynthetic capacity was reduced, and the yield was significantly reduced. In addition, the decreased photosynthetic capacity reduced the overall biomass of the aerial parts, which manifested as decreased dry weights of the sorghum leaves, stems, sheaths, and panicles. However, the effect of root excision at 60 cm underground on the photosynthetic parameters was not significant, indicating that R60 had little effect on photosynthetic performance. From the perspective of root system distribution, the root system was shown to be mainly distributed in the upper 0–30 cm of topsoil, which contained up to approximately 95% of the total number of roots in cotton and maize, respectively [50,51], indicating that roots of taproot and fibrous root crops are mainly distributed in shallow layers, and the surface root system was directly related to photosynthesis [52]. However, in this study, the contribution of roots below 60 cm accounted for more than 30% of the yield, indicating that deeper root activity, which was reflected by the root bleeding intensity and component content, played a critical role in yield production under drought conditions during the filling stage. Schittenhelm et al. [53] indicated that root depth was also a significant feature of drought resistance in sorghum. Common farming fertilization is mainly concentrated in the plow layer, and drought also usually occurs in the cultivated layer, which reduces nutrient availability. Although the root system can penetrate deeper soil depths to obtain water under drought conditions, nutrients are relatively scarce in these deeper layers, leading to a degree of spatial dislocation between nutrients and water. As the root system tends to grow toward fertilizer and water, the spatial distribution of the root system changes accordingly; thus, the role of deep roots will be more obvious under drought conditions.

In brief, R30 and R60 significantly reduced the biological and grain yield of sorghum, especially grain yield. R30 mainly reduced yield by reducing photosynthetic level and root activity, while R60 had no significant effect on photosynthetic characteristics. Although root activity was significantly reduced in R60, it was still significantly higher than that of R30, indicating that the greater vitality of deeper root systems (soil depths below 30 cm) was the reason for their greater contribution to yield, especially under drought conditions. In conclusion, the deep roots of sorghum play a crucial role in yield production, but the roots in different soil depths regulate yield production differently. Our results support the hypothesis that deep roots of sorghum greatly contribute to yield production and thus merit investigation as a potential agronomic trait for more yield under drought conditions.