Effects of Cultivar Type and Node Position on Cadmium Accumulation Characteristics of Ratoon Rice

Abstract

The ratoon rice planting area is gradually expanding, and decreasing Cadmium (Cd) accumulation in ratoon rice is important for food safety and human health. In this study, conventional indica rice (HHZ, Huanghuazhan), three-line indica–japonica hybrid rice (YY-4149, Yongyou 4149), and two-line indica hybrid rice (LY-121, Liangyou 121) were compared regarding ratoon rice yield and Cd uptake, transport, and accumulation. The distribution of Cd at different nodes in the ratoon crop was also examined. The rank-order of the Cd contents in each part (root, stem, leaf, stubble, and spike) of the main and ratoon crops of the tested cultivars was HHZ > LY-121 > YY-4149. The rank-order of the Cd content in each plant part at different nodes in the ratoon crop was HHZ > LY-121 > YY-4149. The Cd content in each plant part increased as the node position (i.e., according to the germination position of regenerated seedlings, the nodes are divided into the second, third, and fourth or fifth node from the top in stubble) was lowered. The redundancy analysis indicated that the low-node brown rice Cd content had the largest effect on the total brown rice Cd content in the ratoon crop. Accordingly, indica–japonica hybrid cultivars should be selected for the production of ratoon rice in mildly Cd-polluted areas, and the height of the main crop stubble should be maximized during harvest.

1. Introduction

Rice (Oryza sativa L.) is one of the most important food crops worldwide, with more than 50% of the global population relying on rice as a staple food [1]. There have recently been considerable decreases in the rural labor force for rice production, while the cost of producing rice has continued to increase. The phenomenon of “double changing to single” is common in regions with a rice-based double-cropping system, with serious consequences for grain security. Because of the associated low production cost, low labor requirements, and relatively high grain quality, there has been increasing interest in ratoon rice. In addition to the extensive ratoon rice planting area in southern China, ratoon rice is also produced in India, Thailand, the United States, Brazil, Ethiopia, Madagascar, and other countries or regions [2,3,4]. There has been considerable physiological and technological research regarding the cultivation of high-yielding and high-quality ratoon rice, which has led to increases in ratoon rice yields [5,6]. Compared with double-cropping rice systems, the production of ratoon rice has greater economic and ecological benefits [7]. Moreover, ratoon rice experiences a relatively low temperature during its grain-filling stage, leading to a popularity in the market associated with its high milling, appearance, and eating quality [8].

Cadmium (Cd) is one of the most toxic and bioavailable heavy metals that can be found in soil. It is not essential to organisms and is potentially toxic [9]. Rice is one of the main food crops that easily accumulates Cd. Accordingly, humans take in Cd primarily from rice [10]. Previous studies developed technical measures to restrict the accumulation of Cd in rice grains. These methods prevented Cd in paddy fields from entering rice roots [11,12]. Alternatively, appropriate agronomic practices were combined with cropping systems to inhibit the accumulation of Cd in rice plants [13]. Earlier research indicated that rice cultivars differ significantly regarding their grain heavy metal content [14]. For example, the rank-order of the brown rice Cd content among indica and japonica rice, as well as conventional and hybrid rice, is as follows: traditional indica rice > hybrid indica rice > traditional japonica rice > hybrid japonica rice [15]. Studies have shown that the difference in Cd accumulation between indica and japonica rice varieties may be caused by the different abilities of Cd transporters involved in xylem loading [16,17]. The OsHMA3 gene is highly expressed in japonica rice varieties. It plays a major role in the vacuolar sequestration of Cd in rice roots, selectively sequestering Cd into the root vacuoles, limiting the root to stem translocation of Cd and alleviating the toxic effects of Cd [18]. Other studies also showed that the brown rice Cd content is significantly higher for indica rice than for japonica rice, but the brown rice Cd content is generally lower for hybrid rice than for conventional rice (this difference is not significant) [19,20]. Hence, there are some inconsistencies in the Cd contents of conventional and hybrid rice cultivars. Based on these findings, we wanted to know whether there were significant differences in the absorption and accumulation of Cd among different types of ratoon rice cultivars. We speculated that, like single-crop or double-crop rice, the Cd content in different types of ratoon rice cultivars would also be greater in indica cultivars than in japonica cultivars.

The study on the accumulation of Cd in ratoon rice is mainly the difference in Cd content between the main crop and the ratoon crop. Yang et al. [21] reported that the differences in grain Cd level between the main crop and the ratoon crop were inconsistent depending on the experimental sites, despite the average value being similar between the two seasons. However, the diversity in the Cd contents of different types of ratoon rice cultivars are relatively unknown. In addition, current research on the node position of ratoon rice mainly focuses on yield [22,23]. We have found that the 4th and 5th node from the top have the highest yield and contribute the most to the regeneration season yield compared to other nodes [23]. The reason for this is that the tillers sprouting from this node position have the highest dry matter accumulation. However, no research has been reported on the differences in Cd accumulation between different nodes for the ratoon crop. A study had shown that because Cd is transported into the grains along with other nutrients, the more dry matter accumulated in the grains, the more Cd will be absorbed and accumulated [24]. Therefore, the trend of Cd content between different node positions in the ratoon crop may be consistent with the yield, both showing an increase with decreasing node position.

Compared with japonica ratoon rice, indica ratoon rice has a higher yield and its stubble height is more suitable for mechanized harvesting [25]. Thus, indica rice is typically used for ratoon rice production. In the study, field experiments were conducted, involving three rice cultivars (conventional indica rice, HHZ; indica–japonica three-line hybrid rice, YY-4149; and indica two-line hybrid rice, LY-121) which were compared in terms of the Cd absorption, translocation, and accumulation in the ratoon rice. Furthermore, the differences in the Cd content between the ratoon crop and different nodes of the main crop were determined. The main objectives in the study were: (1) to compare the Cd contents among cultivars and clarify the mechanism underlying any differences; (2) to reveal the differences in the Cd contents between the ratoon crop and different nodes of the main crop; and (3) to determine the methods and stages suitable for decreasing the Cd content in the main crop and the ratoon crop among the selected cultivars.

2. Materials and Methods

2.1. Experimental Details

The study was performed in a mildly Cd-contaminated paddy field at the Field Experiment Station of Agricultural and Rural Bureau of Hengyang County, Hunan Province, China from March to October 2020 (26°97′ N, 111°37′ E). The soil of the experimental field had the following chemical properties at the upper 20 layer before transplanting in 2020: pH of 5.96, total Cd content of 0.49 mg kg⁻¹, bioavailable Cd content of 0.14 mg kg⁻¹, organic matter content of 24.90 g kg⁻¹, total N content of 1.52 g kg⁻¹, total P content of 0.61 g kg⁻¹, and total K content of 10.01 g kg⁻¹. The ratoon rice cultivars included in this study were conventional indica rice (Huanghuazhan; HHZ), indica–japonica three-line hybrid rice (Yongyou 4149; YY-4149), and indica two-line hybrid rice (Liangyou 121; LY-121). The climatic condition data during the rice growth seasons are shown in Figure S1. The data come from the National Meteorological Science Data Center. Available online: http://data.cma.cn (accessed on 5 May 2024).

A single factor randomized block design was used, with each cultivar analyzed in triplicate; thus, 9 plots, each 40 m², were established. The surrounding area was separated using protective rows 0.35 m wide and 0.35 m high covered with film, and independent irrigation and drainage outlets were installed. All varieties were grown using the traditional water seedling raising method, with sowing on 25 March 2020 and transplanting on 20 April. The transplanting density of the main crop and of ratoon rice was 20 cm × 20 cm, with 3–4 seedlings per hole. The fertilization plan was in accordance with local fertilization practices: compound fertilizer (N:P₂O₅:K₂O is 22:8:12) was applied as base fertilizer in the main crop at a rate of 600 kg ha⁻¹, and urea (containing 46.4% N) was applied at 150 kg ha⁻¹ after the seedlings returned to green. Urea was applied at 150 kg ha⁻¹ 7 to 10 days before the main crop harvest as a bud-promoting fertilizer for the ratoon crop. On the second day after the main crop harvest, apply 150 kg ha⁻¹ of urea and 60 kg ha⁻¹ of potassium oxide as fertilizer for ratoon crop seedlings. Determine the height of the stubble according to the plant height and the height of the axillary buds at the second node from the top. The stubble height for Huanghuazhan and Yongyou 4149 was 30 cm, and for Liangyou 121 it was 35 cm.

2.2. Sampling and Measurements

Soil samples (0–20 cm) were collected from each plot using the five-point sampling method, before fertilization in the main crop and after harvesting in the main and ratoon crop. The soil samples were naturally air-dried and then ground through 20- and 100-mesh sieves. Weigh about 0.2 g of the soil sample (pass through a 100-mesh sieve) into a 50-mL beaker, add 8 mL HNO₃ and 2 mL perchloric acid (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), and digest in a graphite digestion box (China National Analytical Center, Guangzhou, China) until the solution is completely dry and no white smoke is emitted, then dissolve with ultrapure water and dilute to a 50-mL volumetric flask. Filter through a microporous membrane into a 10 mL centrifuge tube for testing. Weigh 5.0 g of soil sample (passed through a 20-mesh sieve) and place it in a 150 mL Erlenmeyer flask. Add 25 mL of DTPA extractant (0.1 mol CaCl₂), seal the flask with plastic wrap, and shake it in a shaker (YCT-80B, Jiecheng Experimental Instrument Co., Ltd., Shanghai, China) at 25 °C and 250 rpm for 2 h. Then, the solution was filtered into a 10 mL centrifuge tube using a microporous filter membrane, and the first 5–6 mL of the filtrate was discarded. The Cd content in the digestion solution and the extract was determined by ICP-AES (ICP 6300, PerkinElmer, Waltham, MA, USA).

Rice plants (main and ratoon crops) were collected at the heading stage, mid-filling stage (15 days after the heading stage), and maturity stage, and 5 rice plants were selected from each plot based on the average tiller number of 40 plants. After being thoroughly rinsed with tap water and deionized water, the plant samples were divided into the stem, leaf, and spike, with the spike further divided into the branches, empty grains and grains in the main crop. Then, use a brown rice machine (JLGJ-45, Beijing, China) to divide the grain into grain husk and brown rice. For the ratoon crop, the regenerated tillers were divided according to their node position (i.e., according to the germination position of regenerated seedlings, the nodes are divided into the second, third, and fourth or fifth node from the top). The tillers at the same node position were divided into the stem, stubble, leaf, and spike, with the spike further divided into the branches, empty grains, grain husk, and brown rice. All samples were dried at 75 °C for 48 h, and ground to fine powders with a stainless-steel grinder (ZYM80, Zhuoya Mining Machinery Co., Ltd., Shanghai, China). Plant samples (0.5 g each) were digested with 10 mL of mixed acid solution (high-purity concentrated HCL and HNO₃, 3:1 of v/v) in a graphite digestion box (MARS6, CEM Microwave logy, Ltd., Matthews, NC, USA). After cooling, the remaining acid was evaporated and the digests were dissolved in 10 mL 2% HNO₃. The Cd content of the plant in the digestion solution was determined by ICP-AES (ICP 6300, PerkinElmer, Waltham, MA, USA).

2.3. Operation Formula

The Cd translocation factor (TF) was calculated using the equation as follows [26]:

TF_{n-to m} = Cdm/Cdn

where Cdm is the Cd content of the upper rice plant tissues, and Cdn is the Cd content of the rice plant lower tissues.

The Cd bioaccumulation factor (BAF) was calculated using the equation as follows:

BAF_i = Cd_i/Cd_soil

where Cdi is the Cd content of specific tissues (i.e., BAFroot, BAFstem, BAFleaf, and BAFbrown rice), and Cdsoil is the total Cd content in the soil.

Cd accumulation (mg ha⁻¹) = Cd content × dry matter weight.

The Cd accumulation of the A stage to the B stage was calculated using the equation as follows:

Cd accumulation_{A to B} (mg ha⁻¹) = Cd accumulation_B − Cd accumulation_A.

2.4. Statistical Analysis

One-way analysis of variance (ANOVA) was used for Duncan’s multiple comparisons (p < 0.05) of different treatments using SPSS 24 for Windows (IBM, Armonk, NY, USA). Graphs were plotted by Origin 2023 (OriginLab, Northampton, MA, USA). Redundancy analysis (RDA) was applied using Canoco 5 for Windows (Microsoft Corporation, Redmond, WA, USA).

3. Results

3.1. Differences in Cd Uptake and Transport among Ratoon Rice Cultivars

3.1.1. Cd Content

The rank-order of the Cd content in various plant parts (root, stem, leaf, spike, and stubble) in the main and ratoon crops of the tested cultivars was as follows (Figure 1): HHZ > LY-121 > YY-4149. The Cd content in each part of HHZ was significantly higher than that of YY-4149. In the main crop, the Cd contents in root, stem, leaf, and spike of HHZ were, respectively, 20.02%, 34.29%, 47.06%, and 46.67% higher than the corresponding contents of YY-4149. For the ratoon crop, the Cd contents in root, stubble, stem, leaf, and spike of HHZ were, respectively, 35.37%, 52.08%, 37.93%, 55.78%, and 47.62% higher than the corresponding contents of YY-4149.

The analysis of the Cd contents in different spike parts of the main and ratoon crops at the maturity stage (

Table 1. Cd contents (mg kg⁻¹) in different spike parts among cultivars.

Season	Cultivars	Branch	Empty Grain	Grain Husk	Brown Rice
Main crop	HHZ	0.30 a	0.22 a	0.19 a	0.17 a
	YY-4149	0.19 b	0.15 b	0.13 b	0.12 b
	LY-121	0.27 a	0.19 b	0.18 a	0.15 a
Ratoon crop	HHZ	0.39 a	0.33 a	0.25 a	0.22 a
	YY-4149	0.28 c	0.23 b	0.17 b	0.15 b
	LY-121	0.33 b	0.25 b	0.22 a	0.19 a

HHZ: Huanghuazhan; YY-4149: Yongyou 4149; LY-121: Liangyou 121. The same lowercase letters indicate no significant differences between the different varieties according to LSD (p < 0.05). Different lowercase letters indicate significant differences between the different varieties according to LSD (p < 0.05).

) indicated the rank-order among the different cultivars was as follows: HHZ > LY-121 > YY-4149. The brown rice Cd contents of HHZ and LY-121 were significantly higher than that of YY-4149. More specifically, for the main crop, the brown rice Cd contents of HHZ and LY-121 were, respectively, 42.17% and 26.03% higher than that of YY-4149. For the ratoon crop, the brown rice Cd contents of HHZ and LY-121 were, respectively, 46.77% and 19.88% higher than that of YY-4149. Among the ratoon rice cultivars, the Cd content in each spike part was highest in the conventional indica rice and lowest in the indica–japonica hybrid rice.

3.1.2. Cd Bioaccumulation (BAF) and Translocation (TF) Factors

The rank-order for the BAF of various plant parts (root, stubble, stem, leaf, and brown rice) in the main and ratoon crops was as follows: HHZ > LY-121 > YY-4149 (

Table 2. Cd bioaccumulation factors in different parts among cultivars at maturity stage.

Treatments	Root	Stem	Leaf	Brown Rice	Stubble
Main crop
HHZ	3.467 a	2.089 a	0.556 a	0.379 a
YY-4149	2.978 b	1.622 b	0.378 b	0.265 b
LY-121	3.222 ab	1.911 ab	0.511 a	0.334 a
Ratoon crop
HHZ	4.422 a	2.667 a	0.867 a	0.484 a	3.244 a
YY-4149	3.267 c	1.933 b	0.556 b	0.330 b	2.133 c
LY-121	3.622 b	2.267 b	0.667 b	0.431 a	2.556 b

HHZ: Huanghuazhan; YY-4149: Yongyou 4149; LY-121: Liangyou 121. The same lowercase letters indicate no significant differences between the different varieties according to LSD (p < 0.05). Different lowercase letters indicate significant differences between the different varieties according to LSD (p < 0.05).

). For the main crop, the root and stem BAFs were higher for HHZ than for YY-4149. Additionally, the leaf and brown rice BAFs were higher for HHZ and LY-121 than for YY-4149. For the ratoon rice, the root, stem, leaf, and stubble BAFs were higher for HHZ than for LY-121 and YY-4149. Moreover, the brown rice BAF was higher for HHZ and LY-121 than for YY-4149.

The rank-order of the Cd TF of different plant parts in the main crop of the examined cultivars was as follows (

Table 3. Cd translocation factors in different parts among cultivars at maturity stage.

Treatments	Root-to- Stem	Root-to- Leaf	Root-to- Brown Rice	Stem-to- Brown Rice	Leaf-to- Brown Rice	Root-to- Stubble	Stubble-to- Brown Rice
Main crop
HHZ	0.603 a	0.160 a	0.109 a	0.181 a	0.680 a
YY-4149	0.545 b	0.127 b	0.090 a	0.164 a	0.657 a
LY-121	0.593 a	0.159 a	0.103 a	0.174 a	0.662 a
Ratoon crop
HHZ	0.623 a	0.196 a	0.111 a	0.183 a	0.584 a	0.734 a	0.151 a
YY-4149	0.592 a	0.170 a	0.102 a	0.172 a	0.560 a	0.653 b	0.156 a
LY-121	0.626 a	0.184 a	0.110 a	0.176 a	0.600 a	0.706 ab	0.157 a

HHZ: Huanghuazhan; YY-4149: Yongyou 4149; LY-121: Liangyou 121. The same lowercase letters indicate no significant differences between the different varieties according to LSD (p < 0.05). Different lowercase letters indicate significant differences between the different varieties according to LSD (p < 0.05).

): HHZ > LY-121 > YY-4149. Specifically, the root-to-stem and leaf TFs were higher for HHZ and LY-121 than for YY-4149, whereas there were no significant differences in the root, stem, and leaf-to-brown rice TFs among the cultivars. In terms of the ratoon crop, the root-to-stubble TF was higher for HHZ than for YY-4149. There were no significant differences in the other TFs among cultivars. Hence, the main reason for the differences in the brown rice Cd contents among the tested rice cultivars was the difference in Cd BAF.

3.1.3. Cd Accumulation

For the main crop, the rank-order of the Cd accumulation in each part, from the transplanting stage to the heading stage, was as follows (Table 4): HHZ > LY-121 > YY-4149; the difference between HHZ and YY-4149 was significant. The accumulation of Cd in each part between the heading stage to the mid-filling stage was also higher in HHZ than in the other two cultivars. The Cd accumulation in the stem from the mid-filling stage to the maturity stage was highest in LY-121, whereas the Cd accumulation in the spike was highest in HHZ. The negative values for the Cd accumulation in the leaf of each cultivar indicated that the translocation of Cd from the leaf exceeded the accumulation of Cd in the leaf at this growth stage. The examination of the Cd accumulation in the plant parts of each cultivar revealed that the accumulation of Cd in the stem and leaf of the main crop mainly occurred between the transplanting stage and the heading stage, while the accumulation of Cd in the spike mainly occurred from the mid-filling stage to the maturity stage.

For the ratoon crop, the accumulation of Cd in each part before the heading stage was highest in HHZ. The Cd accumulation in the spike and aboveground parts from the heading stage to the mid-filling stage was highest and lowest in HHZ and YY-4149, respectively. The Cd accumulation in the spike and aboveground parts from the mid-filling stage to maturity stage was highest in YY-4149. The negative values for the Cd accumulation in the stubble of each cultivar implied the accumulated Cd was transported upward, along with the translocated nutrients. Furthermore, for the ratoon crop, the accumulation of Cd in the stem, leaf, spike, and stubble of each cultivar mainly occurred before the heading stage.

Table 4. Cd accumulation (kg ha⁻¹) in different parts among cultivars at various stages.

Season	Stage	Cultivar	Stem	Leaf	Spike	Stubble	Total Accumulation in Aboveground Parts
Main crop	Transplanting stage to Heading stage	HHZ	1458.13 a	273.66 a	228.37 a		1960.16 a
		YY-4149	1074.74 b	227.80 b	153.65 b		1456.19 c
		LY-121	1244.26 ab	235.76 b	171.15 ab		1651.17 b
	Heading stage to Mid-filling stage	HHZ	250.71 a	163.19 a	494.59 a		908.49 a
		YY-4149	186.93 b	121.17 b	347.79 b		655.89 b
		LY-121	187.47 b	98.18 b	389.38 b		675.03 b
	Mid-filling stage to Maturity stage	HHZ	803.56 b	−139.35 b	956.64 a		1620.85 b
		YY-4149	909.33 ab	−119.47 b	788.56 b		1578.42 b
		LY-121	969.87 a	−30.34 a	933.47 a		1873.00 a
Ratoon crop	Before the Heading stage	HHZ	1577.92 a	378.97 a	885.01 a	1759.44 a	4601.34 a
		YY-4149	1026.03 b	223.47 b	856.49 a	982.28 b	3088.27 b
		LY-121	1177.31 b	360.71 a	800.65 a	1319.67 ab	3658.34 b
	Heading stage to Mid-filling stage	HHZ	210.24 a	219.27 a	591.64 a	315.45 b	1336.60 a
		YY-4149	169.11 b	140.04 b	188.31 c	426.03 a	923.49 b
		LY-121	236.14 a	169.85 b	359.65 b	371.19 ab	1136.83 ab
	Mid-filling stage to Maturity stage	HHZ	935.84 a	−114.64 a	92.75 b	−454.29 b	459.66 b
		YY-4149	640.56 b	−76.01 a	316.00 a	−246.71 a	633.84 a
		LY-121	708.15 b	−125.56 a	215.10 a	−425.86 b	371.83 b

HHZ: Huanghuazhan; YY-4149: Yongyou 4149; LY-121: Liangyou 121. The same lowercase letters indicate no significant differences between the different varieties according to LSD (p < 0.05). Different lowercase letters indicate significant differences between the different varieties according to LSD (p < 0.05).

Table 4. Cd accumulation (kg ha⁻¹) in different parts among cultivars at various stages.

Season	Stage	Cultivar	Stem	Leaf	Spike	Stubble	Total Accumulation in Aboveground Parts
Main crop	Transplanting stage to Heading stage	HHZ	1458.13 a	273.66 a	228.37 a		1960.16 a
		YY-4149	1074.74 b	227.80 b	153.65 b		1456.19 c
		LY-121	1244.26 ab	235.76 b	171.15 ab		1651.17 b
	Heading stage to Mid-filling stage	HHZ	250.71 a	163.19 a	494.59 a		908.49 a
		YY-4149	186.93 b	121.17 b	347.79 b		655.89 b
		LY-121	187.47 b	98.18 b	389.38 b		675.03 b
	Mid-filling stage to Maturity stage	HHZ	803.56 b	−139.35 b	956.64 a		1620.85 b
		YY-4149	909.33 ab	−119.47 b	788.56 b		1578.42 b
		LY-121	969.87 a	−30.34 a	933.47 a		1873.00 a
Ratoon crop	Before the Heading stage	HHZ	1577.92 a	378.97 a	885.01 a	1759.44 a	4601.34 a
		YY-4149	1026.03 b	223.47 b	856.49 a	982.28 b	3088.27 b
		LY-121	1177.31 b	360.71 a	800.65 a	1319.67 ab	3658.34 b
	Heading stage to Mid-filling stage	HHZ	210.24 a	219.27 a	591.64 a	315.45 b	1336.60 a
		YY-4149	169.11 b	140.04 b	188.31 c	426.03 a	923.49 b
		LY-121	236.14 a	169.85 b	359.65 b	371.19 ab	1136.83 ab
	Mid-filling stage to Maturity stage	HHZ	935.84 a	−114.64 a	92.75 b	−454.29 b	459.66 b
		YY-4149	640.56 b	−76.01 a	316.00 a	−246.71 a	633.84 a
		LY-121	708.15 b	−125.56 a	215.10 a	−425.86 b	371.83 b

HHZ: Huanghuazhan; YY-4149: Yongyou 4149; LY-121: Liangyou 121. The same lowercase letters indicate no significant differences between the different varieties according to LSD (p < 0.05). Different lowercase letters indicate significant differences between the different varieties according to LSD (p < 0.05).

3.2. Differences in the Cd Contents of the Ratoon Crop and Different Nodes of the Main Crop

The tillers of the ratoon crop were derived from the germinated axillary buds of the main crop stubble. Because of the differences in plant transpiration or Cd translocation between tissues, there may be significant differences in the Cd contents of different nodes. There were significant differences in the Cd contents of individual plant parts at the same node among the rice cultivars (

Table 5. Cd contents (mg kg⁻¹) in different nodes and tissues of the ratoon crop of each cultivar.

	Cultivar	Stem	Leaf	Branch	Empty Grain	Grain Husk	Brown Rice
The 2nd node from the top	HHZ	0.95 a	0.32 a	0.33 a	0.26 a	0.23 a	0.19 a
	YY-4149	0.78 b	0.21 c	0.22 b	0.18 b	0.15 b	0.13 b
	LY-121	0.88 ab	0.26 b	0.30 a	0.20 b	0.19 ab	0.17 a
The 3rd node from the top	HHZ	1.24 a	0.34 a	0.34 a	0.30 a	0.24 a	0.21 a
	YY-4149	0.80 b	0.24 b	0.29 b	0.25 b	0.19 b	0.14 b
	LY-121	1.14 a	0.31 a	0.32 a	0.29 a	0.20 b	0.17 ab
The 4th and 5th node from the top	HHZ	1.76 a	0.44 a	0.49 a	0.38 a	0.30 a	0.28 a
	YY-4149	1.25 b	0.29 b	0.33 b	0.29 b	0.21 c	0.18 b
	LY-121	1.37 b	0.39 a	0.38 b	0.31 b	0.26 b	0.25 a

HHZ: Huanghuazhan; YY-4149: Yongyou 4149; LY-121: Liangyou 121. The same lowercase letters indicate no significant differences between the different varieties according to LSD (p < 0.05). Different lowercase letters indicate significant differences between the different varieties according to LSD (p < 0.05).

). For the ratoon rice, the rank-order of the Cd content in each plant part at each node was as follows: HHZ > LY-121 > YY-4149; the difference between HHZ and YY-4149 was significant. The brown rice Cd content at the second node from the top was, respectively, 46.15% and 23.53% higher in HHZ and LY-121 than in YY-4149. The brown rice Cd content at the third node from the top was, respectively, 50.14% and 21.43% higher in HHZ and LY-121 than in YY-4149. At the fourth and fifth nodes from the top, the brown rice Cd content was, respectively, 55.54% and 38.89% higher in HHZ and LY-121 than in YY-4149.

There were also some differences in the Cd contents among the nodes in each cultivar. The Cd contents of individual plant parts were highest at the fourth and fifth nodes from the top, followed by the third node from the top, and then the second node from the top. The redundancy analysis showed that the brown rice Cd contents at the fourth and fifth nodes from the top explained most of the variance (90.3%) and contributed the most to the total brown rice Cd content (90.9%) in the ratoon crop (p < 0.01) (Figure 2). Accordingly, the brown rice Cd contents at the fourth and fifth nodes from the top affected the brown rice Cd content of the ratoon crop.

4. Discussion

4.1. Differences in Cd Uptake and Accumulation among Cultivars

The accumulation of Cd varies considerably among rice cultivars. Rice can be divided into indica and japonica rice, with the Cd content generally higher for indica rice than for japonica rice [17]. Rice can also be divided into conventional rice and hybrid rice, but there is no consensus regarding the differences in Cd accumulation between hybrid rice and conventional rice [27,28,29]. Studies have shown that hybrid rice has a greater material production capacity and a higher assimilate transfer rate than conventional rice, which explains the greater accumulation of Cd in hybrid rice than in conventional rice [30,31]. However, some studies indicated that the genetic background, rather than the cultivar type, determines the content of Cd in hybrid rice grains [21,32]. Hybrid rice is mostly indica rice, while conventional rice includes both indica and japonica rice. Notably, the brown rice Cd content is significantly lower in japonica rice cultivars than in indica rice cultivars [33]. Therefore, it is inappropriate to compare the accumulation of Cd in grains between hybrid rice and conventional rice.

Most of the rice cultivars used for producing ratoon crops are indica rice. The analysis of different indica rice cultivars in this study revealed a lack of significant differences in the brown rice Cd content and accumulation between conventional indica rice and hybrid indica rice. However, the brown rice Cd content (Table 1) and accumulation (Table 4) were significantly higher for the indica rice than for the indica–japonica hybrid rice, likely because the Cd BAF of each plant part was lowest for the indica–japonica hybrid rice (Table 2). In addition, in this study, there was no significant difference in soil total Cd content and available Cd content at the maturity stage between the main and ratoon crop among various cultivars (Figure S2).

The Cd BAF is an important indicator of the uptake of Cd by rice. Rice plants absorb nutrients from the soil exclusively through their roots. In this study, there were no differences in soil Cd content and available Cd content among cultivars. Thus, the ability of the root system to absorb Cd reflects the ability of rice to accumulate Cd. The Cd content of rice grains is mainly determined by the uptake of Cd by the root system and the subsequent transport to the shoot [34]. According to earlier reports, the diversity in the Cd uptake by roots among rice varieties is mainly due to genetic differences [35,36]. A study had shown that the Cd content in hybrid rice grains is primarily influenced by genetic effects. In paddy fields mildly polluted with Cd, the genotype affects Cd accumulation more than the soil type; the heritability of Cd accumulation in hybrid rice grains is reportedly as high as 50.8% [24]. Similar to conventional indica rice cultivars, the indica background and the stable inheritance of Cd accumulation may determine the Cd content of hybrid rice grains. Accordingly, there was no difference in the brown rice Cd content between the conventional indica rice and the hybrid indica rice. The Cd content is lower in japonica rice than in indica rice, primarily because the OsHMA3 allele is more highly expressed in japonica rice than in indica rice [37]. The Cd content in hybrid rice is affected by the genetic background of the parents, with the japonica allele decreasing the accumulation of Cd in hybrid rice [38]. Therefore, the indica–japonica hybrid rice inherited genes from its indica and japonica parents. Hence, the Cd accumulation of indica–japonica hybrid rice was lower than that of indica rice. In terms of hybrid rice breeding, inter-subspecies heterosis has been widely studied. Elucidating indica–japonica heterosis may be beneficial for the genetic improvement of low-Cd rice varieties.

The period between the tillering stage and the grain-filling stage is critical for the accumulation of Cd in rice, while the grain-filling stage to the maturity stage is crucial for the translocation of Cd to grains [29]. The results in this study indicated that for the main crop, the main stage for the accumulation of Cd in the spike was between the mid-grain filling stage and the maturity stage, but in the ratoon crop it was mainly concentrated before the heading stage (Table 4). The temperature in the early growth stage of the main crop was low, as was the Cd absorption and translocation efficiency, whereas the temperature, as well as the Cd content and accumulation rate, were high between the mid-grain filling stage and the maturity stage [19]. During the early growth stage of the ratoon crop, the temperature was high and Cd accumulated substantially [39], which may lay the foundation for the subsequent translocation of Cd. Thus, to optimize the production of ratoon rice in Cd-contaminated rice-growing areas, methods for decreasing the Cd level in the ratoon crop should be applied before the heading stage.

4.2. Differences in the Cd Contents of Ratoon Rice and Different Nodes of the Main Crop

In the current study, the rank-order of the Cd content in each plant part at the same node among the analyzed cultivars was HHZ > LY-121 > YY-4149 (Table 5), which was consistent with the Cd contents in the main and ratoon crops of these cultivars (Figure 1). According to earlier studies, in areas with sufficient temperature and light resources, a short stubble after the harvesting of the main crop may increase the yield of the ratoon crop [40]. However, our analyses revealed that the brown rice Cd content of the ratoon crop of each cultivar was highest at the lower node positions. Moreover, the brown rice Cd contents at the fourth and fifth nodes from the top contributed the most to the total brown rice Cd content of the ratoon crop (Figure 2), indicative of the importance of these nodes for the brown rice Cd content. The explanation rate and contribution rate refer to the proportion of variance explained by each factor to the total variance, which is also directly related to the importance of each factor. The explanation rate of redundancy analysis refers to the proportion of the extracted factors in the total variance. An explanation rate of more than 80% is considered an excellent result. These findings suggest that the axillary buds at the lower nodes germinated the earliest, and the regenerated tillers had sufficient time to absorb and accumulate nutrients as well as Cd. In addition, the lower nodes were closer to the roots than the higher nodes, which may have influenced the transport and accumulation of certain compounds. Therefore, low-stubble ratoon rice plants should be carefully developed in Cd-contaminated rice-growing regions. The stubble height should be maximized while harvesting the main crop to increase the number of high-node spikes in the ratoon crop, thereby decreasing the Cd content of ratoon rice.

The limitations of this study include the fact only one representative cultivar was selected for each indica rice type and the experiments were conducted in 1 year. Considering the effects of the soil Cd level and climatic conditions on the Cd accumulation in rice, this study may need to be repeated over several years, using multiple cultivars and regions to screen for differences among diverse rice cultivars. Furthermore, the physiological and molecular mechanisms underlying the observed differences will need to be clarified, which may lead to the development of methods for the stable production of ratoon rice.

5. Conclusions

There were significant differences in the brown rice Cd contents of the main and ratoon crops among different types of rice cultivars (conventional indica rice > hybrid indica rice > indica–japonica hybrid rice). The main factor modulating the Cd content among cultivars was the bioaccumulation factor. Therefore, to optimize the cultivation of ratoon rice in mildly Cd-polluted areas, indica–japonica hybrid cultivars should be selected. The Cd content in each plant part increased as the node position was lowered. After harvesting the main crop, the stubble should ideally be as tall as possible.

In addition, the accumulation of Cd in main crop spike of each cultivar peaked during the mid-grain filling stage to maturity stage. In the ratoon crop, Cd accumulated in the spike mostly before the heading stage. Methods for decreasing Cd accumulation in the ratoon crop should be applicable before the heading stage.