Effects of Combined High Temperature and Waterlogging Stress at Booting Stage on Root Anatomy of Rice (Oryza sativa L.)

In recent years, the alternating occurrence of high temperature and waterlogging disasters in South China has seriously reduced the yield of single cropping rice. Studying the changes in anatomical structure of the rice root system could provide theoretical basis for understanding the mechanisms of high temperature and waterlogging stress. To examine interactions between temperature and waterlogging stress, an experiment was set up in a growth chamber consisting of two temperatures (moderate, 30–34 °C and high, 35–38 °C) with three depths of flooding (0–5, 10 and 15 cm). Treatments commenced at the booting stage and lasted five days, after which all treatments were returned to a 0–5 cm flooding depth and the same temperature regime. Observations were made immediately after cessation of treatments, then after 5, 10 and 20 d to test the effect of treatments on subsequent root anatomical development. The low-stress control (0–5 cm, medium temperature) showed no change with time in aerenchyma area, thickness of the outer root, stele diameter, and the number nor diameter of xylem vessels. Root diameter and stele diameter under the high-stress control (0–5 cm, high temperature) were decreased by 29.09% and 15.28%, respectively, at the booting stage, whereas the high stress control (15 cm, high temperature) affected only the vessel diameter, reducing it by 14.11% compared with that in the low-stress control (0–5 cm, medium temperature). Compared to the high-stress control (0–5 cm, high temperature), the interaction of high temperature and waterlogging stress alleviated the inhibiting effect of the changes in the root system, especially after the end of the stress. We thought that waterlogging could reduce the damage of high temperature on rice root growth. Low water depth waterlogging has little effect on rice root system and aerenchyma area root diameter at 0, 5, 10 and 20 d after the stress ended, and the thickness of the outer root, stele diameter and the number and diameter of vessels at 0 d under M15 (15 cm, medium temperature) had no significant difference compared with M5 (0–5 cm, medium temperature). However, the increase in rice root diameter, stele diameter, thickness of the outer root cortex depth and vessel diameter were inhibited under high temperature stress at the booting stage. Root diameter and stele diameter under H5 (0–5 cm, high temperature) were decreased by 29.09% and 15.28%, respectively, at the booting stage, whereas H15 (15 cm, high temperature) affected only the vessel diameter, reducing it by 14.11% compared with that in the M5. Compared to H5, the interaction of high temperature and waterlogging stress alleviated the inhibiting effect of the changes in the root system, especially after the end of the stress. We thought that waterlogging could lighten the damage of high temperature on rice root growth.


Background
China is one of the major rice producers, accounting for 18.5% of the world's total planting area and 27.7% of world rice production (Zhu et al., 2015). Temperature and moisture are the main factors affecting rice production. Due to global warming, the atmospheric temperature is expected to increase by 0.3-0.7 °C by the middle of this century (IPCC, 2013). Climate change impacts the underground processes in ecosystemsincreased soil temperature may alter the growth of underground plant parts (Oreskes et al, 2004;Xia et al, 2014). High temperatures pose a serious threat to global food security. In rice production, a high temperature (> 35 °C), especially during grain filling, can remarkably reduce both the grain development, quantity and quality (Butardo et al., 2019). High temperature can affect many processes of rice growth and development, including germination, seedling growth, leaf emergence, tillering, heading, and filling (Hakata et Wahid et al., 2007.). The frequency of extremely high temperatures and heat waves in most of the mainland China is likely to continue increasing in the 21st century (Shi et al, 2008). According to Wu Qixia, the middle and lower reaches of the Yangtze River were exposed to mild heat damage and waterlogging-associated stress in summer 15 times between 1960 and 2010, accounting for 44.1% of the total number of heat and waterlogging-associated disasters (Wu et al., 2012). In recent years, high temperature and rainstorms have coincided frequently with the rice reproductive growth stage in Huanghuai and South China (Yang et al., 2013). After heavy summer rainfalls, the water cannot be completely drained in a short period of time and forms a layer on the field surface. As these heavy rains are typically followed by high temperatures, rice is thus subjected to double stress, by high temperature and waterlogging, which affect the absorption and utilization of nutrients by rice roots and lead to the reduction in rice yield. Namely, the root system, as an important organ for plant absorption and metabolism, is the main organ that restricts the growth of aboveground parts. Root growth is a dynamic process, root architecture may change in response to alterations in the immediate environment (Tran et al., 2015). Therefore, study on the combined effect of high temperature and waterlogging stress on microstructure of the rice root system will help to elucidate the growth and development of the root system under double adversity from the microcosmic perspective. This knowledge will provide theoretical reference for improving rice yield stability and stress resistance and ensure future food security (Hubeau et al., 2015).
In a drought or flooded environment, plants can adapt to adversity by changing the homeostasis mechanism and root configuration and shape, which mainly stems from the avoidance of the plant itself (Zhang et al., 2016). In the study of the effect of water stress on rice root architecture, it was found that the milder water stress promoted the growth of rice root system, and its total root length, root surface area, root volume and root bifurcation rate all increased, but the average root diameter decreases. Roots are most susceptible to suffer from oxygen shortage under waterlogging and flooding conditions (Sauter, 2013). Previous studies have shown that 5 days of flooding at the tillering stage triggers the formation of aeration tissues in rice roots and the development of thick-walled cells in the root cortex. At the same time, flooding also inhibits the development of the rice root outer cortex layer (Zhen et al., 2015), thus affecting the absorption and transportation of water and nutrients by rice roots. Root morphological and anatomical adjustments have been revealed to be important to improve oxygen supply to maintain root function under oxygen-deficient conditions. Too high atmospheric temperature will increase the soil temperature. Too high soil temperature will affect the roots' water absorption. High root temperature speeds up the aging process, making the lignified part of the root almost to the tip, the absorption area decreases and the absorption speed also decreases. Accroding to Kang shaozhong, the total water cross-section of roots in the high temperature environment would be smaller than that in the low temperature environment during the jointing stage of maize, so the root hydraulic conductivity was reduced.
There is more research on the water absorption capacity of rice roots under a single stress. However, little research has been done on the effects of high temperature and waterlogging on rice roots, especially on changes in rice root anatomical structure. The anatomical structure of rice root system mainly includes aeration tissue, root diameter, Stele diameter, thickness of thick-walled cells in outer cortex, and cross-sectional area of cells, which all affect the absorption and transportation of water and nutrients in the root system (Huang et al., 2016). In this study, we analyzed the effects of high temperature and waterlogging on the growth and development of rice roots by comparing the root diameter, stele diameter, and the number and diameter of vessels between plants exposed to different temperatures and waterlogging regimes, and examined the interaction of high temperature and waterlogging on the absorption of water and nutrients by rice roots.

Results
Aerenchyma formation in plants exposed to high temperature and waterlogging stress At zero days of stress exposure Aerenchyma was formed in high temperature (T1), high temperature × heavy waterlogging (T3), and light waterlogging (T4) treatments ( Fig. 1), the tissue was especially well-developed in T1 treatment, the number and volume of aerenchymatous cavities were large and connected. This may be due to the increase in rhizosphere temperature caused by high temperature treatment. The resulting hypoxia in the rhizosphere environment triggered the formation of aerenchyma in the root system to enhance oxygen diffusion in the body and maintain appropriate oxygen levels in tissues by long-distance transportation of oxygen into the root tips, which will ensure normal physiological functions. At the same time, oxygen may also be released into the rhizosphere to alleviate the toxicity of reducing substances to plants in the rhizosphere (Fan et al., 2002). The cortex of plants treated with T2 and T5 was composed of cells that were larger, with square shape, and arranged regularly, and the tissue did not form aerenchyma, when compared with the control. Compared with T5, the aerenchyma was formed earlier under T3 treatment, indicating that high temperature × heavy waterlogging (T3) at the booting stage may accelerate the formation of aerenchyma in rice.

At 5 days of stress exposure
Aerenchyma was formed in roots of plants treated with T1, T2, T3, and T4, it was especially developed in plants subjected to T2 and T3 treatments (Fig. 2). The root cortex disintegrated further, and the aerenchyma tissue continued to expand radially. In radial direction, the aerenchyma cavities extended from the stele to the outer cortical thick-walled cells, and some of them were interconnected (as observed in T2 and T3 treatments). The whole cortical structure became loose, and the remaining aerenchyma cavities were separated by thin sheets of residual cell walls and undissolved parenchymal cells. Only a small number of cortex cells in roots treated with T1 and T4 began to expand and deform, became slightly dissociated, and formed a small amount of irregular aerenchyma. No aerenchyma appeared in roots treated with T5. The results showed that the interaction of high temperature and waterlogging stress at the booting stage created certain continuity in the formation of aerenchyma in rice roots.

At 10 days of stress exposure
All treated roots had aerenchyma that was well developed due to the aging of the roots. The large vessels with xylem in the stele were arranged radially. Phloem and metaxylem were arranged alternately (Fig. 3). The gaps between cortex cells were enlarged into cavities, forming aerenchyma for gas transport. Some residual cortex cells were observed only in the roots of plants exposed to T1 and T5 treatments, whereas in the other treatments, the cavities were connected with each other. There were only a few residual cortex cells between the aerenchyma. The results showed that both high temperature and heavy waterlogging alone at the booting stage inhibited and delayed the formation of aerenchyma in rice roots.

At 20 days of stress exposure
In all treatments, aerenchyma was well developed along the circumference and in radial direction of the root. The cavities were connected, facilitating not only the transport of oxygen needed for root respiration in hypoxic environment, but also the release of oxygen along the radial root axis into the rhizosphere (Fig. 4). Only the roots treated with T1 and T5 had some residual parenchyma cells. High temperature or heavy waterlogging alone at the booting stage slowed down the development of aerenchyma in rice roots, while their interaction promoted the formation of aerenchyma in the roots, regardless of the treatments, well-developed aerenchyma were formed in rice roots at the late growth stage.

Effects of combined high temperature and waterlogging stresses on root diameter
The variations in root diameter were different among the treatments at the booting stage (Table 1). After 0 days of stress, compared to CK, the root diameter under T1 treatment (high temperature) decreased by 29.09% (p < 0.05). After 5 days of stress, the largest root diameter was observed in T3 treatment and the smallest in T1 treatment, in the latter, it was reduced significantly by 15.30% compared with that in CK treatment (p < 0.05). After 10 days of stress, the root diameter treated with T4 (light waterlogging) increased by 11.72% compared with that in the CK (p < 0.05). After 20 days of stress, the root diameter treated with T1 decreased by 14.41% compared with that of the CK (p < 0.05). The above results show that high temperature treatment at the booting stage significantly reduced the root diameter and exhibited certain aftereffects. After 20 days of stress, the root diameter under high temperature treatment was still lower than that of the CK, resulting in reduced water conductivity of rice roots.

Effects of combined high temperature and waterlogging stresses on thickness of the outer root layer
There was no significant difference in thickness of the outer layer between all treatments at 0 days of the treatments ( Table 2). After 5 days of stress, compared to CK, the thickness of the outer layer in plants treated with T3 (high temperature × heavy waterlogging), T4 (light waterlogging), and T5 (heavy waterlogging) increased by 48.97%, 29.69%, and 15.71%, respectively, but decreased by 17.78% in T1 treatment. After 10 days of stress, the thickest outer layer was observed in T3 (17.87% thicker than that in the CK), and the thinnest outer root layer was detected in T1 (18.38% thinner than that in the CK). After 20 days of stress, the thickness of the outer root layer treated with T4 (heavy waterlogging) increased by 19.71% compared with that of the CK, whereas that of plants subjected to T1, T2, and T3 was lower compared with its thickness in the CK. These results suggest that high temperature treatment at the booting stage reduced the thickness of the outer root cortex layer. This reduction may be due to restrained formation of structural barriers by high temperature treatment caused by the embolization and lignification of root cells, retarded development of the outer root layer, and the failure to form effective barriers that will prevent oxygen diffusion into the soil. Consequently, rice roots experience shortage in oxygen, which further inhibits root uptake and transport of water and nutrients.

Effects of combined high temperature and waterlogging stresses on stele diameter
Compared with the CK, at 0 days of treatments, light waterlogging (T4) increased the stele diameter by 19.27%, and high temperature (T1) decreased the stele diameter by 15.28% (Table 3). After 5 days of stress, high temperature × heavy waterlogging (T3) treatment increased significantly the stele diameter by 20.26% compared with the CK. After 10 days of stress, the stele diameter increased significantly by 15.08% under heavy waterlogging (T5) when compared with that in CK treatment, and it was similar to that of the CK in other treatments. After 20 days of stress, the largest stele diameter was observed under high temperature, it was 35.30% higher than that of the CK. Therefore, high temperature (T1) inhibited the development of stele diameter, while combination of high temperature and waterlogging (T2, T3) promoted the development of stele diameter. Thus, the interaction between high temperature and waterlogging alleviated the inhibiting effect of high temperature on the development of stele diameter and enhanced the ability of rice roots to absorb water and nutrients.

Effects of high temperature and waterlogging on vessel number and diameter
After 0 days of stress, T3 treatment decreased significantly the vessel diameter by 14.11% compared with that of the CK, but there was no significant difference in the number of vessels (Table 4). After 5 days of stress, root vessel diameter of rice treated with T2 and T4 was increased by 17.36% and 8.57%, respectively, when compared with that of the CK, while root vessel diameter of rice treated with T3 was lower than that of the CK. After 10 days of stress, the vessel diameter in T1 and T5 treatments was much lower than that in CK treatment, but the number of root vessels in all stress treatments was higher than that in the CK, especially in T5 treatment, there were two more vessels compared with the CK. After 20 days of stress, the root vessel diameter of rice treated with T1, T2, and T3 was lower than that of the CK, especially in T2 treatment, there were 1.33 less vessels compared with the CK. These results indicate that high temperature and the combination of high temperature and waterlogging stresses at the booting stage reduced the vessel diameter, whereas the number of vessel was close to or lower than that of the CK. Consequently, high temperature and the interaction between high temperature and waterlogging at the booting stage could reduce water conductivity of the rice root system. Table 4 The number and diameter of vessels in plants exposed to high temperature and waterlogging

Effects of high temperature and waterlogging on anatomical structure parameters of rice roots
Temperature at the booting stage affected significantly the anatomical parameters of rice roots (p < 0.05) ( Table 5). Water had significant effects on root diameter, stele diameter, and vessel diameter, but it had no significant effect on thickness of the outer root layer, whereas temperature × water affected significantly only the vessel diameter. The data in the table are the results of two-factor variance analysis of root anatomical structure parameters at 0 d after stress. "*" means significant difference at 0.05 level.

Discussion
As an important link between aboveground plant parts and soil, the root system is involved in the absorption of water and nutrients, the synthesis and transportation of plant hormones, and the anchorage of plants (Suralta et al., 2016). The morphological and physiological root traits affect the growth and productivity of the plant. Welldeveloped rice roots increase the biomass and yield in response to different conditions in different cultivars (Nada et al.,2019). Root diameter and stele diameter determine the radial transport distance of water and nutrients in roots, and changes in those two parameters affect the absorptive capacity of roots. The external environmental factors impact the root and stele diameter, and roots adapt to the environment by changing their shape (such as reducing their diameter) (Fitter et al., 2002). For example, finer roots and thinner cortex usually have higher absorption rates (Eissenstat et al., 1997). In the present study, root diameter and stele diameter of rice treated with T1 decreased by 29.09% and 15.28%, respectively, compared with that of the CK, suggesting that high temperature inhibits the expansion of rice root diameter by increasing soil temperature and thereby inhibiting root growth. Our results are consistent with other studies (Wu et al., 2014;Majdi et al., 2004). The smaller the root diameter, the larger the proportion of cortex in the root system, and the smaller the water conductivity of the root system, the larger (Niu et al., 2017). This is mainly due to the fact that the root cortex hinders water transport via the roots. Therefore, the thicker the root cortex, the greater the resistance to water transport and the smaller the rate of water absorption (Wang et  , respectively, which values are outside the optimal temperature range for rice root growth of 25-30 °C. When the temperature is too high, the rooting power decreases and the respiration function increases significantly, whereas the reducing substances in the soil are accumulated at an accelerated rate, which harms the root growth and development and ultimately affects the absorption of water and nutrients by plant roots. Simultaneously, higher temperature also reduce dissolved oxygen in water and promote the formation of aerenchyma in rice roots. Our results showed that high temperature (T1), high temperature × light waterlogging (T2), and high temperature × heavy waterlogging (T3) treatments triggered earlier formation of the aerenchyma in roots, especially the T2 and T3 treatments when compared with other treatments. After restoring the natural temperature and normal irrigation conditions, the root diameter and vessel diameter treated with T1 was still lower than CK, which indicated that the effect of high temperature at booting stage on root growth had a certain aftereffect on root growth, while the interaction stress of high temperature and waterlogging had a compensatory effect. Two-way ANOVA showed that temperature had a significant effect on root diameter, thickness of outer root, stele diameter and vessel diameter, while the interaction between high temperature and waterlogging had only an effect on vessel diameter. High temperature, waterlogging, and their interaction at the booting stage inhibited the development of the rice root system. Compared with high temperature treatment, interaction of high temperature and waterlogging stresses weakened the inhibiting effect of high temperature on the root system and water and nutrient transport, thereby preventing large reduction in rice yield. Therefore, during the occasional high temperatures after rainstorms, the water level in rice paddy fields should be maintained at 10-15 cm for 5 days to not only alleviate the effect of high temperature on rice growth, but also reduce the loss of nitrogen and phosphorus.

Conclusion
A combination of high temperature and waterlogging stresses at the booting stage promoted the early formation of aerenchyma in rice roots, and had a certain sustainability.
High temperature (T1) at the booting stage inhibited the expansion of root diameter, stem diameter, thickness of the outer root layer, and vessel diameter in rice; especially, root diameter and stem diameter decreased by 29.09% and 15.28%, respectively, when compared with those of the CK. Even when normal growth conditions were restored, the root anatomical parameters could not be balanced. High temperature treatment at the booting stage had a long-term effect on the inhibition of rice root system, which affected the absorption and transportation of water and nutrients by the root system and subsequently rice yield.
The interaction of high temperature and waterlogging stresses at the booting stage had little effect on rice root diameter, stele diameter, and thickness of the outer root layer. However, high temperature × heavy waterlogging (T3) reduced the rice root vessel diameter by 14.11% compared with the CK, affecting the transport of water and nutrients in rice roots.
Two-way ANOVA showed that temperature and water stress alone at the booting stage affected significantly the root diameter, stele diameter, and vessel diameter, whereas their interaction had a significant effect only on vessel diameter.

Plant material and test site
The experiment was carried out at the National Field Science Observatory of Shangqiu Ecosystem in Henan Province from May to October 2017. The average annual rainfall, annual average evaporation, and daily maximum temperature at the station are 705.1 mm, 1 751 mm, and 32 °C, respectively. The plant material tested was the middle-late maturing conventional japonica rice variety 008, whose growth period lasts about 145 days along the Huaihe River. In this experiment, rice plants were planted in barrels. The bottom diameter of the barrels was 21.5 cm, the upper diameter was 25 cm, and their depth was 29.5 cm. Test soil of the fluvoaquic type was collected from the field tillage layer at the test station. After air-drying and sieving, the soil was loaded into barrels at a volume of 10 kg air-dried soil per barrel. The amount of fertilizer applied per barrel was: 2.20 g CO(NH 2 ) 2 , 0.90 g K 2 SO 4 , 2.50 g KH 2 PO 4 , and 16.7 g organic fertilizer.

Experimental design
Seeds were sown on May 4, 2017. After 40 days (June 13), seedlings with identical growth were selected for transplantation. Three holes were made in each barrel, and two plants were transplanted in each hole and harvested on October 24, 2017. The interaction between high temperature and waterlogging stress and their effect on the plants was tested at the booting stage of rice. Rice is a plant that requires high temperature and humidity, but very high temperature and submergence depth and extended submergence duration will affect its growth and development. Previous studies have shown that during rice growth daily average temperatures higher than 32 °C or daily maximum temperatures higher than 35 °C lead to heat damage of rice (Tian et al., 2008), whereas 5 days of flooding and more than 10 cm of flooding depth affect the growth of rice and the microstructure of its root system (Zhen et al., 2015). Considering the meteorological conditions in the Huaihe River area from July to September, the experiment adopted a completely random design with two factors, high temperature and flooding, resulting in a total of six treatments: 1) high temperature (

Root anatomical observations
Three pots of rice plants with identical growth were selected for each treatment at 0, 5, 10, and 20 days of treatment. Three new, white roots were selected from each pot, and 5-10 mm segments of each root was excised 10-15 mm away from the root tip and dropped immediately into FAA fixative (40% formalin : glacial acetic acid : 70% ethanol, 1:1:18, by vol). After dehydration with graded alcohol series and clearing with xylene, the material was immersed in wax, and the wax-soaked tissues were embedded in the embedding machine. Two wax blocks, each comprising three samples, were prepared for each treatment. The prepared wax blocks were cut into 3 µm thick slices using a microtome. Paraffin sections were then placed in saffron stain for 1-2 h and slightly washed with tap water to remove the excess dye. For discoloration, samples were sequentially placed into 50%, 70%, and 80% gradient alcohols for 3-8 s each. Slices were placed into solid green dyeing solution for 30-60 s and dehydrated (Dehydrator Wuhan Junjie Electronics Co., Ltd., JJ-12J) in anhydrous ethanol. The sections were stained with safranin fixed green dyeing and mounted with neutral gum. Slices were observed with a positive optical microscopy (Nikon, Japan, NIKON ECLIPSE E100) and imaging system (Nikon, Japan, NIKON DS-U3). Relevant indexes of the root system were measured with Case Viewer 2.0 software (3DHISTECH Ltd., Budapest, Hungary), and clear pictures were selected for photography.

Data statistical methods
(1) Root diameter (RD, µm): Six cross-sectional areas of root cells were measured by CaseViewer 2.0 (3DHISTECH Ltd., Hungary) and three measurements of similar value were selected to calculate cross-sectional root diameter using the equation:. The average value of the cross-sectional area was labeled for other parameters.
(2) Thickness of the outer root (µm): Generally, epidermis, outer cortex, thick-walled cells, and adjacent cortical parenchyma cells are collectively referred to as the outer cells layers of the roots. (Meng, 2008). Thickness of the root outer layer at 15 mm from the root tip reflects the development of rice roots. Using the measuring tool in CaseViewer 2.0 (3DHISTECH Ltd., Hungary), three labeled root cells (root cells with the same cross-sectional diameter) were selected. Four values in the direction of 0:00, 3:00, 6:00, and 9:00 were selected for each root cell volume. The thickness of the outer layer was measured in 12 roots, and its average value was calculated.
(3) Stele diameter (SD, µm): Root stele cells refer to the stele parts within the endodermis. The root stele area of three labeled root cells was measured by using the measuring tool in CaseViewer 2.0 and the stele diameter was calculated as follows: , the obtained values were then averaged.
(4) Number and diameter of vessels: When calculating the stele diameter, the number of vessels (VN) and the area of the vessels were recorded, and vessel diameter (VD, µm) was calculated as follows:. The obtained values were then averaged.

Data processing and analysis
Microsoft Excel and SPSS 19.0 software (IBM Corp., Armonk, NY, USA) were used to analyze the data, and significant differences between the means were determined with Duncan's new multiple range test. Cross-sectional map of root anatomy of rice at 5 days after high temperature and waterlogging stress Cross-sectional map of root anatomy of rice at 10 days after high temperature and waterlogging stress Cross-sectional map of root anatomy of rice at 20 days after high temperature and waterlogging stress