3 RESULTS
3.1 Cortical senescence percentage, cortical cell files and cortical lacunae area indicate the progress of RCS
To investigate the spatio-temporal characteristics of cotton RCS, we employed a laser scanning confocal microscope to scrutinize the anatomical structures from the root tip to the root end, as depicted in Figure 1. The lacunae area is the principal indicator of RCS. Drought stress significantly promted the degree of lacunae area. In other words, the lacunae area was larger in tissues far from the root tip under drought stress compared to no-stress treatment. Both varieties exhibited an increasing lacunae area with the increasing distance from the root tip under no-stress conditions (Figure 1). The occurrence of RCS varied in the two varieties. Under drought stress, RCS in ”Guoxin 02” significantly increased compared to ”Ji 228.”.
The cortical senescence percentage, cortical cell files and cortical lacunae area provide the basis for evaluating the progression and patterns of RCS within the root sections (Schneider et al., 2017b). The percentage of the senescent cortex is used to compute the disparity in cross-sectional areas (total cross-sectional area — stele area) at 3 cm from the root apex and at the sample position (Schneider et al., 2017b). Essentially, this determines the difference in the cross-sectional area between the sample and the root tip, providing a quantitative measure of cortical senescence. Among these parameters, cortical senescence percentage is a key indicator for evaluating RCS. RCS was evidenced by the disappearance and absence of the root cortex, which included cortical cell files and cortical lacunae area (Figures 2BC).
Drought stress significantly promoted the occurrence of RCS in cotton. Compared with no-stress, the cortical senescence percentage in ”Guoxin 02” increased significantly by 26.26%-56.69% (Range of all root segments) and by 22.56%-35.48% in ”Ji 228” under drought stress (Figure 2A).
Changes of the cortical senescence percentage in different root segments. Under drought stress, the occurrence of RCS was directly proportional to the distance from the root tip, meaning that the frequency of RCS significantly increased with increasing distance from the root tip (Figure 1). The cortical senescence percentage of ”Guoxin 02” and ”Ji 228” at 12 cm segment from the root tip was 82.43% and 75.64% higher than that at the 6 cm root segment, respectively (Figure 2A).
Significant genotypic differences in RCS were also observed between ”Guoxin 02” and ”Ji 228” (p < 0.01). After 15 days of drought stress, ”Guoxin 02” had a higher occurrence rate of RCS than ”Ji 228”. Specifically, the cortical senescence percentage of the ”Guoxin 02” increased significantly from 14.03% to 20.18% compared to ”Ji 228” under drought stress. (Figure 2A).
Drought stress significantly reduced the cortical cell files (Figure 2B) of cotton but significantly increased the cortical lacunae areas (Figure 2C). Specifically, compared with no-stress treatment, the cortical lacunae area of ”Guoxin 02” and ”Ji 228” significantly increased by 9.45 - 32.65 and 0.51 - 1.79 times, respectively, under drought stress (Figure 2C). The cortical lacunae area significantly increased with increasing distance from the root tip. Specifically, the cortical lacunae area of ”Guoxin 02” and ”Ji 228” increased significantly by 26.13 times and 4.82 times, respectively, in the 12 cm segment compared to the 3 cm segment from the root tip (Figure 2C).
3.2 Association between RCS and reduced root metabolic costs
Schneider et al.(2017b) established a negative correlation between RCS and root metabolic costs. Therefore, to further underscore the occurrence of RCS in cotton crops, we conducted a comprehensive series of experiments involving measuring the changes in root metabolic enzyme activities and root respiration across different root segments (Figure 3).
Compared to no-stress, drought stress reduced the phosphofructokinase, malate dehydrogenase, and glucose-6-phosphate dehydrogenase activities, and root respiration by 0% - 21%, 32% - 34%, 46% - 51%, and 20% - 63% in ”Guoxin 02” and by 5% - 19%, 31% - 39%, 23% - 37%, and 9% - 34% in ”Ji 228”, respectively (Figures 3A-D).
Notably, the trend characteristics at different locations from the root tip to the root end showed that RCS was inversely proportional to phosphofructokinase, malate dehydrogenase, and glucose-6-phosphate dehydrogenase activities, and root respiration. That is, phosphofructokinase, malate dehydrogenase, and glucose-6-phosphate dehydrogenase activities, and root respiration decreased significantly with increasing distance of the root segments from the root tips (Figures 3A-D). Specifically, compared to the 3 cm root segments, phosphofructokinase, malate dehydrogenase, and glucose-6-phosphate dehydrogenase activities, and root respiration significantly decreased by 15.28%, 10.34%, 12.76%, and 71.56% in ”Guoxin 02”, (Figures 3B-D) and by 32.96, 25%, 34.46%, and 57.66% in ”Ji 228”, respectively (Figures 3A-D), in the 12 cm root segments under drought stress.
In addition, root metabolic costs varied significantly among varieties. Compared with ”Ji 228”, root respiration and glucose-6-phosphate dehydrogenase activity of ”Guoxin 02” decreased by 26.78% and 19.39% (whole root segment), respectively, under drought stress (Figures 3DC). Pearson correlation analysis showed that the cortical senescence percentage at 6 cm, 9 cm and 12 cm from the root tip was positively correlated with root respiration (p <0.05, Figures 4D-F). In summary, the trends we observed in root metabolic costs provide further scientific evidence of the presence of RCS in cotton.
3.3 RCS affects thetissue structure of the root cortex
The characteristics of each tissue structure in the different root segments during RCS development were further analyzed (Figure 5A). Drought stress increased the stole/whole ratio (Figure 5B) and lacunae/cortex ratio (Figure 5C) but reduced the cortex/stele ratio (Figure 5D).
Notably, the trend characteristics at different locations from the root tip to the root end showed that RCS was directly proportional to the stele/whole ratio, lacunae/cortex ratio, cross-section area, cross-section diameter, stele area, stele diameter, cortical area, and cortical diameter, but inversely proportional to the cortex/stele ratio. That is, the stele/whole and lacunae/cortex ratio increased significantly (Figures 5HJ), but the cortex/stele ratio decreased significantly with increasing RCS (Figure 5I). Specifically, the stele/whole ratio and the lacunae/cortex ratio increased by 7.14% - 60.60% and 50% - 133.33%, respectively, at the 12 cm segments compared to 3 cm segment from the root tip (Figures 5HJ).
There were genotypic differences in the proportion variation of each tissue structure. Among them, the stele/whole and lacunae/cortex ratios of ”Guoxin 02” were significantly higher (94.22% and 117.02%, respectively) than those of ”Ji 228” under drought stress (Figures 5HJ). However, the cortex/stele ratio was significantly reduced (58.97%) (Figure 5I).
3.4 RCSis inversely proportional to endogenous hormones
To characterize the changes in endogenous hormone levels during RCS development in cotton and the mechanism of their influence, we measured the contents of five endogenous hormones in different root segments. GA, ZR, IAA, and BR contents were decreased under drought stress compared to the no-stress treatment (Figures 6A-D); however, ABA was increased (Figure 6E).
Notably, the trend characteristics at different locations from the root tip to the root end showed that RCS was inversely proportional to GA, ZR, IAA, BR, and ABA contents. That is, GA, ZR, IAA, BR, and ABA decreased significantly with increasing distance from the root tips (Figures 6A-E). Specifically, compared to 3 cm root segments, GA, ZR, IAA, BR, and ABA contents of ”Guoxin 02” significantly decreased by 28.93%, 68.91%, 35.82%, 65.15%, and 60.01%, respectively, in the 12 cm root segments under drought stress (Figures 6B-D). Similarly, the GA, ZR, IAA, BR, and ABA contents of ”Ji 228” significantly decreased by 47.12%, 60.97%, 56.14%, 69.10%, and 40.25% in the 12 cm root segments compared to the 3 cm root segments, respectively, under drought stress (Figures 6B-D). The endogenous hormonal content of each segment differed between the two varieties. Compared with ”Ji 228”, the GA and ABA contents of ”Guoxin 02” decreased by 24.74% - 44.46% (Figure 6A) 31.28% - 54.01% (Figure 6E), respectively, but IAA increased by 10.93% - 69.33% (Figure 6C).
Linear regression analysis showed a significant correlation between RCS and the endogenous hormone content of the root system. GA and ABA contents showed a decreasing trend with the increasing cortical senescence percentage at 6 cm, 9 cm and 12 cm segments from the root tip under drought stress (Figures S1 A-C and G-I). However, IAA showed an increasing trend (Figures S1 D-F). Notably, the cortical senescence percentage at the 12 cm segment from the root tip significantly correlated with GA, ABA, and IAA contents (0.01 < p< 0.05).