4 Discussion
4.1 Characteristics and patterns of RSC in cotton under drought stress
This study reports for the first time the presence of the RCS in cotton, a dicotyledonous crop of the mallow family. RCS has been widely explored in monocotyledons, including wheat, triticale (Triticosecale ), barley (Yeates and Parker, 1986; Liljeroth, 1995), rye (Secale cereale ) (Deacon and Mitchell, 1985; Jupp and Newman, 1987), and oats (Avena sativa ) (Yeates and Parker, 1986). However, there are no reports of RCS in dicotyledonous crops. The study also explored the characteristics and occurrence mechanism of RSC in cotton under drought stress.
To determine the spatio-temporal variation in the occurrence of RCS in cotton, we examined the cortex to determine the absence of acridine orange-stained fluorescent nuclei (Henry and Deacon, 1981). The two cotton varieties showed a similar pattern of RCS, which started in the outer cortical cell region and progressed inward. RCS was triggered mainly by non-apoptotic programmed cell death of cortical cells involving cells between the inner and outer cortex. According to laser scanning confocal microscopy, the gap between the cortical cells increased, and the cortical cells aged and underwent autolysis, with their internal structures gradually disintegrating with the occurrence of RCS. This disintegration gradually led to cortical cell contraction and invagination. When the internal material was depleted, the residual cell walls of the adjacent cells were superimposed, forming air spaces on both sides. Despite the senescence and disintegration of the cortex, the outer epidermis remained intact, and the outer epidermis, the air space, and the stele were clearly distinguishable (Figure 1). Different parts of the cotton root system, from the root tip to the root end, reflect the root developmental processes, including root cell genesis, differentiation, maturation, and death. In the root cross-section of cotton grown under no-stress conditions, the root tip zone (3 cm from the root tip) was almost devoid of RCS, but the root basal zone (12 cm from the root tip) showed partial RCS. In summary, the spatial dimension reveals that RCS shows an increasing trend in cotton with the increasing distance from the root tip; that is, root cortical cells exhibit dynamic changes from nucleated to non-nucleated forms from about the 3 cm region of the root tip to the junction of the taproot (Figure 1).
Analysis of the acridine orange fluorescent nuclei in the cortical area, cortical cell files, and cortical lacunae area also highlighted significant changes in different locations from the root tip to the root end. The cortical lacunae area showed a gradual increase (Figure 2C), while cortical cell files showed an initial increase followed by a decrease (Figure 2B), indicating that RCS mainly occurred in the maturation zone. Notably, the stele remained viable despite the occurrence of RCS, as demonstrated by the acridine orange fluorescent nuclei. The proportion of the root tissues also changed dynamically with increasing distance from the root tips (Figure 5). For example, the stele/whole ratio and lacunae/cortex ratio increased (Figure 5BD), while the cortex/stele ratio decreased (Figure 5C) with increasing root tip distance.
Drought stress induced RCS in cotton. The root profile of cotton grown under drought stress showed different spatio-temporal variations: the root tip zone (3 cm from the root tip) was almost devoid of RCS, while the root middle zone (6 cm from the root tip) showed partial RCS, and the root end zone (12 cm from the root tip) showed the highest RCS rate. However, drought stress did not change the occurrence point of RCS relative to the root tip, but it did increase the degree of RCS occurrence in cotton. After 15 days of drought stress treatment, the cortical cells of cotton roots showed senescence, leaving only root tips (Figure 1). Unlike the no-stress condition, the greater the distance from the root tip, the more severe the RSC degree under drought stress, which seriously affects root function. Therefore, we suggest that the cotton aboveground resilience may exhibit reduced drought tolerance due to premature RCS. Moreover, a previous study demonstrated that under low P and low N conditions, the RCS of barley increased by 13% and 11%, respectively (Schneider et al., 2017b), while shading decreased the RCS of wheat and barley (Lewis and Deacon, 1982). Thus, RCS is a complex phenomenon that depends on environmental factors, plant species, and genetic factors (Liljeroth, 1995; Schneider et al., 2017a).
The extent to which RCS occurs varies among plant species. For example, in 15-day-old wheat roots, 80% - 90% of the root cortical cells were non-nucleated (Liljeroth, 1995), whereas only 20% - 35% were non-nucleated in barley and rye (Liljeroth, 1995). This suggests that wheat has a relatively higher degree of RCS compared to other plants (such as wheat, barley, rye, and oats) (Deacon and Henry, 1980; Deacon and Mitchell, 1985; Henry and Deacon, 1981; Holden, 1976; Liljeroth, 1995; Yeates and Parker, 1986). However, the comparison of RCS between cotton and other crops has not been reported. Our study showed that only 40% - 50% of the root cortical cells of cotton plants grown for 31 days (from planting to harvesting) were non-nucleated (Figure 2B). Thus, the degree of RCS in the 31-day-old cotton roots was reduced by about 20% compared to the 15-day-old wheat. Therefore, the incidence of RCS in cotton was lower than in wheat; however, this should also be compared with other crops (such as triticale, barley, rye, and oats).
RCS exhibits significant genetic variability in cotton. The two cotton varieties (with different drought tolerance) used in this study showed significant differences in RCS. ”Guoxin 02” had a significantly higher rate of RSC than ”Ji 228” under drought stress (Figure 2A), indicating that a higher RCS rate may be a key feature of the plant’s ability to cope with drought stress. Similarly, differences in RCS have been reported between wheat and barley varieties (Henry and Deacon, 1981; Liljeroth, 1995). Therefore, the variation in RCS was mainly influenced by the genotypic differences under drought stress. However, the genetic and molecular mechanisms underlying this variation in cotton need further exploration.
4.2 RCS reduces root respiration
Root respiration is closely related to root maintenance costs. Root respiration, growth, maintenance, and ion uptake are the main components of root metabolic costs (Lambers, 1979; Van Der Werf et al., 1988; Lynch et al., 2005). Root metabolic costs involve changes in the internal configuration of the organelle, where the thickness between the cytoplasm and the vacuole membrane remains constant, such that the increase in RCS is mainly due to increased vacuole size (Gunawardena et al., 2001). Increased vacuole volume relative to the cytoplasm may reduce respiration because metabolic activity is higher in the cytoplasm than in the vacuole (Schussler and Longstreth, 1996). Our study quantified cotton root respiration in the mature region and found that total respiration in this region primarily involves tissue maintenance respiration. By inducing the transition from nucleated to non-nucleated cells, RCS alters the maintenance of respiration in the mature zone, emphasizing the need to maintain metabolic homeostasis in the cotton root system (Lynch et al., 2005; Lynch and Brown, 2008). Since RCS mainly occurs in the mature zone of the cortex, it affects root maintenance costs rather than construction costs. Therefore, we hypothesized that under drought stress, RCS significantly affects maintenance costs rather than construction costs and greatly impacts the ability of plants to adapt to arid environments.
This study revealed, for the first time, a significant negative correlation between RCS and root respiration in cotton under drought stress (p < 0.01, Lynch, 2015). Compared with ”Ji 228”, the root respiration and glucose-6-phosphate dehydrogenase activity (whole-root segment) of drought-resistant variety decreased by 26.78% and 19.39%, respectively. These results support the hypothesis that under drought stress, varieties with lower root metabolic costs seem to possess the ability to develop extensive and deep root systems to fully utilize soil water resources without yield losses (Figure 9). A previous study exploring the utility of RCS in barley under nutrient-deficient conditions indicated that by reducing root respiration, RCS can promote root growth and soil resource acquisition in barley (Schneider et al., 2017b). This is because some metabolic activities may diminish or stagnate as cells senesce, and senescent cells require less energy and nutrients, leading to reduced energy expenditure and metabolic costs (Postma and Lynch, 2011; Jaramillo et al., 2013; Hu et al., 2014b; Saengwilai et al., 2014).
Based on this, we speculate that whether in barley and cotton crops or under nutrient-deficient and drought-stress environments, the reduced metabolic consumption of photosynthetic products caused by RCS may not result from stress damage but rather an adaptive strategy. This complex mechanism helps plants to cope more effectively with environmental stresses while retaining energy for more important survival needs (Chimungu et al., 2014a; Chimungu et al., 2014a; Lynch et al., 2014; Saengwilai et al., 2014). Perhaps, in older root segments, RCS may reduce radial water transport when resources in the root edge have been depleted. Therefore, the loss of cortex means that the resource acquisition function of young root tissues changes to support function, axial transport, and storage of mature root tissue.