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.