1 Introduction
Root anatomical phenes play a pivotal role in capturing soil resources, and the root cortex is a vital anatomical phene found between the epidermis and stele. The root cortex consists of multiple layers of thin-walled cells differentiating from the primary meristem, occupying a significant proportion of the root cross-section volume. The cortex is a lateral pathway for water and solute transport from root hairs to the central cylinder, serving as the primary site for nutrient storage, aeration, and secretion of nutrients and growth regulators (Lynch, 2015). Root cortical senescence (RCS) is a form of programmed cell death occurring in the cortical cells of the root system (Schneider and Lynch, 2018). During RCS, the root external appearance remains healthy and white, but the cortical cells predominantly lose their nuclei (Bingham, 2007), a phenomenon commonly referred to as ”non-pathogenic root cortex death” (Deacon and Henry, 1978).
Accurately quantifying RCS is essential for comprehensively understanding plant growth and physiological status. Histological techniques utilizing cell viability staining have become the predominant approach for studying RCS phenotypes. Commonly used dyes, including neutral red (Beckel, 1956), Feulgen reagent (Holden, 1975), toluidine blue (Brown and Hornby, 1987), acridine orange (Henry and Deacon, 1981), and other cell viability stains, have demonstrated efficacy in RCS assessment. While concerns were raised by Wenzel and McCully (1991) about the effectiveness of acridine orange and other cell viability staining dyes for RCS evaluation, research by Henry and Deacon (1981) indicated a higher potency in RCS detection when assessed using acridine orange staining, Feulgen staining, and single-cell pressure probe techniques (Bingham, 2007). Similar to acridine orange staining, technologies such as single-cell turgor pressure measurements, TUNEL-assay and Nomarski optics revealed comparable RCS patterns and occurrence rates across various plant species (Bingham, 2007; Henry and Deacon, 1981; Liljeroth and Bryngelsson, 2001; Wenzel and McCully, 1991). These research findings confirm the feasibility of using acridine orange and other cell viability staining methods to evaluate RCS.
RCS causes the damage or loss of nuclei in the cortical cells, an early indicator of cell apoptosis and RCS manifestation. Therefore, anucleated cortical cells are the hallmark of RCS, and the number of viable cells gradually diminishes with increasing cortical aging (Liljeroth, 1995). RCS significantly impacts the morphology and physiological characteristics of the root system; for example, during RCS, the cross-sectional area of the root cortex decreases, restricting the radial transport pathways from the cortex to the stele (Liljeroth, 1995). The occurrence of RCS also increases the proportion of aerenchyma in the root cortex of Gramineae crops, thus inhibiting the cortex function and increasing nutrient and water transport resistance (Hu et al., 2014a; Schneider et al., 2017b; Galindo-Castañeda et al., 2018). RCS has been shown to decrease the metabolic consumption of photosynthetic products in the cortex, especially during drought stress, with a substantial portion being utilized for new root growth (Lynch, 2015). This, in turn, promotes root elongation (Chimungu et al., 2015; Schneider et al., 2017b) and enhances water uptake (Lynch et al., 2005). Thus, it is evident that RCS is closely associated with the functionality of the root system under adverse conditions.
The RCS occurrence is influenced by multiple factors., including crop variety, growth stage, culture medium type, position within the root system, stress conditions, and phytohormones (Liljeroth, 1995; Schneider et al., 2017b). For instance, wheat seedling roots exhibited the highest rate of RCS occurrence compared to rye, barley, and oats (Henry and Deacon, 1981). Stress conditions such as low nitrogen, low phosphorus, and drought also induce the occurrence of RCS (Liljeroth, 1995). Compared with the root segments without RCS, the ones with RCS decreased by 19% and 12% under high N and low N conditions, respectively (Schneider et al., 2017b). The occurrence rate of RCS is also regulated by ethylene (Schneider et al., 2018), and exogenous ethylene significantly increases the RCS occurrence in maize seedlings and lateral roots (Schneider et al., 2018). However, the characteristics of RCS occurrence and its response to drought stress in Malvidae cotton are rarely reported.
Drought stress significantly constrains global agricultural productivity (Lynch, 2007), and efficient acquisition of soil moisture is crucial for enhancing plant drought tolerance. Cotton (Gossypium hirsutum L.) is a valuable economic crop. As a straight-rooted crop, cotton exhibits a well-developed root system with a deep tap root and widely distributed lateral roots. The function level and physiological metabolism of the root cortex largely represent the functionality and metabolism of the entire root system. Previous studies on gramineous crops found that unfavorable conditions accelerated RCS, reduced root respiration and decreased the transfer and allocation of photosynthetic products to fast-growing tissues (Schneider et al., 2017b). The anatomical structures of the root systems of dicotyledonous and monocots differ; however, the response mechanisms of RCS to stress conditions have not been reported in dicotyledonous crops. Therefore, there is a need to investigate the RCS responses of dicotyledonous crops to stress conditions. There are several unresolved questions regarding the effects of RCS in cotton: Is the susceptibility of the aboveground parts of cotton to drought due to premature RCS? What are the spatio-temporal variation characteristics and patterns of RCS in cotton? Is there a correlation between the consumption of a substantial quantity of photosynthates by RCS and the resilience of the aboveground parts of cotton?
We hypothesize that endogenous hormones regulate RCS in drought-tolerant varieties under drought stress, leading to reduced root metabolic costs. Consequently, more energy is redirected towards root growth, thereby enhancing drought tolerance. Therefore, this study aimed to explore the causative factors of cotton RCS and its physiological metabolism and endogenous hormonal regulation under drought stress, clarify the physiological role of RCS occurrence in relation to the regulation of the whole root development, and reveal the physiological mechanism of RCS in relation to the drought tolerance of cotton aboveground parts. The study mainly focused on (1) evaluating the existence and occurrence patterns of RCS in cotton and assessing the characteristics of cotton RSC under drought stress; (2) clarifying the characteristics and patterns of metabolic activities and endogenous hormones and their interrelationships during the RCS in cotton; (3) exploring the mechanism of action and effects of RCS on the root system and aboveground parts of cotton. This study is the first to confirm the existence of RCS in a dicotyledonous crop, cotton, and provides an in-depth understanding of the characteristics and patterns of RCS under drought stress. The results form the basis for using RCS to improve drought tolerance in cotton.