To cope with drought stress, plants have evolved complex strategies by modulating drought-responsive signaling and metabolic processes at the cellular, organ, and whole-plant levels. Fine roots are essential for maintaining water balance under drought stress, and this is achieved, in part. Through the regulation of their proteomes. A detailed assessment of the changes in fine root proteomes in response to drought is essential to understand the mechanisms underlying physiological adaptation to stress. This is the first comprehensive proteomic analysis of the fine roots of cotton plants under drought stress.
Soil drought affects physiological and growth characteristics
Several studies have shown that drought stress significantly influences the morphology of cotton plants [15, 16]. In this study, plant height, stem diameter, leaf area, and leaf thickness were significantly reduced under soil drought (Fig. S1). This indicated that drought stress severely inhibits the development of the aboveground portions of plants. In addition, we concluded that the SPAD value and LRWC were closely associated with the duration of drought stress (Fig. 2).
The root system coordinates with the aboveground portion of plants by efficiently utilizing limited resources under drought stress, with synergistic action occurring among the portions of plants that regulate growth [17]. Our results showed that the response of root morphology to drought stress in cotton plants was opposite that of the aboveground portions. Under drought stress, fine roots tended to be thinner and longer, thus promoting elongated root systems and the absorption of water from deep in the soil (Fig. S2).
Effects of soil drought on stress- and defense-related proteins
When plants suffer from drought stress, higher levels of ROS are produced. Furthermore, ROS are used as signal molecules to control programmed cell death, abiotic stress responses, and pathogen defense [18].
Plants have evolved a variety of ROS scavenging strategies to alleviate ROS damage by regulating antioxidant enzyme activities and non-enzyme antioxidant content, which mitigate the adverse effects of drought stress [19]. Among them, SOD provides the first line of defense in antioxidant systems. In this study, the SOD and CAT activities of fine roots began to increase significantly under drought stress compared with CK plants at 30 DAD (Fig. 3). However, the level of DEPs encoding CAT and SOD did not change significantly, indicating that the protein levels were not correlated with their activities. POD is involved in a wide range of physiological processes, including ROS metabolism. Previously, POD levels were shown to be changed under drought stress [18]. In this study, four POD proteins (A0A1U8M415, A0A1U8KFD1, A0A1U8IEZ3, A0A1U8L990) were up-regulated in the ‘DS45 vs CK45’ comparison, consistent with the physiological results observed (Additional File 11: Table S6; Fig. 3b). Ascorbate peroxidase (APX) acts as another type of antioxidant enzyme, and the expression levels of two APX or APX-like proteins (A0A1U8MGX8, A0A1U8L6G4) were significantly up-regulated at 45 DAD (Table S6). Thioredoxin (Trx) is involved in the removal of ROS and is considered a biomarker of oxidative stress [20], and seven Trx or Trx-like proteins (A0A1U8PN19, A0A1U8MVQ7, A0A1U8K8W1, A0A1U8PQ09, A0A1U8IVU7, A0A1U8HYN5, and A0A1U8IT56) were induced at 45 DAD (Table S6). Zhang et al. [21] cloned a Trx superfamily gene TaNRX from common wheat (Triticum aestivum) and found that it functioned as a drought resistance mechanism, consistent with our findings. Thus, these results confirmed that the soil drought treatment induced up-regulation of active oxygen scavenging enzymes in the fine roots of cotton plants, and long-term drought stress assessed at 45 DAD activated more enzymes than were observed at 30 DAD (Table S6).
The abundance of stress-responsive proteins, especially those related to abiotic stress, such as late embryogenesis abundant (LEA) proteins, germin-like proteins (GLPs), annexin, and heat shock proteins (HSPs) also changed significantly. According to the present proteome data, three LEA proteins (P46518, P09441, A0A1U8L687) were found to be highly up-regulated in the ‘DS45 vs CK45’ comparison (Table S6). Owing to their high hydrophilicity, LEA proteins bind a large number of water molecules, and the accumulation of LEA protein was reported to be closely related to dehydration resistance [22]. Garay et al. [23] found that LEA proteins function as a hydrophilic buffer to reduce the rate of cell water loss under drought stress, thereby ensuring sufficient water remains in tissues in order to maintain normal metabolic activity. GLPs are a type of glycoprotein, and many of them possess the manganese-containing SOD activity that catalyzes ROS into H2O2 [24]. Previous studies have shown that GLP gene expression was higher under drought stress [25,26,27]. We found that the abundance of six GLPs (A0A1U8LQB5, A0A1U8PTH1, A0A1U8PBD0, A0A1U8JEA8, A0A1U8P8T2, and A0A1U8L5P7) changed significantly in the ‘DS45 vs CK45’ comparison, while only one (A0A1U8L5P7) was identified in the ‘DS30 vs CK30’ comparison (Table S6). Annexin is a type of protein family that binds to membrane phospholipids in a calcium-dependent manner. Studies have shown that under different stress conditions, the expression of plant annexin will increase and accumulate in large amounts along the cell membrane. It has been speculated that this accumulation may be related to the construction of the ion channel structure, the protection of the cell membrane, and the function of the ROS-induced signal [28,29,30,31]. Eight annexin or annexin-like proteins (A0A1U8J8K4, A0A1U8J7H8, A0A1U8IN71, A0A1U8JFY4, A0A1U8I5D0, S5GFP3, A0A1U8JDH4, and A0A1U8J6E7) were up-regulated in the ‘DS45 vs CK45’ comparison, of which six were already identified as up-regulated DEPs at 30 DAD (A0A1U8IN71, A0A1U8J8K4, A0A1U8I5D0, S5GFP3, A0A1U8J6E7, and A0A1U8JDH4) (Table S6). This implies that the response pattern of annexin in fine roots in the early stages of the full drought treatment and after 45 days of soil drought were consistent, suggesting that this upregulation might be a common strategy by which fine roots cope with drought stress. Qiao et al. [29] found that rice annexin OsAnn1 enhanced the tolerance to drought stress and AtANN1-deletion Arabidopsis mutants showed reduced resistance to drought [28]. In addition, annexin has been reported to be linked to POD activity [32], which was most likely associated with the accumulation of the ROS scavengers APX and POD. As such, annexin likely plays an important role in the process of physiological adaption to drought stress. Furthermore, 13 heat shock protein (HSP) were identified, 5 of which (A0A1U8PG98, A0A1U8MBN0, A0A1U8M2Z7, A0A1U8KRV5, A0A1U8LKC3) were up-regulated (Table S6). HSPs are involved in protein folding and active oxygen clearance, playing a key role in drought stress in cassava (Manihot esculenta Crantz) through transcriptional, post-transcriptional, and translational regulation [33]. HSP70 is involved in many cell processes, conferring plant heat tolerance and drought tolerance in both transgenic tobacco and Arabidopsis [34], and seven DEPs encoding HSP70 or HSP70-like proteins were identified in this study.
Among the identified DEPs, there were some pathogenesis-related proteins (PRs) identified that showed significant changes under soil drought, such as thaumatin, osmotin, glucan endo-1,3-beta -glucosidase, and chitinases [35, 36]. In the ‘DS45 vs CK45’ comparison, ‘pathogenesis-related protein PR-4A-like’ was significantly up-regulated (Table S6). PR proteins can be divided into 18 families (PR-1 to PR-18). Among them, PR-5 protein, also known as thaumatin-like protein (TLP), is accumulated rapidly when plants are subjected to different stresses, with accumulation being significantly correlated with the intensity of plant stress [37]. Osmotin also belongs to the PR-5 family, and it has a structure similar to that of TLP. According to the present study, ‘osmotin-like protein’ (A0A1U8PK21) and ‘osmotin-like protein I’ (Q2HPG3) were 1.48- and 2.20-fold up-regulated at 45 DAD. Glucan endo-1,3-beta-glucindase belongs to the PR-2 family, which is involved in cell division, flower formation, seed maturation, and plant responses to abiotic stress [38]. Our results revealed that three ‘glucan endo-1,3-beta-glucosidase-like’ proteins (A0A1U8N331, A0A1U8HW63, A0A1U8I6K1) were up-regulated (Table S6) at 45 DAD. Chitinase is another kind of well-characterized pathogen-related protein [38]. The chitinase family can be divided into sub-families that include endochitinases, exochitinases, β-N-acetylglucosidases, and chitosidases. These enzymes work together to gradually degrade chitin into monosaccharides and enhance plant defense against abiotic stress [39]. In this study, three DEPs (A0A1U8PCX3, A0A1U8IHA4, Q39799) encoding endochitinases were up-regulated (Table S6).
Taken together, the elevated levels of these antioxidants and pathogenesis-related proteins are likely strategies for cotton plants to cope with the deleterious effects of ROS, and increased stress durations are likely to activate the expression of more related proteins.
Effects of soil drought on ion transport-related proteins
Maintenance and re-establishment of cellular ion homeostasis during stress conditions is extremely important for plant survival and growth, especially for plants under osmotic stressors such as drought [40]. In the current study, it was found that a certain number of DEPs were enriched among the biological process terms related to ion transport, including ‘hydrogen transport’ (GO:0006818), ‘monovalent inorganic cation transport’ (GO:0015672), ‘cation transport’ (GO:0006812), and ‘anion transmembrane transport’ (GO:0098656) (Table S3). This has aroused great interest in the analysis of the DEPs involved. (Additional file 12: Table S7).
V-type ATPases transport hydrogen ions to the vesicles or extracellularly, thus maintaining a stable acid–base environment in cells [41]. Overexpression of AVP1 (a gene encoding a protein that can generate a H+ gradient across the vacuolar membrane similar in magnitude to that of the multisubunit vacuolar H+-ATPase) in transgenic Arabidopsis substantially increased resistance to drought relative to wild-type plants, and it was also found that the resistant phenotypes had increased vacuolar proton gradients, resulting in increased solute accumulation and water retention [42]. V-type ATPase is also induced in the roots of Arabidopsis [43], wheat [44], and cucumber [45] under abiotic stress conditions. Here, the increased abundance of twelve V-type proton ATPases in this study indicates that the increased activities of these enzymes are considered to be a cost-effective strategy for coping with long-term stress (i.e., at 45 DAD) (Table S7). Overexpression of the V-ATPase G subunit in walnut (Juglans regia) effectively improved drought resistance in transgenic plants [46]. In contrast with the 45 DAD results, we found two ‘V-type proton ATPase subunit G’ (A0A1U8KTX7 and A0A1U8NHE1) were down-regulated in the ‘DS30 vs CK30’ comparison, which may indicate that there are two completely opposite regulatory strategies for V-type ATPases between 30 and 45 DAD.
ABC transporters transport stress-related secondary metabolites such as alkaloids, terpenoids, polyphenols, and quinines [47]. In the current study, five ABC transporters (A0A1U8PJL1, A0A1U8N9K0, A0A1U8L5Z3, A0A1U8LYD7, and A0A1U8KBE6) were found to be induced by soil drought in the ‘DS45 vs CK45’ comparison, while in the ‘DS30 vs CK30’ comparison, only one ABC transporter (A0A1U8L5Z3) was identified as a DEP. This confirmed the findings of a previous study in which ABC transporters were shown to improve the resistance of crops to abiotic stresses such as drought [48], and this may indicate that the up-regulation of ABC transporters in the fine roots of cotton plants is an effective regulation strategy under soil drought. Kim et al. [48] also found that overexpression of AtABCG36, an ABC transporter gene in Arabidopsis, can greatly increase the ability of ABC transporter to transport sodium ions and significantly improved the drought resistance of Arabidopsis plants, supporting our hypothesis.
Plants have complex systems used to absorb and transport nitrogenous compounds (e.g., nitrate, ammonium, oligopeptides, and amino acids). The nitrate transport gene family is divided into low- and high-affinity transport families. When the concentration of nitrate in the outside world is less than 0.5 mM, the high-affinity transport family is mainly responsible for its function [49]. In this study, ‘high-affinity nitrate transporter 2.1 (A0A1U8PWK7)’ and ‘high-affinity nitrate transporter 3.2 (A0A1U8NYW8)’ were down-regulated at 30 DAD. In order to better absorb, utilize, and distribute nitrate, plants have evolved different transport vectors or channel proteins to cope with environmental changes. The genes encoding these proteins are mainly divided into four families: NRT1, NRT2, CLC, and SLAV1/SLAH. Of these families, only NRT1 and NRT2 participate in the absorption of nitrate by roots, and they were later collectively renamed NRT1/PRT FAMILY (NPF) proteins according to their evolutionary history [50]. Taochy et al. [51] found that AtNPF2.3 was responsible for transporting nitrate from roots to shoots when plants were subjected to salt stress. In the current study, five NRT1/ PTR FAMILY proteins (A0A1U8PIU8, A0A1U8LJU9, A0A1U8MGW2, A0A1U8MWX1, and A0A1U8M2N9) were down-regulated at 45 DAD. This shows that long-term drought stress may hinder the absorption of nitrate by the fine root system, which in turn leads to a reduction in the nitrates allocated to the aboveground portions of plants. Additionally, some recent studies have revealed that NPF proteins also transport plant hormones, including auxin, abscisic acid, jasmonates, and gibberellin [52, 53].
It is generally believed that the input of drought signals depends on the mechanical load of the membrane. Changes in mechanically sensitive ion channel activity can sense changes in cell membrane tension due to loss of turgidity [54]. In plant cells, such mechanosensitive channels drive an influx of calcium [55]. In this study, two mechanosensitive ion channel proteins (A0A1U8NHU2, A0A1U8LSY1) and one calcium-transporting ATPase were also greatly induced at 45 DAD. It was also observed that five syntaxin-like proteins were exclusively induced by soil drought. Some studies have demonstrated that syntaxin proteins can interact with and coordinate the trafficking of plasma membrane aquaporin to modulate the water permeability of cell membranes [56, 57].
Short-term soil drought affects ‘Cutin, suberin and wax biosynthesis’ in cotton fine roots
The most enriched KEGG pathway in the ‘DS30 vs CK30’ comparison was ‘Cutin, suberin and wax biosynthesis,’ and it was not enriched at 45 DAD (Fig. 6), indicating that this pathway likely plays an important role when cotton fine roots respond to early drought and attracting our focus.
During the evolution of land plants, epidermal tissues evolved to prevent the loss of water and nutrients [58]. These epidermal tissues are composed of cutin, wax, and inner suberin layers [59]. Suberin is ubiquitous in specific internal root-tissues, where it controls water and ion uptake and also play roles in protecting plants from abiotic stresses and establishing plant morphology [60]. In the current study, the three up-regulated DEPs enriched in the ‘Cutin, suberin and wax biosynthesis’ pathway were ‘peroxygenase-like’ (A0A1U8HRH9), ‘probable peroxygenase 5 isoform X2’ (A0A1U8K2T6), and ‘omega-hydroxypalmitate O-feruloyl transferase-like’ (A0A1U8HNM9). Suberin is composed of suberin polyphenolic and polyaliphatic domains. Omega-hydroxypalmitate O-feruloyl transferase (HHT) is the main enzyme that regulates the phenylpropane-ferulic acid pathway, which directly or indirectly affects the expression of ferulic acid, thus affecting the structural composition of the suberin polyphenolic and polyaliphatic domains. Lotfy et al. [61] have shown that HHT can promote suberin formation in potato; Arabidopsis esb1 (enhanced suberin 1) mutants have increased suberin content and increased their water use efficiency during their vegetative growth stage, resulting in increased resistance to wilt relative to wild-type plants under drought stress [62]. Our TMT results suggest that the response of fine roots of cotton plants to the 30-days soil drought treatment is likely to increase the content of suberin by up-regulating HHT expression, thereby reducing water loss. Peroxygenase is a key enzyme involved in the formation of cutin [63]. Maize treatment with peroxygenase inhibitor caused cuticle changes, resulting in increased permeability to pesticides [63]. Therefore, it is likely that proteins belonging to the ‘Cutin, suberin and wax biosynthesis’ pathway were activated in the fine roots of cotton plants during the early stages of soil drought, thus promoting increased suberization of epidermal tissues, so as to protect internal vascular tissues from drought stress. This, in turn, would maintain the vascular connection between the root system and shoots, helping plants resist short-term drought. However, we did not find that this pathway was enriched in the ‘DS45 vs CK45’ comparison, suggesting that drought stress caused functional damage to the fine root epidermis at this stage.
Long-term soil drought activated more phytohormone-related DEPs than short-term drought
According to the physiological and morphological results of our experiment, 45 days of soil drought caused serious damage to cotton plants. To explore the mechanisms of drought stress in fine roots at 45 DAD, we performed differential proteomic analysis between fine roots under the CK and DS treatments. Compared with the ‘DS30 vs CK30’ comparison, there were more identified DEPs and more enriched metabolic pathways at 45 DAD. We classified these pathways into five categories based on their first-level KEGG classification, including ‘Lipid metabolism,’ ‘Secondary metabolism,’ ‘Energy metabolism,’ ‘Carbohydrate metabolism,’ and ‘Amino acid metabolism,’ each of which has been widely studied in previous articles. Some DEPs that play an important role are listed in Table S8 (Additional file 13).
A series of adaptive responses produced by plants under drought stress are controlled by many phytohormones, and they are thus the basic mediators for tolerating or avoiding the negative effects of water deficit. In the current study, some important DEPs were identified to be involved in the regulation of phytohormones at 45 DAD, and only a small part of these DEPs appeared in the ‘DS30 vs CK30’ comparison (Additional file 14: Table S9).
One of the proteins markedly up-regulated by drought at 45 DAD was identified as indole-3-acetic acid-amido synthetase GH3.17-like protein (Table S9). The proteins of the GH3 family have hormone amide synthetase activity, catalyzing the binding of free auxin (IAA) to amino acids [64]. OsGH3.13 encodes indole-3-acetic acid-amino synthetase in rice, which improves plant drought resistance [65]. S-adenosylmethionine synthetase (SAMS) functions as one of the key enzymes in the ethylene synthesis pathway [66]. Four SAMS (A0A1U8P2T2, A0A1U8L5H6, A0A1U8JUM7, A0A1U8NVJ7) and five 1-aminocyclopropane-1-carboxylate oxidases (A0A1U8NWE4, A0A1U8MU28, A0A1U8JC48, A0A1U8JY55, A0A1U8PRG1) were identified as down-regulated DEPs at 45 DAD. As the last enzyme in the ethylene pathway, 1-aminocyclopropane-1-carboxylate oxidase (ACO), is generally considered the rate-limiting enzyme in ethylene biosynthesis [67]. A large amount of ACO is induced under drought conditions, which decomposes 1-aminocyclopropane-1-carboxylate into ethylene, eventually leading to an increase in the expression of ACO genes and ethylene production [68, 69]. However, the results we obtained were contrary to earlier research, suggesting that the fine roots of cotton plants activated a response mechanism when challenged by drought, which led to a decline in ACO levels.
It has been shown that the interaction between IAA and ABA promotes the development of lateral roots in plants, and the morphology of roots is a necessary element of plant responses to drought stress [70]. Some proteins involved in abscisic acid (ABA) metabolic were also identified as DEPs in this study. Abscisic acid 8′-hydroxylase (ABAH) acts as the key enzyme in the ABA oxidative inactivation pathway [71]. Takeuchi et al. [72] reported that ABAH inhibitors can significantly improve drought tolerance in Arabidopsis. In the current study, it was determined that two ABAH proteins (A0A1U8NTQ2, A0A1U8N913) were down-regulated at 45 DAD (Table S9), indicating that the fine roots of cotton plants activate a corresponding drought resistance mechanism by down-regulating ABAH protein expression. ABSCISIC ACID-INSENSITIVE 5 (ABI5) is a key factor involved in ABA response, and its protein stability and protein phosphorylation are all regulated by ABA, with different degrees of increases exhibited under abiotic stresses [73,74,75]. In this study, two ABI5 proteins (A0A1U8PG85, A0A1U8P7L8) were found to be significantly up-regulated at 45 DAD, confirming previous proteomic studies [73,74,75].
The above results indicated that the fine roots of cotton were activated across a series of signal transmission pathways under long-term drought stress, some of which are involved in the regulation of phytohormones, and may therefore eventually lead to changes in phytohormone levels.
Based on our results and previous studies, strategies to minimize the harm of drought stress on cotton plants or improve the resistance of cotton plants to drought stress can be determined. In Fig. 7, we summarize the response of cotton fine roots to 45 days of soil drought based on the above morphological, physiological, and proteomic results. First, selecting cotton varieties with longer root systems and growing cotton in soil types that facilitate root penetration are effective strategies for enhancing the adaptability of cotton plants to drought conditions. Second, exogenous application of plant hormones or growth regulators with similar effects can be an effective method of improving drought resistance of cotton plants. Finally, based on results from different stress stages, appropriate proteins can be identified for the purpose of altering the genetics of crops through traditional artificial selection or genetic transformation. In short, our results enhance the current understanding of the protein expression mechanism in the fine roots of cotton plants under drought stress and provide new targets for genetic improvement and enhanced agronomic management practices.