Introduction

Citrus is a genus from the Geraniales order, Rutaceae family and Aurantioideae subfamily comprising trees typically cultivated in tropical and subtropical regions. Phylogenetic studies clustered the Citrus groups into six related genera, including Citrus, Fortunella, Poncirus, Clymenia, Eremocitrus, and Microcitrus (Swingle and Reece 1967). Citrus species initially originated in Southeast Asia and were then distributed to different global regions where they underwent further selection and domestication, which led to a wide range of ecological varieties with substantially different traits (de Ollas et al. 2019). At present, citrus is one of the most popular fruit crops worldwide with high nutritional and economic value. In recent years, the incorporation of agricultural sciences and biotechnology has dramatically improved the development of the citrus industry worldwide (Reen et al. 2020; Ziogas et al. 2021). Accordingly, the annual global production of citrus fruits increased from 126 million tons in 2011 to 143 million tons in 2021 (FAO 2021).

As a sessile organism, citrus needs to cope with several abiotic stress combinations that are exacerbated by changes in atmospheric temperature and precipitation patterns due to climate change (de Ollas et al. 2019). In this context, water deficiency, extreme temperatures, and high soil salinity are major abiotic factors that commonly affect plant growth and development, restricting citrus production and geographical distribution (Xiong et al. 2002; Ferrarezi et al. 2019; Peng et al. 2020). Notably, citrus cultivars have been domesticated by improving traits related to fruit yield and quality while ignoring environmental traits related to plant performance in the field (Rao et al. 2021b). Thus, most citrus varieties that produce high-quality fruit are not optimally stress tolerant. Therefore, cultivated scions are routinely grafted onto rootstocks to propagate the desired trees (Huang et al. 2021). However, environmental cues are diverse and continuously changing. Therefore, a citrus rootstock appropriate for all stressful conditions does not exist (Huang et al. 2011; Raveh et al. 2020). It is well known that abiotic factors, including soil salinity, drought, and extreme temperatures, induce different stress signals that stimulate the expression and magnitude of stress-related genes related to osmoprotectant production and oxidation scavenging (Fu et al. 2011; Krasensky and Jonak 2012; Peng et al. 2012; Dahro et al. 2016, 2021). However, knowledge related to the signaling cascades and regulatory networks that coordinate the response of citrus to multiple forms of abiotic stress is largely elusive. Thus, investigating tolerance against multiple forms of abiotic stress is an effective way to accelerate the breeding program of citrus.

The response to multiple abiotic stresses in plants comes from the integration of complex crosstalk between various molecular pathways at the levels of stress signal perception, the regulation of stress-responsive genes and the adjustment of metabolism for morphological and physiological adaptation (Tanou et al. 2010, 2012; Verslues and Juenger 2011). Consequently, constructing citrus cultivars/rootstocks that tolerate multiple forms of abiotic stress is hampered by the complexity of the integrated molecular mechanisms that underly the tolerance to combinations of different types of abiotic stress. Hence, in this review, we attempt to provide a comprehensive understanding of the adverse impacts of the major types of abiotic stress (i.e. drought, salinity and low temperatures) on citrus trees and how citrus responds to these abiotic stress conditions. Moreover, we summarized the current knowledge about the signaling events and the molecular mechanisms involved in the response to abiotic cues that could facilitate the genetic improvement of stress resistance in citrus.

Definition of stress conditions and their effects on citrus

Abiotic stress is defined generally as conditions that deviate beyond the threshold considered normal for plant physiology and that impede plant growth and development due to abiotic factors, such as extreme temperatures, water deficiency, and soil salinity.

Influences of low temperatures on citrus

For extreme temperatures, cold stress is categorized into chilling stress occurring at cool temperatures above 0°C and freezing stress occurring when the ambient temperatures drop below 0°C (Jiang et al. 2021a). The drop below suitable growth temperatures impairs plant growth, reduces crop production, and limits the geographic distribution of plants (Tuteja et al. 2011). At the individual plant level, freezing initially occurs in the large vessels of the stem and leaf xylem and in substomatal cavities. At the cellular level, chilling temperatures reduce the fluidity of cellular membranes, which in turn influences membrane permeability, causing membrane leakiness and cellular malfunctions (Janská et al. 2010; Theocharis et al. 2012). Moreover, further decreasing the temperature generates ice crystals in the apoplastic space with relatively lower osmotic potential, leading to the outward movement of cytoplasmic water (Theocharis et al. 2012; Perotti et al. 2015). During freezing, 90% of the unfrozen water is removed from the cell, causing loss in cell turgor and intense cellular dehydration (Verslues et al. 2006; Peng et al. 2014). Thus, in addition to the physical impact of ice nucleation damaging the cell membrane, freezing-induced cellular dehydration shrinks the cell, disrupts cellular osmotic homeostasis and increases cellular ionic leakage (Thomashow 2001; Xiong et al. 2002; Verslues et al. 2006; Peng et al. 2014; Barnes et al. 2016). Citrus trees are widely grown in tropical and subtropical zones, where adequate fruit production relies on a low risk of frost. Initially, chilling temperatures inhibit cellular metabolism and cause leaf wilting (Santos et al. 2011). Further reduction in temperatures impairs tree growth, reduces fruit quality and quantity and causes fruit pitting and dropping (Perotti et al. 2015; Primo-Capella et al. 2021). However, the severity of frost damage depends on the degree of freezing (duration and degree of temperature), the environmental conditions (nutrient availability, water content, and characteristics of the soil), and plant status (variety, rootstock, age, and health status) (Welling and Palva 2006; Perotti et al. 2015). Flowers and fruits are very sensitive to frost (i.e. when temperatures fall below -2°C for 4 h leading to the nucleation of ice crystals in the extracellular spaces of citrus fruits), which causes serious losses in yield (Huang et al. 2011). Furthermore, frost stress irreversibly damages plant metabolic processes, which manifest as chlorosis and necrosis (Usadel et al. 2008; Ding et al. 2020).

Impacts of drought stress on citrus

For drought stress, soil water deficiency occurs in orchards without irrigation systems when the annual precipitation is significantly reduced to less than the normal recorded levels, leading to an increase in osmotic pressure and a reduction in soil water availability (Tuteja et al. 2011). In the field, drought stress can occur simultaneously with other environmental constraints, including extreme temperatures, intense sunlight and nutrient deficiencies, which aggravate the deleterious effects of water deficiency on plants. The interactions among these abiotic factors can synergistically increase the damage to the plant and further reduce its ability to grow and produce fruits (Shafqat et al. 2021). Desiccation stress decreases cell turgor, disorganizes integral membrane proteins and damages cellular compartments, thereby interrupting many aspects of cellular metabolism and osmotic equilibrium (Verslues et al. 2006). Citrus trees have a large canopy with low hydraulic conductivities; thus, irrigating at appropriate frequencies is required to reduce fruit abscission during fruit development (Pérez-Pérez et al. 2008). However, the shrinking water resources available for irrigation systems due to climate change along with the drought sensitivity of citrus threatens the productivity and development of citrus fruits, particularly in late spring and summer (de Ollas et al. 2019). Although citrus trees abundantly bloom in spring, many flower buds drop during fruit development. Indeed, only a small fraction of flowers reach the mature fruit stage. High temperature and low rainfall occurring during spring and early summer are also correlated with fruitlet drop (Pérez-Pérez et al. 2008). In addition, water stress during rapid fruit growth influences fruit size and quality in different citrus cultivars (Rodríguez-Gamir et al. 2011), and drought stress during fruit maturation has major impacts on fruit quality by affecting total soluble solids and acidity (Gonçalves et al. 2016; Santana-Vieira et al. 2016).

Impacts of salt stress on citrus

A soil Na+ concentration above 1500 ppm or 25 mM and chloride concentrations greater than 355 ppm constrained citrus growth (Ziogas et al. 2021). High concentrations of soil salts increase the soil osmotic pressure and then reduce the soil water potential, leading to physiological drought stress (Verslues et al. 2006). Moreover, soil salinity leads to higher accumulation of Na+ and Cl− ions inside plant cells, causing ion toxicity (Fu et al. 2011). Under salt stress, the accumulation of Na+ causes toxicity in the woody roots and trunk, while Cl− causes necrotic lesions mainly in young shoots and leaves of citrus; therefore, salinized citrus plants tend to shed old leaves that accumulate excessive salt ions to avoid salt toxicity (Fu et al. 2011). Under such conditions, the resultant inhibition of photosynthetic activity reduces the supply of energy and thus, leads to impaired growth (López-Climent et al. 2008). The symptoms of salinity stress manifest as leaf chlorosis, necrosis and even abscission are accompanied by inhibition of cellular division, growth retardation and yield loss (Ferguson and Grattan 2005; Hussain et al. 2012; Deng et al. 2013). However, the capacity of citrus to sustain growth and production in salty soil varies among varieties and species, planting systems, developmental stage, age and other climatic factors (Ferguson and Grattan 2005).

Physiological response of citrus to abiotic stresses

Two major strategies (stress avoidance and tolerance) are used by citrus germplasm to cope with different abiotic cues. Initially, citrus avoids stress by implementing abundant protective mechanisms that delay the detrimental impacts of stress. The capacity to adjust the membrane system, modify the cell wall architecture, alter metabolism, and change the cell cycle and cell division, which determines the stress tolerance of citrus varieties (Ziogas et al. 2021). Understanding the physiological traits is a prerequisite for unraveling the genetic basis of the citrus plant’s response to multiple forms of abiotic stress (Krasensky and Jonak 2012). Therefore, in this paragraph, we focused on the physiological adjustments that render citrus germplasm tolerant to low temperature, drought, and high salinity.

Physiological acclimation for freezing tolerance

Citrus genotypes vary in their tolerance to low temperatures from sensitive to moderately tolerant to tolerant. It has been documented that freezing tolerance and the depth of dormancy are strongly associated with the survival rate of citrus plants during cold stress (Welling and Palva 2006). The depth of dormancy during winter varies among citrus species. P. trifoliata is deciduous and has a deep dormancy. Mandarin, orange, and grapefruit are moderately dormant. Lemon, citron and lime are less dormant during winter (Champ et al. 2007; Bowman and Joubert 2020). P. trifoliata and its hybrids are usually used as cold-tolerant rootstocks because their dormancy in low temperature conditions renders them the most resistant to damage from freezing temperatures (Bowman and Joubert 2020). Given that citrus trees are sensitive to freezing stress when they are actively growing, rootstocks primarily improve freezing tolerance by regulating the vegetative growth of scions (Huang et al. 2011). Furthermore, the transmission of cold tolerance from citrus rootstocks to cultivated scions was associated with an enhanced detoxification system and osmotic adjustment (Oustric et al. 2017). In addition, several physiological and biochemical changes are induced during cold acclimation to sustain the integrity of cellular membranes and compartments (Theocharis et al. 2012; Jiang et al. 2021a). Cold acclimation (4°C treatment for 3 d) enhanced sucrose (Suc), glucose (Glc), fructose (Frc), and total sugar levels, which were more positively correlated with higher soluble invertase (INV) and hexokinase activities in cold-tolerant Citrus species than in sensitive species (Dahro et al. 2021). Similarly, the cold-tolerant citrus rootstock Citrus junos elicited more than 400 proteins related to phenylpropanoid, carbohydrate, and specialized metabolite metabolism, which were associated with higher accumulation of Suc, Glc, Fcr, and phenols in response to cold stress (Jiang et al. 2021a). In another field experiment, cold-tolerant Citrus species were shown to maintain photosynthesis and to detoxify oxidative radicals in response to seasonal changes in climate (Santini et al. 2012). Therefore, the cold tolerance of citrus is partially determined by photosynthetic performance and oxidative stress tolerance (Santini et al. 2012; Jiang et al. 2021a). In the field, C. reticulata (mandarin) and C. maxima (pummelo) maintained the efficiency of their photosynthesis and antioxidant systems, and were demonstrated to be more cold tolerant than C. medica and F. japonica (Santini et al. 2012).

Physiological changes supporting drought tolerance

The uptake of water from the soil is more difficult during drought stress because the free energy of water decreases, which in turn elicits a wide range of responses that aim to prevent water loss and to increase the tolerance for reduced water content (Verslues et al. 2006; de Souza et al. 2017). In response to low water potential, citrus plants organize changes at the cellular and organism levels to increase water-use efficiency and limit water loss that maintain cellular water retention close to the unstressed level (Verslues et al. 2006). Initially, citrus plants can avoid drought stress by reducing shoot growth and shedding their old leaves to decrease their surface area and minimize water loss (Woo et al. 2008). Citrus plants also enhance root growth under water deficit conditions to facilitate water absorbance from deeper soil (Verslues et al. 2006; Bhatnagar-Mathur et al. 2008). The tradeoff of this response is the reduction in photosynthesis and carbon assimilation caused by decreased stomatal CO2 uptake (Bresson et al. 2015). These mechanisms of drought avoidance shift the energy products toward root growth rather than the photosynthetic and reproductive tissues, leading to inhibition of vegetative and reproductive growth (Thalmann et al. 2016). As water depletion becomes more severe, additional mechanisms are involved in sustaining the cellular water content. The accumulation of compatible solutes and cell wall hardening are important mechanisms to increase the water potential of plant tissue (Gonçalves et al. 2016; Santana-Vieira et al. 2016). The accumulation of compatible solutes in root tissues plays an essential role in dehydration tolerance by alleviating the cellular osmotic potential and maintaining the root’s capability to uptake soil water (de Ollas et al. 2013; Zaher-Ara et al. 2016). Simultaneously, solute accumulation plays an osmoprotective role by protecting protein and membrane structures, regulating redox status and acting as reactive oxygen species (ROS) scavengers (de Campos et al. 2011). Thus, tolerant citrus cultivars accumulate several types of compatible solutes, such as proline (Pro), sugars and glycine-betaine (GB), in response to low water content (de Campos et al. 2011; Shafqat et al. 2021). According to the aforementioned insights, the capability of citrus plants to cope with the deleterious impact of water depletion is genotype-dependent. Mandarin (Citrus reticulata spp.), Rangpur lime, rough lemon, and sour orange are drought resistant; lemon, trifoliate orange, and citrange hybrids are moderately tolerant; and sweet orange and grapefruit are poorly tolerant (Romero et al. 2006; Rodríguez-Gamir et al. 2011; Gonçalves et al. 2016).

Physiological modulation of salinity tolerance

Adverse impacts and salinity tolerance have been extensively studied in commonly grown citrus genotypes (Ziogas et al. 2021). Generally, grafting is a common practice to impart salt tolerance on citrus cultivars. However, the interplay between rootstocks and scions determines salinity tolerance. The root hydraulic conductivity of rootstocks and the transpiration and growth rates of scions are important factors that control the movement of ions throughout the plant (Moya et al. 2003; Simpson et al. 2014). Generally, citrus plants possess the ability to avoid Na+ toxicity by accumulating Na+ in woody roots and basal stems and excluding Na+ from foliage (Martínez-Alcántara et al. 2015). Several reports have demonstrated that citrus genotypes vary in their ability to exclude Cl− from foliage; therefore, Cl− ion content is used as an essential indicator for salt tolerance of citrus genotypes (López-Climent et al. 2008). However, this statement does not imply that Cl− is metabolically more toxic than Na+; rather, citrus is better at excluding Na+ from the leaf blades than Cl− (Brumós et al. 2009). Cl− appears to damage citrus more than other ions because the accumulation of Cl−in leaves was positively correlated with salinity damage (Brumós et al. 2009, 2010). Furthermore, high levels of Cl− in leaves directly decreased chlorophyll content and impaired the maximum quantum efficiency of PSII photochemistry(Fv/Fm). Thus, chlorophyll content and Fv/Fm are useful proxies for detecting salt stress in citrus (López-Climent et al. 2008; Tanou et al. 2012). Therefore, it is believed that the physiological basis of salt tolerance is typically related to the capacity of citrus rootstocks to restrict Cl− uptake and to translocate only small portions of Cl− from roots to leaves (Simpson et al. 2014). Several studies have summarized distinct mechanisms by which citrus genotypes endure salinity, including sodium sequestration, Cl− exclusion, ion chelation, cellular water retention, and osmolyte accumulation (Storey and Walker 1998; Ferguson and Grattan 2005). Generally, Sunki mandarin (C. sunki Hort. ex Tan.), Rangpur lime (C. limonia Osbeck), and Cleopatra mandarin (C. reshni Hort. ex Tan.) are good Cl− excluders and thus, were demonstrated to be salt-tolerant rootstocks. In contrast, P. trifoliata and its hybrid Carrizo citrange (C. sinensis (L.) Osbeck x P. trifoliata (L.) Raf.) have good Na+ exclusion but poor Cl− exclusion and are thus, salt-sensitive rootstocks (Hussain et al. 2012; Syvertsen and Garcia-Sanchez 2014). The salt-sensitive genotypes of citrus were demonstrated to accumulate excess salt ions, leading to oxidative damage. In contrast, salt-tolerant citrus genotypes accumulated fewer Cl− ions in the foliage and reduced their photosynthetic rates and stomatal conductance to avoid osmotic stress-induced oxidative damage (Moya et al. 2003; López-Climent et al. 2008; Brumós et al. 2009; Hussain et al. 2012). Taken together, the data indicate that mechanisms of salt tolerance vary among different citrus genotypes and that these mechanisms are heritable traits that can be transmitted by hybridization and from rootstocks to grafted scions.

Molecular mechanisms underlying abiotic stress tolerance in citrus

Despite our advanced knowledge of the physiological basis of tolerance of citrus to different types of abiotic stress, the genetic mechanisms are still not fully understood. Plants, as sessile organisms, respond continuously to changing environmental cues, which promote the evolution of delicate adaptive mechanisms that dynamically coordinate abundant molecular components (Mittler et al. 2004; Liu et al. 2013). Citrus, similar to other plant species, has stress memories that lead to epigenetic, transcriptional, proteomic, and metabolomic modifications, enabling dynamic responses to abiotic factors (Molassiotis et al. 2016; Santana-Vieira et al. 2016; Liu et al. 2021). Intriguingly, it was found that stress priming elicits stress memory that adequately optimizes tolerance against multiple types of abiotic stress (Pérez-Pérez et al. 2007; Molassiotis et al. 2016). In addition, many studies have demonstrated that the expression patterns of several stress-responsive genes in citrus overlap when plants experience drought, cold and salinity stress (Şahin-Çevik and Moore 2013). Furthermore, the overexpression of stress-responsive genes involved in one type of stress enhanced tolerance to multiple abiotic stresses, further revealing the potential for the cross-regulation of tolerance to multiple abiotic stresses (Wang et al. 2011; Xian et al. 2013; Lo Cicero et al. 2015; Dahro et al. 2016). Here, we summarize the current knowledge of stress signal transduction and the regulatory pathways used to coordinate metabolite levels when citrus is subjected to different types of abiotic stress.

Molecular basis of cold tolerance

Low temperatures activate distinct molecular mechanisms in cold-tolerant and cold-sensitive citrus species (Champ et al. 2007; Crifò et al. 2011; He et al. 2012). During cold stress, plants respond initially by inducing alterations in the cellular membranes, which in turn trigger the production of several secondary messengers. After perceiving the stress signal, plants stimulate a rapid and temporary accumulation of high intracellular Ca2+ concentrations, which can be detected by different receptors and protein kinases (Theocharis et al. 2012; Zhu 2016). Then, these regulatory proteins relay the signal by activating several secondary signaling pathways, such as ROS-responsive and abscisic acid (ABA)-responsive pathways (Xiong et al. 2002). When stress signals reach the nucleus, various epigenetic (DNA methylation and histone modifications), transcriptional (transcription factors), posttranscriptional (microRNA, lncRNA), translational, and posttranslational modifications participate in the cold response (Long et al. 2011; Zhang et al. 2013; Wang et al. 2015). Consequently, a vast number of low temperature-responsive genes are transcriptionally reprogrammed, leading directly or indirectly to induce different physiological, morphological and biochemical changes to protect plant cells from the harmful effects of low temperatures (Crifò et al. 2011; Wang et al. 2015). These stress-inducible genes are generally categorized into functional and regulatory genes based on their functions (Dahro et al. 2016; Wang et al. 2019; Ming et al. 2020; Khan et al. 2021; Zhang et al. 2021). Because P. trifoliata evolves in a cold climate and tolerates temperatures down to -20°C when fully acclimated, it evolves several genetic polymorphisms leading to morphological and biochemical modifications that allow P. trifoliata to tolerate freezing stress (Champ et al. 2007; Peng et al. 2020). Therefore, several groups have made extensive efforts to illustrate the cold-induced molecular mechanisms responsible for the freezing tolerance of P. trifoliata (Ṣahin-Çevik and Moore 2006; Huang et al. 2013; Sahin-Cevik 2013; Zhang et al. 2013; Wang et al. 2019; Ming et al. 2020; Peng et al. 2020). Similar to other species, the transcriptional reprogramming in the cold-adapted P. trifoliata involves C-repeat binding factor (CBF)-dependent and CBF-independent cold signaling pathways (Fig. 1) (Champ et al. 2007; Wang et al. 2015; Wang et al. 2019). The CBF-dependent pathway in citrus was demonstrated to regulate the expression of a large spectrum of cold-responsive (COR) genes during cold acclimation (Champ et al. 2007). Consistently, several osmoprotectant-related genes were revealed as target genes of the CBFs, which enhance cold tolerance by adjusting osmotic homeostasis and scavenging the excessive accumulation of ROS (Peng et al. 2014; Song et al. 2022a, b). For example, the β-amylase-coding gene PtrBAM1 was demonstrated to be a PtrCBF regulon member that plays an important role in cold tolerance by modulating the levels of maltose and soluble sugars that act as osmolytes and antioxidants. Similarly, CsCBF1 of C. sinensis enhanced the freezing tolerance of transgenic plants by influencing the expression of arginine decarboxylase (PtrADC), which ultimately affected polyamine (PA) levels (Song et al. 2022a, b). On the other hand, the CBF-independent pathway was also involved in the freezing tolerance of P. trifoliata. Interestingly, the expression of ubiquitin E3 ligase PtrHOS1, which promotes the inactivation of ICE1 and negatively regulates CBF-induced cold-responsive genes, was repressed in response to cold and ABA treatments, which provides evidence for PtrHOS1 participating in the integration of CBF-dependent and CBF-independent cold signaling (Liu et al. 2009). Recently, many TFs that belong to large gene families and that contribute to different regulatory modules were revealed to modulate cold tolerance in P. trifoliata or its related genera by coordinating osmolyte accumulation and ROS detoxification. A new cold-responsive signaling module that consists of AT-Hook motif containing nuclear localized PtrAHL14/17 transcription factors-histone acetyltransferases-PtrA/NINV7 was demonstrated to orchestrate Suc catabolism by influencing the transcriptional regulation of the alkaline/neutral invertase PtrA/NINV7 gene in P. trifoliata (Dahro et al. 2022). Furthermore, the transcriptional regulatory module PtrMYC2-PtrBADH-l was verified to transduce jasmonic acid (JA) signaling for GB accumulation and freezing tolerance in P. trifoliata (Ming et al. 2020). Basic helix-loop-helix (PtrbHLH) transcription factors were found to regulate the transcription of peroxidase (PtrPOD) and catalase (PtrCAT) genes for hydrogen peroxide (H2O2) scavenging and cold tolerance (Huang et al. 2013; Geng and Liu 2018; Geng et al. 2019). Additionally, the plant ethylene-responsive transcription factors PtrERF109, PtrERF108, and PtrERF09 were demonstrated to confer freezing tolerance to cold-sensitive citrus transgenic lines by regulating the transcription of genes encoding a class III peroxidase-encoding (PtrPrx1), glutathione S-transferase U17 (PtrGSTU17), and raffinose synthase (PtrRafS) (Wang et al. 2019; Khan et al. 2021; Zhang et al. 2021). Furthermore, several findings provide evidence that epigenetic factors have vital roles in promoting freezing tolerance in P. trifoliata. Global DNA methylation profiling demonstrated that autotetraploids of P. trifoliata with enhanced freezing tolerance underwent more extensive DNA demethylation than the diploids (Wang et al. 2022). Another study on cold acclimation illustrated the involvement of conserved and novel miRNAs in regulating cold stress responses in P. trifoliata by modulating ethylene and PA levels (Zhang et al. 2013; Zhang et al. 2016). Overall, despite the progressive achievement in revealing the transcriptional reprogramming components of the cold stress response, there is still a great paucity of information on how the cold stress signal is perceived and then transduced to TFs.

Fig. 1
figure 1

A typical molecular model illustrates cold tolerance in citrus and its related genera. Cold stress signals alter the fluidity of the cellular membranes, triggering the rapid accumulation of Ca2+ that can be detected by several signaling proteins, such as kinase and phosphatase proteins, which in turn relay the stress signals downstream to induce several transduction cascades. Several secondary signals and phytohormones orchestrate the delicate molecular mechanisms that activate a wide range of regulatory components, such as transcription factors (indicated by yellow rounded rectangles), microRNAs and histone acetyltransferases (indicated by blue ellipses), to induce the global transcriptional reprogramming of various cold-responsive genes (indicated by blue rounded rectangles). Ultimately, several physiological and biochemical changes (indicated as brown rounded rectangles) occurred in response to the freezing tolerance signaled by cold. The dashed lines refer to uncharacterized gene functions and pathways in citrus. The solid lines indicate functionally characterized genes and pathways in citrus

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Molecular mechanisms of drought stress tolerance

Recently, transcriptome and protein interactomic analysis identified the molecular basis of drought tolerance in grafted sweet orange scion transmitted from the drought-tolerant Rangpur lime rootstock (de Souza et al. 2017; Gonçalves et al. 2019). This analysis showed that the rootstock-induced drought tolerance of sweet orange scions involves the transcriptional activation of genes related to soluble sugars, antioxidant metabolism, cell wall, both biotic and abiotic stress responses, TFs, protein kinases, ABA signaling and the downregulation of genes which implicated in the light reactions of photosynthesis, starch metabolism, and ethylene signaling (Gonçalves et al. 2019). As expected, the expression of genes encoding ABA signaling components of citrus, including six cytoplasmic ABA receptors (PAR/PAL/PCAR), five clade-A type 2C protein phosphatases (PP2CA), and seven subclass III SNF-1 related protein kinases2 (SnRK2) was induced in response to drought, indicating the importance of the ABA pathway for drought tolerance in citrus (Fig. 2) (Romero et al. 2012; Song et al. 2022a, b). Furthermore, drought-induced ABA-responsive element (ABRE)-binding factor (PtrABF3) contributed to drought tolerance by regulating several physiological responses. PtrABF was demonstrated to interact with PtrICE1 to modulate stomatal development. Moreover, the overexpression of PtrABF in trifoliate orange resulted in higher antioxidant enzyme activities and free PA levels but lower levels of ROS and malondialdehyde (MDA) by leading to the upregulated expression of peroxidase (PtrPOD) and PtrADC (Zhang et al. 2015b). Recently, the new regulatory module SnRK2.4-ABF2-ADC emphasized the responsibility of ABA for fine-tuning putrescine accumulation under drought stress (Song et al. 2022a, b). In parallel, the ectopic overexpression of the PtrADC gene, which produces putrescine (Put) indirectly from arginine (Arg), was reported to confer enhanced tolerance to multiple abiotic stresses, including drought stress in Arabidopsis (Wang et al. 2011). Interestingly, investigating the transcriptional regulation of the PtrADC gene in citrus showed that several TFs could elevate the transcript abundance of PtrADC and PA biosynthesis in response to drought stress (Fig. 2). The stress-responsive PtrMYB of trifoliate orange was reported to positively regulate the expression of PtADC by binding a sequence in the PtADC promoter (Sun et al. 2014). Additionally, WRKY70 was also demonstrated to interact with W-box elements in the promoter of the F. crassifolia ADC gene, leading to the activation of ADC-mediated Put biosynthesis and enhanced drought tolerance (Gong et al. 2015). In contrast, PtrNAC72 from trifoliate orange was demonstrated to serve as a negative regulator of PtADC that represses Put biosynthesis and thus impairs drought tolerance (Wu et al. 2016). Furthermore, several other TFs, including nuclear factor Y (CsNF-YA5), histone acetyltransferases (HATs) and histone deacetylase (HDAC), were reported to fulfill critical roles in drought tolerance in citrus plants (Pereira et al. 2018; Shu et al. 2020; Shu et al. 2021). Interestingly, the transcriptional cascade composed of ABF4 and ABR1 was demonstrated to work synergistically to upregulate BAM3 expression and starch catabolism in response to drought conditions (Zhang et al. 2023). Moreover, several proteins were reported to exert distinct roles during drought tolerance in citrus. It is well established that dehydrins and late-embryogenesis abundant (LEA) proteins are protective proteins that accumulate in response to decreases in tissue water content (Zhang et al. 2015a). Dehydrins and LEA proteins function as chaperones that protect proteins and membrane structures and prevent the inactivation of enzymes. Dehydrin proteins were also found to bind water molecules and thus to have a role in retaining water in addition to their role in protecting cellular structures (Zhang et al. 2015b). Furthermore, the cellular membrane protein channels, aquaporins, are abundantly expressed in roots to regulate plant hydraulic conductance and water uptake. Downregulation of aquaporin PIP1 and PIP2 in drought-treated citrus roots decreases membrane permeability, which in turn preserves cellular water content and maintains proper water balance in citrus plants (Rodríguez-Gamir et al. 2011). In parallel, the aquaporin NOD26-like intrinsic protein CsNIP5;1 was demonstrated to participate in regulating water permeability through relocalization of water channel PIPs (Zhang et al. 2021). Although the most efficient mechanisms of drought tolerance in citrus rely on sustaining cellular structures and metabolic processes, it is noteworthy that the signaling, regulatory and functional genes conferring drought tolerance are still not fully understood in citrus.

Fig. 2
figure 2

A schematic model of the molecular mechanisms involved in drought tolerance in citrus and its related genera. The rapid accumulation of abscisic acid (ABA) during drought stress induces pyrabactin resistance 1-like (PYLs) to form complexes with the PP2Cs, releasing SnRK2s that activate downstream signaling transduction. The stress signal then activates a wide-range of regulatory components such as transcription factors (indicated by yellow rectangles) to induce global transcriptional reprogramming of various water stress-responsive genes (indicated by blue rectangles) derived by ABA-dependent and independent pathways. Ultimately, several morphological, physiological and biochemical changes (indicated by brown rounded rectangles) occurred to confer drought tolerance. The dashed lines refer to uncharacterized functions of genes and pathways in citrus. The solid lines indicate functionally characterized genes and pathways in citrus

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Molecular mechanisms of salt tolerance

The molecular mechanisms of salinity tolerance in the model plant A. thaliana were attributed to Na+ compartmentalization and thus, several genes encoding Na+ transporters and regulators are well documented (Rus et al. 2001; Wang et al. 2014). In contrast to the model plant, chloride toxicity rather than sodium toxicity was demonstrated to be highly effective in citrus trees (Brumós et al. 2009). Consistently, salt-tolerant citrus genotypes were found to efficiently exclude Cl− from their leaves, whereas salt-sensitive citrus genotypes are poor Cl− excluders, despite their capability to exclude Na+ (Hussain et al. 2012; Syvertsen and Garcia-Sanchez 2014). These different mechanisms of citrus genotypes in response to salt stress allow biologists to unravel the molecular components controlling the accumulation of Cl− and Na+ in plant cells. Interestingly, the leaf transcriptome comparison between a salt-tolerant and efficient Cl− excluder (Cleopatra mandarin) and a salt-sensitive and poor Cl− excluder (Carrizo citrange) found that the former showed a high correlation between transcript levels and leaf chloride accumulation, consistent with a role for transcriptional regulation in the response to salt stress (Brumós et al. 2009). The differentially expressed genes between Cleopatra mandarin and Carrizo citrange subjected to salinity stress were functionally categorized into stress sensing and stress responsive, and carbon metabolism and photosynthesis, indicating the importance of regulating these processes for salt tolerance (Brumós et al. 2009). Moreover, the molecular mechanism of the H2O2-primed salt tolerance of citrus was illustrated by modulating NaCl-responsive proteins that participate in photosynthesis and carbon metabolism, providing more evidence for the relevance of maintaining photosynthesis for salt tolerance (Tanou et al. 2012). In addition, the highly induced expression of NRT1-2, which encodes a nitrate transporter, in the efficient Cl− excluder genotype provides evidence that NRT1-2 contributes to Cl− homeostasis (Brumós et al. 2009). Additionally, the chloride-conductance regulatory protein CcICln1 was also reported to influence chloride exclusion from the salt-tolerant Cleopatra mandarin by reducing net Cl− loading into the root xylem (Brumós et al. 2010). On the other hand, the difference in Na+ tolerance between Cleopatra mandarin and trifoliate orange was illustrated by the enhanced retrieval of Na+ from the xylem stream and the concentration of Na+ in root vacuoles; thus, this ion was partially translocated to the shoot tissues of trifoliate orange. This pattern of Na+ disturbance was associated with high-level expression of the plasma membrane-localized Na+/H+ antiporter SOS1 in roots to promote Na+ efflux from root tips into the external medium, which restricts the net Na+ uptake and limits Na+ loading in the xylem sap of roots to reduce Na+ damage to leaves. Furthermore, the induced expression of genes encoding the high-affinity potassium transporter (HKT1) and Na+/H+ antiporter (NHX1) in both roots and shoots accompanied by enhanced activity of the tonoplast proton pumps (V-ATPase, V-PPiase) led to preferential sequestration of Na+ in the root vacuoles of trifoliate orange relative to Cleopatra mandarin (Martínez-Alcántara et al. 2015). Gene expression profiling of roots from the salt-tolerant C. junos germplasm after 24 h of salt treatment identified a total of 1831 differentially expressed genes that included genes associated with ROS signaling and Salt Overly Sensitive (SOS) pathways. Additionally, genes involved in phytohormone signaling, metabolism and cell wall structure were speculated to play important roles in salt stress tolerance (Xie et al. 2017). Accordingly, a model was made for the signals associated with salt stress tolerance in citrus (Fig. 3). First, salt stress signals alter the structure of the plasma membrane and increase its permeability. Consequently, the increased influx of Ca2+ into the cytoplasm induces Ca2+ signaling sensors (e.g. CAMs, CMLs, and CBLs) and activates mitogen-activated protein kinase (MAPK) cascades that influence downstream stress signaling mechanisms to regulate the transcription of a range of salt stress-inducible genes that promote salt tolerance in citrus (Xie et al. 2017). Similar to other plant species, the SOS model also plays a critical role in coordinating salinity stress signaling cascades in Citrus and its related genera. In a previous report, the F. crassifolia transcription factor FcWRKY40 was reported to enhance salt tolerance by activating the expression of SOS2 and D-1-pyrroline-5-carboxylate synthetase 1 (P5CS1), thus regulating ion homeostasis and Pro biosynthesis. Intriguingly, this functional model is involved in the ABA-dependent signaling pathway, as the transcription factor FcABF2 positively regulates FcWRKY40 expression (Dai et al. 2018). The contribution of ABA to salt tolerance was further demonstrated by the overexpression of the citrus CrNCED1 gene, which induces ABA biosynthesis and renders CrNCED1-overexpressing tobacco seedlings more salt tolerant (Xian et al. 2013). In addition, several studies indicate the importance of manipulating PA biosynthesis and inducing the expression of genes encoding antioxidant enzymes (Mn-SOD, GR, cAPX and GST) for salt tolerance (Tanou et al. 2014). In parallel, a multitude of TFs, including NAC, AP2/ERF, WRKY, GATA, MYB, bZIP, SPL, bHLH, ZFP, CBF, and CAMTA transcription factors were implicated in the transcriptional regulation of the response to salt stress (Xie et al. 2017). However, in comparison with the progress made in understanding the physiological basis of salt tolerance, the molecular mechanisms and the regulatory pathways that control salt tolerance in citrus need further investigation.

Fig. 3
figure 3

The proposed model illuminating the characterized components involved in citrus salinity tolerance. Salt stress alters plasma membrane permeability to increase Ca2+ influx into the cytoplasm. The resultant Ca2+ influx induces Ca2+ signaling components (CAMs, CMLs, and CBLs) and mitogen-activated protein kinase (MAPK) cascades, which further regulate the transcripts of a wide range of salt stress-induced genes. The three interacting SOS pathway components (SOS1, SOS2 and SOS3) transduce Ca2+ signaling and then efflux excess Na+ from the cytoplasm. Additionally, the abscisic acid (ABA) signaling pathway triggers the activation of FcABF2, which acts upstream of FcWRYK40, which in turn regulates FcSOS2 and FcP5CS1. Upregulation of the SOS3-SOS2-SOS1 and WRKY40-SOS2/P5CS1 modules promotes Na+ exclusion and proline biosynthesis, alleviating salt-induced damage by maintaining ion homeostasis and osmotic adjustment. The dashed lines refer to uncharacterized functions of genes and pathways in citrus. The solid lines indicate functionally characterized genes and pathways in citrus

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Redox adjustment and osmotic homeostasis are essential for tolerance to multiple types of abiotic stress in citrus

Different types of abiotic stress affect the redox balance in mitochondria, peroxisomes and chloroplasts and thus, lead to the production of singlet oxygen (1O2), superoxide (O2.−), hydroxyl radical (·OH), and hydrogen peroxide (H2O2). This increase in ROS levels elicits a broad range of responses during the response to multiple types of abiotic stress. Accordingly, the pretreatment of sour orange plants (Citrus aurantium L.) with H2O2 primed salt tolerance by transducing oxidative signals to regulate cellular adjustments (Tanou et al. 2012). However, excessive accumulation of ROS during abiotic cues causes irreversible damage by oxidizing proteins, lipids, organic molecules, and nucleic acids (Couee et al. 2006; Hartmann et al. 2015). Consequently, tolerant citrus genotypes have developed robust ROS detoxification systems that are coordinated with ROS production when plants are subjected to stressful conditions (Rao et al. 2021a). In this regard, ample evidence has demonstrated the essential role of osmoprotectants in detoxifying ROS during abiotic stress responses (Couee et al. 2006; Morsy et al. 2007; Bolouri-Moghaddam et al. 2010; Keunen et al. 2013; Hartmann et al. 2015). It is well known that abiotic stress induced by soil salinity, drought, and extreme temperatures activate different stress signaling mechanisms and thus, lead to different levels of stress-related gene expression that produce several types of osmoprotectants (Fig. 4) (Fu et al. 2011; Krasensky and Jonak 2012; Peng et al. 2012; Dahro et al. 2016, 2021). Suc, Glc and Frc are compatible solutes that synchronously accumulate in abiotic stress-exposed cells to maintain their hydrated state and cell turgor. Sugar molecules were found to substitute water molecules to form hydrophilic interactions with membrane-localized proteins and lipids and thus maintain the integrity of the cellular membranes under water deficit stress (Hare et al. 1998). Furthermore, sugars function as ROS scavengers and thus, attenuate oxidation damage (Koch 2004; Couee et al. 2006; Qi et al. 2007; Bolouri-Moghaddam et al. 2010; Ruan et al. 2010; Keunen et al. 2013). The crosslink between plant phytohormones and sugars in response to osmotic stress also manifested the critical role of sugars as hormone-like regulators that induce tolerance to multiple types of abiotic stress (Ruan et al. 2010; Hartmann et al. 2015). Furthermore, these osmolytes sustain the cellular redox balance, which in turn protects the photosynthetic apparatus (Hare et al. 1998; Khan et al. 2015; Dahro et al. 2016). Recently, the tolerance of citrus genotypes to multiple types of abiotic stress was directly correlated with the high-level accumulation of Suc, Glc, and Frc and increases in total sugar content, concomitant with high-levels of soluble INV activity (Wei et al. 2019; Jiang et al. 2021a). The potential role of PtrA/NINV7-mediated sucrose hydrolysis in abiotic stress tolerance was further emphasized by the finding that the overexpression of PtrA/NINV7 in tobacco (Nicotiana nudicaulis) led to enhanced tolerance to multiple types of abiotic stress (Dahro et al. 2016). Consequently, it is plausible that transformation with genes associated with sugar metabolism could modulate sugar biosynthesis and alter sugar content to simultaneously confer tolerance to multiple types of abiotic stress in transgenic plants.

Fig. 4
figure 4

A speculative model reflecting the common mechanism of citrus response to multiple abiotic stresses. a The physiological response of citrus to multiple abiotic cues. Cold, salinity and drought stresses can synergistically occur in the field and share in impairing citrus growth, reduce fruit quality and quantity, and cause fruit pitting and dropping. Under salt stress, Na+ ions cause toxicity in woody roots and trunks, while Cl− ions cause necrotic lesions mainly in the foliage of citrus. Typically, abscisic acid (ABA) is an essential stimulus that is initially elicited in citrus roots to coordinate long-distance abiotic stress signaling and adaptation mechanisms. In addition, the maintenance of photosynthesis will sustain sucrose allocation that promotes root growth for deeper water absorption. b ABA is a key phytohormone that regulates a wide range of physiological processes involved in various aspects of multiple abiotic stress tolerance. The initial response of citrus to multiple stressful conditions is stomatal closure to retain water content. Several physio-biochemical changes are stimulated against multiple abiotic stress factors, such as sugars, antioxidants, and osmoprotectants, to sustain the integrity of cellular membranes and scavenge the excess accumulation of reactive oxygen species (ROS). c The molecular mechanism that possibly controls citrus tolerance to multiple abiotic cues. The regulatory pathways of phytohormones such as ABA, gibberellins (GAs), ethylene, and jasmonic acid (JA) crosstalk together to maximize the response of citrus to the combined stressful conditions. The signaling cascades are fine-tuned by complex regulatory networks, where ABA is the backbone of this response. The interplay of these signaling cascades will be transduced generally to activate abundant transcription factors (TFs) for transcriptional regulation of functional genes. TFs that belong to the WRKY, NAC, MYB, ABF, ERF and bHLH families are good candidates for conferring tolerance against multiple abiotic stressors. However, it is noteworthy that no report has identified any TF for multiple abiotic tolerances, and the molecular components integrated in these regulations are largely uncharacterized in citrus

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Pro is a major factor contributing to osmotic adjustments in response to different types of abiotic stress (Yelenosky 1979; Mohammadrezakhani et al. 2019). Pro is a compatible solute and plays an essential role in ROS scavenging and the stabilization of DNA and proteins (Krasensky and Jonak 2012). Previously, the transformation of sensitive citrus species with genes that can manipulate the endogenous content of Pro increased their tolerance to drought, cold and salinity by mediating osmotic adjustment and gas exchange and ameliorating the deleterious effects of oxidative stress (Molinari et al. 2004; de Campos et al. 2011; Dai et al. 2018). In addition, it was evident that PAs also play several protective and ROS scavenging roles in response to multiple types of abiotic stresses in citrus (Liu et al. 2007; Huang et al. 2015). PAs were defined as positively charged nitrogenous aliphatic molecules with low molecular weights. It has been well established that multiple abiotic factors induce the high-level accumulation of PA, including Put, spermidine (Spd) and spermine (Spm), in citrus (Wang et al. 2011). Merging evidence has demonstrated that the concentration of PAs is tightly coordinated during adverse environmental conditions by balancing biosynthesis, catabolism, transportation and conjugation (Alhag et al. 2021). Interestingly, the transformation of sensitive citrus plants with genes that coordinate the endogenous level of PAs and the exogenous application of PAs improved tolerance to cold, drought and salinity stress by modulating ROS production and redox status concomitant with regulating the activities of various antioxidant enzymes, revealing the significance of PA manipulation for constructing citrus germplasm to endure climate change (Shi et al. 2010; Wang et al. 2011; Tanou et al. 2014; Mahdavian et al. 2021; Mohammadrezakhani et al. 2021). However, there is still no clear view about how these molecules exert these functions. Moreover, anthocyanins are also considered protective molecules that play an essential role in ROS detoxification in response to multiple types of abiotic stress (Lo Piero 2015). The importance of anthocyanins for cold tolerance was specifically demonstrated in red citrus varieties that raised anthocyanin levels in response to low temperatures (Crifò et al. 2011; Huang et al. 2019). Taken together, manipulating the expression of genes implicated in osmotic homeostasis and redox adjustment is a promising strategy for establishing citrus germplasm that can adapt to climate change.

Essential roles of phytohormones in coordinating tolerance to multiple types of abiotic stress in citrus

Phytohormones such as ABA, gibberellins (GAs), ethylene and JA are integral regulators of abiotic stress signaling in citrus (Fig. 4). Typically, ABA is a key stimulus that regulates many physiological processes involved in various aspects of abiotic stress tolerance (Danquah et al. 2014; Lee and Seo 2015). The tolerance of citrus plants to multiple types of abiotic stress generally involves ABA-dependent and ABA-independent pathways (Xian et al. 2013; Wang et al. 2019). It is not surprising that the signaling cascades in response to multiple types of abiotic stresses are fine-tuned by complex regulatory networks and that ABA is the backbone of this response. This was supported by the induced expression of genes associated with ABA biosynthesis and signaling in the cold-hardy P. trifoliata during cold acclimation, indicating the importance of ABA to freezing tolerance (Wang et al. 2015). Furthermore, salt and drought signaling cascades are generally integrated with ABA-dependent pathways. Indeed, most salinity- and dehydration-responsive genes have ABA-responsive cis-acting regulatory elements in their promoters (Dai et al. 2018).

On the other hand, the ABA-independent pathway is also involved in the citrus response to abiotic stress. Several studies have demonstrated that most low temperature- and dehydration-responsive genes possess C-repeat/drought-responsive elements (CRTs/DREs) in their promoters and are regulated by CBFs, which are involved in the ABA-independent pathway (Xiong et al. 2002; Lee and Seo 2015). Furthermore, the increased levels of transcripts from ethylene-related genes in response to abiotic cues and the enhancement of abiotic stress tolerance by ethylene indicate that the ethylene is also important for the cold acclimation of P. trifoliata (Wang et al. 2019; Zhang et al. 2021). Furthermore, GAs and JA hormones control various aspects of growth and development during stressful conditions (Huerta et al. 2008; Ming et al. 2020; Xiong et al. 2020). Notably, integration of these regulatory pathways was found to maximize the response to combinations of abiotic stress (de Ollas et al. 2013; Lee and Seo 2015). The integration of the phytohormone pathways, including ABA, ethylene and JA, was shown to play an essential role in the response of citrus to multiple types of abiotic stress (de Ollas et al. 2013). However, the molecular components required to integrate these mechanisms are largely uncharacterized in citrus. Furthermore, phytohormone perception, signal transduction, regulated gene expression and interactions with different hormones that modulate the response to multiple abiotic factors are still elusive in citrus (Fig. 4).

Polyploid citrus genotypes provide unique germplasm for tolerance to multiple types of abiotic stress

Emerging evidence based on several agronomical, physiological and molecular reports illustrates that tetraploid rootstocks manifest particularly interesting behavior for resilience and resistance to abiotic stress. Even though citrus cultivars are typically diploid, genomic duplication produces polyploidy citrus genotypes (Podda et al. 2013). Several studies on polyploidy in citrus have demonstrated that genomic duplication of citrus is accompanied by several physiological, biochemical and anatomical changes and by distinct capacities to endure multiple types of abiotic stress (Allario et al. 2011; Tan et al. 2017; Wei et al. 2019; Khalid et al. 2021). Tetraploid citrus plants have significantly lower transpiration rates, which is associated with the smaller size of tetraploid compared to diploid plants. Moreover, in citrus tetraploids, the roots, stems and leaves are typically thicker and more succulent than in diploid plants. In roots, citrus tetraploids have a unique root structure that includes fewer root branches, fewer root tips, and a thicker exodermis and that is reflected in an increased ability to alleviate water loss and enhance root conductivity (Saleh et al. 2008; Allario et al. 2011, 2013; Tan et al. 2015). In addition, the tetraploid Carrizo citrange rootstock enhanced the cold hardiness of the clementine scion by strengthening the antioxidant system (Oustric et al. 2017). Additionally, transcriptome analysis and global DNA methylation profiling demonstrated that the whole‐genome duplication of the cold-hardy P. trifoliata was associated with higher expression levels of a range of well‐characterized cold stress‐responsive genes, especially those involved in unsaturated fatty acid (UFA) and JA biosynthesis (Wang et al. 2022). Likewise, the P. trifoliata autotetraploids exceeded the diploid lines in drought tolerance by sustaining a robust system of ROS detoxification and sugar accumulation relative to their diploid counterpart (Wei et al. 2019). Furthermore, the proteomic analysis of two citrus genotypes, Cleopatra and Willow leaf mandarins, which differ in their Na+ and Cl− accumulation, and their autotetraploids accumulated higher constitutive levels of antioxidant enzymes and heat shock proteins in salinized 4 × genotypes compared with the cognate 2 × genotypes, illustrating the greater tolerance of the tetraploids (Podda et al. 2013). Consistently, the enhanced tolerance of citrus tetraploids to high saline toxicity was attributed to the enhanced Cl− exclusion capacity of roots (due to the histological and morphological characters of citrus tetraploids), leading to lower levels of toxic ions in the foliage because of the lower transpiration rate (Saleh et al. 2008; Ruiz et al. 2016; Khalid et al. 2021). Physiological experiments have revealed that the ABA signaling pathway is implicated in root‒shoot communication for transmitting water deficit tolerance from autotetraploid Rangpur lime rootstocks to the diploid Valencia Delta sweet orange scion (Allario et al. 2013). This enhanced abiotic stress tolerance was transmitted to grafted scions, indicating the economic importance of using tetraploid rootstocks to confer enhanced tolerance to multiple types of abiotic stress (Allario et al. 2013; Khalid et al. 2021). On the other hand, it is noteworthy that the development of triploid citrus varieties is considered an essential method for producing seedless citrus varieties with enhanced tolerance to abiotic factors (Lourkisti et al. 2020; Lourkisti et al. 2021). Taken together, the data indicate that the physiological, morphological, biochemical and molecular differences that are obtained by genomic duplication contribute greatly to the enhanced tolerance of citrus polyploids to multiple types of abiotic stress and indicate the great potential for using polyploidization to develop new varieties of citrus with improved stress tolerance.

Integration of multiomics is a promising approach for the genetic improvement of citrus

The plant phenotype is a consequence of the combination of multiple intertwined and dynamic interactions among DNA, transcripts, proteins and metabolites with environmental conditions during the developmental stages. Currently, citrus biologists have implemented advanced molecular tools to fully understand how citrus trees respond to their environments. However, despite the recent identification of many abiotic stress-responsive genes in citrus (Ling et al. 2000; Long et al. 2011; Dahro et al. 2016; Wang et al. 2019; Ming et al. 2020), a full understanding of the regulatory networks influencing the expression of these genes and the molecular mechanisms underlying abiotic stress tolerance has not been achieved (Caruso et al. 2020; Vincent et al. 2020). In this context, the natural diversity of citrus germplasm with a broad distribution of physiological and morphological traits could be advantageous for the genetic breeding of citrus (Zheng et al. 2019; Biswas et al. 2020). Abiotic stress tolerance is a complex quantitative trait that is controlled by the expression of an unknown number of genes. Thus, the causal genetic polymorphisms in the abiotic stress-tolerant germplasm support a wide variety of biochemical pathways and networks that lead to the accumulation of several abiotic stress-related metabolites. Furthermore, phenotypic performance under abiotic cues also depends on protein translational and posttranslational modifications (Podda et al. 2013; Dutra de Souza et al. 2017). In such complicated regulatory scenarios, the integration of epigenomics, genomics, transcriptomics, proteomics, and metabolomics with network analysis would illustrate a comprehensive and reliable view of the biochemical and physiological changes that participate in citrus varieties that tolerate multiple types of abiotic stress (Fig. 5) (Jiang et al. 2021b). In the last decade, genomic sequencing of sweet orange and several related genera has offered genetic information for hundreds of citrus accessions, leading to the identification of a vast number of genes associated with valuable horticultural traits (Xu et al. 2013; Wang et al. 2018; Zhu et al. 2019; Huang et al. 2021). With the development of genomic sequencing, the application of current knowledge on horticultural trait-linked genetic markers could hamper the power of quantitative trait loci (QTLs) and genome-wide association studies (GWAS) for genotyping citrus varieties and describing their responses to abiotic factors (Jiang et al. 2021b). On the other hand, RNA sequencing is also an essential strategy that has been particularly used to identify differentially expressed genes associated with abiotic stress tolerance (Silva Santos et al. 2021). Using this approach, crucial salt stress-responsive genes in citrus rootstocks were unraveled by the transcriptomics comparison of the salt-tolerant Cleopatra mandarin and the salt-sensitive Carrizo citrange (Brumós et al. 2009). In parallel, transcriptome analysis demonstrated the significant role of ABA in the drought tolerance of the tetraploid Rangpur lime (C. limonia) (Allario et al. 2013) and showed rootstock-induced molecular responses associated with drought tolerance in grafted sweet orange (Gonçalves et al. 2019). Unfortunately, marker-assisted selection is strongly influenced by the precise evaluation of abiotic stress tolerance in citrus. Notably, metabolites are closer to the phenotype, as they reflect the association between several regulatory levels, including epigenomic, genomic, transcriptomic, protein interaction and regulatory pathways (Wentzell et al. 2007; de Souza et al. 2017). Therefore, the integration between genetic polymorphisms and metabolic phenotypes using metabolomic genome-wide association studies (mGWAS) and metabolite quantitative trait locus (mQTL) mapping could facilitate the identification of causal genes regulating the candidate metabolites that provide tolerance to multiple types of abiotic stress (Jiang et al. 2021b). In crops, extensive efforts have been invested in several multiomics approaches that combined genetic variations, gene expression, protein functions, enzyme activities and metabolite analyses to construct eQTL, eGWAS, mQTL, and mGWASs, which contribute to the genetic improvement of cultivars and thus, to enhance productivity in stressful conditions (Chen et al. 2014). Interestingly, the combination of eQTL with mQTL successfully identified the causal element underpinning the natural variation of red coloration in citrus peel (Zheng et al. 2019). Similarly, the exploitation of this knowledge would accelerate the identification of candidate genes and proteins for the genetic improvement of elite rootstocks combining tolerance/resistance against different environmental stressors and devastating diseases.

Fig. 5
figure 5

Integrated omics would pave the way to identify candidate genes and proteins that coordinate the metabolites responsible for tolerance to combined abiotic stresses. The development of epigenomics, genomics, transcriptomics, and metabolomics technologies provides a comprehensive strategy to understand the biology of abiotic stress tolerance. The organization of biomolecular information from DNA to phenotype is shown on the horizontal axis. Expression quantitative trait locus and expression genome-wide association study (eQTL, eGWAS); proteomic QTL and proteomic GWAS (pQTL and pGWAS); metabolomics QTL and metabolomics GWAS (mQTL and mGWAS) integrated interdisciplinary knowledge to facilitate the genetic improvement of citrus

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Conclusion remarks and future prospects

The constraints posed by a fluctuating climate limit global food production. Therefore, breeding new citrus varieties that are more resilient to multiple types of abiotic stress with improved productivity traits is an important objective. Efficient citrus improvement programs need precise phenotyping and genotyping of citrus germplasm to identify essential characters linked with enhanced productivity and tolerance to abiotic factors. Although the integration of multiomics succeeded in the genetic improvement of staple crops, the technological gap between staple crops and fruit trees narrows this achievement in citrus breeding to abiotic stress tolerance. The integration of epigenomics, genomics, transcriptomics, proteomics, and metabolomics technologies could accelerate the identification of polymorphisms that are significantly associated with key agronomic traits, thereby facilitating powerful strategies for the genetic improvement of citrus. Therefore, the investment of multiomics is an essential tool that can implement the heterozygosity of natural populations to elucidate the mechanistic basis underpinning abiotic stress tolerance. In addition, the recent development of genome editing using the clustered regularly interspaced short palindromic repeat (CRISPR) tool opened an avenue to efficiently engineer targeted modifications in the genome without transferring foreign genes (Jiang et al. 2021b), thus simplifying the precise introduction of single or multiple genes for genetic improvement of citrus to cope with dynamic changes in the climate. Redesigned citrus plants with stress memory provide a new means to safeguard citrus production and engineer climate-smart citrus varieties for the future.