Liping Huang ^1* Xing Liu ¹ Qianqian Wang ¹ Wen Chen ¹ Wenxuan Fu ¹ Yongjun Guo ^1,2

• ¹ Foshan University, Foshan, China
• ² Foshan ZhiBao Ecological Technology Co. Ltd, Foshan, China

in plants is highly complex due to the involvement of several key players in regulating ST in plants (Khan et al., 2020;Tanveer and Ahmed, 2020). The SS reduces plant growth by inducing membrane depolarization, which later increases ROS production in the cytosol and activated voltage-gated and ROS-activated K + -outward rectifying channel and transporter at the plasma membrane (PM) (Fig. 1B) (Wegner et al. 2011;Shabala et al. 2015). Thus, in this context, the activation of PM-H + -ATPase and Ca 2+ signaling is critical in regulating cytosolic K + homeostasis and ST in plants.Compared with alkalization, higher activation of H + -ATPase is required to reduce the SS-induced activation of voltage-gated K + outward rectifying channels (e.g., GORK) at the plasma membrane (PM) (Shabala et al., 2016). SS results in a significant membrane depolarization leading to a considerable disturbance in cell ionic balance and metabolism. Plants' ability to maintain highly negative membrane potential (MP) values has been firmly associated with their tolerance to SS (Chen et al., 2007;Bose et al., 2015;Chakraborty et al., 2016). In this context, RALF proteins are important for alkalization and may play an important role in regulating PM-H + -ATPase to regulate ST in plants. RALF regulates the activity of H + -ATPase (Gjetting et al., 2020), which later imposes a massive implication to the regulation of plant ionic homeostasis via controlling cell membrane potential (MP) at the plasma membrane. Though H + -ATPase activity is critical in determining cell MP (Palmgren and Nissen, 2011), more negative MPs are required for the operation of voltage-gated ion channels and ion transporters. The H + gradients between extracellular and intracellular space create an H + motive force for the secondary active transport of other ions (e.g., K + , NH4 + , NO3 -; PO4 2-; SO4 2-) via H + -coupled co-transport systems (Shabala et al., 2016). Thus, RALF-induced modulation of H + -ATPase activity may be an essential factor in controlling cellular MP and, ultimately, cell metabolism under stress conditions. However, it has been argued that higher H + -ATPase activity may lead to ATP reduction in the cells, which later affects the ability of plants to survive under salinity stress (Rubio et al. 2020). Thus, tight regulation between the activation of RALF-mediated H + -ATPase and ATP reduction is required. However, there is also less evidence to prove that RALF as an upstream inhibitor of H + -ATPase blocks some channels or transporters (e.g. SOS1, HAK family) that are driven by H + gradient.Moreover, due to the diverse ability of RALFs to interact with other receptor proteins, it can be suggested that different RALFs may affect different signal pathways to activate H + transporters/channels in the cell and regulate ST in plants. The LRR domains of LRX3 and LRX4 can interact with both AtRALF22/23 and FER to regulate salt stress response (Zhao et al., 2018). Recently (Gjetting et al., 2020) showed that the H + pump activity was increased three times after the application of 1 µM RALF33/36, while, the aha mutant was hypersensitive to AtRALF1 which was some unknown H + transporters or channels led to the net influx of H + in the cytoplasm and the increase of extracellular pH in highly possible (Li et al., 2022). However, how cells accurately perceive the extracellular environmental pH and release RALF to regulate it is a problem that needs to be solved. SS also triggers apoplastic alkalization and thereby inhibits plant growth (Kesten et al., 2019), however, it can also induce the formation of mature RALFs (Zhao et al., 2018). In two halophytes, higher activation of H + -ATPases under SS contributes to Na + efflux from cytosol and low apoplastic pH associated with higher Na + /H + exchanger at PM (Bose et al., 2015). Such SS induced apoplastic alkalization could later be mediated by RALF-FER-AHA2 and apoplastic acidification is important for ST. This aspect needs to be examined in future studies.Ca 2+ being as an important component of the cell wall and membrane structure (Bascom et al., 2018), and the oscillations of the cytoplasmic Ca 2+ concentration as a second messenger to be involved in various physiological reactions and signal transmission processes (Thor, 2019;Sanders et al., 1999). So far, only a few extracellular mediators have been found to affect cytoplasmic Ca 2+ "signatures". In Arabidopsis, AtRALF1 increased the cytoplasmic Ca 2+ level by promoting the influx of extracellular Ca 2+ and reducing efflux of intracellular Ca 2+ (Haruta et al., 2008).Later, the relationship between RALF induced Ca 2+ signal and pH was confirmed as RALF-induced extracellular pH change depends on Ca 2+ signal which occurs before alkalinization (Gjetting et al., 2020). AtRALF1 also interacts in a Ca 2+ and pHdependent manner with calmodulin-like 38 for regulating root growth (Campos et al., 2018). Furthermore, RALF33 treatment did not affect the characteristics of Ca 2+ and H + while RALFL36 treatment showed some effects in the fer Arabidopsis mutant (Gjetting et al.,2020), again suggesting different RALF peptides may bind with different receptors to trigger intracellular Ca 2+ , which later may activate H + pumping at PM. Therefore, exploring other unknown RALF receptor-induced Ca 2+ oscillations and testing whether their signal pathways overlap is a direction noteworthy in the future (Tanveer et al., 2018;Choudhary et al., 2021).RALFs proteins regulate the overall plant growth and redox homeostasis by regulating ROS production (Zhang et al., 2020). For instance, FER positively regulates root hair polar growth by regulating auxin-mediated ROS production (Yu et al. 2012). Likewise, FER and related proteins regulate ROS production by regulating the transcription of respiratory burst oxidase homologs (RBOH) (Franck et al. 2018).For instance, FER-LLG1-Rop-Guanine Nucleotide Exchange factor complex regulates RBOH dependence ROS production (Duan et al., 2010;Li et al., 2015).ANX1 and ANX2 also maintain ROS production and regulate cell wall integrity during pollen tube growth (Boisson-Dernier et al., 2013), thus RALFs protein complex requires ROS as important signaling molecules for regulating cell growth (Zhang et al. 2020). Nonetheless, the overproduction of ROS under salinity stress is inevitable, and salinity tolerance is linked with maintaining an equilibrium between overall ROS production and ROS scavenging; thus, tight regulation is required (Shabala and Tanveer 2018;Khan et al. 2020). Moreover, the effects of salinity stress on the overall redox state are highly tissue-specific and NaCl dose-specific (Shah et al.). This should be explored in future studies.Cell wall (CW) biosynthesis is a very complex mechanism in plants and to examine the CW status, plants exhibit CW integrity maintenance (CWIM) system.The CWMI system assists plants in adapting to stress conditions without compromising the integrity and organization of CW (Liu et al., 2021). SS can adversely affect CWI, thus plants' ability to maintain CWIM system is essential for ST. Having said that, Ca 2+ being a universal secondary messenger, is actively involved in the operation of CWIM system in plants. However, the maintenance of balance between Ca 2+ concentrations in CW, apoplast, and cytoplasm raises the question relating to the validity of this concept. In this context, Feng et al. (2018) showed that FER is required for the activation of Ca 2+ influx and maintenance of CWI under salt stress (Fig. 1B). FER is an important CWIM sensor and required for Ca2+ influx into cytoplasm under salinity stress (Feng et al., 2018). FER contains two malectin domains that directly bind with de-methyl-esterified HG in vitro and in vivo (Feng et al., 2018;Lin et al., 2018;Liu et al., 2021). This suggests that FER probably senses the CW changes directly via its extracellular domain and then transduces the CW signals to the cytoplasm via its cytoplasmic kinase domain. Later, it was shown that SS may dissociate the LRX3/4/5-RALFs complex via the SS-induced ROS and pH changes in the apoplast, and the released RALFs bind to LLG1-FER complex and thereby allow the transduction of CW signals (Zhao et al., 2018). The mechanism behind the dissociation of LRX3/4/5 and RALFs under SS needs to be further investigated. Moreover, FEI1 and FEI2 are other two important LRR-RLK complexes that regulate cellulose synthesis in the cell wall (Xu et al., 2008) and loss of function mutants of FEI1 and FEI2 showed roots with reduced cellulose contents (Basu et al., 2015), thus indicating the role of FEIs in CWI sensing.Hormonal regulation under SS is also important, as different phytohormones regulate different physiological processes (Tanveer et al., 2018;Choudhary et al., 2021). Abscisic acid (ABA) is an essential hormone of plant stress resistance and tolerance. The signal crosstalk between RALF and ABA is involved in the response of plants to abiotic stresses including SS and water deficit (Chen et al., 2016). Studies showed that RALF1-FER signaling pathway activates ABI2 (ABA Insensitive 2) phosphatase by GEF1/4/10-ROP11 pathway and further inhibits ABA response (Yu et al., 2012;Chen et al., 2016). The LRX is an important receptor of CWI signal and The LRX3/4/5 triple mutants as well as FER mutants displayed salt hypersensitivity, which was mimicked by overexpression of RALF22/23 (Zhao et al., 2018). In Arabidopsis, LRX3/4/5-RALF22/23-FER regulated ST by regulating equilibrium between ROS production and accumulation of phytohormones (ABA, JA, SA) (Zhao et al., 2021).Moreover, RALF acts as upstream of ROS regulatory pathway and has been shown to interact with ABA to regulate the growth of plant roots (Chen et al., 2016). AGB1-a G protein β-subunit involved in ABA mediating stomatal opening and FER-ABG1 are reported to be involved in salt stress responses (Yu and Assmann, 2018). Mutants lacking FER and ABG1 showed hypersensitive phenotype to salt stress (Yu and Assmann, 2018) even the application of AtRALF was not effective in reducing hypersensitivity to salt stress (Zhao et al., 2018). Contrarily, AtRALF1 mutants did not showed any response to higher salt stress levels (Feng et al., 2018).Taken an example of non-vascular plants, PpRALF3 knockout lines showed higher resistant under SS and ROS stress in moss (Physcomitrium patens), implying the functional role of RALFs in regulating ST (Solís-Miranda et al., 2023). However, the relationship of RALFs with other phytohormones-e.g., melatonin should be considered in future studies. As melatonin-induced enhancement of PM H + -ATPase activity may negate salinity-induced MP depolarization preventing activation of outward K + channels, thereby leading to higher ST (Yu et al., 2018). Moreover, the abiotic stress regulatory role of MEL has also been reported elsewhere (Tanveer and Shabala, 2020;Huang et al., 2022;Huang et al., 2024).Given that, the RALFs gene expression are highly plant species-and tissuespecific (Kim et al., 2021. For instance, five homologs of RALFs were observed in popular (Haruta et al., 2003) while 37 homologs were in Arabidopsis (Abarca et al., 2021). A total of 765 RALF proteins were identified from 51 plant species (Campbell and Turner, 2017). Recently, a genome-wide association study revealed 163 RALF genes in seven species from Rosaceae family, including 45 mature RALF genes (Zhang et al., 2020). A phylogenetic tree analysis showed the diversification of different RALF genes in different plant species (Fig 1C), thus indicating the genetic differences among different plant species could also govern the regulation of RALFs genes in plants. RALFs are widely distributed in plants and RALFs induced cell expansion is the result of the interaction of the changes of intra-and extra-cellular ions, deposition of new cell wall materials, and the rearrangement of existing cell walls. During the transition from vegetative growth to reproductive growth, RALFs have different functions in maintaining the normal life activities of plants. This is achievable because RALF forms a complex signal network to complete these complex functions.For instance, RALF-FER signal transduction pathway is highly conserved in plants and is very important for mediating RALF signaling. Thus, the physiological significance of the regulatory activities of RALF and some membrane receptors is still unknown and several outstanding question should be focused during examining the role of RALFs in regulating salinity tolerance in plants. For instance,• how does RALF-induced FER-specific internalization conduct Ca 2+ transduction in cell wall is also a focus of research.• Equally interesting is the question of intracellular signal transduction of RALF-induced FER-specific internalization. While it is widely accepted that RALFs regulate plant growth and development together with cell wall components and PM receptors, spatio-temporal aspects of such regulation remain largely unknown, in the light of the apparent dual function of RALF.• It is also worth noting that some plant hormones such as ABA or melatonin are also involved in ST, however whether RALF is directly or indirectly crosstalk with these phytohormones for mediating ST is not fully comprehended.• Moreover, whether RALF is directly or indirectly involved in ion rebalance and transport under SS via regulating Ca 2+ signaling and H + -ATPase is not yet clear. Therefore, exploring the mechanism of RALF under SS is also an important direction and great challenge for future research in developing ST crops.• Given that RALFs peptides family is very diverse and binds to large arrays of receptors, thus mechanisms regulating the specificity of RALFs-receptor interaction under different growth conditions should be examined. NaCl induced Ca 2+ is sensed by the extracellular domain of FERONIA (FER) and co-receptor LLGs, which also senses the cell wall perturbation and initiates cell wall repair (Feng et al., 2018). Higher Na + entry into cytosol induces cell wall perturbation, dissociates RALF22/23 from LRXs and promotes RALF22/23-induced internalization of FER, and finally, RALF inhibits the signal transduction ability of FER (Zhao et al., 2018).Moreover, RALF complex with LRX and FER induces cell death under salt stress through loss of ABA homeostasis, higher Na + accumulation, and ROS production mediated by the Respiratory burst oxidase homolog (RBOH) gene (Zhao et al., 2021). Phylogenetic tree of different RALFs genes in Arabidopsis, rice, and soybean. We considered one model plant species (Arabidopsis thaliana L.), one cereal (rice-Oryza sativa L.), and one legume (soybean-Glycine max L.). We first found the number of RALFs genes, for example, 27 soybeans, 41 ric,e and 34 Arabidopsis RALF protein sequences were downloaded from the phytozome (https://phytozome-next.jgi.doe.gov/), rice Genomics Network (https://rice.uga.edu/cgi-bin/gbrowse/rice/) and TAIR (www.arabidopsis.org/) databases, respectively. The phylogenetic tree was produced using MEGA 11 software via the Neighbor-Joining (NJ) method with 1000 bootstrap replicates.