Rapid alkalinization factor: function, regulation, and potential applications in agriculture

Abstract

Rapid alkalinization factor (RALF) is widespread throughout the plant kingdom and controls many aspects of plant life. Current studies on the regulatory mechanism underlying RALF function mainly focus on Arabidopsis, but little is known about the role of RALF in crop plants. Here, we systematically and comprehensively analyzed the relation between RALF family genes from five important crops and those in the model plant Arabidopsis thaliana. Simultaneously, we summarized the functions of RALFs in controlling growth and developmental behavior using conservative motifs as cues and predicted the regulatory role of RALFs in cereal crops. In conclusion, RALF has considerable application potential in improving crop yields and increasing economic benefits. Using gene editing technology or taking advantage of RALF as a hormone additive are effective way to amplify the role of RALF in crop plants.

Keywords: Crops; Fertilization; Molecular regulatory network; RALF; Stress.

Fig. 1

Phylogenetic analysis and conserved domain analysis of RALFs in five cereals and Arabidopsis. A The maximum-likelihood phylogenetic tree of RALF family members. The bootstrap value has been marked on the node by stars of different sizes. According to the motif differences and genetic relationship, RALFs can be divided into four subfamilies and denoted as Subfamilies -I, II, III and IV, respectively. B The logos of conserved motif sequences. The logos of conserved domain sequences were obtained from the MEME suite website (https://meme-suite.org/meme/). The bit score represents information content of each position in the amino acid sequence. The conserved motif was marked in different colors and displayed on the periphery of the phylogenetic tree. The evolutionary tree is modified with ITOL (https://itol.embl.de/)

Fig. 2

RALFs play a precise molecular gating role in different stages of plant reproduction. A On the pollen-stigma surface, RALF23/33 competes with PCP-Bs to bind to the receptor complex FER/ANJ/LLGs, regulates ROS production through the downstream GTPase activation pathway, thus affects pollen hydration (Liu, et al. 2021). B At the pollen tube extension process, RALF4/19 activates GTPase and the cytoplasmic receptor-like kinase MARIS through the receptor complex. Pollen tube integrity is maintained by maintaining exocytosis at the pollen tube tip and Ca²⁺ concentration gradient (Boisson-Dernier, et al. ; Gao, et al. ; Ge, et al. 2017). In pear, PbrRALF2 binds to the ectodomain of PbrCrRLK1L13 and induces phosphorylation of PbrCrRLK1L13, which induces ROS production through downstream PbrMPK18, and excess ROS negatively regulates pollen tube elongation (Kou, et al. 2021). C At the pollen tube-synergids interface, RALF4/19 sensed by the receptor complex recruit membrane channel protein NTA, trigger Ca²⁺ oscillation and participate in the process of pollen tube reception. CaM has an inhibitory feedback mechanism on the activity of NTA, thereby preventing the excessive Ca²⁺ influx activated by RALFs (Gao, et al. 2022). The receptor complex induces the rupture of pollen tubes reaching female gametophytes through activation of GTPase-RBOHs resulting in the increasing of ROS (Duan, et al. 2014). The substitution of RALF34 for RALF4/19 induced pollen tube rupture (Ge, et al. 2017). RALF6/7/16/36/37 maintain high-efficiency double fertilization by binding to the FER/ANJ/HERK1 receptor complex (Zhong, et al. 2022). Annotated Arabidopsis RALF homologs in other species

Fig. 3

RALFs act as endogenous signaling peptides to regulate root development by controlling plant cell expansion. RALFs phosphorylate AHA2 on the plasma membrane through receptor complex, inhibiting proton transport, thereby inducing alkalinization of the extracellular matrix (Haruta, et al. 2014). RALFs–FER promotes the expression of auxin synthesis genes YUCs, thereby inducing TIR1/AFB transcription and persistently inhibiting root elongation (Li, et al. 2022). Increased pH promotes the dissociation of BRI and BAK1, allowing AtRALF1 and CML38 to bind to BAK1 (Campos, et al. ; Dressano, et al. 2017), inhibiting BL-induced cell elongation. RALFs activate FER by increasing the phosphorylation level of FER. The FER–RopGEF–ROP/RAC complex interacts with ABI2, phosphorylates and activates ABI2, and negatively regulates ABA signaling pathways (Chen, et al. ; Yu, et al. 2012). Activated FER mediates signal transduction from the cell membrane surface to the nucleus by phosphorylating EBP1, eIF4E, and RIPK. EBP1 appears to repress the transcription of CML38 (Li, et al. ; Liu, et al. ; Zhu, et al. 2020). RALF1 mediates endocytosis of FER and BRI into the vacuole (Yu, et al. 2020). In root cells, RALFs–FER probably triggers the increasing of cytoplasmic Ca²⁺ concentration in response to receptor-like cytoplasmic kinase (RLCK) and Ca²⁺-binding protein (CBP) (Fuglsang, et al. 2007). Annotated Arabidopsis RALF homologs in other species

Fig. 4

RALFs exhibit different responses in the face of external stimuli. The upper side of the cell shows the activity of RALFs under biotic stress: FER acts as a scaffold to mediate the immune complex formation and sense the stimulation of RALFs. RALFs respond to the stimulation of pathogenic microorganisms, and show positive or negative regulation of plant immunity (Stegmann, et al. 2017a). The RALF-receptor complex induces phosphorylation of downstream BIK1 and participates in MYC2-regulated jasmonic acid signaling (Guo, et al. ; Shen, et al. 2017). RALF is likely to regulate plant immunity by activating or inhibiting immune-related responses such as Ca²⁺ oscillations, MAPK cascades, and ROS bursts. The influx of Ca²⁺ regulates ROS production during immune signaling and controls stomatal responses to pathogenic microorganisms through the activation of calcium-dependent protein kinases (CPKs) (Li, et al. 2014). A range of fungi and nematodes can also secrete plant RALF homologs to suppress host immune responses and increase host disease susceptibility (Duan, et al. 2022). The lower side of the cell shows the activity of RALFs under abiotic stress: under salt stress, the extracellular domain of FER and co-receptor LLGs sense the cell wall perturbation caused by Na⁺, trigger intracellular Ca²⁺ transients, and initiate cell wall repair (Feng, et al. 2018). Increased Na⁺ induces perturbation of the cell wall, dissociation of 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). The RALF–LRX–FER module likely induces cell death under salt stress through loss of ABA homeostasis, accumulation of ROS, and increased ion concentration (Zhao, et al. 2021). Annotated Arabidopsis RALF homologs in other species

Fig. 5

Application potential of RALFs in agriculture. Traditional transgenic tools or gene editing techniques were used to create transgenic plants of RALFs, for relieving the growth inhibition caused by RALFs and amplifying the positive regulatory role of RALFs. We can also develop plant growth additives related to RALFs: (1) applying RALFs to stigma to improve plant reproduction, (2) applying RALFs to root system to improve the adaptation to acidic culture substrates and cultivate crops with excellent root traits, and (3) applying antimicrobial RALFs to plants to increase the resistance of crops