Update on the Roles of Rice MAPK Cascades

The mitogen-activated protein kinase (MAPK) cascades have been validated playing critical roles in diverse aspects of plant biology, from growth and developmental regulation, biotic and abiotic stress responses, to phytohormone signal transduction or responses. A classical MAPK cascade consists of a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK), and a MAPK. From the 75 MAPKKKs, eight MAPKKs, and 15 MAPKs of rice, a number of them have been functionally deciphered. Here, we update recent advances in knowledge of the roles of rice MAPK cascades, including their components and complicated action modes, their diversified functions controlling rice growth and developmental responses, coordinating resistance to biotic and abiotic stress, and conducting phytohormone signal transduction. Moreover, we summarize several complete MAPK cascades that harbor OsMAPKKK-OsMAPKK-OsMAPK, their interaction with different upstream components and their phosphorylation of diverse downstream substrates to fulfill their multiple roles. Furthermore, we state a comparison of networks of rice MAPK cascades from signal transduction crosstalk to the precise selection of downstream substrates. Additionally, we discuss putative concerns for elucidating the underlying molecular mechanisms and molecular functions of rice MAPK cascades in the future.


Introduction
The mitogen-activated protein kinase (MAPK) cascades have been designated to be highly conserved signal transduction modules in eukaryotes with diverse functions by linking different extracellular stimuli to a wide range of intracellular responses [1,2]. A complete MAPK cascade mainly consists of three kinases, including a MAPK kinase kinase (MAPKKK or MEKK), a MAPK kinase (MAPKK or MEK), and a MAPK (MPK). Upon sensing external stimulus signal, MAPKKKs phosphorylate and activate MAPKKs, the activated MAPKKs subsequently phosphorylate MAPKs, and finally the activated MAPKs phosphorylate a large number of specific downstream substrates, such as transcription factors, chromatin remodeling factors, kinases or other enzymes, leading to reprogramming of transcriptome and proteome in the whole cell. The sequential phosphorylation is fundamental for MAPK cascade-mediated signal transduction and interactions between MAPK proteins and their substrates [3].
In plants, the MAPK cascades play essential roles in growth and developmental regulation, biotic and abiotic stress responses, phytohormone signal transduction or responses [1,[4][5][6][7]. After receiving external signals, plant MAPKKKs mostly phosphorylate the two conserved serine (S) and threonine (T) residues in the S/T-X5-S/T (X is any amino acid) motif of MAPKKs and activate MAPKKs. The activated MAPKKs in turn phosphorylate both the threonine (T) and the tyrosine (Y) in the T-D-Y or T-E-Y motif of MAPKs and activate MAPKs. However, plant MAPK cascade-mediated signal transduction needs to be precisely regulated, as continuous activation or suppression of MAPK signaling cause side-effects for the normal growth of plants. Thus, plant MAPKs can reversely phosphorylate MAPKKKs to regulate the MAPK cascade, precisely controlling signal transduction or responses [8].

Component of Rice MAPK Cascades
The rice genome contains 75 OsMAPKKKs, 8 OsMAPKKs and 15 OsMAPKs [9,10]. The OsMAPKKKs, occupying the largest group of rice MAPK cascade proteins, are divided into three families, including 43 Raf family OsMAPKKKs, 22 MEKK family OsMAPKKKs, and 10 ZIK family OsMAPKKKs [10]. Although the rice genome harbors eight OsMAPKKs, two of them could not be detected on transcriptional levels in different rice tissues, and are considered as pseudogenes, thus there are only six functional MAPKKs in rice [11]. The MAPKs are divided into two subtypes, T-E-Y and T-D-Y, according to the conserved T-X-Y motif in their active loop that specifically phosphorylated by MAPKKs. Of these, T-E-Y subtype contains five OsMAPKs, T-D-Y subtype has 10 OsMAPKs [11].

Complicated Action Mode of Rice MAPK Cascades
Compared with 75 OsMAPKKKs and 15 OsMAPKs, rice contains six functional OsMAPKKs, implying that an OsMAPKK can be phosphorylated by multiple upstream OsMAPKKKs, and similarly a OsMAPKK can phosphorylate and activate several downstream OsMAPKs as its substrate. It seems that OsMAPKKs function as key nodes or hubs in MAPK cascades [59]. Of the five functionally deciphered OsMAPKKs, OsMAPKK4 typically acts as a hub of MAPK cascades, since OsMAPKKK10, OsMAPKKK11, OsMAPKKK18, and OsMAPKKK24 can separately phosphorylate OsMAPKK4 [20,21]. OsMAPKK4, in turn, can simultaneously phosphorylate and activate both OsMAPK3 and OsMAPK6 [33]. When phosphorylated by different OsMAPKKKs after rice sensing different external signals, OsMAPKK4 can select different downstream OsMAPKs for subsequential signal transduction. After rice sensing chitin-triggered signal or recognizing fungal pathogen M. oryzae invasion, OsMAPKK4 is rapidly phosphorylated by OsMAPKKK11, OsMAPKKK18 or OsMAPKKK24, then OsMAPKK4 subsequently phosphorylates OsMAPK3 and OsMAPK6 to transfer signals to downstream transcription factors, promoting rice resistance to M. oryzae [20,21]. However, when rice senses a BR signal, OsMAPKK4 is phosphorylated by OsMAPKKK10, then OsMAPKK4 phosphorylates OsMAPK6 for downstream signal transduction [17][18][19].

Controlling Growth and Development by Rice MAPK Cascades
Like MAPK cascade regulates cell proliferation and cell differentiation to influence plant growth or development, some members of rice MAPK cascades control embryogene-sis, fertility, seed development, grain performance, panicle morphogenesis, and architecture (Table 1).
MAPK cascades play critical roles in rice embryogenesis. Functional analysis of lossof-function mutants of OsMAPK6 reveals that OsMAPK6 affects the differentiation of L1 layer cells during early embryogenesis to arrest the embryonic development at the globular stage via influencing GA and auxin synthesis [45]. By screening of a series of osmapk mutants generated via CRISPR-Cas9 technology, heterozygous osmapk6 mutants can produce homozygous osmapk6 seeds but with abnormal embryo [42], while heterozygous osmapk4 mutants do not produce homozygous osmapk4 seeds, implying OsMAPK6 and OsMAPK4 influence seed development [42].
MAPK cascades play key roles in rice grain size and panicle morphogenesis. By screening mutants with altered grain size, smg1 mutant with multiple phenotypes, including small grains, erect leaves, dense and erect panicles has been identified. Genetic analysis indicates that smg1 is loss-of-function of OsMAPKK4, which influences cell proliferation and BR signal [32]. Meanwhile, a natural mutant, dsg1 with pleiotropic phenotypes, including significant dwarfism, small grains, erect and dark-green leaves has been identified. Complement genetic assay indicates that pleiotropic phenotypes of dsg1 are caused by loss of OsMAPK6. Subsequently, genetic analysis indicates that OsMAPKK4 acts upstream of OsMAPK6, by phosphorylating and activating OsMAPK6 to influence cell proliferation [62]. Recently, OsMAPKKK10 has been validated to regulate rice grain size and panicle development via activating OsMAPKK4-OsMAPK6 cascade by a series of genetic and biochemical analysis [17,18]. The OsMAPKKK10-OsMAPKK4-OsMAPK6 is so far the only completely known MAPK cascade, which regulates rice growth and development. The latest data have uncovered that plasma membrane localized receptor kinase OsER1 acts directly upstream of OsMAPKKK10-OsMAPKK4-OsMAPK6 cascade. The phosphorylated OsMAPK6 can subsequently phosphorylate OsDST1, then the phosphorylated OsDST1 binds to the promoter of OsCKX2 and promotes the transcription of OsCKX2 [17][18][19]. The whole signal transduction pathway, from plasma membrane OsER1 to cytoplasm OsMAPKKK10-OsMAPKK4-OsMAPK6, then to nucleus OsCKX2, uncovers a practically perfect genetic regulating network which regulates rice panicle morphogenesis, except the only gap between OsER1 and OsMAPKKK10 ( Figure 1). MAPK cascades function in rice architecture formation via modulating leaf morphology and plant height. The osmapkkk43 mutant caused by a T-DNA insertion shows an increased leaf angle. Following cell biology and genetic assays indicate that OsMAPKKK43 regulates mechanical tissue formation to modify leaf lamina joint by modulating secondary wall synthesis [22,23].

Coordinating Biotic Stress Response by Rice MAPK Cascades
A great number of plant MAPK cascades, especially of Arabidopsis MAPK cascades, have positive or negative effects on pathogens or insects invasion. Several rice MAPK cascades have been validated to coordinate biotic response and trigger resistance to bacterial and fungal pathogens or herbivores (Table 1).
MAPK cascades confer resistance to fungal pathogens. At least two OsMAPKKKs, two OsMAPKKs, and four OsMAPKs have been reported to be involved in resistance to fungal pathogen M. oryzae. Both OsMAPKKK1 and OsMAPKKK24 play positive roles in resistance to M. oryzae, while employing different molecular mechanisms. OsMAPKKK1 triggers resistance to M. oryzae by modulating ET biosynthesis to inhibit fungi penetration into rice cells, and OsMAPKKK24 by activating OsMAPKK4-OsMAPK6 cascade [12,13,21]. Both OsMAPKKK11 and OsMAPKKK18 are activated by chitin, the fungal microbial-associated molecular pattern. However, there is no direct evidence to confirm these two genes enhancing rice resistance to M. oryzae [20]. Of the four OsMAPKs to be involved in resistance to fungal pathogen, OsMAPK3 and OsMAPK16 negatively regulate resistance to M. oryzae [36,37,48], while OsMAPK20-5 positively confers resistance to M. oryzae [53]. OsMAPK6 is transcriptionally induced by sphingolipid elicitor and chitin, implying that OsMAPK6 possibly plays role in rice-M. oryzae interactions [33,34]. OsMAPKK10-2 can phosphorylate OsMAPK6, causing activated OsMAPK6 to subsequently phosphorylate and enhance the biochemical activity of downstream transcription factor OsWRKY45 to trigger rice resistance to M. oryzae [27]. Similarly, OsMAPKK4 phosphorylates and activates OsMAPK3 and OsMAPK6 to confer resistance to M. oryzae, through accumulation of diterpenoid phytoalexin, momilactones and phytocassanes [33]. However, the underlying mechanisms, why phosphorylated OsMAPK3 and OsMAPK6 by different upstream OsMAPKKs, cause susceptibility and confer resistance to M. oryzae, respectively, are unclear. Apart from being involved in resistance to fungal pathogen M. oryzae, OsMAPK20-5 has been reported simultaneously to be involved in resistance to fungal pathogen R. solani [53]. By integrating the characterized MAPK cascades, OsMAPKKK11/18/24-OsMAPKK4/5-OsMAPK3/6 cascades are the complete MAPK cascades, which mediate M. oryzae-triggered signal transduction and promote rice resistance to M. oryzae (Figure 2). MAPK cascades trigger resistance to bacterial pathogens. Up to now, one OsMAPKKK, two OsMAPKKs, and six OsMAPKs have been referenced to be involved in resistance to bacterial pathogens, Xoo, Xoc or B. glumae. OsMAPKKK1 negatively regulates resistance to Xoo by modulating accumulation of JA and SA [13]. OsMAPKK10-2 functions as a positive regulator in response to Xoc by activating downstream OsMAPK6 [28]. OsMAPKK3 also functions as a positive regulator but in response to Xoo by activating downstream OsMAPK7, with the signal transduction that the activated OsMAPK7 phosphorylates and activates the transcription factor OsWRKY30 to enhance rice resistance to Xoo [30]. Of the six OsMAPKs conferring resistance to bacterial pathogen, OsMAPK3 and OsMAPK16 play negative roles in response to Xoo [37,48], while OsMAPK7 and OsMAPK17-1 play positive roles in resistance to Xoo [30,49]. Interestingly, OsMAPK4 positively confers resistance to Xoo by promoting the accumulation of JA and SA, while it negatively influences resistance to Xoo by negatively regulating systemic acquired resistance, because both OsMAPK4 overexpressing plants and osmapk4 mutant exhibit enhanced resistance to Xoo [40,41]. In addition, OsMAPK3 is also involved in resistance to B. glumae, a soil bacterium [36].

Conferring Resistance to Abiotic Stress by Rice MAPK Cascades
In addition to biotic stress, rice MAPK cascades have also been confirmed conferring abiotic stress responses, under such as salt, drought, cold, or submergence. For example, OsMAPK3 is the fully characterized MAPK cascade protein which kinase activity is induced by a series of abiotic stress including drought, salt, cold and submergence. The Os-MAPK3 overexpressing plants show enhanced resistance to these different abiotic stress [36]. The following research indicates that OsMAPKK6 which acts upstream of OsMAPK3 enhances rice cold tolerance [34,35]. The mechanism of OsMAPKK6-OsMAPK3 cascade being involved in cold tolerance is recently been deciphered, with that the activated Os-MAPK3 interacts with and phosphorylates OsbHLH002/OsICE1, in turn phosphorylated OsbHLH002/OsICE1 binds and promotes the expression of OsTPP1 to cause trehalose accumulation, thereby increasing cold tolerance for rice plants [66]. Whereas, OsMAPK3 has roles in drought tolerance by acting as substrate for OsMAPKKK10-2, the underlying molecular mechanism is unclear [28]. Furthermore, OsMAPK3 has positive effect on salt tolerance by attenuating the reactive oxygen species accumulation [67]. These results demonstrate that OsMAPK3 confers tolerance to salt, drought, or cold stress probably by phosphorylating different substrates (Figure 3).
For other MAPKs, OsMAPKK1, its kinase activity is induced by salinity, plays positive roles towards salt stress by phosphorylating and activating downstream substrate OsMAPK4 [26]. Recently, OsMAPKKK63 is found to associate with OsMAPKK1 to enhance rice resistance to salt stress [25]. Thus, a complete MAPK cascade consisting of Os- MAPK cascades also have roles in resistance to herbivores. Although a number of rice MAPK genes show diverse transcriptional patterns upon herbivores BPH and SSB infection, only one OsMAPKK and three OsMAPKs have been validated exhibiting resistance to BPH or SSB. OsMAPKK3 functions as a positive regulator in rice-BPH interactions by modulating herbivory-induced phytohormone dynamics [31]. In line with OsMAPKK3, OsMAPK3 and OsMAPK4 also act as positive regulators conferring resistance to SSB with partly similar mechanisms. OsMAPK3 triggers resistance to SSB by regulating JA signaling pathway and promoting accumulation of herbivory-induced trypsin protease inhibitors [39], and OsMAPK4 confers resistance to SSB by regulating JA, ET and SA signaling pathways [43]. Additionally, OsMAPK20-5 which transcriptionally induced by gravid female BPH, negatively regulates rice resistance to BPH via suppressing the accumulation of ET and NO [52]. It seems that these three OsMAPKs largely regulate resistance to herbivores by modulating phytohormone signaling pathway.

Conferring Resistance to Abiotic Stress by Rice MAPK Cascades
In addition to biotic stress, rice MAPK cascades have also been confirmed conferring abiotic stress responses, under such as salt, drought, cold, or submergence. For example, OsMAPK3 is the fully characterized MAPK cascade protein which kinase activity is induced by a series of abiotic stress including drought, salt, cold and submergence. The OsMAPK3 overexpressing plants show enhanced resistance to these different abiotic stress [36]. The following research indicates that OsMAPKK6 which acts upstream of OsMAPK3 enhances rice cold tolerance [34,35]. The mechanism of OsMAPKK6-OsMAPK3 cascade being involved in cold tolerance is recently been deciphered, with that the activated OsMAPK3 interacts with and phosphorylates OsbHLH002/OsICE1, in turn phosphorylated OsbHLH002/OsICE1 binds and promotes the expression of OsTPP1 to cause trehalose accumulation, thereby increasing cold tolerance for rice plants [66]. Whereas, OsMAPK3 has roles in drought tolerance by acting as substrate for OsMAPKKK10-2, the underlying molecular mechanism is unclear [28]. Furthermore, OsMAPK3 has positive effect on salt tolerance by attenuating the reactive oxygen species accumulation [67]. These results demonstrate that OsMAPK3 confers tolerance to salt, drought, or cold stress probably by phosphorylating different substrates (Figure 3). MAPKKK63-OsMAPKK1-OsMAPK4 is identified, which positively promotes rice for salinity tolerance (Figure 3). OsMAPKKK6 functions as a positive regulator towards drought stress by regulating reactive oxygen species scavenging, while its downstream OsMAPKK or OsMAPK are unidentified till now [16]. Figure 3. Rice MAPK cascades are activated by different abiotic stress signals and confer tolerance to diverse abiotic stress in rice. A variety of abiotic stress, such as cold, salt, drought, and submergence could activate diverse rice MAPK cascades, which play critical roles in triggering rice resistance to these stresses [16,[25][26][27][28][34][35][36]67,68]. OsMAPKKK63-OsMAPKK1-OsMAPK4 is the only known cascade conferring salt tolerance, while its downstream substrates have not been identified.

Conducting Phytohormone Signal Transduction by Rice MAPK Cascades
As key molecules linking extracellular and intracellular signal transduction, MAPK cascades have been widely reported to participate in phytohormone accumulation, signaling pathways or response, such as ABA, SA, JA, CK, BR or ET. MAPK cascade-mediated phytohormone signal transduction largely contributes to their diverse roles in growth and developmental responses, or biotic and abiotic stress responses. For example, SA treatment can upregulate the transcription of OsMAPKK10-2, the activated Os-MAPKK10-2 phosphorylates and enhances the activity of OsMAPK6, triggering the SA signaling pathway to improve rice resistance to bacterial pathogen Xoc and fungal pathogen M. oryzae. Reversely, ABA treatment can induce the transcription of both OsPTP1 and OsPTP2, encoding two tyrosine phosphatases, which two can dephosphorylate the tyrosine residues at the T-E-Y motif of OsMAPK6 and cause the inactivation of OsMAPK6, resulting in decreased resistance to fungal pathogen M. oryzae and increased sensitivity to drought stress [27,28]. Lately, OsMAPK6 is reported to be a substrate of OsMAPKKK10-OsMAPKK4 cascade being involved in BR signal transduction, modulating rice architecture and grain size [17][18][19]. Furthermore, activated OsMAPK6 by OsMAPKKK10-Os-MAPKK4 cascade can also regulate CK metabolism to alter rice panicle development. Os-MAPK6 interacts with and phosphorylates OsDST1, then the phosphorylated OsDST1 binds and promotes the transcription of OsCKX2, which encodes the cytokinin oxidase/dehydrogenase. Thereby, activated OsCKX2 accelerates catalyzing the degradation of active CK to alter the number of rice spikelets [19]. Thus, the OsMAPKKK10-Os-MAPKK4-OsMAPK6 cascade is closely associated with CK homeostasis in determining rice panicle development (Figure 4). The series of results suggest that different phytohormone signaling pathways can modulate OsMAPK6 function in diverse physiological processes, and OsMAPK6 could also phosphorylate different downstream substrates to regulate phytohormone homeostasis, fine-tuning rice growth and developmental responses as well as biotic and abiotic stress responses. Rice MAPK cascades are activated by different abiotic stress signals and confer tolerance to diverse abiotic stress in rice. A variety of abiotic stress, such as cold, salt, drought, and submergence could activate diverse rice MAPK cascades, which play critical roles in triggering rice resistance to these stresses [16,[25][26][27][28][34][35][36]67,68]. OsMAPKKK63-OsMAPKK1-OsMAPK4 is the only known cascade conferring salt tolerance, while its downstream substrates have not been identified.
For other MAPKs, OsMAPKK1, its kinase activity is induced by salinity, plays positive roles towards salt stress by phosphorylating and activating downstream substrate OsMAPK4 [26]. Recently, OsMAPKKK63 is found to associate with OsMAPKK1 to enhance rice resistance to salt stress [25]. Thus, a complete MAPK cascade consisting of OsMAPKKK63-OsMAPKK1-OsMAPK4 is identified, which positively promotes rice for salinity tolerance (Figure 3). OsMAPKKK6 functions as a positive regulator towards drought stress by regulating reactive oxygen species scavenging, while its downstream OsMAPKK or OsMAPK are unidentified till now [16].

Conducting Phytohormone Signal Transduction by Rice MAPK Cascades
As key molecules linking extracellular and intracellular signal transduction, MAPK cascades have been widely reported to participate in phytohormone accumulation, signaling pathways or response, such as ABA, SA, JA, CK, BR or ET. MAPK cascade-mediated phytohormone signal transduction largely contributes to their diverse roles in growth and developmental responses, or biotic and abiotic stress responses. For example, SA treatment can upregulate the transcription of OsMAPKK10-2, the activated OsMAPKK10-2 phosphorylates and enhances the activity of OsMAPK6, triggering the SA signaling pathway to improve rice resistance to bacterial pathogen Xoc and fungal pathogen M. oryzae. Reversely, ABA treatment can induce the transcription of both OsPTP1 and OsPTP2, encoding two tyrosine phosphatases, which two can dephosphorylate the tyrosine residues at the T-E-Y motif of OsMAPK6 and cause the inactivation of OsMAPK6, resulting in decreased resistance to fungal pathogen M. oryzae and increased sensitivity to drought stress [27,28]. Lately, OsMAPK6 is reported to be a substrate of OsMAPKKK10-OsMAPKK4 cascade being involved in BR signal transduction, modulating rice architecture and grain size [17][18][19]. Furthermore, activated OsMAPK6 by OsMAPKKK10-OsMAPKK4 cascade can also regulate CK metabolism to alter rice panicle development. OsMAPK6 interacts with and phosphorylates OsDST1, then the phosphorylated OsDST1 binds and promotes the transcription of OsCKX2, which encodes the cytokinin oxidase/dehydrogenase. Thereby, activated OsCKX2 accelerates catalyzing the degradation of active CK to alter the number of rice spikelets [19]. Thus, the OsMAPKKK10-OsMAPKK4-OsMAPK6 cascade is closely associated with CK homeostasis in determining rice panicle development (Figure 4). The series of results suggest that different phytohormone signaling pathways can modulate OsMAPK6 function in diverse physiological processes, and OsMAPK6 could also phosphorylate different downstream substrates to regulate phytohormone homeostasis, fine-tuning rice growth and developmental responses as well as biotic and abiotic stress responses.  [12][13][14][15]27,28,40,43,48,49]. The established OsMAPKKK62-Os-MAPKK3-OsMAPK7/14 cascades are regulated by ABA, while the OsMAPKKK10-OsMAPKK4-Os-MAPK6 cascade can regulate BR and CK signal transduction [17][18][19]24].
In the same way, other MAPK cascades are involved in phytohormone response. For example, the OsMAPKKK62-OsMAPKK3-OsMAPK7/14 and OsMAPKK10-2-OsMAPK3 cascades are associated with ABA signal transduction, regulating rice seed dormancy [24,28]. OsMAPKKK1 acts as a positive regulator in ABA and ET signaling pathways, while as a negative regulator in JA and SA signaling pathways [12][13][14][15], implying that Os-MAPKKK1 probably interacts with different proteins or phosphorylates different downstream OsMAPKKs to play roles in diverse phytohormone signaling pathways. Similarly, OsMAPK4 positively regulates the accumulation of JA and SA, and OsMAPK17-1 positively regulates the accumulation of SA, while OsMAPK16 negatively regulates the accumulation of JA and SA, and OsMAPK20-5 negatively affects the synthesis of ET [40,43,48,49]. These data demonstrate that rice MAPK cascades regulate or involve in complex phytohormone accumulation, signaling pathways or response ( Figure 4).
In the same way, other MAPK cascades are involved in phytohormone response. For example, the OsMAPKKK62-OsMAPKK3-OsMAPK7/14 and OsMAPKK10-2-OsMAPK3 cascades are associated with ABA signal transduction, regulating rice seed dormancy [24,28]. OsMAPKKK1 acts as a positive regulator in ABA and ET signaling pathways, while as a negative regulator in JA and SA signaling pathways [12][13][14][15], implying that OsMAPKKK1 probably interacts with different proteins or phosphorylates different downstream OsMAP-KKs to play roles in diverse phytohormone signaling pathways. Similarly, OsMAPK4 positively regulates the accumulation of JA and SA, and OsMAPK17-1 positively regulates the accumulation of SA, while OsMAPK16 negatively regulates the accumulation of JA and SA, and OsMAPK20-5 negatively affects the synthesis of ET [40,43,48,49]. These data demonstrate that rice MAPK cascades regulate or involve in complex phytohormone accumulation, signaling pathways or response ( Figure 4).

Complex Substrates of Rice MAPK Cascades
MAPK cascades play roles relying on phosphorylating a variety of downstream substrates, which include transcription factors, chromatin remodeling factors, kinases or other enzymes, and other proteins. So far, nine substrates for OsMAPK3, six substrates for OsMAPK6, one substrate for both OsMAPK4 and OsMAPK7, two substrates for both OsMAPK14 and OsMAPK17-1 have been identified and functionally characterized. Of which majority of substrates are composed of transcription factors (TF), such as WRKY or bHLH, few of them are kinase or other proteins (Table 2). For example, OsMAPK3 phosphorylates OsCDPK18 and OsRAI1 to confer rice resistance to fungal pathogen M. oryzae [38,64], acts on OsWRKY30 to confer resistance to bacterial pathogen Xoo [69], while phosphorylates OsbHLH002, OsZFP213, SUB1A1 and OsWRKY30 to alter stress tolerance, such as cold, salt, submergence and drought, respectively [66][67][68][69]. Occasionally, an OsMAPK could phosphorylate different substrates to regulate the same signaling pathway or play the same roles. OsMAPK3 phosphorylates kinase OsCDPK18 and TF OsRAI1, totally improving rice resistance to M. oryzae [38,64]. In the same way, OsMAPK6 acts on three different TFs, OsWRKY53, OsWRKY45, and OsRAI1, to collectively trigger rice resistance to M. oryzae [29,64,65]. Reversely, different OsMAPKs target the same substrate participating in the same physiological processes. For example, both OsMAPK3 and OsMAPK6 phosphorylate OsRAI1 to positively confer rice resistance to fungal pathogen M. oryzae [64], and phosphorylate OsWRKY70 to enhance rice resistance to herbivores, BPH and SSB [69,73]. OsMAPK3, OsMAPK7, and OsMAPK14 all can phosphorylate OsWRKY30 to promote rice resistance to bacterial pathogen Xoo and modulate rice drought tolerance [69,73]. These data suggest the complex relationships between OsMAPKs and their diverse substrates.

Conclusions
Tremendous progress has been made to decipher the multiple functions of rice MAPK cascades, with several complete MAPK cascades have been uncovered, including OsMAPKKK11/18/24-OsMAPKK4/5-OsMAPK3/6 cascades, OsMAPKKK10-OsMAPKK4-OsMAPK6 cascade, OsMAPKKK63-OsMAPKK1-OsMAPK4 cascade, and OsMAPKKK62-OsMAPKK3-OsMAPK7/14 cascades. However, due to over 98 MAPK genes in rice, a large number of them have not been functionally characterized. The gaps need to be filled, including which proteins target OsMAPKKKs, which OsMAPKKs can be phosphorylated by OsMAPKKKs, which OsMAPKs can be phosphorylated by OsMAPKKs, and which proteins can be subsequently phosphorylated by OsMAPKs. Previously, yeast two hybrid and in vitro phosphorylation assay are the main methods for MAPK substrates identification, while these two methods may produce false negatives and positives [1,60]. Thus, quantitative phosphoproteomic and immunoprecipitation-mass spectrometry methods have recently been used to identify protein kinase substrates and study the function of protein kinases [78,79]. Therefore, the combination of quantitative phosphoproteomic, immunoprecipitation-mass spectrometry, in vitro phosphorylation, and genetic assays would be alternative strategies to uncover the function of MAPKs and identify their substrates. Furthermore, the same MAPK cascade can sense and mediate different signal transduction, playing roles in diverse physiological processes. However, the underlying mechanisms that a MAPK cascade precisely activates and phosphorylates different downstream substrates after sensing different upstream signals are still unclear. Resolving these putative concerns would fully accelerate to elucidate the underlying molecular mechanisms and molecular functions of rice MAPK cascades.