Genome-Wide Identification of the MPK Gene Family and Expression Analysis under Low-Temperature Stress in the Banana

Mitogen-activated protein kinases (MAPKs and MPKs) are important in the process of resisting plant stress. In this study, 21, 12, 18, 16, and 10 MPKs were identified from Musa acuminata, Musa balbisiana, Musa itinerans, Musa schizocarpa, and Musa textilis, respectively. These MPKs were divided into Group A, B, C, and D. Phylogenetic analysis revealed that this difference in number was due to the gene shrinkage of the Group B subfamily of Musa balbisiana and Musa textilis. KEGG annotations revealed that K14512, which is involved in plant hormone signal transduction and the plant–pathogen interaction, was the most conserved pathway of the MPKs. The results of promoter cis-acting element prediction and focTR4 (Fusarium oxysporum f. sp. cubense tropical race 4) transcriptome expression analysis preliminarily confirmed that MPKs were relevant to plant hormone and biotic stress, respectively. The expression of MPKs in Group A was significantly upregulated at 4 °C, and dramatically, the MPKs in the root were affected by low temperature. miR172, miR319, miR395, miR398, and miR399 may be the miRNAs that regulate MPKs during low-temperature stress, with miR172 being the most critical. miRNA prediction and qRT-PCR results indicated that miR172 may negatively regulate MPKs. Therefore, we deduced that MPKs might coordinate with miR172 to participate in the process of the resistance to low-temperature stress in the roots of the banana. This study will provide a theoretical basis for further analysis of the mechanism of MPKs under low-temperature stress of bananas, and this study could be applied to molecular breeding of bananas in the future.


Introduction
MAPK cascades are conserved in eukaryotes, which play an important role in plant development [1][2][3] and the stress-response mechanism [4][5][6]. Upstream signals of MPKs include mitogen-activated protein kinase kinase (MAPKK, MKK, and MEK) and mitogenactivated protein kinase kinase kinase (MAPKKK, MKKK, and MEKK) [7]. In general, MPKs relate to a variety of kinases with similar functions, which diversify the signal transduction pathways in plants to meet the needs of plant growth and development in complex environments. MKKKs, MKKs, and MPKs are phosphorylated in the signal transduction pathways. MPKs are activated and transferred to the nucleus or cytoplasm [8], where they then promote the expression of transcription factors or phosphorylate downstream substrates [9]. Eventually, these phosphorylation processes enable plant cells to respond to external signals.

Colinearity Analysis of the MPK Gene Family
Collinear analysis of the whole genome was used to understand gene duplication events of the MPK gene family. Meanwhile, unknown base content, GC content, and gene density data were supplemented as references. Unfortunately, the Musa itinerans and Musa textilis genomes were not assembled to the pseudochromosome level, meaning only Musa acuminata, Musa balbisiana, and Musa schizocarpa were analyzed ( Figure 3). There

Colinearity Analysis of the MPK Gene Family
Collinear analysis of the whole genome was used to understand gene duplication events of the MPK gene family. Meanwhile, unknown base content, GC content, and gene density data were supplemented as references. Unfortunately, the Musa itinerans and Musa textilis genomes were not assembled to the pseudochromosome level, meaning only Musa acuminata, Musa balbisiana, and Musa schizocarpa were analyzed ( Figure 3). There

Colinearity Analysis of the MPK Gene Family
Collinear analysis of the whole genome was used to understand gene duplication events of the MPK gene family. Meanwhile, unknown base content, GC content, and gene density data were supplemented as references. Unfortunately, the Musa itinerans and Musa textilis genomes were not assembled to the pseudochromosome level, meaning only Musa acuminata, Musa balbisiana, and Musa schizocarpa were analyzed ( Figure 3). There were eight, six, and three segmental duplications and eight, zero, and three tandem

KEGG Annotation Analysis of the MPK Gene Family
To further verify the functional similarities and differences of the MPK gene fam functional annotations were made on their protein sequences (as shown in Figure 5). M of the MPKs were annotated to K04371, K14512, K20535, K20536, and K20600, and ot e f

KEGG Annotation Analysis of the MPK Gene Family
To further verify the functional similarities and differences of the MPK gene family, functional annotations were made on their protein sequences (as shown in Figure 5). Most of the MPKs were annotated to K04371, K14512, K20535, K20536, and K20600, and others e f

KEGG Annotation Analysis of the MPK Gene Family
To further verify the functional similarities and differences of the MPK gene family, functional annotations were made on their protein sequences (as shown in Figure 5). Most of the MPKs were annotated to K04371, K14512, K20535, K20536, and K20600, and others were not annotated. It is noteworthy that K14512 is the most conserved in bananas and existed in all species. K14512 is divided into three pathways, which plays an important were not annotated. It is noteworthy that K14512 is the most conserved in bananas and existed in all species. K14512 is divided into three pathways, which plays an important role in plant growth and development. These pathways contains plant hormone signal transduction (map04075), plant-pathogen interaction (map04626), and MAPK signaling pathway-plant (map04016).

Prediction Analysis of the MPKs Promoter Cis-Acting Elements
The results of promoter analysis showed that MPKs promoter cis-acting elements were related to plant hormones, the stress response, biological clocks, and plant development ( Figure 6). In terms of plant hormones, it mainly contained abscisic acid, auxin, gibberellin, methyl jasmonate, and salicylic acid response elements, which, to some extent, confirmed the conclusion that MPK participated in plant hormone signal transduction pathways in KEGG. The stress response is mainly involved in anaerobic induction, stress defense, drought inducibility, low-temperature responsiveness, and wound responsiveness. Elements of cell cycle regulation and circadian control response are related to the biological clock regulation system. Finally, endosperm expression, light responsiveness, meristem expression, seed-specific regulation, and zein metabolism regulation elements are involved in plant development.
Overall, the most responsive element in the promoters was light responsiveness, while the least responsive element was wound responsiveness. This result suggested that MPKs may mainly rely on light, abscisic acid, and the MeJA metabolic pathways, which was found to be similar across different species of bananas. However, wound responsiveness was only found in the promoters of MaMPK7 and MiMPK2 (Figure 6a,b), but not in the others (Figure 6c,d,e). Seed-specific regulation did not exist in Musa schizocarpa ( Figure  6d) but was found in all the others. In summary, the promoters of MPKs are both conserved and specific. A variety of promoter cis-acting elements could provide the MPKs with more functions.

Prediction Analysis of the MPKs Promoter Cis-Acting Elements
The results of promoter analysis showed that MPKs promoter cis-acting elements were related to plant hormones, the stress response, biological clocks, and plant development ( Figure 6). In terms of plant hormones, it mainly contained abscisic acid, auxin, gibberellin, methyl jasmonate, and salicylic acid response elements, which, to some extent, confirmed the conclusion that MPK participated in plant hormone signal transduction pathways in KEGG. The stress response is mainly involved in anaerobic induction, stress defense, drought inducibility, low-temperature responsiveness, and wound responsiveness. Elements of cell cycle regulation and circadian control response are related to the biological clock regulation system. Finally, endosperm expression, light responsiveness, meristem expression, seed-specific regulation, and zein metabolism regulation elements are involved in plant development.
Overall, the most responsive element in the promoters was light responsiveness, while the least responsive element was wound responsiveness. This result suggested that MPKs may mainly rely on light, abscisic acid, and the MeJA metabolic pathways, which was found to be similar across different species of bananas. However, wound responsiveness was only found in the promoters of MaMPK7 and MiMPK2 (Figure 6a,b), but not in the others (Figure 6c-e). Seed-specific regulation did not exist in Musa schizocarpa ( Figure 6d) but was found in all the others. In summary, the promoters of MPKs are both conserved and specific. A variety of promoter cis-acting elements could provide the MPKs with more functions.

Prediction of miRNA Regulation on MaMPKs
A total of 119 types of miRNAs regulating the MPKs were predicted by psRNATarge ( Figure 7). Interestingly, sixteen miRNAs were found to regulate three genes simultaneously. MaMPK3, MaMPK14, and MaMPK16 were regulated by miR5303; MaMPK14, MaMPK16, and MaMPK19 were regulated by miR395; MaMPK3, MaMPK14, and MaMPK19 were regulated by the other 14 miRNAs. Notably, the regulation of miR172 on MaMPK3, MaMPK14, and MaMPK19 was found to be the most significant. Therefore, three miR172 family members were screened from the banana miRNA database for expression analysis under low-temperature stress ( Figure 8). The results showed that mac-miR172a was significantly downregulated, while mac-miR172b and mac-miR172d were hardly affected by the low temperature. The expression level of mac-miR172a was about 4-fold higher than that of mac-miR172b and mac-miR172d at 28 °C. The expression of mac-miR172a was downregulated at 4 °C, indicating that mac-miR172a plays a major role in low-temperature stress. These results indicate that miR172 has a functional redundancy in bananas. In addition, miRNAs associated with cold stress, such as miR319, miR395, miR398, and miR399, were also predicted. This suggested that the regulatory mechanisms of miRNAs on MaMPKs were diverse.

Prediction of miRNA Regulation on MaMPKs
A total of 119 types of miRNAs regulating the MPKs were predicted by psRNATarge ( Figure 7). Interestingly, sixteen miRNAs were found to regulate three genes simultaneously. MaMPK3, MaMPK14, and MaMPK16 were regulated by miR5303; MaMPK14, MaMPK16, and MaMPK19 were regulated by miR395; MaMPK3, MaMPK14, and MaMPK19 were regulated by the other 14 miRNAs. Notably, the regulation of miR172 on MaMPK3, MaMPK14, and MaMPK19 was found to be the most significant. Therefore, three miR172 family members were screened from the banana miRNA database for expression analysis under low-temperature stress ( Figure 8). The results showed that mac-miR172a was significantly downregulated, while mac-miR172b and mac-miR172d were hardly affected by the low temperature. The expression level of mac-miR172a was about 4-fold higher than that of mac-miR172b and mac-miR172d at 28 • C. The expression of mac-miR172a was downregulated at 4 • C, indicating that mac-miR172a plays a major role in low-temperature stress.
These results indicate that miR172 has a functional redundancy in bananas. In addition, miRNAs associated with cold stress, such as miR319, miR395, miR398, and miR399, were also predicted. This suggested that the regulatory mechanisms of miRNAs on MaMPKs were diverse. The interaction network between miRNAs and MaMPKs. The yellow rectangles represent genes, and the other ellipses represent miRNAs. The orange and red ellipses represent miRNAs that regulate three genes simultaneously. The pink, blue, green, and purple ellipses represent miRNAs that regulate one gene. This interaction network was visualized using Cytoscape.

Expression Patterns of MPKs under Biotic and Abiotic Stress
The transcriptome FPKM data were used to analyze the expression patterns of MPKs, including pathogen stress and low-temperature stress (Figure 9). Thirteen genes were identified with low expression levels after 0, 1, 4, and 7 days of Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4) infection (Figure 9a), and their expression decreased with the extension of the infection time. The expression levels of the other MPKs were found to be higher than these 13 genes. Among them, the expression of MaMPK8 and MiMPK14

Expression Patterns of MPKs under Biotic and Abiotic Stress
The transcriptome FPKM data were used to analyze the expression patterns of MPKs, including pathogen stress and low-temperature stress (Figure 9). Thirteen genes were identified with low expression levels after 0, 1, 4, and 7 days of Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4) infection (Figure 9a), and their expression decreased with the extension of the infection time. The expression levels of the other MPKs were found to be higher than these 13 genes. Among them, the expression of MaMPK8 and MiMPK14

Expression Patterns of MPKs under Biotic and Abiotic Stress
The transcriptome FPKM data were used to analyze the expression patterns of MPKs, including pathogen stress and low-temperature stress (Figure 9). Thirteen genes were identified with low expression levels after 0, 1, 4, and 7 days of Fusarium oxysporum f. sp. The expression levels of the other MPKs were found to be higher than these 13 genes. Among them, the expression of MaMPK8 and MiMPK14 belonging to the Group B subfamily decreased most significantly. It is noteworthy that most of these MPKs are highly expressed. The expression of MiMPK12, MaMPK16, and MbMPK9 increased most significantly following pathogen induction. MsMPK11, MtMPK3, MiMPK2, MaMPK15, and MbMPK8 were downregulated from 0-1 d and upregulated from 1-14d. These results indicate that most MPKs can respond to focTR4, especially MiMPK12, MaMPK16, and MbMPK9, and proves that MPKs play a crucial role in resisting pathogen stress in bananas. In addition, MPKs may rely on a large signal transduction network to either directly or indirectly regulate the expression of disease resistance genes to resist pathogen infection. These results also prove the correctness of the KEGG annotations.
cially MiMPK12, MaMPK16, and MbMPK9, and proves that MPKs play a crucial role in resisting pathogen stress in bananas. In addition, MPKs may rely on a large signal transduction network to either directly or indirectly regulate the expression of disease resistance genes to resist pathogen infection. These results also prove the correctness of the KEGG annotations.
On the whole, about half of the MPKs were highly expressed under low-temperature stress, and only a few were lowly expressed (Figure 9b). We found that several MPKs were highly expressed at each temperature, but also that the expression of some MPKs was induced to increase through low temperatures. For example, the expression levels of MiMPK7, MaMPK20, and MsMPK15 were the highest at each temperature, while the expression levels of MiMPK12, MaMPK16, and MbMPK9 were increased after low temperature induction. Under low-temperature stress, a few genes were differentially expressed from 28 °C to 13 °C. However, the expression of all genes was found to be significantly different from 13 °C to 4 °C, and the situation was also similar from 4 °C to 0 °C. Interestingly, while the expression levels of MiMPK12, MaMPK16, and MbMPK9 were upregulated from 28 °C to 4 °C, they were downregulated at 0 °C in the high expression region. MiMPK15, MaMPK10, and MsMPK6 were found to be downregulated and upregulated at 0 °C in the low expression region. From 13 °C to 4 °C, the expression levels of all genes at 4 °C were markedly different from 13 °C, which indicates that for the banana plant, the key temperature of low-temperature stress was 4 °C.

The Expression Analysis of Group A under Low-Temperature Stress
Phylogenetic tree, KEGG annotation analysis, and transcriptome results all suggested that Group A may participate in cold resistance, resulting in our focus on MaMPK3, MaMPK14, MaMPK16, and MaMPK19, which belong to Group A in Musa acuminata. The On the whole, about half of the MPKs were highly expressed under low-temperature stress, and only a few were lowly expressed (Figure 9b). We found that several MPKs were highly expressed at each temperature, but also that the expression of some MPKs was induced to increase through low temperatures. For example, the expression levels of MiMPK7, MaMPK20, and MsMPK15 were the highest at each temperature, while the expression levels of MiMPK12, MaMPK16, and MbMPK9 were increased after low temperature induction. Under low-temperature stress, a few genes were differentially expressed from 28 • C to 13 • C. However, the expression of all genes was found to be significantly different from 13 • C to 4 • C, and the situation was also similar from 4 • C to 0 • C. Interestingly, while the expression levels of MiMPK12, MaMPK16, and MbMPK9 were upregulated from 28 • C to 4 • C, they were downregulated at 0 • C in the high expression region. MiMPK15, MaMPK10, and MsMPK6 were found to be downregulated and upregulated at 0 • C in the low expression region. From 13 • C to 4 • C, the expression levels of all genes at 4 • C were markedly different from 13 • C, which indicates that for the banana plant, the key temperature of low-temperature stress was 4 • C.

The Expression Analysis of Group A under Low-Temperature Stress
Phylogenetic tree, KEGG annotation analysis, and transcriptome results all suggested that Group A may participate in cold resistance, resulting in our focus on MaMPK3, MaMPK14, MaMPK16, and MaMPK19, which belong to Group A in Musa acuminata. The four genes in the genome of Musa acuminata was expressed in the roots, pseudostems, and leaves (Figures 10 and 11). These results showed that the expression levels of MaMPK3 and MaMPK19 were upregulated at 4 • C for 24 h. It also indicated that MaMPK3 and MaMPK19 were more sensitive to low temperatures. In contrast, the expression of MaMPK14 did not change significantly in the pseudostems and leaves but was upregulated in the roots. The expression of MaMPK16 decreased in the pseudostems and leaves while it was increased in the roots. The expression level in the roots was significantly enhanced. The expression levels of MaMPK3 and MaMPK19 were more intense compared to the other genes, which were upregulated about 20-fold and 35-fold compared with the control group, respectively. The expression levels of MaMPK14 and MaMPK16 in the roots increased about 4-fold. Together, these results indicate that the regulatory mechanisms of MPKs under low-temperature stress are diversified in bananas. four genes in the genome of Musa acuminata was expressed in the roots, pseudostems, and leaves ( Figures 10 and 11). These results showed that the expression levels of MaMPK3 and MaMPK19 were upregulated at 4 °C for 24 h. It also indicated that MaMPK3 and MaMPK19 were more sensitive to low temperatures. In contrast, the expression of MaMPK14 did not change significantly in the pseudostems and leaves but was upregulated in the roots. The expression of MaMPK16 decreased in the pseudostems and leaves while it was increased in the roots. The expression level in the roots was significantly enhanced. The expression levels of MaMPK3 and MaMPK19 were more intense compared to the other genes, which were upregulated about 20-fold and 35-fold compared with the control group, respectively. The expression levels of MaMPK14 and MaMPK16 in the roots increased about 4-fold. Together, these results indicate that the regulatory mechanisms of MPKs under low-temperature stress are diversified in bananas.

Interspecific Specificity and Genome Quality Together Determine the Number of MPKs in Bananas
An interesting phenomenon was that the number of Group B species was associated with the species themselves, while Group A, C, and D were almost unaffected by the species. The gene shrinkage of the Group B subfamily of Musa balbisiana and Musa textilis was the reason why their MPKs were significantly less than Musa acuminata, Musa schizocarpa, and Musa itinerans. Combined with the results of collinearity analysis, Musa balbisiana did not display many tandem duplications in Chr04: 3-10 MB, which was different from Musa acuminata and Musa schizocarpa. The results showed that the number of MPKs was contracted when Musa balbisiana experienced WGD (whole genome duplication) three times [23,24]. However, since the genomes of Musa itinerans and Musa textilis have not been assembled to the pseudochromosome level in this study [25,26], they were unable to verify the tandem duplication events in Chr04: 3-10 MB. This inference needs to be proven with the aid of more detailed genomic data of Musa itinerans and Musa textilis. Therefore, it is also an interesting research direction to further verify the differences of the MPKs in bananas through designing genetic complementation experiments for the genes in Chr04: 3-10 MB.
Genome-wide identification results showed that there were differences in the number of MPKs in bananas. These differences were closely related to the species, genome sequencing quality, and assembly quality. Five chromosomes achieved a complete telomere-to-telomere assembly of Musa acuminata for long-term improvement and optimization of the genome [27][28][29]. A high-quality genome typically indicates that more complete genes have been discovered, which may be the reason why the number of MPKs in Musa acuminata was found to be more than the others. The Musa acuminata and Musa schizocarpa genomes were assembled based on nanopore sequencing [30], which had higher continuity and integrity than the Musa balbisiana genomes. Overall, the assembly quality of Musa acuminata, Musa balbisiana, and Musa schizocarpa was better than of Musa itinerans and Figure 11. The expression of MaMPKs in different tissues. Red represents high expression and white represents low expression. These cartoon heat maps were visualized using TBtools.

Interspecific Specificity and Genome Quality Together Determine the Number of MPKs in Bananas
An interesting phenomenon was that the number of Group B species was associated with the species themselves, while Group A, C, and D were almost unaffected by the species. The gene shrinkage of the Group B subfamily of Musa balbisiana and Musa textilis was the reason why their MPKs were significantly less than Musa acuminata, Musa schizocarpa, and Musa itinerans. Combined with the results of collinearity analysis, Musa balbisiana did not display many tandem duplications in Chr04: 3-10 MB, which was different from Musa acuminata and Musa schizocarpa. The results showed that the number of MPKs was contracted when Musa balbisiana experienced WGD (whole genome duplication) three times [23,24]. However, since the genomes of Musa itinerans and Musa textilis have not been assembled to the pseudochromosome level in this study [25,26], they were unable to verify the tandem duplication events in Chr04: 3-10 MB. This inference needs to be proven with the aid of more detailed genomic data of Musa itinerans and Musa textilis. Therefore, it is also an interesting research direction to further verify the differences of the MPKs in bananas through designing genetic complementation experiments for the genes in Chr04: 3-10 MB.
Genome-wide identification results showed that there were differences in the number of MPKs in bananas. These differences were closely related to the species, genome sequencing quality, and assembly quality. Five chromosomes achieved a complete telomereto-telomere assembly of Musa acuminata for long-term improvement and optimization of the genome [27][28][29]. A high-quality genome typically indicates that more complete genes have been discovered, which may be the reason why the number of MPKs in Musa acuminata was found to be more than the others. The Musa acuminata and Musa schizocarpa genomes were assembled based on nanopore sequencing [30], which had higher continuity and integrity than the Musa balbisiana genomes. Overall, the assembly quality of Musa acuminata, Musa balbisiana, and Musa schizocarpa was better than of Musa itinerans and Musa textilis. According to the current genomic data, the number of MPKs is typically determined through interspecific specificity and genome quality. In future studies, more advanced genome sequencing techniques will be used to obtain high-quality genomes. It is an essential subject to completely eliminate the interference of sequencing and assembly techniques, which will not only benefit the comprehension of the influence of interspecific differences on the number of MPKs but will also facilitate the bioinformatics analysis and functional verification of the key genes. This will make outstanding contributions to the breeding of bananas using molecular biotechnology.

MPKs Retain the Most Critical Functions during Evolution in the Banana
A large number of studies have shown that Group A and B are involved in abiotic stress and biotic stress [31][32][33]. The number of K14152 pathways is roughly equal across all species of bananas, and all genes of K14152 belong to the Group A subfamily. Notably, the K14152 is closely associated with plant hormone signal transduction and the plant-pathogen interaction. In terms of plant immunity, the resistance of rice blast can be enhanced using the key immune component OsWRKY31 of the OsMPK cascade [34]. AtMPK3/6 can be phosphorylated through activating the extracellular ATP receptor P2K1, which improves the defense ability of plants to pathogens [35]. AtMPK3/6 is involved in the regulation of plant immunity by SUMO (small ubiquitin-related modifier protein) and RNA-binding proteins [36]. In addition, the immune response of mechanosensory trichome cells can be activated through AtMPK3/6 [37,38].
MPKs play a vital role in plant immunity depending on MAPKKK3/MAPKKK5-MKK4/ MKK5-MPK3/MPK6 [39,40]. The interaction between upstream and downstream genes forms a huge signal transduction network and regulates gene expression. MPKs associate with plant hormone synthesis, transport, and signal transduction [41]. The MKK4/5-MPK6 pathway regulates the polar auxin transport in Arabidopsis thaliana [42]. AtMPK3/6 can phosphorylate ACS in the ethylene synthesis pathway and participate in the process of response to abiotic stress or pathogen infection [43]. LeMPK1 and LeMPK2 are involved in the JA synthesis pathway to enhance tomato resistance to pests and diseases [44]. Similarly, AtMPK3/6 participates in the immune response in the SA signal transduction pathway [45]. The promoter cis-acting elements showed that 76.62%, 25.97%, 88.31%, and 35.06% of the members contained the ABA, Aux, MeJA, and SA response elements, respectively; it indicated that MaMPKs were closely related to plant hormones. In conclusion, K14152 is the most conserved entry in MaMPKs, and it retains the plant hormone signal transduction pathway and the plant-pathogen interaction pathway under long-term evolution and natural selection. Promoter cis-acting element prediction and transcriptome data can preliminarily prove that MPKs play an important role in plant hormone signal transduction and in the plant-pathogen interaction in bananas.

MaMPKs Are Negatively Regulated by miR172 in Low-Temperature Stress
In this study, the expression levels of MaMPK3, MaMPK14, MaMPK16, and MaMPK19 were significantly upregulated under low-temperature stress. In contrast, mac-miR172a was downregulated, but there was no significant change in the expression levels of mac-miR172b and mac-miR172d. This indicated that mac-miR172a negatively regulates MaMPKs. The ICE1-CBF-COR pathway has been widely studied in the cold resistance of bananas. It found that the cold resistance of Dajiao (Musa spp. ABB Group) was enhanced after MpICE1 overexpression, while weakened after RNA interference with MpMAPK3 [46,47]. It indicated that the MAPK cascade played a positive regulatory role in the cold resistance network of bananas.
A noteworthy phenomenon was that the expression of MaMPKs in the leaves was at its highest at 28 • C, and in the roots was highest at 4 • C. The roots were most markedly affected by low temperature. A previous study found that roots improved the ability of the stem cell niche to resist low-temperature stress through auxin accumulation and programmed cell death [48]. AtMPK6 and its substrate EB1c (end-binding protein 1c) regulated the mitosis of root epidermal cells in Arabidopsis [49]. The RGF1-RGI1 ligand-receptor complex regulated downstream PLT1 and PLT2 through YDA-MKK4/5-MPK3/6 and then regulated root meristem development, which has been demonstrated by researchers from different perspectives [50,51]. The stem tip of Arabidopsis thaliana converts high-temperature signals into biochemical signals and transmits them to the roots to stimulate the response of the whole plant [52]. From what has been discussed above, MaMPKs expression is activated through cold stress and may mainly act on the roots. Therefore, it has been speculated that leaves may transmit low-temperature signals to the roots through the connection between aboveground and underground or the signal transduction pathways. In this way, the roots can provide nutrients or water for the overground to alleviate cold stress.

Plant Materials
The test-tube plantlet of 'Brazilian' (Musa AAA, Cavendish) was placed at 28 • C for 5 days and were then transplanted into the substrate. The cultural conditions were as follows: temperature 26 ± 2 • C, relative humidity 60-80%, light intensity 2500 Lux, and 12 h light/12 dark. Bananas were moved into the incubator at 4.0 ± 0.5 • C for 24 h, and the other conditions were the same as above. The leaves, pseudostems, and roots were quickly cut into pieces, frozen in liquid nitrogen, and then stored at −80 • C.

Identification of MPK Gene Family in Bananas
The genomic data of Musa acuminata, Musa balbisiana, Musa itinerans, Musa schizocarpa, and Musa textilis were downloaded from the Banana Genome Database [53] (https:// banana-genome-hub.southgreen.fr/content/download, accessed on 15 April 2022). The gene and amino acid sequences of AtMPKs were downloaded from the TAIR database (https://www.arabidopsis.org/index.jsp, accessed on 21 April 2022). The candidate gene sequences of MPKs were screened using BLAST. The CDD database (https://www.ncbi. nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 22 April 2022) was used to screen the domains, and the genes containing the STKc_TEY_MAPK and STKc_TDY_MAPK domains were identified as the MPK gene family in bananas. Finally, Tbtools [54] was used to visualize the domains.

Protein Physicochemical Properties and Domain Analysis of MPKs
ExPAsy (https://web.expasy.org/protparam/, accessed on 28 April 2022) was used to predict the number of amino acids, molecular weight (Mw), theoretical pI, instability index, aliphatic index, and the grand average of hydropathicity (GRAVY) of the MPK proteins.

Phylogenetic Tree, Collinearity and Chromosome Localization Analysis
The phylogenetic tree containing Arabidopsis thaliana, Musa acuminata, Musa balbisiana, Musa itinerans, Musa schizocarpa, and Musa textilis was constructed through neighbor-joining of MEGA-X and beautified with iTOL (https://itol.embl.de/, accessed on 2 May 2022). Based on the gene location in the genomes, collinearity analysis was shown using Tbtools.

KEGG Annotation Analysis and Promoter Cis-Acting Element Prediction
The protein sequences of MPKs were uploaded to KofamKOALA (https://www. genome.jp/tools/kofamkoala/, accessed on 29 August 2022) to predict their KO numbers in KEGG (Kyoto Encyclopedia of Genes and Genomes). The upstream 2000 bp sequences of the CDS were extracted for promoter cis-acting element prediction with Plant CARE (http: //bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 29 April 2022).

Expression Analysis of MPKs and Prediction of miRNA
FPKM (fragments per kilobase million) values of MPKs were extracted from focTR4 [55] and low temperature [56] transcriptome data, which were displayed using Tbtools. The psRNATarget [57] (https://www.zhaolab.org/psRNATarget/, accessed on 12 November 2022) was used to predict the miRNA that regulates the MPKs, and then Cytoscape 3.8.2 was used to draw the interaction network between the miRNAs and genes.

RNA Extraction and qRT-PCR
Total RNA was extracted with the RNAprep Pure Plant Plus Kit (TIANGEN, Beijing, China). The RNA reverse transcription used the 1st strand cDNA synthesis superMix for qPCR (Yeasen, Shanghai, China). The primers for qRT-PCR have been listed in Table S2. The reaction system was as follows: 10 µL qPCR SYBR green master mix (Herui, Fuzhou, China), 0.4 µL forward primer (10 µM), 0.4 µL reverse primer (10 µM), 1 µL cDNA, and 8.2 µL ddH 2 O. The CAC was used as a reference gene. The reverse transcription of miRNAs used the 1st strand cDNA synthesis Kit by stem-loop (Vazyme, Nanjing, China). The sequences of miR172 were derived from NCBI [58] (https://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE77590, accessed on 12 December 2022). The miR172 reverse transcription and qRT-PCR primers have been displayed in Table S2. The reaction system was as follows: 10 µL 2 × miRNA Universal SYBR qPCR master mix (Vazyme, Nanjing, China), 0.4 µL specific primer (10 µM), 0.4 µL mQ primer R (10 µM), 1 µL cDNA, and 8.2 µL ddH 2 O. The qPCR programs were as follows: 95 • C for 5min, 40 cycles of 95 • C for 10 s, and 60 • C for 30 s. The U6 was used as a reference gene. The relative expression levels of MaMPKs and miRNAs were calculated using the 2 −∆∆Ct . SPSS26 was used to analyze the significance test between samples. The instrument used for this experiment was the Roche LightCycler 480.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12162926/s1, Figure S1: Protein conserved domains of MPKs in bananas, Table S1: Basic information and protein physicochemical properties of the MPK family in bananas, Table S2: qRT-PCR and reverse transcription primers of MPKs and miRNAs, Table S3: annotation of MPKs in KEGG.