Soil potassium deficiency had become an important factor affecting crop yield and quality. Therefore, how to make bananas use potassium resources in the soil more effectively was a problem that needs to be solved urgently. According to the laboratory previous data, we screened ‘Guijiao No.1’ and ‘Brazilian banana’ as low-K tolerant and low-K sensitive genotypes, respectively. The response difference between the two genotypes of bananas was first manifested at the physiological level. The low-K tolerant variety ‘Guijiao No.1’ had better plant height, biomass, K concentration and accumulation under low-K stress. Some abiotic stresses affected H+/K+-ATPase activity. The H+/K+-ATPase and Ca2+/Mg2+ -ATPase of ‘Guijiao No. 1’ in root that showed an extremely significant increase under low-K stress. The tolerance of banana varieties under low-K+ conditions may be related to enzyme activity Ye et al(2017).
The difference in genotype response was secondly manifested at the mRNA level. ‘Guijiao No.1’ (low-K tolerant) had more DEG responses under low-K+ conditions. The first three functions of ‘Guijiao No.1’ GO analysis mainly focus on cellular anatomical entity, catalytic activity, binding, etc. This was consistent with studies in tomatoes and Chinese yam [45, 46]. The results of KEGG showed that the first three types of pathways in ‘Guijiao No.1’ were phenylpropanoid biosynthesis (30), MAPK signaling pathway-plants (31), and protein processing in the endoplasmic reticulum (22). Phenylpropanoid compounds acted as biological signals for plants to sense external stimuli and participate in biotic and abiotic stress responses. In recent years, studies in Arabidopsis have shown that leucine-rich repeat sequences receptor protein kinases (RLK) were at least 600, most of which were involved in the growth and development of plants. The MAPK signal transduction pathway was located downstream of RLKs and played a central role in growth, development and biological and abiotic stress signaling . Protein processing in the endoplasmic reticulum could confer the function of protein signal transduction. DEGs in ‘Brazilian banana’ were involved in the MAPK signal pathway, plant hormone signal transduction, starch and sucrose metabolism, and other related pathways. Starch and sucrose metabolism were involved in cell wall biosynthesis, cell proliferation, cell expansion, nutrient accumulation, primary metabolism and hormone signal transduction.
MiRNA plays a role of post-transcriptional regulation through targeted shearing of target mRNA, and its role in regulating plant nutrient stress had been extensively studied. In this study, ‘Guijiao No.1’ showed more response differentially expressed miRNAs(DEMs). The first three GO classifications of the target genes of ‘Guijiao No.1’ and ‘Brazilian banana’ DEMs were the same, and they were binding, cell, and cell parts. The KEGG analysis of target genes showed that ‘Guijiao No. 1’ was mainly enriched in plant hormone signal transduction, while ‘Brazilian banana’ was enriched in metabolic pathways and tyrosine metabolism. Phytohormones were small molecular compounds produced in plants, but they could affect cell division, differentiation, elongation, germination and rooting, plant height, branching, flowering and fruiting, etc., and played an important regulatory role in regulating plant growth and development. The tyrosine metabolic pathway was the starting point for plants to produce a variety of natural compounds with diverse structures, such as tocopherol, plastoquinone, ubiquitin, betaine, salidroside, and benzyl isoquinoline alkaloids. Among them, tyrosine metabolites, tocopherols, plastoquinone and ubiquitinone were necessary for plant survival.
Based on the data of mRNA and miRNA, a miRNA-mRNA regulation network was constructed. In this regulation network, 4 miRNAs and 6 target mRNAs form 8 pairs of negative regulatory effects. Four known miRNAs and 11 genes formed 14 pairs of miRNA-mRNAs in the yam tuber regulatory network. Among them, novel_mir3 acted on the SPL17.The Squamosa Promoter-Binding Protein-Like (SPL) gene encoded a plant-specific transcription factor, which played a role in plant phase transition, flower and fruit development, plant configuration, gibberellin signaling, sporogenesis, and response to copper and mycotoxins important role. The feedback interaction of miR156-SPLs ran through the entire plant development process and may also exist in other plants. In Arabidopsis, many SPL genes were regulated by miR156 after transcription, and AtSPL9 in turn positively regulated the expression of the second miRNA miR172. Among them, miR160a-5p acts on ARF18. In salt-tolerant beet (Beta vulgaris) seedlings, miR160 acted on the target gene ARF17/ARF18 to cope with salt stress.MiR319_1 acted on the three target genes of GATA26, RAV1, and PCF6. GATA transcription factors were a type of transcription factors that were widely present in eukaryotes. They played an important role in the biological processes of plant light response regulation, chlorophyll synthesis, cytokinin response, and carbon and nitrogen metabolism. In addition, GATA also played an important role in plant response to stress, such as nitrogen stress, cold stress, drought stress, etc., which had been proved by research[1, 2, 42]. RAV-like proteins were a subclass of the AP2 protein family and played a key regulatory role in plant stress responses and hormone responses. TCP gene family members could be divided into two major branches: TCP-P (consisting of the PCF subfamily) and TCP-C (consisting of the CIN and CYC/TB1 subfamilies)[22, 25]. The interaction between the TCP-P branch and key proteins in the hormone signal transduction pathway was involved in the regulation of hormone signals such as gibberellin, cytokinin, abscisic acid, jasmonic acid, and auxin. MiR319a_1 acted on the three target genes of RAVI, PCF6 and GAMYB. Gamyb-like genes may mediated the response of GA signaling pathway during plant growth and flowering. Among all the negatively regulated miRNA-mRNA pairs, the expression difference of miR160a was the most significant. Its target gene ARF was involved in the auxin pathway and may respond to low-K stress by regulating root growth. Therefore, miR160a was selected as a key miRNA for further functional validation.
In order to verify the regulatory effect of miR160a on potassium, an overexpressing miR160a vector was constructed and a miR160a overexpressed Arabidopsis line was obtained. Compared with WT, the TG strain showed lower dry weight, shorter root length, and lower potassium content. This indicated that overexpression of miR160a made Arabidopsis more intolerant to low-K stress. The study founded that miR160a acted on three target genes of atARF10, atARF16, and atARF17. In our study, RT-qPCR was used to verify that the target genes of miR160a in Arabidopsis were atARF10 and atARF17. In Arabidopsis, ARF17 was involved in the auxin signal transduction pathway, affecting its embryo and root development, vegetative growth and reproductive growth. miR160 degraded the target genes ARF16 and ARF10 by cutting them, regulating root tip growth and geotropism, and overexpressing miR160c in Arabidopsis can cause root tip development defects. miR160a/b was a key regulator in potatoes, which affected the root structure of plants by cutting the mRNA of StARF10 and StARF16. That miR160 was involved in the formation of adventitious roots of apple rootstocks induced by auxin. miR160 directed soybean nodule development in soybeans. miR160-ARF18-mediated peanut (Arachis hypogaea L.) response to salt stress. In
Alfalfa(Medicago Sativa L.), overexpression of miRNA160a significantly inhibited root length. In apple (Malus pumila Mill.), the Mdm-miR160-MdARF17-MdHYL1 module regulated the development of adventitious roots to regulate the drought tolerance of apples. In peanuts, the down-regulation of miR160a may promote the growth of primary roots and lateral roots under potassium-deficient conditions. To analyze the differences in their genotype responses, and the key difference miRNA160a was screened out by constructing a miRNA-mRNA regulatory network.miR160a had been subjected to a preliminary gene function validation. The results showed that miR160a overexpression inhibited the growth and potassium absorption of transgenic Arabidopsis thaliana, and played an important regulatory role in potassium absorption. We further verified the target genes ARF10, ARF17, and ARF18 of miRNA160a in transgenic Arabidopsis thaliana by RT-qPCR, and found that the expression of ARF10 and ARF17 was severely inhibited. This indicated that miR160a may regulate potassium absorption by regulating ARF transcription factors to participate in the auxin metabolism pathway.
In conclusion, this present study demonstrated that miR160a had an important regulatory effect on banana low-K stress. Overexpression of miR160a inhibited the growth and development of Arabidopsis thaliana through the target gene ARF10/17, and reduced the low-K tolerance of Arabidopsis thaliana by inhibiting root growth. Although the complex regulation mechanism of miR160a in low-K stress was still unclear, our results provided a possible mechanism for miR160a-ARFs module-mediated regulation of low-K stress response, and provided a new insights into the molecular mechanism of banana tolerance to low-K stress.