Identification And Functional Analysis Of Light-Induced MiRNAs Associated With Glycoalkaloids Metabolism In Potato

Steroidal glycoalkaloids?SGAs?are secondary metabolites in solanaceae and some liliaceae plants.Although SGAs have diverse function such as plant resistance against pathogen and pest attacks,resistance to osmotic stress and allelopathic effect,accumulation of SGAs in tuber can influence the food flavor and safety in the tuber of Solonum tuberosum L.To enhance stress tolerance in potato plants and meet food safety requirements,it is viral to breed cultivars with low SGAs content in tuber and high content in leaf.The SGAs biosynthesis are determined by the complex gene regulatory network.Some genes are frequently transcriptionally coregulated and existed as a cluster on chromosome.But current knowledge on post-transcriptional gene regulatory mechanism of steroidal-alkaloids genes is still limited.As we know,accumulation of steroidal glycoalkaloids are sensitive to red light stimulus.Therefore,the cultivated and wild potato was studied under darkness and red light condition.To determine miRNAs associated with SGAs biosynthesis pathway,light responsive miRNAs and biological functions of targets were analyzed by small RNA-Seq,RNA-Seq and degradome-Seq.In addition,key miRNAs were overexpressed in green tissues of potato to modulate SGAs spatial distribution.The main results as follows:1.In cultivated potato?S.tuberosum,2n=4X=48?,we identied 277 known and 120 novel miRNAs.Under red light stimulus,a total of 31 and 48 miRNAs were identified to be differentially expressed in the leaf and tuber,respectively.The RNA-seq analysis indicated that a total of 1353 and 1841 genes were upregulated in the leaf and tuber,while 1595 and 897 genes were downregulated by light in the leaf and tuber respectively.q RT-PCR analysis showed that the data of sRNA-seq and RNA-seq is reliable.2.In the tuber,biological process analysis showed that enrichment of GO terms were related to ‘secondary metabolism',‘heterocycle biosynthesis',‘tetrapyrrole metabolism',‘chlorophyll biosynthetic process' and ‘isoprenoid biosynthesis'.In the leaf and tuber,Mapman enrichment analyses showed that genes associated with major CHO metabolism were repressed,while genes related to secondary metabolism were upregulated.Moreover,genes related to the first step of SGAs biosynthesis?MVA/ isoprenoid pathway?were significantly upregulated.Two larger families of enzymes,cytochromes P450 and UDP-dependent glycosyl transferases were obviously altered in the leaf and tuber.Furthermore,continuous illumination also upregulated the transcription levels of steroidal alkaloid genes.3.Integrated miRNAs and m RNAs profiles revealed that a total of 41 and 73 inversed DE-miRNA/m RNA modules were regulated by red light,respectively.In the leaf,lightresponsive miRNAs are important regulators in glucan catabolism,jasmonic acid-mediated signaling pathway and UMP salvage.For tuber,transcripts were enriched at carbohydrate metabolism,NAD?P?H dehydrogenase complex assembly,sugar-mediated signaling pathway and nc RNA processing.Integrated analysis showed that several miRNAs appear to target b ZIP and b HLH transcription factors in alkaloids metabolism.In this study,upregulated miR482b-3p appears to target uridine kinase?uridine kinase appears to increase UDP-glucose content and UDP-glucose is glycoside donor for SGT2?.miR479 appears to be target Cytochrome P450?CYP71D7?which may participate in glycoalkaloid biosynthesis.4.Solanum.chacoense?2n=2X=24?is a diploid wild potato species which contains high contents of SGAs,and appears to be rich in disease resistance,insect resistance and stress tolerance genes.Under red light stimulus,we identified 187 known miRNAs and 163 p5/p3 miRNAs in Group 1 by high-throughout sequencing.In addition,204 known miRNAs and 220 p5/p3 miRNAs were identified in Group 2.Among these families,24 highly conserved miRNA families were identified.The largest conserved family was miR156.Moreover,a total of 337 unique mature miRNAs were identified in three libraries.The result of K-means cluster indicated that light responsive-miRNAs can be categorized into nine temporally related clusters?designed k1-k9?.And these nine temporal clusters have four distinct expression patterns as follows: consistently upregulated miRNAs?k2,k3,k4,k5?,consistently downregulated miRNAs?k1,k8,k9?,early upregulated miRNAs?k6?and early downregulated miRNAs?k7?.The result of k-means cluster indicated that specific miRNAs can be dynamic regulated by light,suggesting these miRNAs may be involved in various biological process.5.In total,1319 unique target transcripts cleaved by miRNAs were identified using degradome-Seq.GO enrichment revealed that more GO terms were found to be enriched in the TD24 and TD72 degradome libraries.In TD24 library,some biological processes were associated with ‘apoptotic process',‘two-component signal transduction system',‘defense response' and ‘chromatin remodeling'.In addition,GO terms associated with ‘apoptotic process',‘potassium ion transport',‘riboflavin biosynthetic process' and ‘steroid metabolic process' were over-represented in the TD72 library.6.Taken together,combined sRNA-Seq and degradome-Seq analysis demonstrated that the ability of light-responsive miRNAs to modulate transcript abundance of MYB4,HSPs and EBF1/EBF2 may get their function in cross-tolerance responses to abiotic and biotic stresses.In addition,we identified light-responsive miRNAs and target transcripts that were associated with primary and secondary metabolic pathway.We identified the homology genes of CPO and CHLP targeted by stu-miR390-5p and ath-miR8175_L-2₁ss12AG in the light stimulus,respectively.A MYB4 transcription factor was detected to be targeted by stu-miR8033-3p_L-1₁ss17AG,indicating that this miRNA might be involved in flavonol biosynthesis.Furthermore,several light-responsive miRNAs were identified to cleave target transcripts related to steroidal glycoalkaloid/steroid biosynthesis.isomiRs of ath-miR8175/gma-6300 family could cleave target transcripts AMP-activated protein kinase can inhibit isoprenoid synthesis by phosphorylation of HMG-Co A reductase.miR156-targeted SPLs appear to be involved in sequiterpenes biosynthesis.The 3-beta hydroxysteroid dehydrogenase/isomerase family gene related to steroid biosynthesis was targeted by stu-miR482 c.In addition,we identified steroidal glycoalkaloid/steroid biosynthesis genes Cytochrome P450 and CYP72A58 targeted by stu-miR390-5p and ath-miR8175_L-2₁ss12AG,respectively.7.Based on the template p RSA-4 vector?derivative vector of p RS300?,we constructed 35 s promoter-drived plant expression vector p CE35s-miR408 and multiple cistronic vector p CE35s-dmiR408.Next,we got rbc S promoter-drived plant expression vector p CEr-miR408 and multiple cistronic vector p CEr-dmiR408.Internodal explants from L-3,L-6 and Favorita genotype was used for transformation by Agrobacterium-mediated genetic transformation.A total of 27?p CEr-miR408?and?p CEr-dmiR408?16 resistant plantlets were obtained by glyphosate selection.Next,we got 18 and 13 transformed plantlets by PCR-based selection,respectively.q RT-PCR analysis indicated that the relative expression level of miR408 in transformed plantlets?p CEr-miR408 and p CEr-dmiR408?was higher than untransformed plantlets.Whereas the relative expression level of target gene VS1 in transformed plantlets?p CEr-miR408 and p CEr-dmiR408?was lower than untransformed plantlets.Although miR408 and targets VS1 showed inversed expression pattern,the content of SGAs in transformed plantlets needs to be analysed and validated in the future.