Genome-wide Association Study Dissects Biosynthesis And Regulation Of Phenolamides And Their Uv-protection Role In Rice And Qingke

Radiation from the sun is a critical factor for the survival of plants and indeed all life on earth.However,the dependency of plants on sunlight also renders them exposed to UV light,including UV-B light(at a wavelength of 280-315 nm).This wavelength range leads to increased production of reactive oxygen species(ROS)and is documented to be damaging to DNA,RNA and proteins,eventually causing significant reduction in the biomass accumulation and yield of many plant species including our major food crops.To counter these UV effects,plants have adopted a range of important and sophisticated strategies,one of which is to increase the accumulation of UV-absorbing metabolites.Phenolamides(PAs),also referred to as hydroxycinnamic acid amides,are a diverse group of specialized metabolites found in many plants.As their chemical structures consist of several(one to four)benzene rings with strong UV absorption capacity,phenolamides are therefore regarded as UV-B phytoprotectants.In this study,we performed forward genetic analysis in the monocot model plant-rice and identified eight BAHD acyltransferases controlling the accumulation of different phenolamides,especially aromatic phenolamides.Our research demonstrated that aromatic PAs play positive role on enhancing UV-B tolerance for rice.Furthermore,our work focus on Tibetan hulless barley,also called “Qingke” in Chinese,which is a specific crop grown in Tibetan plateau.We characterized four BAHD acyltransferases and one MYB transcription factor involved in the biosynthesis and regulation of phenolamides,providing direct evidence on mechanism for how Qingke adapts to strong UV-B irradiation in Tibet.The results are listed as below.1.A total of 11 PAs,including six aliphatic PAs and five aromatic PAs were identified in rice leaf and grain.Metabolic profiling of these PAs using a worldwide collection of480 rice accessions showed that most of PAs display higher accumulation pattern in japonica than indica subspecies in both leaf and grain.A metabolite-based genome-wide association study(m GWAS)was performed using a worldwide collection of 480 rice accessions for 11 PAs.Meanwhile,a metabolite-based quantitative trait locus(m QTL)analysis was performed using a recombinant inbred line(RIL)population generated from Zhenshan 97(ZS97)and Minghui 63(MH63)for 6 PAs.m GWAS results showed that natural variation of agmatine derived PAs was controlled by one major locus on chromosome 4.Similar results were obtained from the content of variation of putrescine-derived PAs,in which another major locus on chromosome 9 was observed.In addition,two significant loci were detected for tryptamine and serotonin derived PAs.Two of these loci mapped by m GWAS were shared with the loci detected by m QTL.2.Combined in vitro enzyme assays with over-expression transgenic technology in rice,we identified eight BAHD acyltransferases catalyzing the biosynthesis of PAs.Four of them(Os THT1/2 and Os TBT1/2)were found to be bifunctional tryptamine/tyramine N-acyltransferases,playing the major role in the biosynthesis of aromatic PAs in rice.BAHD family of tryptamine/tyramine N-acyltransferases evolved conservatively in Gramineae,whereas no homologous protein was found in core Eudicotyledons.Four tandem arginine residues were identified to a key determinant for agmatine specificity of Os THT1 and Os TBT1.Natural variation analysis of Os TBT1/2 showed that four SNPs in the coding sequence of Os TBT1 and nine SNPs in the coding sequence of Os TBT2 were significant associated with the content of benzoyl tryptamine.Sequence alignments of Os TBT1/2 alleles between ZS97 and MH63 revealed a 7-bp deletion that results in a frame shift in Os TBT1 of MH63 allele,together with 19 non-synonymous SNPs that led to changes in amino acid polarity in Os TBT2 allele in MH63 could be responsible for the almost complete absence of benzoyl tryptamine and benzoyl serotonin in this accession.3.Both accumulations of aromatic PAs and transcription levels of their biosynthetic genes-BAHD acyltransferases were strongly induced by UV-B irradiation.Compared with wild type,transgenic rice over-expressing Os TBT2 showed significantly enhanced UV-B tolerance,possibly through the increased ability of ROS scavenging under the UV-B treatment.4.A total of eight PAs,including five aliphatic PAs and three aromatic PAs were identified in Qingke leaf and grain.Metabolic profiling on these PAs using 142 Qingke(hulless barley)accessions and 54 barley(hulled barley)accessions showed that most of PAs display higher accumulation pattern in Qingke than that of in barley in both leaf and grain.A metabolite-based genome-wide association study(m GWAS)was performed using a worldwide collection of 196 Qingke and barley accessions for 8 PAs.m GWAS mapping result showed that natural variation of feruloyl tyramine was controlled by three loci with large effects.Both in vitro enzymatic assay and tobacco transient expression system illustrated all of HvTHT1/2/3 function in utilizing tyramine as substrate and were therefore regarded as key BAHD acyltransferases catalyzing the biosynthesis of tyramine derived aromatic PAs in Qingke.Natural variation analysis on HvTHT3 revealed a 97-bp deletion in the second exon of this allele may lead to the lowest production of feruloyl tyramine among these accessions.5.Both a BAHD acyltransferase and a MYB transcription factor were identified for feruloyl tryptamine by m GWAS in Qingke study.As BAHD acyltransferase,HvACT exhibited tryptamine hydroxycinnamoyl transferase activity both in vitro and in tobacco.As MYB transcription factor,HvMYB up-regulates transcription levels of phenylpropanoid pathway genes and acyltransferase genes,thus playing a positive role in the biosynthesis of PAs in tobacco.Yeast one-hybrid showed HvMYB could bind to the promoter of Hv4CL1(4-hydroxycinnamoyl-Co A ligase).Our study provides phytochemical insights into the mechanism of plant adaptation to UV-B stress and a potential strategy of “metabolomic-assisted breeding” aiming at producing stress-tolerant plants.