Study On Transport Protein-mediated Flavonoid Transport And Ferrum Homeostasis In Glycine Max And Medicago Truncatula

Secondary metabolites transport and nutrient elements homeostasis is not only the hotspot of plant biological research,but also the focus of stress-resistance and quality improvement in crop breeding.Flavonoid,comprises diversity compounds including isoflavonoids,anthocyanins,and proanthocyanins,is one of the most important secondary metabolites in plants,but its transport mechanism is poorly understood in legumes,especially in Glycine max.Legume plants such as soybean and Medicago truncatula defend against pathogens or rhizobia symbiosis by secreting specific isoflavones,either phytoalexins or genistein and daidzein,apoplasts at the infection site or root rhizosphere.However,the transport mechanism of root isoflavone secretions remains unclear.The seed coat of black soybean accumulates a lot of flavonoid pigments including anthocyanins and proanthocyanidins,previous studies focused on the genetic loci of pigmentation in soybean seed coat,only a few studies reported the transport mechanism of anthocyanins and proanthocyanidins or other flavonoid pigments in soybean.Ferrum is indispensable for plant life,generally,ferrum homeostasis is related to metabolites transport,but the mechanism of Fe homeostasis in legume is still elusive.Our research interests focus on the roles of transport proteins in flavonoid transport and metal homeostasis in legumes,main findings are as follows:By comparison of the ABCG transporters involved in either vacuolar sequestration or plasma membrane export of isoflavones in elicited Medicago and soybean roots by phylogenetic and RNA-Seq analysis,we identified a subgroup of ABCG transporters putatively responsible for genistein and daidzein transport upon fungal elicitor treatment,we identified a subgroup of ABCG transporters putatively responsible for genistein and daidzein transport upon fungal elicitor treatment.A GmABCG18 was found to be involved in the root exudation of isoflavones and required for nodulation.GmABCG18 was highly expressed in the roots and up-regulated by fungal elicitor treatment or rhizobial infection.Overexpression or knockdown of GmABCG18 in soybean hairy roots not only enhanced and reduced the root secretion of genistein and daidzein,respectively,but also resulted in an increased and reduced nodule number in their corresponding transgenic chimeric plants,respectively.GmABCG18 expression was immediately increased in response to rhizobia infection,together with up-regulation of early nodulation genes,whose transcripts were altered also in the GmABCG18-OE or-KD hairy root lines.Thus,GmABCG18 is an ABCG transporter involved in secretion of the nodule signaling isoflavones genistein and daidzein and plays important role in the soybean root-rhizobia interaction and subsequent nodulation.The study provides insight into the active soybean root excretion of signaling isoflavones by a plasma membrane ABCG transporter coupled with symbiotic nodule formation and nitrogen fixation.Three GSTs and two MATEs putatively related to anthocyanins or proanthocyanidins transport have been selected based on phylogenic analysis with other identified transport proteins.Their functions in flavonoids transport was characterized by over-expression of orthologous in Arabidopsis mutants.Three GSTs played distinct roles in flavonoid transport,GmGSTF8 not only saved phenotype deficiency due to the lack of proanthocyanidins in mature Arabidopsis seeds,but also restored the anthocyanins in Arabidopsis seedlings.This result indicates the function of GmGSTF8 is as same as TT19.However,GmGSTF11 can only patially improve the proanthocyanidins deficient in mature Arabidopsis seeds,although GmGSTF11 complemented the anthocyanins in Arabidopsis seedlings.Only a small number of anthocyanins were detected in GmGSTF13 overexpressed Arabidopsis mutants but GmGSTF13 is not capable of proanthocyanidins accumulation under sugar induction.Two MATE transporters GmMATE60 and GmMATE117 were also identified in this study and similar function in flavonoid transport of these two MATE transporters was conducted by over-expression in Arabidopsis mutants.Comparison of phenotype of immature seeds between MATE transporters over-expressed lines in Arabidopsis mutant fft and pab1 indicates that both of them were involved in anthocyanins accumulation in immature seeds.Ruthenium red staining indicated the mucilage layer surrounding seeds of mutant fft was absent or disrupted,seeds size of mutant fft and pab1 was also altered.Seeds harvested from double mutant pab1 showed irregular sharp(no study reported this before).The deficient phenotypes were saved by overexpression of GmMATE60 and GmMATE117 in mutants fft and pab1.In a word,both GSTs and MATEs from soybean are involved in flavonoids transport.This study provides insight into the roles of transport proteins involved in flavonoid pigments transport from Glycine max.The MATE transporter family comprises 70 members in the Medicago truncatula genome,and they play seemingly important,yet mostly uncharacterized,physiological functions.Here,we employed bioinformatics and molecular genetics to identify and characterize MATE transporters involved in citric acid export and Fe translocation.MtMATE69 is a citric acid transporter induced by Fe-deficiency.Overexpression of MtMATE69 in hairy roots altered Fe homeostasis and hormone levels under Fe-deficient or Fe-oversupplied conditions.MtMATE66 is a plasma membrane citric acid transporter primarily expressed in root.The mtmate66 mutant seedlings were chlorotic under Fedeficient conditions.MtMATE55 is involved in seedling development and iron homeostasis,as well as hormone signaling.The mtmate55 mutant had delayed development and chlorotic leaves in mature plants.Both knockout and overexpression mutants of MtMATE55 showed altered Fe accumulation and abnormal hormone levels compared to the wild type.This study reveals roles of MATE transporters in metal homeostasis and hormone signaling under various stresses.