Effect Of Symbiotic Nitrogen Fixation On Flowering Time And Its Regulation Mechanism In Soybean

Under nitrogen-limiting conditions,legume crops interact with soil nitrogen-fixing rhizobia to produce specialized root organs called nodules.Bacteria in nodule cells convert atmospheric nitrogen into ammonia that can be used by plants.It has been assumed that fixed nitrogen to promote legume growth and increase crop yield.However,it remains unknown whether there are other symbiotic signals directly involved in the regulation of legume growth and development besides nitrogen.Like other crops,the precise timing of flowering time is critical for successful reproduction and seed yield in legumes.It is well-known that the number of nodules and the amount of fixed nitrogen in a nodulated soybean plant reach a peak at the flowering stage.Theoretically,symbiotic nitrogen fixation（e.g.,nodulation and nitrogen supplies）and developmental timing（e.g.,flowering time）interwine to affect plant reproductive growth,which determines the yield and quality of soybean.Whether and how symbiotic nitrogen fixation are coordinated with developmental timing remain to be uncovered.In this work,we first conducted experiments with four distantly related legumes,including soybean to determine whether and how symbiotic nitrogen fixation influences growth and development of legumes.We found that rhizobia inoculation significantly accelerated flowering in legumes,which is a common phenomenon.Further experiments showed that nitrogen can promoted the flowering time of uninoculated plants,suggesting that soybean has a nitrogen-controlled flowering pathway.Next,we inoculated DN50with a mutant Sinorhizobium fredii CCBAU45436 mutant of pts P(encoding EI^Ntr)-Δpts P strain that formed ineffective nodules and failed to fix nitrogen,and found that these plants also flowered earlier than the uninoculated control plants.These results strongly suggest that there is a symbiosis-specific regulatory pathway of flowering time in addition to the nitrogen of flowering pathway of nodulated soybean plants.Next,we investigated whether symbiosis-specific micro RNA-micro RNA172c（mi R172c）produced in infected roots and nodules directly links symbiotic nitrogen fixation and flowering,given that mi R172 is a central regulator of age-flowering time in plants.We quantified the temporal and spatial expression changes of mi R172c in uninoculated plants with different levels of nitrogen and in plants inoculated with wild-type rhizobia and mutantΔpts P strains under low nitrogen conditions.The results showed that:the expression pattern of symbiotic-specific mi R172c in root was correlated with the abundance of mi R172c in leaves and flowering time;symbiotic specific pathway-induced mi R172c and nitrogen-induced mi R172c together led to increased abundance of mi R172c in leaves and earlier flowering;and symbiotic-induced high levels of mi R172c in leaves was not due to increased transcription.Further combinatorial approaches confirmed that mi R172c produced during symbiotic nodulation can be transmitted from roots to leaves as a long-distance signal to promote flowering.Further gene expression and biochemical analyses showed that TARGET OF EAT1-like 4a（Gm TOE4a）is the main target of mi R172c in nodulation-accelerated flowering.Under low nitrogen conditions,the abundance of mi R172c in the leaves of uninoculated plants remained at a stable low level,and Gm TOE4a directly bound to the promoter of Gm FT2a/5a and inhibiting its expression,resulting in delayed flowering;while soybeans were inoculated with rhizobia,the expression of mi R172c in leaves was induced,thereby reducing the expression of the target gene Gm TOE4a and relieving its transcriptional inhibition of Gm FT2a/5a,and promoting flowering.We also analyzed the mi R172expression patterns in L.japonicus and M.sativa and found that symbiotic-induced mi R172a were also correlated with the mi R172 peaks in leaves and the timing of flowering of inoculated L.japonicus and M.sativa plants,suggesting that the mi R172-centered nodulation and nitrogen flowering pathway are conserved in legumes.In summary,this study discovers a novel pathway of symbiotic nitrogen fixation to promote flowering in legumes.Our findings show that mi R172c levels are systematically up-regulated in leaves by rhizobial symbiosis and fixed nitrogen.Elevated levels of mi R172c in the leaf reduce transcription repreression of Gm TOE4a on both Gm FT2a and Gm FT5a,and increased levels of the florigen Gm FT2a and Gm FT5a subsequently trigger floral initiation and the developmental transition.Our findings expand our understanding of genetic basis of plant flowering regulation,and provide novel insight into breeding new legume crops with high-efficiency nitrogen fixation and high-yield.