Ribosomal Protein S6 Kinase Is Mediated By TOR/RAPTOR2 To Regulate Thylakoid Membrane Biogenesis And Plant Growth In Rice

Target of rapamycin(TOR)is a Ser/Thr protein kinase belonging to the phosphoinositide 3-kinase-related kinase family.In yeast and mammals,TOR forms two complexes,target of rapamycin complexl(TORC1)and TORC2,through interacting with different proteins.Mammal TORC1 consists of mTOR,Regulatory associate protein of TOR(Raptor)and lethal sec-13 protein8(mLST8).Raptor is a core component of TORC1 complex to recruit substrates to TORC1 for phosphorylation.TORC1 is a master regulator in modulating cell growth and metabolic process in response to nutrition,growth factors,energy supply,and stress.Ribosomal protein S6 kinase(S6K),the key downstream component of TORC1 signaling pathway,is involved in various biological processes such as ribosomal RNA synthesis,protein translation,cell cycle,transcriptional regulation,and nutrient transport.It was showed that loss of TOR,Raptor,or S6K resulted in embryonic lethality and growth arrest.The knowledge of TOR/Raptor/S6K in plants is still limited.It would be interesting to clarify the roles of TOR/Raptor/S6K post-embryonic growth and development in plants.Plants are autotrophic organisms,the regulation and molecular mechanism in growth and development may be distinguishable from yeast and animals.To determine the roles of rice TOR/Raptor/S6K in plant growth and development,multiple approaches such as molecular manipulation,protein expression,mutation,RNA silence,protein interaction,metabolic analysis,pharmacological treatment,cell ultrastructural observation,and transcriptional analysis were performed in this study.The results showed that TOR/Raptor2/S6K1 coordinately regulates thylakoid membrane biognesis,lipid homeostasis,and thylakoid grana architecture.The specific results in this study are as follows:Rice genome contains two S6Ks,S6K1 and S6K2,which is still unclear and yet to be clarified.To investigate the subcellular localization and enzyme activity of S6Ks,the S6Ks genes were amplified from rice.The expression level of S6K1 in the roots remained relatively consistent across various stages.By comparison,S6K1 transcript level in leaves was higher than that of S6K2 in all stages tested,and was highly expressed at the early stage,and then gradually decreased as plant development progressed.GFP-S6K1 was detected in both the cytoplasm and nucleus,whereas GFP-S6K2 was mainly localized in the nucleus.The distinct temporal and spatial distributions between S6K1 and S6K2 suggest the functional difference of the two S6Ks.Rice S6K1 purified by affinity chromatography indicated high activity toward a human ribosomal protein S6 consensus polypeptide(KRRRLASLR),suggesting that rice S6K1 encodes an active S6 kinase that is closely related to mammalian p70S6K.To elucidate the biological functions of rice S6K1 in postembryonic growth and development,a mutant designated s6kl,caused by a T-DNA insertion into the 5'-UTR of S6K1 was isolated.The S6K1 expression level in s6k1 plants was?20%of the WT level.The s6kl plants exhibited pale yellow-green leaves and growth arrest under normal growth conditions.The chlorophyll a content in the s6kl leaves was reduced by 29%.The photosynthetic rate in s6kl plants was only 27%that in WT plants.The contents of starch and soluble sugar in the leaves of s6k1 were significantly reduced to 57%and 85%of those of WT,respectively.Ultrastructural observation showed that the architecture of the thylakoid grana in the chloroplasts was defective in s6kl.The thickness of the cylindrical grana stacks in s6k1 was dramatically reduced,and the shape of the thylakoid grana was hardly visualized in the chloroplasts of s6kl,whereas the intact,tightly organized and much more lamellaes of thylakoid were detected in the chloroplasts of WT leaves during seedling stage.Instead of intact cylindrical grana stacks,18 plastoglobules on average were detected within each chloroplast in s6K1,whereas only-3 droplets per chloroplast were found in WT.The loss of well-organized thylakoid grana membrane in s6kl plants resulted in yellow pale-green leaves.These data suggest that S6K1 plays a positive role in thylakoid membrane biogenesis,thus enhances photosynthesis.Pull-down and CO-IP analysis showed than S6K1 can directly interact with Raptor2,and the activity of S6K1 was regulated by Raptor2.The raptor2 mutant exhibited pale or striped-pale leaves with substantially reduced chlorophyll content during the seedling and tillering stages.The pale leaves exhibited defective thylakoid grana structure in the raptor2 mutant,indicating S6K1 interact with Raptor2 to regulate thylakoid membrane biogenesis.To elucidate whether the involvement of Raptor2/S6K1 in thylakoid membrane biogenesis is regulated by TOR signaling,TOR activity indicated phosphorylated S6K level was suppressed by TOR specific inhibitors.The phosphorylated S6K was dramatically suppressed by TOR inhibitor,in a concentration-dependent manner,and leaves became pale yellow-green after TOR inhibitor treatment.Loss of TOR,Raptor2,or S6K led to pale leaf color and reduced chlorophyll content suggest that TOR,Raptor2,and S6K function in the same pathway to coordinately regulate thylakoid grana lamella architecture.Lipid profiling revealed that a significant reduction in galactolipids MGDG and DGDG,and a corresponding increase phospholipid in s6kl,raptor2 mutant or TOR suppressed plants,which is accompanied by a downregulation of MGD1,DGD1,and LPPa5 involved in galactolipid synthesis.These results demonstrate that TOR/Raptor2/S6K functions as upstream signaling to modulate the biosynthesis of thylakoid membrane,and the thylakoid grana modeling to ensure normal photosynthesis.Loss of S6K1 also caused the growth arrest.The tiller number,plant height,shoot dry weight,and thousand-grain weight in the s6kl mutant were significantly reduced than in WT.The pale leaves and the multiple defects could be rescused by the genetic compensation with S6K1 under its native promoter or maize ubiqutin promoter.The results suggest that rice S6K1 is important for normal green leaf and plant growth regulation.In addition,S6K1 also exhibited multiple defects in root growth,root hair development,pollen viability,and cells around vascular bundle in the s6k1 mutant.Moreover,S6K1 also interacts with E2F3 transcriptor involved in cell cycle regulation.The S5K1-E2F3 interaction promotes the nuclear localization of E2F3.