| Ferredoxin(Fd)are widely exist in plants,animals,and microorganisms to participate in electron transfer.Fd in higher plants are classified into leaf-type and root-type.In addition,there is a class of Fd homologues with C-terminal extension named FdC.In the photosynthetic light reaction,leaf-type Fd receive electrons from PSI and transport these electrons to downstream Fd-dependent metabolic processes,such as chlorophyll metabolism,phytochrome synthesis,fatty acids synthesis,and several steps in the pathways for assimilation of carbon,sulfur,and nitrogen.This makes Fds crucial regulators of the distribution of photosynthetic electrons to downstream enzymes dependent on these electrons.5 Fd and2 FdC genes have been annotated in the rice genome.However,the molecular mechanism of Fd participation in photosynthetic electron transport in rice and the effect of Fd on rice growth and development are not yet fully understood.Further research on the molecular mechanism of rice Fd participate in photosynthetic electron transport is helpful to deepen the understanding of the molecular mechanism of photosynthesis and then lay a theoretical foundation for the use of photosynthesis to provide more clean and sustainable energy and photosynthetic products for human beings through artificial synthetic biology and other methods.Our previous study showed that FdC2 plays an important role in rice heading date and development.This study provides multiple lines of evidence that Fd1 and FdC2 are two leaf-type Fd in rice,and Fd1 is the primary leaf-type Fd that participates in rice photosynthetic electron transport.Loss of the photosynthetic electron transport ability in fd1 mutants disrupted the Calvin-Benson cycle,resulting in a chlorotic and lethal phenotype at the three-leaf stage.The main conclusions of this paper are as follows:1.Homology alignment suggested that the rice genome encode 5 Fd proteins and 2 FdC proteins.Phylogenetic analysis showed that only OsFd1 may be a leaf-type Fd.RT-PCR analysis of seven Fd genes showed that the transcript level of Fd1 is highest in all of Fd genes,and the transcript level of FdC2 is about 15%that of Fd1,while the transcript levels of other Fds are very low and almost undetectable.Moreover,Fd1 was expressed in all tissues,but preferentially expressed in developing leaves and not expressed at high levels in roots.These results indicate that there are two leaf-type Fd in rice,and Fd1 is the primary leaf-type Fd.2.Cytochrome c and NADP~+photoreduction experiments proved that Fd1 donate photosynthetic electron from PSI to FNR to generate NADPH,which provides reduction power for rice carbon assimilation in vitro.However,FdC2 can also accept photosynthetic electron from PSI,but cannot contribute to FNR to generate NADPH.It is indicating that FdC2 does not directly participate in rice photosynthetic carbon assimilation,but undertake other biological functions necessary for rice growth and development.The true electron acceptor of FdC2 is unclear.Subcellular localization results indicated that Fd1 is located in the chloroplasts of rice.These results suggest that,as the two primary leaf-type Fd in rice,Fd1 is mainly involved in rice photosynthetic carbon assimilation,while FdC2 may supplement the function of Fd1 or contribute photosynthetic electrons to other metabolic pathways to regulate rice growth and development.3.We generated several independent Fd1 mutant lines using a CRISPR/Cas9 strategy in the NPB background.The results of predicted 3D structures of WT and selected threeΔFd1 mutant proteins by Swiss-Model suggested that the Fd1 gene had a loss-of-function mutation in different transgenic lines.The expression level of Fd1 in the fd1 mutants was significantly lower than WT.Upon germination,none of the fd1 mutants had obvious abnormalities.However,the mutants showed gradual chlorosis at the two-leaf stage and began to rapidly desiccate,then died at the three-leaf stage.The measurement results of endogenous sugar and NADPH content showed that after the fd1 mutation,the accumulation of endogenous sugar and NADPH was much lower than that of WT at three-leaf stage.Exogenous applied sugar can partially prolong the growth of the mutant,and as the concentration of externally applied sugar increases,the growth time of the mutant also increases accordingly.The expression levels of genes involved in Calvin-Benson cycle were all reduced in the fd1 mutant.These results indicate that Fd1 affects rice endogenous carbon assimilation and accumulation of carbon assimilation products,which is necessary for growth and development of rice.4.We found that compared with the WT,the chlorophyll content in fd1 leaves was significantly decreased,and the number of chloroplasts was reduced.The chloroplasts of WT cells had multiple starch granules and well-arranged grana lamellae,whereas the fd1 mutant chloroplasts had few grana lamellae and no visible starch granules.These results indicated that the WT began to undergo photosynthetic carbon assimilation after the three-leaf stage,accumulating carbon assimilation products in the chloroplast and providing energy for rice growth.However,due to the damage of photosynthesis,the fd1 mutant could not produce enough photosynthetic products,resulting in the rapid degradation of photosynthetic pigments in the fd1 mutant and the rapid chlorotic of the leaves and subsequent death.5.The measurement results of chlorophyll fluorescence parameters show that F_V/F_M andφII were predominantly reduced in the fd1 mutants compared with WT indicating that PSII was inhibited.On the basis of no obvious change in non-photochemical quenching(NPQ),the photochemical quenching(qP)is significantly reduced,indicating that the photon number used for photosynthesis in the mutant is significantly reduced compared to the WT,and the photon number for non-photosynthesis is almost unchanged.The electron transport rate is also significantly reduced.These results indicate that after Fd1mutation,the photosynthetic efficiency of the mutant did decrease significantly.6.Measuring the levels of all Fd transcripts in fd1 and hdy1 mutants showed that the level of transcripts of FdC2 in fd1 mutant decreased,and the level of transcripts of Fd1 in hdy1 mutants also decreased.Moreover,the expression trends of Fd-dependent genes are quite similar in fd1 and hdy1mutants.These results suggest that Fd1 and FdC2,which are two primary leaf-type Fd in rice,the function of Fd1 and FdC2 are coordinated in rice rather than compensated.Although both Fd1 and FdC2 are participated in rice photosynthetic electron transport,but their biological pathways do not overlap,and they are both essential electron transporters for rice growth and development... |
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