The nuclear-encoded factors and the photosynthetic apparatus have been studied extensively during chloroplast biogenesis.However,many questions regarding these processes remain unanswered,particularly in perennial woo...The nuclear-encoded factors and the photosynthetic apparatus have been studied extensively during chloroplast biogenesis.However,many questions regarding these processes remain unanswered,particularly in perennial woody plants.As a model material of woody plants,poplar not only has very significant value of research,but also possesses economic and ecological properties.This study reports the Populus trichocarpa DJ-1C(PtrDJ1C)factor,encoded by a nuclear gene,and a member of the DJ-1 superfamily.PtrDJ1C knock-out with the CRISPR/Cas9 system resulted in different albino phenotypes.Chlorophyll fluorescence and immunoblot analyses showed that the levels of photosynthetic complex proteins decreased significantly.Moreover,the transcript level of plastid-encoded RNA polymerase-dependent genes and the splicing efficiency of several introns were affected in the mutant line.Furthermore,rRNA accumulation was abnormal,leading to developmental defects in chloroplasts and affecting lignin accumulation.We concluded that the PtrDJ1C protein is essential for early chloroplast development and lignin deposition in poplar.展开更多
Calcium(Ca^(2+))is an essential nutrient and a cellular signal in plants.The level of free Ca^(2+)in the apoplast is~10,000-fold higher than that in the cytoplasm at the resting state.This large Ca^(2+)level gradient ...Calcium(Ca^(2+))is an essential nutrient and a cellular signal in plants.The level of free Ca^(2+)in the apoplast is~10,000-fold higher than that in the cytoplasm at the resting state.This large Ca^(2+)level gradient across the membrane between the interior and exterior can cause Ca^(2+)overload and toxicity.Plants have evolved mechanisms to export Ca^(2+)out of the cytoplasm or store it in intracellular organelles,such as endoplasmic reticulum(ER),mitochondria,and vacuole.Additionally,Ca^(2+)-binding proteins,including those containing the most common Ca^(2+)-binding EF-hand domains,maintain the cytosolic Ca^(2+)level by directly binding them,thus participating in Ca^(2+)homeostasis.Ca^(2+)/H+exchangers(CAXs,antiporter)and Ca^(2+)-ATPases(ACAs)are responsible for transferring Ca^(2+)from the cytosol to either the external space or intracellular stores(Bose et al.,2011).However,it is still unknown how these Ca^(2+)-scavenging transporters are activated when the cytosolic Ca^(2+)level is elevated to threshold levels.An“auto-regulatory”mechanism to scavenge excess cytosolic Ca^(2+),providing a guarding system to maintain cytosolic Ca^(2+)homeostasis in plants.展开更多
The endoplasmic reticulum(ER)and the plasma membrane(PM)form ER–PM contact sites(EPCSs)that allow the ER and PM to exchange materials and information.Stress-induced disruption of protein folding triggers ER stress,an...The endoplasmic reticulum(ER)and the plasma membrane(PM)form ER–PM contact sites(EPCSs)that allow the ER and PM to exchange materials and information.Stress-induced disruption of protein folding triggers ER stress,and the cell initiates the unfolded protein response(UPR)to resist the stress.However,whether EPCSs play a role in ER stress in plants remains unclear.VESICLE-ASSOCIATED MEMBRANE PROTEIN(VAMP)-ASSOCIATED PROTEIN 27-1(VAP27-1)functions in EPCS tethering and is encoded by a family of 10 genes(VAP27-1–10)in Arabidopsis thaliana.Here,we used CRISPR-Cas9-mediated genome editing to obtain a homozygous vap27-1 vap27-3 vap27-4(vap27-1/3/4)triple mutant lacking three of the key VAP27 family members in Arabidopsis.The vap27-1/3/4 mutant exhibits defects in ER–PM connectivity and EPCS architecture,as well as excessive UPR signaling.We further showed that relocation of VAP27-1 to the PM mediates specific VAP27-1-related EPCS remodeling and expansion under ER stress.Moreover,the spatiotemporal dynamics of VAP27-1 at the PM increase ER–PM connectivity and enhance Arabidopsis resistance to ER stress.In addition,we revealed an important role for intracellular calcium homeostasis in the regulation of UPR signaling.Taken together,these results broaden our understanding of the molecular and cellular mechanisms of ER stress and UPR signaling in plants,providing additional clues for improving plant broad-spectrum resistance to different stresses.展开更多
Manganese （Mn） is an essential catalytic metal in the Mn-cluster that oxidizes water to produce oxygen dur- ing photosynthesis. However, the transport protein（s） responsible for Mn2＋ import into the chloroplast r...Manganese （Mn） is an essential catalytic metal in the Mn-cluster that oxidizes water to produce oxygen dur- ing photosynthesis. However, the transport protein（s） responsible for Mn2＋ import into the chloroplast re- mains unknown. Here, we report the characterization ofArabidopsis CMT1 （Chloroplast Manganese Trans- porter 1）, an evolutionarily conserved protein in the Uncharacterized Protein Family 0016 （UPFO016）, that is required for manganese accumulation into the chloroplast. CMT1 is expressed primarily in green tissues, and its encoded product is localized in the inner envelope membrane of the chloroplast. Disruption of CMT1 in the T-DNA insertional mutant cmtl-1 resulted in stunted plant growth, defective thylakoid stacking, and severe reduction of photosystem II complexes and photosynthetic activity. Consistent with reduced oxy- gen evolution capacity, the mutant chloroplasts contained less manganese than the wild-type ones. In sup- port of its function as a Mn transporter, CMT1 protein supported the growth and enabled Mn2＋ accumula- tion in the yeast cells of Mn2＋-uptake deficient mutant （3smfl）. Taken together, our results indicate that CMT1 functions as an inner envelope Mn transporter responsible for chloroplast Mn2＋ uptake.展开更多
In multicellular and even single-celled organisms,individual components are interconnected at multiscale levels to produce enormously complex biological networks that help these systems maintain homeostasis for develo...In multicellular and even single-celled organisms,individual components are interconnected at multiscale levels to produce enormously complex biological networks that help these systems maintain homeostasis for development and environmental adaptation.Systems biology studies initially adopted network analysis to explore how relationships between individual components give rise to complex biological processes.Network analysis has been applied to dissect the complex connectivity of mammalian brains across different scales in time and space in The Human Brain Project.In plant science,network analysis has similarly been applied to study the connectivity of plant components at the molecular,subcellular,cellular,organic,and organism levels.Analysis of these multiscale networks contributes to our understanding of how genotype determines phenotype.In this review,we summarized the theoretical framework of plant multiscale networks and introduced studies investigating plant networks by various experimental and computational modalities.We next discussed the currently available analytic methodologies and multi-level imaging techniques used to map multiscale networks in plants.Finally,we highlighted some of the technical challenges and key questions remaining to be addressed in this emerging field.展开更多
The plant cytoskeleton undergoes dynamic remodeling in response to diverse developmental and environmental cues. Remodeling of the cytoskeleton coordinates growth in plant cells, including trafficking and exocytosis o...The plant cytoskeleton undergoes dynamic remodeling in response to diverse developmental and environmental cues. Remodeling of the cytoskeleton coordinates growth in plant cells, including trafficking and exocytosis of membrane and wall components during cell expansion, and regulation of hypocotyl elongation in response to light. Cytoskeletal remodeling also has key functions in disease resistance and abiotic stress responses. Many stimuli result in altered activity of cytoskeleton-associatedproteins,microtubuleassociated proteins(MAPs) and actin-binding proteins(ABPs). MAPs and ABPs are the main players determining the spatiotemporally dynamic nature of the cytoskeleton, functioning in a sensory hub that decodes signals to modulate plant cytoskeletal behavior. Moreover, MAP and ABP activities and levels are precisely regulated during development and environmental responses, but our understanding of this process remains limited. In this review, we summarize the evidence linking multiple signaling pathways, MAP and ABP activities and levels, and cytoskeletal rearrangements in plant cells. We highlight advances in elucidating the multiple mechanisms that regulate MAP and ABP activities and levels, including calcium and calmodulin signaling, ROP GTPase activity, phospholipid signaling, and post-translational modifications.展开更多
Three-dimensional electron microscopy(3 D-EM) has attracted considerable attention because of its ability to provide detailed information with respect to developmental analysis. However, large-scale high-resolution 3 ...Three-dimensional electron microscopy(3 D-EM) has attracted considerable attention because of its ability to provide detailed information with respect to developmental analysis. However, large-scale high-resolution 3 D reconstruction of biological samples remains challenging. Herein, we present a 3 D view of a Picea wilsonii Mast. pollen grain with 100 nm axial and38.57 nm lateral resolution using AutoCUTS-SEM(automatic collector of ultrathin sections-scanning electron microscopy). We established a library of 3,127 100 nm thick serial sections of pollen grains for preservation and observation, demonstrating that the protocol can be used to analyze large-volume samples. After obtaining the SEM images, we reconstructed an entire pollen grain comprising 734 serial sections. The images produced by 3D reconstruction clearly revealed the main components of the P.wilsonii pollen grain, i.e., two sacci and pollen corpus, tube cell, generative cell, and two degenerated prothallial cells, and their internal organization. In addition, we performed a quantitative analysis of the different pollen grain cells, including sacci, and found that there were 202 connections within a saccus SEM image. Thus, for the first time, this study provided a global 3D view of the entire pollen grain, which will be useful for analyzing pollen development and growth.展开更多
Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether thes...Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fuorescent protein （GFP） tag and expressed in Escherichia coil and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgenic A. thaliana lines revealed that 35S：：GFP-PRF1 formed a filamentous network, while 35S：：GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profllin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive Iocalizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.展开更多
基金supported by the National Natural Science Foundation of China(Grant Nos.32201516,91954202)the Youth Top-notch Talent Program of Hebei Education Department(BJK2022028)+1 种基金National Training Program of Innovation and Entrepreneurship for Undergraduates(Grant Nos.S202110022037,G202010022075)the funding of Hebei North University(XJ2021013)。
文摘The nuclear-encoded factors and the photosynthetic apparatus have been studied extensively during chloroplast biogenesis.However,many questions regarding these processes remain unanswered,particularly in perennial woody plants.As a model material of woody plants,poplar not only has very significant value of research,but also possesses economic and ecological properties.This study reports the Populus trichocarpa DJ-1C(PtrDJ1C)factor,encoded by a nuclear gene,and a member of the DJ-1 superfamily.PtrDJ1C knock-out with the CRISPR/Cas9 system resulted in different albino phenotypes.Chlorophyll fluorescence and immunoblot analyses showed that the levels of photosynthetic complex proteins decreased significantly.Moreover,the transcript level of plastid-encoded RNA polymerase-dependent genes and the splicing efficiency of several introns were affected in the mutant line.Furthermore,rRNA accumulation was abnormal,leading to developmental defects in chloroplasts and affecting lignin accumulation.We concluded that the PtrDJ1C protein is essential for early chloroplast development and lignin deposition in poplar.
基金supported by the Natural Science Foundation of Jiangsu Province(BK20211319)the National Natural Science Foundation of China(W2433065,32000201)the Jiangsu Outstanding Post-doctoral Program(2023ZB863).
文摘Calcium(Ca^(2+))is an essential nutrient and a cellular signal in plants.The level of free Ca^(2+)in the apoplast is~10,000-fold higher than that in the cytoplasm at the resting state.This large Ca^(2+)level gradient across the membrane between the interior and exterior can cause Ca^(2+)overload and toxicity.Plants have evolved mechanisms to export Ca^(2+)out of the cytoplasm or store it in intracellular organelles,such as endoplasmic reticulum(ER),mitochondria,and vacuole.Additionally,Ca^(2+)-binding proteins,including those containing the most common Ca^(2+)-binding EF-hand domains,maintain the cytosolic Ca^(2+)level by directly binding them,thus participating in Ca^(2+)homeostasis.Ca^(2+)/H+exchangers(CAXs,antiporter)and Ca^(2+)-ATPases(ACAs)are responsible for transferring Ca^(2+)from the cytosol to either the external space or intracellular stores(Bose et al.,2011).However,it is still unknown how these Ca^(2+)-scavenging transporters are activated when the cytosolic Ca^(2+)level is elevated to threshold levels.An“auto-regulatory”mechanism to scavenge excess cytosolic Ca^(2+),providing a guarding system to maintain cytosolic Ca^(2+)homeostasis in plants.
基金supported by the National Natural Science Foundation of China(32170689,91954202,32030010)National Key Research and Development Program of China(2022YFF0712500)+1 种基金the Program of Introducing Talents of Discipline to Universities(111 Project,B13007)Beijing Forestry University Outstanding Postgraduate Mentoring Team Building(YJSY-DSTD2022005).
文摘The endoplasmic reticulum(ER)and the plasma membrane(PM)form ER–PM contact sites(EPCSs)that allow the ER and PM to exchange materials and information.Stress-induced disruption of protein folding triggers ER stress,and the cell initiates the unfolded protein response(UPR)to resist the stress.However,whether EPCSs play a role in ER stress in plants remains unclear.VESICLE-ASSOCIATED MEMBRANE PROTEIN(VAMP)-ASSOCIATED PROTEIN 27-1(VAP27-1)functions in EPCS tethering and is encoded by a family of 10 genes(VAP27-1–10)in Arabidopsis thaliana.Here,we used CRISPR-Cas9-mediated genome editing to obtain a homozygous vap27-1 vap27-3 vap27-4(vap27-1/3/4)triple mutant lacking three of the key VAP27 family members in Arabidopsis.The vap27-1/3/4 mutant exhibits defects in ER–PM connectivity and EPCS architecture,as well as excessive UPR signaling.We further showed that relocation of VAP27-1 to the PM mediates specific VAP27-1-related EPCS remodeling and expansion under ER stress.Moreover,the spatiotemporal dynamics of VAP27-1 at the PM increase ER–PM connectivity and enhance Arabidopsis resistance to ER stress.In addition,we revealed an important role for intracellular calcium homeostasis in the regulation of UPR signaling.Taken together,these results broaden our understanding of the molecular and cellular mechanisms of ER stress and UPR signaling in plants,providing additional clues for improving plant broad-spectrum resistance to different stresses.
文摘Manganese （Mn） is an essential catalytic metal in the Mn-cluster that oxidizes water to produce oxygen dur- ing photosynthesis. However, the transport protein（s） responsible for Mn2＋ import into the chloroplast re- mains unknown. Here, we report the characterization ofArabidopsis CMT1 （Chloroplast Manganese Trans- porter 1）, an evolutionarily conserved protein in the Uncharacterized Protein Family 0016 （UPFO016）, that is required for manganese accumulation into the chloroplast. CMT1 is expressed primarily in green tissues, and its encoded product is localized in the inner envelope membrane of the chloroplast. Disruption of CMT1 in the T-DNA insertional mutant cmtl-1 resulted in stunted plant growth, defective thylakoid stacking, and severe reduction of photosystem II complexes and photosynthetic activity. Consistent with reduced oxy- gen evolution capacity, the mutant chloroplasts contained less manganese than the wild-type ones. In sup- port of its function as a Mn transporter, CMT1 protein supported the growth and enabled Mn2＋ accumula- tion in the yeast cells of Mn2＋-uptake deficient mutant （3smfl）. Taken together, our results indicate that CMT1 functions as an inner envelope Mn transporter responsible for chloroplast Mn2＋ uptake.
基金supported by the National Natural Science Foundation of China(31530084,32000558,32000483,and31800504)the Programme of Introducing Talents of Discipline to Universities(111 project,B13007)the China Postdoctoral Science Foundation Grant(2019M660494)。
文摘In multicellular and even single-celled organisms,individual components are interconnected at multiscale levels to produce enormously complex biological networks that help these systems maintain homeostasis for development and environmental adaptation.Systems biology studies initially adopted network analysis to explore how relationships between individual components give rise to complex biological processes.Network analysis has been applied to dissect the complex connectivity of mammalian brains across different scales in time and space in The Human Brain Project.In plant science,network analysis has similarly been applied to study the connectivity of plant components at the molecular,subcellular,cellular,organic,and organism levels.Analysis of these multiscale networks contributes to our understanding of how genotype determines phenotype.In this review,we summarized the theoretical framework of plant multiscale networks and introduced studies investigating plant networks by various experimental and computational modalities.We next discussed the currently available analytic methodologies and multi-level imaging techniques used to map multiscale networks in plants.Finally,we highlighted some of the technical challenges and key questions remaining to be addressed in this emerging field.
基金supported by the National Natural Science Foundation of China(31771493, 32030010)the Program of Introducing Talents of Discipline to Universities (111 Project, B13007)the China Postdoctoral Science Foundation (2018M641219,2019T120057)。
文摘The plant cytoskeleton undergoes dynamic remodeling in response to diverse developmental and environmental cues. Remodeling of the cytoskeleton coordinates growth in plant cells, including trafficking and exocytosis of membrane and wall components during cell expansion, and regulation of hypocotyl elongation in response to light. Cytoskeletal remodeling also has key functions in disease resistance and abiotic stress responses. Many stimuli result in altered activity of cytoskeleton-associatedproteins,microtubuleassociated proteins(MAPs) and actin-binding proteins(ABPs). MAPs and ABPs are the main players determining the spatiotemporally dynamic nature of the cytoskeleton, functioning in a sensory hub that decodes signals to modulate plant cytoskeletal behavior. Moreover, MAP and ABP activities and levels are precisely regulated during development and environmental responses, but our understanding of this process remains limited. In this review, we summarize the evidence linking multiple signaling pathways, MAP and ABP activities and levels, and cytoskeletal rearrangements in plant cells. We highlight advances in elucidating the multiple mechanisms that regulate MAP and ABP activities and levels, including calcium and calmodulin signaling, ROP GTPase activity, phospholipid signaling, and post-translational modifications.
基金supported by grants from Fundamental Research Funds for the Central Universities(BLX201617)the Program of Introducing Talents of Discipline to Universities(111 projects,B13007)+1 种基金the National Natural Science Foundation of China(31700250,31530084,31761133009,31670182)supported by the Center for Biological Imaging,Institute of Biophysics,Chinese Academy of Sciences。
文摘Three-dimensional electron microscopy(3 D-EM) has attracted considerable attention because of its ability to provide detailed information with respect to developmental analysis. However, large-scale high-resolution 3 D reconstruction of biological samples remains challenging. Herein, we present a 3 D view of a Picea wilsonii Mast. pollen grain with 100 nm axial and38.57 nm lateral resolution using AutoCUTS-SEM(automatic collector of ultrathin sections-scanning electron microscopy). We established a library of 3,127 100 nm thick serial sections of pollen grains for preservation and observation, demonstrating that the protocol can be used to analyze large-volume samples. After obtaining the SEM images, we reconstructed an entire pollen grain comprising 734 serial sections. The images produced by 3D reconstruction clearly revealed the main components of the P.wilsonii pollen grain, i.e., two sacci and pollen corpus, tube cell, generative cell, and two degenerated prothallial cells, and their internal organization. In addition, we performed a quantitative analysis of the different pollen grain cells, including sacci, and found that there were 202 connections within a saccus SEM image. Thus, for the first time, this study provided a global 3D view of the entire pollen grain, which will be useful for analyzing pollen development and growth.
基金Supported by the State Key Basic Research and Development Plan of China (2006CB100101 and 2007CB108700)the National Natural Science Foundation of China (30421002,30370707,30570925 and 30630005)a Grant from Deutsche Forschungsgemeinschaft (DFG,SA 1564/2-1)
文摘Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fuorescent protein （GFP） tag and expressed in Escherichia coil and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgenic A. thaliana lines revealed that 35S：：GFP-PRF1 formed a filamentous network, while 35S：：GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profllin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive Iocalizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.
