Pollen tubes usually exhibit a prominent region at their apex called the "clear zone" because it lacks light refracting amyloplasts. A robust, long clear zone often associates with fast growing pollen tubes, and thu...Pollen tubes usually exhibit a prominent region at their apex called the "clear zone" because it lacks light refracting amyloplasts. A robust, long clear zone often associates with fast growing pollen tubes, and thus serves as an indicator of pollen tube health. Nevertheless we do not understand how it arises or how it is maintained. Here we review the structure of the clear zone, and attempt to explain the factors that contribute to its formation. While amyloplasts and vacuolar elements are excluded from the clear zone, virtually all other organelles are present including secretory vesicles, mitochondria, Golgi dictyosomes, and the endoplas-mic reticulum (ER). Secretory vesicles aggregate into an inverted cone appressed against the apical plasma membrane. ER elements move nearly to the extreme apex, whereas mitochondria and Golgi dictyosomes move less far forward. The cortical actin fringe assumes a central position in the control of clear zone formation and maintenance, given its role in generating cytoplasmic streaming. Other likely factors include the tip-focused calcium gradient, the apical pH gradient, the influx of water, and a host of signaling factors (small G-proteins). We think that the clear zone is an emergent property that depends on the interaction of several factors crucial for polarized growth.展开更多
The pollen receptor kinases (PRK) are critical regulators of pollen tube growth. The Arabidopsis genome encodes eight PRK genes, of which six are highly expressed in pollen tubes. The potential functions of AtPRK1 thr...The pollen receptor kinases (PRK) are critical regulators of pollen tube growth. The Arabidopsis genome encodes eight PRK genes, of which six are highly expressed in pollen tubes. The potential functions of AtPRK1 through AtPRK5, but not of AtPRK6,in pollen growth were analyzed in tobacco. Herein, AtPRK6 was cloned, and its function was identified. AtPRK6 was expressed specifically in pollen tubes. A yeast two-hybrid screen of AtPRK6 against 14 Arabidopsis Rop guanine nucleotide exchange factors (RopGEFs) showed that AtPRK6 interacted with AtRopGEF8 and AtRopGEF12. These interactions were confirmed in Arabidopsis mesophyll protoplasts. The interactions between AtPRK6 and AtRopGEF8/12 were mediated by the C-termini of AtRopGEF8/12 and by the juxtamembrane and kinase domain of AtPRK6, but were not dependent on the kinase activity. In addition, transient overexpression of AtPRK6::GFP in Arabidopsis protoplasts revealed that AtPRK6 was localized to the plasma membrane. Tobacco pollen tubes overexpressing AtPRK6 exhibited shorter tubes with enlarged tips. This depolarized tube growth required the kinase domain of AtPRK6 and was not dependent on kinase activity. Taken together, the results show that AtPRK6,through its juxtamembrane and kinase domains (KD), interacts with AtRopGEF8/12 and plays crucial roles in polarized growth of pollen tubes.展开更多
In this article, the functions of D-myo-inositol-1,4,5-trisphosphate (Ins [1,4,5] P3) in regulating pollen tube polar growth were investigated by application of caged version of the phosphoinositides. To increase the ...In this article, the functions of D-myo-inositol-1,4,5-trisphosphate (Ins [1,4,5] P3) in regulating pollen tube polar growth were investigated by application of caged version of the phosphoinositides. To increase the intracellular Ins(1,4,5)P3 concentration at the apical region of pollen tube, the caged Ins(1,4,5)P3 loaded by osmotic shock was activated by 10 s 360 nm UV flash at this domain (10 μm from the tip). More than 70% pollen tubes were induced swelling at apex and/or growth axis reorientation, accompanying temporarily growth arrest, by localized increase of Ins(1,4,5)P3 concentration (n=21). While, pollen tubes without being loaded with caged Ins(1,4,5)P3 had not response to the same dosage UV flash. With FM 1-43 fluorescent staining, it was found that growth perturbation by UV activated caged Ins(1,4,5)P3 had tight link with membrane trafficking at the apical zone of pollen tubes. Upon UV pulse, the apical V-shaped bright area where was full of secretory vesicles spread to a much broader region, which implied that actin filaments at the apical region were remodeled. It was also observed that the FM 1-43 fluorescence intensity at tip remarkably increased than that before UV flash, which demonstrated that more secretory vesicles were accumulated at this region. To estimate the role of Ins(1,4,5)P3 in modulating intracellular calcium concentration and distribution, dextran conjugated fluorescent dye Calcium Green-1 and Rhodamine B were microinjected into pollen tubes together with caged Ins(1,4,5)P3. The results showed that calcium concentration at the subapical region increased upon UV released Ins(1,4,5)P3. Consequently, the tip-focused calcium gradient under the apical dome of pollen tube was disturbed. Simultaneously, the pollen tube bulged at the apical region and its growth rate decreased. As the tip-focused calcium gradient was reestablished, the pollen tube morphology and growth recovered to the normal level. Therefore, Ins(1,4,5)P3 can modulate pollen tube growth rate and direction through mobilizing intracellular calcium and regulating vesicle trafficking during pollen tube finding its way to the ovary.展开更多
The ROP1 GTPase-based signaling network controls tip growth in Arabidopsis pollen tubes. Our previous studies imply that ROP1 might be directly activated by RopGEF1, which belongs to a plant-specific family of Rho gua...The ROP1 GTPase-based signaling network controls tip growth in Arabidopsis pollen tubes. Our previous studies imply that ROP1 might be directly activated by RopGEF1, which belongs to a plant-specific family of Rho guanine nucleotide exchange factors (RopGEFs) and in turn may be activated by an unknown factor through releasing RopGEFI's auto-inhibition. In this study, we found that RopGEF1 forms a complex with ROP1 and AtPRK2, a receptor-like protein kinase previously shown to interact with RopGEFs. AtPRK2 phosphorylated RopGEF1 in vitro and the atprkl,2,5 tri- ple mutant showed defective pollen tube growth, similar to the phenotype of the ropgef1,9,12,14 quadruple mutant. Overexpression of a dominant negative form of AtPRK2 (DN-PRK2) inhibited pollen germination in Arabidopsis and reduced pollen elongation in tobacco. The DN-PRK2-induced pollen germination defect was rescued by overexpressing a constitutively active form of RopGEF1, RopGEF1(90-457), implying that RopGEF1 acts downstream of AtPRK2. Moreover, AtPRK2 increased ROP1 activity at the apical plasma membrane whereas DN-PRK2 reduced ROP1 activity. Finally, two mutations at the C-terminal putative phosphorylation sites of RopGEF1 (RopGEF1S460A and RopGEF1S480A) eliminated the function of RopGEF1 in vivo. Taken together, our results support the hypothesis that AtPRK2 acts as a positive regula- tor of the ROP1 signaling pathway most likely by activating RopGEF1 through phosphorylation.展开更多
Although pollen tube growth is a prerequisite for higher plant fertilization and seed production, the processes leading to pollen tube emission and elongation are crucial for understanding the basic mechanisms of tip ...Although pollen tube growth is a prerequisite for higher plant fertilization and seed production, the processes leading to pollen tube emission and elongation are crucial for understanding the basic mechanisms of tip growth. It was generally accepted that pollen tube elongation occurs by accumulation and fusion of Golgi-derived secretory vesicles (SVs) in the apical region, or clear zone, where they were thought to fuse with a restricted area of the apical plasma membrane (PM), defining the apical growth domain. Fusion of SVs at the tip reverses outside cell wall material and provides new segments of PM. However, electron microscopy studies have clearly shown that the PM incorporated at the tip greatly exceeds elongation and a mechanism of PM retrieval was already postulated in the mid-nineteenth century. Recent studies on endocytosis during pollen tube growth showed that different endocytic pathways occurred in distinct zones of the tube, including the apex, and led to a new hypothesis to explain vesicle accumulation at the tip; namely, that endocytic vesicles contribute substantially to V-shaped vesicle accumulation in addition to SVs and that exocytosis does not involve the entire apical domain. New insights suggested the intriguing hypothesis that modulation between exo- and endocytosis in the apex contributes to maintain PM polarity in terms of lipid/protein composition and showed distinct degradation pathways that could have different functions in the physiology of the cell. Pollen tube growth in vivo is closely regulated by interaction with style molecules. The study of endocytosis and membrane recycling in pollen tubes opens new perspectives to studying pollen tube-style interactions in vivo.展开更多
The molecular links between extracellular signals and the regulation of localized protein synthesis in plant cells are poorly understood.Here,we show that in Arabidopsis thaliana,the extracellular peptide RALF1 and it...The molecular links between extracellular signals and the regulation of localized protein synthesis in plant cells are poorly understood.Here,we show that in Arabidopsis thaliana,the extracellular peptide RALF1 and its receptor,the FERONIA receptor kinase,promote root hair(RH)tip growth by modulating protein synthesis.We found that RALF1 promotes FERONIA-mediated phosphorylation of elF4E1,a eukaryotic translation initiation factor that plays a crucial role in the control of mRNA translation rate.Phosphorylated elF4E1 increases mRNA affinity and modulates mRNA translation and,thus,protein synthesis.The mRNAs targeted by the RALF1-FERONIA-elF4E1 module include ROP2 and RSL4,which are important regulators of RH cell polarity and growth.RALF1 and FERONIA are expressed in a polar manner in RHs,which facilitate elF4E1 polar丨ocalization and thus may control local f?OP2 translation.Moreover,we demonstrated that high-level accumulation of RSL4 exerts negative-feedback regulation of RALF1 expression by directly binding the RALF1 gene promoter,determining the final RH size.Our study reveals that the link between RALF1-FERONIA signaling and protein synthesis constitutes a novel component regulating cell expansion in these polar growing cells.展开更多
The genetic identities of Ca2+ channels in root hair (RH) tips essential for constitutive RH growth have remained elusive for decades. Here, we report the identification and characterization of three cyclicnucleotide-...The genetic identities of Ca2+ channels in root hair (RH) tips essential for constitutive RH growth have remained elusive for decades. Here, we report the identification and characterization of three cyclicnucleotide-gated channel (CNGC) family members, CNGC5, CNGC6, and CNGC9, as Ca2+ channels essential for constitutive RH growth in Arabidopsis. We found that the cngc5-1cngc6-2cngc9-1 triple mutant(designated shrh1) showed significantly shorter and branching RH phenotypes as compared with thewild type. The defective RH growth phenotype of shrh1 could be rescued by either the expression ofCNGC5, CNGC6, or CNGC9 single gene or by the supply of high external Ca2+, but could not be rescuedby external K+ supply. Cytosolic Ca2+ imaging and patch-clamp data in HEK293T cells showed that thesethree CNGCs all function as Ca2+-permeable channels. Cytosolic Ca2+ imaging in growing RHs furthershowed that the Ca2+ gradients and their oscillation in RH tips were dramatically attenuated in shrh1compared with those in the wild type. Phenotypic analysis revealed that these three CNGCs are Ca2+ channels essential for constitutive RH growth, with different roles in RHs from the conditional player CNGC14.Moreover, we found that these three CNGCs are involved in auxin signaling in RHs. Taken together, ourstudy identified CNGC5, CNGC6, and CNGC9 as three key Ca2+ channels essential for constitutive RHgrowth and auxin signaling in Arabidopsis.展开更多
基金supported by a grant from the USA National Science Foundation (MCB-0847876)
文摘Pollen tubes usually exhibit a prominent region at their apex called the "clear zone" because it lacks light refracting amyloplasts. A robust, long clear zone often associates with fast growing pollen tubes, and thus serves as an indicator of pollen tube health. Nevertheless we do not understand how it arises or how it is maintained. Here we review the structure of the clear zone, and attempt to explain the factors that contribute to its formation. While amyloplasts and vacuolar elements are excluded from the clear zone, virtually all other organelles are present including secretory vesicles, mitochondria, Golgi dictyosomes, and the endoplas-mic reticulum (ER). Secretory vesicles aggregate into an inverted cone appressed against the apical plasma membrane. ER elements move nearly to the extreme apex, whereas mitochondria and Golgi dictyosomes move less far forward. The cortical actin fringe assumes a central position in the control of clear zone formation and maintenance, given its role in generating cytoplasmic streaming. Other likely factors include the tip-focused calcium gradient, the apical pH gradient, the influx of water, and a host of signaling factors (small G-proteins). We think that the clear zone is an emergent property that depends on the interaction of several factors crucial for polarized growth.
基金supported by the National Natural Science Foundation of China (31300247)
文摘The pollen receptor kinases (PRK) are critical regulators of pollen tube growth. The Arabidopsis genome encodes eight PRK genes, of which six are highly expressed in pollen tubes. The potential functions of AtPRK1 through AtPRK5, but not of AtPRK6,in pollen growth were analyzed in tobacco. Herein, AtPRK6 was cloned, and its function was identified. AtPRK6 was expressed specifically in pollen tubes. A yeast two-hybrid screen of AtPRK6 against 14 Arabidopsis Rop guanine nucleotide exchange factors (RopGEFs) showed that AtPRK6 interacted with AtRopGEF8 and AtRopGEF12. These interactions were confirmed in Arabidopsis mesophyll protoplasts. The interactions between AtPRK6 and AtRopGEF8/12 were mediated by the C-termini of AtRopGEF8/12 and by the juxtamembrane and kinase domain of AtPRK6, but were not dependent on the kinase activity. In addition, transient overexpression of AtPRK6::GFP in Arabidopsis protoplasts revealed that AtPRK6 was localized to the plasma membrane. Tobacco pollen tubes overexpressing AtPRK6 exhibited shorter tubes with enlarged tips. This depolarized tube growth required the kinase domain of AtPRK6 and was not dependent on kinase activity. Taken together, the results show that AtPRK6,through its juxtamembrane and kinase domains (KD), interacts with AtRopGEF8/12 and plays crucial roles in polarized growth of pollen tubes.
文摘In this article, the functions of D-myo-inositol-1,4,5-trisphosphate (Ins [1,4,5] P3) in regulating pollen tube polar growth were investigated by application of caged version of the phosphoinositides. To increase the intracellular Ins(1,4,5)P3 concentration at the apical region of pollen tube, the caged Ins(1,4,5)P3 loaded by osmotic shock was activated by 10 s 360 nm UV flash at this domain (10 μm from the tip). More than 70% pollen tubes were induced swelling at apex and/or growth axis reorientation, accompanying temporarily growth arrest, by localized increase of Ins(1,4,5)P3 concentration (n=21). While, pollen tubes without being loaded with caged Ins(1,4,5)P3 had not response to the same dosage UV flash. With FM 1-43 fluorescent staining, it was found that growth perturbation by UV activated caged Ins(1,4,5)P3 had tight link with membrane trafficking at the apical zone of pollen tubes. Upon UV pulse, the apical V-shaped bright area where was full of secretory vesicles spread to a much broader region, which implied that actin filaments at the apical region were remodeled. It was also observed that the FM 1-43 fluorescence intensity at tip remarkably increased than that before UV flash, which demonstrated that more secretory vesicles were accumulated at this region. To estimate the role of Ins(1,4,5)P3 in modulating intracellular calcium concentration and distribution, dextran conjugated fluorescent dye Calcium Green-1 and Rhodamine B were microinjected into pollen tubes together with caged Ins(1,4,5)P3. The results showed that calcium concentration at the subapical region increased upon UV released Ins(1,4,5)P3. Consequently, the tip-focused calcium gradient under the apical dome of pollen tube was disturbed. Simultaneously, the pollen tube bulged at the apical region and its growth rate decreased. As the tip-focused calcium gradient was reestablished, the pollen tube morphology and growth recovered to the normal level. Therefore, Ins(1,4,5)P3 can modulate pollen tube growth rate and direction through mobilizing intracellular calcium and regulating vesicle trafficking during pollen tube finding its way to the ovary.
基金a MOST 973 project,National Institute of General Medical Research,DOE (DE-FG02-04ER15555,which supported F.C.and the biochemical experiments described in this work) to Z.Y.,and a National Science Foundation of China (31070274) to F.C
文摘The ROP1 GTPase-based signaling network controls tip growth in Arabidopsis pollen tubes. Our previous studies imply that ROP1 might be directly activated by RopGEF1, which belongs to a plant-specific family of Rho guanine nucleotide exchange factors (RopGEFs) and in turn may be activated by an unknown factor through releasing RopGEFI's auto-inhibition. In this study, we found that RopGEF1 forms a complex with ROP1 and AtPRK2, a receptor-like protein kinase previously shown to interact with RopGEFs. AtPRK2 phosphorylated RopGEF1 in vitro and the atprkl,2,5 tri- ple mutant showed defective pollen tube growth, similar to the phenotype of the ropgef1,9,12,14 quadruple mutant. Overexpression of a dominant negative form of AtPRK2 (DN-PRK2) inhibited pollen germination in Arabidopsis and reduced pollen elongation in tobacco. The DN-PRK2-induced pollen germination defect was rescued by overexpressing a constitutively active form of RopGEF1, RopGEF1(90-457), implying that RopGEF1 acts downstream of AtPRK2. Moreover, AtPRK2 increased ROP1 activity at the apical plasma membrane whereas DN-PRK2 reduced ROP1 activity. Finally, two mutations at the C-terminal putative phosphorylation sites of RopGEF1 (RopGEF1S460A and RopGEF1S480A) eliminated the function of RopGEF1 in vivo. Taken together, our results support the hypothesis that AtPRK2 acts as a positive regula- tor of the ROP1 signaling pathway most likely by activating RopGEF1 through phosphorylation.
基金Supported by the FIRST Research Program of the University of Milan
文摘Although pollen tube growth is a prerequisite for higher plant fertilization and seed production, the processes leading to pollen tube emission and elongation are crucial for understanding the basic mechanisms of tip growth. It was generally accepted that pollen tube elongation occurs by accumulation and fusion of Golgi-derived secretory vesicles (SVs) in the apical region, or clear zone, where they were thought to fuse with a restricted area of the apical plasma membrane (PM), defining the apical growth domain. Fusion of SVs at the tip reverses outside cell wall material and provides new segments of PM. However, electron microscopy studies have clearly shown that the PM incorporated at the tip greatly exceeds elongation and a mechanism of PM retrieval was already postulated in the mid-nineteenth century. Recent studies on endocytosis during pollen tube growth showed that different endocytic pathways occurred in distinct zones of the tube, including the apex, and led to a new hypothesis to explain vesicle accumulation at the tip; namely, that endocytic vesicles contribute substantially to V-shaped vesicle accumulation in addition to SVs and that exocytosis does not involve the entire apical domain. New insights suggested the intriguing hypothesis that modulation between exo- and endocytosis in the apex contributes to maintain PM polarity in terms of lipid/protein composition and showed distinct degradation pathways that could have different functions in the physiology of the cell. Pollen tube growth in vivo is closely regulated by interaction with style molecules. The study of endocytosis and membrane recycling in pollen tubes opens new perspectives to studying pollen tube-style interactions in vivo.
基金grants from the National Natural Science Foundation of China(NSFC-31400232,31871396,31571444)Young Elite Scientist Sponsorship program of CAST(YESS20160001)+1 种基金the Open Research Fund of the State Key Laboratory of Hybrid Rice(Hunan Hybrid Rice Research Center)to F.Y.and from ANPCyT(PICT2016-0132 and PICT2017-0066),ICGEB(CRP/ARG16-03)Instituto Milenio iBio-Iniciativa Cientffica Milenio MINECON to J.M.E.
文摘The molecular links between extracellular signals and the regulation of localized protein synthesis in plant cells are poorly understood.Here,we show that in Arabidopsis thaliana,the extracellular peptide RALF1 and its receptor,the FERONIA receptor kinase,promote root hair(RH)tip growth by modulating protein synthesis.We found that RALF1 promotes FERONIA-mediated phosphorylation of elF4E1,a eukaryotic translation initiation factor that plays a crucial role in the control of mRNA translation rate.Phosphorylated elF4E1 increases mRNA affinity and modulates mRNA translation and,thus,protein synthesis.The mRNAs targeted by the RALF1-FERONIA-elF4E1 module include ROP2 and RSL4,which are important regulators of RH cell polarity and growth.RALF1 and FERONIA are expressed in a polar manner in RHs,which facilitate elF4E1 polar丨ocalization and thus may control local f?OP2 translation.Moreover,we demonstrated that high-level accumulation of RSL4 exerts negative-feedback regulation of RALF1 expression by directly binding the RALF1 gene promoter,determining the final RH size.Our study reveals that the link between RALF1-FERONIA signaling and protein synthesis constitutes a novel component regulating cell expansion in these polar growing cells.
基金This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB27020102)the National Natural Science Foundation of China(91635301,31570262,and 31770292).
文摘The genetic identities of Ca2+ channels in root hair (RH) tips essential for constitutive RH growth have remained elusive for decades. Here, we report the identification and characterization of three cyclicnucleotide-gated channel (CNGC) family members, CNGC5, CNGC6, and CNGC9, as Ca2+ channels essential for constitutive RH growth in Arabidopsis. We found that the cngc5-1cngc6-2cngc9-1 triple mutant(designated shrh1) showed significantly shorter and branching RH phenotypes as compared with thewild type. The defective RH growth phenotype of shrh1 could be rescued by either the expression ofCNGC5, CNGC6, or CNGC9 single gene or by the supply of high external Ca2+, but could not be rescuedby external K+ supply. Cytosolic Ca2+ imaging and patch-clamp data in HEK293T cells showed that thesethree CNGCs all function as Ca2+-permeable channels. Cytosolic Ca2+ imaging in growing RHs furthershowed that the Ca2+ gradients and their oscillation in RH tips were dramatically attenuated in shrh1compared with those in the wild type. Phenotypic analysis revealed that these three CNGCs are Ca2+ channels essential for constitutive RH growth, with different roles in RHs from the conditional player CNGC14.Moreover, we found that these three CNGCs are involved in auxin signaling in RHs. Taken together, ourstudy identified CNGC5, CNGC6, and CNGC9 as three key Ca2+ channels essential for constitutive RHgrowth and auxin signaling in Arabidopsis.