The major brassinosteroid （BR） receptor of Arabidopsis BRASSINOSTEROID INSENSITIVE1 （BRI1） plays fundamental roles in BR signaling, but the molecular mechanisms underlying the effects of BR on BRI1 internalization...The major brassinosteroid （BR） receptor of Arabidopsis BRASSINOSTEROID INSENSITIVE1 （BRI1） plays fundamental roles in BR signaling, but the molecular mechanisms underlying the effects of BR on BRI1 internalization and assembly state remain unclear. Here, we applied variable angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy to analyze the dynamics of GFP-tagged BRII. We found that, in response to BR, the degree of co-localization of BRI1-GFP with AtFIotl-mCherry increased, and especially BR stimulated the membrane microdomain-associated pathway of BRI1 internalization. We also verified these observations in endocytosis-defective chc2-1 mutants and the AtFIotl amiRNA 15-5 lines. Furthermore, examination of the phosphorylation status of bril-EMS-suppressor 1 and measurement of BR-responsive gene expression revealed that membrane microdomains affect BR signaling. These results suggest that BR promotes the partitioning of BRI1 into functional membrane microdomains to activate BR signaling.展开更多
Growth and immunity are opposing processes that compete for cellular resources,and proper resource allocation is crucial for plant survival.BSK1 plays a key role in the regulation of both growth and immunity by associ...Growth and immunity are opposing processes that compete for cellular resources,and proper resource allocation is crucial for plant survival.BSK1 plays a key role in the regulation of both growth and immunity by associating with BRI1 and FLS2,respectively.However,it remains unclear how two antagonistic signals co-opt BSK1 to induce signal-specific activation.Here we show that the dynamic spatial reorganiization of BSK1 within the plasma membrane underlies the mechanism of signal-specific activation for growth or immunity.Resting BSK1 localizes to membrane rafts as complexes.Unlike BSK1-associated FLS2 and BRI1,flg22 or exogenous brassinosteroid(BR)treatment did not decrease BSK1 levels at the plasma membrane(PM)but rather induced BSK1 multimerization and dissociation from FLS2/BSK1 or BRI1/BSK1,respectively.Moreover,flg22-activated BSK1 translocated from membrane rafts to non-membrane-raft regions,whereas BR-activated BSK1 remained in membrane rafts.When applied together with flg22,BR suppressed various flg22-induced BSK1 activities such as BSK1 dissociation from FLS2/BSK1,BSK1 interaction with MAPKKK5,and BSK translocation together with MAPKKK5.Taken together,this study provides a unique insight into how the precise control of BSK1 spatiotemporal organization regulates the signaling specificity to balance plant growth and immunity.展开更多
The most obvious symptom of Al toxicity is the inhibition of root growth. However, the mechanism of Al-inhibiting root growth remains to be elucidated. In this study, auxin transport and vesicle movement of an auxin-e...The most obvious symptom of Al toxicity is the inhibition of root growth. However, the mechanism of Al-inhibiting root growth remains to be elucidated. In this study, auxin transport and vesicle movement of an auxin-efflux carrier (PIN2) were investigated in Arabidopsis roots in response to Al stress. Results indicated that Al inhibited the apical transport of auxin in root tips of Arabidopsis significantly. The severe inhibition was localized in the cells of transition zone, where the concentration of auxin was only 34% that of the control. Brefeldin A (BFA), an inhibitor of vesicle transport, induced the dot-like structure of PIN2 vesicle significantly. Al decreased the size of dot-like structure of PIN2 vesicles. Re- sults of real-time RT-PCR and Western-blotting analysis showed that Al increased the transcript level of PIN2 and the accumulation of PIN2 protein in horizontal direction of plasma membrane, but decreased its distribution in endosomes, suggesting that Al inhibited the transport of PIN2 vesicles from plasma membrane to endosomes. Results of cytoskeleton-depolymering drugs indicated that it was via the pathway of disruption of actin microfilaments that Al inhibited the transport of PIN2 vesicles. Exposed to Al stress, the cells of elongation zone had less Al uptake and less transport frequency of vesicles than cells of transition zone. Taken together, our results suggested that Al inhibited root growth mainly by modulating the transport of PIN2 vesicles between plasma membrane and endosomes, thus block- ing auxin transport and root growth.展开更多
Phototropin （phot）-mediated signaling initiated by blue light （BL） plays a critical role in optimizing photosyn- thetic light capture at the plasma membrane （PM） in plants. However, the mechanisms underlying the...Phototropin （phot）-mediated signaling initiated by blue light （BL） plays a critical role in optimizing photosyn- thetic light capture at the plasma membrane （PM） in plants. However, the mechanisms underlying the regu- lation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and stepwise photobleaching analysis of photl-GFP proteins we demonstrated that in the dark photl proteins remain in an inactive state and mostly exist as monomers. Dimerization and the diffusion rate of photl-GFP increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive photlD806N-GFP but did enhance its dimerization, suggesting that photl dimerization is independent of phosphorylation. Forster resonance energy transfer-fluorescence life- time imaging microscopy analysis revealed that the interaction between photl-GFP and a marker of sterol- rich lipid environments, AtRem1.3-mCherry, was enhanced with increased time of BL treatment. However, this BL-dependent interaction was not obvious in plants co-expressing phot1D806N-GFP and AtRem1.3- mCherry, indicating that BL facilitates the translocation of functional photl-GFP into AtRem1.3-1abeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated photl-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane micro- domains act as organizing platforms essential for the proper function of activated photl at the PM.展开更多
Arabidopsis synaptotagmin 2 （SYT2） has been reported to participate in an unconventional secretory pathway in somatic cells. Our results showed that SYT2 was expressed mainly in the pollen ofArabidopsis thaliana. Th...Arabidopsis synaptotagmin 2 （SYT2） has been reported to participate in an unconventional secretory pathway in somatic cells. Our results showed that SYT2 was expressed mainly in the pollen ofArabidopsis thaliana. The pollen of syt2 T-DNA and RNA interference mutant lines exhibited reduced total germination and impeded pollen tube growth. Analysis of the expression of SYT2-GFP fusion protein in the pollen tube indicates that SYT2 was localized to distinct, patchy compartments but could co-localize with the Golgi markers, BODIPY TR C5 ceramide and GmManl-mCherry. However, SYT2-DsRed-E5 was localized to the plasma membrane in Arabidopsis suspension cells, in addition to the Golgi apparatus. The localization of SYT2 at the plasma membrane was further supported by immunofluorescence staining in pollen tubes. Moreover, brefeldin A treatment inhibited the transport of SYT2 to the plasma membrane and caused SYT2 to aggregate and form enlarged compartments. Truncation of the SYT2-C2AB domains also resulted in retention of SYT2 in the Golgi apparatus. An in vitro phospholipid-binding assay showed that SYT2-C2AB domains bind to the phospholipid membrane in a calcium-dependent manner. Take together, our results indicated that SYT2 was required for pollen germination and pollen tube growth, and was involved in conventional exocytosis.展开更多
Phototropin 1 （photl） is a photoreceptor for phototropism, chloroplast movement, stomatal opening, leaf expansion, and solar tracking in response to blue light. Following earlier work with PHOTI：：GFP （Sakamoto an...Phototropin 1 （photl） is a photoreceptor for phototropism, chloroplast movement, stomatal opening, leaf expansion, and solar tracking in response to blue light. Following earlier work with PHOTI：：GFP （Sakamoto and Briggs, 2002）, we investigated the pattern of cellular and subcellular localization of photl in 3-4 d old etiolated seedlings of Arabidopsis thalinana. As expressed from native upstream sequences, the PHOTT：GFP fusion protein is expressed strongly in the abaxial tissues of the cotyledons and in the elongating regions of the hypocotyl. It is moderately expressed in the shoot/root transition zone and in cells near the root apex. A fluorescence signal is undetectable in the root epidermis, root cap, and root apical meristem itself. The plasma membranes of mesophyll cells near the cotyledon margin appear labeled uniformly but cross-walls created by recent cell divisions are more strongly labeled. The pattern of labeling of individual cell types varies with cell type and developmental stage. Blue-light treatment causes PHOTI：：GFP, initially relatively evenly distributed at the plasma membrane, to become reorganized into a distinct mosaic with strongly labeled punctate areas and other areas completely devoid of fluorescenco a phenomenon best observed in cortical cells in the hypocotyl elongation region. Concomitant with or following this reorganization, PHOTT：GFP moves into the cytoplasm in all cell types investigated except for guard cells. It disappears from the cytoplasm by an unidentified mechanism after several hours in darkness. Neither its appearance in the cytoplasm nor its eventual disappearance in darkness is prevented by the translation inhibitor cycloheximide, although the latter process is retarded. We hypothesize that blue-light-induced photl relocalization modulates blue-light-activated signal transduction.展开更多
Unlike actively moving animals,plants actively change their body architecture via growth of their diverse organs in response to environmental clues and stresses.For example,changes in Arabidopsis thaliana root apex zo...Unlike actively moving animals,plants actively change their body architecture via growth of their diverse organs in response to environmental clues and stresses.For example,changes in Arabidopsis thaliana root apex zonation under phosphate(Pi)deficiency is considered to be such an active response,including inhibition of primary root elongation and increasing densities of lateral roots and root hairs(Abel,2017).However,a recent study by Zheng et al.(2019)modifies this hypothesis,which is based on the transparent Petri dish-based culture method of young Arabidopsis seedlings causing illumination of roots.In this issue,Zheng et al.(2019)report that Pi deficiency not only increases malate secretion from Arabidopsis root apices via the ALMT1 transporter but also that blue light(BL)triggers a malate-mediated photo-Fenton reaction in the rhizosphere,increasing the OH radical levels.展开更多
文摘The major brassinosteroid （BR） receptor of Arabidopsis BRASSINOSTEROID INSENSITIVE1 （BRI1） plays fundamental roles in BR signaling, but the molecular mechanisms underlying the effects of BR on BRI1 internalization and assembly state remain unclear. Here, we applied variable angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy to analyze the dynamics of GFP-tagged BRII. We found that, in response to BR, the degree of co-localization of BRI1-GFP with AtFIotl-mCherry increased, and especially BR stimulated the membrane microdomain-associated pathway of BRI1 internalization. We also verified these observations in endocytosis-defective chc2-1 mutants and the AtFIotl amiRNA 15-5 lines. Furthermore, examination of the phosphorylation status of bril-EMS-suppressor 1 and measurement of BR-responsive gene expression revealed that membrane microdomains affect BR signaling. These results suggest that BR promotes the partitioning of BRI1 into functional membrane microdomains to activate BR signaling.
基金This work was supported by the Program of Introducing Talents of Discipline to Universities(111 Project,B13007 to J.L.)the National Natural Science Foundation of China(32030010 and 31530084 to J.L.,31871424 to X.S.).
文摘Growth and immunity are opposing processes that compete for cellular resources,and proper resource allocation is crucial for plant survival.BSK1 plays a key role in the regulation of both growth and immunity by associating with BRI1 and FLS2,respectively.However,it remains unclear how two antagonistic signals co-opt BSK1 to induce signal-specific activation.Here we show that the dynamic spatial reorganiization of BSK1 within the plasma membrane underlies the mechanism of signal-specific activation for growth or immunity.Resting BSK1 localizes to membrane rafts as complexes.Unlike BSK1-associated FLS2 and BRI1,flg22 or exogenous brassinosteroid(BR)treatment did not decrease BSK1 levels at the plasma membrane(PM)but rather induced BSK1 multimerization and dissociation from FLS2/BSK1 or BRI1/BSK1,respectively.Moreover,flg22-activated BSK1 translocated from membrane rafts to non-membrane-raft regions,whereas BR-activated BSK1 remained in membrane rafts.When applied together with flg22,BR suppressed various flg22-induced BSK1 activities such as BSK1 dissociation from FLS2/BSK1,BSK1 interaction with MAPKKK5,and BSK translocation together with MAPKKK5.Taken together,this study provides a unique insight into how the precise control of BSK1 spatiotemporal organization regulates the signaling specificity to balance plant growth and immunity.
基金the National Natural Science Foundation of China (Grant Nos. 30771294 and 30471040)the International Foundation for Science of Sweden (Grant Nos. C/3042-1,2), and the DAAD (Ref423)
文摘The most obvious symptom of Al toxicity is the inhibition of root growth. However, the mechanism of Al-inhibiting root growth remains to be elucidated. In this study, auxin transport and vesicle movement of an auxin-efflux carrier (PIN2) were investigated in Arabidopsis roots in response to Al stress. Results indicated that Al inhibited the apical transport of auxin in root tips of Arabidopsis significantly. The severe inhibition was localized in the cells of transition zone, where the concentration of auxin was only 34% that of the control. Brefeldin A (BFA), an inhibitor of vesicle transport, induced the dot-like structure of PIN2 vesicle significantly. Al decreased the size of dot-like structure of PIN2 vesicles. Re- sults of real-time RT-PCR and Western-blotting analysis showed that Al increased the transcript level of PIN2 and the accumulation of PIN2 protein in horizontal direction of plasma membrane, but decreased its distribution in endosomes, suggesting that Al inhibited the transport of PIN2 vesicles from plasma membrane to endosomes. Results of cytoskeleton-depolymering drugs indicated that it was via the pathway of disruption of actin microfilaments that Al inhibited the transport of PIN2 vesicles. Exposed to Al stress, the cells of elongation zone had less Al uptake and less transport frequency of vesicles than cells of transition zone. Taken together, our results suggested that Al inhibited root growth mainly by modulating the transport of PIN2 vesicles between plasma membrane and endosomes, thus block- ing auxin transport and root growth.
基金This work is supported by the National Natural Science Foundation of China （31530084, 31270224） and the Program of Introducing Talents of Discipline to Universities （111 project, B13007）.
文摘Phototropin （phot）-mediated signaling initiated by blue light （BL） plays a critical role in optimizing photosyn- thetic light capture at the plasma membrane （PM） in plants. However, the mechanisms underlying the regu- lation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and stepwise photobleaching analysis of photl-GFP proteins we demonstrated that in the dark photl proteins remain in an inactive state and mostly exist as monomers. Dimerization and the diffusion rate of photl-GFP increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive photlD806N-GFP but did enhance its dimerization, suggesting that photl dimerization is independent of phosphorylation. Forster resonance energy transfer-fluorescence life- time imaging microscopy analysis revealed that the interaction between photl-GFP and a marker of sterol- rich lipid environments, AtRem1.3-mCherry, was enhanced with increased time of BL treatment. However, this BL-dependent interaction was not obvious in plants co-expressing phot1D806N-GFP and AtRem1.3- mCherry, indicating that BL facilitates the translocation of functional photl-GFP into AtRem1.3-1abeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated photl-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane micro- domains act as organizing platforms essential for the proper function of activated photl at the PM.
文摘Arabidopsis synaptotagmin 2 （SYT2） has been reported to participate in an unconventional secretory pathway in somatic cells. Our results showed that SYT2 was expressed mainly in the pollen ofArabidopsis thaliana. The pollen of syt2 T-DNA and RNA interference mutant lines exhibited reduced total germination and impeded pollen tube growth. Analysis of the expression of SYT2-GFP fusion protein in the pollen tube indicates that SYT2 was localized to distinct, patchy compartments but could co-localize with the Golgi markers, BODIPY TR C5 ceramide and GmManl-mCherry. However, SYT2-DsRed-E5 was localized to the plasma membrane in Arabidopsis suspension cells, in addition to the Golgi apparatus. The localization of SYT2 at the plasma membrane was further supported by immunofluorescence staining in pollen tubes. Moreover, brefeldin A treatment inhibited the transport of SYT2 to the plasma membrane and caused SYT2 to aggregate and form enlarged compartments. Truncation of the SYT2-C2AB domains also resulted in retention of SYT2 in the Golgi apparatus. An in vitro phospholipid-binding assay showed that SYT2-C2AB domains bind to the phospholipid membrane in a calcium-dependent manner. Take together, our results indicated that SYT2 was required for pollen germination and pollen tube growth, and was involved in conventional exocytosis.
基金We thank Tong-Seung Tseng for verifying the efficacy of the cycloheximide treatment in stopping protein synthesis and Inseob Han and Margaret Olney for testing the phototropic sensitivity of the plants expressing PHOT：：GFP in the double mutant background. We also thank all three of them for many useful discussions related to this study. This work was supported by National Science Foundation Grant 0444504. The authors are grateful for this support.
文摘Phototropin 1 （photl） is a photoreceptor for phototropism, chloroplast movement, stomatal opening, leaf expansion, and solar tracking in response to blue light. Following earlier work with PHOTI：：GFP （Sakamoto and Briggs, 2002）, we investigated the pattern of cellular and subcellular localization of photl in 3-4 d old etiolated seedlings of Arabidopsis thalinana. As expressed from native upstream sequences, the PHOTT：GFP fusion protein is expressed strongly in the abaxial tissues of the cotyledons and in the elongating regions of the hypocotyl. It is moderately expressed in the shoot/root transition zone and in cells near the root apex. A fluorescence signal is undetectable in the root epidermis, root cap, and root apical meristem itself. The plasma membranes of mesophyll cells near the cotyledon margin appear labeled uniformly but cross-walls created by recent cell divisions are more strongly labeled. The pattern of labeling of individual cell types varies with cell type and developmental stage. Blue-light treatment causes PHOTI：：GFP, initially relatively evenly distributed at the plasma membrane, to become reorganized into a distinct mosaic with strongly labeled punctate areas and other areas completely devoid of fluorescenco a phenomenon best observed in cortical cells in the hypocotyl elongation region. Concomitant with or following this reorganization, PHOTT：GFP moves into the cytoplasm in all cell types investigated except for guard cells. It disappears from the cytoplasm by an unidentified mechanism after several hours in darkness. Neither its appearance in the cytoplasm nor its eventual disappearance in darkness is prevented by the translation inhibitor cycloheximide, although the latter process is retarded. We hypothesize that blue-light-induced photl relocalization modulates blue-light-activated signal transduction.
文摘Unlike actively moving animals,plants actively change their body architecture via growth of their diverse organs in response to environmental clues and stresses.For example,changes in Arabidopsis thaliana root apex zonation under phosphate(Pi)deficiency is considered to be such an active response,including inhibition of primary root elongation and increasing densities of lateral roots and root hairs(Abel,2017).However,a recent study by Zheng et al.(2019)modifies this hypothesis,which is based on the transparent Petri dish-based culture method of young Arabidopsis seedlings causing illumination of roots.In this issue,Zheng et al.(2019)report that Pi deficiency not only increases malate secretion from Arabidopsis root apices via the ALMT1 transporter but also that blue light(BL)triggers a malate-mediated photo-Fenton reaction in the rhizosphere,increasing the OH radical levels.
