Understanding how plant cells adapt dynamically to changes in the environment is a fundamental problem of plant biology. Under many conditions, plant cells respond to environmental changes by modifying their intracell...Understanding how plant cells adapt dynamically to changes in the environment is a fundamental problem of plant biology. Under many conditions, plant cells respond to environmental changes by modifying their intracellular organization. A critical example of intracellular reorganization is chloroplast photo-relocation, which is required for optimal energy harvesting and avoiding photodamage. A key system responsible for the spatial organization of intracellular components is the microtubule cytoskeleton and its associated motor proteins, kinesins. Here we tested the hypothesis that members of the kinesin 4II subfamily are important for chloroplast photo-relocation in the moss Physcomitrella patens. Most land plants, including P. patens, use an actin cytoskeleton-dependent mechanism to transport chloroplasts in response to light. In addition to the actin-based system, P. patens can also transport chloroplasts via a microtubule-dependent mechanism, which is absent in flowering plants. Here, we used a P. patens line that contains an inducible RNAi system to silence all three kinesin 4-II genes present in this moss and evaluated their participation in the microtubule-dependent chloroplast light avoidance response. Because we found a significant effect on cell growth when kinesin 4IIs are silenced, we took advantage of the inducible system to establish a reproducible and quantitative assay to evaluate chloroplast photo-relocation in full-grown cells. Using a laser scanning confocal-based chloroplast light avoidance response assay, we found a reduction in chloroplast motility when kinesin 4IIs were silenced. Hence, in addition to identifying a role for kinesin 4II proteins in protonemal cell growth, our results strongly support the hypothesis that these kinesins play an important role in the chloroplast light avoidance response.展开更多
In plants, light determines chloroplast position; these organelles show avoidance and accumulation re- sponses in high and low fluence-rate light, respectively. Chloroplast motility in response to light is driven by c...In plants, light determines chloroplast position; these organelles show avoidance and accumulation re- sponses in high and low fluence-rate light, respectively. Chloroplast motility in response to light is driven by cytoskeletal elements. The actin cytoskeleton mediates chloroplast photorelocation responses in Arabidopsis thali- ana. In contrast, in the moss Physcomitrella patens, both, actin filaments and microtubules can transport chloroplasts. Because of the surprising evidence that two kinesin-like proteins （called KACs） are important for actin-dependent chloroplast photorelocation in vascular plants, we wanted to determine the cytoskeletal system responsible for the function of these proteins in moss. We performed gene- specific silencing using RNA interference in P. patens. We confirmed existing reports using gene knockouts, that PpKAC1 and PpKAC2 are required for chloroplast dispersion under uniform white light conditions, and that the two proteins are functionally equivalent. To address the specificcytoskeletal elements responsible for motility, this loss-of- function approach was combined with cytoskeleton-targeted drug studies. We found that, in P. patens, these KACs mediate the chloroplast light-avoidance response in an actin filament- dependent, rather than a microtubule-dependent manner. Using correlation-decay analysis of cytoskeletal dynamics, we found that PpKAC stabilizes cortical actin filaments, but has no effect on microtubule dynamics.展开更多
文摘Understanding how plant cells adapt dynamically to changes in the environment is a fundamental problem of plant biology. Under many conditions, plant cells respond to environmental changes by modifying their intracellular organization. A critical example of intracellular reorganization is chloroplast photo-relocation, which is required for optimal energy harvesting and avoiding photodamage. A key system responsible for the spatial organization of intracellular components is the microtubule cytoskeleton and its associated motor proteins, kinesins. Here we tested the hypothesis that members of the kinesin 4II subfamily are important for chloroplast photo-relocation in the moss Physcomitrella patens. Most land plants, including P. patens, use an actin cytoskeleton-dependent mechanism to transport chloroplasts in response to light. In addition to the actin-based system, P. patens can also transport chloroplasts via a microtubule-dependent mechanism, which is absent in flowering plants. Here, we used a P. patens line that contains an inducible RNAi system to silence all three kinesin 4-II genes present in this moss and evaluated their participation in the microtubule-dependent chloroplast light avoidance response. Because we found a significant effect on cell growth when kinesin 4IIs are silenced, we took advantage of the inducible system to establish a reproducible and quantitative assay to evaluate chloroplast photo-relocation in full-grown cells. Using a laser scanning confocal-based chloroplast light avoidance response assay, we found a reduction in chloroplast motility when kinesin 4IIs were silenced. Hence, in addition to identifying a role for kinesin 4II proteins in protonemal cell growth, our results strongly support the hypothesis that these kinesins play an important role in the chloroplast light avoidance response.
基金supported by WPI startup funds to LV and ETthe Eppley Foundation for Research
文摘In plants, light determines chloroplast position; these organelles show avoidance and accumulation re- sponses in high and low fluence-rate light, respectively. Chloroplast motility in response to light is driven by cytoskeletal elements. The actin cytoskeleton mediates chloroplast photorelocation responses in Arabidopsis thali- ana. In contrast, in the moss Physcomitrella patens, both, actin filaments and microtubules can transport chloroplasts. Because of the surprising evidence that two kinesin-like proteins （called KACs） are important for actin-dependent chloroplast photorelocation in vascular plants, we wanted to determine the cytoskeletal system responsible for the function of these proteins in moss. We performed gene- specific silencing using RNA interference in P. patens. We confirmed existing reports using gene knockouts, that PpKAC1 and PpKAC2 are required for chloroplast dispersion under uniform white light conditions, and that the two proteins are functionally equivalent. To address the specificcytoskeletal elements responsible for motility, this loss-of- function approach was combined with cytoskeleton-targeted drug studies. We found that, in P. patens, these KACs mediate the chloroplast light-avoidance response in an actin filament- dependent, rather than a microtubule-dependent manner. Using correlation-decay analysis of cytoskeletal dynamics, we found that PpKAC stabilizes cortical actin filaments, but has no effect on microtubule dynamics.
