Periodic Reporting for period 1 - THyM (Why is Transparent Hypocotyl Mutant showed reduced phototropic response?)
Período documentado: 2020-03-01 hasta 2022-02-28
Plants capture sunlight efficiently by bending towards favorable light conditions by a mechanism known as phototropism. The phototropic response is ubiquitously conserved across the plant kingdom, contributing to the optimal seedling establishment and higher yields in crop plants.
The directional light leads to a light gradient across the photosensory tissue, such as coleoptiles in monocots or hypocotyls in dicot plants. Blue light photoreceptors-phototropin detect the light gradient to activate downstream signaling, establishing auxin gradient leading to asymmetric growth of the photo-stimulated stem. Despite progress in understanding the molecular mechanisms of phototropism, the connecting link between light perception and auxin redistribution remain unclear. Thus, the Fankhauser group conducted a genetic screen for altered phototropism mutants and identified a gene encoding an ABC (ATP-binding cassette) transporter as essential for phototropism. The project's primary objective is to identify the function of AtABC in phototropism. The Atabc mutant is characterized by a transparent hypocotyl which allowed us to test the importance of the light gradient across photo-stimulated hypocotyl for phototropic bending.
Our results indicate that the Atabc mutant is specifically defective for hypocotyl phototropism as gravitopic hypocotyl reorientation remains normal in the mutant. Normal early light signaling events in the Atabc mutant and the requirement of active photoreceptor phot1 to explain the aberrant growth re-orientation in the Atabc mutant suggest that AtABC functions upstream of light perception. The study of optical properties of etiolated seedlings and shallow light gradient experiments allowed us to conclude that the light gradient in the photosensory hypocotyl is altered in the Atabc mutant.
The directional light leads to a light gradient across the photosensory tissue, such as coleoptiles in monocots or hypocotyls in dicot plants. Blue light photoreceptors-phototropin detect the light gradient to activate downstream signaling, establishing auxin gradient leading to asymmetric growth of the photo-stimulated stem. Despite progress in understanding the molecular mechanisms of phototropism, the connecting link between light perception and auxin redistribution remain unclear. Thus, the Fankhauser group conducted a genetic screen for altered phototropism mutants and identified a gene encoding an ABC (ATP-binding cassette) transporter as essential for phototropism. The project's primary objective is to identify the function of AtABC in phototropism. The Atabc mutant is characterized by a transparent hypocotyl which allowed us to test the importance of the light gradient across photo-stimulated hypocotyl for phototropic bending.
Our results indicate that the Atabc mutant is specifically defective for hypocotyl phototropism as gravitopic hypocotyl reorientation remains normal in the mutant. Normal early light signaling events in the Atabc mutant and the requirement of active photoreceptor phot1 to explain the aberrant growth re-orientation in the Atabc mutant suggest that AtABC functions upstream of light perception. The study of optical properties of etiolated seedlings and shallow light gradient experiments allowed us to conclude that the light gradient in the photosensory hypocotyl is altered in the Atabc mutant.
To understand the role of AtABC in hypocotyl growth re-orientation we analyzed gravitropism and phototropism in Atabc mutants. Our results showed that random bending of the mutant is specific to light as a gravi-reorientation response is normal. This result suggests that the Atabc mutant is not defective in auxin signaling events that occur downstream of both gravi- and photo-stimulation. Blue light excitation results in autophosphorylation of phototropin, leading to the initiation of phot-mediated signaling. Our analysis of a double mutant phot1 Atabc showed that random bending of the Atabc mutant requires active phot1. Our studies of the phosphorylation status of phot1, NPH3 and PKS4 in response to blue light suggest normal light signaling in the Atabc mutant.
We focused on understanding the reason behind the reduced phototropism in the Atabc mutant. It is known that the upper region of hypocotyl acts as a site of light perception. The scattering and absorption of light while passing through plant tissue leads to a drop in light intensity across photosensory tissue, establishing a light gradient. Interestingly, the hypocotyl of Atabc mutant is transparent, and the analyses of optical properties of etiolated seedlings using an integrating sphere showed that scattering and reflection are reduced in the Atabc mutant. Based on these results, we hypothesized that the amount of light received at the lid side and shaded side would lead to a shallow light gradient in the Atabc mutant compared to WT. To test this hypothesis, we exposed etiolated seedlings to blue light from two sides and found that WT plants efficiently bend towards stronger light while the Atabc mutant fails to respond to small differences in light intensity. These results suggest that the light gradient is altered in the Atabc mutant. We tried to restore the light gradient by expressing the red pigment betalain, which has an absorption peak in the blue region. The over-accumulation of betalain leads to inhibition of phototropic bending in WT and Atabc mutant, pointing toward the importance of light scattering in establishing a light gradient.
We explore the molecular function of AtABC by checking its sub-cellular and tissue-specific localization. We found that AtABC is a plasma membrane-localized protein. The overexpression lines (Pro35S: GFP-ABC) failed to recover the transparent hypocotyl phenotype, whereas complementation lines (ProABC: GFP-ABC) rescued the mutant phenotype. This result implies the requirement of AtABC expression in the ovules and developing embryos for normal hypocotyl development. To understand the reason behind the transparent hypocotyl phenotype of the Atabc mutant, we compared the optical properties of water infiltrated WT seedlings with the Atabc mutant. Our results showed that the optical properties of water infiltrated WT seedlings and Atabc mutant are similar, suggesting a possible role of air spaces between cells in promoting light scattering. These results were also supported by our TEM and CryoSEM examinations of transverse hypocotyl cuts, highlighting the requirement of AtABC in maintaining the air spaces between cells in the cortex region of the hypocotyl.
It is essential to communicate our project results to a vast audience to boost the impact of the work. I presented these results in departmental seminars and international scientific conferences. To reach the broader scientific community, we have planned to submit these results to an open-access journal. We shared our results outside the scientific community, such as with school students and families curious about science. We prepared some online interactive activities (https://wp.unil.ch/mysteres/) and small videos (https://www.youtube.com/watch?v=T-WoGsoLk5g&t=70s) to attract and create awareness in people toward the unique plants' world.
We focused on understanding the reason behind the reduced phototropism in the Atabc mutant. It is known that the upper region of hypocotyl acts as a site of light perception. The scattering and absorption of light while passing through plant tissue leads to a drop in light intensity across photosensory tissue, establishing a light gradient. Interestingly, the hypocotyl of Atabc mutant is transparent, and the analyses of optical properties of etiolated seedlings using an integrating sphere showed that scattering and reflection are reduced in the Atabc mutant. Based on these results, we hypothesized that the amount of light received at the lid side and shaded side would lead to a shallow light gradient in the Atabc mutant compared to WT. To test this hypothesis, we exposed etiolated seedlings to blue light from two sides and found that WT plants efficiently bend towards stronger light while the Atabc mutant fails to respond to small differences in light intensity. These results suggest that the light gradient is altered in the Atabc mutant. We tried to restore the light gradient by expressing the red pigment betalain, which has an absorption peak in the blue region. The over-accumulation of betalain leads to inhibition of phototropic bending in WT and Atabc mutant, pointing toward the importance of light scattering in establishing a light gradient.
We explore the molecular function of AtABC by checking its sub-cellular and tissue-specific localization. We found that AtABC is a plasma membrane-localized protein. The overexpression lines (Pro35S: GFP-ABC) failed to recover the transparent hypocotyl phenotype, whereas complementation lines (ProABC: GFP-ABC) rescued the mutant phenotype. This result implies the requirement of AtABC expression in the ovules and developing embryos for normal hypocotyl development. To understand the reason behind the transparent hypocotyl phenotype of the Atabc mutant, we compared the optical properties of water infiltrated WT seedlings with the Atabc mutant. Our results showed that the optical properties of water infiltrated WT seedlings and Atabc mutant are similar, suggesting a possible role of air spaces between cells in promoting light scattering. These results were also supported by our TEM and CryoSEM examinations of transverse hypocotyl cuts, highlighting the requirement of AtABC in maintaining the air spaces between cells in the cortex region of the hypocotyl.
It is essential to communicate our project results to a vast audience to boost the impact of the work. I presented these results in departmental seminars and international scientific conferences. To reach the broader scientific community, we have planned to submit these results to an open-access journal. We shared our results outside the scientific community, such as with school students and families curious about science. We prepared some online interactive activities (https://wp.unil.ch/mysteres/) and small videos (https://www.youtube.com/watch?v=T-WoGsoLk5g&t=70s) to attract and create awareness in people toward the unique plants' world.
The results obtained in this project provide strong evidence for the old thought of the scientific community that plants detect the light direction by measuring light gradients across tissues. The air spaces within hypocotyl tissue are essential for light scattering will draw the interest of developmental biologists to follow the question, "how did air spaces develop in plant tissue?" Understanding plant architecture responsible for light scattering and the establishment of the light gradient will influence plant scientists and physicists to develop new technologies to harvest solar energy. Moreover, our efforts to communicate results to common people will help draw their interest in plants' fascinating ways to adapt to their surrounding environment.