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Journal articles on the topic ""light harvesting stress related protein 1""
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Girolomoni, Laura, Stefano Cazzaniga, Alberta Pinnola, Federico Perozeni, Matteo Ballottari, and Roberto Bassi. "LHCSR3 is a nonphotochemical quencher of both photosystems inChlamydomonas reinhardtii." Proceedings of the National Academy of Sciences 116, no. 10 (February 19, 2019): 4212–17. http://dx.doi.org/10.1073/pnas.1809812116.
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Photosynthetic organisms prevent oxidative stress from light energy absorbed in excess through several photoprotective mechanisms. A major component is thermal dissipation of chlorophyll singlet excited states and is called nonphotochemical quenching (NPQ). NPQ is catalyzed in green algae by protein subunits called LHCSRs (Light Harvesting Complex Stress Related), homologous to the Light Harvesting Complexes (LHC), constituting the antenna system of both photosystem I (PSI) and PSII. We investigated the role of LHCSR1 and LHCSR3 in NPQ activation to verify whether these proteins are involved in thermal dissipation of PSI excitation energy, in addition to their well-known effect on PSII. To this aim, we measured the fluorescence emitted at 77 K by whole cells in a quenched or unquenched state, using green fluorescence protein as the internal standard. We show that NPQ activation by high light treatment inChlamydomonas reinhardtiileads to energy quenching in both PSI and PSII antenna systems. By analyzing quenching properties of mutants affected on the expression of LHCSR1 or LHCSR3 gene products and/or state 1–state 2 transitions or zeaxanthin accumulation, namely,npq4,stt7,stt7 npq4,npq4 lhcsr1,lhcsr3-complementednpq4 lhcsr1andnpq1, we showed that PSI undergoes NPQ through quenching of the associated LHCII antenna. This quenching event is fast-reversible on switching the light off, is mainly related to LHCSR3 activity, and is dependent on thylakoid luminal pH. Moreover, PSI quenching could also be observed in the absence of zeaxanthin or STT7 kinase activity.
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Wu, Pei, Qiusheng Kong, Jirong Bian, Golam Jalal Ahammed, Huimei Cui, Wei Xu, Zhifeng Yang, Jinxia Cui, and Huiying Liu. "Unveiling Molecular Mechanisms of Nitric Oxide-Induced Low-Temperature Tolerance in Cucumber by Transcriptome Profiling." International Journal of Molecular Sciences 23, no. 10 (May 17, 2022): 5615. http://dx.doi.org/10.3390/ijms23105615.
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Cucumber (Cucumis sativus L.) is one of the most popular cultivated vegetable crops but it is intrinsically sensitive to cold stress due to its thermophilic nature. To explore the molecular mechanism of plant response to low temperature (LT) and the mitigation effect of exogenous nitric oxide (NO) on LT stress in cucumber, transcriptome changes in cucumber leaves were compared. The results showed that LT stress regulated the transcript level of genes related to the cell cycle, photosynthesis, flavonoid accumulation, lignin synthesis, active gibberellin (GA), phenylalanine metabolism, phytohormone ethylene and salicylic acid (SA) signaling in cucumber seedlings. Exogenous NO improved the LT tolerance of cucumber as reflected by increased maximum photochemical efficiency (Fv/Fm) and decreased chilling damage index (CI), electrolyte leakage and malondialdehyde (MDA) content, and altered transcript levels of genes related to phenylalanine metabolism, lignin synthesis, plant hormone (SA and ethylene) signal transduction, and cell cycle. In addition, we found four differentially expressed transcription factors (MYB63, WRKY21, HD-ZIP, and b-ZIP) and their target genes such as the light-harvesting complex I chlorophyll a/b binding protein 1 gene (LHCA1), light-harvesting complex II chlorophyll a/b binding protein 1, 3, and 5 genes (LHCB1, LHCB3, and LHCB5), chalcone synthase gene (CSH), ethylene-insensitive protein 3 gene (EIN3), peroxidase, phenylalanine ammonia-lyase gene (PAL), DNA replication licensing factor gene (MCM5 and MCM6), gibberellin 3 beta-dioxygenase gene (GA3ox), and regulatory protein gene (NPRI), which are potentially associated with plant responses to NO and LT stress. Notably, HD-ZIP and b-ZIP specifically responded to exogenous NO under LT stress. Taken together, these results demonstrate that cucumber seedlings respond to LT stress and exogenous NO by modulating the transcription of some key transcription factors and their downstream genes, thereby regulating photosynthesis, lignin synthesis, plant hormone signal transduction, phenylalanine metabolism, cell cycle, and GA synthesis. Our study unveiled potential molecular mechanisms of plant response to LT stress and indicated the possibility of NO application in cucumber production under LT stress, particularly in winter and early spring.
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Kondo, Toru, Jesse B. Gordon, Alberta Pinnola, Luca Dall’Osto, Roberto Bassi, and Gabriela S. Schlau-Cohen. "Microsecond and millisecond dynamics in the photosynthetic protein LHCSR1 observed by single-molecule correlation spectroscopy." Proceedings of the National Academy of Sciences 116, no. 23 (May 17, 2019): 11247–52. http://dx.doi.org/10.1073/pnas.1821207116.
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Biological systems are subjected to continuous environmental fluctuations, and therefore, flexibility in the structure and function of their protein building blocks is essential for survival. Protein dynamics are often local conformational changes, which allows multiple dynamical processes to occur simultaneously and rapidly in individual proteins. Experiments often average over these dynamics and their multiplicity, preventing identification of the molecular origin and impact on biological function. Green plants survive under high light by quenching excess energy, and Light-Harvesting Complex Stress Related 1 (LHCSR1) is the protein responsible for quenching in moss. Here, we expand an analysis of the correlation function of the fluorescence lifetime by improving the estimation of the lifetime states and by developing a multicomponent model correlation function, and we apply this analysis at the single-molecule level. Through these advances, we resolve previously hidden rapid dynamics, including multiple parallel processes. By applying this technique to LHCSR1, we identify and quantitate parallel dynamics on hundreds of microseconds and tens of milliseconds timescales, likely at two quenching sites within the protein. These sites are individually controlled in response to fluctuations in sunlight, which provides robust regulation of the light-harvesting machinery. Considering our results in combination with previous structural, spectroscopic, and computational data, we propose specific pigments that serve as the quenching sites. These findings, therefore, provide a mechanistic basis for quenching, illustrating the ability of this method to uncover protein function.
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Correa-Galvis, Viviana, Petra Redekop, Katharine Guan, Annika Griess, Thuy B. Truong, Setsuko Wakao, Krishna K. Niyogi, and Peter Jahns. "Photosystem II Subunit PsbS Is Involved in the Induction of LHCSR Protein-dependent Energy Dissipation in Chlamydomonas reinhardtii." Journal of Biological Chemistry 291, no. 33 (June 29, 2016): 17478–87. http://dx.doi.org/10.1074/jbc.m116.737312.
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Non-photochemical quenching of excess excitation energy is an important photoprotective mechanism in photosynthetic organisms. In Arabidopsis thaliana, a high quenching capacity is constitutively present and depends on the PsbS protein. In the green alga Chlamydomonas reinhardtii, non-photochemical quenching becomes activated upon high light acclimation and requires the accumulation of light harvesting complex stress-related (LHCSR) proteins. Expression of the PsbS protein in C. reinhardtii has not been reported yet. Here, we show that PsbS is a light-induced protein in C. reinhardtii, whose accumulation under high light is further controlled by CO2 availability. PsbS accumulated after several hours of high light illumination at low CO2. At high CO2, however, PsbS was only transiently expressed under high light and was degraded after 1 h of high light exposure. PsbS accumulation correlated with an enhanced non-photochemical quenching capacity in high light-acclimated cells grown at low CO2. However, PsbS could not compensate for the function of LHCSR in an LHCSR-deficient mutant. Knockdown of PsbS accumulation led to reduction of both non-photochemical quenching capacity and LHCSR3 accumulation. Our data suggest that PsbS is essential for the activation of non-photochemical quenching in C. reinhardtii, possibly by promoting conformational changes required for activation of LHCSR3-dependent quenching in the antenna of photosystem II.
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Pinnola, Alberta, Leonardo Ghin, Elisa Gecchele, Matilde Merlin, Alessandro Alboresi, Linda Avesani, Mario Pezzotti, Stefano Capaldi, Stefano Cazzaniga, and Roberto Bassi. "Heterologous Expression of Moss Light-harvesting Complex Stress-related 1 (LHCSR1), the Chlorophylla-Xanthophyll Pigment-protein Complex Catalyzing Non-photochemical Quenching, inNicotianasp." Journal of Biological Chemistry 290, no. 40 (August 10, 2015): 24340–54. http://dx.doi.org/10.1074/jbc.m115.668798.
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Dinc, Emine, Lijin Tian, Laura M. Roy, Robyn Roth, Ursula Goodenough, and Roberta Croce. "LHCSR1 induces a fast and reversible pH-dependent fluorescence quenching in LHCII in Chlamydomonas reinhardtii cells." Proceedings of the National Academy of Sciences 113, no. 27 (June 22, 2016): 7673–78. http://dx.doi.org/10.1073/pnas.1605380113.
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To avoid photodamage, photosynthetic organisms are able to thermally dissipate the energy absorbed in excess in a process known as nonphotochemical quenching (NPQ). Although NPQ has been studied extensively, the major players and the mechanism of quenching remain debated. This is a result of the difficulty in extracting molecular information from in vivo experiments and the absence of a validation system for in vitro experiments. Here, we have created a minimal cell of the green alga Chlamydomonas reinhardtii that is able to undergo NPQ. We show that LHCII, the main light harvesting complex of algae, cannot switch to a quenched conformation in response to pH changes by itself. Instead, a small amount of the protein LHCSR1 (light-harvesting complex stress related 1) is able to induce a large, fast, and reversible pH-dependent quenching in an LHCII-containing membrane. These results strongly suggest that LHCSR1 acts as pH sensor and that it modulates the excited state lifetimes of a large array of LHCII, also explaining the NPQ observed in the LHCSR3-less mutant. The possible quenching mechanisms are discussed.
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Allorent, Guillaume, Linnka Lefebvre-Legendre, Richard Chappuis, Marcel Kuntz, Thuy B. Truong, Krishna K. Niyogi, Roman Ulm, and Michel Goldschmidt-Clermont. "UV-B photoreceptor-mediated protection of the photosynthetic machinery inChlamydomonas reinhardtii." Proceedings of the National Academy of Sciences 113, no. 51 (December 5, 2016): 14864–69. http://dx.doi.org/10.1073/pnas.1607695114.
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Life on earth is dependent on the photosynthetic conversion of light energy into chemical energy. However, absorption of excess sunlight can damage the photosynthetic machinery and limit photosynthetic activity, thereby affecting growth and productivity. Photosynthetic light harvesting can be down-regulated by nonphotochemical quenching (NPQ). A major component of NPQ is qE (energy-dependent nonphotochemical quenching), which allows dissipation of light energy as heat. Photodamage peaks in the UV-B part of the spectrum, but whether and how UV-B induces qE are unknown. Plants are responsive to UV-B via the UVR8 photoreceptor. Here, we report in the green algaChlamydomonas reinhardtiithat UVR8 induces accumulation of specific members of the light-harvesting complex (LHC) superfamily that contribute to qE, in particular LHC Stress-Related 1 (LHCSR1) and Photosystem II Subunit S (PSBS). The capacity for qE is strongly induced by UV-B, although the patterns of qE-related proteins accumulating in response to UV-B or to high light are clearly different. The competence for qE induced by acclimation to UV-B markedly contributes to photoprotection upon subsequent exposure to high light. Our study reveals an anterograde link between photoreceptor-mediated signaling in the nucleocytosolic compartment and the photoprotective regulation of photosynthetic activity in the chloroplast.
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Zada, Ahmad, Ahmad Ali, Dalal Nasser Binjawhar, Usama K. Abdel-Hameed, Azhar Hussain Shah, Shahid Maqsood Gill, Irtiza Hussain, et al. "Molecular and Physiological Evaluation of Bread Wheat (Triticum aestivum L.) Genotypes for Stay Green under Drought Stress." Genes 13, no. 12 (November 30, 2022): 2261. http://dx.doi.org/10.3390/genes13122261.
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Water availability is considered as the main limiting factor of wheat growth illuminating the need of cultivars best adapted to drought situations for better wheat production and yield. Among these, the stay-green trait is thought to be related to the ability of wheat plants to maintain photosynthesis and CO2 assimilation, and a detailed molecular understanding of this trait may help in the selection of high-yielding, drought-tolerant wheats. The current study, therefore, evaluated the physiological responses of the selected wheat genotypes under pot-induced water stress conditions through different field capacities. The study also focused on exploring the molecular mechanisms involved in drought tolerance conferred due to the stay-green trait by studying the expression pattern of the selected PSI-associated light-harvesting complex I (LHC1) and PSII-associated LHCII gene families related to pigment-binding proteins. The results revealed that the studied traits, including relative water content, membrane stability index and chlorophyll, were variably and negatively affected, while the proline content was positively enhanced in the studied wheats under water stress treatments. Molecular diagnosis of the selected wheat genotypes using the expression profile of 06 genes, viz. TaLhca1, TaLhca2, TaLhca3, TaLhcb1, TaLhcb4 and TaLhcb6 that encodes for the LHCI and LHCII proteins, indicated variable responses to different levels of drought stress. The results obtained showed the relation between the genotypes and the severity of the drought stress condition. Among the studied genotypes, Chirya-1 and SD-28 performed well with a higher level of gene expression under drought stress conditions and may be used in genetic crosses to enrich the genetic background of common wheat against drought stress.
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Bailleul, B., A. Rogato, A. de Martino, S. Coesel, P. Cardol, C. Bowler, A. Falciatore, and G. Finazzi. "An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light." Proceedings of the National Academy of Sciences 107, no. 42 (October 4, 2010): 18214–19. http://dx.doi.org/10.1073/pnas.1007703107.
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Pinnola, Alberta. "The rise and fall of Light-Harvesting Complex Stress-Related proteins as photoprotection agents during evolution." Journal of Experimental Botany 70, no. 20 (August 2, 2019): 5527–35. http://dx.doi.org/10.1093/jxb/erz317.
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This review on the evolution of quenching mechanisms for excess energy dissipation focuses on the role of Light-Harvesting Complex Stress-Related (LHCSR) proteins versus Photosystem II Subunit S (PSBS) protein, and the reasons for the redundancy of LHCSR in vascular plants as PSBS became established.
Dissertations / Theses on the topic ""light harvesting stress related protein 1""
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Ghin, Leonardo. "Prokaryotic and eukaryotic expression systems for the production of recombinant proteins and nanoparticles for research and bio-industry." Doctoral thesis, 2015. http://hdl.handle.net/11562/915587.
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Gli organismi viventi sono in grado di produrre complesse strutture biologiche con funzioni specifiche per il loro metabolismo utilizzabili in settori industrali e di ricerca. In questo contesto le moderne tecnologie biotecnologiche sfruttano sistemi procariotici ed eucariotici per l'espressione di proteine ricombinanti, vaccini, anticorpi e nanoparticelle. In particolare il settore delle nanotecnologie sta conoscendo in questi ultimi anni un forte sviluppo e prodotti a base di nanoparticelle sono già stati approvati per la diagnostica e la terapia mentre altre sono attualmente in fase di sperimentazione clinica.
In questa tesi, è stata analizzata la possibilità di utilizzare diversi sistemi biologici per l’ espressione di proteine ricombinanti o di nanoparticelle sfuttando sia sistemi procariotici che eucariotici.
La prima parte del presente eleborato di tesi riguarda la purificazione di nanoparticelle magnetiche chiamate magnetosomi da Magnetospirillum gryphiswaldense , un batterio magnetotattico microaerofilo, nonché la successiva applicazione delle stesse come possibili agenti di contrasto in analisi NMR o come agenti terapeutici per termoterapia anti-tumorale. Nel presente elaborato di tesi è stato ottimizzato il metodo di coltura di M.gryphiswaldense, realizzando un'efficiente purificazione dei magnetosomi in lunghe catene, successivamente analizzati nelle loro caratteristiche chimico-fisiche (analisi DLS, TEM, misure di relassività e MRI) confermando la bontà come mezzi di contrasto e succesivamente impiegate in vivo come efficaci agenti terapeutici per ipertermia.
Nella seconda parte della tesi invece è stato sviluppato un sistema eucariotico per la produzione di un’importante proteina di membrana, LHCSR1, coinvolta nel delicato processo di regolazione della fotoprotezione in alghe e muschi. In particolare la proteina LHCSR1 del muschio Physcomitrella patens è stata espressa in due sistemi eucariotici ,Nicotiana benthamiana e Nicotiana tabacum realizzando l’isolamento della proteina e la preliminare determinazione delle principali proprietà spettroscopiche e biochimiche.Living organisms can produce complex structures with specific functions for their metabolism that are used in a range of bio-industry and research activities. Biotechnology exploits prokaryotic and eukaryotic systems for expression of recombinant proteins, vaccines and antibodies as well as nano-structures. Indeed a number of nanoparticle-based products have been approved for diagnostics and therapeutics and more are currently under clinical trials. In this thesis work, the possibility of using different expression systems for the preparation of bio-products has been exploited. The first part of this thesis concerns the purification of magnetic nanoparticles called magnetosomes from Magnetospirillum.gryphiswaldense, a magnetotactic microaerophilic bacterium. Subsequently these nanoparticles have been tested as contrast agents in NMR analysis or as therapeutic agents for tumor thermotherapy. The chemical-physical properties of magnetosomes efficiently purified have been studied, confirming the goodness of these nanoparticles as contrast agents. Then magnetosomes has been tested in thermotherapy in vitro and in vivo against two cancer cell lines. In the second part of the thesis instead has been developed a system for the production of an important eukaryotic membrane protein, LHCSR, involved in the delicate process of regulation photoprotection in algae and mosses. In particular the protein LHCSR1 from the moss Physcomitrella patens has been efficiently expressed in two eukaryotic systems, Nicotiana benthamiana and Nicotiana tabacum realizing its isolation from thylakoid membrane and the preliminary determination of its biochemical and spectroscopic properties.
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DIKAIOS, IOANNIS. "Analysis of Moss Light-Harvesting Complex Stress-Related (LHCSR1) Protein Function Upon Heterologous Expression in Arabidopsis thaliana." Doctoral thesis, 2017. http://hdl.handle.net/11562/965064.
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Non-photochemical quenching (NPQ) of chlorophyll fluorescence is a process essential for the regulation of photosynthesis and plant protection from light stress. In vascular plants this process is triggered by a luminal pH sensor, the PSBS protein, which transduces chloroplast lumen acidification, induced by excess light, into a quenching reaction occurring within specific interacting chromophore-bound lightharvesting proteins (LHC). In algae, such as Chlamydomonas reinhardtii, stress-related light-harvesting proteins (LHCSR) fulfill both pH sensing and quenching reactions, due to their capacity of binding chlorophylls and xanthophylls. The moss Physcomitrella patens, an evolutionary intermediate between algae and plants, has both PSBS and LHCSR active in quenching with LHCSR working in a direct zeaxanthin-dependent manner. Plants and mosses have a very similar organization of thylakoid membranes thus suggesting LHCSR might be active in plants. To verify this hypothesis, we overexpressed lhcsr1 gene into Arabidopsis thaliana PSBS mutant, npq4, and screened transformants by fluorescence video-imaging, resulting to the isolation of A. thaliana plants, which accumulate a pigment-binding, NPQ-active LHCSR1 in thylakoid membranes. In the context of functional and structural analysis of LHCSR1 protein, a series of in vivo transformations was performed using A. thaliana mutants altered in xanthophyll content or lacking specific LHC subunits. For this reason the double mutant npq1npq4 - unable to convert violaxathin into zeaxanthin - was complemented in order to verify the direct dependence of LHCSR1 on zeaxanthin, mutant lut2npq4 was used due to its complete lack of lutein and antenna mutants NoMnpq4 and ch1lhcb5 were used due to their lack of either minor antennas or the complete antenna system respectively; all of them overexpressing LHCSR1 in different levels. Finally, a first approach for the in vivo mutational analysis of P. patens LHCSR1 has been initiated, using A. thaliana as a tool for heterologous protein expression.
Conference papers on the topic ""light harvesting stress related protein 1""
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Pšenčík, J., M. Vácha, F. Adamec, M. Ambrož, J. Dian, J. Boček, and J. Hála. "Spectral Hole Burning in Chlorosomes of Green Photosynthetic Bacteria Chlorobium limicola." In Spectral Hole-Burning and Luminescence Line Narrowing: Science and Applications. Washington, D.C.: Optica Publishing Group, 1992. http://dx.doi.org/10.1364/shbl.1992.tub19.
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Chlorobium limicola is a green sulfur photosynthetic bacteria. Its light harvesting system consists of huge extra-membrane ellipsoidal structures (100-150 nm long and 30-40 nm in diameter) called chlorosomes. Each chlorosome contains several thousands of bacteriochlorophyll-c (BChl-c) molecules (organized in long rod-like pigment-protein complexes) together with a significant amount of carotenoids. The whole chlorosome is enveloped with a BChl-a containing layer and attached to the cytoplasmic membrane through a water soluble BChl-a polypeptide link complex. Light energy absorbed in BChl-c is transfered via BChl-a in the envelope and via BChl-a in the link polypeptide complex to the BChl-a containing core antenna which surrounds the reaction center and is located within the cytoplasmic membrane. In the last few years, the dynamics of the excited energy transport (EET) in the whole light harvesting system of Chlorobium limicola and related species have been extensively studied. Fast EET from BChl-c to BChl-a was studied by means of ps absorption [1] and ps fluorescence decay [2]. Both methods yield similar values of the shortest decay components which characterize the EET: 21 ps and 26 ps, respectively. Detailed hole burning study of the BChl-a containing link antenna of Prosthecochloris aestuarii revealed excitonic structure of the antenna subunits and provided the decay times (on the order of 100 fs) of upper excitonic levels [3].
Reports on the topic ""light harvesting stress related protein 1""
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Chamovitz, Daniel, and Albrecht Von Arnim. Translational regulation and light signal transduction in plants: the link between eIF3 and the COP9 signalosome. United States Department of Agriculture, November 2006. http://dx.doi.org/10.32747/2006.7696515.bard.
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The COP9 signalosome (CSN) is an eight-subunit protein complex that is highly conserved among eukaryotes. Genetic analysis of the signalosome in the plant model species Arabidopsis thaliana has shown that the signalosome is a repressor of light dependent seedling development as mutant Arabidopsis seedlings that lack this complex develop in complete darkness as if exposed to light. These mutant plants die following the seedling stage, even when exposed to light, indicating that the COP9 signalosome also has a central role in the regulation of normal photomorphogenic development. The biochemical mode of action of the signalosome and its position in eukaryotic cell signaling pathways is a matter of controversy and ongoing investigation, and recent results place the CSN at the juncture of kinase signaling pathways and ubiquitin-mediated protein degradation. We have shown that one of the many CSN functions may relate to the regulation of translation through the interaction of the CSN with its related complex, eukaryotic initiation factor (eIF3). While we have established a physical connection between eIF3 subunits and CSN subunits, the physiological and developmental significance of this interaction is still unknown. In an effort to understand the biochemical activity of the signalosome, and its role in regulating translation, we originally proposed to dissect the contribution of "h" subunit of eIF3 (eIF3h) along the following specific aims: (i) Isolation and phenotypic characterization of an Arabidopsis loss-of-function allele for eIF3h from insertional mutagenesis libraries; (ii) Creation of designed gain and loss of function alleles for eIF3h on the basis of its nucleocytoplasmic distribution and its yeast-two-hybrid interactions with other eIF3 and signalosome partner proteins; (iii) Determining the contribution of eIF3h and its interaction with the signalosome by expressing specific mutants of eIF3h in the eIF3h- loss-of function background. During the course of the research, these goals were modified to include examining the genetic interaction between csn and eif3h mutations. More importantly, we extended our effort toward the genetic analysis of mutations in the eIF3e subunit, which also interacts with the CSN. Through the course of this research program we have made several critical scientific discoveries, all concerned with the apparent diametrically opposed roles of eIF3h and eIF3e. We showed that: 1) While eIF3e is essential for growth and development, eIF3h is not essential for growth or basal translation; 2) While eIF3e has a negative role in translational regulation, eIF3h is positively required for efficient translation of transcripts with complex 5' UTR sequences; 3) Over-accumulation of eIF3e and loss-of-function of eIF3h both lead to cop phenotypes in dark-grown seedlings. These results were published in one publication (Kim et al., Plant Cell 2004) and in a second manuscript currently in revision for Embo J. Are results have led to a paradigm shift in translation research – eIF3 is now viewed in all systems as a dynamic entity that contains regulatory subuits that affect translational efficiency. In the long-term agronomic outlook, the proposed research has implications that may be far reaching. Many important plant processes, including developmental and physiological responses to light, abiotic stress, photosynthate, and hormones operate in part by modulating protein translation [23, 24, 40, 75]. Translational regulation is slowly coming of age as a mechanism for regulating foreign gene expression in plants, beginning with translational enhancers [84, 85] and more recently, coordinating the expression of multiple transgenes using internal ribosome entry sites. Our contribution to understanding the molecular mode of action of a protein complex as fundamental as eIF3 is likely to lead to advances that will be applicable in the foreseeable future.
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Locy, Robert D., Hillel Fromm, Joe H. Cherry, and Narendra K. Singh. Regulation of Arabidopsis Glutamate Decarboxylase in Response to Heat Stress: Modulation of Enzyme Activity and Gene Expression. United States Department of Agriculture, January 2001. http://dx.doi.org/10.32747/2001.7575288.bard.
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Most plants accumulate the nonprotein amino acid, g-aminobutyric acid (GABA), in response to heat stress. GABA is made from glutamate in a reaction catalyzed by glutamate decarboxylase (GAD), an enzyme that has been shown by the Israeli PI to be a calmodulin (CaM) binding protein whose activity is regulated in vitro by calcium and CaM. In Arabidopsis there are at least 5 GAD genes, two isoforms of GAD, GAD1 and GAD2, are known to be expressed, both of which appear to be calmodulin-binding proteins. The role of GABA accumulation in stress tolerance remains unclear, and thus the objectives of the proposed work are intended to clarify the possible roles of GABA in stress tolerance by studying the factors which regulate the activity of GAD in vivo. Our intent was to demonstrate the factors that mediate the expression of GAD activity by analyzing the promoters of the GAD1 and GAD2 genes, to determine the role of stress induced calcium signaling in the regulation of GAD activity, to investigate the role of phosphorylation of the CaM-binding domain in the regulation of GAD activity, and to investigate whether ABA signaling could be involved in GAD regulation via the following set of original Project Objectives: 1. Construction of chimeric GAD1 and GAD2 promoter/reporter gene fusions and their utilization for determining cell-specific expression of GAD genes in Arabidopsis. 2. Utilizing transgenic plants harboring chimeric GAD1 promoter-luciferase constructs for isolating mutants in genes controlling GAD1 gene activation in response to heat shock. 3. Assess the role of Ca2+/CaM in the regulation of GAD activity in vivo in Arabidopsis. 4. Study the possible phosphorylation of GAD as a means of regulation of GAD activity. 5. Utilize ABA mutants of Arabidopsis to assess the involvement of this phytohormone in GAD activation by stress stimuli. The major conclusions of Objective 1 was that GAD1 was strongly expressed in the elongating region of the root, while GAD2 was mainly expressed along the phloem in both roots and shoots. In addition, GAD activity was found not to be transcriptionally regulated in response to heat stress. Subsequently, The Israeli side obtained a GAD1 knockout mutation, and in light of the objective 1 results it was determined that characterization of this knockout mutation would contribute more to the project than the proposed Objective 2. The major conclusion of Objective 3 is that heat-stress-induced changes in GAD activity can be explained by heat-stress-induced changes in cytosolic calcium levels. No evidence that GAD activity was transcriptionally or translationally regulated or that protein phosphorylation was involved in GAD regulation (objective 4) was obtained. Previously published data by others showing that in wheat roots ABA regulated GABA accumulation proved not to be the case in Arabidopsis (Objective 5). Consequently, we put the remaining effort in the project into the selection of mutants related to temperature adaptation and GABA utilization and attempting to characterize events resulting from GABA accumulation. A set of 3 heat sensitive mutants that appear to have GABA related mutations have been isolated and partially characterized, and a study linking GABA accumulation to growth stimulation and altered nitrate assimilation were conducted. By providing a better understanding of how GAD activity was and was not regulated in vivo, we have ruled out the use of certain genes for genetically engineering thermotolerance, and suggested other areas of endeavor related to the thrust of the project that may be more likely approaches to genetically engineering thermotolerance.
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Sadot, Einat, Christopher Staiger, and Mohamad Abu-Abied. Studies of Novel Cytoskeletal Regulatory Proteins that are Involved in Abiotic Stress Signaling. United States Department of Agriculture, September 2011. http://dx.doi.org/10.32747/2011.7592652.bard.
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In the original proposal we planned to focus on two proteins related to the actin cytoskeleton: TCH2, a touch-induced calmodulin-like protein which was found by us to interact with the IQ domain of myosin VIII, ATM1; and ERD10, a dehydrin which was found to associate with actin filaments. As reported previously, no other dehydrins were found to interact with actin filaments. In addition so far we were unsuccessful in confirming the interaction of TCH2 with myosin VIII using other methods. In addition, no other myosin light chain candidates were found in a yeast two hybrid survey. Nevertheless we have made a significant progress in our studies of the role of myosins in plant cells. Plant myosins have been implicated in various cellular activities, such as cytoplasmic streaming (1, 2), plasmodesmata function (3-5), organelle movement (6-10), cytokinesis (4, 11, 12), endocytosis (4, 5, 13-15) and targeted RNA transport (16). Plant myosins belong to two main groups of unconventional myosins: myosin XI and myosin VIII, both closely related to myosin V (17-19). The Arabidopsis myosin family contains 17 members: 13 myosin XI and four myosin VIII (19, 20). The data obtained from our research of myosins was published in two papers acknowledging BARD funding. To address whether specific myosins are involved with the motility of specific organelles, we cloned the cDNAs from neck to tail of all 17 Arabidopsis myosins. These were fused to GFP and used as dominant negative mutants that interact with their cargo but are unable to walk along actin filaments. Therefore arrested organelle movement in the presence of such a construct shows that a particular myosin is involved with the movement of that particular organelle. While no mutually exclusive connections between specific myosins and organelles were found, based on overexpression of dominant negative tail constructs, a group of six myosins (XIC, XIE, XIK, XI-I, MYA1 and MYA2) were found to be more important for the motility of Golgi bodies and mitochondria in Nicotiana benthamiana and Nicotiana tabacum (8). Further deep and thorough analysis of myosin XIK revealed a potential regulation by head and tail interaction (Avisar et al., 2011). A similar regulatory mechanism has been reported for animal myosin V and VIIa (21, 22). In was shown that myosin V in the inhibited state is in a folded conformation such that the tail domain interacts with the head domain, inhibiting its ATPase and actinbinding activities. Cargo binding, high Ca2+, and/or phosphorylation may reduce the interaction between the head and tail domains, thus restoring its activity (23). Our collaborative work focuses on the characterization of the head tail interaction of myosin XIK. For this purpose the Israeli group built yeast expression vectors encoding the myosin XIK head. In addition, GST fusions of the wild-type tail as well as a tail mutated in the amino acids that mediate head to tail interaction. These were sent to the US group who is working on the isolation of recombinant proteins and performing the in vitro assays. While stress signals involve changes in Ca2+ levels in plants cells, the cytoplasmic streaming is sensitive to Ca2+. Therefore plant myosin activity is possibly regulated by stress. This finding is directly related to the goal of the original proposal.
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Shomer, Ilan, Ruth E. Stark, Victor Gaba, and James D. Batteas. Understanding the hardening syndrome of potato (Solanum tuberosum L.) tuber tissue to eliminate textural defects in fresh and fresh-peeled/cut products. United States Department of Agriculture, November 2002. http://dx.doi.org/10.32747/2002.7587238.bard.
Full textAbstract:
The project sought to understand factors and mechanisms involved in the hardening of potato tubers. This syndrome inhibits heat softening due to intercellular adhesion (ICA) strengthening, compromising the marketing of industrially processed potatoes, particularly fresh peeled-cut or frozen tubers. However, ICA strengthening occurs under conditions which are inconsistent with the current ideas that relate it to Ca-pectate following pectin methyl esterase (PME) activity or to formation of rhamnogalacturonan (RG)-II-borate. First, it was necessary to induce strengthening of the middle lamellar complex (MLX) and the ICA as a stress response in some plant parenchyma. As normally this syndrome does not occur uniformly enough to study it, we devised an efficient model in which ICA-strengthening is induced consistently under simulated stress by short-chain, linear, mono-carboxylic acid molecules (OAM), at 65 oC [appendix 1 (Shomer&Kaaber, 2006)]. This rapid strengthening was insufficient for allowing the involved agents assembly to be identifiable; but it enabled us to develop an efficient in vitro system on potato tuber parenchyma slices at 25 ºC for 7 days, whereas unified stress was reliably simulated by OAMs in all the tissue cells. Such consistent ICA-strengthening in vitro was found to be induced according to the unique physicochemical features of each OAM as related to its lipophilicity (Ko/w), pKa, protonated proportion, and carbon chain length by the following parameters: OAM dissociation constant (Kdiss), adsorption affinity constant (KA), number of adsorbed OAMs required for ICA response (cooperativity factor) and the water-induced ICA (ICAwater). Notably, ICA-strengthening is accompanied by cell sap leakage, reflecting cell membrane rupture. In vitro, stress simulation by OAMs at pH<pKa facilitated the consistent assembly of ICAstrengthening agents, which we were able to characterize for the first time at the molecular level within purified insoluble cell wall of ICA-strengthened tissue. (a) With solid-state NMR, we established the chemical structure and covalent binding to cell walls of suberin-like agents associated exclusively with ICA strengthening [appendix 3 (Yu et al., 2006)]; (b) Using proteomics, 8 isoforms of cell wall-bound patatin (a soluble vacuolar 42-kDa protein) were identified exclusively in ICA-strengthened tissue; (c) With light/electron microscopy, ultrastructural characterization, histochemistry and immunolabeling, we co-localized patatin and pectin in the primary cell wall and prominently in the MLX; (d) determination of cell wall composition (pectin, neutral sugars, Ca-pectate) yielded similar results in both controls and ICA-strengthened tissue, implicating factors other than PME activity, Ca2+ or borate ions; (e) X-ray powder diffraction experiments revealed that the cellulose crystallinity in the cell wall is masked by pectin and neutral sugars (mainly galactan), whereas heat or enzymatic pectin degradation exposed the crystalline cellulose structure. Thus, we found that exclusively in ICA-strengthened tissue, heat-resistant pectin is evident in the presence of patatin and suberinlike agents, where the cellulose crystallinity was more hidden than in fresh control tissue. Conclusions: Stress response ICA-strengthening is simulated consistently by OAMs at pH< pKa, although PME and formation of Ca-pectate and RG-II-borate are inhibited. By contrast, at pH>pKa and particularly at pH 7, ICA-strengthening is mostly inhibited, although PME activity and formation of Ca-pectate or RG-II-borate are known to be facilitated. We found that upon stress, vacuolar patatin is released with cell sap leakage, allowing the patatin to associate with the pectin in both the primary cell wall and the MLX. The stress response also includes formation of covalently bound suberin-like polyesters within the insoluble cell wall. The experiments validated the hypotheses, thus led to a novel picture of the structural and molecular alterations responsible for the textural behavior of potato tuber. These findings represent a breakthrough towards understanding of the hardening syndrome, laying the groundwork for potato-handling strategies that assure textural quality of industrially processed particularly in fresh peeled cut tubers, ready-to-prepare and frozen preserved products.