Progress 10/01/02 to 09/30/07
Outputs
The var2 variegation mutant of Arabidopsis arises as a consequence of a lesion in the nuclear gene for a chloroplast AtFtsH metalloprotease. The mutant plants have green- and white-sectored leaves, and they are hypersusceptible to photoinhibition (photosynthesis-induced light stress). Consistent with this phenotype, VAR2 is involved in the D1 repair cycle of Photosystem II, likely by affecting turnover of the photodamaged D1 polypeptide. Because VAR2 plays a central role in protecting plants against light stress, it is critical to understand the function of this protein in photosynthesis and light stress, and how its activity and expression are regulated. In this project we applied a genetic suppressor screen to address this question. These sorts of analyses allow one to define proteins that interact with a protein-of-interest to control its function (directly or indirectly). Second-site suppressor screens of var2 yielded several lines in which the variegation
phenotype of var2 is significantly altered. Some of these lines have a "central yellow" (CY) phenotype, in which the basal part of younger leaves on the rosette are pale-green or yellow, then turn fully-green as they develop and expand. Two suppressor lines with a CY phenotype, 2484 and 24107, were chosen for further analysis. Map-based cloning revealed that the suppressor gene in 2484 encodes a plastid Polyribonucleotide Phosphorylase (PNPase), which is involved in RNA processing and chloroplast protein translation. We designated this gene CY1. Isolation of the cy1 single mutant showed that it has a phenotype similar to the cy1/var2 double mutant, which suggests that cy1 is epistatic to var2. Because var2 and cy1 are recessive, the results suggest that VAR2 and CY1 act antagonistically in chloroplast biogenesis, and that down-regulation of CY1 reduces the requirement for VAR2 in photosynthesis and plastid development. Map-based cloning has also been undertaken to clone the suppressor
gene in line 24107, and these experiments showed that suppression is due to a lesion in At5g18820, which encodes a chloroplast GroEL-like chaperone, termed "chaperonin 60 alpha-like protein" (or "Cpn60 alpha-like"). Previous studies have shown that this protein mediates the folding (and/or unfolding) of many chloroplast proteins, and that it is important in assembling the photosynthetic apparatus during early plant development. The gene for this protein has been designated CY2. Independent lines with cy2 single mutations have been generated, and like cy1, cy2 single mutants have a phenotype similar to the cy2/var2 double mutant, suggesting that cy2 is epistatic to var2. Genetic complementation and further molecular analyses are underway to elucidate the relationships between CY1, CY2 and VAR2. Isolation of the cy1 and cy2 mutants represents an important advance in the generation of tools to understand photosynthesis, variegation mechanisms and photoprotection in plants.
Impacts
VAR2 is particularly important because it mediates the plant's response to excess light, preventing photooxidative damage and plant death. In this role VAR2 degrades proteins in the photosynthetic membrane that become oxidized as a consequence of too much light exposure. VAR2 normally degrades these proteins, allowing their replacement by newly synthesized copies. Our studies have shown that other activities in the chloroplast are closely linked to VAR2 function and serve to modify the activity of this protein. Over the course of this project, these have included a chaperone and a plastid Polyribonucleotide Phosphorylase (PNPase). These novel findings imply that the ability of the plant to perceive and respond to photooxidative damage can be modified significantly by these proteins. Hence, our results set the stage for experiments whereby these factors might be used to modify the light gathering ability (and hence photosynthetic capacity) of crop plants using
techniques of classical breeding or transgenics.
Publications
- Aluru, M.R., D.J. Stessman, M.H. Spalding and S.R. Rodermel. 2007. Alterations in photosynthesis in Arabidopsis lacking IMMUTANS, a chloroplast terminal oxidase. Photosynthesis Research 91:11-23.
- Yu, F., A. Fu, M. Aluru, S. Park, Y. Xu, H. Liu, X. Liu, A. Foudree, M. Nambogga and S. Rodermel. 2007. Variegation mutants and mechanisms of chloroplast biogenesis. Plant Cell & Environment 30:350-365. (Refereed review) (Cover Article).
- Bogorad, L. and S. Rodermel. 2007. Evolution of early eukayotic cells: genomes, proteomes and compartments. Photosynthesis Research (in press)
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Progress 01/01/06 to 12/31/06
Outputs
The Arabidopsis AtFtsH metalloprotease gene family comprises three pairs of closely-related genes that are targeted to chloroplasts (AtFtsH2 and 8; AtFtsH1 and 5; and AtFtsH7 and 9). Mutations in AtFtsH5 (var1) and AtFtsH2 (var2) give rise to variegated plants with green- and white-sectored leaves. During the past year we have applied genetic suppressor analysis to understand the variegation mechanism of var2. One suppressor of var2, TAG-FN, was cloned and the responsible gene was found to encode a chloroplast-localized pseudouridine synthase. Pseudouridine synthases are enzymes that mediate the post-transcriptional isomerization of uridines to pseudouridines in various RNA species. Compared to both wild type and var2, the TAG-FN mutant does not display a gross change in nuclear and chloroplast gene expression, nor in chloroplast protein accumulation, with the exception of a consistent reduction in the abundance of Rubisco. However, aberrant chloroplast rRNA
processing was observed, and this correlates with the presence of abnormal ribosomal fractions containing 23S rRNA. Thus, TAG-FN represents a previously unknown component of the chloroplast rRNA processing process. We further demonstrated that a defect in chloroplast translation in TAG-FN is the reason for the suppression of variegation. We concluded that the disruption in pseudouridine synthase in TAG-FN causes abnormal rRNA metabolism and ribosomal assembly, and that these events affect translation and, ultimately, photosynthesis.
Impacts
FtsH metalloproteases, such as VAR2, play an important role in the process of thylakoid membrane biogenesis and the devlopemnt of normal chloroplasts. Further insight into the function of this protein in photosynthesis, plant development and plant stress responses might lead to the design of strategies to manipulate the photosynthetic capacity and quality of crop plants.
Publications
- Aluru, M.R., F. Yu, A. Fu and S. Rodermel. 2006. Arabidopsis variegation mutants: new insights into chloroplast biogenesis. J. Experimental Botany 57: 1871-1881. (Refereed review).
- Rosso, D. A.G. Ivanov, A. Fu, J. Geisler-Lee, L. Hendrickson, M. Geisler, G. Stewart, M. Krol, V. Hurry, S.R. Rodermel, D.P. Maxwell and N.P.A. Huner. 2006. IMMUTANS does not act as a stress-induced safety valve in the protection of the photosynthetic apparatus of Arabidopsis during steady state photosynthesis. Plant Physiol. 142: 574-585.
- Yu, F., A. Fu, M. Aluru, S. Park, Y. Xu, H. Liu, X. Liu, A. Foudree, M. Nambogga and S. Rodermel. 2006. Variegation mutants and mechanisms of chloroplast biogenesis. Plant Cell & Environment (online Jan 2007) (Refereed review).
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Progress 01/01/05 to 12/31/05
Outputs
The thylakoid membranes of higher plants, algae and some cyanobacteria contain a terminal oxidase (PTOX, the product of the IMMUTANS gene) that functions in the reduction of the plastoquinone pool. PTOX is distantly-related to the alternative oxidase (AOX) of mitochondrial inner membranes. Structural models of AOX and PTOX have been proposed based on the crystal structures of diiron carboxylate proteins in which the ligation sphere of the diiron center is composed of six conserved histidine and glutamate residues. We have tested the functional role of these residues in PTOX by site-directed mutagenesis using an in vitro activity assay in which mutant proteins are expressed in E. coli, and quinone/oxygen oxidoreductase activities are measured using isolated membranes and NADH as an electron donor. We found that conservative and non-conservative substitutions in all six ligands abolish cyanide-resistant oxygen uptake, indicating that these amino acids are essential for
PTOX activity. Mutation of other histidine or glutamate residues in the active site have no impact on activity. Although PTOX and AOX share many functional domains, PTOX has a conserved 16 amino acid domain near the active site that is not present in AOX. Mutant proteins lacking this domain accumulate normally in E. coli but lack in vitro activity. An Arabidopsis immutans allele was isolated whose PTOX lacks the 16 amino acid domain, and in contrast to E. coli, this mutant did not accumulate PTOX. Because the mutant contained normal PTOX mRNA levels, we conclude that the 16 amino acid domain is required for PTOX mRNA translatability or protein stability in vivo.
Impacts
It is clear that PTOX plays an important role early in the process of thylakoid membrane biogenesis. Further insight into the function of this protein in photosynthesis, plant development and plant stress responses might lead to the design of strategies to manipulate the photosynthetic capacity and quality of crop plants. Understanding the genetic basis for stress tolerance and the evolution of stress tolerant lines will require suitable screening protocols by which to compare inbreds and populations with putative differential tolerance to low temperature stress. Such protocols should provide the basis for evolving stress tolerant lines and thereby improving yield stability and expanding adaptational ranges without an increased need for external inputs.
Publications
- Rodermel, S.R., Viret, J. and Krebbers, E. 2005. Lawrence Bogorad (1921-2003), a pioneer in photosynthesis research: a tribute (Invited tribute). Photosynthesis Research 83:17-124.
- Baerr, J.N., Thomas, J.D., Taylor, B.G., Rodermel, S.R. and Gray, G.R. 2005. Differential photosynthetic compensatory mechanisms exist in the immutans mutant of Arabidopsis thaliana. Physiologia Plantarum 124:390-402 11 (Cover Article).
- Yu, F., Park, S. and Rodermel, S.R. 2005. Functional redundancy of AtFtsH metalloproteases in thylakoid membrane complexes. Plant Physiol. 138:1957-1966.
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Progress 01/01/04 to 12/31/04
Outputs
The Arabidopsis AtFtsH metalloprotease gene family comprises three pairs of closely-related genes that are targeted to chloroplasts (AtFtsH2 and 8; AtFtsH1 and 5; and AtFtsH7 and 9). Mutations in AtFtsH5 (var1) and AtFtsH2 (var2) give rise to variegated plants with green- and white-sectored leaves. We found by two-dimensional green gel and gel filtration analyses that AtFtsH2/VAR2 forms oligomeric complexes. Two bands in the 2-D green gels that correspond to AtFtsH5/VAR1 + AtFtsH1 and AtFtsH2/VAR2 + AtFtsH8 have been identified, and these bands are coordinately reduced in amount in var1 and var2 thylakoids that lack AtFtsH5/VAR1 and AtFtsH2/VAR2, respectively. Overexpression of AtFtsH8 in var2-4 (a putative null allele) normalizes the variegation phenotype of the mutant and restores the two bands to their wild type levels. These results suggest that AtFtsH8 is interchangeable with AtFtsH2/VAR2 in AtFtsH-containing oligomers, and that the two proteins have redundant
functions. Consistent with this hypothesis, AtFtsH2 and AtFtsH8 have similar expression patterns, as monitored by promoter-GUS fusion and RT-PCR experiments. Based on our findings, we propose that AtFtsH1, AtFtsH2/VAR2, AtFtsH5/VAR1 and AtFtsH8 interact to form oligomeric structures, and that subunit stoichiometry is controlled post-transcriptionally in var1 and var2, perhaps by turnover. This is consistent with a threshold model to explain the pattern of variegation in var2 in which AtFtsH8 provides a compensating activity in the green sectors of the mutant. The apparent photosynthesis (APSN) and chlorophyll fluorescence (fv/fm) of three maize inbred lines, Co255, B73, and A619, were compared with and without an acclimational treatment, after a subsequent stress, and finally after ca. 2 da. recovery at 25 C. The results indicate a that interpretation of PSN rate and PSII efficiency relative to stress tolerance is complex. While APSN and fv/fm of Co255 (putatively cold tolerant) was
reduced most dramatically by stress treatments, its response to acclimation was also most pronounced. Further research is required to establish appropriate screening protocols for low temperature stress tolerance.
Impacts
It is clear that FtsH metalloproteases, such as VAR2, play an important role early in the process of thylakoid membrane biogenesis. Further insight into the function of this protein in photosynthesis, plant development and plant stress responses might lead to the design of strategies to manipulate the photosynthetic capacity and quality of crop plants Understanding the genetic basis for stress tolerance and the evolution of stress tolerant lines will require suitable screening protocols by which to compare inbreds and populations with putative differential tolerance to low temperature stress. Such protocols should provide the basis for evolving stress tolerant lines and thereby improving yield stability and expanding adaptational ranges without an increased need for external inputs.
Publications
- Barr, J., White, W.S., Chen, L., Bae, H. and Rodermel, S. 2004. The GHOST terminal oxidase regulates developmental programming in tomato fruit. Plant, Cell & Environment 27:840-852 (Cover article).
- Lonosky, P., Zhang, X., Honavar, V., Dobbs, D., Fu, A. and Rodermel, S. 2004. A proteomic analysis of maize chloroplast biogenesis. Plant Physiology 134:560-574.
- Yu, F., Park, S. and Rodermel, S.R. 2004. The Arabidopsis FtsH metalloprotease gene family: interchangeability of subunits in chloroplast oligomeric complexes. Plant Journal 37:864-876.
- Mazarei, M., Lennon, K.A., Puthoff, D.P., Rodermel, S.R. and Baum, T.J. 2004. Expression of an Arabidopsis phosphoglycerate mutase homologue is localized to apical meristems, regulated by hormones, and induced by sedentary plant-parasitic nematodes. Plant Mol. Biol. 53:513-530.
- Mazarei, M., Lennon, K.A., Puthoff, D.P., Rodermel, S.R. and Baum, T.J. 2004. Homologous soybean and Arabidopsis genes share responsiveness to cyst nematode infection. Molecular Plant Pathology 5:409-423.
- Park, S. and Rodermel, S. 2004. Mutations in ClpC2/Hsp100 suppress the requirement for FtsH in thylakoid membrane biogenesis. Proc. Natl. Acad. Sci. USA 101:12765-12770.
- Grote, K. 2004. Characterization of inducible cold tolerance in photosynthetic maize seedlings and the behavior of selected xanthophyll compounds. M.S Thesis. Iowa State Univ., Ames.
- Arwatchanakarn, A. 2004. Dehydrin-like protein expression profiles of maize seedlings during germination, low temperature stress, and genotype. M.S. Thesis. Iowa State Univ., Ames.
- Aluru, M.R. and Rodermel, S.R. 2004. Control of chloroplast redox by the IMMUTANS terminal oxidase. Physiologia Plantarum 120:4-11 (Cover Article).
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Progress 01/01/03 to 12/31/03
Outputs
We have been studying the immutans (im) variegation mutant of Arabidopsis as a means of gaining insight into the control of thylakoid membrane biogenesis and the mechanisms that coordinate gene expression between the nucleus-cytoplasm and the plastid. The IMMUTANS (IM) protein is a terminal oxidase in plastid membranes and is an electron acceptor for the desaturase reactions of carotenoid biosynthesis. We have previously proposed that IM might play a more general role in plastid metabolism, and consistent with this notion, we found during the past year that IM mRNAs and proteins are up-regulated in high light stress conditions, as well as in double antisense mutants that lack ascorbate peroxidase and catalase. These mutants are less sensitive to oxidative stress than single antisense plants lacking ascorbate peroxidase or catalase alone. These results suggest that IM is a component of the high light stress response in plants, perhaps by acting as an alternate electron
sink to dissipate excess electrons generated by high light treatment. To gain insight into factors that interact with IM and that regulate its activity, we initiated a suppressor screen during the past year. In this screen we mutagenized im plants and screened for mutants that display less variegation than im. Albinos were discarded because mutations in a large number of genes are predicted to give rise to abnormal, non-pigmented plastids that have nothing to do with the mechanism of im variegation. We isolated three putative suppressor mutations and confirmed that these are lesions in single genes. Steps to clone the suppressor genes by map-based methods have been undertaken. A second aspect of this study involves the investigation of mechanisms of low temperature stress tolerance in maize. Our initial investigations have been aimed at evaluating stress screening protocols and general associations between various measurements of low temperature stress and seed/seedling performance.
At this point, comparisons of potassium leakage, electrical conductivity of leaf leachates as a percentage of total leaf electrolytes from maize inbred lines A619, B73, and CO255, indicate that the duration of stress can change the cold tolerance rankings of inbreds. These differential rankings occur both immediately following stress and after a recovery period at optimal temperatures. Studies, involving photosynthetic rates, chlorophyll fluorescence, xanthophyl levels, and inbred responses to seedling acclimation treatments, are completed and will be reported this spring.
Impacts
It is clear that IM plays an important role early in the process of thylakoid membrane biogenesis. Further insight into the function of this protein in photosynthesis, plant development and plant stress responses might lead to the design of strategies to manipulate the photosynthetic capacity and quality of important crop plants. Understanding the genetic basis for stress tolerance and the evolution of stress tolerant lines will require suitable screening protocols by which to compare inbreds and populations with putative differential tolerance to low temperature stress. Such protocols should provide the basis for evolving stress tolerant lines and thereby improving yield stability and expanding adaptational ranges without an increased need for external inputs.
Publications
- Rodermel, S. and Park, S. 2003. Pathways of intracellular communication: tetrapyrroles and plastid-to-nucleus signaling. BioEssays 25:631-636.
- Shurney, S.N. 2003. Screening for low temperature stress in maize: Protocol development and testing. M.S. Thesis. Iowa State Univ., Ames.
- Aluru, M. and Rodermel, S.R. 2003. Identification of IMMUTANS as a plastid terminal oxidase: its role in differentiation, carotenoid biosynthesis and chlororespiration. Recent Res. Devel. Plant Mol. Biol. 1:39-55.
- Zheng, P., Ammar, K., Girard, A.-M., Rodermel, S., Thomas, D.R., Ning, L., Callis, J.B., Edwards, G.E. and Daley, L. 2003. Test of an in vivo method to detect chloroplast division in crop plants. Part III: Statistical proofs of observation and general utility of the method. Spectroscopy 18(12):102-105.
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