Photosynthetic light reactions rely on the proper function of large protein complexes (including photosystems I and II) that reside in the thylakoid membrane. Although their composition, structure, and function are known, the repertoire of assembly and maintenance factors is still being determined. Here we show that an immunophilin of the cyclophilin type, CYP38, plays a critical role in the assembly and maintenance of photosystem II (PSII) supercomplexes (SCs) in Arabidopsis. Mutant plants with the CYP38 gene interrupted by T-DNA insertion showed stunted growth and were hypersensitive to high light. Leaf chlorophyll fluorescence analysis and thylakoid membrane composition indicated that cyp38 mutant plants had defects in PSII SCs. Sucrose supplementation enabled the rescue of the mutant phenotype under low-light conditions, but failed to mitigate hypersensitivity to high-light stress. Protein radiolabeling assays showed that, although individual thylakoid proteins were synthesized equally in mutant and wild type, the assembly of the PSII SC was impaired in the mutant. In addition, the D1 and D2 components of the mutant PSII had a short half-life under high-light stress. The results provide evidence that CYP38 is necessary for the assembly and stabilization of PSII.thylakoid lumen ͉ immunophilin ͉ photosynthesis ͉ protein folding ͉ chaperone T he light reactions and attendant evolution of oxygen in photosynthesis are carried out by four multisubunit protein complexes residing in the chloroplast thylakoid membranes: photosystems I (PSI) and II (PSII), cytochrome b 6 f complex, and CF O -CF 1 complex (1-3). For a complete understanding of the photosynthetic process, it is essential to understand the biogenesis and maintenance of the participating complexes. Earlier studies on thylakoid protein supercomplex (SC) assembly, especially PSII, concentrated on the role of stromal factors, such as the translation and import machinery (4), because only a limited number of proteins were known to reside in the thylakoid lumen. However, recent proteomic findings suggest a population of 80-100 proteins in that compartment (5-7). The immunophilin family is one of the predominant groups identified.Immunophilins were originally discovered in their capacity as cellular receptors for immunosuppressive drugs: cyclosporin A and FK506 (8, 9). The receptors for cyclosporin A and FK506, named cyclophilins (CYPs) and FK506-binding proteins (FKBPs), respectively, were collectively designated as immunophilins. A common feature of most immunophilins is the associated peptidyl-prolyl cis-trans isomerase activity that catalyzes the cis-trans conversion of X-Pro peptide bonds, a rate-limiting step in protein folding (8). These proteins are now known to occur widely in organisms ranging from bacteria and fungi to animals and plants. Studies in animal and plant systems have uncovered diverse functions of immunophilins, such as protein foldases, chaperones, and scaffolding facilitators. They also possibly have unknown catalytic capabilities (10, ...

Very-long-chain fatty acids (VLCFAs) are essential for many aspects of plant development and necessary for the synthesis of seed storage triacylglycerols, epicuticular waxes, and sphingolipids. Identification of the acetyl-CoA carboxylase PASTICCINO3 and the 3-hydroxy acyl-CoA dehydratase PASTICCINO2 revealed that VLCFAs are important for cell proliferation and tissue patterning. Here, we show that the immunophilin PASTICCINO1 (PAS1) is also required for VLCFA synthesis. Impairment of PAS1 function results in reduction of VLCFA levels that particularly affects the composition of sphingolipids, known to be important for cell polarity in animals. Moreover, PAS1 associates with several enzymes of the VLCFA elongase complex in the endoplasmic reticulum. The pas1 mutants are deficient in lateral root formation and are characterized by an abnormal patterning of the embryo apex, which leads to defective cotyledon organogenesis. Our data indicate that in both tissues, defective organogenesis is associated with the mistargeting of the auxin efflux carrier PIN FORMED1 in specific cells, resulting in local alteration of polar auxin distribution. Furthermore, we show that exogenous VLCFAs rescue lateral root organogenesis and polar auxin distribution, indicating their direct involvement in these processes. Based on these data, we propose that PAS1 acts as a molecular scaffold for the fatty acid elongase complex in the endoplasmic reticulum and that the resulting VLCFAs are required for polar auxin transport and tissue patterning during plant development.

Cyclophilins belong to a large family of enzymes called “peptidyl prolyl isomerases” that assist protein folding and assembly. The cyclophilin CYP20–3 (also known as “ROC4”) is the only member of this group located in the stroma (soluble phase) of chloroplasts. In the present study we isolated mutant Arabidopsis plants defective in the CYP20–3 gene and found them to be hypersensitive to oxidative stress conditions created by high light levels, rose bengal, high salt levels, and osmotic shock. Chloroplast serine acetyltransferase (SAT1), a rate-limiting enzyme in cysteine biosynthesis, was identified as an interacting partner for CYP20–3 by protein interaction analyses. In the present experiments, SAT1 activity increased significantly under conditions of light and oxidative stress in concert with total thiols in wild-type plants. By contrast, these parameters changed only marginally in experiments with the cyp20–3 mutant, suggesting that CYP20–3 links light and stress to SAT1 activity and cysteine biosynthesis. In further support of this conclusion, our analyses showed that the salt-hypersensitive phenotype of the mutant developed under illumination and not in the dark. Together with the earlier report that CYP20–3 foldase activity is enhanced by thioredoxin-mediated reduction, our findings suggest that CYP20–3 links photosynthetic electron transport and redox regulation to the folding of SAT1, thereby enabling the cysteine-based thiol biosynthesis pathway to adjust to light and stress conditions.

Photosystem II (PSII) catalyzes the first of two photosynthetic reactions that convert sunlight into chemical energy. Native PSII is a supercomplex consisting of core and light-harvesting chlorophyll proteins. Although the structure of PSII has been resolved by x-ray crystallography, the mechanism underlying its assembly is poorly understood. Here, we report that an immunophilin of the chloroplast thylakoid lumen is required for accumulation of the PSII supercomplex in Arabidopsis thaliana. The immunophilin, FKBP20-2, belongs to the FK-506 binding protein (FKBP) subfamily that functions as peptidyl-prolyl isomerases (PPIases) in protein folding. FKBP20-2 has a unique pair of cysteines at the C terminus and was found to be reduced by thioredoxin (Trx) (itself reduced by NADPH by means of NADP-Trx reductase). The FKBP20-2 protein, which contains only two of the five amino acids required for catalysis, showed a low level of PPIase activity that was unaffected on reduction by Trx. Genetic disruption of the FKBP20-2 gene resulted in reduced plant growth, consistent with the observed lower rate of PSII activity determined by fluorescence (using leaves) and oxygen evolution (using isolated chloroplasts). Analysis of isolated thylakoid membranes with blue native gels and immunoblots showed that accumulation of the PSII supercomplex was compromised in mutant plants, whereas the levels of monomer and dimer building blocks were elevated compared with WT. The results provide evidence that FKBP20-2 participates specifically in the accumulation of the PSII supercomplex in the chloroplast thylakoid lumen by means of a mechanism that has yet to be determined.chloroplast thylakoid lumen ͉ protein folding ͉ photosynthetic electron transport M uch of life on Earth is sustained by oxygenic photosynthesis, a process that utilizes sunlight to produce oxygen and organic carbon from water and carbon dioxide. The absorption of light and its conversion into chemical energy is brought about by two photosystems [photosystem II (PSII) and photosystem I (PSI)] acting sequentially. PSII catalyzes the lightdependent oxidation of water that results in the evolution of oxygen. The electrons released in this reaction are transferred along a photosynthetic electron transport chain that leads, by means of PSI, to the production of NADPH and ATP, the chemical energy currency used for carbon fixation.The chloroplast PSII core complex consists of Ϸ17 protein subunits that include the D1 and D2 reaction centers for chlorophyll (Chl) P680 binding, cytochrome b559, CP43 and CP47 for building the Chl antennae, and other proteins whose function is less well characterized (1-4). The native form of PSII residing in the thylakoid membrane is believed to be a supercomplex consisting of the core and peripheral light-harvesting complex II (LHCII) components. The light harvested by LHCII is transferred to the core complex that brings about charge separation, thereby driving the transfer of electrons from water to plastoquinone and initiating photosynthetic electr...

SummaryMercury is one of the most hazardous heavy metals and is a particular problem in aquatic ecosystems, where organic mercury is biomagnified in the food chain. Previous studies demonstrated that transgenic model plants expressing a modified mercuric ion reductase gene from bacteria could detoxify mercury by converting the more toxic and reductive ionic form [Hg( II )] to less toxic elemental mercury [Hg (0)]. To further investigate if a genetic engineering approach for mercury phytoremediation can be effective in trees with a greater potential in riparian ecosystems, we generated transgenic Eastern cottonwood ( Populus deltoides ) trees expressing modified merA9 and merA18 genes. Leaf sections from transgenic plantlets produced adventitious shoots in the presence of 50 µ M Hg( II ) supplied as HgCl 2 , which inhibited shoot induction from leaf explants of wild-type plantlets.Transgenic shoots cultured in a medium containing 25 µ M Hg( II ) showed normal growth and rooted, while wild-type shoots were killed. When the transgenic cottonwood plantlets were exposed to Hg( II ), they evolved 2 -4-fold the amount of Hg(0) relative to wild-type plantlets.

SummaryA cDNA expression library from Brassica juncea was introduced into the fission yeast Schizosaccharomyces pombe to select for transformants tolerant to cadmium. Transformants expressing OXIDATIVE STRESS 3 (OXS3) or OXS3-Like cDNA exhibited enhanced tolerance to a range of metals and oxidizing chemicals. OXS3 belongs to a family of proteins that share a highly conserved domain corresponding to a putative Nacetyltransferase or thioltransferase catalytic site. Mutations within this conserved domain abolished the ability of Arabidopsis thaliana OXS3 to enhance stress tolerance in S. pombe, indicating a role in stress tolerance for the presumptive catalytic domain. A stress-sensitive mutant of AtOXS3 and enhanced tolerance of overexpression lines support the role of OXS3 in stress tolerance. The expression of tagged B. juncea and A. thaliana OXS3 proteins in plant cells revealed a subnuclear speckling pattern related to the nucleosome in discrete parts of the chromatin. It is possible that OXS3 might act as a chromatin remodeling factor for the stress response.

Black willow (Salix nigra Marsh.) is the largest and only commercially important willow species in North America. It is a candidate for phytoremediation of polluted soils because it is fast-growing and thrives on floodplains throughout eastern USA. Our objective was to develop a protocol for the in vitro regeneration of black willow plants that could serve as target material for gene transformation. Unexpanded inflorescence explants were excised from dormant buds collected from three source trees and cultured on woody plant medium (WPM) supplemented with one of: (1) 0.1 mg l(-1) thidiazuron (TDZ); (2) 0.5 mg l(-1) 6-benzoaminopurine (BAP); or (3) 1 mg l(-1) BAP. All plant growth regulator (PGR) treatments induced direct adventitious bud formation from the genotypes. The percentage of explants producing buds ranged from 20 to 92%, depending on genotype and treatment. Although most of the TDZ-treated inflorescences produced buds, these buds failed to elongate into shoots. Buds on explants treated with BAP elongated into shoots that were easily rooted in vitro and further established in potting mix in high humidity. The PGR treatments significantly affected shoot regeneration frequency (P < 0.01). The highest shoot regeneration frequency (36%) was achieved with Genotype 3 cultured on 0.5 mg l(-1) BAP. Mean number of shoots per explant varied from one to five. The ability of black willow inflorescences to produce adventitious shoots makes them potential targets for Agrobacterium-mediated transformation with heavy-metal-resistant genes for phytoremediation.

Arsenic is a metalloid that occurs naturally at parts per million (ppm) levels in the earth's crust. Natural and human activities have contributed to arsenic mobilization and increased concentration in the environment, such that World Health Organization guidelines for arsenic levels in drinking water are exceeded at many locations, worldwide. This translates into an increased risk of arsenic-related illnesses for millions of people. Recent studies demonstrate that increasing thiol-sinks in transgenic plants by overexpressing the bacterial gamma-glutamylcysteine synthetase (ECS) gene results in a higher tolerance and accumulation of metals and metalloids such as cadmium, mercury, and arsenic. We used Agrobacterium-mediated transformation to genetically engineer eastern cottonwood with a bacterial ECS gene. Eastern cottonwood plants expressing ECS had elevated thiol group levels, consistent with increased ECS activity. In addition, these ECS-expressing plants had enhanced growth on levels of arsenate toxic to control plants in vitro. Furthermore, roots of ECS-expressing plants accumulated significantly more arsenic than control roots (approximately twice as much), while shoots accumulated significantly less arsenic than control shoots (approximately two-thirds as much). We discuss potential mechanisms for shifting the balance of plant arsenic distribution from root accumulation to shoot accumulation, as it pertains to arsenic phytoremediation.