Stromal Enzyme Research Articles

Redox regulation in chloroplast thylakoid lumen: The pmf changes everything, again.

Photosynthesis is the foundation of life on Earth. However, if not well regulated, it can also generate excessive reactive oxygen species (ROS), which can cause photodamage. Regulation of photosynthesis is highly dynamic, responding to both environmental and metabolic cues, and occurs at many levels, from light capture to energy storage and metabolic processes. One general mechanism of regulation involves the reversible oxidation and reduction of protein thiol groups, which can affect the activity of enzymes and the stability of proteins. Such redox regulation has been well studied in stromal enzymes, but more recently, evidence has emerged of redox control of thylakoid lumenal enzymes. This review/hypothesis paper summarizes the latest research and discusses several open questions and challenges to achieving effective redox control in the lumen, focusing on the distinct environments and regulatory components of the thylakoid lumen, including the need to transport electrons across the thylakoid membrane, the effects of pH changes by the proton motive force (pmf) in the stromal and lumenal compartments, and the observed differences in redox states. These constraints suggest that activated oxygen species are likely to be major regulatory contributors to lumenal thiol redox regulation, with key components and processes yet to be discovered.

Chloroplastic ascorbate peroxidases targeted to stroma or thylakoid membrane: The chicken or egg dilemma.

Ascorbate peroxidases (APXs) are heme peroxidases that remove hydrogen peroxide in different subcellular compartments with concomitant ascorbate cycling. Here, we analysed and discussed phylogenetic and molecular features of the APX family. Ancient APX originated as a soluble stromal enzyme, and early during plant evolution, acquired both chloroplast-targeting and mitochondrion-targeting sequences and an alternative splicing mechanism whereby it could be expressed as a soluble or thylakoid membrane-bound enzyme. Later, independent duplication and neofunctionalization events in some angiosperm groups resulted in individual genes encoding stromal, thylakoidal and mitochondrial isoforms. These data reaffirm the complexity of plant antioxidant defenses that allow diverse plant species to acquire new means to adapt to changing environmental conditions.

Stromal NADH supplied by PHOSPHOGLYCERATE DEHYDROGENASE3 is crucial for photosynthetic performance.

During photosynthesis, electrons travel from light-excited chlorophyll molecules along the electron transport chain to the final electron acceptor nicotinamide adenine dinucleotide phosphate (NADP) to form NADPH, which fuels the Calvin–Benson–Bassham cycle (CBBC). To allow photosynthetic reactions to occur flawlessly, a constant resupply of the acceptor NADP is mandatory. Several known stromal mechanisms aid in balancing the redox poise, but none of them utilizes the structurally highly similar coenzyme NAD(H). Using Arabidopsis (Arabidopsis thaliana) as a C3-model, we describe a pathway that employs the stromal enzyme PHOSPHOGLYCERATE DEHYDROGENASE 3 (PGDH3). We showed that PGDH3 exerts high NAD(H)-specificity and is active in photosynthesizing chloroplasts. PGDH3 withdrew its substrate 3-PGA directly from the CBBC. As a result, electrons become diverted from NADPH via the CBBC into the separate NADH redox pool. pgdh3 loss-of-function mutants revealed an overreduced NADP(H) redox pool but a more oxidized plastid NAD(H) pool compared to wild-type plants. As a result, photosystem I acceptor side limitation increased in pgdh3. Furthermore, pgdh3 plants displayed delayed CBBC activation, changes in nonphotochemical quenching, and altered proton motive force partitioning. Our fluctuating light-stress phenotyping data showed progressing photosystem II damage in pgdh3 mutants, emphasizing the significance of PGDH3 for plant performance under natural light environments. In summary, this study reveals an NAD(H)-specific mechanism in the stroma that aids in balancing the chloroplast redox poise. Consequently, the stromal NAD(H) pool may provide a promising target to manipulate plant photosynthesis.

Redox regulation of chloroplast metabolism.

Regulation of enzyme activity based on thiol-disulfide exchange is a regulatory mechanism in which the protein disulfide reductase activity of thioredoxins (TRXs) plays a central role. Plant chloroplasts are equipped with a complex set of up to 20 TRXs and TRX-like proteins, the activity of which is supported by reducing power provided by photosynthetically reduced ferredoxin (FDX) with the participation of a FDX-dependent TRX reductase (FTR). Therefore, the FDX–FTR–TRXs pathway allows the regulation of redox-sensitive chloroplast enzymes in response to light. In addition, chloroplasts contain an NADPH-dependent redox system, termed NTRC, which allows the use of NADPH in the redox network of these organelles. Genetic approaches using mutants of Arabidopsis (Arabidopsis thaliana) in combination with biochemical and physiological studies have shown that both redox systems, NTRC and FDX-FTR-TRXs, participate in fine-tuning chloroplast performance in response to changes in light intensity. Moreover, these studies revealed the participation of 2-Cys peroxiredoxin (2-Cys PRX), a thiol-dependent peroxidase, in the control of the reducing activity of chloroplast TRXs as well as in the rapid oxidation of stromal enzymes upon darkness. In this review, we provide an update on recent findings regarding the redox regulatory network of plant chloroplasts, focusing on the functional relationship of 2-Cys PRXs with NTRC and the FDX–FTR–TRXs redox systems for fine-tuning chloroplast performance in response to changes in light intensity and darkness. Finally, we consider redox regulation as an additional layer of control of the signaling function of the chloroplast.

Chloroplast Redox Regulatory Mechanisms in Plant Adaptation to Light and Darkness.

Light is probably the most important environmental stimulus for plant development. As sessile organisms, plants have developed regulatory mechanisms that allow the rapid adaptation of their metabolism to changes in light availability. Redox regulation based on disulfide-dithiol exchange constitutes a rapid and reversible post-translational modification, which affects protein conformation and activity. This regulatory mechanism was initially discovered in chloroplasts when it was identified that enzymes of the Calvin-Benson cycle (CBC) are reduced and active during the day and become rapidly inactivated by oxidation in the dark. At present, the large number of redox-sensitive proteins identified in chloroplasts extend redox regulation far beyond the CBC. The classic pathway of redox regulation in chloroplasts establishes that ferredoxin (Fdx) reduced by the photosynthetic electron transport chain fuels reducing equivalents to the large set of thioredoxins (Trxs) of this organelle via the activity of a Fdx-dependent Trx reductase (FTR), hence linking redox regulation to light. In addition, chloroplasts harbor an NADPH-dependent Trx reductase with a joint Trx domain, termed NTRC. The presence in chloroplasts of this NADPH-dependent redox system raises the question of the functional relationship between NTRC and the Fdx-FTR-Trx pathways. Here, we update the current knowledge of these two redox systems focusing on recent evidence showing their functional interrelationship through the action of the thiol-dependent peroxidase, 2-Cys peroxiredoxin (2-Cys Prx). The relevant role of 2-Cys Prxs in chloroplast redox homeostasis suggests that hydrogen peroxide may exert a key function to control the redox state of stromal enzymes. Indeed, recent reports have shown the participation of 2-Cys Prxs in enzyme oxidation in the dark, thus providing an explanation for the long-lasting question of photosynthesis deactivation during the light-dark transition.

Chloroplast function and ion regulation in plants growing on saline soils: lessons from halophytes.

Salt stress impacts multiple aspects of plant metabolism and physiology. For instance it inhibits photosynthesis through stomatal limitation, causes excessive accumulation of sodium and chloride in chloroplasts, and disturbs chloroplast potassium homeostasis. Most research on salt stress has focused primarily on cytosolic ion homeostasis with few studies of how salt stress affects chloroplast ion homeostasis. This review asks the question whether membrane-transport processes and ionic relations are differentially regulated between glycophyte and halophyte chloroplasts and whether this contributes to the superior salt tolerance of halophytes. The available literature indicates that halophytes can overcome stomatal limitation by switching to CO2 concentrating mechanisms and increasing the number of chloroplasts per cell under saline conditions. Furthermore, salt entry into the chloroplast stroma may be critical for grana formation and photosystem II activity in halophytes but not in glycophytes. Salt also inhibits some stromal enzymes (e.g. fructose-1,6-bisphosphatase) to a lesser extent in halophyte species. Halophytes accumulate more chloride in chloroplasts than glycophytes and appear to use sodium in functional roles. We propose the molecular identities of candidate transporters that move sodium, chloride and potassium across chloroplast membranes and discuss how their operation may regulate photochemistry and photosystem I and II activity in chloroplasts.

P-class pentatricopeptide repeat protein PTSF1 is required for splicing of the plastid pre-tRNA(I) (le) in Physcomitrella patens.

Pentatricopeptide repeat (PPR) proteins are widely distributed in eukaryotes and are mostly localized in mitochondria or plastids. PPR proteins play essential roles in various RNA processing steps in organelles; however, the function of the majority of PPR proteins remains unknown. To examine the function of plastid PPR proteins, PpPPR_4 gene knock-out mutants were characterized in Physcomitrella patens. The knock-out mosses displayed severe growth retardation and reduced effective quantum yield of photosystem II. Immunoblot analysis showed that knock-out of PpPPR_4 resulted in a strongly reduced level of plastid-encoded proteins, such as photosystem II reaction center protein D1, the β subunit of ATP synthase, and the stromal enzyme, Rubisco. To further investigate whether knock-out of the PpPPR_4 gene affects plastid gene expression, we analyzed steady-state transcript levels of protein- and rRNA-coding genes by quantitative RT-PCR. This analysis showed that the level of many protein-coding transcripts increased in the mutants. In contrast, splicing of a spacer tRNA(I) (le) precursor encoded by the rrn operon was specifically impaired in the mutants, whereas the accumulation of other plastid tRNAs and rRNAs was not largely affected. Thus, the defect in tRNA(I) (le) splicing leads to a considerable reduction of mature tRNA(I) (le) , which may be accountable for the reduced protein level. An RNA mobility shift assay showed that the recombinant PpPPR_4 bound preferentially to domainIII of the tRNA(I) (le) group-II intron. These results provide evidence that PpPPR_4 functions in RNA splicing of the tRNA(I) (le) intron, and hence PpPPR_4 was named plastid tRNA splicing factor1 (PTSF1).

Abstract 4842: The stem cell niche detoxifies chemotherapy and protects malignant hematopoietic cells via expression of cytochrome P450 enzymes

Abstract The bone marrow (BM) microenvironment plays a critical role in drug resistance of malignant cells. Although both, direct contact as well as soluble factors have been proposed, the mechanisms responsible for this microenvironment-mediated protection remain unclear. We have recently shown that BM stroma protects human hematopoietic stem cells from the pro-differentiating effects of retinoid via cytochrome P450 (CYP) 26 mediated degradation (Ghiaur G et al., PNAS 2013). Here, we hypothesized that drug metabolism by BM stroma via CYP enzymes is a general cell-extrinsic mechanism of protection of malignant cells from chemotherapy. First, we have designed a qPCR expression array platform and found that human BM stroma expresses a variety of drug metabolizing enzymes to levels comparable to human hepatocytes. Next, using qRT-PCR, we confirmed that CYP3A4, a major drug metabolizing enzyme, is expressed in primary BM stroma but not in multiple myeloma (MM) or acute myeloid leukemia (AML) cells. We have found that BM stroma protects against Bortezomib mediated kill of MM cells [H929 (p<0.01)] and VP16 mediated kill of AML cells [KG1 (p0.01)] (Table). Since CYP3A4 plays a major role in the metabolism of aforementioned drugs, we studied if stromal CYP3A4 contributes to this protection. We found that ketoconazole, a potent CYP3A4 antagonist, overcomes bone marrow stroma-mediated resistance to Bortezomib (p=0.01), and VP16 (p=0.03). Ketoconazole had no effects in stroma free cultures. In addition, shRNA-mediated knockdown of stromal CYP3A4 renders these cells unable to protect malignant cells from chemotherapy (p=0.01). These studies open the possibilities that the stem cell niche serves as a drug-free sanctuary, where malignant stem cells can survive conventional chemotherapy. Targeting stromal P450 enzymes hold promise of improved efficiency of stem cell directed therapies and decrease rate of relapse in hematological malignancies. Table.Effects of VP16 and Bortezomib on cellular and clonogenic recovery of KG1 and H929 cells, respectively. “Stroma-CYP3A4” represents experiments performed on CYP3A4 knockdown stroma. Control shRNA infected stroma showed no differences compared to untransduced cells. Data are presented as mean±SEM from three independent experiments.VP16: KG1 cellular recoveryBortezomib: H929 clonogenic (CFU-C) recoveryLiquidStromaStroma+ketoconazoleStroma- CYP3A4LiquidStromaStroma +ketoconazoleStroma-CYP3A48.10 ± 1.5529.38 ± 0.515.7 ± 2.719.35 ± 0.8315.77 ± 3.289.5 ± 2.549.33 ± 860.35 ± 4.2 Citation Format: Salvador Alonso, Meng Su, Richard Jones, Gabriel Ghiaur. The stem cell niche detoxifies chemotherapy and protects malignant hematopoietic cells via expression of cytochrome P450 enzymes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4842. doi:10.1158/1538-7445.AM2014-4842

Multimeric states of starch phosphorylase determine protein–protein interactions with starch biosynthetic enzymes in amyloplasts

Protein–protein interactions between starch phosphorylase (SP) and other starch biosynthetic enzymes were investigated using isolated maize endosperm amyloplasts and a recombinant maize enzyme. Plastidial SP is a stromal enzyme existing as a multimeric protein in amyloplasts. Biochemical analysis of the recombinant maize SP indicated that the tetrameric form was catalytically active in both glucan-synthetic and phosphorolytic directions. Protein–protein interaction experiments employing the recombinant SP as an affinity ligand with amyloplast extracts showed that the multimeric state of SP determined interactions with other enzymes of the starch biosynthetic pathway. The monomeric form of SP interacts with starch branching enzyme I (SBEI) and SBEIIb, whereas only SBEI interacts with the tetrameric form of SP. In all cases, protein–protein interactions were broken when amyloplast lysates were dephosphorylated in vitro, and enhanced following pre-treatment with ATP, suggesting a mechanism of protein complex formation regulated by protein phosphorylation. In vitro protein phosphorylation experiments with [γ-32P]-ATP show that SP is phosphorylated by a plastidial protein kinase. Evidence is presented which suggests SBEIIb modulates the catalytic activity of SP through the formation of a heteromeric protein complex.

Exercise and nutrition in myocardial matrix metabolism, remodeling, regeneration, epigenetics, microcirculation, and muscle.

Remodeling and myocardial matrix metabolism contributes to cardiac endothelium-myocyte (perivascular fibrosis), myocyte-myocyte (interstitial fibrosis), and mitochondrion-myocyte (fusion and fission) coupling. Matrix metalloproteinases (MMPs), and tissue inhibitor of metalloproteinases (TIMPs) play differential roles in different tissues and diseases. For example, although present in the heart, MMP-3 is known as stromelysin (i.e., stromal tissue enzyme). Interestingly, TIMP-3 causes apoptosis. Exercise and nutrition are synergistic in the mitigation of diseases: exercise releases exosomes containing miRNAs. Nutrition/vitamins B6 and B12 regulate the metabolism of homocysteine (an epigenetic byproduct of DNA/RNA/protein methylation). Thus, epigenetic silencing is an important therapeutic target. The statistical analysis of cohorts may be less indicative for the treatment of a disease, particularly if the 2 twins are different in terms of responding to the medicine for the same disease, therefore, personalized medicine is the future of therapy.

A Chloroplast Light-Regulated Oxidative Sensor for Moderate Light Intensity in Arabidopsis

The transition from dark to light involves marked changes in the redox reactions of photosynthetic electron transport and in chloroplast stromal enzyme activity even under mild light and growth conditions. Thus, it is not surprising that redox regulation is used to dynamically adjust and coordinate the stromal and thylakoid compartments. While oxidation of regulatory proteins is necessary for the regulation, the identity and the mechanism of action of the oxidizing pathway are still unresolved. Here, we studied the oxidation of a thylakoid-associated atypical thioredoxin-type protein, ACHT1, in the Arabidopsis thaliana chloroplast. We found that after a brief period of net reduction in plants illuminated with moderate light intensity, a significant oxidation reaction of ACHT1 arises and counterbalances its reduction. Interestingly, ACHT1 oxidation is driven by 2-Cys peroxiredoxin (Prx), which in turn eliminates peroxides. The ACHT1 and 2-Cys Prx reaction characteristics in plants further indicated that ACHT1 oxidation is linked with changes in the photosynthetic production of peroxides. Our findings that plants with altered redox poise of the ACHT1 and 2-Cys Prx pathway show higher nonphotochemical quenching and lower photosynthetic electron transport infer a feedback regulatory role for this pathway.

Differential epithelial and stromal protein profiles in keratoconus and normal human corneas

The purpose of the study was to identify epithelial and stromal proteins that exhibit up- or down-regulation in keratoconus (KC) vs. normal human corneas. Because previous proteomic studies utilized whole human corneas or epithelium alone, thereby diluted the specificity of the proteome of each tissue, we selectively analyzed the epithelium and stromal proteins. Individual preparations of epithelial and stromal proteins from KC and age-matched normal corneas were analyzed by two independent methods, i.e., a shotgun proteomic using a Nano-Electrospray Ionization Liquid Chromatography Tandem Mass Spectrometry [Nano-ESI-LC-MS (MS) 2] and two-dimensional-difference gel electrophoresis (2D-DIGE) coupled with mass spectrometric methods. The label-free Nano-ESI-LC-MS (MS) 2 method identified 104 epithelial and 44 stromal proteins from both normal and KC corneas, and also quantified relative changes in levels of selected proteins, in both the tissues using spectral counts in a proteomic dataset. Relative to normal corneal epithelial proteins, six KC epithelial proteins (lamin-A/C, keratin type I cytoskeletal 14, tubulin beta chain, heat shock cognate 71 kDa protein, keratin type I cytoskeletal 16 and protein S100-A4) exhibited up-regulation and five proteins (transketolase, pyruvate kinase, 14-3-3 sigma isoform, phosphoglycerate kinase 1, and NADPH dehydrogenase (quinone) 1) showed down-regulation. A similar relative analysis showed that three KC stromal proteins (decorin, vimentin and keratocan) were up-regulated and five stromal proteins (TGF-betaig h3 (Bigh3), serotransferrin, MAM domain-containing protein 2 and isoforms 2C2A of collagen alpha-2[VI] chain) were down-regulated. The 2D-DIGE-mass spectrometry followed by Decyder software analysis showed that relative to normal corneas, the KC corneal epithelium exhibited up-regulation of four proteins (serum albumin, keratin 5, l-lactate dehydrogenase and annexin A8) and down-regulation of four proteins (FTH1 [Ferritin heavy chain protein 1], calpain small subunit 1, heat shock protein beta 1 and annexin A2). A similar relative analysis of stroma by this method also showed up-regulation of aldehyde dehydrogenase 3A1 (ALDH3A1), keratin 12, apolipoprotein A-IV precursor, haptoglobin precursor, prolipoprotein and lipoprotein Gln in KC corneas. Together, the results suggested that the Nano-ESI-LC-MS(MS) 2 method was superior than the 2D-DIGE method as it identified a greater number of proteins with altered levels in KC corneas. Further, the epithelial and stromal structural proteins of KC corneas exhibited altered levels compared to normal corneas, suggesting that they are affected due to structural remodeling during KC development and progression. Additionally, because several epithelial and stromal enzymes exhibited up- or down-regulation in the KC corneas relative to normal corneas, the two layers of KC corneas were under metabolic stress to adjust their remodeling.

Is light quality involved in the regulation of the photosynthetic apparatus in attached rice leaves?

The regulatory effect of light quality on the photosynthetic apparatus in attached leaves of rice plants was investigated by keeping rice plants under natural light, in complete darkness, or under illumination with light of different colors. When leaves were left in darkness and far-red (FR)-light conditions for 6 days at 30 degrees C, there was an initial lag in chlorophyll (Chl) content, Chl a/b ratio, and maximum photosystem (PS) II photochemistry that lasted until the second day; these then rapidly decreased on the fourth day. In contrast, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) rapidly disappeared with no lag under low or zero light conditions. By using spectrophotometric quantitation, it was determined that the PSII and PSI reaction centers were regulated by light quality, but cytochrome (Cyt) f was regulated by light intensity. However, the PSII heterogeneity was also strongly modified by the light intensity; PSIIalpha with the large antenna decreased markedly both in content and in antenna size. Consequently, the PSIIalpha/PSI ratio declined under FR-light because the low intensity of FR-light dominated over its quality in the modulation of the PSIIalpha/PSI ratio. An imbalance between them induced the generation of reactive oxygen species (ROS), although the ROS were scavenged by stromal enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR). The activities of these stromal enzymes are also regulated by light quality. Thus, although the photosynthetic apparatus is regulated differently depending on light quality, light quality may play an important role in the regulation of the photosynthetic apparatus.

Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function

The light-dependent regulation of stromal enzymes by thioredoxin (Trx)-catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that Trx targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Trx-mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis. However, the extent of thiol-dependent redox regulation in the thylakoid lumen has not been previously systematically explored. In this study, we addressed Trx-linked redox control in the chloroplast lumen of Arabidopsis thaliana. Using complementary proteomics approaches, we identified 19 Trx target proteins, thus covering more than 40% of the currently known lumenal chloroplast proteome. We show that the redox state of thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II. Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase, which participates in thermal dissipation of excess absorbed light. Our results indicate that redox-controlled reactions in the chloroplast lumen play essential roles in the function of photosystem II and the regulation of adaptation to light intensity.

Variable Photosynthetic Roles of Two Leaf‐type Ferredoxins in Arabidopsis, as Revealed by RNA Interference†

Ferredoxin (Fd) is the soluble protein that accepts electrons from photosystem I (PSI) and makes them available to stromal enzymes in higher plant chloroplasts. In linear electron flow, Fd mainly donates electrons to Fd:NADPH reductase (FNR) which generates NADPH for use in the Calvin cycle, but Fd may also return electrons to the thylakoid plastoquinone pool, forming a cyclic electron flow. Many higher plants contain two different photosynthetic Fd proteins, but there are no conserved sequence differences that allow their division into evolutionary groups. In the model C3 photosynthesizing dicot, Arabidopsis thaliana, there are two such photosynthetic Fds, and we have exploited RNA interference (RNAi) techniques to specifically decrease transcript abundance of different Fds in this plant. Surprisingly, the perturbation of photosynthesis, as measured by cholorophyll fluorescence, in RNAi lines of the two different photosynthetic Fds shows opposite trends. Linear electron flow is retarded in lines with lower Fd2 (the most abundant Fd species) levels and under certain circumstances enhanced in lines with lower Fd1 (the minor isoprotein) levels. These data are evidences for at least partially differentiated roles of Fd1 and Fd2 in photosynthetic electron transfer, possibly in the partition of electrons into linear and cyclic electron flow.

Altered photosynthetic electron channelling into cyclic electron flow and nitrite assimilation in a mutant of ferredoxin:NADP(H) reductase

The mechanism by which plants regulate channelling of photosynthetically derived electrons into different areas of chloroplast metabolism remains obscure. Possible fates of such electrons include use in carbon assimilation, nitrogen assimilation and redox signalling pathways, or return to the plastoquinone pool through cyclic electron flow. In higher plants, these electrons are made accessible to stromal enzymes, or for cyclic electron flow, as reduced ferredoxin (Fd), or NADPH. We investigated how knockout of an Arabidopsis (Arabidopsis thaliana) ferredoxin:NADPH reductase (FNR) isoprotein and the loss of strong thylakoid binding by the remaining FNR in this mutant affected the channelling of photosynthetic electrons into NADPH- and Fd-dependent metabolism. Chlorophyll fluorescence data show that these mutants have complex variation in cyclic electron flow, dependent on light conditions. Measurements of electron transport in isolated thylakoid and chloroplast systems demonstrated perturbed channelling to NADPH-dependent carbon and Fd-dependent nitrogen assimilating metabolism, with greater competition in the mutant. Moreover, mutants accumulate greater biomass than the wild type under low nitrate growth conditions, indicating that such altered chloroplast electron channelling has profound physiological effects. Taken together, our results demonstrate the integral role played by FNR isoform and location in the partitioning of photosynthetic reducing power.

Phase 2 study of talabostat/gemcitabine in Stage IV pancreatic cancer

4616 Background: Talabostat is an oral small molecule inhibitor of fibroblast activation protein (FAP), a stromal enzyme with collagenase and dipeptidyl peptidase activity. Talabostat also upregulates cytokine and chemokine production, resulting in immune stimulation. Talabostat is active in pancreatic tumor xenograft models and enhances the activity of gemcitabine in mice. Therefore, a clinical trial in patients with metastatic pancreatic cancer was initiated. Methods: Open-label, single-arm, Phase 2 study in 60 evaluable patients with Stage IV pancreatic cancer. Study treatment is administered in 4 x 4-week cycles; gemcitabine 1g/m2 weekly for 4 weeks in Cycle 1, then once weekly for 3 of 4 weeks. Talabostat 200mcg tablets are given BID for 6 days following each gemcitabine infusion; dose-escalation to 300mcg BID is allowed post-Cycle 1. Either agent alone or in combination can be continued beyond 4 cycles depending on tolerability. Eligible patients have measureable Stage IV pancreatic adenocarcinoma (per RECIST) are chemotherapy-naive, have a KPS ≥50, no CNS metastases, transaminases < 3 X ULN, and total bilirubin < 1.5 X ULN. Primary endpoint is 6-month survival with secondary endpoints of overall survival, PFS, pain, and quality-of-life. Tumor response or PD is assessed per RECIST. Results: As of the cut-off date, 46 patients (30 men, 16 women, median age 66 [range 43–88 years]) have received study treatment. Most patients (76%) were Stage IV at original diagnosis, and 72% have a KPS of 80 or higher. Ten of 21 evaluable patients treated as of June 30, 2006 meet 6-month survival. Median overall survival is currently estimated at 4.8 months (95% CI: 2.9, NE) in all 46 patients, and median PFS at 3.5 months (95% CI: 2.0, 4.9). Tumor responses have been reported in 3 patients: one CR and 2 PRs. Pain and QOL have not yet been analyzed. AEs are consistent with those of gemcitabine, with the exception of edema in 28.3% of patients. Grade 3 anemia, neutropenia, hyponatremia hyperbilirubinemia, and increased alk phos are reported in 2 patients each. No Grade 4 AEs have been reported in more than one patient. Conclusions: Talabostat/gemcitabine shows activity in metastatic pancreatic cancer and can be safety administered. Enrollment completed in early January 2007, and final results will be presented at the annual meeting. No significant financial relationships to disclose.

A Pentatricopeptide Repeat Protein Is Required for RNA Processing of clpP Pre-mRNA in Moss Chloroplasts

Pentatricopeptide repeat (PPR) proteins are encoded by the nuclear genome as a large gene family in land plants. PPR proteins play essential roles in organelle-related functions, mostly in RNA-processing steps in plastids and mitochondria. In the moss Physcomitrella patens, there is also a large gene family, but the moss PPR proteins are likely to be divergent from those of higher plants. To investigate the function of plastid PPR proteins, we have generated and characterized a PPR protein gene disruptant of P. patens. The PPR531-11-disrupted mosses displayed abnormal phenotypic characteristics, such as a significantly smaller protonemal colony, different chloroplast morphology, and incomplete thylakoid membrane formation. In addition, the quantum yield of photosystem II was reduced in the disrupted mosses. To further investigate whether disruption of the PPR531-11 gene affects chloroplast gene expression, we performed Northern blot and reverse transcription polymerase chain reaction analyses. These analyses revealed that PPR531-11 has a role in intergenic RNA cleavage between clpP and 5'-rps12 and in the splicing of clpP pre-mRNA. Western blot analysis showed that disruption of PPR531-11 resulted in a reduced level of ClpP, photosystem II reaction center protein D1, and the stromal enzyme, ribulose-bisphosphate carboxylase/oxygenase. These reductions might result in the severely retarded growth of the protonemal colony. Taken together, we propose a model where PPR531-11 function affects the steady-state level of ClpP, which regulates the formation and maintenance of thylakoid membranes in chloroplasts. This is the first evidence of a PPR protein controlling the protein expression level of ClpP.

DNA binding and partial nucleoid localization of the chloroplast stromal enzyme ferredoxin:sulfite reductase.

Sulfite reductase (SiR) is an important enzyme catalyzing the reduction of sulfite to sulfide during sulfur assimilation in plants. This enzyme is localized in plastids, including chloroplasts, and uses ferredoxin as an electron donor. Ferredoxin-dependent SiR has been found in isolated chloroplast nucleoids, but its localization in vivo or in intact plastids has not been examined. Here, we report the DNA-binding properties of SiRs from pea (PsSiR) and maize (ZmSiR) using an enzymatically active holoenzyme with prosthetic groups. PsSiR binds to both double-stranded and single-stranded DNA without significant sequence specificity. DNA binding did not affect the enzymatic activity of PsSiR, suggesting that ferredoxin and sulfite are accessible to SiR molecules within the nucleoids. Comparison of PsSiR and ZmSiR suggests that ZmSiR does indeed have DNA-binding activity, as was reported previously, but the DNA affinity and DNA-compacting ability are higher in PsSiR than in ZmSiR. The tight compaction of nucleoids by PsSiR led to severe repression of transcription activity in pea nucleoids. Indirect immunofluorescence microscopy showed that the majority of SiR molecules colocalized with nucleoids in pea chloroplasts, whereas no particular localization to nucleoids was detected in maize chloroplasts. These results suggest that SiR plays an essential role in compacting nucleoids in plastids, but that the extent of association of SiR with nucleoids varies among plant species.

The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach.

14C-Labelled octulose phosphates were formed during photosynthetic 14CO2 fixation and were measured in spinach leaves and chloroplasts. Because mono- and bisphosphates of d-glycero-d-ido-octulose are the active 8-carbon ketosugar intermediates of the L-type pentose pathway, it was proposed that they may also be reactants in a modified Calvin–Benson–Bassham pathway reaction scheme. This investigation therefore initially focussed only on the ido-epimer of the octulose phosphates even though 14C-labelled d-glycero-d-altro-octulose mono- and bisphosphates were also identified in chloroplasts and leaves. 14CO2 predominantly labelled positions 5 and 6 of d-glycero-d-ido-octulose 1,8-P2 consistent with labelling predictions of the modified scheme. The kinetics of 14CO2 incorporation into ido-octulose was similar to its incorporation into some traditional intermediates of the path of carbon, while subsequent exposure to 12CO2 rapidly displaced the 14C isotope label from octulose with the same kinetics of label loss as some of the confirmed Calvin pathway intermediates. This is consistent with octulose phosphates having the role of cyclic intermediates rather than synthesized storage products. (Storage products don’t rapidly exchange isotopically labelled carbons with unlabelled CO2.)A spinach chloroplast extract, designated stromal enzyme preparation (SEP), catalysed and was used to measure rates of CO2 assimilation with Calvin cycle intermediates and octulose and arabinose phosphates. Only pentose (but not arabinose) phosphates and sedoheptulose 7-phosphate supported CO2 fixation at rates in excess of 120 μmol h−1 mg−1 Chl. Rates for octulose, sedoheptulose and fructose bisphosphates, octulose, hexose and triose monophosphates were all notably less than the above rate and arabinose 5-phosphate was inactive. Altro-octulose phosphates were more active than phosphate esters of the ido-epimer. The modified scheme proposed a specific phosphotransferase and SEP unequivocally catalysed reversible phosphate transfer between sedoheptulose bisphosphate and d-glycero-d-ido-octulose 8-phosphate. It was also initially hypothesized that arabinose 5-phosphate, an L-Type pentose pathway reactant, may have a role in a modified Calvin pathway. Arabinose 5-phosphate is present in spinach chloroplasts and leaves. Radiochromatography showed that 14C-arabinose 5-phosphate with SEP, but only in the presence of an excess of unlabelled ribose 5-phosphate, lightly labelled ribulose 5-phosphate and more heavily labelled hexose and sedoheptulose mono- and bisphosphates. However, failure to demonstrate any CO2 fixation by arabinose 5-phosphate as sole substrate suggested that the above labelling may have no metabolic significance. Despite this arabinose and ribose 5-phosphates are shown to exhibit active roles as enzyme co-factors in transaldolase and aldolase exchange reactions that catalyse the epimeric interconversions of the phosphate esters of ido- and altro-octulose. Arabinose 5-phosphate is presented as playing this role in a New Reaction Scheme for the path of carbon, where it is concluded that slow reacting ido-octulose 1,8 bisphosphate has no role. The more reactive altro-octulose phosphates, which are independent of the need for phosphotransferase processing, are presented as intermediates in the new scheme. Moreover, using the estimates of phosphotransferase activity with altro-octulose monophosphate as substrate allowed calculation of the contributions of the new scheme, that ranged from 11% based on the intact chloroplast carboxylation rate to 80% using the carboxylation rate required for the support of octulose phosphate synthesis and its role in the phosphotransferase reaction.