PEP Activity Research Articles

Characterization and Fine Mapping of a Yellow-Virescent Gene Regulating Chlorophyll Biosynthesis and Early Stage Chloroplast Development in Brassica napus

Chlorophyll biosynthesis and chloroplast development are crucial to photosynthesis and plant growth, but their regulatory mechanism remains elusive in many crop species. We isolated a Brassica napus yellow-virescent leaf (yvl) mutant, which exhibited yellow-younger-leaf and virescent-older-leaf with decreased chlorophyll accumulation and delayed chloroplast development. We mapped yvl locus to a 70-kb interval between molecular markers yvl-O10 and InDel-O6 on chromosome A03 in BC2F2 population using whole genome re-sequencing and bulked segregant analysis. The mutant had a ‘C’ to ‘T’ substitution in the coding sequence of BnaA03.CHLH, which encodes putative H subunit of Mg-protoporphyrin IX chelatase (CHLH). The mutation resulted in an imperfect protein structure and reduced activity of CHLH. It also hampered the plastid encoded RNA polymerase which transcribes regulatory genes of photosystem II and I. Consequently, the chlorophyll a/b and carotenoid contents were reduced and the chloroplast ultrastructure was degraded in yvl mutant. These results explain that a single nucleotide mutation in BnaA03.CHLH impairs PEP activity to disrupt chloroplast development and chlorophyll biosynthesis in B. napus.

An isoform of the plastid RNA polymerase-associated protein FSD3 negatively regulates chloroplast development

BackgroundPlastid-encoded RNA polymerase (PEP) plays an essential role in chloroplast development by governing the expression of genes involved in photosynthesis. At least 12 PEP-associated proteins (PAPs), including FSD3/PAP4, regulate PEP activity and chloroplast development by modulating formation of the PEP complex.ResultsIn this study, we identified FSD3S, a splicing variant of FSD3; the FSD3 and FSD3S transcripts encode proteins with identical N-termini, but different C-termini. Characterization of FSD3 and FSD3S proteins showed that the C-terminal region of FSD3S contains a transmembrane domain, which promotes FSD3S localization to the chloroplast membrane but not to nucleoids, in contrast to FSD3, which localizes to the chloroplast nucleoid. We also found that overexpression of FSD3S negatively affects photosynthetic activity and chloroplast development by reducing expression of genes involved in photosynthesis. In addition, FSD3S failed to complement the chloroplast developmental defects in the fsd3 mutant.ConclusionThese results suggest FSD3 and FSD3S, with their distinct localization patterns, have different functions in chloroplast development, and FSD3S negatively regulates expression of PEP-dependent chloroplast genes, and development of chloroplasts.

NCP activates chloroplast transcription by controlling phytochrome-dependent dual nuclear and plastidial switches

Phytochromes initiate chloroplast biogenesis by activating genes encoding the photosynthetic apparatus, including photosynthesis-associated plastid-encoded genes (PhAPGs). PhAPGs are transcribed by a bacterial-type RNA polymerase (PEP), but how phytochromes in the nucleus activate chloroplast gene expression remains enigmatic. We report here a forward genetic screen in Arabidopsis that identified NUCLEAR CONTROL OF PEP ACTIVITY (NCP) as a necessary component of phytochrome signaling for PhAPG activation. NCP is dual-targeted to plastids and the nucleus. While nuclear NCP mediates the degradation of two repressors of chloroplast biogenesis, PIF1 and PIF3, NCP in plastids promotes the assembly of the PEP complex for PhAPG transcription. NCP and its paralog RCB are non-catalytic thioredoxin-like proteins that diverged in seed plants to adopt nonredundant functions in phytochrome signaling. These results support a model in which phytochromes control PhAPG expression through light-dependent double nuclear and plastidial switches that are linked by evolutionarily conserved and dual-localized regulatory proteins.

The implications of sympathetic and parasympathetic regulatory coordination for understanding child adjustment.

The autonomic nervous system (ANS) is comprised of sympathetic and parasympathetic branches that control core adaptive systems, including cardiac regulation, across periods of rest, reactivity, and recovery. Despite their heavily intertwined functions, research examining the coordination of parasympathetic and sympathetic ANS regulation is limited. This study examined the effects of 6-year-olds' (N=198; 49.5% female; 46% Latinx) capacity for ANS reactivity and recovery in both sympathetic (i.e., pre-ejection period [PEP]) and parasympathetic (i.e., respiratory sinus arrhythmia [RSA]) systems on their caregiver- and examiner-reported adaptability and attention problems at age 8. Results indicated that children's later adaptation was better accounted for by the coordination of their PEP and RSA activity than by either system in isolation. Children who evidenced optimal reactivity and recovery patterns, which entail reciprocal sympathetic and parasympathetic activity, evidenced more adaptability and fewer attention problems at age 8. In contrast, children who displayed discoordinated ANS reactivity patterns (e.g., high activation of both systems) or a total failure to recover (e.g., short PEP connoting high sympathetic activity and low RSA connoting low parasympathetic activity) evidenced poorer adjustment. These findings illustrate the incremental knowledge afforded by the joint consideration of both sympathetic and parasympathetic branches of ANS regulation in concert, as well as the importance of considering both ANS reactivity and recovery capacities for understanding adaptation.

Effects of the Dietary Probiotic Clostridium butyricum on Intestine Digestive and Metabolic Capacities, SCFA Content and Body Composition in Marsupenaeus japonicus

A 56-day feeding trial was performed to evaluate the effects of dietary probiotic Clostridium butyricum (CB) on intestine digestive and metabolic capacities, intestine short-chain fatty acids (SCFA) content and body composition of kuruma shrimp Marsupenaeus japonicus. Shrimps were randomly allocated into 9 tanks, 30 each, and fed with diets containing different levels of C. butyricum (1 × 109 cfu g−1): 0 mg g−1 feed (Control), 100 mg g−1 feed (CB-100), 200 mg g−1 feed (CB-200), while each level was triplicated. The results indicated that compared with the control group, the intestine pepsin (Pep) activity and 5-hydroxytryptamine (5-HT) concentration of two C. butyricum groups were both increased. Amylase (AMY) and lipase (LPS) activities were only induced in CB-200 group. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities of two C. butyricum groups showed no significant change. The α-amylase (AMY) gene expression was induced in CB-200 group, and trypsin gene expression of two C. butyricum treated groups were both induced. Intestine SCFA content and body composition analysis showed that the contents of propionic acid, butyric acid and the crude protein of two C. butyricum groups were all higher than those of control. These results revealed that C. butyricum can modulate intestine digestive and metabolic capacities, improve intestine SCFA content and body crude protein content in M. japonicus.

Redox regulation of PEP activity during seedling establishment in Arabidopsis thaliana

Activation of the plastid-encoded RNA polymerase is tightly controlled and involves a network of phosphorylation and, as yet unidentified, thiol-mediated events. Here, we characterize PLASTID REDOX INSENSITIVE2, a redox-regulated protein required for full PEP-driven transcription. PRIN2 dimers can be reduced into the active monomeric form by thioredoxins through reduction of a disulfide bond. Exposure to light increases the ratio between the monomeric and dimeric forms of PRIN2. Complementation of prin2-2 with different PRIN2 protein variants demonstrates that the monomer is required for light-activated PEP-dependent transcription and that expression of the nuclear-encoded photosynthesis genes is linked to the activity of PEP. Activation of PEP during chloroplast development likely is the source of a retrograde signal that promotes nuclear LHCB expression. Thus, regulation of PRIN2 is the thiol-mediated mechanism required for full PEP activity, with PRIN2 monomerization via reduction by TRXs providing a mechanistic link between photosynthetic electron transport and activation of photosynthetic gene expression.

A propeptide toolbox for secretion optimization of Flavobacterium meningosepticum endopeptidase in Lactococcus lactis

BackgroundLactic acid bacteria are a family of “generally regarded as safe” organisms traditionally used for food fermentation. In recent years, they have started to emerge as potential chassis for heterologous protein production. And more recently, due to their beneficial properties in the gut, they have been examined as potential candidates for mucosal delivery vectors, especially for acid-sensitive enzymes. One such application would be the delivery of gluten-digesting endopeptidases for the treatment of celiac disease. To facilitate these applications, an efficient recombinant protein expression toolbox is required, especially for recombinant protein secretion. While current tools for enhancing protein secretion consist mainly of signal peptides, secretion propeptides have also been observed to play a crucial role for protein secretion and improved yields.ResultsTo expand the propeptide library for secretion optimization, we have mined and characterized three naturally occurring propeptides from the sequenced genomes of 109 Lactococcus species. These newly-mined propeptides were introduced after the N-terminal USP45 secretion signal to characterize and compare their effects on the secretion of Escherichia coli thioredoxin (TRX) and Flavobacterium meningosepticum prolyl endopeptidase (Fm PEP) in Lactococcus lactis NZ9000. All three propeptides, along with the positive control LEISSTCDA, improved volumetric secretion yields by 1.4–2.3-folds. However, enhancement of secretion yield is dependent on protein of interest. For TRX, the optimal combination of USP45 signal peptide and LEISSTCDA produced a 2.3-fold increase in secretion yields. Whilst for Fm PEP, propeptide 1 with USP45 signal peptide improved volumetric secretion yields by 2.2-fold compared to a 1.4-fold increase by LEISSTCDA. Similar trends in Fm PEP activity and protein yield also demonstrated minimal effect of the negative charged propeptides on PEP activity and thus folding.ConclusionsOverall, we have characterized three new propeptides for use in L. lactis secretion optimization. From success of these propeptides for improvement of secretion yields, we anticipate this collection to be valuable to heterologous protein secretion optimisation in lactic acid bacteria. We have also demonstrated for the first time, secretion of Fm PEP in L. lactis for potential use as a therapy agent in celiac disease.

Effects of UPR and ERAD pathway on the prolyl endopeptidase production in Pichia pastoris by controlling of nitrogen source.

Prolyl endopeptidase (PEP) is very useful in various industries, while the high cost of enzyme production remains a major obstacle for its industrial applications. Pichia pastoris has been used for the PEP production; however, the fermentation process has not be investigated and little is known about the impact of excessive PEP production on the host cell physiology. Here, we optimized the nitrogen source to improve the PEP expression level and further evaluated the cellular response including UPR and ERAD. During methanol induction phase the PEP activity (1583 U/L) was increased by 1.48-fold under the optimized nitrogen concentration of NH4+ (300mmol/L) and casamino acids [1.0% (w/v)] in a 3-L bioreactor. Evaluated by RT-PCR the UPR and ERAD pathways were confirmed to be activated. Furthermore, a strong decrease of ERAD-related gene transcription was observed with the addition of nitrogen source, which contributed to a higher PEP expression level.

High-throughput Screening of Small Molecule Inhibitors of the Streptococcus Quorum-sensing Signal Pathway

The main components of the quorum-sensing system are expected to be favorable targets for drug development to combat various chronic infectious diseases. ComA of Streptococcus is an ATP-binding cassette transporter containing a peptidase domain (PEP), which is essential for the quorum-sensing signal production. Using high-throughput screening, we found a potent small molecule that suppressed the S. mutans quorum-sensing pathway through inhibition of PEP activity. The compound effectively attenuated the biofilm formation and competence development of S. mutans without inhibiting cell growth. The kinetic and structural studies with this molecule and a related compound unexpectedly revealed an allosteric site of PEP. This relatively hydrophobic site is thought to undergo large structural changes during the catalytic process. These compounds inhibit PEP activity by binding to and suppressing the structural changes of this site. These results showed that PEP is a good target for inhibitors of the Streptococcus quorum-sensing system.

Lethal albinic seedling, encoding a threonyl-tRNA synthetase, is involved in development of plastid protein synthesis system in rice.

An albinic rice is caused by mutation of threonyl-tRNA synthetase, which is essential for plant development by stabilizing of NEP and PEP gene expressions and chloroplast protein synthesis. Chloroplast biogenesis and development depend on complex genetic mechanisms. Apart from their function in translation, aminoacyl-tRNA synthetases (aaRSs) play additional role in gene expression regulation, RNA splicing, and cytokine activity. However, their detailed functions in plant development are still poorly understood. We isolated a lethal albinic seedling (las) mutant in rice. Physiological and ultrastructural analysis of las mutant plants revealed weak chlorophyll fluorescence, negligible chlorophyll accumulation, and defective thylakoid membrane development. By map based cloning we determined that the LAS allele gene encodes threonyl-tRNA synthetase (ThrRS). LAS was constitutively expressed with relatively high level in leaves. NEP-dependent gene transcripts accumulated in the developing chloroplasts, while PEP-dependent transcripts were reduced in the las mutant. This result indicated that PEP activity was impaired. Chloroplast-encoded protein levels were sharply reduced in the las mutant. Biogenesis of chloroplast rRNAs (16S and 23S rRNA) was arrested, leading to impaired translation and protein synthesis. Together, our findings indicated that LAS is essential not only for chloroplast development by stabilizing the NEP and PEP gene expression, but also for protein synthesis and construction of the ribosome system in rice chloroplasts.

Rubisco accumulation is important for the greening of the fln2-4 mutant in Arabidopsis

The fructokinase-like protein2 (FLN2) is a component of the PEP complex. FLN2 knockout mutants displayed a delayed greening phenotype on sucrose-containing medium. Our previous work indicated that partial PEP activity is essential for its greening phenotype. In this study, we further report that sufficient Rubisco accumulation is critical for fln2-4 greening. Sugar serves many important functions, such as an energy source and signaling molecule. Through pharmacological experiments using a sugar analog and sugar signaling inhibitor, we demonstrate that sugar serves as energy to support the fln2-4 greening. Seed-reserve and photosynthetic CO2-fixation are the primary energy sources for early seedling growth. No obvious differences were observed in the seed-reserve of the wild-type and fln2-4 by comparing their seed size and dark-germination, indicating that the defective carbon fixation may account for the energy deficit in fln2-4 during its early seedling growth. The Rubisco content was low in fln2-4, but it rapidly accumulated during the greening of fln2-4. Expression of a nuclear-encoded rbcL gene facilitates Rubisco accumulation and partially complements the mutant defects. These results suggest that the Rubisco accumulation is critical for fln2-4 greening. In summary, the rapid Rubisco accumulation that depends on sufficient PEP activity is important for normal seedling growth.

Characterization and ontogenetic development of digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae.

The major digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae were characterized, and the physiological characteristics of the enzymes during early ontogeny were clarified using biochemical and molecular approaches. The maximum activity of trypsin (Try), chymotrypsin (Ct) and amylase (Amy) was observed at pH 6-11, 8-11 and 6-9, respectively. Maximum activity of Try, Ct and Amy occurred at 50 °C, that of lipase (Lip) was at 60 °C and that of pepsin (Pep) was at 40-50 °C. These pH and thermal profiles were similar to those for other fish species but differed from those previously reported for adult bluefin tuna. Enzyme activity for all enzymes assayed was found to decrease at high temperatures (Try, Ct, Amy and Pep: 50 °C; Lip: 40 °C), which is similar to findings for other fish species with one marked exception-increased Try activity was observed at 40 °C. Lip activity appeared to be dependent on bile salts under our assay conditions, resulting in a significant increase in activity in the presence of bile salts. Ontogenetic changes in pancreatic digestive enzymes showed similar gene expression patterns to those of other fish species, whereas marked temporal increases in enzyme activities were observed at 10-12 days post hatching (dph), coinciding with previously reported timing of the development of the pyloric caeca in bluefin tuna larvae. However, complete development of digestive function was indicated by the high pep gene expression from 19 dph, which contradicts the profile of Pep activity and previously reported development timing of the gastric gland. These findings contribute to the general knowledge of bluefin tuna larval digestive system development.

Increased prolyl endopeptidase activity in human neoplasia

Prolyl endopeptidase (EC 3.4.21.26) (PEP) is a serine peptidase that converts several biologically active peptides. This enzyme has been linked to several neurological, digestive, cardiovascular and infectous disorders. However, little is known about its involvement in neoplastic processes. This study analyzes fluorimetrically cytosolic and membrane-bound PEP activity in a large series ( n = 122) of normal and neoplastic tissues from the kidney, colon, oral cavity, larynx, thyroid gland and testis. Cytosolic PEP activity significantly increased in clear cell renal cell carcinoma, urothelial carcinoma of the renal pelvis and head and neck squamous cell carcinoma. Both cytosolic and membrane-bound PEP activity were also increased in colorectal adenomatous polyps. These data suggest the involvement of PEP in some mechanisms that underlie neoplastic processes.

Plasma Activity of Prolyl Endopeptidase in Relation to Psychopathology During Immunotherapy with IFN-α in Patients with Renal Cell Carcinoma

Abnormal activity in peripheral blood of the cytosolic enzyme prolyl endopeptidase (PEP, EC 3.4.21.26, post prolyl cleaving enzyme, prolyl oligopeptidase) has been found in patients with a variety of psychiatric disorders, most consistently in mood disorders. Mood disturbance is a well-known side effect of immunotherapy with interferon-alpha (IFN-alpha). Earlier, we documented a decrease in serum PEP activity in the first 4 weeks of treatment with IFN-alpha. In 24 patients (16 men, 8 women, median age 60.5 years, range 47-72 years) with metastatic renal cell carcinoma (RCC), psychiatric assessment and blood sampling were performed before and at 4 and 8 weeks and at 6 months after initiation of treatment with IFN-alpha. No episodes of depression were observed, and the sum score and the scores on the subscales for depression and hostility of the Symptom Check List-90 (SCL-90) did not change during follow-up, whereas the anxiety scores were somewhat lower at 4 and 8 weeks compared with baseline. No change in plasma PEP activity and no relationships between change in psychiatric parameters and change in plasma PEP activity were found. As more subtle relationships between PEP activity and psychiatric status could have easily been obscured, a role for PEP in the pathophysiology of IFN-alpha-induced mood disturbance can neither be confirmed nor excluded.

Prolyl Endopeptidase Inhibitory Activity of Unsaturated Fatty Acids

Prolyl endopeptidase (PEP, EC 3.4.21.26) is widely distributed in various organs, particularly in the brains of amnestic patients. Evaluation of PEP levels in postmortem brains of Alzheimer's disease patients revealed significant increases in PEP activity, suggesting that a specific PEP inhibitor can be a good candidate for an antiamnestic drug. In this study, mono- and polyunsaturated fatty acids were investigated to determine their role as PEP inhibitors. Oleic, linoleic, and arachidonic acids, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) showed PEP inhibitory activities (IC50 values of 23.6 +/- 0.4, 43.8 +/- 1.8, 53.4 +/- 1.2, 99.4 +/- 1.2, and 46.2 +/- 1.0 microM, respectively), indicating that they were effective PEP inhibitors, with inhibition constant (Ki) values of 26.7 +/- 0.3, 51.0 +/- 0.7, 91.3 +/- 3.1, 247.5 +/- 2.6, and 89.0 +/- 2.3 microM, respectively. Oleic acid showed the highest PEP inhibitory activity. Dixon plots of PEP inhibition showed oleic, linoleic, and arachidonic acids, EPA, and DHA are noncompetitive inhibitors; despite higher IC50 values of these unsaturated fatty acids than strong natural inhibitors, they may have potential use in preventing memory loss.

Alterations of prolyl endopeptidase activity in the plasma of children with autistic spectrum disorders

BackgroundProlyl Endopeptidase (PEP, EC 3.4.21.26), a cytosolic endopeptidase, hydrolyses peptide bonds on the carboxyl side of proline residue in proteins with a relatively small molecular weight. It has been shown that altered PEP activity is associated with various psychological diseases such as schizophrenia, mania and depression. Autistic Spectrum Disorders (ASD) are neuropsychiatric and behavioural syndromes affecting social behaviours and communication development. They are classified as developmental disorders. The aim of this study was to examine the hypothesis that PEP activity is also associated with ASDs.MethodsFluorometric assay was used to measure PEP activity in EDTA plasma in children with ASD (n = 18) aged 4–12 years (mean ± SD: 7.9 ± 2.5). These results were then compared to PEP activity in a control group of non-ASD children (n = 15) aged 2–10 years (mean ± SD: 6.4 ± 2.2).ResultsAn alteration in PEP activity was found in the children with ASD compared to the control group. There was much greater variation of PEP activity in the group of ASD children when compared to the controls (SD= 39.9 and SD 9.6, respectively). This variation was significant (p < 0.0005), although the mean level of PEP activity in the group of ASD children was slightly higher than in the control group (124.4 and 134.1, respectively).ConclusionOur preliminary finding suggests a role for PEP enzyme in the pathophysiology of autism but further research should be conducted to establish its role in the aetiology of psychiatric and neurological disorders, including autism and related spectrum disorders.

A comparison of psychophysiological and self‐report measures of BAS and BIS activation

The behavioral approach (BAS) and behavioral inhibition (BIS) systems are central to theories of both personality and psychopathology, yet agreement on methods of assessing BAS and BIS sensitivity has yet to emerge. We compare the Carver and White (1994) BIS/BAS scales with putative physiological markers of BAS (pre-ejection period [PEP], respiratory sinus arrhythmia [RSA]) and BIS (electrodermal responding) reactivity during reward and extinction among 50 undergraduates. PEP, RSA, and electrodermal activity each responded strongly to one or more task conditions, but correlations with BIS/BAS scores were stronger for measures of affectivity than for any physiological marker. Finally, PEP reactivity was the only autonomic index that responded only to reward. These findings suggest that (a) self-report and physiological measures of BAS and BIS reactivity are independent, and (b) PEP may be superior to RSA as an index of approach motivation.

Factors influencing analysis of prolyl endopeptidase in human blood and cerebrospinal fluid: increase in assay sensitivity

Prolyl endopeptidase (EC 3.4.21.26) (PEP) is present in nearly all investigated mammalian cells and biological fluids and might be involved in the degradation of physiologically important neuropeptides. To be able to investigate the variation of PEP in blood and cerebrospinal fluid (CSF) in human disease, the factors influencing analysis of PEP in these body fluids must be determined. The purpose of the present work was to study the influence of storage conditions, anticoagulation additives, freezing and thawing and substrate solvent on determination of PEP in blood plasma/serum and CSF. It was found that the PEP activity was about 10% higher in plasma (with EDTA and heparinate for anticoagulation) than in serum. Storage at room temperature (20°C) caused a rapid decline in enzyme activity, which was smaller but still considerable at 4°C. Storage at −20°C and −70°C did not decrease the PEP activity. Freezing and thawing of plasma/serum samples showed that the first freeze‐thawing cycle produced a 20% reduction in enzyme activity but little further decrease was observed during subsequent cycles of freeze‐thawing. In conclusion, PEP activity should preferably be measured within one hour after sampling using EDTA‐ or heparinate plasma. For long‐term storage, samples should be immediately frozen and stored at −20°C or colder. The selection and amount of the organic solvent used to dissolve the fluorogenic substrate strongly influenced the sensitivity of the assay. By developing an optimal solvent system an increase in assay sensitivity of about 400% could be obtained, which for the first time allowed measurement of the PEP activity in CSF.

Effect of S 17092, a novel prolyl endopeptidase inhibitor, on substance P and alpha-melanocyte-stimulating hormone breakdown in the rat brain.

In the present study, we have investigated the effects of a novel prolyl endopeptidase (EC 3.4.21.26, PEP) inhibitor, compound S 17092, on substance P (SP) and alpha-melanocyte-stimulating hormone (alpha-MSH) metabolism in the rat brain. In vitro experiments revealed that S 17092 inhibits in a dose-dependent manner PEP activity in rat cortical extracts (IC50 = 8.3 nm). In addition, S 17092 totally abolished the degradation of SP and alpha-MSH induced by bacterial PEP. In vivo, a significant decrease in PEP activity was observed in the medulla oblongata after a single oral administration of S 17092 at doses of 10 and 30 mg/kg (-78% and -82%, respectively) and after chronic oral treatment with S 17092 at doses of 10 and 30 mg/kg per day (-75% and -88%, respectively). Concurrently, a single administration of S 17092 (30 mg/kg) caused a significant increase in SP- and alpha-MSH-like immunoreactivity (LI) in the frontal cortex (+41% and +122%, respectively) and hypothalamus (+84% and +49%, respectively). In contrast, chronic treatment with S 17092 did not significantly modify SP- and alpha-MSH-LI in the frontal cortex and hypothalamus. Collectively, the present results show that S 17092 elevates SP and alpha-MSH concentrations in the rat brain by inhibiting PEP activity. These data suggest that the effect of S 17092 on memory impairment can be accounted for, at least in part, by inhibition of catabolism of promnesic neuropeptides such as SP and alpha-MSH.

Analysis of promoter selectivity of plastid sigma factors in Arabidopsis thaliana

In higher plants, chloroplasts have at least two types of RNA polymerases(RNAPs), the bacterial-type RNAP (PEP; plastid-encoded RNAP) and the T7 bacteriophage type RNAP (NEP; nuclear-encoded RNAP). PEP transcribes preferentially the genes encoding the proteins implicated in the characteristic functions of chloroplasts and is composed of plastid-encoded core enzyme subunits and a nuclear-encoded sigma factor, which presumably confer promoter-specific transcription initiation on RNAP. In higher plant genome analysis, a number of putative sigma factors have been identified. These multiple sigma factors are considered to recognize different sets of promoter sequences and regulate the activity of PEP in response to the developmental stage and various environmental clues. To analyze the promoter selectivity of sigma factors in Arabidopsis thaliana, each of six sig genes was expressed transiently in protoplasts and the transcription of 12 plastid genes in each transformant was measured by means of the run-on transcription assay. In this assay, three sigma factors were found to elevate the PEP activity for different plastid genes, indicating that each sigma factor has different promoter recognition property. The regulation mechanism of transcription of plastid genes through sigma factors will be discussed.