The translocon protein FtsHi1 is an ATP-dependent DNA/RNA helicase that prevents R-loop accumulation in chloroplasts.

R-loops, three-stranded nucleic acid structures consisting of a DNA: RNA hybrid and displaced single-stranded DNA, play critical roles in gene expression and genome stability. How R-loop homeostasis is integrated into chloroplast gene expression remains largely unknown. We found an unexpected function of FtsHi1, an inner envelope membrane-bound AAA-ATPase in chloroplast R-loop homeostasis of Arabidopsis thaliana. Previously, this protein was shown to function as a component of the import motor complex for nuclear-encoded chloroplast proteins. However, this study provides evidence that FtsHi1 is an ATP-dependent helicase that efficiently unwinds both DNA-DNA and DNA-RNA duplexes, thereby preventing R-loop accumulation. Over-accumulation of R-loops could impair chloroplast transcription but not necessarily genome integrity. The dual function of FtsHi1 in both protein import and chloroplast gene expression may be important to coordinate the biogenesis of nuclear- and chloroplast-encoded subunits of multi-protein photosynthetic complexes. This study suggests a mechanical link between protein import and R-loop homeostasis in chloroplasts of higher plants.

Autophagy targets Hd1 for vacuolar degradation to regulate rice flowering.

Flowering time (heading date) is a critical agronomic trait that determines the yield and regional adaptability of crops. Heading date 1 (Hd1) is a central regulator of photoperiodic flowering in rice (Oryza sativa). However, how the homeostasis of Hd1 protein is achieved is poorly understood. Here, we report that the nuclear autophagy pathway mediates Hd1 degradation in the dark to regulate flowering. Loss of autophagy function results in an accumulation of Hd1 and delays flowering under both short-day and long-day conditions. In the dark, nucleus-localized Hd1 is recognized as a substrate for autophagy and is subjected to vacuolar degradation via the autophagy protein OsATG8. The Hd1-OsATG8 interaction is required for autophagic degradation of Hd1 in the dark. Our study reveals a new mechanism by which Hd1 protein homeostasis is regulated by autophagy to control rice flowering. Our study also indicates that the regulation of flowering by autophagic degradation of Hd1 orthologs may have arisen over the course of mesangiosperm evolution, which would have increased their flexibility and adaptability to the environment by modulating flowering time.

Plastid Deficient 1 Is Essential for the Accumulation of Plastid-Encoded RNA Polymerase Core Subunit ß and Chloroplast Development in Arabidopsis.

Plastid-encoded RNA polymerase (PEP)-dependent transcription is an essential process for chloroplast development and plant growth. It is a complex event that is regulated by numerous nuclear-encoded proteins. In order to elucidate the complex regulation mechanism of PEP activity, identification and characterization of PEP activity regulation factors are needed. Here, we characterize Plastid Deficient 1 (PD1) as a novel regulator for PEP-dependent gene expression and chloroplast development in Arabidopsis. The PD1 gene encodes a protein that is conserved in photoautotrophic organisms. The Arabidopsis pd1 mutant showed albino and seedling-lethal phenotypes. The plastid development in the pd1 mutant was arrested. The PD1 protein localized in the chloroplasts, and it colocalized with nucleoid protein TRXz. RT-quantitative real-time PCR, northern blot, and run-on analyses indicated that the PEP-dependent transcription in the pd1 mutant was dramatically impaired, whereas the nuclear-encoded RNA polymerase-dependent transcription was up-regulated. The yeast two-hybrid assays and coimmunoprecipitation experiments showed that the PD1 protein interacts with PEP core subunit ß (PEP-ß), which has been verified to be essential for chloroplast development. The immunoblot analysis indicated that the accumulation of PEP-ß was barely detected in the pd1 mutant, whereas the accumulation of the other essential components of the PEP complex, such as core subunits α and ß', were not affected in the pd1 mutant. These observations suggested that the PD1 protein is essential for the accumulation of PEP-ß and chloroplast development in Arabidopsis, potentially by direct interaction with PEP-ß.

Analysis of the changes of electron transfer and heterogeneity of photosystem II in Deg1-reduced Arabidopsis plants.

Deg1 protease functions in protease and chaperone of PSII complex components, but few works were performed to study the effects of Deg1 on electron transport activities on the donor and acceptor side of PSII and its correlation with the photoprotection of PSII during photoinhibition. Therefore, we performed systematic and comprehensive investigations of electron transfers on the donor and acceptor sides of photosystem II (PSII) in the Deg1-reduced transgenic lines deg1-2 and deg1-4. Both the maximal quantum efficiency of PSII photochemistry (Fv/Fm) and the actual PSII efficiency (ΦPSII) decreased significantly in the transgenic plants. Increases in nonphotochemical quenching (NPQ) and the dissipated energy flux per reaction center (DI0/RC) were also shown in the transgenic plants. Along with the decreased D1, CP47, and CP43 content, these results suggested photoinhibition under growth light conditions in transgenic plants. Decreased Deg1 caused inhibition of electron transfer on the PSII reducing side, leading to a decline in the number of QB-reducing centers and accumulation of QB-nonreducing centers. The Tm of the Q band shifted from 5.7 °C in the wild-type plant to 10.4 °C and 14.2 °C in the deg1-2 and deg1-4 plants, respectively, indicating an increase in the stability of S2QA¯ in transgenic plants. PSIIα in the transgenic plants largely reduced, while PSIIß and PSIIγ increased with the decline in the Deg1 levels in transgenic plants suggesting PSIIα centers gradually converted into PSIIß and PSIIγ centers in the transgenic plants. Besides, the connectivity of PSIIα and PSIIß was downregulated in transgenic plants. Our results reveal that downregulation of Deg1 protein levels induced photoinhibition in transgenic plants, leading to loss of PSII activities on both the donor and acceptor sides in transgenic plants. These results give a new insight into the regulation role of Deg1 in PSII electron transport.

OsSWEET14 cooperates with OsSWEET11 to contribute to grain filling in rice.

The grain-filling process is crucial for cereal crop yields, but how the caryopsis of such plants is supplied with sugars, which are produced by photosynthesis in leaves and then transported long distance, is largely unknown. In rice (Oryza sativa), various SWEET family sucrose transporters are thought to have important roles in grain filling. Here, we report that OsSWEET14 plays a crucial part in this process in rice. ossweet14 knockout mutants did not show any detectable phenotypic differences from the wild type, whereas ossweet14;ossweet11 double-knockout mutants had much more severe phenotypes than ossweet11 single-knockout mutants, including strongly reduced grain weight and yield, reduced grain-filling rate, and increased starch accumulation in the pericarp. Both OsSWEET14 and OsSWEET11 exhibited distinct spatiotemporal expression patterns between the early stage of caryopsis development and the rapid grain-filling stage. During the rapid grain-filling stage, OsSWEET14 and OsSWEET11 localized to four key sites: vascular parenchyma cells, the nucellar projection, the nucellar epidermis, and cross cells. These results demonstrate that OsSWEET14 plays an important role in grain filling, and they suggest that four major apoplasmic pathways supply sucrose to the endosperm during the rapid grain-filling stage via the sucrose effluxers SWEET14 and SWEET11.

mTERF5 Acts as a Transcriptional Pausing Factor to Positively Regulate Transcription of Chloroplast psbEFLJ.

RNA polymerase transcriptional pausing represents a major checkpoint in transcription in bacteria and metazoans, but it is unknown whether this phenomenon occurs in plant organelles. Here, we report that transcriptional pausing occurs in chloroplasts. We found that mTERF5 specifically and positively regulates the transcription of chloroplast psbEFLJ in Arabidopsis thaliana that encodes four key subunits of photosystem II. We found that mTERF5 causes the plastid-encoded RNA polymerase (PEP) complex to pause at psbEFLJ by binding to the +30 to +51 region of double-stranded DNA. Moreover, we revealed that mTERF5 interacts with pTAC6, an essential subunit of the PEP complex, although pTAC6 is not involved in the transcriptional pausing at psbEFLJ. We showed that mTERF5 recruits additional pTAC6 to the transcriptionally paused region of psbEFLJ, and the recruited pTAC6 proteins could be assembled into the PEP complex to regulate psbEFLJ transcription. Taken together, our findings shed light on the role of transcriptional pausing in chloroplast transcription in plants.

PPR-SMR protein SOT1 has RNA endonuclease activity.

Numerous attempts have been made to identify and engineer sequence-specific RNA endonucleases, as these would allow for efficient RNA manipulation. However, no natural RNA endonuclease that recognizes RNA in a sequence-specific manner has been described to date. Here, we report that SUPPRESSOR OF THYLAKOID FORMATION 1 (SOT1), an Arabidopsis pentatricopeptide repeat (PPR) protein with a small MutS-related (SMR) domain, has RNA endonuclease activity. We show that the SMR moiety of SOT1 performs the endonucleolytic maturation of 23S and 4.5S rRNA through the PPR domain, specifically recognizing a 13-nucleotide RNA sequence in the 5' end of the chloroplast 23S-4.5S rRNA precursor. In addition, we successfully engineered the SOT1 protein with altered PPR motifs to recognize and cleave a predicted RNA substrate. Our findings point to SOT1 as an exciting tool for RNA manipulation.

The Phytol Phosphorylation Pathway Is Essential for the Biosynthesis of Phylloquinone, which Is Required for Photosystem I Stability in Arabidopsis.

Phytyl-diphosphate, which provides phytyl moieties as a common substrate in both tocopherol and phylloquinone biosynthesis, derives from de novo isoprenoid biosynthesis or a salvage pathway via phytol phosphorylation. However, very little is known about the role and origin of the phytyl moiety for phylloquinone biosynthesis. Since VTE6, a phytyl-phosphate kinase, is a key enzyme for phytol phosphorylation, we characterized Arabidopsis vte6 mutants to gain insight into the roles of phytyl moieties in phylloquinone biosynthesis and of phylloquinone in photosystem I (PSI) biogenesis. The VTE6 knockout mutants vte6-1 and vte6-2 lacked detectable phylloquinone, whereas the phylloquinone content in the VTE6 knockdown mutant vte6-3 was 90% lower than that in wild-type. In vte6 mutants, PSI function was impaired and accumulation of the PSI complex was defective. The PSI core subunits PsaA/B were efficiently synthesized and assembled into the PSI complex in vte6-3. However, the degradation rate of PSI subunits in the assembled PSI complex was more rapid in vte6-3 than in wild-type. In vte6-3, PSI was more susceptible to high-light damage than in wild-type. Our results provide the first genetic evidence that the phytol phosphorylation pathway is essential for phylloquinone biosynthesis, and that phylloquinone is required for PSI complex stability.

Glutathione reductase 2 maintains the function of photosystem II in Arabidopsis under excess light.

Glutathione reductase plays a crucial role in the elimination of H(2)O(2) molecules via the ascorbate-glutathione cycle. In this study, we used transgenic Arabidopsis plants with decreased glutathione reductase 2 (GR2) levels to investigate whether this GR2 activity protects the photosynthetic machinery under excess light. The transgenic plants were highly sensitive to excess light and accumulated high levels of H(2)O(2). Photosystem II (PSII) activity was significantly decreased in transgenic plants. Flash-induced fluorescence relaxation and thermoluminescence measurements demonstrated inhibition of electron transfer between Q(A) and Q(B) and decreased redox potential of Q(B) in transgenic plants. Immunoblot and blue native gel analysis showed that the levels of PSII proteins and PSII complexes were decreased in transgenic plants. Analyses of the repair of photodamaged PSII and in vivo pulse labeling of thylakoid proteins showed that the repair of photodamaged PSII is inhibited due to the inhibition of the synthesis of the D1 protein de novo in transgenic plants. Taken together, our results suggest that under excess light conditions, GR2 plays an important role in maintaining both the function of the acceptor side of PSII and the repair of photodamaged PSII by preventing the accumulation of H(2)O(2). In addition, our results provide details of the role of H(2)O(2) in vivo accumulation in photoinhibition in plants.

Decreased glutathione reductase2 leads to early leaf senescence in Arabidopsis.

Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) and participates in the ascorbate-glutathione cycle, which scavenges H2 O2 . Here, we report that chloroplastic/mitochondrial GR2 is an important regulator of leaf senescence. Seed development of the homozygous gr2 knockout mutant was blocked at the globular stage. Therefore, to investigate the function of GR2 in leaf senescence, we generated transgenic Arabidopsis plants with decreased GR2 using RNAi. The GR2 RNAi plants displayed early onset of age-dependent and dark- and H2 O2 -induced leaf senescence, which was accompanied by the induction of the senescence-related marker genes SAG12 and SAG13. Furthermore, transcriptome analysis revealed that genes related to leaf senescence, oxidative stress, and phytohormone pathways were upregulated directly before senescence in RNAi plants. In addition, H2 O2 accumulated to higher levels in RNAi plants than in wild-type plants and the levels of H2 O2 peaked in RNAi plants directly before the early onset of leaf senescence. RNAi plants showed a greater decrease in GSH/GSSG levels than wild-type plants during leaf development. Our results suggest that GR2 plays an important role in leaf senescence by modulating H2 O2 and glutathione signaling in Arabidopsis.

Enhanced Sucrose Loading Improves Rice Yield by Increasing Grain Size.

Yield in cereals is a function of grain number and size. Sucrose (Suc), the main carbohydrate product of photosynthesis in higher plants, is transported long distances from source leaves to sink organs such as seeds and roots. Here, we report that transgenic rice plants (Oryza sativa) expressing the Arabidopsis (Arabidopsis thaliana) phloem-specific Suc transporter (AtSUC2), which loads Suc into the phloem under control of the phloem protein2 promoter (pPP2), showed an increase in grain yield of up to 16% relative to wild-type plants in field trials. Compared with wild-type plants, pPP2::AtSUC2 plants had larger spikelet hulls and larger and heavier grains. Grain filling was accelerated in the transgenic plants, and more photoassimilate was transported from the leaves to the grain. In addition, microarray analyses revealed that carbohydrate, amino acid, and lipid metabolism was enhanced in the leaves and grain of pPP2::AtSUC2 plants. Thus, enhancing Suc loading represents a promising strategy to improve rice yield to feed the global population.

Purine biosynthetic enzyme ATase2 is involved in the regulation of early chloroplast development and chloroplast gene expression in Arabidopsis.

To investigate the molecular mechanism of chloroplast biogenesis and development, we characterized an Arabidopsis mutant (dg169, delayed greening 169) which showed growth retardation and delayed greening phenotype in leaves. Newly emerged chlorotic leaves recovered gradually with leaf development in the mutant, and the mature leaves showed similar phenotype to those of wild-typewild-type plants. Compared with wild-type, the chloroplasts were oval-shaped and smaller and the thylakoid membranes were less abundant in yellow section of young leaves of dg169. In addition, the functions of photosystem II (PSII) and photosystem I (PSI) were also impaired. Furthermore, the amount of core subunits of PSII and PSI, as well as PSII and PSI complexes reduced in yellow section of young leaves of dg169. Map-based positional cloning identified that phenotype of dg169 was attributed to a point mutation of ATase2 which converts the conserved Ile-155 residue to Asn. ATase2 catalyzes the first step of de novo purine biosynthesis. This mutation resulted in impaired purine synthesis and a significant decrease in ATP, ADP, GTP and GDP contents. The analysis of ATase2-GFP protein fusion showed that ATase2 was localized to nucleoid of chloroplasts. Our results further demonstrated that the levels of PEP-dependent transcripts in yellow section of young leaves of dg169 were decreased while NEP-dependent and both PEP- and NEP-dependent transcripts and chloroplast DNA replications were increased. The results in this study suggest that ATase2 plays an essential role in early chloroplast development through maintaining PEP function.

Identification and characterization of chloroplast casein kinase II from Oryza sativa (rice).

Plastid casein kinase II is an important regulator of transcription, posttranscriptional processes, and, most likely, different metabolic functions in dicotyledonous species. Here we report the identification and characterization of pCKII from the monocotyledonous species Oryza sativa. OspCKII activity was enriched from isolated rice chloroplasts using heparin-Sepharose chromatography, in which it co-elutes with the transcriptionally active chromosome (TAC) and several ribosomal proteins. Inclusion mass scanning of the kinase-active fraction identified the gene model for OspCKII. Transient expression of GFP fused to the 184 N-terminal amino acids of the OspCKII sequence in rice confirmed the chloroplastic localization of the kinase. OspCKII activity shows the characteristic features of casein kinase II, such as the utilization of GTP as phosphate donor, inhibition by low concentrations of heparin and poly-lysine, and utilization of the canonical pCKII motif E-S-E-G-E in the model substrate RNP29. Phosphoproteome analysis of a protein extract from rice leaves combined with a meta-analysis with published phosphoproteomics data revealed differences in the target protein spectrum between rice and Arabidopsis. Consistently, several pCKII phosphorylation sites in dicotyledonous plants are not conserved in monocots and algae, suggesting that details of pCKII regulation in plastids have changed during evolution.

Expression and significance of biglycan in endometrial cancer.

OBJECTIVE: This study aimed to determine the expression level of biglycan in different lesion properties of endometrium and to investigate the possible function and prognostic value of biglycan in endometrial cancer. METHODS: Immunohistochemical staining (IHC) and quantitative realtime reverse transcription polymerase chain reaction (qRT-PCR) were used to determine the protein and mRNA levels of biglycan in human normal endometrium, atypical hyperplasia endometrium, and endometrial cancer tissue samples. The expression of biglycan in serum and peritoneal washings was detected by ELISA method. Then we analyzed the correlation of biglycan expression with clinicopathological parameters in endometrial cancer. RESULTS: (1) Biglycan was overexpressed in endometrial cancer, especially in cancerous mesenchyme. Moreover, biglycan expression was significantly correlated with histopathological grade and FIGO stage of endometrial cancer; (2) Biglycan expression level in sera and peritoneal washings was significantly higher in endometrial cancer patients; otherwise, Serum expression correlated with clinicopathological parameters of endometrial cancer; (3) Higher level expression of biglycan in cancerous mesenchyme correlated with poor prognosis of endometrial cancer. CONCLUSIONS: Biglycan might play a role in the progression of human endometrial cancer and it might be a useful molecular marker for the prognosis of endometrial cancer. This research is an initial step towards biglycan as a potential prognosis marker in endometrial cancer.

Characterization of photosystem I in rice (Oryza sativa L.) seedlings upon exposure to random positioning machine.

To gain a better understanding of how photosynthesis is adapted under altered gravity forces, photosynthetic apparatus and its functioning were investigated in rice (Oryza sativa L.) seedlings grown in a random positioning machine (RPM). A decrease in fresh weight and dry weight was observed in rice seedlings grown under RPM condition. No significant changes were found in the chloroplast ultrastructure and total chlorophyll content between the RPM and control samples. Analyses of chlorophyll fluorescence and thermoluminescence demonstrate that PSII activity was unchanged under RPM condition. However, PSI activity decreased significantly under RPM condition. 77 K fluorescence emission spectra show a blue-shift and reduction of PSI fluorescence emission peak in the RPM seedlings. In addition, RPM caused a significant decrease in the amplitude of absorbance changes of P700 at 820 nm (A 820) induced by saturated far-red light. Moreover, the PSI efficiency (Φ I) decreased significantly under RPM condition. Immunoblot and blue native gel analyses further illustrate that accumulation of PSI proteins was greatly decreased in the RPM seedlings. Our results suggest that PSI, but not PSII, is down-regulated under RPM condition.

Chloroplast small heat shock protein HSP21 interacts with plastid nucleoid protein pTAC5 and is essential for chloroplast development in Arabidopsis under heat stress.

Compared with small heat shock proteins (sHSPs) in other organisms, those in plants are the most abundant and diverse. However, the molecular mechanisms by which sHSPs are involved in cell protection remain unknown. Here, we characterized the role of HSP21, a plastid nucleoid-localized sHSP, in chloroplast development under heat stress. We show that an Arabidopsis thaliana knockout mutant of HSP21 had an ivory phenotype under heat stress. Quantitative real-time RT-PCR, run-on transcription, RNA gel blot, and polysome association analyses demonstrated that HSP21 is involved in plastid-encoded RNA polymerase (PEP)-dependent transcription. We found that the plastid nucleoid protein pTAC5 was an HSP21 target. pTAC5 has a C4-type zinc finger similar to that of Escherichia coli DnaJ and zinc-dependent disulfide isomerase activity. Reduction of pTAC5 expression by RNA interference led to similar phenotypic effects as observed in hsp21. HSP21 and pTAC5 formed a complex that was associated mainly with the PEP complex. HSP21 and pTAC5 were associated with the PEP complex not only during transcription initiation, but also during elongation and termination. Our results suggest that HSP21 and pTAC5 are required for chloroplast development under heat stress by maintaining PEP function.

Sterol regulatory element-binding protein 1 is required for ovarian tumor growth.

Re-programming of lipogenic signaling is one of the most significant alterations of tumor cell pathology. Consistent with a large demand for lipids, tumor cells express high levels of lipogenic enzymes, most of which are transcriptional targets of sterol regulatory element-binding protein 1 (SREBP1). However, the expression levels and the function of SREBP1 in ovarian cancer are largely unknown. Our study aimed to assess the oncogenic potential of SREBP1 in ovarian cancer. In this study, we showed that the SREBP1 protein expression was significantly higher in human ovarian cancer compared to benign and borderline ovarian tumors by immunohistochemical staining. Knockdown of SREBP1 by small hairpin RNA (shRNA) in ovarian cancer cells retarded cell growth, migration and invasion and enhanced cell apoptosis without significant effects on cell cycle distribution. In a xenograft SCID mouse model, SREBP1 silencing inhibited tumor growth in vivo and reduced the expression of SREBP1 downstream lipogenic genes at both the protein and mRNA levels. Taken together, the results from this study demonstrate a crucial role of SREBP1 in ovarian cancer growth, which establish SREBP1 as a novel therapeutic target for antitumor therapy.

PsbP-domain protein1, a nuclear-encoded thylakoid lumenal protein, is essential for photosystem I assembly in Arabidopsis.

To gain insights into the molecular details of photosystem I (PSI) biogenesis, we characterized the PsbP-domain protein1 (ppd1) mutant of Arabidopsis thaliana that specifically lacks PSI activity. Deletion of PPD1 results in an inability of the mutant to grow photoautotrophically and a specific loss of the stable PSI complex. Unaltered transcription and translation of plastid-encoded PSI genes indicate that PPD1 acts at the posttranslational level. In vivo protein labeling experiments reveal that the rate of synthesis of PSI reaction center proteins PsaA/B in ppd1 is comparable to that of wild-type plants, whereas the rate of turnover of PsaA/B proteins is higher in ppd1 than in wild-type plants. With increasing leaf age, PPD1 content decreases considerably, while PSI content remains constant. PPD1 is a nuclear-encoded thylakoid lumenal protein and is associated with PSI but is not an integral subunit of PSI. Biochemical and molecular analyses reveal that PPD1 interacts directly and specifically with PsaB and PsaA. Yeast two-hybrid experiments show that PPD1 interacts with some lumenal loops of PsaB and PsaA. Our results suggest that PPD1 is a PSI assembly factor that assists the proper folding and integration of PsaB and PsaA into the thylakoid membrane.

Enhanced sensitivity and characterization of photosystem II in transgenic tobacco plants with decreased chloroplast glutathione reductase under chilling stress.

Chloroplast glutathione reductase (GR) plays an important role in protecting photosynthesis against oxidative stress. We used transgenic tobacco (Nicotiana tabacum) plants with severely decreased GR activities by using a gene encoding tobacco chloroplast GR for the RNAi construct to investigate the possible mechanisms of chloroplast GR in protecting photosynthesis against chilling stress. Transgenic plants were highly sensitive to chilling stress and accumulated high levels of H2O2 in chloroplasts. Spectroscopic analysis and electron transport measurements show that PSII activity was significantly reduced in transgenic plants. Flash-induced fluorescence relaxation and thermoluminescence measurements demonstrate that there was a slow electron transfer between Q(A) and Q(B) and decreased redox potential of Q(B) in transgenic plants, whereas the donor side function of PSII was not affected. Immunoblot and blue native gel analyses illustrate that PSII protein accumulation was decreased greatly in transgenic plants. Our results suggest that chloroplast GR plays an important role in protecting PSII function by maintaining the electron transport in PSII acceptor side and stabilizing PSII complexes under chilling stress. Our results also suggest that the recycling of ascorbate from dehydroascorbate in the ascorbate-glutathione cycle in the chloroplast plays an essential role in protecting PSII against chilling stress.

Increased expression of dachshund homolog 1 in ovarian cancer as a predictor for poor outcome.

OBJECTIVE: This study aimed to determine the functional relationship between the levels of dachshund homolog 1 (DACH1) expression and different subtypes of ovarian cancer and to investigate the possible prognostic value of DACH1 in ovarian cancer. METHODS: Immunohistochemical staining was deployed to determine the protein levels of DACH1. Staining was performed on patient samples, for whom the detailed follow-up data have been acquired during the last 10 years. Normal, benign, borderline, cancer, and metastatic ovarian cancer samples were included in this study. RESULTS: The results of our study show that DACH1 protein levels increase with the invasiveness of the ovarian cancer. As the cancer progresses from benign and borderline to metastatic, DACH1 protein expression increases as well. Moreover, with the increase in expression, the subcellular distribution of DACH1 changes from nucleus in normal tissue to cytoplasm in cancer. Finally, DACH1 expression levels were compared with estrogen receptor α (ERα) levels, and the results showed that overall DACH1 levels were higher, whereas also DACH1 exhibited increased cytoplasmic expression in ERα-positive ovarian cancer samples. CONCLUSIONS: These results indicate that DACH1 is highly expressed in metastatic ovarian cancer compared with that of normal, benign, and borderline ovarian tissues and that it could play an important role in cancer growth.