Effect of pulmonary vein isolation on rotor/multiple wavelet dynamics in persistent atrial fibrillation, association with vagal response and implications for adjunctive ablation.

Persistent atrial fibrillation (PeAF) may develop arrhythmogenic substrates of rotors/multiple wavelets. However, the ways in which pulmonary vein isolation (PVI) affects the dynamics of rotor/multiple wavelets in PeAF patients remain elusive. Real-time phase-mapping (ExTRa mapping, EXT) in the whole left atrium (LA) was performed during PeAF before and after PVI (n = 111). The percentage of time in which rotor/multiple wavelets (phase singularities) was observed during each 5-s phase-mapping recording (non-passive activation ratio, %NP) was measured as an index of its burden. The mapping areas showing %NP ≥ 50% were defined as rotor/multiple-wavelet substrates (RSs). Before PVI, RSs were globally distributed in the LA. After PVI, %NP decreased (< 50%) in many RSs (PVI-modifiable RSs) but remained high (≥ 50%) in some RSs, especially localized in the anterior/septum/inferior regions (PVI-unmodifiable RSs, 2.3 ± 1.0 areas/patient). Before PVI, vagal response (VR) to high-frequency stimulation was observed in 23% of RSs, especially localized in the inferior region. VR disappearance after PVI was more frequently observed in PVI-modifiable RSs (79%) than in PVI-unmodifiable RSs (55%, p < 0.05), suggesting that PVI affects autonomic nerve activities and rotor/multiple wavelet dynamics. PVI-unmodifiable RSs were adjunctively ablated in 104 patients. The 1-year AT/AF-free survival rate was 70% in those with PVI alone (n = 115), and 86% in patients with the adjunctive ablation (log-rank test = 7.65, p < 0.01). PVI suppresses not only ectopic firing but also rotor/multiple wavelets partly via modification of autonomic nerve activities. The adjunctive ablation of PVI-unmodifiable RSs improved the outcome in PeAF patients and might be a novel ablation strategy beyond PVI.

DOMINANT AWN INHIBITOR Encodes the ALOG Protein Originating from Gene Duplication and Inhibits AWN Elongation by Suppressing Cell Proliferation and Elongation in Sorghum.

The awn, a needle-like structure extending from the tip of the lemma in grass species, plays a role in environmental adaptation and fitness. In some crops, awns appear to have been eliminated during domestication. Although numerous genes involved in awn development have been identified, several dominant genes that eliminate awns are also known to exist. For example, in sorghum (Sorghum bicolor), the dominant awn-inhibiting gene has been known since 1921; however, its molecular features remain uncharacterized. In this study, we conducted quantitative trait locus analysis and a genome-wide association study of awn-related traits in sorghum and identified DOMINANT AWN INHIBITOR (DAI), which encodes the ALOG family protein on chromosome 3. DAI appeared to be present in most awnless sorghum cultivars, likely because of its effectiveness. Detailed analysis of the ALOG protein family in cereals revealed that DAI originated from a duplication of its twin paralog (DAIori) on chromosome 10. Observations of immature awns in near-isogenic lines revealed that DAI inhibits awn elongation by suppressing both cell proliferation and elongation. We also found that only DAI gained a novel function to inhibit awn elongation through an awn-specific expression pattern distinct from that of DAIori. Interestingly, heterologous expression of DAI with its own promoter in rice inhibited awn elongation in the awned cultivar Kasalath. We found that DAI originated from gene duplication, providing an interesting example of gain-of-function that occurs only in sorghum but shares its functionality with rice and sorghum.

Selvester QRS score predicts improvement of LVEF in atrial fibrillation patients with systolic heart failure.

BACKGROUND: Left-ventricular systolic dysfunction (LVSD) comorbid with atrial fibrillation is reversible, but recovery is limited in a subset of patients. The Selvester QRS (S-QRS) score is an electrocardiogram-based assessment that reportedly reflects myocardial scar/damage. We evaluated the predictability of S-QRS score for the recovery of left-ventricular ejection fraction (LVEF) in persistent AF (PeAF) patients with LVSD undergoing catheter ablation (CA). METHOD: CA was performed in 51 PeAF patients with reduced LVEF (<40%); S-QRS scores were measured after restoration of sinus rhythm. LVEF was re-evaluated at one year after CA; LVEF recovery was related to the S-QRS score. RESULTS: The median [interquartile range] S-QRS score was 1 point [0-2]. LVEF increased from 32% [28-37] at baseline to 56% [49-57] at 1 year after CA. Thirty-seven patients achieved normalization of LVEF (≥50%, Group A); 14 patients did not (Group B). Group A had significantly lower S-QRS scores than Group B (0 point [0-2] vs. 2 points [2-3], p < .05). In univariate/multivariate analyses, S-QRS score was an independent predictor of LVEF normalization. In the receiver operating characteristic curve, the cut-off value of S-QRS score was 2 points for prediction of the LVEF normalization (AUC = 0.79). Patients with low S-QRS score (<2 points) had greater LVEF improvement than those with high S-QRS score (≥2 points, ΔLVEF: 23% [17-28] vs. 17% [12-24], p < .05). CONCLUSION: S-QRS scoring noninvasively assesses the improvement of LVEF in PeAF patients with LVSD after CA. A high S-QRS score may indicate underlying myocardial scar/damage associated with unknown etiologies for LVSD other than PeAF.

Wall thickness-based adjustment of ablation index improves efficacy of pulmonary vein isolation in atrial fibrillation: Real-time assessment by intracardiac echocardiography.

BACKGROUND: Ablation index (AI) linearly correlates with lesion depth and may yield better therapeutic performance in pulmonary vein isolation (PVI) when tailored to a patient's wall thickness (WT) in the left atrium (LA). METHODS AND RESULTS: First study: In paroxysmal atrial fibrillation patients (PAF; n = 20), the average LA WT (mm) in each anatomical segment for PVI was measured by intracardiac echocardiography (ICE) placed in the LA; the optimal AI for creating 1-mm transmural lesion (AI/mm) was calculated. Second study: PAF (n = 80) patients were randomly assigned either to a force-time integral protocol (FTI; 400 g·s, n = 40) or a tailored-AI protocol (TAI; n = 40). In TAI, the LA WT in each segment was individually measured by ICE before starting ablation; a target AI was adjusted according to the individual WT in each segment (AI/mm × WT). The acute procedure outcomes and the 1-year AF-recurrence rate were compared between FTI and TAI. TAI had higher success rate of first-pass isolation (88% vs. 65%) and had lower incidence of residual PV-potentials/conduction-gaps after a circular ablation than FTI (15% vs. 45%). The procedure time to complete PVI decreased in TAI compared to FTI (52 vs. 83 min), being attributed to the increased radiofrequency power and the decreased radiofrequency application time in each point in TAI. TAI had a lower 1-year AF-recurrence rate than FTI. CONCLUSION: TAI increased acute procedure success, decreased time for PVI, and reduced the 1-year AF-recurrence rate, compared to FTI. Understanding the precise ablation target and tailoring AI would improve the efficacy of PVI.

Factors associated with silent cerebral events during atrial fibrillation ablation in patients on uninterrupted oral anticoagulation.

INTRODUCTION: Silent cerebral events (SCEs) are related to the potential thromboembolic risk in atrial fibrillation (AF) ablation. Periprocedural uninterrupted oral anticoagulation (OAC) reportedly reduced the risk of SCEs, but the incidence still remains. METHODS AND RESULTS: AF patients undergoing catheter ablation were eligible. All patients took non-vitamin K antagonist oral anticoagulants (NOACs; n = 248) or vitamin K antagonist (VKA; n = 37) for periprocedural OAC (>4 weeks) without interruption during the procedure. Brain magnetic resonance imaging was performed within 2 days after the procedure to detect SCEs. Clinical characteristics and procedure-related parameters were compared between patients with and without SCEs. SCEs were detected in 66 patients (23.1%; SCE[+]) but were not detected in 219 patients (SCE[-]). Age was higher in SCE[+] than in SCE[-] (66 ± 10 vs. 62 ± 12 years; p < .05). Persistent AF prevalence, CHADS2 /CHA2 DS2 -VASc scores, serum NT-ProBNP levels, left atrial dimension (LAD), and spontaneous echo contrast prevalence in transesophageal echocardiography significantly increased in SCE[+] versus SCE[-]. SCE[+] had lower baseline activated clotting time (ACT) before heparin injection and longer time to reach optimal ACT (>300 s) than SCE[-] (146 ± 27 vs. 156 ± 29 s and 44 ± 30 vs. 35 ± 25 min; p < .05, respectively). In multivariate analysis, age, LAD, baseline ACT, and time to reach the optimal ACT were predictors for SCEs. The average values of the ACT parameters were significantly different among NOACs/VKA. CONCLUSION: Age, LAD, and intraprocedural ACT kinetics significantly affect SCEs during AF ablation. Different anticoagulants have different impacts on ACT during the procedure, which should be considered when estimating the risk of SCEs.

Association between inflammation and skeletal muscle proteolysis, skeletal mass and strength in elderly heart failure patients and their prognostic implications.

BACKGROUND: Inflammation and skeletal muscle wasting often coexist in elderly populations, but few studies have examined their relationship in elderly heart failure (HF) patients. This study examined the relationship between inflammation and increased skeletal muscle proteolysis, reduced skeletal mass and strength, and their prognostic implications in elderly HF patients (> 65 years) using a random forest approach. METHODS: We prospectively enrolled consecutive elderly HF patients (n = 78) and age- and sex-matched control subjects (n = 83). We measured the interleukin (IL)-6, C-reactive protein (CRP), and B-type natriuretic peptide (BNP) levels, lower limb muscle mass and strength, and 6-min walk distance. The amount of muscle proteolysis was determined by urinary 3-methylhystidine, normalized by creatinine (3-MH/Cr). The composite endpoint was defined as all-cause death or hospitalizations due to worsening HF. RESULTS: Compared to controls, elderly HF patients had a significantly higher IL-6, CRP, BNP, and 3-MH/Cr, and exhibited a reduced lower limb muscle mass and strength. A correlation analysis demonstrated significant positive correlations between the inflammatory cytokine levels and 3-MH/Cr and BNP, and negative correlations with the lower limb muscle mass and strength, and 6-min walk distance. During a median follow-up of 2.4-years, 24 patients reached the endpoint. A random forest model revealed that inflammatory cytokines, skeletal muscle wasting, and the BNP had greater effects on the risk prediction. The algorithm achieved an area under the receiver operating characteristic curve of 0.887 (95% CI, 0.772-1.000). CONCLUSION: This study provided evidence of the association between inflammation and increased skeletal muscle proteolysis, reduced skeletal mass and strength, and their prognostic roles in elderly HF patients.

Applications of Plant-Made Fibroblast Growth Factor for Human Pluripotent Stem Cells.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) hold great potential in regenerative medicine. These cells can be expanded indefinitely in theory and are able to differentiate into different types of cells for cell therapies, drug screening, and basic biology studies. The reliable and effective propagation of hESCs and hiPSCs is important for their downstream applications. Basic fibroblast growth factor (bFGF) is critical to hESCs and hiPSCs for maintaining their pluripotency. Plant-produced growth factors are safe to use without potential contamination of infectious viruses and are less expensive to produce. In this study, we used rice cell-made basic fibroblast growth factor (RbFGF) to propagate hESCs and hiPSCs for at least eight passages. Both hESCs and hiPSCs cultured with RbFGF not only maintained the morphology but also the specific expression (OCT4, SSEA4, SOX2, and TRA-1-60) of PSCs, similar to those cultured with the commercial Escherichia coli-produced bFGF. Furthermore, both gene chip-based PluriTest and TaqMan hPSC Scorecard pluripotency analysis demonstrated the pluripotent expression profile of the hESCs cultured with RbFGF. In vitro trilineage assays further showed that these hESCs and hiPSCs cultured on RbFGF were capable of giving rise to cell derivatives of ectoderm, mesoderm, and endoderm, further demonstrating their pluripotency. Finally, chromosome stability was also maintained in hESCs cultured with RbFGF as demonstrated by normal karyotypes. This study suggests broad applications for plant-made growth factors in stem cell culture and regenerative medicine.

Circulating miR-20b-5p and miR-330-3p are novel biomarkers for progression of atrial fibrillation: Intracardiac/extracardiac plasma sample analysis by small RNA sequencing.

BACKGROUND: Circulating microRNAs (miRNAs, miR) have been considered as biomarkers reflecting the underlying pathophysiology in atrial fibrillation (AF). Nevertheless, miRNA expression in the peripheral blood samples might not reflect a cardiac phenomenon since most miRNAs are expressed in numerous organs. This study aimed to identify the cardiac-specific circulating miRNAs as biomarkers for AF. METHODS: Plasma samples were obtained from a luminal coronary sinus catheter (CS, cardiac-specific samples) and femoral venous sheath (FV, peripheral samples) in patients with AF and paroxysmal supraventricular tachycardia (control, CTL) undergoing catheter ablation. The circulating miRNA profiles were analyzed by small RNA sequencing. Differently expressed miRNAs between AF and CTL were identified in each sample of the CS and FV; miRNAs exhibiting similar expression patterns in the CS and FV samples were selected as candidates for cardiac-specific biomarkers. The selected miRNAs were related to the outcome of catheter ablation of AF. RESULTS: Small RNA sequencing detected 849 miRNAs. Among the top 30 most differently expressed miRNAs between AF and CTL, circulating hsa-miR-20b-5p, hsa-miR-330-3p, and hsa-miR-204-5p had a similar pattern in the CS and FV samples. Another set of peripheral blood samples was obtained from AF patients undergoing catheter ablation (n = 141). The expression of the miR-20b-5p and miR-330-3p, but not the miR-204-5p, negatively correlated with the echocardiographic left-atrial dimension and was decreased in patients with AF recurrence as compared to those without AF recurrence during a 1-year follow-up. CONCLUSION: Circulating miR-20b-5p and miR-330-3p can be cardiac-specific biomarkers for atrial remodeling progression and arrhythmia recurrence after catheter ablation in AF patients.

Metagenomic mining and structure-function studies of a hyper-thermostable cellobiohydrolase from hot spring sediment.

Enzymatic breakdown is an attractive cellulose utilisation method with a low environmental load. Its high temperature operation could promote saccharification and lower contamination risk. Here we report a hyper-thermostable cellobiohydrolase (CBH), named HmCel6A and its variant HmCel6A-3SNP that were isolated metagenomically from hot spring sediments and expressed in Escherichia coli. They are classified into glycoside hydrolases family 6 (GH6). HmCel6A-3SNP had three amino acid replacements to HmCel6A (P88S/L230F/F414S) and the optimum temperature at 95 °C, while HmCel6A did it at 75 °C. Crystal structure showed conserved features among GH6, a (ß/α)8-barrel core and catalytic residues, and resembles TfCel6B, a bacterial CBH II of Thermobifida fusca, that had optimum temperature at 60 °C. From structure-function studies, we discuss unique structural features that allow the enzyme to reach its high thermostability level, such as abundance of hydrophobic and charge-charge interactions, characteristic metal bindings and disulphide bonds. Moreover, structure and surface plasmon resonance analysis with oligosaccharides suggested that the contribution of an additional tryptophan located at the tunnel entrance could aid in substrate recognition and thermostability. These results may help to design efficient enzymes and saccharification methods for cellulose working at high temperatures.

Agrobacterium Transformation in the Rice Genome.

Modification of the rice genome by Agrobacterium-mediated transformation is a general technique that can be easily performed today. Successful methods were established by vigorous studies on the culture system and the elucidation of Agrobacterium transformation mechanisms. This section provides a detailed description of routine and efficient rice transformation protocols by Agrobacterium. This method uses mature seeds as a material and can be applied to many japonica and some other varieties of rice. According to this method, it is even possible for beginners to obtain rice transformants.

Comparison of effectiveness and safety between uninterrupted direct oral anticoagulants with and without switching to dabigatran in atrial fibrillation ablation.

INTRODUCTION: Recent studies have demonstrated the feasibility of uninterrupted direct oral anticoagulants (DOACs) with a temporary switch to dabigatran ("dabigatran bridge") for atrial fibrillation (AF) ablation. We compared the effectiveness and safety between uninterrupted DOACs with and without the "dabigatran bridge" in patients taking factor Xa inhibitors. METHODS: AF patients on factor Xa inhibitors (rivaroxaban/apixaban/edoxaban) undergoing catheter ablation were eligible (n = 348). Brain MRI was performed within 2 days after the procedure to detect silent cerebral events (SCEs). Rivaroxaban/apixaban/edoxaban were uninterruptedly used in 153 patients (Group 1); these DOACs were switched to dabigatran on the day of AF ablation in 195 patients (Group 2). After propensity score matching, the unfractionated heparin (UFH) amount and the activated clotting time (ACT) kinetics during the procedure, the SCE incidence, and the follow-up complications (30 days, thromboembolism and major/minor bleeding) in the two groups were compared. RESULTS: Group 2 had higher initial ACT value and shorter time to optimal ACT (>300 seconds) than Group 1 (184 ± 36 s vs 145 ± 22 s, and 34 ± 29 s vs 43 ± 34 s, P < .05, respectively). Group 2 tended to require less amount of UFH to achieve optimal ACT than Group 1, but the total amount of UFH for the procedure was comparable. Group 2 had lower SCE incidence than Group 1 (16.2% vs 26.4%, P < .05). The prevalence of follow-up complications was unchanged between the two groups. CONCLUSIONS: Switching to dabigatran on the day of AF ablation decreases preclinical thromboembolic events with similar bleeding risk to uninterrupted factor Xa inhibitors.

Isolation of highly thermostable ß-xylosidases from a hot spring soil microbial community using a metagenomic approach.

The DNA extracted from a high-temperature environment in which micro-organisms are living will be a good source for the isolation of thermostable enzymes. Using a metagenomic approach, we aimed to isolate thermostable ß-xylosidases that will be exploited for biofuel production from lignocellulosic biomass. DNA samples obtained from the soil near a spout of a hot spring (70°C, pH7.2) were subjected to sequencing, which generated a total of 84.2 Gbp with 967,925 contigs of >500 bp in length. Similarity search for ß-xylosidase in the contigs revealed the presence of 168 candidate sequences, each of which may have arisen from more than one gene. Individual genes were amplified by PCR using sequence-specific primers. The resultant DNA fragments were cloned and introduced into Escherichia coli BL21 Star(DE3). Consequently, 269 proteins were successfully expressed in the E. coli cells and then examined for ß-xylosidase activity. A total of 82 proteins exhibited ß-xylosidase activity at 50°C, six of which retained the activity even at 90°C. Out of the six, three proteins were originated from a single candidate sequence, AR19M-311. An amino acid sequence comparison suggested the amino acid residues that appeared to be crucial for thermal stability of the enzymes.

Symmetrically dividing cell specific division axes alteration observed in proteasome depleted C. elegans embryo.

A fertilised Caenorhabditis elegans embryo shows an invariable pattern of cell division and forms a multicellular body where each cell locates to a defined position. Mitotic spindle orientation is determined by several preceding events including the migration of duplicated centrosomes on a nucleus and the rotation of nuclear-centrosome complex. Cell polarity is the dominant force driving nuclear-centrosome rotation and setting the mitotic spindle axis in parallel with the polarity axis during asymmetric cell division. It is reasonable that there is no nuclear-centrosome rotation in symmetrically dividing blastomeres, but the mechanism(s) which suppress rotation in these cells have been proposed because the rotations occur in some polarity defect embryos. Here we show the nuclear-centrosome rotation can be induced by depletion of RPN-2, a regulatory subunit of the proteasome. In these embryos, cell polarity is established normally and both asymmetrically and symmetrically dividing cells are generated through asymmetric cell divisions. The nuclear-centrosome rotations occurred normally in the asymmetrically dividing cell lineage, but also induced in symmetrically dividing daughter cells. Interestingly, we identified RPN-2 as a binding protein of PKC-3, one of critical elements for establishing cell polarity during early asymmetric cell divisions. In addition to asymmetrically dividing cells, PKC-3 is also expressed in symmetrically dividing cells and a role to suppress nuclear-centrosome rotation has been anticipated. Our data suggest that the expression of RPN-2 is involved in the mechanism to suppress nuclear-centrosome rotation in symmetrically dividing cells and it may work in cooperation with PKC-3.

A protocol for Agrobacterium-mediated transformation in rice.

Agrobacterium-mediated transformation of rice is an important method that has been widely adopted by many laboratories. However, because current approaches rely on culture systems, routine protocols have been established only in japonica rice, especially those varieties with higher regeneration potential. Some very efficient methods have been developed for japonica varieties that enable high-throughput functional analysis in rice; however, many elite japonica, and most indica, varieties are difficult to regenerate, leading to low transformation efficiencies. Much effort has been devoted to improving transformation efficiency for all rice genotypes. Here, we describe an Agrobacterium-mediated rice transformation method that is applicable to easily cultured varieties in addition to elite japonica varieties that are more difficult to culture. Using this method, transgenic rice plants can be obtained in about 2-3 months with a transformation frequency of 30-50%, both in easily cultured varieties and recalcitrant elite japonica rice.

Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems.

Regeneration of plant organs is often the essential step in genetic transformation; however, the regeneration ability of a plant varies depending on the genetic background. By conventional crosses of low-regeneration rice strain Koshihikari with high-regeneration rice strain Kasalath, we identified some quantitative trait loci, which control the regeneration ability in rice. Using a map-based cloning strategy, we isolated a main quantitative trait loci gene encoding ferredoxin-nitrite reductase (NiR) that determines regeneration ability in rice. Molecular analyses revealed that the poor regeneration ability of Koshihikari is caused by lower expression than in Kasalath and the specific activity of NiR. Using the NiR gene as a selection marker, we succeeded in selectively transforming a foreign gene into rice without exogenous marker genes. Our results demonstrate that nitrate assimilation is an important process in rice regeneration and also provide an additional selectable marker for rice transformation.

Cytokinin oxidase regulates rice grain production.

Most agriculturally important traits are regulated by genes known as quantitative trait loci (QTLs) derived from natural allelic variations. We here show that a QTL that increases grain productivity in rice, Gn1a, is a gene for cytokinin oxidase/dehydrogenase (OsCKX2), an enzyme that degrades the phytohormone cytokinin. Reduced expression of OsCKX2 causes cytokinin accumulation in inflorescence meristems and increases the number of reproductive organs, resulting in enhanced grain yield. QTL pyramiding to combine loci for grain number and plant height in the same genetic background generated lines exhibiting both beneficial traits. These results provide a strategy for tailormade crop improvement.

OsPNH1 regulates leaf development and maintenance of the shoot apical meristem in rice.

The Arabidopsis PINHEAD/ZWILLE (PNH/ZLL) gene is thought to play an important role in the formation of the shoot apical meristem (SAM) and in leaf adaxial cell specification. To investigate the molecular mechanisms of rice development, we have isolated a rice homologue of PNH/ZLL, called OsPNH1. Around the SAM, OsPNH1 was strongly expressed in developing leaf primordia, specifically in the presumptive vascular domains, developing vascular tissues, a few cell-layers of the adaxial region, and future bundle sheath extension cells. In the SAM, only weak expression was observed in the central region, whereas strong expression was detected in the mid-vein region of leaf founder cells in the peripheral SAM domain. We produced transgenic rice plants containing the antisense OsPNH1 strand. The antisense OsPNH1 plants developed malformed leaves with an altered vascular arrangement and abnormal internal structure. These plants also formed an aberrant SAM with reduced KNOX gene expression. We examined the subcellular localization of the OsPNH1-GFP fusion protein and found that it was localized in the cytoplasm. On the basis of these observations, we propose that OsPNH1 functions not only in SAM maintenance as previously thought, but also in leaf formation through vascular development.

Rice globular embryo 4 (gle4) mutant is defective in radial pattern formation during embryogenesis.

In higher plants, the main elements of the fundamental body plan, the apical-basal and radial patterns, are established during embryogenesis. We have isolated several globular embryo (gle) mutants of rice that fail to develop any embryonic organs. We expected that these gle mutants might include mutants defective in their radial pattern formation ability. We developed two markers specifically staining the L2 and L3 layers (OsSCR and OsPNH1, respectively) and characterized the gle mutants by using these markers in addition to the already developed markers Roc1 (marker for the L1 layer), Ramy1A (marker of the L1 layer of the epithelium), and OSH1 (marker of the apical region). One of the gle mutants, gle4, expressed Roc1 and Ramy1A at the normal positions, but other markers exhibited an abnormal expression pattern; that is, both OsPNH1 and OsSCR were expressed in the central region of the embryo and OSH1 expression was not observed. Calli from the gle4 epithelium regenerated plants with abnormal morphologies. These results indicate that the GLE4 gene is involved in radial pattern formation during rice embryogenesis to differentiate the L2 and L3 layers, but is not involved in the establishment of the L1 layer or in the formation of embryonic organs.

Position dependent expression of GL2-type homeobox gene, Roc1: significance for protoderm differentiation and radial pattern formation in early rice embryogenesis.

In early plant embryogenesis, the determination of cell fate in the protodermal cell layer is considered to be the earliest event in radial pattern formation. To elucidate the mechanisms of epidermal cell fate determination and radial pattern formation in early rice embryogenesis, we have isolated a GL2-type homeobox gene Roc1 (Rice outermost cell-specific gene1), which is specifically expressed in the protoderm (epidermis). In early rice embryogenesis, cell division occurs randomly and the morphologically distinct layer structure of the protoderm cannot be observed until the embryo reaches more than 100 microm in length. Nonetheless, in situ hybridization analyses revealed that specific expression of Roc1 in the outermost cells is established shortly after fertilization, much earlier than protoderm differentiation. In the regeneration process from callus, the Roc1 gene is also expressed in the outermost cells of callus in advance of tissue and organ differentiation, and occurs independently of whether the cells will differentiate into epidermis in the future or not. Furthermore, this cell-specific Roc1 expression could be induced flexibly in the newly produced outermost cells when we cut the callus. These findings suggest that the expression of Roc1 in the outermost cells may be dependent on the positional information of cells in the embryo or callus prior to the cell fate determination of the protoderm (epidermis). Furthermore, the Roc1 expression is downregulated in the inner cells of ligule, which have previously been determined as protodermal cells, also suggesting that the Roc1 expression is position dependent and that this position dependent Roc1 expression is important also in post-embryonic protoderm (epidermis) differentiation.