A diRNA-protein scaffold module mediates SMC5/6 recruitment in plant DNA repair.

In eukaryotes, the STRUCTURAL MAINTENANCE OF CHROMOSOME 5/6 (SMC5/6) complex is critical to maintaining chromosomal structures around double-strand breaks (DSBs) in DNA damage repair. However, the recruitment mechanism of this conserved complex at DSBs remains unclear. In this study, using Arabidopsis thaliana as a model, we found that SMC5/6 localization at DSBs is dependent on the protein scaffold containing INVOLVED IN DE NOVO 2 (IDN2), CELL DIVISION CYCLE 5 (CDC5), and ALTERATION/DEFICIENCY IN ACTIVATION 2B (ADA2b), whose recruitment is further mediated by DNA-damage-induced RNAs (diRNAs) generated from DNA regions around DSBs. The physical interactions of protein components including SMC5-ADA2b, ADA2b-CDC5, and CDC5-IDN2 result in formation of the protein scaffold. Further analysis indicated that the DSB localization of IDN2 requires its RNA-binding activity and ARGONAUTE 2 (AGO2), indicating a role for the AGO2-diRNA complex in this process. Given that most of the components in the scaffold are conserved, the mechanism presented here, which connects SMC5/6 recruitment and small RNAs, will improve our understanding of DNA repair mechanisms in eukaryotes.

SUMOylation facilitates the assembly of a Nuclear Factor-Y complex to enhance thermotolerance in Arabidopsis.

Heat stress (HS) has serious negative effects on plant development and has become a major threat to agriculture. A rapid transcriptional regulatory cascade has evolved in plants in response to HS. Nuclear Factor-Y (NF-Y) complexes are critical for this mechanism, but how NF-Y complexes are regulated remains unclear. In this study, we identified NF-YC10 (NF-Y subunit C10), a central regulator of the HS response in Arabidopsis thaliana, as a substrate of SUMOylation, an important post-translational modification. Biochemical analysis showed that the SUMO ligase SIZ1 (SAP AND MIZ1 DOMAIN-CONTAINING LIGASE1) interacts with NF-YC10 and enhances its SUMOylation during HS. The SUMOylation of NF-YC10 facilitates its interaction with and the nuclear translocation of NF-YB3, in which the SUMO interaction motif (SIM) is essential for its efficient association with NF-YC10. Further functional analysis indicated that the SUMOylation of NF-YC10 and the SIM of NF-YB3 are critical for HS-responsive gene expression and plant thermotolerance. These findings uncover a role for the SIZ1-mediated SUMOylation of NF-YC10 in NF-Y complex assembly under HS, providing new insights into the role of a post-translational modification in regulating transcription during abiotic stress responses in plants.

A SWI/SNF subunit regulates chromosomal dissociation of structural maintenance complex 5 during DNA repair in plant cells.

DNA damage decreases genome stability and alters genetic information in all organisms. Conserved protein complexes have been evolved for DNA repair in eukaryotes, such as the structural maintenance complex 5/6 (SMC5/6), a chromosomal ATPase involved in DNA double-strand break (DSB) repair. Several factors have been identified for recruitment of SMC5/6 to DSBs, but this complex is also associated with chromosomes under normal conditions; how SMC5/6 dissociates from its original location and moves to DSB sites is completely unknown. In this study, we determined that SWI3B, a subunit of the SWI/SNF complex, is an SMC5-interacting protein in Arabidopsis thialiana Knockdown of SWI3B or SMC5 results in increased DNA damage accumulation. During DNA damage, SWI3B expression is induced, but the SWI3B protein is not localized at DSBs. Notably, either knockdown or overexpression of SWI3B disrupts the DSB recruitment of SMC5 in response to DNA damage. Overexpression of a cotranscriptional activator ADA2b rescues the DSB localization of SMC5 dramatically in the SWI3B-overexpressing cells but only weakly in the SWI3B knockdown cells. Biochemical data confirmed that ADA2b attenuates the interaction between SWI3B and SMC5 and that SWI3B promotes the dissociation of SMC5 from chromosomes. In addition, overexpression of SMC5 reduces DNA damage accumulation in the SWI3B knockdown plants. Collectively, these results indicate that the presence of an appropriate level of SWI3B enhances dissociation of SMC5 from chromosomes for its further recruitment at DSBs during DNA damage in plant cells.

Profile of gene expression changes during estrodiol-17ß-induced feminization in the Takifugu rubripes brain.

BACKGROUND: As the critical tissue of the central nervous system, the brain has been found to be involved in gonad development. Previous studies have suggested that gonadal fate may be affected by the brain. Identifying brain-specific molecular changes that occur during estrodiol-17ß (E2) -induced feminization is crucial to our understanding of the molecular control of sex differentiation by the brains of fish. RESULTS: In this study, the differential transcriptomic responses of the Takifugu rubripes larvae brain were compared after E2 treatment for 55 days. Our results showed that 514 genes were differentially expressed between E2-treated-XX (E-XX) and Control-XX (C-XX) T. rubripes, while 362 genes were differentially expressed between E2-treated-XY (E-XY) and Control-XY (C-XY). For example, the expression of cyp19a1b, gnrh1 and pgr was significantly up-regulated, while st, sl, tshß, prl and pit-1, which belong to the growth hormone/prolactin family, were significantly down-regulated after E2 treatment, in both sexes. The arntl1, bhlbe, nr1d2, per1b, per3, cry1, cipc and ciart genes, which are involved in the circadian rhythm, were also found to be altered. Differentially expressed genes (DEGs), which were identified between E-XX and C-XX, were significantly enriched in neuroactive ligand-receptor interaction, arachidonic acid metabolism, cytokine-cytokine receptor interaction and the calcium signaling pathway. The DEGs that were identified between E-XY and C-XY were significantly enriched in tyrosine metabolism, phenylalanine metabolism, arachidonic acid metabolism and linoleic acid metabolism. CONCLUSION: A number of genes and pathways were identified in the brain of E2-treated T. rubripes larvae by RNA-seq. It provided the opportunity for further study on the possible involvement of networks in the brain-pituitary-gonadal axis in sex differentiation in T. rubripes.

Quantitative Fluorescence Resonance Energy Transfer Analysis on the Direct Interaction of Activation-2b with Histone H3/Switch-3B Protein in Arabidopsis Mesophyll Protoplasts.

Interaction between the alteration/deficiency in activation-2b (ADA2b) and histone H3/switch-3B (SWI3B) proteins was evaluated in arabidopsis mesophyll protoplasts by quantitative fluorescence resonance energy transfer (FRET) analysis. Microscopic image showed that ADA2b, SWI3B and H3 proteins colocalized in nucleus, and quantitative FRET measurements showed 0.31 of FRET efficiency (E) for the protoplasts coexpressing ECFP-ADA2b and EYFP-SWI3B, and 0.285 of E for the protoplasts coexpressing ECFP-H3 and EYFP-ADA2b, demonstrating the direct interaction of ADA2b with SWI3B/H3 protein. Collectively, SWI3B and H3 proteins are the inherent components of the ADA2b complex in which ADA2b directly interacts with SWI3B/H3 protein.

Universal and Naked-Eye Gene Detection Platform Based on the Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a/13a System.

Gold-nanoparticles-based colorimetric assay is an attractive detection format, but is limited by the tedious and ineffective posthybridization manipulations for genomic analysis. Here, we present a new design for a colorimetric gene-sensing platform based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. In this strategy, programmable recognition of DNA by Cas12a/crRNA and RNA by Cas13a/crRNA with a complementary target activates the trans-ssDNA or -ssRNA cleavage. Target-induced trans-ssDNA or ssRNA cleavage triggers an aggregation behavior change for the designed AuNPs-DNA probes pair, enabling the completion of naked-eye gene detection (transgenic rice, African swine fever virus, and miRNAs as the models) within 1 h. This platform is also showing promise as a fast and inexpensive tool for bacteria identification using 16S rDNA or 16S rRNA. A CRISPR/Cas-based colorimetric platform shows superior characteristics, such as probe universality, compatibility with isothermal reaction conditions, on-site detection capability, and high sensitivity, thus, demonstrating its use as a robust next-generation gene detection platform.

Nuclear Prohibitin3 Maintains Genome Integrity and Cell Proliferation in the Root Meristem through Minichromosome Maintenance 2.

The nucleo-mitochondrial dual-localized proteins can act as gene expression regulators; however, few instances of these proteins have been described in plants. Arabidopsis (Arabidopsis thaliana) PROHIBITIN 3 (PHB3) is involved in stress responses and developmental processes, but it is unknown how these roles are achieved at the molecular level in the nucleus. In this study, we show that nucleo-mitochondrial PHB3 plays an essential role in regulating genome stability and cell proliferation. PHB3 is up-regulated by DNA damage agents, and the stress-induced PHB3 proteins accumulate in the nucleus. Loss of function of PHB3 results in DNA damage and defective maintenance of the root stem cell niche. Subsequently, the expression patterns and levels of the root stem cell regulators are altered and down-regulated, respectively. In addition, the phb3 mutant shows aberrant cell division and altered expression of cell cycle-related genes, such as CycB1 and Cyclin dependent kinase 1 Moreover, the minichromosome maintenance (MCM) genes, e.g. MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7, are up-regulated in the phb3 mutant. Reducing the MCM2 expression level substantially recovers the DNA damage in the phb3 mutant and partially rescues the altered cell proliferation and root deficiency of phb3 seedlings. PHB3 acts as a transcriptional coregulator that represses MCM2 expression by competitively binding to the promoter E2F-cis-acting elements with E2Fa so as to modulate primary root growth. Collectively, these findings indicate that nuclear-localized PHB3 acts as a transcriptional coregulator that suppresses MCM2 expression to sustain genome integrity and cell proliferation for stem cell niche maintenance in Arabidopsis.

The Transcriptional Coactivator ADA2b Recruits a Structural Maintenance Protein to Double-Strand Breaks during DNA Repair in Plants.

DNA damage occurs in all cells and can hinder chromosome stability and cell viability. Structural Maintenance of Chromosomes5/6 (SMC5/6) is a protein complex that functions as an evolutionarily conserved chromosomal ATPase critical for repairing DNA double-strand breaks (DSBs). However, the mechanisms regulating this complex in plants are poorly understood. Here, we identified the transcriptional coactivator ALTERATION/DEFICIENCY IN ACTIVATION2B (ADA2b) as an interactor of SMC5 in Arabidopsis (Arabidopsis thaliana). ADA2b is a conserved component of the Spt-Ada-Gcn5 acetyltransferase complex, which functions in transcriptional regulation. Characterization of mutant and knockdown Arabidopsis lines showed that disruption of either SMC5 or ADA2b resulted in enhanced DNA damage. Both SMC5 and ADA2b were associated with γ-H2AX, a marker of DSBs, and the recruitment of SMC5 onto DSBs was dependent on ADA2b. In addition, overexpression of SMC5 in the ada2b mutant background stimulated cell death. Collectively, our results show that the interaction between ADA2b and SMC5 mediates DNA repair in plant cells, suggesting a functional association between these conserved proteins and further elucidating mechanisms of DNA damage repair in plants.

The Arabidopsis SUMO E3 Ligase AtMMS21 Dissociates the E2Fa/DPa Complex in Cell Cycle Regulation.

Development requires the proper execution and regulation of the cell cycle via precise, conserved mechanisms. Critically, the E2F/DP complex controls the expression of essential genes during cell cycle transitions. Here, we discovered the molecular function of the Arabidopsis thaliana SUMO E3 ligase METHYL METHANESULFONATE SENSITIVITY GENE21 (AtMMS21) in regulating the cell cycle via the E2Fa/DPa pathway. DPa was identified as an AtMMS21-interacting protein and AtMMS21 competes with E2Fa for interaction with DPa. Moreover, DPa is a substrate for SUMOylation mediated by AtMMS21, and this SUMOylation enhances the dissociation of the E2Fa/DPa complex. AtMMS21 also affects the subcellular localization of E2Fa/DPa. The E2Fa/DPa target genes are upregulated in the root of mms21-1 and mms21-1 mutants showed increased endoreplication. Overexpression of DPa affected the root development of mms21-1, and overexpression of AtMMS21 completely recovered the abnormal phenotypes of 35S:E2Fa-DPa plants. Our results suggest that AtMMS21 dissociates the E2Fa/DPa complex via competition and SUMOylation in the regulation of plant cell cycle.

A SUMO Ligase AtMMS21 Regulates the Stability of the Chromatin Remodeler BRAHMA in Root Development.

Chromatin remodeling is essential for gene expression regulation in plant development and response to stresses. Brahma (BRM) is a conserved ATPase in the SWI/SNF chromatin remodeling complex and is involved in various biological processes in plant cells, but the regulation mechanism on BRM protein remains unclear. Here, we report that BRM interacts with AtMMS21, a SUMO ligase in Arabidopsis (Arabidopsis thaliana). The interaction was confirmed in different approaches in vivo and in vitro. The mutants of BRM and AtMMS21 displayed a similar defect in root development. In the mms21-1 mutant, the protein level of BRM-GFP was significantly lower than that in wild type, but the RNA level of BRM did not change. Biochemical evidence indicated that BRM was modified by SUMO3, and the reaction was enhanced by AtMMS21. Furthermore, overexpression of wild-type AtMMS21 but not the mutated AtMMS21 without SUMO ligase activity was able to recover the stability of BRM in mms21-1 Overexpression of BRM in mms21-1 partially rescued the developmental defect of roots. Taken together, these results supported that AtMMS21 regulates the protein stability of BRM in root development.

OsAGSW1, an ABC1-like kinase gene, is involved in the regulation of grain size and weight in rice.

Grain shape and weight are two determining agronomic traits of rice yield. ABC1 (Activity of bc1 complex) is a newly found atypical kinase in plants. Here, we report on an ABC1 protein kinase gene, OsAGSW1 (ABC1-like kinase related to Grain size and Weight). Expression of OsAGSW1-GFP in rice revealed that OsAGSW1 is localized to the chloroplasts in rice. Analysis of OsAGSW1 promoter::ß-glucuronidase transgenic rice indicated that this gene was highly expressed in vascular bundles in shoot, hull and caryopsis. Furthermore, OsAGSW1-RNAi and overexpressed transgenic rice lines were generated. Stable transgenic lines overexpressing OsAGSW1 exhibited a phenotype with a significant increase in grain size, grain weight, grain filling rate and 1000-grain weight compared with the wild-type and RNAi transgenic plants. Microscopy analysis showed that spikelet hulls just before heading were different in the OsAGSW1-overexpressed plants compared with wild-type and OsAGSW1 RNAi rice. Further cytological analysis showed that the number of external parenchyma cells in rice hulls of OsAGSW1-overexpressed plants increased, leading to wider and longer spikelet hulls than those of the wild-type and OsAGSW1-RNAi plants. The vascular cross-sectional area in lemma, carpopodium and ovules also strikingly increased and area of both xylem and phloem were enlarged in the OsAGSW1-overexpressed plants. Thus, our results demonstrated that OsAGSW1 plays an important role in seed shape and size of rice by regulating the number of external parenchyma cells and the development of vascular bundles, providing a new insight into the functions of ABC1 genes in plants.

SIZ1-mediated SUMOylation of CPSF100 promotes plant thermomorphogenesis by controlling alternative polyadenylation.

Under warm temperatures, plants adjust their morphologies for environmental adaption via precise gene expression regulation. However, the function and regulation of alternative polyadenylation (APA), an important fine-tuning of gene expression, remains unknown in plant thermomorphogenesis. In this study, we found that SUMOylation, a critical post-translational modification, is induced by a long-term treatment at warm temperatures via a SUMO ligase SIZ1 in Arabidopsis. Disruption of SIZ1 altered the global usage of polyadenylation signals and affected the APA dynamic of thermomorphogenesis-related genes. CPSF100, a key subunit of the CPSF complex for polyadenylation regulation, is SUMOylated by SIZ1. Importantly, we demonstrated that SUMOylation is essential for the function of CPSF100 in genome-wide polyadenylation site choice during thermomorphogenesis. Further analyses revealed that the SUMO conjugation on CPSF100 attenuates its interaction with two isoforms of its partner CPSF30, increasing the nuclear accumulation of CPSF100 for polyadenylation regulation. In summary, our study uncovers a regulatory mechanism of APA via SIZ1-mediated SUMOylation in plant thermomorphogenesis.

Food production and agricultural systems on the southwestern frontier of the Han Empire: archaeobotanical remains from the 2016 excavation of Hebosuo, Yunnan.

Dian Basin in Yunnan province is an important center for both early agricultural production and centralized state formation. Settled agricultural villages are present in the province since at least the third millennium BC, and by the first millennium BC, the Dian Culture, a highly specialized bronze polity, flourished in the Dian Basin and surrounding area, until it was conquered by the Han in 109 BC. The increased deployment of flotation at recent archaeological excavations in Yunnan allowed the reconstruction of agricultural practices from the Neolithic to the early Bronze Age, documented at Baiyangcun, Haimenkou, and Xueshan among others. However, archaeobotanical evidence relating to the pivotal period right before and after the Han conquest have so far been lacking, with only limited written records about agricultural production in the Shiji by Sima Qian. Here we present for the first time direct archaeobotanical evidence relating to this transitional period as revealed by rich Han period deposits found during the 2016 excavation of Hebosuo, the largest Dian settlement investigated in Yunnan so far, dated by direct AMS on charred cereal grains and artefactual evidence as spanning from between 850 BC-220 AD. Following the Han conquest, the main components of the agricultural system did not undergo radical changes, but the weedy flora indicates a heavier reliance of wet-land rice systems, evidencing a higher level of water management or even irrigation practices, and the consequent intensification of the agricultural production. These findings on shifting agricultural regimes in Yunnan also contribute to current debates about the interplay between intensification, food risk, and ecology in times of political instability. Supplementary Information: The online version contains supplementary material available at 10.1007/s12520-023-01766-9.

Functional characterization of three maize SIZ/PIAS-type SUMO E3 ligases.

SUMOylation is a critical post-translational modification that regulates the nature and activity of protein substrates. The reaction is usually enhanced by a SIZ/PIAS-type of SUMO E3 ligase, but the functions of its homologs in maize have not yet been reported. In this study, we functionally characterized three members of this family of SUMO ligases, ZmSIZ1a, ZmSIZ1b, and ZmSIZ1c, from Zea mays. These maize SIZ1 homologs harbor conserved domains and structures with AtSIZ1, suggesting that they are potential functional SUMO ligases, which is supported by further biochemical data. The expression of these maize SIZ1 genes was detectable ubiquitously in different maize tissues and was usually induced by abiotic stresses. Expression of ZmSIZ1 members complements the leaf developmental defects of the AtSIZ1 mutant, suggesting their conserved function in development regulation. Interestingly, overexpression of ZmSIZ1c, but not ZmSIZ1a or ZmSIZ1b, in the wild-type Arabidopsis resulted in early flowering, implying that these members differ in terms of flowering control. Besides, overexpression of these ZmSIZ1 genes also improved salt tolerance in Arabidopsis. Collectively, our functional characterization of the ZmSIZ1 members provides hints for further investigation on the functions of SUMOylation in the development and stress responses in maize.

A SUMO ligase OsMMS21 regulates rice development and auxin response.

SUMOylation, which transfers the Small Ubiquitin-related Modifier (SUMO) polypeptides to target proteins, regulates diverse cellular processes in eukaryotes. The SUMO conjugation reaction is usually promoted by SUMO E3 ligases, but the molecular functions of this type of enzymes remain unclear in cereal crops. Here, OsMMS21, a SUMO E3 ligase, was functionally characterized in rice (Oryza sativa). Bioinformatics analysis showed that OsMMS21 harbors a conserved SP-RING domain that is essential for the activity of SUMO ligases. Biochemical data indicated that this protein is auto-SUMOylated. Besides, overexpression of OsMMS21 rescued the developmental defects of the AtMMS21 mutant, supporting that OsMMS21 is a functional homolog of the Arabidopsis SUMO ligase AtMMS21. The OsMMS21 rice T-DNA mutant displays a short-root and dwarfism phenotype. RNA-seq data revealed that the expression levels of many genes involved in signaling transduction of hormones, including auxin, are altered in the OsMMS21 mutant. Further results under the auxin treatment showed that the OsMMS21 mutant is insensitive to auxin. Collectively, our results demonstrated the molecular features of OsMMS21 and uncovered the roles of this SUMO ligase in development and auxin response, providing hints for further studies on protein SUMOylation in rice.

Gene Expression of Takifugu rubripes Gonads During AI- or MT-induced Masculinization and E2-induced Feminization.

Elucidating the global molecular changes that occur during aromatase inhibitor (AI)- or 17α-methyltestosterone (MT)-induced masculinization and estradiol-17ß (E2)-induced feminization is critical to understanding the roles that endocrine and genetic factors play in regulating the process of sex differentiation in fish. Here, fugu larvae were treated with AI (letrozole), MT, or E2 from 25 to 80 days after hatching (dah), and gonadal transcriptomic analysis at 80 dah was performed. The expression of dmrt1, gsdf, foxl2, and other key genes (star, hsd3b1, cyp11c1, cyp19a1a, etc.) involved in the steroid hormone biosynthesis pathway were found be altered. The expression of dmrt1, gsdf, cyp19a1a, and foxl2 was further verified by quantitative polymerase chain reaction. In the control group, the expression of dmrt1 and gsdf was significantly higher in XY larvae than in XX larvae, while the expression of foxl2 and cyp19a1a was significantly higher in XX larvae than in XY larvae (P < .05). AI treatment suppressed the expression of foxl2 and cyp19a1a, and induced the expression of dmrt1 and gsdf in XX larvae. MT treatment suppressed the expression of foxl2, cyp19a1a, dmrt1, and gsdf in XX larvae. E2 treatment suppressed the expression of dmrt1 and gsdf, but did not restore the expression of foxl2 and cyp19a1a in XY larvae. The shared response following AI, MT, and E2 treatment suggested that these genes are essential for sex differentiation. This finding offers some insight into AI or MT-induced masculinization, and E2-induced femininization in fugu.

Identification of sex differentiation-related microRNA and long non-coding RNA in Takifugu rubripes gonads.

Although sex determination and differentiation are key developmental processes in animals, the involvement of non-coding RNA in the regulation of this process is still not clarified. The tiger pufferfish (Takifugu rubripes) is one of the most economically important marine cultured species in Asia, but analyses of miRNA and long non-coding RNA (lncRNA) at early sex differentiation stages have not been conducted yet. In our study, high-throughput sequencing technology was used to sequence transcriptome libraries from undifferentiated gonads of T. rubripes. In total, 231 (107 conserved, and 124 novel) miRNAs were obtained, while 2774 (523 conserved, and 2251 novel) lncRNAs were identified. Of these, several miRNAs and lncRNAs were predicted to be the regulators of the expression of sex-related genes (including fru-miR-15b/foxl2, novel-167, novel-318, and novel-538/dmrt1, novel-548/amh, lnc_000338, lnc_000690, lnc_000370, XLOC_021951, and XR_965485.1/gsdf). Analysis of differentially expressed miRNAs and lncRNAs showed that three mature miRNAs up-regulated and five mature miRNAs were down-regulated in male gonads compared to female gonads, while 79 lncRNAs were up-regulated and 51 were down-regulated. These findings could highlight a group of interesting miRNAs and lncRNAs for future studies and may reveal new insights into the function of miRNAs and lncRNAs in sex determination and differentiation.

Effects of the Thyroid Endocrine System on Gonadal Sex Ratios and Sex-Related Gene Expression in the Pufferfish Takifugu rubripes.

To examine the effect and mechanism of thyroid hormone on gonadal sex differentiation, Takifugu rubripes larvae were treated with goitrogen (methimazole, MET, 1000 g/g), and thyroxine (T4, 2nM) from 25 to 80 days after hatching (dah). Gonadal histology and sex ratios of fish were then determined at 80 dah. MET treatment induced masculinization, but T4 treatment did not induce feminization in T. rubripes larvae. Transcriptomic analysis of gonads at 80 dah was then conducted. Among the large number of differentially expressed genes between the groups, the expression of foxl2, cyp19a1a, and dmrt1 was altered. The expression of foxl2, cyp19a1a, dmrt1 and gsdf at 25, 40, 55 days after treatment (dat) was further analyzed by qPCR. MET treatment suppressed the expression of foxl2 and cyp19a1a, and induced the expression of dmrt1 in genetic females (p < 0.05). Additionally, T4 treatment induced an increase in the expression of cyp19a1a in genetic XY gonads only at 25 dat. However, the increase in cyp19a1a expression did not continue to 40 and 55 dat. This may explain why feminization of larvae was not found in the T4-treated group. Thus, the present study provides the first evidence that MET treatment causes masculinization in teleost fish. The effects of MET-induced masculinization in T. rubripes may act primarily via suppression of the expression of foxl2 and cyp19a1a, and stimulation of the expression of dmrt1. Moreover, the effects of higher concentrations of T4 or different concentrations of T3, on sex differentiation require further testing.

Overexpression of a CPYC-Type Glutaredoxin, OsGrxC2.2, Causes Abnormal Embryos and an Increased Grain Weight in Rice.

Glutaredoxins (Grxs) are a ubiquitous group of oxidoreductase enzymes that are important in plant growth and development; however, the functions of rice Grxs have not been fully elucidated. In this paper, we showed that one of the Grxs, encoded by OsGrxC2.2, exhibited Grx activity. Furthermore, we demonstrated that OsGrxC2.2 was able to regulate embryo development during embryogenesis. Transgenic rice lines overexpressing OsGrxC2.2 unexpectedly exhibited degenerate embryos as well as embryoless seeds. Our data indicated that the embryonic abnormalities occurred at an early stage during embryogenesis. We found that the expression of several endodermal layer marker genes for embryo development, such as OSH1 (apical region marker), OsSCR (L2 ground tissue marker), and OsPNH1 (L3 vascular tissue marker), were significantly decreased in the OsGrxC2.2-overexpressed transgenic rice lines. In contrast, the transcript levels of the majority of protodermal layer markers, including HAZ1, ROC2, ROC3, and RAmy1A, and the shoot apical meristem marker HB, showed little change between the wild-type (WT) and OsGrxC2.2-overexpressing embryos. Surprisingly, the seed weight of the overexpressed transgenic rice was remarkably increased in comparison to that of the WT. These results indicate that the overexpression of OsGrxC2.2 interferes with the normal embryogenesis of rice embryos and leads to increased grain weight. To the best of our knowledge, this is the first report that OsGrxC2.2 is a rice embryo development-associated gene.