Thermo-Sensitive Spikelet Defects 1 acclimatizes rice spikelet initiation and development to high temperature.

Crop reproductive development is vulnerable to heat stress, and the genetic modulation of thermotolerance during the reproductive phase, especially the early stage, remains poorly understood. We isolated a Poaceae-specific FAR-RED ELONGATED HYPOCOTYLS3 (FHY3)/FAR-RED IMPAIRED RESPONSE1 (FAR1)family transcription factor, Thermo-sensitive Spikelet Defects 1 (TSD1), derived from transposase in rice (Oryza sativa) TSD1 was highly expressed in spikelets, induced by heat, and specifically enhanced the thermotolerance of spikelet morphogenesis. Disrupting TSD1 did not affect vegetative growth but markedly retarded spikelet initiation and development, as well as caused varying degrees of spikelet degeneration, depending on the temperature. Most tsd1 spikelets were normal at low temperature but gradually degenerated as temperature increased, and all disappeared at high temperature, leading to naked branches. TSD1 directly promoted the transcription of YABBY1 and YABBY3 and could physically interact with YABBY1 and three TOB proteins, YABBY5, YABBY4, and YABBY3. These YABBY proteins can form either homodimers or heterodimers and play an important role in spikelet morphogenesis, similar to TSD1. Notably, the knockout mutant yab5-ko and double mutant tsd1 yab5-ko resembled tsd1 in spikelet appearance and response to temperature, indicating that these genes likely participate in spikelet development through the cooperative TSD1-YABBY module. These findings reveal a distinctive function of FHY3/FAR1 family genes and a unique TSD1-YABBY complex to acclimate spikelet development to high temperature in rice, providing insight into the regulating pathway of enhancing thermotolerance in plant reproductive development.

Identification and candidate analysis of a new brown planthopper resistance locus in an Indian landrace of rice, paedai kalibungga.

The brown planthopper (Nilaparvata lugens Stål, BPH) is the most destructive pest of rice (Oryza sativa L.). Utilizing resistant rice cultivars that harbor resistance gene/s is an effective strategy for integrated pest management. Due to the co-evolution of BPH and rice, a single resistance gene may fail because of changes in the virulent BPH population. Thus, it is urgent to explore and map novel BPH resistance genes in rice germplasm. Previously, an indica landrace from India, Paedai kalibungga (PK), demonstrated high resistance to BPH in both in Wuhan and Fuzhou, China. To map BPH resistance genes from PK, a BC1F2:3 population derived from crosses of PK and a susceptible parent, Zhenshan 97 (ZS97), was developed and evaluated for BPH resistance. A novel BPH resistance locus, BPH39, was mapped on the short arm of rice chromosome 6 using next-generation sequencing-based bulked segregant analysis (BSA-seq). BPH39 was validated using flanking markers within the locus. Furthermore, near-isogenic lines carrying BPH39 (NIL-BPH39) were developed in the ZS97 background. NIL-BPH39 exhibited the physiological mechanisms of antibiosis and preference toward BPH. BPH39 was finally delimited to an interval of 84 Kb ranging from 1.07 to 1.15 Mb. Six candidate genes were identified in this region. Two of them (LOC_Os06g02930 and LOC_Os06g03030) encode proteins with a similar short consensus repeat (SCR) domain, which displayed many variations leading to amino acid substitutions and showed higher expression levels in NIL-BPH39. Thus, these two genes are considered reliable candidate genes for BPH39. Additionally, transcriptome sequencing, DEGs analysis, and gene RT-qPCR verification preliminary revealed that BPH39 may be involved in the jasmonic acid (JA) signaling pathway, thus mediating the molecular mechanism of BPH resistance. This work will facilitate map-based cloning and marker-assisted selection for the locus in breeding programs targeting BPH resistance. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01485-6.

Construction of a High-Density Recombination Bin-Based Genetic Map Facilitates High-Resolution Mapping of a Major QTL Underlying Anthocyanin Pigmentation in Eggplant.

High-density genetic maps can significantly improve the resolution of QTL mapping. We constructed a high-density recombination bin-based genetic map of eggplant based on 200 F2 plants from an interspecific cross (Solanum melongena × S. incanum) using the whole genome resequencing strategy. The map was 2022.8 cM long, covering near 99% of the eggplant genome. The map contained 3776 bins, with 3644 (96.5%) being effective (position non-redundant) ones, giving a nominal average distance of 0.54 cM and an effective average distance of 0.56 cM between adjacent bins, respectively. Using this map and 172 F2:3 lines, a major QTL with pleiotropic effects on two anthocyanin pigmentation-related traits, leaf vein color (LVC) and fruit pericarp color (FPC), was steadily detected in a bin interval of 2.28 cM (or 1.68 Mb) on chromosome E10 in two cropping seasons, explaining ~65% and 55% of the phenotypic variation in LVC and FPC, respectively. Genome-wide association analysis in this population validated the QTL and demonstrated the correctness of mapping two bins of chromosome E02 onto E10. Bioinformatics analysis suggested that a WDR protein gene inside the bin interval with reliable effective variation between the two parents could be a possible candidate gene of the QTL.

BRM: a statistical method for QTL mapping based on bulked segregant analysis by deep sequencing.

MOTIVATION: Bulked segregant analysis by deep sequencing (BSA-seq) has been widely used for quantitative trait locus (QTL) mapping in recent years. A number of different statistical methods for BSA-seq have been proposed. However, determination of significance threshold, the key point for QTL identification, remains to be a problem that has not been well solved due to the difficulty of multiple testing correction. In addition, estimation of the confidence interval is also a problem to be solved. RESULTS: In this paper, we propose a new statistical method for BSA-seq, named Block Regression Mapping (BRM). BRM is robust to sequencing noise and is applicable to the case of low sequencing depth. Significance threshold can be reasonably determined by taking multiple testing correction into account. Meanwhile, the confidence interval of QTL position can also be estimated. AVAILABILITY AND IMPLEMENTATION: The R scripts of our method are open source under GPLv3 license at https://github.com/huanglikun/BRM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RETINOBLASTOMA-RELATED Genes Specifically Control Inner Floral Organ Morphogenesis and Pollen Development in Rice.

RETINOBLASTOMA-RELATED (RBR) is an essential gene in plants, but its molecular function outside of its role in cell cycle entry remains poorly understood. We characterized the functions of OsRBR1 and OsRBR2 in plant growth and development in rice using both forward- and reverse-genetics methods. The two genes were coexpressed and performed redundant roles in vegetative organs but exhibited separate functions in flowers. OsRBR1 was highly expressed in the floral meristem and regulated the expression of floral homeotic genes to ensure floral organ formation. Mutation of OsRBR1 caused loss of floral meristem identity, resulting in the replacement of lodicules, stamens, and the pistil with either a panicle-like structure or whorls of lemma-like organs. OsRBR2 was preferentially expressed in stamens and promoted pollen formation. Mutation of OsRBR2 led to deformed anthers without pollen. Similar to the protein interaction between AtRBR and AtMSI1 that is essential for floral development in Arabidopsis, OsMSI1 was identified as an interaction partner of OsRBR1 and OsRBR2. OsMSI1 was ubiquitously expressed and appears to be essential for development in rice (Oryza sativa), as the mutation of OsMSI1 was lethal. These results suggest that OsRBR1 and OsRBR2 function with OsMSI1 in reproductive development in rice. This work characterizes further functions of RBRs and improves current understanding of specific regulatory pathways of floral specification and pollen formation in rice.

Mapping of QTLs conferring high grain length-breadth relative expansion during cooking in rice cultivar Paw San Hmwe.

Paw San Hmwe (PSH) is a high-quality rice cultivar from Myanmar. PSH has short and broad grains, but the grains become slender after cooking. This desirable feature can be described as a high value of grain length-breadth relative expansion index (GREI). To understand the genetic basis of high GREI in PSH, we crossed PSH with Guang 8B (G8B), a rice cultivar from China with low GREI, to develop an F2 population and a subsequent F2:3 population. Based on the phenotypes of these two populations measured in two years and using the method of sequencing-based bulked segregant analysis followed by verification with conventional linkage-based QTL mapping method, we mapped three QTLs for GREI. The three QTLs were located on chromosomes 3, 5 and 12, respectively, with the trait-increasing alleles all from PSH, and could explain a total of 62.5% of the phenotypic variance and 84.1% of the additive genetic variance. The results suggest that the three QTLs would be useful for the genetic improvement of GREI in rice, and the linked markers will facilitate the selection of the favorable alleles from PSH in breeding.

Estimation of QTL heritability based on pooled sequencing data.

Motivation: Bulked segregant analysis combined with next generation sequencing has proven to be a simple and efficient approach for fast mapping of quantitative trait loci (QTLs). However, how to estimate the proportion of phenotypic variance explained by a QTL (or termed QTL heritability) in such pooled QTL mapping is an unsolved problem. Results: In this paper, we propose a method called PQHE to estimate QTL heritability using pooled sequencing data obtained under different experimental designs. Simulation studies indicated that our method is correct and feasible. Four practical examples from rice and yeast are demonstrated, each representing a different situation. Availability and implementation: The R scripts of our method are open source under GPLv3 license at http://genetics.fafu.edu.cn/PQHE or https://github.com/biotangweiqi/PQHE. The R scripts require the R package rootSolve. Contact: wuwr@fafu.edu.cn. Supplementary information: Supplementary data are available at Bioinformatics online.

Teaching practice and experiences of verifying the three laws of genetics based on the SSLP marker analysis.

We explored the practical effect of the genetic analysis of simple sequence length polymorphism (SSLP) molecular markers in rice in the genetics lab course. Two parents and their F2 population were analyzed and detected with three SSLP molecular markers that located on two chromosomes of the rice genome. The markers' genotype data were used to verify the three laws of genetics, including segregation, independent assortment and linkage and crossing-over. Our practice has proved not only beneficial to deepen students' understandings about the three laws of genetics, but also conducive to cultivate students' interests in research and innovation and improve their skills and comprehensive analysis abilities. At the same time, the application scope of the experiment was discussed. This comprehensive experiment is also useful for the transformation of scientific research achievements into undergraduate experimental teaching.

A and D genomes spatial separation at somatic metaphase in tetraploid cotton: evidence for genomic disposition in a polyploid plant.

Chromosomal dispositions were analyzed on the metaphase plate of tetraploid cotton (AADD). At metaphase, the two subgenomes, A and D, were separated in a radial pattern in which the small D subgenome chromosomes tended to concentrate at the center and the large A subgenome chromosomes were scattered about the periphery on the metaphase plate. Although the ordered chromosome arrangement was disturbed in an artificial hexaploid (AADDGG), the separation pattern could be recovered after the majority of the additional genome (GG) chromosomes were removed by backcrossing the artificial hexaploid with the tetraploid cotton (AADD). A similar genome separation phenomenon was also found in synthesized tetraploid cotton (AAGG). These results indicate that the genome separation pattern could be established immediately after tetraploid cotton formation and could be stably inherited in tetraploid cotton. Given the evidence of parental genome separation in other plants and animals, we speculated that genome separation might be a normal phenomenon in diploid and polyploid species. These finding will shed light on the chromosome conformation in plant cells.

Dwarf and deformed flower 1, encoding an F-box protein, is critical for vegetative and floral development in rice (Oryza sativa L.).

Recent studies have shown that F-box proteins constitute a large family in eukaryotes, and play pivotal roles in regulating various developmental processes in plants. However, their functions in monocots are still obscure. In this study, we characterized a recessive mutant dwarf and deformed flower 1-1 (ddf1-1) in Oryza sativa (rice). The mutant is abnormal in both vegetative and reproductive development, with significant size reduction in all organs except the spikelet. DDF1 controls organ size by regulating both cell division and cell expansion. In the ddf1-1 spikelet, the specification of floral organs in whorls 2 and 3 is altered, with most lodicules and stamens being transformed into glume-like organs and pistil-like organs, respectively, but the specification of lemma/palea and pistil in whorls 1 and 4 is not affected. DDF1 encodes an F-box protein anchored in the nucleolus, and is expressed in almost all vegetative and reproductive tissues. Consistent with the mutant floral phenotype, DDF1 positively regulates B-class genes OsMADS4 and OsMADS16, and negatively regulates pistil specification gene DL. In addition, DDF1 also negatively regulates the Arabidopsis LFY ortholog APO2, implying a functional connection between DDF1 and APO2. Collectively, these results revealed that DDF1, as a newly identified F-box gene, is a crucial genetic factor with pleiotropic functions for both vegetative growth and floral organ specification in rice. These findings provide additional insights into the molecular mechanism controlling monocot vegetative and reproductive development.

[Genetic analysis and gene mapping for a salt tolerant mutant at seedling stage in rice].

A salt tolerant mutant at seedling stage was obtained from an M2 population of radiation mutagenesis of an indica rice cultivar R401. The mutant seedlings could survive under the treatment of sodium chloride solution at the concentration of 150 mmol/L, while the wild-type control seedlings withered and died. An F2 population was developed from a cross between a japonica cultivar Nipponbare and the salt tolerant mutant. By investigating the performance of the F2 population under the stress of 150 mmol/L NaCl solution, we found that the mutant phenotype was caused by the recessive mutation of a single gene, temporarily designated SST(t). Bulked segregant analysis (BSA) based on the F2 mapping population revealed that SST(t) is located on chromosome 6. By analyzing 137 typical salt-tolerant F2 plants using molecular markers, SST(t) was mapped in a 2.3 cM (or 406 kb) interval between InDel markers ID26847 and ID27253, with genetic distances of 1.2 cM and 1.1 cM to the two markers, respectively.

Glucan Synthase-like 2 is Required for Seed Initiation and Filling as Well as Pollen Fertility in Rice.

BACKGROUND: The Glucan synthase-like (GSL) genes are indispensable for some important highly-specialized developmental and cellular processes involving callose synthesis and deposition in plants. At present, the best-characterized reproductive functions of GSL genes are those for pollen formation and ovary expansion, but their role in seed initiation remains unknown. RESULTS: We identified a rice seed mutant, watery seed 1-1 (ws1-1), which contained a mutation in the OsGSL2 gene. The mutant produced seeds lacking embryo and endosperm but filled with transparent and sucrose-rich liquid. In a ws1-1 spikelet, the ovule development was normal, but the microsporogenesis and male gametophyte development were compromised, resulting in the reduction of fertile pollen. After fertilization, while the seed coat normally developed, the embryo failed to differentiate normally. In addition, the divided endosperm-free nuclei did not migrate to the periphery of the embryo sac but aggregated so that their proliferation and cellularization were arrested. Moreover, the degeneration of nucellus cells was delayed in ws1-1. OsGSL2 is highly expressed in reproductive organs and developing seeds. Disrupting OsGSL2 reduced callose deposition on the outer walls of the microspores and impaired the formation of the annular callose sheath in developing caryopsis, leading to pollen defect and seed abortion. CONCLUSIONS: Our findings revealed that OsGSL2 is essential for rice fertility and is required for embryo differentiation and endosperm-free nucleus positioning, indicating a distinct role of OsGSL2, a callose synthase gene, in seed initiation, which provides new insight into the regulation of seed development in cereals.

Genome-wide association study of cooking-caused grain expansion in rice (Oryza sativa L.).

Cooking-caused rice grain expansion (CCRGE) is a critical trait for evaluating the cooking quality of rice. Previous quantitative trait locus (QTL) mapping studies on CCRGE have been limited to bi-parental populations, which restrict the exploration of natural variation and mapping resolution. To comprehensively and precisely dissect the genetic basis of CCRGE, we performed a genome-wide association study (GWAS) on three related indices: grain breadth expansion index (GBEI), grain length expansion index (GLEI), and grain length-breadth ratio expansion index (GREI), using 345 rice accessions grown in two years (environments) and 193,582 SNP markers. By analyzing each environment separately using seven different methods (3VmrMLM, mrMLM, FASTmrMLM, FASTmrEMMA, pLARmEB, pKWmEB, ISIS EM-BLASSO), we identified a total of 32, 19 and 27 reliable quantitative trait nucleotides (QTNs) associated with GBEI, GLEI and GREI, respectively. Furthermore, by jointly analyzing the two environments using 3VmrMLM, we discovered 19, 22 and 25 QTNs, as well as 9, 5 and 7 QTN-by-environment interaction (QEIs) associated with GBEI, GLEI and GREI, respectively. Notably, 12, 9 and 15 QTNs for GBEI, GLEI and GREI were found within the intervals of previously reported QTLs. In the vicinity of these QTNs or QEIs, based on analyses of mutation type, gene ontology classification, haplotype, and expression pattern, we identified five candidate genes that are related to starch synthesis and endosperm development. The five candidate genes, namely, LOC_Os04g53310 (OsSSIIIb, near QTN qGREI-4.5s), LOC_Os05g02070 (OsMT2b, near QTN qGLEI-5.1s), LOC_Os06g04200 (wx, near QEI qGBEI-6.1i and QTNs qGREI-6.1s and qGLEI-6.1t), LOC_Os06g12450 (OsSSIIa, near QTN qGLEI-6.2t), and LOC_Os08g09230 (OsSSIIIa, near QTN qGBEI-8.1t), are predicted to be involved in the process of rice grain starch synthesis and to influence grain expansion after cooking. Our findings provide valuable insights and will facilitate genetic research and improvement of CCRGE.

Characterization of Osmads6-5, a null allele, reveals that OsMADS6 is a critical regulator for early flower development in rice (Oryza sativa L.).

AGL6-clade genes are a subfamily of MADS-box genes and preferentially expressed in floral organs. OsMADS6 and OsMADS17 are two AGL6-like genes in rice. OsMADS17 has been shown to play a minor role in floral development and appears to result from a duplication of OsMADS6. OsMADS6 was initially named as MFO1 for mosaic floral organs based on its moderate mutant phenotypes. So far, four moderate or weak mutant alleles of OsMADS6 have been described, providing valuable insights into its role in flower development. Here, we report a null allele of OsMADS6 (Osmads6-5), which exhibited a strong mutant phenotype in spikelet without affecting vegetative traits, causing all floral organs except lemma homeotically transformed into lemma-like organs (LLOs) as well as an indeterminate floral meristem, thus resulting in a mutant floret consisting of reiterating whorls of lemma and LLOs. In consistently, over-expression of OsMADS6 led to additional lodicule-, stamen- and carpel-like organs. Expression analysis showed that OsMADS6 controls the formation of the incipient primordia of lodicule, stamen and carpel via regulating the expression of class B, C and SEP-like MADS-box genes. Taken together, our results revealed that OsMADS6 acts as a critical regulator for early flower development in rice and provide novel insights into the molecular mechanism of OsMADS6.

[Mapping of Sanming dominant genic male sterility gene in rice].

The mutant of "Sanming Dominant Genic Male Sterile Rice" was found from an F2 population of cross "SE2lS/Basmati370" by Sanming Institute of Agricultural Science in 2001. It has proven that the male sterility of this mutant is controlled by a dominant gene (named as SMS). By multiple backcrosses, this dominant male sterile allele was introduced into the genetic background of an indica rice cultivar Jiafuzhan (which was known as Jiabuyu). In order to map SMS, a mapping population was constructed by crossing Jiabuyu with a japonica cultivar Nipponbare and further crossing the F1 with Jiafuzhan. By bulked segregant analysis and linkage analysis using SSR and INDEL markers, SMS was mapped to a 99 kb interval between INDEL markers ZM30 and ZM9 on chromosome 8. This result will facilitate cloning of SMS.

Optimization of BSA-seq experiment for QTL mapping.

Deep sequencing-based bulked segregant analysis (BSA-seq) has become a popular approach for quantitative trait loci (QTL) mapping in recent years. Effective statistical methods for BSA-seq have been developed, but how to design a suitable experiment for BSA-seq remains unclear. In this paper, we show in theory how the major experimental factors (including population size, pool proportion, pool balance, and generation) and the intrinsic factors of a QTL (including heritability and degree of dominance) affect the power of QTL detection and the precision of QTL mapping in BSA-seq. Increasing population size can improve the power and precision, depending on the QTL heritability. The best proportion of each pool in the population is around 0.25. So, 0.25 is generally applicable in BSA-seq. Small pool proportion can greatly reduce the power and precision. Imbalance of pool pair in size also causes decrease of the power and precision. Additive effect is more important than dominance effect for QTL mapping. Increasing the generation of filial population produced by selfing can significantly increase the power and precision, especially from F2 to F3. These findings enable researchers to optimize the experimental design for BSA-seq. A web-based program named BSA-seq Design Tool is available at http://124.71.74.135/BSA-seqDesignTool/ and https://github.com/huanglikun/BSA-seqDesignTool.

LUX ARRHYTHMO Interacts With ELF3a and ELF4a to Coordinate Vegetative Growth and Photoperiodic Flowering in Rice.

The evening complex (EC) plays a critical role in photoperiod flowering in Arabidopsis. Nevertheless, the underlying functions of individual components and coordinate regulation mechanism of EC genes in rice flowering remain to be elucidated. Here, we characterized the critical role of LUX ARRHYTHMO (LUX) in photoperiod perception and coordinating vegetative growth and flowering in rice. Non-functional alleles of OsLUX extremely extended vegetative phase, leading to photoperiod-insensitive late flowering and great increase of grain yield. OsLUX displayed an obvious diurnal rhythm expression with the peak at dusk and promoted rice flowering via coordinating the expression of genes associated with the circadian clock and the output integrators of photoperiodic flowering. OsLUX combined with OsELF4a and OsELF3a or OsELF3b to form two ECs, of which the OsLUX-OsELF3a-OsELF4a was likely the dominant promoter for photoperiodic flowering. In addition, OsELF4a was also essential for promoting rice flowering. Unlike OsLUX, loss OsELF4a displayed a marginal influence under short-day (SD) condition, but markedly delayed flowering time under long-day (LD) condition. These results suggest that rice EC genes share the function of promoting flowering. This is agreement with the orthologs of SD plant, but opposite to the counterparts of LD species. Taken together, rice EC genes display similar but not identical function in photoperiodic flowering, probably through regulating gene expression cooperative and independent. These findings facilitate our understanding of photoperiodic flowering in plants, especially the SD crops.

Transcriptome Analyses Indicate Significant Association of Increased Non-Additive and Allele-Specific Gene Expression with Hybrid Weakness in Rice (Oryza sativa L.).

The heterosis in hybrid rice is highly affected by the environment and hybrid weakness occurs frequently depending on the genotypes of the hybrid and its parents. Hybrid weakness was also observed in our field experiments on nine rice hybrids produced by 3 × 3 incomplete diallel crosses. Among the nine hybrids, five displayed mid-parent heterosis (MPH) for grain yield per plant, while four showed mid-parent hybrid weakness (MPHW). A sequencing analysis of transcriptomes in panicles at the seed-filling stage revealed a significant association between enhanced non-additive gene expression (NAE) and allele-specific gene expression (ASE) with hybrid weakness. High proportions of ASE genes, with most being of mono-allele expression, were detected in the four MPHW hybrids, ranging from 22.65% to 45.97%; whereas only 4.80% to 5.69% of ASE genes were found in the five MPH hybrids. Moreover, an independence test indicated that the enhancements of NAE and ASE in the MPHW hybrids were significantly correlated. Based on the results of our study, we speculated that an unfavorable environment might cause hybrid weakness by enhancing ASE and NAE at the transcriptome level.

AVR1-CO39 is a predominant locus governing the broad avirulence of Magnaporthe oryzae 2539 on cultivated rice (Oryza sativa L.).

Magnaporthe oryzae 2539 was previously found to be avirulent to most rice cultivars and, therefore, was assumed to carry many avirulence (AVR) genes. However, only one AVR gene, AVR1-CO39, which corresponds to a resistance (R) gene Pi-CO39(t) in rice cv. CO39, has been found from 2539 thus far. In order to identify more AVR genes, we isolated 228 progeny strains from a cross between 2539 and Guy11, an M. oryzae strain with strong virulence on rice, and inoculated these strains onto 23 rice accessions (22 individual cultivars and a mixture of 14 cultivars) that are all resistant to 2539 but susceptible to Guy11. Unexpectedly, the experimental results indicated that the avirulence of 2539 on these rice cultivars appeared to be controlled only by the AVR1-CO39 locus. Consistent with this result, we further found that all except one of the rice cultivars were resistant to two transformed Guy11 strains carrying a 1.05-kb fragment containing the AVR1-CO39 gene from 2539. These results suggest that AVR1-CO39 is a predominant locus controlling the broad avirulence of 2539 on cultivated rice. Based on the results of this study and other previous studies, we infer that AVR1-CO39 is a species-wise rather than a cultivar-wise host-specific AVR locus of M. oryzae for rice.

Knockdown of NtMed8, a Med8-like gene, causes abnormal development of vegetative and floral organs in tobacco (Nicotiana tabacum L.).

Med8, a subunit of mediator complex, has proved to possess crucial functions in many organisms from yeast to human. In plant, the med8 mutant of Arabidopsis thaliana displayed delayed anthesis and increased number of leaves during the vegetative period. However, the roles of Med8 in other flowering plants are still unknown. To investigate the function of Med8 ortholog in tobacco (Nicotiana tabacum L.; named as NtMed8), we created transgenic tobacco plants with repressed NtMed8 expression mediated by RNA interference (RNAi). Compared with the wild type, the NtMed8-RNAi plants exhibited: more leaves with smaller but thicker blades; larger cells and vascular bundles with lower stomata density in leaves; swelled chloroplasts with thicker and lumen-enlarged thylakoids; weaker root system with fewer lateral roots; larger flowers and floral organs; flowering earlier under long day, but later under short day conditions; and male sterile with larger but less germinable pollens. In addition, quantitative RT-PCR indicated that NtMed8 is expressed in both vegetative and floral tissues. Subcellular localization analysis by transient expression of fusion protein in Nicotiana benthamiana leaves showed that NtMed8 was located in both plasma membrane and nucleus. These results suggest that NtMed8 plays important roles in both vegetative and reproductive development, and the function of Med8 appears to be, at least partially, conserved in flowering plants.