Customized Lanthanide Nanobiohybrids for Noninvasive Precise Phototheranostics of Pulmonary Biofilm Infection.

A noninvasive strategy for in situ diagnosis and precise treatment of bacterial biofilm infections is highly anticipated but still a great challenge. Currently, no in vivo biofilm-targeted theranostic agent is available. Herein, we fabricated intelligent theranostic alginate lyase (Aly)-NaNdF4 nanohybrids with a 220 nm sunflower-like structure (NaNdF4@DMS-Aly) through an enrichment-encapsulating strategy, which exhibited excellent photothermal conversion efficiency and the second near-infrared (NIR-II) luminescence. Benefiting from the site-specific targeting and biofilm-responsive Aly release from NaNdF4@DMS-Aly, we not only enabled noninvasive diagnosis but also realized Aly-photothermal synergistic therapy and real-time evaluation of therapeutic effect in mice models with Pseudomonas aeruginosa biofilm-induced pulmonary infection. Furthermore, such nanobiohybrids with a sheddable siliceous shell are capable of delaying the NaNdF4 dissolution and biodegradation upon accomplishing the therapy, which is highly beneficial for the biosafety of theranostic agents.

OsOFP6 Overexpression Alters Plant Architecture, Grain Shape, and Seed Fertility.

OVATE family proteins (OFPs) play important roles in plant growth and development, hormone signaling, and stress response pathways. However, the functions of OsOFPs in rice are largely unknown. In this study, a novel gain-of-function rice mutant, Osofp6-D, was identified. This mutant exhibited decreased plant height, erect leaves, reduced panicle size, short and wide seeds, delayed seed germination time, and reduced fertility. These phenotypic changes were attributed to the increased expression of OsOFP6, which was caused by a T-DNA insertion. Complementation of the Osofp6-D phenotype by knockout of OsOFP6 using the CRISPR/Cas9 system confirmed that the Osofp6-D phenotype was caused by OsOFP6 overexpression. In addition, transgenic plants overexpressing OsOFP6 with the 35S promoter mimicked the Osofp6-D phenotype. Cytological observations of the glumes showed that OsOFP6 overexpression altered the grain shape, mainly by altering the cell shape. Hormone response experiments showed that OsOFP6 was involved in the gibberellin (GA) and brassinolide (BR) signaling responses. Further studies revealed that OsOFP6 interacts with E3BB, which is orthologous to the Arabidopsis central organ size-control protein BIG BROTHER (BB). This study further elucidates the regulation mechanism of the rice OFP family on plant architecture and grain shape.

A multi-functional nano-system combining PI3K-110α/ß inhibitor overcomes P-glycoprotein mediated MDR and improves anti-cancer efficiency.

P-glycoprotein (P-gp/ABCB1)-mediated multidrug resistance (MDR) in cancers severely limit chemotherapeutic efficacy. We recently reported that phosphatidylinositol-3-kinase (PI3K) 110α and 110ß subunits can be novel targets for reversal of P-gp mediated MDR in cancers, and BAY-1082439 as an inhibitor specific for PI3K 110α and 110ß subunits could reverse P-gp-mediated MDR by downregulating P-gp expression in cancer cells. However, BAY-1082439 has very low solubility, short half-life and high in-vivo clearance rate. Till now, nano-system with the functions to target PI3K P110α and P110ß and reverse P-gp mediated MDR in cancers has not been reported. In our study, a tumor targeting drug delivery nano-system PBDF was established, which comprised doxorubicin (DOX) and BAY-1082439 respectively encapsulated by biodegradable PLGA-SH nanoparticles (NPs) that were grafted to gold nanorods (Au NRs) modified with FA-PEG-SH, to enhance the efficacy to reverse P-gp mediated MDR and to target tumor cells, further, to enhance the efficiency to inhibit MDR tumors overexpressing P-gp. In-vitro experiments indicated that PBDF NPs greatly enhanced uptake of DOX, improved the activity to reverse MDR, inhibited the cell proliferation, and induced S-phase arrest and apoptosis in KB-C2 cells, as compared with free DOX combining free BAY-1082439. In-vivo experiments further demonstrated that PBDF NPs improved the anti-tumor ability of DOX and inhibited development of KB-C2 tumors. Notably, the metastasis of KB-C2 cells in livers and lungs of nude mice were inhibited by treatment with PBDF NPs, which showed no obvious in-vitro or in-vivo toxicity.

Molecular engineering to red-shift the absorption band of AIE photosensitizers and improve their ROS generation ability.

Photodynamic therapy (PDT) with aggregation-induced emission photosensitizers (AIE-PSs) has attracted increasing attention for their enhanced fluorescence and reactive oxygen species (ROS) generation abilities upon aggregation. However, it is difficult for AIE-PSs to simultaneously achieve long-wavelength excitation (>600 nm) and high singlet oxygen quantum yield, which restricts their application in deep-tissue PDT. In this study, four novel AIE-PSs were developed by appropriate molecular engineering, and their absorption peaks shifted from 478 to 540 nm with a tail extending to 700 nm. Meanwhile, their emission peaks were also moved from 697 nm to 779 nm with a tail extending over 950 nm. Importantly, their singlet oxygen quantum yields successfully increased from 0.61 to 0.89. Moreover, TBQ, the best photosensitizer developed by us, has been successfully applied to image-guided PDT in BALB/C mice bearing 4T1 breast cancer under red light (605 ± 5 nm) irradiation, with IC50 less than 2.5 µM at a low light dose (10.8 J cm-2). The success of this molecular engineering indicates that increasing the number of acceptors is more effective at red-shifting the absorption band of AIE-PSs than increasing the number of donors, and extending the π-conjugation of acceptors will red-shift the absorption-emission band, increase the maximum molar extinction coefficient, and improve the ROS generation ability of AIE-PSs, thus providing a new strategy for the design of advanced AIE-PSs for deep-tissue PDT.

High-brightness and high-efficiency diode laser module based on double wavelength division multiplexing external cavities.

In this Letter, a new, to the best of our knowledge, external cavity structure based on double wavelength division multiplexing external cavities is proposed and demonstrated. The electro-optical conversion efficiency is improved and the brightness of the spectral beam combining diode lasers is enhanced. One wavelength division multiplexing external cavity is placed on the rear-side of the laser emitters to provide the strong optical feedback for wavelength locking and the other wavelength division multiplexing external cavity is placed on the front-side of laser emitters to combine three emitter beams to one beam. A maximum output power of up to 7.5 W is obtained and the brightness of the laser diode is 100 MW cm-2 sr-1 with an electro-optical conversion efficiency of 46.5%. Compared with a standard cavity for spectral beam combining, the use of double wavelength division multiplexing external cavities results in an electro-optical conversion efficiency improvement of 6.5%. The whole structure provides a new technology to achieve high-brightness and high electro-optical conversion efficiency for a laser diode source.

Single-chip hybrid integrated silicon photonics transmitter based on passive alignment.

A single-chip hybrid integrated silicon photonics transmitter based on passive alignment flip-chip bonding technology has been demonstrated. The transmitter is developed by the hybrid integration of a C-band slotted laser with 1 mm cavity length and a Mach-Zehnder modulator with 2 mm long phase shifter. A 3 dB bandwidth of the small signal response is 16.35 GHz at 5.99 VPP superimposed with a reverse bias voltage of 2.43 V. A 25 Gbps data transmission experiment of the hybrid integrated transmitter is performed at 25°C.

Restricting Bond Rotations by Ring Fusion: A Novel Molecular Design Strategy to Improve Photodynamic Antibacterial Efficacy of AIE Photosensitizers.

In recent years, aggregation-induced emission photosensitizers (AIE-PSs) for antibacterial photodynamic therapy (aPDT) have received increasing attention because of their ability to increase reactive oxygen species (ROS) generation in the aggregation state. However, their antibacterial effect still has great room for improvement. Herein, we propose that if the rotation of some bonds in AIE-PSs is restricted, the nonradiative decay could be further suppressed to boost the generation of fluorescence and ROS, so as to improve their antibacterial efficacy. Following this molecular design strategy, we developed a new class of carbazole group-based AIE-PSs (CPVBA, CPVBP, CPVBP2, and CPVBP3), in which the rotation of phenyl-N bonds is restricted in the carbazole ring. Compared with diphenylamine group-based AIE-PSs with free rotation of phenyl-N bonds, carbazole group-based AIE-PSs showed stronger fluorescence, ROS generation, and antibacterial abilities, demonstrating the feasibility of this new design strategy. Notably, CPVBP3 can enter the entire cell of E. coli to exert its antibacterial effect, and there are few reports of photosensitizers with similar functions. Furthermore, to the best of our knowledge, the light dose (1.2 J/cm2) we used for CPVBP2 to kill Staphylococcus aureus is much lower than that of many reported photosensitizers, indicating great prospects for AIE antimicrobial photosensitizers.

Synergistic Lysozyme-Photodynamic Therapy Against Resistant Bacteria based on an Intelligent Upconversion Nanoplatform.

The rapid emergence of drug-resistant bacteria has raised a great social concern together with the impetus for exploring advanced antibacterial ways. NIR-triggered antimicrobial photodynamic therapy (PDT) by lanthanide-doped upconversion nanoparticles (UCNP) as energy donor exhibits the advantages of high tissue penetration, broad antibacterial spectrum and less acquired resistance, but is still limited by its low efficacy. Now we designed a bio-inorganic nanohybrid and combined lysozyme (LYZ) with UCNP-PDT system to enhance the efficiency against resistant bacteria. Benefiting from the rapid adhesion to bacteria, intelligently bacteria-responsive LYZ release and synergistic LYZ-PDT effect, the nanoplatform achieves an exceptionally strong bactericidal capacity and conspicuous bacteriostasis on methicillin-resistant S. aureus. These findings pave the way for designing efficiently antibacterial nanomaterials and provide a new strategy for combating deep-tissue bacterial infection.

[Species composition and community structure of coniferous-broadleaved mixed forest in Xiaolong Mountain, Gansu Province, Northwest China]. / 甘肃小陇山暖温带针阔混交林物种组成和群落结构.

Xiaolong Mountain, located in warm temperate subtropical transition zone, is one of the important biodiversity conservation areas in China. We analyzed species composition, community structure, and habitat preferences of all woody plant species with DBH (diameter at breast height)≥1 cm in a 6 hm2 plot in Xiaolong Mountain, Gansu Province, Northwest China. A total of 29251 individuals (41735 stems) belonging to 33 families, 65 genera, and 124 species were recorded. The 28 species with importance value ≥1 contributed 82.9% to the total abundance. The top four species with the highest importance value were Quercus aliena var. acuteserrata, Betula platyphylla, Lindera aggregata var. playfairii and Corylus heterophylla. The structure of DBH size class of all stems showed an inverse 'J' type, indicating a successful regeneration tendency in the understory. Results from the indicator species analysis showed that 11 species had significant habitat prefe-rences, an two species (Ostrya japonica and Acer stachyophyllum subsp. betulifolium) had the most obvious preferences. Results from the redundancy analysis and partial methods showed that topographic factors played a dominant role in determining species distribution.

Suppression of cancer proliferation and metastasis by a versatile nanomedicine integrating photodynamic therapy, photothermal therapy, and enzyme inhibition.

Cancer therapeutics are varied and target diverse processes in cancer progression. Photodynamic therapy (PDT), photothermal therapy (PTT), and the inhibition of pro-cancer proteases are non-invasive anticancer therapeutics that attract increasing attentions for their enhanced specificities and milder systemic toxicities compared to traditional therapeutics. These modalities offer advantages to compensate for the shortcomings of their counterparts. For instance, PDT or PTT efficiently eliminates locally confined tumor cells while exhibiting no effect on metastatic tumor cells. In contrast, the inhibition of pro-cancer proteases systemically suppresses the proliferation and metastasis of cancer cells but does not eradicate existing cancer cells. To synergize these therapeutics, we hereby report a versatile nanoparticle that integrates the effects of PDT, PTT, and enzyme-inhibition. This nanoparticle (CIKP-NP) was synthesized by covalently or non-covalently modifying a photothermal nanoparticle with a photosensitizer, a pro-cancer protease inhibitor, and an albumin-binding molecule. After confirming the PDT, PTT, albumin-binding, and enzyme-inhibition properties at the molecular level, we demonstrated that CIKP-NP killed tumor cells through PDT or PTT and suppressed tumor cell invasion through enzyme-inhibition. In addition, through a breast cancer xenograft mouse model, we demonstrated that CIKP-NP suppressed tumor growth by PDT or PTT effect. Notably, the synergism of PDT and PTT significantly enhanced its anticancer efficiency. Furthermore, CIKP-NP significantly suppressed cancer metastasis in a lung metastatic mouse model. Last, biodistribution and the in vivo retention of CIKP-NP confirmed the tumor-targeting property. Beyond demonstrating the anti-tumor and anti-metastatic efficacy of CIKP-NP, our study also suggests a new strategy to synergize multiple anticancer therapeutics.

Near-infrared-excited upconversion photodynamic therapy of extensively drug-resistant Acinetobacter baumannii based on lanthanide nanoparticles.

Extensively drug-resistant Acinetobacter baumannii (XDR-AB) has raised considerable concerns due to its mortal damage to humans and its high transmission rate of infections in hospitals. However, current antibiotics not only show poor anti-infection effects in vivo but also frequently cause high nephrotoxicity and neurotoxicity. Herein, we report a near-infrared (NIR) light-initiated antimicrobial photodynamic therapy (aPDT) to effectively treat in vivo XDR-AB infections based on photosensitizer (PS) loaded upconversion nanoparticles (UCNPs, LiYF4:Yb/Er). Such nanoagents feature robust NIR triggered UC luminescence and high-efficiency energy transfer from UCNPs to the loaded PS, thereby allowing NIR-triggered generation of reactive oxygen species (ROS) for destroying the bacterial cell membrane. This strategy permits a high antibacterial activity against XDR-AB, resulting in a decline of 4.72 log10 in viability at a dose of 50 µg mL-1 UCNPs-PVP-RB with 980 nm laser irradiation (1 W cm-2). More significantly, we can achieve excellent therapeutic efficacy against deep-tissue (about 5 mm) XDR-AB infections without causing any side effects in the murine model. In brief, such NIR-activated aPDT may open up new avenues for treating various deep-tissue intractable infections.

Specific inhibition of plasminogen activator inhibitor 1 reduces blood glucose level by lowering TNF-a.

AIMS: The study aims to investigate the effect of plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of fibrinolytic process, on blood glucose in type 2 diabetes mellitus (T2DM) and its mechanism. MATERIALS AND METHODS: We developed a highly potent and highly specific PAI-1 inhibitor, named PAItrap3, based on the inactivated urokinase. Meanwhile, a single point mutation of PAItrap3 (i.e., PAItrapNC) was parallelly prepared as negative control. PAItrap3 was intravenously injected into type 2 diabetic (T2D) mice and its effect on metabolic system was evaluated by measuring the levels of blood glucose, PAI-1, and tumor necrosis factor alpha (TNF-α) in T2D mice. KEY FINDINGS: PAItrap3 significantly reduced the high blood glucose level and PAI-1 level in streptozotocin-induced T2D mice. PAItrapNC did not have any hypoglycemic effect at all on T2D mice. Mechanistically, both PAI-1 and TNF-α levels were attenuated by the administration of PAItrap3. In addition, we observed that PAItrap3 reduced the amount of fat droplets in adipocytes. SIGNIFICANCE: These findings provide clear evidence for PAI-1 to participate in inflammation and obesity mediated hyperglycemia, and open up a new prospect for the treatment of T2DM by PAI-1 inhibition.

Near-infrared-triggered antibacterial and antifungal photodynamic therapy based on lanthanide-doped upconversion nanoparticles.

An alarming worldwide increase in microbial resistance to traditional drugs and classical pharmacophores has spurred the search for new antimicrobial compounds. Antimicrobial photodynamic therapy (aPDT) has recently emerged as an effective modality for the selective destruction of bacteria and other pathogenic microorganisms. However, some of the factors, including the aggregation of the hydrophobic photosensitizer (PS) in aqueous media and the inefficient biodistribution of PS limit its expansion to clinical conditions. In addition, the photoactivation under visible-light irradiation limits the therapeutic effect of aPDT for deep-tissue infection. To overcome these limitations, a PS (ß-carboxyphthalocyanine zinc, CPZ) delivery system with lanthanide-doped upconversion nanoparticles (UCNPs, LiYF4:Yb/Er) and polyvinylpyrrolidone (PVP) was prepared and its antimicrobial (antibacterial and antifungal) activities were investigated. Such a near-infrared (NIR) triggered UCNPs-CPZ-PVP system significantly reduced the aggregation of CPZ and presented a high anti-infectious activity against multi-drug resistant (MDR) bacteria (methicillin-resistant Staphylococcus aureus by 4.7 log10 and MDR Escherichia coli by 2.1 log10) post aPDT (at 50 µg mL-1 UCNPs-CPZ-PVP with 0.5 W cm-2 980 nm light). In particular, UCNPs-CPZ-PVP showed high antifungal efficacy against Candida albicans. In vivo aPDT experiments were further carried out using an MDR bacterial infection murine model in the presence of 5 mm thick tissue specimens, demonstrating the great potential of UCNPs-CPZ-PVP against infections in deep tissue. Altogether, we reveal an efficient NIR-triggered nano-photosensitizer with promising antifungal and antibacterial efficacy for clinical antimicrobial therapy.

Rice Flowering Locus T 1 plays an important role in heading date influencing yield traits in rice.

Important role of flowering genes in enhancing grain productivity in rice has become well recognized for a number of key genes regulating the florigen production, but little has been known for the two florigen genes themselves. In this study, pleiotropism of Rice Flowering Locus T 1 (RFT1), one of the two florigen genes in rice, was firstly evaluated using near isogenic lines (NILs) carrying RFT1 alleles from the indica rice cultivars Zhenshan 97 (ZS97) and Milyang 46, respectively, and then determined by transformation of the RFT1 ZS97 allele into a japonica rice variety, Zhonghua 11. The RFT1 ZS97 allele was shown to delay heading and increase plant height, grain weight, grain number and grain yield, indicating that RFT1 plays an important role in the growth and development of rice. This study has also validated the potential of using a new type of genetic resource, sequential residual heterozygotes (SeqRHs), for QTL fine-mapping. A step-by-step approach was employed for SeqRHs identification, NIL development and QTL fine-mapping. The heterozygous segments and candidate QTL regions were gradually narrowed down. Eventually, the QTL region was delimited to a 1.7 kb region containing a single gene.

Newly identified CSP41b gene localized in chloroplasts affects leaf color in rice.

A rice mutant with light-green leaves was discovered from a transgenic line of Oryza sativa. The mutant has reduced chlorophyll content and abnormal chloroplast morphology throughout its life cycle. Genetic analysis revealed that a single nuclear-encoded recessive gene is responsible for the mutation, here designated as lgl1. To isolate the lgl1 gene, a high-resolution physical map of the chromosomal region around the lgl1 gene was made using a mapping population consisting of 1984 mutant individuals. The lgl1 gene was mapped in the 76.5kb region between marker YG4 and marker YG5 on chromosome 12. Sequence analysis revealed that there was a 39bp deletion within the fourth exon of the candidate gene Os12g0420200 (TIGR locus Os12g23180) encoding a chloroplast stem-loop-binding protein of 41kDa b (CSP41b). The lgl1 mutation was rescued by transformation with the wild type CSP41b gene. Accordingly, the CSP41b gene is identified as the LGL1 gene. CSP41b was transcribed in various tissues and was mainly expressed in leaves. Expression of CSP41b-GFP fusion protein indicated that CSP41b is localized in chloroplasts. The expression levels of some key genes involved in chlorophyll biosynthesis and photosynthesis, such as ChlD, ChlI, Hema1, Ygl1, POR, Cab1R, Cab2R, PsaA, and rbcL, was significantly changed in the lgl1 mutant. Our results demonstrate that CSP41b is a novel gene required for normal leaf color and chloroplast morphology in rice.

Novel rice mutants overexpressing the brassinosteroid catabolic gene CYP734A4.

KEY MESSAGE: Moderate overexpression of CYP734A4 improves grain number per main panicle and seed setting rate. Brassinosteroid (BR) homeostasis and signaling are crucial for plant growth and development. CYP734A genes encode cytochrome P450 monooxygenases that control the level of bioactive BRs by degrading BRs. However, fertile plants overexpressing CYP734As have not been reported in rice. Here, we isolated a novel semi-dominant mutant brd3-D, in which T-DNA was inserted approximately 4 kb upstream of the CYP734A4 gene (GenBank Accession AB488667), causing its overexpression. The mutant is characterized by dwarfism, small grains, and erect leaves and is less sensitive to brassinolide-induced lamina joint inclination and primary root elongation. However, increased grain number per main panicle and improved seed setting rate were also found in heterozygous brd3-D. To our knowledge, these traits have not been reported in other BR deficient mutants. Quantitative real-time PCR analysis indicated that phenotypic severity of the brd3-D mutant is positively correlated with the CYP734A4 transcription level. In accordance with the increased expression of CYP734A4, a lower castasterone (a rice BR) content was detected in the brd3-D mutants. Knockout of brd3-D by using the CRISPR/Cas9 system rescued the mutation. In addition, transgenic plants overexpressing CYP734A4 with the 35S enhancer mimicked the brd3-D phenotypes, confirming that moderate overexpression of the CYP734A4 gene can improve grain number per main panicle and the seed setting rate in rice. Further studies showed that overexpression of CYP734A4 influences the expressions of multiple genes involved in the BR pathway, and the expression of CYP734A4 is induced by exogenous brassinolide, confirming the negative regulatory role of CYP734A4 in the BR pathway. CYP734A4 might provide a useful gene resource for developing new high-yielding rice varieties.

Interspecific associations of dominant tree populations in a virgin old-growth oak forest in the Qinling Mountains, China.

BACKGROUND: Understanding interspecific associations in old-growth forests will help to reveal mechanisms of interspecific replacement in the process of forest development and provide a theoretical basis for vegetation restoration and reestablishment. In this study, we analyzed interspecific associations of eleven dominant tree populations of varying development stages in an old-growth oak forest stand in the Qinling Mountains, China. We examined overall interspecific associations (multiple species) and pairwise interspecific associations (two species). RESULTS: Interspecific competition was intense during forest development and was the main factor driving succession. Community structure appears to become more stable over time which supports the harsh-benign hypothesis that interspecific competition is more common in stable sites. CONCLUSION: Old growth oak (Quercus spp.) forests are distributed widely around the world in part due to oak being a typical K-selected species. K-selected species produce fewer, high-quality offspring with higher survival rates, strong competitive ability, and longevity. The resulting distribution shifted from clumped to random, likely as a result of intense interspecific competition creating ecological niche differentiation.

Genetic polymorphisms of Trim5a are associated with disease progression in acutely and chronically HIV-infected patients.

BACKGROUND: The tripartite interaction motif 5a (Trim5a) plays critical roles in restricting various kinds of retroviruses in different species. It has been shown that Trim5a could inhibit HIV-1 inhibition in vitro. METHODS: In this study, 16 SNPs of Trim5a gene were screened in 236 acutely HIV-infected patients (169 common type (CT) patients and 67 patients with rapid disease progression). In addition, they were screened in 162 chronically HIV-infected patients (147 common type patients and 15 long-term non-progressors (LTNP)). The potential effects of polymorphisms at Trim5a genes on HIV-infection disease progression were analyzed. RESULTS: Among all tested SNP sites, 3 SNPs (rs3824949, rs2291841 and rs11038628) were identified to be associated with rapid disease progression in acutely HIV-infected patients. Carriage of rs3824949 allele G, rs2291841 allele C or rs11038628 allele T associated with rapid disease progression. In chronically HIV-infected patients, Patients carrying rs3802981 allele C or rs3802980 allele A had increased opportunity to be LTNP. We also found that greater age was associated with disease deterioration. CONCLUSIONS: Different genetic polymorphisms of Trim5a may have an impact on the clinical course of both acute and chronic stages of HIV-infection.

Characterization and fine mapping of a novel rice narrow leaf mutant nal9.

A narrow leaf mutant was isolated from transgenic rice (Oryza sativa L.) lines carrying a T-DNA insertion. The mutant is characterized by narrow leaves during its whole growth period, and was named nal9 (narrow leaf 9). The mutant also has other phenotypes, such as light green leaves at the seedling stage, reduced plant height, a small panicle and increased tillering. Genetic analysis revealed that the mutation is controlled by a single recessive gene. A hygromycin resistance assay showed that the mutation was not caused by T-DNA insertion, so a map-based cloning strategy was employed to isolate the nal9 gene. The mutant individuals from the F2 generations of a cross between the nal9 mutant and Longtepu were used for mapping. With 24 F2 mutants, the nal9 gene was preliminarily mapped near the marker RM156 on the chromosome 3. New INDEL markers were then designed based on the sequence differences between japonica and indica at the region near RM156. The nal9 gene was finally located in a 69.3 kb region between the markers V239B and V239G within BAC OJ1212_C05 by chromosome walking. Sequence and expression analysis showed that an ATP-dependent Clp protease proteolytic subunit gene (ClpP) was most likely to be the nal9 gene. Furthermore, the nal9 mutation was rescued by transformation of the ClpP cDNA driven by the 35S promoter. Accordingly, the ClpP gene was identified as the NAL9 gene. Our results provide a basis for functional studies of NAL9 in future work.