Genome-wide identification of R2R3-MYB transcription factor subfamily genes involved in salt stress in rice (Oryza sativa L.).

BACKGROUND: R2R3-MYB transcription factors belong to one of the largest gene subfamilies in plants, and they are involved in diverse biological processes. However, the role of R2R3-MYB transcription factor subfamily genes in the response of rice (Oryza sativa L.) to salt stress has been rarely reported. RESULTS: In this study, we performed a genome-wide characterization and expression identification of rice R2R3-MYB transcription factor subfamily genes. We identified a total of 117 R2R3-MYB genes in rice and characterized their gene structure, chromosomal location, and cis-regulatory elements. According to the phylogenetic relationships and amino acid sequence homologies, the R2R3-MYB genes were divided into four groups. qRT-PCR of the R2R3-MYB genes showed that the expression levels of 10 genes significantly increased after 3 days of 0.8% NaCl treatment. We selected a high expression gene OsMYB2-115 for further analysis. OsMYB2-115 was highly expressed in the roots, stem, leaf, and leaf sheath. OsMYB2-115 was found to be localized in the nucleus, and the yeast hybrid assay showed that OsMYB2-115 has transcriptional activation activity. CONCLUSION: This result provides important information for the functional analyses of rice R2R3-MYB transcription factor subfamily genes related to the salt stress response and reveals that OsMYB2-115 may be an important gene associated with salt tolerance in rice.

The OsNAC24-OsNAP protein complex activates OsGBSSI and OsSBEI expression to fine-tune starch biosynthesis in rice endosperm.

Starch accounts for up to 90% of the dry weight of rice endosperm and is a key determinant of grain quality. Although starch biosynthesis enzymes have been comprehensively studied, transcriptional regulation of starch-synthesis enzyme-coding genes (SECGs) is largely unknown. In this study, we explored the role of a NAC transcription factor, OsNAC24, in regulating starch biosynthesis in rice. OsNAC24 is highly expressed in developing endosperm. The endosperm of osnac24 mutants is normal in appearance as is starch granule morphology, while total starch content, amylose content, chain length distribution of amylopectin and the physicochemical properties of the starch are changed. In addition, the expression of several SECGs was altered in osnac24 mutant plants. OsNAC24 is a transcriptional activator that targets the promoters of six SECGs; OsGBSSI, OsSBEI, OsAGPS2, OsSSI, OsSSIIIa and OsSSIVb. Since both the mRNA and protein abundances of OsGBSSI and OsSBEI were decreased in the mutants, OsNAC24 functions to regulate starch synthesis mainly through OsGBSSI and OsSBEI. Furthermore, OsNAC24 binds to the newly identified motifs TTGACAA, AGAAGA and ACAAGA as well as the core NAC-binding motif CACG. Another NAC family member, OsNAP, interacts with OsNAC24 and coactivates target gene expression. Loss-of-function of OsNAP led to altered expression in all tested SECGs and reduced the starch content. These results demonstrate that the OsNAC24-OsNAP complex plays key roles in fine-tuning starch synthesis in rice endosperm and further suggest that manipulating the OsNAC24-OsNAP complex regulatory network could be a potential strategy for breeding rice cultivars with improved cooking and eating quality.

Mapping and validation of quantitative trait loci associated with dorsal aleurone thickness in rice (Oryza sativa).

KEY MESSAGE: Mapping of QTLs for dorsal aleurone thickness (DAT) was performed using chromosome segment substitution lines in rice. Three QTLs, qDAT3.1, qDAT3.2, and qDAT7.1, were detected in multiple environments. As a specified endosperm cell type, the aleurone has an abundance of various nutrients. Increasing the number of aleurone layers is a practicable way of developing highly nutritious cereals. Identifying genes that can increase aleurone thickness is useful for the breeding of aleurone traits to improve the nutritional and health values of rice. Here, we found that iodine staining could efficiently distinguish the aleurone layers, which revealed great variation of the aleurone thickness in rice, especially at the dorsal side of the seed. Therefore, we used a population of chromosome segmental substitution lines (CSSLs) derived from Koshihikari and Nona Bokra for quantitative trait locus (QTL) analysis of the dorsal aleurone thickness (DAT). Three QTLs, qDAT3.1, qDAT3.2, and qDAT7.1, were detected in multiple seasons. Among these, qDAT3.2 colocalizes with Hd6 and Hd16, two QTLs previously identified to regulate the heading date of Koshihikari, explaining the negative correlation between the DAT and days to heading (DTH) in rice. We also provide evidence that early-heading ensures the filling of rice seed under a relatively high temperature to promote aleurone thickening. qDAT7.1, the most stable QTL expressed in different environments, functions independently from heading date. Although Nona Bokra has a lower DAT, its qDAT7.1 allele significantly increased DAT in rice, which was further validated using two near-isogenic lines (NILs). These findings pave the way for further gene cloning of aleurone-related QTLs and may aid the development of highly nutritious rice.

GRAIN SIZE AND NUMBER1 Negatively Regulates the OsMKKK10-OsMKK4-OsMPK6 Cascade to Coordinate the Trade-off between Grain Number per Panicle and Grain Size in Rice.

Grain number and size are interactive agronomic traits that determine grain yield. However, the molecular mechanisms responsible for coordinating the trade-off between these traits remain elusive. Here, we characterized the rice (Oryza sativa) grain size and number1 (gsn1) mutant, which has larger grains but sparser panicles than the wild type due to disordered localized cell differentiation and proliferation. GSN1 encodes the mitogen-activated protein kinase phosphatase OsMKP1, a dual-specificity phosphatase of unknown function. Reduced expression of GSN1 resulted in larger and fewer grains, whereas increased expression resulted in more grains but reduced grain size. GSN1 directly interacts with and inactivates the mitogen-activated protein kinase OsMPK6 via dephosphorylation. Consistent with this finding, the suppression of mitogen-activated protein kinase genes OsMPK6, OsMKK4, and OsMKKK10 separately resulted in denser panicles and smaller grains, which rescued the mutant gsn1 phenotypes. Therefore, OsMKKK10-OsMKK4-OsMPK6 participates in panicle morphogenesis and acts on a common pathway in rice. We confirmed that GSN1 is a negative regulator of the OsMKKK10-OsMKK4-OsMPK6 cascade that determines panicle architecture. The GSN1-MAPK module coordinates the trade-off between grain number and grain size by integrating localized cell differentiation and proliferation. These findings provide important insights into the developmental plasticity of the panicle and a potential means to improve crop yields.

DCA1 Acts as a Transcriptional Co-activator of DST and Contributes to Drought and Salt Tolerance in Rice.

Natural disasters, including drought and salt stress, seriously threaten food security. In previous work we cloned a key zinc finger transcription factor gene, Drought and Salt Tolerance (DST), a negative regulator of drought and salt tolerance that controls stomatal aperture in rice. However, the exact mechanism by which DST regulates the expression of target genes remains unknown. In the present study, we demonstrated that DST Co-activator 1 (DCA1), a previously unknown CHY zinc finger protein, acts as an interacting co-activator of DST. DST was found to physically interact with itself and to form a heterologous tetramer with DCA1. This transcriptional complex appears to regulate the expression of peroxidase 24 precursor (Prx 24), a gene encoding an H2O2 scavenger that is more highly expressed in guard cells. Downregulation of DCA1 significantly enhanced drought and salt tolerance in rice, and overexpression of DCA1 increased sensitivity to stress treatment. These phenotypes were mainly influenced by DCA1 and negatively regulated stomatal closure through the direct modulation of genes associated with H2O2 homeostasis. Our findings establish a framework for plant drought and salt stress tolerance through the DCA1-DST-Prx24 pathway. Moreover, due to the evolutionary and functional conservation of DCA1 and DST in plants, engineering of this pathway has the potential to improve tolerance to abiotic stress in other important crop species.

A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control.

Abiotic stresses, such as drought and salinity, lead to crop growth damage and a decrease in crop yields. Stomata control CO(2) uptake and optimize water use efficiency, thereby playing crucial roles in abiotic stress tolerance. Hydrogen peroxide (H(2)O(2)) is an important signal molecule that induces stomatal closure. However, the molecular pathway that regulates the H(2)O(2) level in guard cells remains largely unknown. Here, we clone and characterize DST (drought and salt tolerance)-a previously unknown zinc finger transcription factor that negatively regulates stomatal closure by direct modulation of genes related to H(2)O(2) homeostasis-and identify a novel pathway for the signal transduction of DST-mediated H(2)O(2)-induced stomatal closure. Loss of DST function increases stomatal closure and reduces stomatal density, consequently resulting in enhanced drought and salt tolerance in rice. These findings provide an interesting insight into the mechanism of stomata-regulated abiotic stress tolerance, and an important genetic engineering approach for improving abiotic stress tolerance in crops.

The miR156-SPL9-DFR pathway coordinates the relationship between development and abiotic stress tolerance in plants.

Young organisms have relatively strong resistance to diseases and adverse conditions. When confronted with adversity, the process of development is delayed in plants. This phenomenon is thought to result from the rebalancing of energy, which helps plants to coordinate the relationship between development and stress tolerance; however, the molecular mechanism underlying this phenomenon remains mysterious. In this study, we found that miR156 integrates environmental signals to ensure timely flowering, thus enabling the completion of breeding. Under stress conditions, miR156 is induced to maintain the plant in the juvenile state for a relatively long period of time, whereas under favorable conditions, miR156 is suppressed to accelerate the developmental transition. Blocking the miR156 signaling pathway in Arabidopsis thaliana with 35S::MIM156 (via target mimicry) increased the sensitivity of the plant to stress treatment, whereas overexpression of miR156 increased stress tolerance. In fact, this mechanism is also conserved in Oryza sativa (rice). We also identified downstream genes of miR156, i.e. SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) and DIHYDROFLAVONOL-4-REDUCTASE (DFR), which take part in this process by influencing the metabolism of anthocyanin. Our results uncover a molecular mechanism for plant adaptation to the environment through the miR156-SPLs-DFR pathway, which coordinates development and abiotic stress tolerance.

A rice quantitative trait locus for salt tolerance encodes a sodium transporter.

Many important agronomic traits in crop plants, including stress tolerance, are complex traits controlled by quantitative trait loci (QTLs). Isolation of these QTLs holds great promise to improve world agriculture but is a challenging task. We previously mapped a rice QTL, SKC1, that maintained K(+) homeostasis in the salt-tolerant variety under salt stress, consistent with the earlier finding that K(+) homeostasis is important in salt tolerance. To understand the molecular basis of this QTL, we isolated the SKC1 gene by map-based cloning and found that it encoded a member of HKT-type transporters. SKC1 is preferentially expressed in the parenchyma cells surrounding the xylem vessels. Voltage-clamp analysis showed that SKC1 protein functions as a Na(+)-selective transporter. Physiological analysis suggested that SKC1 is involved in regulating K(+)/Na(+) homeostasis under salt stress, providing a potential tool for improving salt tolerance in crops.

A rice high-affinity potassium transporter (HKT) conceals a calcium-permeable cation channel.

Plant high-affinity K(+) transport (HKT) proteins are so named because of their relation to bacterial and fungal transporters that mediate high-affinity K(+) uptake. The view that HKT family members are sodium-selective uniporters or sodium-potassium symporters is widely held. We have found that one of the rice HKT proteins also functions as a Ca(2+)-permeable cation channel that conducts current carried by a wide range of monovalent and divalent cations. The HKT rice gene, named OsHKT2;4, is expressed in several cell types, including root hairs and vascular parenchyma cells. The protein is localized to the plasma membrane, thereby providing a mechanism for cation uptake and extrusion. This finding goes against firmly entrenched dogma in showing that HKT proteins can function as both ion carriers and channels. The study further extends the function of HKT proteins to Ca(2+)-linked processes and, in so doing, defines a previously undescribed type of Ca(2+)-permeable cation channels in plants. The work also raises questions about the evolutionary changes in this protein family following the divergence of monocots and dicots.

Rice carotenoid ß-ring hydroxylase CYP97A4 is involved in lutein biosynthesis.

Lutein is the most abundant plant carotenoid and plays essential roles in photosystem assembly and stabilization, as well as protection against photostress. To date, only a few lutein biosynthesis genes have been identified in crop plants. In this study, the rice Cyt P450 gene CYP97A4 encoding a carotenoid ß-ring hydroxylase was shown to be involved in lutein biosynthesis. The results revealed that CYP97A4 was preferentially expressed in leaf compared with spikelet, sheath, stalk and root, and encoded a protein localized at the subcellular level to the chloroplasts. Compared with the wild type, the three allelic mutants of CYP97A4 displayed lutein reductions of 12-24% with substantially increased α-carotene, while Chl a/b levels were unaltered. The increased α-carotene in the mutants led to greater sensitivity under high light stress. Similarly, reactive oxygen species (ROS) imaging of leaves treated with intense light showed that the mutants generally accumulated greater levels of ROS compared with wild-type plants, which probably caused detrimental effects to the plant photosystem. In conclusion, this study demonstrated the important role of CYP97A4 in α-carotene hydroxylation in rice, and knock-out of the gene reduced lutein and increased α-carotene, contributing to sensitivity to intense light.

OsNAC129 Regulates Seed Development and Plant Growth and Participates in the Brassinosteroid Signaling Pathway.

Grain size and the endosperm starch content determine grain yield and quality in rice. Although these yield components have been intensively studied, their regulatory mechanisms are still largely unknown. In this study, we show that loss-of-function of OsNAC129, a member of the NAC transcription factor gene family that has its highest expression in the immature seed, greatly increased grain length, grain weight, apparent amylose content (AAC), and plant height. Overexpression of OsNAC129 had the opposite effect, significantly decreasing grain width, grain weight, AAC, and plant height. Cytological observation of the outer epidermal cells of the lemma using a scanning electron microscope (SEM) revealed that increased grain length in the osnac129 mutant was due to increased cell length compared with wild-type (WT) plants. The expression of OsPGL1 and OsPGL2, two positive grain-size regulators that control cell elongation, was consistently upregulated in osnac129 mutant plants but downregulated in OsNAC129 overexpression plants. Furthermore, we also found that several starch synthase-encoding genes, including OsGBSSI, were upregulated in the osnac129 mutant and downregulated in the overexpression plants compared with WT plants, implying a negative regulatory role for OsNAC129 both in grain size and starch biosynthesis. Additionally, we found that the expression of OsNAC129 was induced exclusively by abscisic acid (ABA) in seedlings, but OsNAC129-overexpressing plants displayed reduced sensitivity to exogenous brassinolide (BR). Therefore, the results of our study demonstrate that OsNAC129 negatively regulates seed development and plant growth, and further suggest that OsNAC129 participates in the BR signaling pathway.

High-resolution quantitative trait locus mapping for rice grain quality traits using genotyping by sequencing.

Rice is a major food crop that sustains approximately half of the world population. Recent worldwide improvements in the standard of living have increased the demand for high-quality rice. Accurate identification of quantitative trait loci (QTLs) for rice grain quality traits will facilitate rice quality breeding and improvement. In the present study, we performed high-resolution QTL mapping for rice grain quality traits using a genotyping-by-sequencing approach. An F2 population derived from a cross between an elite japonica variety, Koshihikari, and an indica variety, Nona Bokra, was used to construct a high-density genetic map. A total of 3,830 single nucleotide polymorphism markers were mapped to 12 linkage groups spanning a total length of 2,456.4 cM, with an average genetic distance of 0.82 cM. Seven grain quality traits-the percentage of whole grain, percentage of head rice, percentage of area of head rice, transparency, percentage of chalky rice, percentage of chalkiness area, and degree of chalkiness-of the F2 population were investigated. In total, 15 QTLs with logarithm of the odds (LOD) scores >4 were identified, which mapped to chromosomes 6, 7, and 9. These loci include four QTLs for transparency, four for percentage of chalky rice, four for percentage of chalkiness area, and three for degree of chalkiness, accounting for 0.01%-61.64% of the total phenotypic variation. Of these QTLs, only one overlapped with previously reported QTLs, and the others were novel. By comparing the major QTL regions in the rice genome, several key candidate genes reported to play crucial roles in grain quality traits were identified. These findings will expedite the fine mapping of these QTLs and QTL pyramiding, which will facilitate the genetic improvement of rice grain quality.

Fine mapping and candidate gene analysis of spd6, responsible for small panicle and dwarfness in wild rice (Oryza rufipogon Griff.).

Identification of genes in rice that affect production and quality is necessary for improving the critical global food source. CSSL58, a chromosome segment substitution line (CSSL) containing a chromosome segment of Oryza rufipogon in the genetic background of the indica cultivar Teqing showed significantly smaller panicles, fewer grains per panicle, smaller grains and dwarfness compared with the recurrent parent Teqing. Genetic analysis of the BC(4)F(1) and BC(4)F(2) generations, derived from a cross between CSSL58 and Teqing, showed that these traits are controlled by the recessive gene spd6, which mapped to the short arm of chromosome 6. Fine mapping and high-resolution linkage analysis using 24,120 BC(4)F(3) plants and markers flanking spd6 were carried out, and the gene was localized to a 22.4 kb region that contains four annotated genes according to the genome sequence of japonica Nipponbare. Phenotypic evaluation of the nearly isogenic line NIL(spd6) revealed that spd6 from wild rice has pleiotropic effects on panicle number per plant, grain size, grain weight, grain number per panicle and plant height, suggesting that this gene might play an important role in the domestication of rice. The discovery of spd6 may ultimately be useful for the design and breeding of crops with high grain yield and quality.

Identification of quantitative trait loci for rice quality in a population of chromosome segment substitution lines.

The demand for high quality rice represents a major issue in rice production. The primary components of rice grain quality include appearance, eating, cooking, physico-chemical, milling and nutritional qualities. Most of these traits are complex and controlled by quantitative trait loci (QTLs), so the genetic characterization of these traits is more difficult than that of traits controlled by a single gene. The detection and genetic identification of QTLs can provide insights into the genetic mechanisms underlying quality traits. Chromosome segment substitution lines (CSSLs) are effective tools used in mapping QTLs. In this study, we constructed 154 CSSLs from backcross progeny (BC(3)F(2)) derived from a cross between 'Koshihikari' (an Oryza sativa L. ssp. japonica variety) as the recurrent parent and 'Nona Bokra' (an O. sativa L. ssp. indica variety) as the donor parent. In this process, we carried out marker-assisted selection by using 102 cleaved amplified polymorphic sequence and simple sequence repeat markers covering most of the rice genome. Finally, this set of CSSLs was used to identify QTLs for rice quality traits. Ten QTLs for rice appearance quality traits were detected and eight QTLs concerned physico-chemical traits. These results supply the foundation for further genetic studies and breeding for the improvement of grain quality.

Translational Regulation of Plant Response to High Temperature by a Dual-Function tRNAHis Guanylyltransferase in Rice.

As sessile organisms, plants have evolved numerous strategies to acclimate to changes in environmental temperature. However, the molecular basis of this acclimation remains largely unclear. In this study we identified a tRNAHis guanylyltransferase, AET1, which contributes to the modification of pre-tRNAHis and is required for normal growth under high-temperature conditions in rice. Interestingly, AET1 possibly interacts with both RACK1A and eIF3h in the endoplasmic reticulum. Notably, AET1 can directly bind to OsARF mRNAs including the uORFs of OsARF19 and OsARF23, indicating that AET1 is associated with translation regulation. Furthermore, polysome profiling assays suggest that the translational status remains unaffected in the aet1 mutant, but that the translational efficiency of OsARF19 and OsARF23 is reduced; moreover, OsARF23 protein levels are obviously decreased in the aet1 mutant under high temperature, implying that AET1 regulates auxin signaling in response to high temperature. Our findings provide new insights into the molecular mechanisms whereby AET1 regulates the environmental temperature response in rice by playing a dual role in tRNA modification and translational control.

OsHAL3, a Blue Light-Responsive Protein, Interacts with the Floral Regulator Hd1 to Activate Flowering in Rice.

In flowering plants, photoperiodic flowering is controlled by a complicated network. Light is one of the most important environmental stimuli that control the timing of the transition from vegetative growth to reproductive development. Several photoreceptors, including PHYA, PHYB, CRY2, and FKF1 in Arabidopsis and their homologs (OsPHYA, OsPHYB, OsPHYC, and OsCRY2) in rice, have been identified to be related to flowering. Our previous study suggests that OsHAL3, a flavin mononucleotide-binding protein, may function as a blue-light sensor. Here, we report the identification of OsHAL3 as a positive regulator of flowering in rice. OsHAL3 overexpression lines exhibited an early flowering phenotype, whereas downregulation of OsHAL3 expression by RNA interference delayed flowering under an inductive photoperiod (short-day conditions). The change in flowering time was not accompanied by altered Hd1 expression but rather by reduced accumulation of Hd3a and MADS14 transcripts. OsHAL3 and Hd1 colocalized in the nucleus and physically interacted in vivo under the dark, whereas their interaction was inhibited by white or blue light. Moreover, OsHAL3 directly bound to the promoter of Hd3a, especially before dawn. We conclude that OsHAL3, a novel light-responsive protein, plays an essential role in photoperiodic control of flowering time in rice, which is probably mediated by forming a complex with Hd1. Our findings open up new perspectives on the photoperiodic flowering pathway.

Effects of Fluoride on DNA Damage and Caspase-Mediated Apoptosis in the Liver of Rats.

Fluoride compounds are abundant and widely distributed in the environment at a variety of concentrations. Further, fluoride induces toxic effects in target organs such as the liver. In this study, we investigated liver histopathology, DNA damage, apoptosis, and the mRNA and protein expressions of caspase-3 and -9 in the rat livers by administering varying concentrations of fluoride (0, 50, 100, 200 mg/L ) for 120 days. The results showed fluoride-induced morphological changes and significantly increased apoptosis and DNA damage in rats exposed to fluoride, especially in response to higher doses. The immunohistochemical and qRT-PCR results indicated that caspase-3, caspase-9 protein positive expression and mRNA relative expression enhanced with increasing NaF concentration. In summary, our findings suggest that chronic exposure to fluoride causes damages to liver histopathology and leads to liver apoptosis through caspase-mediated pathways.

Natural alleles of a proteasome α2 subunit gene contribute to thermotolerance and adaptation of African rice.

Global warming threatens many aspects of human life, for example, by reducing crop yields. Breeding heat-tolerant crops using genes conferring thermotolerance is a fundamental way to help deal with this challenge. Here we identify a major quantitative trait locus (QTL) for thermotolerance in African rice (Oryza glaberrima), Thermo-tolerance 1 (TT1), which encodes an α2 subunit of the 26S proteasome involved in the degradation of ubiquitinated proteins. Ubiquitylome analysis indicated that OgTT1 protects cells from heat stress through more efficient elimination of cytotoxic denatured proteins and more effective maintenance of heat-response processes than achieved with OsTT1. Variation in TT1 has been selected for on the basis of climatic temperature and has had an important role in local adaptation during rice evolution. In addition, we found that overexpression of OgTT1 was associated with markedly enhanced thermotolerance in rice, Arabidopsis and Festuca elata. This discovery may lead to an increase in crop security in the face of the ongoing threat of global warming.

Toxic effects of sodium fluoride on cell proliferation and apoptosis of Leydig cells from young mice.

The biological effects of fluoride on human health are often extensive, either beneficial or detrimental. Among the various effects of fluoride exposure in different organs, the reproductive tract is particularly susceptible to disruption by fluoride at a sufficient concentration. It has attracted much attention to the effect of sodium fluoride on male fertility, gestational female, and offspring. Herein, we applied a widespread natural compound sodium fluoride (NaF) and investigated the effects of acute NaF exposure on Leydig cells, including their proliferation, apoptosis, and signal pathway changes. Our results demonstrated that high dosage of NaF could inhibit cell proliferation by stress-induced apoptosis, which was confirmed by cellular and molecular evidences. We found that fluoride exposure affected the expression levels of stress response factors, signal transduction components, and apoptosis-related proteins, including caspase-3/caspase-9, B-cell lymphoma 2 (Bcl-2), and Bax. This study suggests that the complex effects of fluoride on Leydig cells are closely related to its dosage.

Sodium fluoride induces apoptosis in the kidney of rats through caspase-mediated pathways and DNA damage.

Long-term excessive sodium fluoride (NaF) intake can cause many bone diseases and nonskeletal fluorosis. The kidneys are the primary organs involved in the excretion and retention of NaF. The objective of the present study was to determine the effects of NaF treatment on renal cell apoptosis, DNA damage, and the protein expression levels of cytosolic cytochrome C (Cyt C) and cleaved caspases 9, 8, and 3 in vivo. Male Sprague-Dawley rats were divided randomly into four groups (control, low fluoride, medium fluoride, and high fluoride) and administered 0, 50, 100, and 200 mg/L of NaF, respectively, via drinking water for 120 days. Histopathological changes in the kidneys were visualized using hematoxylin and eosin staining. Renal cell apoptosis was examined using flow cytometry, and renal cell DNA damage was detected using the comet assay. Cytosolic Cyt C and cleaved caspases 9, 8, and 3 protein expression levels were visualized using immunohistochemistry and Western blotting. The results showed that NaF treatment increased apoptosis and DNA damage. In addition, NaF treatment increased the protein expression levels of cytosolic Cyt C and cleaved caspases 9, 8, and 3. These results indicated that NaF induces apoptosis in the kidney of rats through caspase-mediated pathway, and DNA damage may be involved in this process.