Regulation of carbohydrate metabolism during anther development in a thermo-sensitive genic male-sterile wheat line.

Bainong sterility (BNS) is a thermo-sensitive genic male sterile wheat line, characterised by anther fertility transformation in response to low temperature (LT) stress during meiosis, the failure of vacuole decomposition and the absence of starch accumulation in sterile bicellular pollen. Our study demonstrates that the late microspore (LM) stage marks the transition from the anther growth to anther maturation phase, characterised by the changes in anther structure, carbohydrate metabolism and the main transport pathway of sucrose (Suc). Fructan is a main storage polysaccharide in wheat anther, and its synthesis and remobilisation are crucial for anther development. Moreover, the process of pollen amylogenesis and the fate of the large vacuole in pollen are closely intertwined with fructan synthesis and remobilisation. LT disrupts the normal physiological metabolism of BNS anthers during meiosis, particularly affecting carbohydrate metabolism, thus determining the fate of male gametophytes and pollen abortion. Disruption of fructan synthesis and remobilisation regulation serves as a decisive event that results in anther abortion. Sterile pollen exhibits common traits of pollen starvation and impaired starch accumulation due to the inhibition of apoplastic transport starting from the LM stage, which is regulated by cell wall invertase TaIVR1 and Suc transporter TaSUT1.

GhBEE3-Like gene regulated by brassinosteroids is involved in cotton drought tolerance.

Brassinosteroids (BRs) are important phytohormones that play a vital role in plant drought tolerance, but their mechanisms in cotton (Gossypium hirsutum L.) are poorly understood. Numerous basic helix-loop-helix (bHLH) family genes are involved in the responses to both BRs and drought stress. GhBEE3-Like, a bHLH transcription factor, is repressed by both 24-epi-BL (an active BR substance) and PEG8000 (drought simulation) treatments in cotton. Moreover, GhBZR1, a crucial transcription factor in BR signaling pathway, directly binds to the E-box element in GhBEE3-Like promoter region and inhibits its expression, which has been confirmed by electrophoretic mobility shift assay (EMSA) and dual luciferase reporter assay. Functional analysis revealed that Arabidopsis with GhBEE3-Like overexpression had drought sensitive phenotype, while GhBEE3-Like knock-down cotton plants obtained by virus-induced gene silencing (VIGS) technology were more tolerant to drought stress. Furthermore, the expression levels of three stress-related genes, GhERD10, GhCDPK1 and GhRD26, were significantly higher in GhBEE3-Like knock-down cotton than in control cotton after drought treatment. These results suggest that GhBEE3-Like is inhibited by BRs which elevates the expressions of stress-related genes to enhance plant drought tolerance. This study lays the foundation for understanding the mechanisms of BR-regulated drought tolerance and establishment of drought-resistant cotton lines.

OsABT Is Involved in Abscisic Acid Signaling Pathway and Salt Tolerance of Roots at the Rice Seedling Stage.

Rice is a staple cereal crop worldwide, and increasing its yields is vital to ensuring global food security. Salinity is a major factor that affects rice yield. Therefore, it is necessary to investigate salt tolerance mechanisms in rice. Proteins containing WD40 repeats play important roles in eukaryotic development and environmental adaptation. Here, we showed that overexpression of OsABT, a gene encoding a WD40-repeat protein, enhanced salt tolerance in rice seedlings by regulating root activity, relative conductivity, malondialdehyde and H2O2 content, and O2•- production rate. Root ion concentrations indicated that OsABT overexpression lines could maintain lower Na+ and higher K+/Na+ ratios and upregulated expression of salt-related genes OsSOS1 and OsHAK5 compared with the wild-type (WT) Nipponbare plants. Furthermore, Overexpression of OsABT decreased the abscisic acid (ABA) content, while downregulating the ABA synthesis genes OsNCED3 and OsNCED4 and upregulating the ABA catabolic gene OsABA8ox2. The yeast two-hybrid and bimolecular fluorescence complementation analyses showed that OsABT interacted with the ABA receptor proteins OsPYL4, OsPYL10, and PP2C phosphatase OsABIL2. A transcriptome analysis revealed that the differentially expressed genes between OsABT overexpression lines and WT plants were enriched in plant hormone signal transduction, including ABA signaling pathway under salt stress. Thus, OsABT can improve the salt tolerance in rice seedling roots by inhibiting reactive oxygen species accumulation, thereby regulating the intracellular Na+/K+ balance, ABA content, and ABA signaling pathway.

A rapid, simple, and highly efficient method for VIGS and in vitro-inoculation of plant virus by INABS applied to crops that develop axillary buds and can survive from cuttings.

BACKGROUND: Virus-induced gene silencing (VIGS) is one of the most convenient and powerful methods of reverse genetics. In vitro-inoculation of plant virus is an important method for studying the interactions between viruses and plants. Agrobacterium-based infiltration has been widely adopted as a tool for VIGS and in vitro-inoculation of plant virus. Most agrobacterium-based infiltration methods applied to VIGS and virus inoculation have the characteristics of low transformation efficiencies, long plant growth time, large amounts of plant tissue, large test spaces, and complex preparation procedures. Therefore, a rapid, simple, economical, and highly efficient VIGS and virus inoculation method is in need. Previous studies have shown that the selection of suitable plant tissues and inoculation sites is the key to successful infection. RESULTS: In this study, Tobacco rattle virus (TRV) mediated VIGS and Tomato yellow leaf curl virus (TYLCV) for virus inoculation were developed in tomato plants based on the agrobacterium tumefaciens-based infiltration by injection of the no-apical-bud stem section (INABS). The no-apical-bud stem section had a "Y- type" asymmetric structure and contained an axillary bud that was about 1-3 cm in length. This protocol provides high transformation (56.7%) and inoculation efficiency (68.3%), which generates VIGS transformants or diseased plants in a very short period (8 dpi). Moreover, it greatly reduces the required experimental space. This method will facilitate functional genomic studies and large-scale disease resistance screening. CONCLUSIONS: Overall, a rapid, simple, and highly efficient method for VIGS and virus inoculation by INABS was developed in tomato. It was reasonable to believe that it can be used as a reference for the other virus inoculation methods and for the application of VIGS to other crops (such as sweet potato, potato, cassava and tobacco) that develop axillary buds and can survive from cuttings.

A Ricin B-Like Lectin Protein Physically Interacts with TaPFT and Is Involved in Resistance to Fusarium Head Blight in Wheat.

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, has become one of the most serious diseases that damage wheat. The TaPFT (pore-forming toxin-like) and TaHRC (histidine-rich calcium-binding protein) genes at the quantitative trait locus Fhb1 were identified to confer resistance to FHB in the wheat cultivar Sumai 3. In this study, a wheat ricin B-like lectin gene (designated TaRBL) that interacted with TaPFT was isolated by a yeast two-hybrid screen of a wheat cDNA library. A yeast two-hybrid and bimolecular fluorescence complementation study further verified that TaRBL interacted with TaPFT but not with TaHRC. Gene expression studies showed that upon F. graminearum infection, TaRBL expression was upregulated in resistant cultivars but downregulated in susceptible cultivars. Furthermore, knockdown of TaRBL expression by barley stripe mosaic virus-induced gene silencing significantly reduced the resistance of wheat to FHB in both the resistant cultivar Sumai 3 and the susceptible cultivar Jimai 22. Thus, we conclude that TaRBL encodes a ricin B-like lectin protein that interacts with TaPFT and is involved in resistance to FHB in wheat.

Biological Characteristics and Molecular Mechanism of Fludioxonil Resistance in Botrytis cinerea From Henan Province of China.

The gray mold caused by Botrytis cinerea has a significant impact on tomato production throughout the world. Although the synthetic fungicide fludioxonil can effectively control B. cinerea, there have been several reports of resistance to this fungicide. This study indicated that all of the fludioxonil-resistant strains tested, including one field-resistant isolate and four laboratory strains, had reduced fitness relative to sensitive isolates. In addition to having reduced growth, sporulation, and pathogenicity, the resistant strains were more sensitive to osmotic stress and had significantly (P < 0.05) higher peroxidase activity. BOs1, a kinase in the high-osmolarity glycerol stress response signal transduction pathway, is believed to harbor mutations related to fludioxonil resistance. Sequence analysis of their BOs1 sequences indicated that the fludioxonil-resistant field isolate, XXtom1806, had four point mutations resulting in four amino acid changes (I365S, S531G, T565N, and T1267A) and three amino acids (I365S, S531G, and T565N) in the histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis receptors, and phosphatases domain, which associated with fludioxonil binding. Similarly, two of the laboratory strains, XXtom-Lab1 and XXtom-Lab4, had three (Q846S, I1126S, and G415D) and two (P1051S and V1241M) point mutations, respectively. A third strain, XXtom-lab3, had a 52-bp insertion that included a stop codon at amino acid 256. Interestingly, the BOs1 sequence of the fourth laboratory strain, XXtom-lab5, was identical to those of the sensitive isolates, indicating that an alternative resistance mechanism exists. The study also found evidence of positive cross-resistance between fludioxonil and the dicarboximide fungicides procymidone and iprodione, but no cross-resistance was detected with any other fungicides tested, including boscalid, carbendazim, tebuconazole, and fluazinam.

BR deficiency causes increased sensitivity to drought and yield penalty in cotton.

BACKGROUND: Brassinosteroids (BRs) play crucial roles in drought tolerance, but the underlying molecular mechanisms remain unclear in the important oilseed and fiber crop, cotton (Gossypium hirsutum L.). RESULTS: To elucidate how BRs mediate drought tolerance in cotton, a cotton brassinosteroid (BR)-deficient mutant, pag1 (pagoda1), was employed for analysis. Importantly, the pag1 mutant showed increased sensitivity to drought stress, with shorter primary roots and fewer lateral roots. The number of stomata was significantly increased in the mutant, and the stomata aperture was much wider than that of the control plants. These mutant plants therefore showed an increased water loss rate. Furthermore, the abscisic acid (ABA) content, photosynthetic efficiency and starch content of the mutant were significantly lower than those of the wild type. The overall performance of the mutant plants was worse than that of the wild-type control under both normal and drought conditions. Moreover, Proteomic analysis revealed reduced levels of stress-related proteins in pag1 plants. CONCLUSIONS: These results suggest that BRs may modulate the drought tolerance of cotton by regulating much genes that related to drought stress and multiple organ responses to drought, including root growth, stomata development, the stomata aperture and photosynthesis. This study provides an important basis for understanding drought resistance regulated by BRs and cultivating drought-resistant cotton lines.

Overexpressed BRH1, a RING finger gene, alters rosette leaf shape in Arabidopsis thaliana.

Leaves are the most important plant parts for photosynthesis and respiration. Many genes are involved in determining leaf shape; however, little is known about the effects of brassinosteroid (BR) signaling-pathway genes on the development of leaf shape. Here, the brassinosteroid-responsive RING-H2 (BRH1) gene, which is suppressed by 24-epi-brassinolide treatment, was isolated from Arabidopsis thaliana. The amino acid sequence contained a highly conserved RING finger domain. In a phylogenetic analysis, BRH1 clustered closely with GLYMA11G02470.1. The leaves of brh1 mutant plants were not much different to those of the wild-type, while transgenic plants with high BRH1 expression levels had rounder rosette leaves. Mutants of the BR synthesis pathway also had a similar round leaf phenotype, and greater BRH1 expression levels. Moreover, the related marker genes KNAT1, AtHB13 and ROT4, which are known to control leaf shape, altered transcriptional levels in both transgenic BRH1 and BR-synthesis mutant lines. Thus, BRH1 may be involved in the BR signaling pathway and regulate the growth and development of rosette leaves. Research on BRH1 may prove valuable for understanding the regulatory mechanism of leaf shape and improving the leaf shapes of ornamental plants.

A Phi-Class Glutathione S-Transferase Gene for Verticillium Wilt Resistance in Gossypium arboreum Identified in a Genome-Wide Association Study.

Verticillium wilt disease is one of the most destructive biotic stresses faced by cotton plants. Here, we performed a genome-wide association study (GWAS) in 215 Chinese Gossypium arboreum accessions inoculated as seedlings with Verticillium dahliae to identify candidate loci involved in wilt resistance. We identified 309 loci that had a significant association with Verticillium wilt resistance and - log(P) values >5.0; the highest signal appeared on Ca3 in a 74 kb haplotype block. Five genes were also located within this haplotype block. One of these genes, CG05, was positioned close to the most significant SNP Ca3_23037225 (14 kb); expression of the gene was induced by V. dahliae or by treatment with salicylic acid (SA). Therefore, we suggest that CG05 may respond to invasion by V. dahliae via an SA-related signaling pathway, and we designated this gene as GaGSTF9. We showed that GaGSTF9 was a positive regulator of Verticillium wilt through the use of virus-induced gene silencing (VIGS) and overexpression in Arabidopsis. In addition, the glutathione S-transferase (GST) mutant gstf9 of Arabidopsis was found to be more susceptible to Verticillium wilt than wild-type plants. The levels of endogenous SA and hydrogen peroxide had a significant effect on Arabidopsis plants that overexpressed GaGSTF9, indicating that GST may regulate reactive oxygen species content via catalytic reduction of the tripeptide glutathione (GSH), and then affect SA content. Our data demonstrated that GaGSTF9 was a key regulator mediating cotton responses to V. dahliae and a potential candidate gene for cotton genetic improvement.

GaMYB85, an R2R3 MYB gene, in transgenic Arabidopsis plays an important role in drought tolerance.

BACKGROUND: MYB transcription factors (TFs) are one of the largest families of TFs in higher plants and are involved in diverse biological, functional, and structural processes. Previously, very few functional validation studies on R2R3 MYB have been conducted in cotton in response to abiotic stresses. In the current study, GaMYB85, a cotton R2R3 MYB TF, was ectopically expressed in Arabidopsis thaliana (Col-0) and was functionally characterized by overexpression in transgenic plants. RESULTS: The in-silico analysis of GaMYB85 shows the presence of a SANT domain with a conserved R2R3 MYB imperfect repeat. The GaMYB85 protein has a 257-amino acid sequence, a molecular weight of 24.91 kD, and an isoelectric point of 5.58. Arabidopsis plants overexpressing GaMYB85 exhibited a higher seed germination rate in response to mannitol and salt stress, and higher drought avoidance efficiency than wild-type plants upon water deprivation. These plants had notably higher levels of free proline and chlorophyll with subsequent lower water loss rates and higher relative water content. Germination of GaMYB85 transgenics was more sensitive to abscisic acid (ABA) and extremely liable to ABA-induced inhibition of primary root elongation. Moreover, when subjected to treatment with different concentrations of ABA, transgenic plants with ectopically expressed GaMYB85 showed reduced stomatal density, with greater stomatal size and lower stomatal opening rates than those in wild-type plants. Ectopic expression of GaMYB85 led to enhanced transcript levels of stress-related marker genes such as RD22, ADH1, RD29A, P5CS, and ABI5. CONCLUSIONS: Our results indicate previously unknown roles of GaMYB85, showing that it confers good drought, salt, and freezing tolerance, most probably via an ABA-induced pathway. These findings can potentially be exploited to develop improved abiotic stress tolerance in cotton plants.

Genome-wide identification and characterization of SnRK2 gene family in cotton (Gossypium hirsutum L.).

BACKGROUND: Sucrose non-fermenting-1-related protein kinase 2 (SnRK2) is a plant-specific serine/threonine kinase family involved in the abscisic acid (ABA) signaling pathway and responds to osmotic stress. A genome-wide analysis of this protein family has been conducted previously in some plant species, but little is known about SnRK2 genes in upland cotton (Gossypium hirsutum L.). The recent release of the G. hirsutum genome sequence provides an opportunity to identify and characterize the SnRK2 kinase family in upland cotton. RESULTS: We identified 20 putative SnRK2 sequences in the G. hirsutum genome, designated as GhSnRK2.1 to GhSnRK2.20. All of the sequences encoded hydrophilic proteins. Phylogenetic analysis showed that the GhSnRK2 genes were classifiable into three groups. The chromosomal location and phylogenetic analysis of the cotton SnRK2 genes indicated that segmental duplication likely contributed to the diversification and evolution of the genes. The gene structure and motif composition of the cotton SnRK2 genes were analyzed. Nine exons were conserved in length among all members of the GhSnRK2 family. Although the C-terminus was divergent, seven conserved motifs were present. All GhSnRK2s genes showed expression patterns under abiotic stress based on transcriptome data. The expression profiles of five selected genes were verified in various tissues by quantitative real-time RT-PCR (qRT-PCR). Transcript levels of some family members were up-regulated in response to drought, salinity or ABA treatments, consistent with potential roles in response to abiotic stress. CONCLUSIONS: This study is the first comprehensive analysis of SnRK2 genes in upland cotton. Our results provide the fundamental information for the functional dissection of GhSnRK2s and vital availability for the improvement of plant stress tolerance using GhSnRK2s.

A novel GhBEE1-Like gene of cotton causes anther indehiscence in transgenic Arabidopsis under uncontrolled transcription level.

Male-sterile lines are very important for selective breeding, and anther dehiscence defect is an effective way to generate male-sterile lines. Although several bHLH-family proteins in Arabidopsis have been characterized, little is known about the role of bHLH-family proteins in cotton. Here, we isolated a novel bHLH protein from cotton (Gossypium hirsutum), named GhBEE1-Like. Protein domain analysis showed that GhBEE1-Like contained a basic domain and an HLH domain. Subcellular localization analysis revealed that GhBEE1-Like was a nuclear-localized protein. Expression pattern analysis showed GhBEE1-Like was highly expressed in floral organs, and its expression was induced by the active brassinosteroid (BR) substance 24-epi-BL. GhBEE1-Like overexpression in Arabidopsis resulted in two types of transgenic lines, one with normal anther dehiscence and the other with defective anther dehiscence. Semi-qRT-PCR and qRT-PCR analyses revealed that GhBEE1-Like transcript levels acted as a check-point determining how anther dehiscence proceeds in these transgenic lines; regulated transcript levels result in normal anther dehiscence, whereas uncontrolled transcript levels lead to anther indehiscence. These results suggest that GhBEE1-Like plays an important role via its accumulation in regulating anther dehiscence. Therefore, controlling the level of GhBEE1-Like expression in cotton could be a convenient tool for generating male-sterile lines to use in selective breeding.

Salicylic acid-related cotton (Gossypium arboreum) ribosomal protein GaRPL18 contributes to resistance to Verticillium dahliae.

BACKGROUND: Verticillium dahliae is a phytopathogenic fungal pathogen that causes vascular wilt diseases responsible for considerable decreases in cotton yields. The complex mechanism underlying cotton resistance to Verticillium wilt remains uncharacterized. Identifying an endogenous resistance gene may be useful for controlling this disease. RESULTS: We cloned the ribosomal protein L18 (GaRPL18) gene, which mediates resistance to Verticillium wilt, from a wilt-resistant cotton species (Gossypium arboreum). We then characterized the function of this gene in cotton and Arabidopsis thaliana plants. GaRPL18 encodes a 60S ribosomal protein subunit important for intracellular protein biosynthesis. However, previous studies revealed that some ribosomal proteins are also inhibitory toward oncogenesis and congenital diseases in humans and play a role in plant disease defense. Here, we observed that V. dahliae infections induce GaRPL18 expression. Furthermore, we determined that the GaRPL18 expression pattern is consistent with the disease resistance level of different cotton varieties. GaRPL18 expression is upregulated by salicylic acid (SA) treatments, suggesting the involvement of GaRPL18 in the SA signal transduction pathway. Virus-induced gene silencing technology was used to determine whether the GaRPL18 expression level influences cotton disease resistance. Wilt-resistant cotton species in which GaRPL18 was silenced became more susceptible to V. dahliae than the control plants because of a significant decrease in the abundance of immune-related molecules. We also transformed A. thaliana ecotype Columbia (Col-0) plants with GaRPL18 according to the floral dip method. The plants overexpressing GaRPL18 were more resistant to V. dahliae infections than the wild-type Col-0 plants. The enhanced resistance of transgenic A. thaliana plants to V. dahliae is likely mediated by the SA pathway. CONCLUSION: Our findings provide new insights into the role of GaRPL18, indicating that it plays a crucial role in resistance to cotton "cancer", also known as Verticillium wilt, mainly regulated by an SA-related signaling pathway mechanism.

Genome-wide analysis of the HD-ZIP IV transcription factor family in Gossypium arboreum and GaHDG11 involved in osmotic tolerance in transgenic Arabidopsis.

HD-ZIP IV proteins belong to the homeodomain-leucine zipper (HD-ZIP) transcription factor family and are involved in trichome development and drought stress in plants. Although some functions of the HD-ZIP IV group are well understood in Arabidopsis, little is known about their function in cotton. In this study, HD-ZIP genes were identified from three Gossypium species (G. arboreum, G. raimondii and G. hirsutum) and clustered into four families (HD-ZIP I, II, III and IV) to separate HD-ZIP IV from the other three families. Systematic analyses of phylogeny, gene structure, conserved domains, and expression profiles in different plant tissues and the expression patterns under osmotic stress in leaves were further conducted in G. arboreum. More importantly, ectopic overexpression of GaHDG11, a representative of the HD-ZIP IV family, confers enhanced osmotic tolerance in transgenic Arabidopsis plants, possibly due to elongated primary root length, lower water loss rates, high osmoprotectant proline levels, significant levels of antioxidants CAT, and/or SOD enzyme activity with reduced levels of MDA. Taken together, these observations may lay the foundation for future functional analysis of cotton HD-ZIP IV genes to unravel their biological roles in cotton.

Functional Characterization of Cotton GaMYB62L, a Novel R2R3 TF in Transgenic Arabidopsis.

Drought stress can trigger the production of ABA in plants, in response to adverse conditions, which induces the transcript of stress-related marker genes. The R2R3 MYB TFs are implicated in regulation of various plants developmental, metabolic and multiple environmental stress responses. Here, a R2R3-MYB cloned gene, GaMYB62L, was transformed in Arabidopsis and was functionally characterized. The GaMYB62L protein contains two SANT domains with a conserved R2R3 imperfect repeats. The GaMYB62L cDNA is 1,017 bp with a CDS of 879, encodes a 292-residue polypeptide with MW of 38.78 kD and a pI value of 8.91. Overexpressed GaMYB62L transgenic Arabidopsis have increased proline and chlorophyll content, superior seed germination rate under salt and osmotic stress, less water loss rate with reduced stomatal apertures, high drought avoidance as compared to WT on water deprivation and also significant plant survival rates at low temperature. In addition, overexpressed GaMYB62L lines were more sensitive to ABA mediated germination and root elongation assay. Moreover, ABA induced GaMYB62L overexpression, enhanced the expression of ABA stress related marker genes like RD22, COR15A, ADH1, and RD29A. Together, overexpression of GaMYB62L suggested having developed better drought, salt and cold tolerance in transgenic Arabidopsis and thus presented it as a prospective candidate gene to achieve better abiotic stress tolerance in cotton crop.

Genome-wide identification and functional analysis of the TIFY gene family in response to drought in cotton.

Jasmonates control many aspects of plant biological processes. They are important for regulating plant responses to various biotic and abiotic stresses, including drought, which is one of the most serious threats to sustainable agricultural production. However, little is known regarding how jasmonate ZIM-domain (JAZ) proteins mediate jasmonic acid signals to improve stress tolerance in cotton. This represents the first comprehensive comparative study of TIFY transcription factors in both diploid A, D and tetraploid AD cotton species. In this study, we identified 21 TIFY family members in the genome of Gossypium arboretum, 28 members from Gossypium raimondii and 50 TIFY genes in Gossypium hirsutum. The phylogenetic analyses indicated the TIFY gene family could be divided into the following four subfamilies: TIFY, PPD, ZML, and JAZ subfamilies. The cotton TIFY genes have expanded through tandem duplications and segmental duplications compared with other plant species. Gene expression profile revealed temporal and tissue specificities for TIFY genes under simulated drought conditions in Gossypium arboretum. The JAZ subfamily members were the most highly expressed genes, suggesting that they have a vital role in responses to drought stress. Over-expression of GaJAZ5 gene decreased water loss, stomatal openings, and the accumulation of H2O2 in Arabidopsis thaliana. Additionally, the results of drought tolerance assays suggested that this subfamily might be involved in increasing drought tolerance. Our study provides new data regarding the genome-wide analysis of TIFY gene families and their important roles in drought tolerance in cotton species. These data may form the basis of future studies regarding the relationship between drought and jasmonic acid.

Genome-wide analysis of microRNAs and their target genes related to leaf senescence of rice.

Grain production of rice (Oryza sativa L.) is a top priority in ensuring food security for human beings. One of the approaches to increase yield is to delay leaf senescence and to extend the available time for photosynthesis. MicroRNAs (miRNAs) are key regulators of aging and cellular senescence in eukaryotes. Here, to help understand their biological role in rice leaf senescence, we report identification of miRNAs and their putative target genes by deep sequencing of six small RNA libraries, six RNA-seq libraries and two degradome libraries from the leaves of two super hybrid rice, Nei-2-You 6 (N2Y6, age-resistant rice) and Liang-You-Pei 9 (LYP9, age-sensitive rice). In total 372 known miRNAs, 162 miRNA candidates and 1145 targets were identified. Compared with the expression of miRNAs in the leaves of LYP9, the numbers of miRNAs up-regulated and down-regulated in the leaves of N2Y6 were 47 and 30 at early stage of grain-filling, 21 and 17 at the middle stage, and 11 and 37 at the late stage, respectively. Six miRNA families, osa-miR159, osa-miR160 osa-miR164, osa-miR167, osa-miR172 and osa-miR1848, targeting the genes encoding APETALA2 (AP2), zinc finger proteins, salicylic acid-induced protein 19 (SIP19), auxin response factors (ARF) and NAC transcription factors, respectively, were found to be involved in leaf senescence through phytohormone signaling pathways. These results provided valuable information for understanding the miRNA-mediated leaf senescence of rice, and offered an important foundation for rice breeding.