Advances in the Research on Plant WRKY Transcription Factors Responsive to External Stresses.

The WRKY transcription factors are a class of transcriptional regulators that are ubiquitous in plants, wherein they play key roles in various physiological activities, including responses to stress. Specifically, WRKY transcription factors mediate plant responses to biotic and abiotic stresses through the binding of their conserved domain to the W-box element of the target gene promoter and the subsequent activation or inhibition of transcription (self-regulation or cross-regulation). In this review, the progress in the research on the regulatory effects of WRKY transcription factors on plant responses to external stresses is summarized, with a particular focus on the structural characteristics, classifications, biological functions, effects on plant secondary metabolism, regulatory networks, and other aspects of WRKY transcription factors. Future research and prospects in this field are also proposed.

Advances in Research on the Regulation of Floral Development by CYC-like Genes.

CYCLOIDEA (CYC)-like genes belong to the TCP transcription factor family and play important roles associated with flower development. The CYC-like genes in the CYC1, CYC2, and CYC3 clades resulted from gene duplication events. The CYC2 clade includes the largest number of members that are crucial regulators of floral symmetry. To date, studies on CYC-like genes have mainly focused on plants with actinomorphic and zygomorphic flowers, including Fabaceae, Asteraceae, Scrophulariaceae, and Gesneriaceae species and the effects of CYC-like gene duplication events and diverse spatiotemporal expression patterns on flower development. The CYC-like genes generally affect petal morphological characteristics and stamen development, as well as stem and leaf growth, flower differentiation and development, and branching in most angiosperms. As the relevant research scope has expanded, studies have increasingly focused on the molecular mechanisms regulating CYC-like genes with different functions related to flower development and the phylogenetic relationships among these genes. We summarize the status of research on the CYC-like genes in angiosperms, such as the limited research conducted on CYC1 and CYC3 clade members, the necessity to functionally characterize the CYC-like genes in more plant groups, the need for investigation of the regulatory elements upstream of CYC-like genes, and exploration of the phylogenetic relationships and expression of CYC-like genes with new techniques and methods. This review provides theoretical guidance and ideas for future research on CYC-like genes.

CmHY5 functions in apigenin biosynthesis by regulating flavone synthase II expression in chrysanthemum flowers.

MAIN CONCLUSION: The predominant flavones in the ray florets of chrysanthemum flowers are apigenin and its derivatives. CmHY5 participates in apigenin biosynthesis by directly regulating the expression of FNSII-1 in chrysanthemum. Chrysanthemum (Chrysanthemum morifolium) flowers have been used for centuries as functional food and in herbal tea and traditional medicine. The chrysanthemum flower contains significant amounts of the biologically active compound flavones, which has medicinal properties. However, the mechanism regulating flavones biosynthesis in chrysanthemum flowers organs is still unclear. Here, we compared the transcriptomes and metabolomes of different floral organs between two cultivars with contrasting flavone levels in their flowers. We identified 186 flavonoids by metabolome analysis. The predominant flavones in the ray florets of chrysanthemum flowers are apigenin and its derivatives, of which the contents are highly correlated with the expression of flavones synthase II gene CmFNSII-1. We also determined that CmHY5 is a direct upstream regulator of CmFNSII-1 transcription. We showed that CmHY5 RNAi interference lines in chrysanthemum have lower contents of apigenin compared to wild-type chrysanthemum. Our results demonstrated that CmHY5 participates in flavone biosynthesis by directly regulating the expression of FNSII-1 in chrysanthemum.

CRISPR/Cas9 Technology and Its Utility for Crop Improvement.

The rapid growth of the global population has resulted in a considerable increase in the demand for food crops. However, traditional crop breeding methods will not be able to satisfy the worldwide demand for food in the future. New gene-editing technologies, the most widely used of which is CRISPR/Cas9, may enable the rapid improvement of crop traits. Specifically, CRISPR/Cas9 genome-editing technology involves the use of a guide RNA and a Cas9 protein that can cleave the genome at specific loci. Due to its simplicity and efficiency, the CRISPR/Cas9 system has rapidly become the most widely used tool for editing animal and plant genomes. It is ideal for modifying the traits of many plants, including food crops, and for creating new germplasm materials. In this review, the development of the CRISPR/Cas9 system, the underlying mechanism, and examples of its use for editing genes in important crops are discussed. Furthermore, certain limitations of the CRISPR/Cas9 system and potential solutions are described. This article will provide researchers with important information regarding the use of CRISPR/Cas9 gene-editing technology for crop improvement, plant breeding, and gene functional analyses.

Purification and characterization of ZmRIP1, a novel reductant-inhibited protein tyrosine phosphatase from maize.

Protein tyrosine phosphatases (PTPases) have long been thought to be activated by reductants and deactivated by oxidants, owing to the presence of a crucial sulfhydryl group in their catalytic centers. In this article, we report the purification and characterization of Reductant-Inhibited PTPase1 (ZmRIP1) from maize (Zea mays) coleoptiles, and show that this PTPase has a unique mode of redox regulation and signaling. Surprisingly, ZmRIP1 was found to be deactivated by a reductant. A cysteine (Cys) residue (Cys-181) near the active center was found to regulate this unique mode of redox regulation, as mutation of Cys-181 to arginine-181 allowed ZmRIP1 to be activated by a reductant. In response to oxidant treatment, ZmRIP1 was translocated from the chloroplast to the nucleus. Expression of ZmRIP1 in Arabidopsis (Arabidopsis thaliana) plants and maize protoplasts altered the expression of genes encoding enzymes involved in antioxidant catabolism, such as At1g02950, which encodes a glutathione transferase. Thus, the novel PTPase identified in this study is predicted to function in redox signaling in maize.

Chitosan-pullulan films enriched with Artemisia annua essential oil: Characterization and application in grape preservation.

Composite films were prepared using a flow casting method, with chitosan and pullulan as film-forming agents and Artemisia annua essential oil as the UV absorber. The utility of the composite films for preserving grape berries was assessed. The effect of the added Artemisia annua essential oil on the physicochemical properties of the composite film was investigated to determine the optimal amount of essential oil that should be added to the composite film. When the Artemisia annua essential oil content was 0.8 %, the elongation at break of the composite film increased to 71.25 ± 2.87 % and the water vapor transmission rate decreased to 0.378 ± 0.007 g‧mm/(m2‧h‧kpa). The transmittance of the composite film was almost 0 % in the UV region (200-280 nm) and <30 % in the visible light region (380-800 nm), reflecting the UV absorption by the composite film. Additionally, the composite film extended the storage time of the grape berries. Therefore, the composite film containing Artemisia annua essential oil may be a promising fruit packaging material.

Cold tolerance identification of nine Rosa L. materials and expression patterns of genes related to cold tolerance in Rosa hybrida.

Members of the Rosa genus have a high ornamental value, but their cultivation area is limited by their sensitivity to cold temperatures. The aim of this study was to evaluate the cold tolerance of a range of Rosa materials, and then determine which genes were related to cold tolerance. Nine Rosa materials were subjected to a cold treatment. To identify genes related to cold tolerance, R. hybrida was treated at -15°C for 10 min, and leaves collected before and after this treatment were collected for RNA-Seq analyses. The transcript profiles of four DEGs (POD17, NDUFA9, PMA1, and b-Amy1) in R. hybrida were determined by qRT-PCR at 0 h, 1 h, 2 h, and 3 h at -15°C. Nine Rosa materials were subjected to a cold treatment, and the most cold-tolerant materials were identified as those that showed the lowest levels of electrolyte leakage and the best recovery after 30 d of growth. The most cold-tolerant materials were Rosa hybrida, Rosa rugosa 'Pingyin 12', and Rosa rugosa. In total, 204 significantly differentially expressed genes (DEGs) were identified, of which 88 were significantly up-regulated and 116 were significantly down-regulated under cold conditions. Gene Ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that the DEGs were enriched in 57 pathways, especially starch and sucrose metabolism, phenylpropane biosynthesis, MAPK signaling, fructose and mannose metabolism, and oxidative phosphorylation. By transcriptional analysis, PMA1, which was related to H+ ATPase activity, was continuously up-regulated, but the transcript levels of POD17, NDUFA9, and ß-Amy1 fluctuated during the freezing treatment. This research uncovered scarce cold-resistant materials and layed the foundation for further research on the cold tolerance mechanism of Rosa plants and the breeding of cold-tolerant varieties.

Research advance on cold tolerance in chrysanthemum.

Chrysanthemums are one of the top ten most well-known traditional famous flowers in China and one of the top four cut flowers worldwide, holding a significant position in landscape gardening. The cold temperatures of winter restrict the cultivation, introduction, and application of chrysanthemum, resulting in high costs for year-round production. This severely impacts the ornamental and economic value of chrysanthemum. Therefore, research on cold tolerance is of vital importance for guiding chrysanthemum production and application. With the development of genomics, transcriptomics, metabolomics, and other omics approaches, along with high-throughput molecular marker technologies, research on chrysanthemum cold tolerance has been continuously advancing. This article provides a comprehensive overview of the progress in cold tolerance research from various aspects, including chrysanthemum phenotype, physiological mechanisms, the forward genetics, molecular mechanisms, and breeding. The aim is to offer insights into the mechanisms of cold tolerance in chrysanthemum and provide reference for in-depth research and the development of new cold tolerance chrysanthemum varieties.

Current Achievements and Future Prospects in Virus Elimination Technology for Functional Chrysanthemum.

Chrysanthemum is an important functional plant that is used for food, medicine and tea. Functional chrysanthemums become infected with viruses all around the world, seriously lowering their quality and yield. Viral infection has become an important limiting factor in chrysanthemum production. Functional chrysanthemum is often propagated asexually by cutting during production, and viral infection of seedlings is becoming increasingly serious. Chrysanthemums can be infected by a variety of viruses causing different symptoms. With the development of biotechnology, virus detection and virus-free technologies for chrysanthemum seedlings are becoming increasingly effective. In this study, the common virus species, virus detection methods and virus-free technology of chrysanthemum infection are reviewed to provide a theoretical basis for virus prevention, treatment and elimination in functional chrysanthemum.

Bioinspired Superhydrophobic/Superhydrophilic Janus Copper Foam for On-Demand Oil/Water Separation.

Superwettable Janus membranes with unique interfacial characteristics have versatile applications in oil/water separation, microfluid transportation, and membrane distillation. However, it remains a significant challenge to simply fabricate three-dimensional (3D) metallic foams with Janus superwettability using a facile and environment-friendly method. In this study, a novel method is present to construct a Janus copper foam (CF) by combining superhydrophobicity and superhydrophilicity into CF. Based on gravity, the water in the light oil (LO)/water mixture can be transported from the superhydrophilic (SHL) side to the superhydrophobic (SHB) side, while the heavy oil (HO) in the HO/water/mixture can be transported from the SHB side to the SHL side. Therefore, cylindrical Janus oil/water separation devices with superior separation efficiency and excellent repeatability can achieve on-demand oil/water separation effortlessly. This design and fabrication method offers a novel avenue for the preparation of Janus interface materials for practical applications in liquid transportation, sensor devices, energy materials, and oil spills.

Evaluation of Rosa germplasm resources and analysis of floral fragrance components in R. rugosa.

Rosa rugosa (Rosaceae) is an important functional plant used in food products, tea, and aromatherapy. Characteristics of R. rugosa varieties based on the biological traits and floral fragrant components were studied by applying an analytic hierarchy process, headspace solid-phase microextraction gas chromatography-mass spectrometry, and metabolomic analysis. The 77 Rosa accessions (comprising 27 R. rugosa varieties, 43 scented R. hybrida cultivars, and seven fragrant R. species) were grouped into nine classes based on 17 morphological characters and 16 targeted fragrant substances by cluster analysis. Three R. rugosa cultivars differing in fragrance type were selected for volatile metabolomics analysis at four stages of flower development. In total, 156 differential volatile organic compounds (VOC) were detected and the VOC content patterns were further investigated in two important metabolic pathways (the monoterpenoid biosynthetic pathway, and the phenylalanine, tyrosine, and tryptophan biosynthesis pathway). The results provide a foundation for efficient use of Rosa germplasm and insights into the utilization of R. rugosa as a functional flower.

Gas chromatography-mass spectrometry analysis of floral fragrance-related compounds in scented rose (Rosa hybrida) varieties and a subsequent evaluation on the basis of the analytical hierarchy process.

Scented rose (Rosa hybrida) varieties are valued as ornamentals, but they also contain volatile organic compounds (VOCs) that produce pleasant aromas. In plants, aromas are produced via metabolism during growth, and each aroma compound has a unique function. In this study, the floral aroma compounds of diverse scented rose varieties were analyzed and classified. The VOCs of different rose varieties were qualitatively and quantitatively analyzed via headspace solid-phase microextraction combined with gas chromatography and mass spectrometry. The test materials were the mature flowers of 55 scented rose varieties that were cultivated under identical conditions. Seventeen important aroma compounds were selected and an analytical hierarchy process (AHP)-based method was developed to identify the most suitable essential oil resources, aromatherapy resources, and healthcare resources. A floral fragrance evaluation model was established for the comprehensive evaluation of the scented rose varieties. The 55 varieties were classified into three grades according to their suitability for each use. 'Soeur Emmanuelle', 'Wollerton Old Hall', 'Accademia', and 'Tianmidemeng' were revealed to be suitable essential oil, aromatherapy, and healthcare resources. On the basis of their aroma compound types, the fifty-five rose varieties were divided into eight groups. The results of this study provide the theoretical basis for the classification of rose flower aromas as well as the rational use of diverse rose varieties to further develop the rose industry.

Whole-transcriptome analysis of differentially expressed genes between ray and disc florets and identification of flowering regulatory genes in Chrysanthemum morifolium.

Chrysanthemum morifolium has ornamental and economic values. However, there has been minimal research on the morphology of the chrysanthemum florets and related genes. In this study, we used the leaves as a control to screen for differentially expressed genes between ray and disc florets in chrysanthemum flowers. A total of 8,359 genes were differentially expressed between the ray and disc florets, of which 3,005 were upregulated and 5,354 were downregulated in the disc florets. Important regulatory genes that control flower development and flowering determination were identified. Among them, we identified a TM6 gene (CmTM6-mu) that belongs to the Class B floral homeotic MADS-box transcription factor family, which was specifically expressed in disc florets. We isolated this gene and found it was highly similar to other typical TM6 lineage genes, but a single-base deletion at the 3' end of the open reading frame caused a frame shift that generated a protein in which the TM6-specific paleoAP3 motif was missing at the C terminus. The CmTM6-mu gene was ectopically expressed in Arabidopsis thaliana. Petal and stamen developmental processes were unaffected in transgenic A. thaliana lines; however, the flowering time was earlier than in the wild-type control. Thus, the C-terminal of paleoAP3 appears to be necessary for the functional performance in regulating the development of petals or stamens and CmTM6-mu may be involved in the regulation of flowering time in chrysanthemum. The results of this study will be useful for future research on flowering molecular mechanisms and for the breeding of novel flower types.

Analysis of terpenoids and their gene regulatory networks on the basis of the transcriptome and metabolome of Opisthopappus longilobus.

Opisthopappus longilobus, which is a unique wild plant resource in China, produces leaves and flowers with distinct aromas. However, there have been relatively few molecular studies on its floral aroma, which has hindered the research on this plant species at the molecular level and the breeding of novel varieties. In this study, transcriptome and metabolome analyses were performed using O. longilobus leaves, buds, and inflorescences at the exposure, initial opening, and blooming stages. Using high-quality reads and assembly software, a total of 45,674 unigenes were annotated according to the Nr, Swiss-Prot, KOG, and KEGG databases. Additionally, a GC-MS system and a self-built database were used to detect 1,371 metabolites in the leaves, buds, and inflorescences. Terpene metabolites were the most common compounds (308 in total). We analyzed the gene network regulating terpenoid accumulation in O. longilobus and identified 56 candidate genes related to terpenoid synthesis. The expression of OlPMK2, OlMVK1, OlTPS1, and OlTPS3 may lead to the accumulation of 11 different terpenoids specifically in the inflorescences at the exposure, initial opening, and blooming stages. The generated data may be useful for future research on O. longilobus genetic resources and the molecular mechanism regulating aroma formation in this plant species. The findings of this study may be used to accelerate the breeding of new O. longilobus varieties with enhanced aromatic traits.

Terpenoids and their gene regulatory networks in Opisthopappus taihangensis 'Taihang Mingzhu' as detected by transcriptome and metabolome analyses.

'Taihang Mingzhu' is the hybrid offspring of Opisthopappus taihangensis, and it has an excellent characteristic of whole-plant fragrance. At present, the genes and metabolites involved in the synthesis of its aromatic compounds are unknown because of the paucity of molecular biology studies on flowering in the Opisthopappus Shih genus. To elucidate the biosynthetic pathways of terpenoids, the main aromatic compounds in 'Taihang Mingzhu', we conducted transcriptome and metabolite analyses on its leaves and bud, inflorescences at the color-development, flowering, and full-bloom stages. A total of 82,685 unigenes were obtained, of which 43,901 were annotated on the basis of information at the protein databases Nr, SwissProt, KEGG, and COG/KOG (e-value<0.00001). Using gas headspace solid-phase microextraction chromatography - mass spectrometry (HS-SPME-GC/MS), 1350 metabolites were identified, the most abundant of which were terpenoids (302 metabolites). Analyses of the gene regulatory network of terpenoids in 'Taihang Mingzhu' identified 52 genes potentially involved in the regulation of terpenoid synthesis. The correlations between genes related to terpenoid metabolism/regulation and metabolite abundance were analyzed. We also extracted the essential oil from the leaves of 'Taihang Mingzhu' by hydrodistillation, and obtained 270 aromatic compounds. Again, the most abundant class was terpenoids. These results provide guidance for the extraction of essential oil from 'Taihang Mingzhu' leaves and flowers. In addition, our analyses provide valuable genetic resources to identify genetic targets to manipulate the aromatic profiles of this plant and other members the Opisthopappus Shih genus by molecular breeding.

Effect of inoculation with the endophyte Clavibacter sp. strain Enf12 on chilling tolerance in Chorispora bungeana.

Endophytic bacteria have been shown to increase resistance against biotic stress and tolerance to abiotic stress in many plants. The objective of this study was to evaluate the effect of an endophytic bacterium, Clavibacter sp. strain Enf12, in regenerated plantlets of Chorispora bungeana subjected to chilling stress (0°C). Aerial biomass and physiological markers for chilling stress, such as electrolyte leakage, lipid peroxidation, reactive oxygen species (ROS) accumulation, proline content and activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), guaiacol peroxidase (EC 1.11.1.7) and ascorbate peroxidase (EC 1.11.1.11), were assessed. We demonstrated that Clavibacter sp. strain Enf12 was capable of colonizing internal tissues of regenerated plantlets of C. bungeana and maintained stable population densities under both normal (20°C) and chilling (0°C) conditions. Inoculation enhanced plantlet growth under both conditions and significantly attenuated the chilling-induced electrolyte leakage, lipid peroxidation and ROS accumulation. The endophyte significantly increased the activities of antioxidant enzymes and proline content in C. bungeana plantlets under chilling stress. These findings suggest that Clavibacter sp. strain Enf12 inoculation stimulates the growth of C. bungeana plantlets and improves their tolerance to chilling stress through enhancing the antioxidant defense system.

Transcriptome and Metabolome Analyses of the Flowers and Leaves of Chrysanthemum dichrum.

Chrysanthemum dichrum is an important wild species in the family Asteraceae. However, because of a lack of genetic information, there has been relatively little research conducted on the molecular mechanisms in C. dichrum. There is no report describing the transcriptome and metabolome of C. dichrum flowers and leaves at different developmental stages. In this study, high-throughput sequencing and RNA-seq analyses were used to investigate the transcriptome of C. dichrum leaves, flower buds, and blooming flowers. Additionally, these three tissues also underwent a metabolomics analysis. A total of 447,313,764 clean reads were assembled into 77,683 unigenes, with an average length of 839 bp. Of the 44,204 annotated unigenes, 42,189, 28,531, 23,420, and 17,599 were annotated using the Nr, Swiss-Prot, KOG, and KEGG databases, respectively. Furthermore, 31,848 differentially expressed genes (DEGs) were detected between the leaves and flower buds, whereas 23,197 DEGs were detected between the leaves and blooming flowers, and 11,240 DEGs were detected between the flower buds and blooming flowers. Finally, a quantitative real-time Polymerase Chain Reaction (qRT-PCR) assay was conducted to validate the identified DEGs. The metabolome data revealed several abundant metabolites in C. dichrum leaves, flower buds, and blooming flowers, including raffinose, 1-kestose, asparagine, glutamine, and other medicinal compounds. The expression patterns of significant DEGs revealed by the transcriptome analysis as well as the data for the differentially abundant metabolites in three C. dichrum tissues provide important genetic and metabolic information relevant for future investigations of the molecular mechanisms in C. dichrum. Moreover, the results of this study may be useful for the molecular breeding, development, and application of C. dichrum resources.

Whole-transcriptome analysis of differentially expressed genes in the mutant and normal capitula of Chrysanthemum morifolium.

BACKGROUND: Chrysanthemum morifolium is one of the most economically important and popular floricultural crops in the family Asteraceae. Chrysanthemum flowers vary considerably in terms of colors and shapes. However, the molecular mechanism controlling the development of chrysanthemum floral colors and shapes remains an enigma. We analyzed a cut-flower chrysanthemum variety that produces normal capitula composed of ray florets with normally developed pistils and purple corollas and mutant capitula comprising ray florets with green corollas and vegetative buds instead of pistils. RESULTS: We conducted a whole-transcriptome analysis of the differentially expressed genes (DEGs) in the mutant and normal capitula using third-generation and second-generation sequencing techniques. We identified the DEGs between the mutant and normal capitula to reveal important regulators underlying the differential development. Many transcription factors and genes related to the photoperiod and GA pathways, floral organ identity, and the anthocyanin biosynthesis pathway were differentially expressed between the normal and mutant capitula. A qualitative analysis of the pigments in the florets of normal and mutant capitula indicated anthocyanins were synthesized and accumulated in the florets of normal capitula, but not in the florets of mutant capitula. These results provide clues regarding the molecular basis of the replacement of Chrysanthemum morifolium ray florets with normally developed pistils and purple corollas with mutant ray florets with green corollas and vegetative buds. Additionally, the study findings will help to elucidate the molecular mechanisms underlying floral organ development and contribute to the development of techniques for studying the regulation of flower shape and color, which may enhance chrysanthemum breeding. CONCLUSIONS: The whole-transcriptome analysis of DEGs in mutant and normal C. morifolium capitula described herein indicates the anthocyanin deficiency of the mutant capitula may be related to the mutation that replaces ray floret pistils with vegetative buds. Moreover, pistils may be required for the anthocyanin biosynthesis in the corollas of chrysanthemum ray florets.

Cold-inducible expression of an Arabidopsis thaliana AP2 transcription factor gene, AtCRAP2, promotes flowering under unsuitable low-temperatures in chrysanthemum.

Flowering time is regulated by biotic and abiotic stresses and affected by the ambient temperature. For chrysanthemum, a low ambient growth temperature can cause a flowering delay, which limits the annual commercial production. Therefore, it is important to improve the low-temperature flowering capability of chrysanthemum through genetic modifications. Here, we isolated a natural variation of a CRT/DRE-binding factor (CBF/DREB) 3 gene, CRAP2, from the Arabidopsis thaliana accession Condara (190AV) that encodes a stop codon at position 151 of the CBF3 protein. Unlike AtCBF3, the overexpression AtCRAP2 in Arabidopsis did not cause detectable growth retardation nor delayed flowering and it conferred cold tolerance. The cold-inducible expression of AtCRAP2 in chrysanthemum promoted flowering under short-day conditions with a low 15 °C nighttime temperature. RNA-sequencing of rd29A:AtCRAP2 and qRT-PCR assays of flowering time-related genes and AtCRAP2 expressed at an ambient temperature revealed that AtCRAP2 positively affected SOC1 and FTL3, thereby promoting flowering under low temperature stress and short-day conditions. These results indicate that DREB genes can be used in the genetic engineering of crop plants without accompanying negative effects by modifying the encoded proteins' C termini.

Nucleotide variation in the NCED3 region of Arabidopsis thaliana and its association study with abscisic acid content under drought stress.

Drought tolerance is a comprehensive quantitative trait that is being understood further at the molecular genetic level. Abscisic acid (ABA) is the main drought-induced hormone that regulates the expression of many genes related to drought responses. 9-cis-epoxycarotenoid dioxygenase (NCED3) is thought to be a key enzyme in ABA biosynthesis. In this paper, we measured the ABA content increase under drought stress, and sequenced and compared the sequence of AtNCED3 among 22 Arabidopsis thaliana accessions. The results showed that the fold of ABA content increase under drought stress was highly variable among these accessions. High density single nucleotide polymorphism (SNP) and insertion/deletion (indel) were found in the AtNCED3 region, on average one SNP per 87.4 bp and one indel per 502 bp. Nucleotide diversity was significantly lower in the coding region than that in non-coding regions. The results of an association study with anova analysis suggested that the 274th site (P<-->S) and the 327th site (P<-->R) amino acid variations might be the cause of ABA content increase of 163av accession under drought stress.