Verification of DNA motifs in Arabidopsis using CRISPR/Cas9-mediated mutagenesis.

Transcription factors (TFs) and chromatin-modifying factors (CMFs) access chromatin by recognizing specific DNA motifs in their target genes. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) has been widely used to discover the potential DNA-binding motifs for both TFs and CMFs. Yet, an in vivo method for verifying DNA motifs captured by ChIP-seq is lacking in plants. Here, we describe the use of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) to verify DNA motifs in their native genomic context in Arabidopsis. Using a single-guide RNA (sgRNA) targeting the DNA motif bound by REF6, a DNA sequence-specific H3K27 demethylase in plants, we generated stable transgenic plants where the motif was disrupted in a REF6 target gene. We also deleted a cluster of multiple motifs from another REF6 target gene using a pair of sgRNAs, targeting upstream and downstream regions of the cluster, respectively. We demonstrated that endogenous genes with motifs disrupted and/or deleted become inaccessible to REF6. This strategy should be widely applicable for in vivo verification of DNA motifs identified by ChIP-seq in plants

Proteomic and functional analyses of Nelumbo nucifera annexins involved in seed thermotolerance and germination vigor

Annexins are multifunctional proteins characterized by their capacity to bind calcium ions and negatively charged lipids. Although there is increasing evidence implicating their importance in plant stress responses, their functions in seeds remain to be further studied. In this study, we identified a heat-induced annexin, NnANN1, from the embryonic axes of sacred lotus (Nelumbo nucifera Gaertn.) using comparative proteomics approach. Moreover, the expression of NnANN1 increased considerably in response to high-temperature treatment. Quantitative real-time PCR (qRT-PCR) revealed that the transcripts of NnANN1 were detected predominantly during seed development and germination in sacred lotus, implicating a role for NnANN1 in plant seeds. Ectopic expression of NnANN1 in Arabidopsis resulted in enhanced tolerance to heat stress in transgenic seeds. In addition, compared to the wild-type seeds, transgenic seeds ectopically expressing NnANN1 exhibited improved resistance to accelerated aging treatment used for assessing seed vigor. Furthermore, transgenic seeds showed enhanced peroxidase activities, accompanied with reduced lipid peroxidation and reduced ROS release levels compared to the wild-type seeds. Taken together, these results indicate that NnANN1 plays an important role in seed thermotolerance and germination vigor.Peer reviewed: YesNRC publication: Ye

NnHSP17.5, a cytosolic class II small heat shock protein gene from Nelumbo nucifera, contributes to seed germination vigor and seedling thermotolerance in transgenic Arabidopsis

In plants, small heat shock proteins (sHSPs) are unusually abundant and diverse proteins involved in various abiotic stresses, but their functions in seed vigor remain to be fully explored. In this study, we report the isolation and functional characterization of a sHSP gene, NnHSP17.5, from sacred lotus (Nelumbo nucifera Gaertn.) in seed germination vigor and seedling thermotolerance. Sequence alignment and phylogenetic analysis indicate that NnHSP17.5 is a cytosolic class II sHSP, which was further supported by the cytosolic localization of the NnHSP17.5-YFP fusion protein. NnHSP17.5 was specifically expressed in seeds under normal conditions, and was strongly up-regulated in germinating seeds upon heat and oxidative stresses. Transgenic Arabidopsis seeds ectopically expressing NnHSP17.5 displayed enhanced seed germination vigor and exhibited increased superoxide dismutase activity after accelerated aging treatment. In addition, improved basal thermotolerance was also observed in the transgenic seedlings. Taken together, this work highlights the importance of a plant cytosolic class II sHSP both in seed germination vigor and seedling thermotolerance.Peer reviewed: YesNRC publication: Ye

Overexpression of AtOGG1, a DNA glycosylase/AP lyase, enhances seed longevity and abiotic stress tolerance in Arabidopsis

Reactive oxygen species (ROS) are toxic by-products generated continuously during seed desiccation, storage, and germination, resulting in seed deterioration and therefore decreased seed longevity. The toxicity of ROS is due to their indiscriminate reactivity with almost any constituent of the cell, such as lipids, proteins, and DNA. The damage to the genome induced by ROS has been recognized as an important cause of seed deterioration. A prominent DNA lesion induced by ROS is 7,8-dihydro-8-oxoguanine (8-oxo-G), which can form base pairs with adenine instead of cytosine during DNA replication and leads to GC\u2192TA transversions. In Arabidopsis, AtOGG1 is a DNA glycosylase/apurinic/apyrimidinic (AP) lyase that is involved in base excision repair for eliminating 8-oxo-G from DNA. In this study, the functions of AtOGG1 were elaborated. The transcript of AtOGG1 was detected in seeds, and it was strongly up-regulated during seed desiccation and imbibition. Analysis of transformed Arabidopsis protoplasts demonstrated that AtOGG1\u2013yellow fluorescent protein fusion protein localized to the nucleus. Overexpression of AtOGG1 in Arabidopsis enhanced seed resistance to controlled deterioration treatment. In addition, the content of 8-hydroxy-2\u2032-deoxyguanosine (8-oxo-dG) in transgenic seeds was reduced compared to wild-type seeds, indicating a DNA damage-repair function of AtOGG1 in vivo. Furthermore, transgenic seeds exhibited increased germination ability under abiotic stresses such as methyl viologen, NaCl, mannitol, and high temperatures. Taken together, our results demonstrated that overexpression of AtOGG1 in Arabidopsis enhances seed longevity and abiotic stress tolerance.Peer reviewed: YesNRC publication: Ye

Overexpression of Nelumbo nucifera metallothioneins 2a and 3 enhances seed germination vigor in Arabidopsis

Metallothioneins (MTs) are small, cysteine-rich and metal-binding proteins which are involved in metal homeostasis and scavenging of reactive oxygen species. Although plant MTs have been intensively studied, their roles in seeds remain to be clearly established. Here, we report the isolation and characterization of NnMT2a, NnMT2b and NnMT3 from sacred lotus (Nelumbo nucifera Gaertn.) and their roles in seed germination vigor. The transcripts of NnMT2a, NnMT2b and NnMT3 were highly expressed in developing and germinating sacred lotus seeds, and were dramatically up-regulated in response to high salinity, oxidative stresses and heavy metals. Analysis of transformed Arabidopsis protoplasts showed that NnMT2a-YFP and NnMT3-YFP were localized in cytoplasm and nucleoplasm. Transgenic Arabidopsis seeds overexpressing NnMT2a and NnMT3 displayed improved resistance to accelerated aging (AA) treatment, indicating their significant roles in seed germination vigor. These transgenic seeds also exhibited higher superoxide dismutase activity compared to wild-type seeds after AA treatment. In addition, we showed that NnMT2a and NnMT3 conferred improved germination ability to NaCl and methyl viologen on transgenic Arabidopsis seeds. Taken together, these data demonstrate that overexpression of NnMT2a and NnMT3 in Arabidopsis significantly enhances seed germination vigor after AA treatment and under abiotic stresses.Peer reviewed: YesNRC publication: Ye