Chloroplastic Sec14-like proteins modulate growth and phosphate deficiency responses in Arabidopsis and rice.

Phosphorus is an essential nutrient acquired from soil as phosphate (Pi), and its deficiency severely reduces plant growth and crop yield. Here, we show that single nucleotide polymorphisms (SNPs) at the PHOSPHATIDYLINOSITOL TRANSFER PROTEIN7 (AtPITP7) locus, which encodes a chloroplastic Sec14-like protein, are associated with genetic diversity regarding Pi uptake activity in Arabidopsis (Arabidopsis thaliana). Inactivation of AtPITP7 and its rice (Oryza sativa) homolog (OsPITP6) through T-DNA insertion and CRISPR/Cas9-mediated gene editing, respectively, decreased Pi uptake and plant growth, regardless of Pi availability. By contrast, overexpression of AtPITP7 and OsPITP6 enhanced Pi uptake and plant growth, especially under limited Pi supply. Importantly, overexpression of OsPITP6 increased the tiller number and grain yield in rice. Targeted metabolome analysis of glycerolipids in leaves and chloroplasts revealed that inactivation of OsPITP6 alters phospholipid contents, independent of Pi availability, diminishing the reduction in phospholipid content and increase in glycolipid content induced by Pi deficiency; meanwhile, overexpression of OsPITP6 enhanced Pi deficiency-induced metabolic alterations. Together with transcriptome analysis of ospitp6 rice plants and phenotypic analysis of grafted Arabidopsis chimeras, these results suggest that chloroplastic Sec14-like proteins play an essential role in growth modulations in response to changes in Pi availability, although their function is critical for plant growth under any Pi condition. The superior traits of OsPITP6-overexpressing rice plants also highlight the potential of OsPITP6 and its homologs in other crops as additional tools for improving Pi uptake and plant growth in low Pi environments.

Real-Time Monitoring of Key Gene Products Involved in Rice Photoperiodic Flowering.

Flowering is an important biological process through which plants determine the timing of reproduction. In rice, florigen mRNA is induced more strongly when the day length is shorter than the critical day length through recognition of 30-min differences in the photoperiod. Grain number, plant height, and heading date 7 (Ghd7), which encodes a CCT-domain protein unique to monocots, has been identified as a key floral repressor in rice, and Heading date 1 (Hd1), a rice ortholog of the Arabidopsis floral activator CONSTANS (CO), is another key floral regulator gene. The Hd1 gene product has been shown to interact with the Ghd7 gene product to form a strong floral repressor complex under long-day conditions. However, the mRNA dynamics of these genes cannot explain the day-length responses of their downstream genes. Thus, a real-time monitoring system of these key gene products is needed to elucidate the molecular mechanisms underlying accurate photoperiod recognition in rice. Here, we developed a monitoring system using luciferase (LUC) fusion protein lines derived from the Ghd7-LUC and Hd1-LUC genes. We successfully obtained a functionally complemented gene-targeted line for Ghd7-LUC. Using this system, we found that the Ghd7-LUC protein begins to accumulate rapidly after dawn and reaches its peak more rapidly under a short-day condition than under a long-day condition. Our system provides a powerful tool for revealing the accurate time-keeping regulation system incorporating these key gene products involved in rice photoperiodic flowering.

Gene regulatory network and its constituent transcription factors that control nitrogen-deficiency responses in rice.

Increase in the nitrogen (N)-use efficiency and optimization of N response in crop species are urgently needed. Although transcription factor-based genetic engineering is a promising approach for achieving these goals, transcription factors that play key roles in the response to N deficiency have not been studied extensively. Here, we performed RNA-seq analysis of root samples of 20 Asian rice (Oryza sativa) accessions with differential nutrient uptake. Data obtained from plants exposed to N-replete and N-deficient conditions were subjected to coexpression analysis and machine learning-based pathway inference to dissect the gene regulatory network required for the response to N deficiency. Four transcription factors, including members of the G2-like and bZIP families, were predicted to function as key regulators of gene transcription within the network in response to N deficiency. Cotransfection assays validated inferred novel regulatory pathways, and further analyses using genome-edited knockout lines suggested that these transcription factors are important for N-deficiency responses in planta. Many of the N deficiency-responsive genes, including those encoding key regulators within the network, were coordinately regulated by transcription factors belonging to different families. Transcription factors identified in this study could be valuable for the modification of N response and metabolism.

Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants.

Gene targeting (GT) enables precise genome modification-e.g., the introduction of base substitutions-using donor DNA as a template. Combined with clean excision of the selection marker used to select GT cells, GT is expected to become a standard, generally applicable, base editing system. Previously, we demonstrated marker excision via a piggyBac transposon from GT-modified loci in rice. However, piggyBac-mediated marker excision has the limitation that it recognizes only the sequence TTAA. Recently, we proposed a novel and universal precise genome editing system consisting of GT with subsequent single-strand annealing (SSA)-mediated marker excision, which has, in principle, no limitation of target sequences. In this study, we introduced base substitutions into the microRNA miR172 target site of the OsCly1 gene-an ortholog of the barley Cleistogamy1 gene involved in cleistogamous flowering. To ensure efficient SSA, the GT vector harbors 1.2-kb overlapped sequences at both ends of a selection marker. The frequency of positive-negative selection-mediated GT using the vector with overlapped sequences was comparable with that achieved using vectors for piggyBac-mediated marker excision without overlapped sequences, with the frequency of SSA-mediated marker excision calculated as ~40% in the T0 generation. This frequency is thought to be adequate to produce marker-free cells, although it is lower than that achieved with piggyBac-mediated marker excision, which approaches 100%. To date, introduction of precise substitutions in discontinuous multiple bases of a targeted gene using base editors and the prime editing system based on CRISPR/Cas9 has been quite difficult. Here, using GT and our SSA-mediated marker excision system, we succeeded in the precise base substitution not only of single bases but also of artificial discontinuous multiple bases in the miR172 target site of the OsCly1 gene. Precise base substitution of miRNA target sites in target genes using this precise genome editing system will be a powerful tool in the production of valuable crops with improved traits.

NSR1/MYR2 is a negative regulator of ASN1 expression and its possible involvement in regulation of nitrogen reutilization in Arabidopsis.

Nitrogen (N) is a major macronutrient that is essential for plant growth. It is important for us to understand the key genes that are involved in the regulation of N utilization. In this study, we focused on a GARP-type transcription factor known as NSR1/MYR2, which has been reported to be induced under N-deficient conditions. Our results demonstrated that NSR1/MYR2 has a transcriptional repression activity and is specifically expressed in vascular tissues, especially in phloem throughout the plant under daily light-dark cycle regulation. The overexpression of NSR1/MYR2 delays nutrient starvation- and dark-triggered senescence in the mature leaves of excised whole aerial parts of Arabidopsis plants. Furthermore, the expression of asparagine synthetase 1 (ASN1), which plays an important role in N remobilization and reallocation, i.e. N reutilization, in Arabidopsis, is negatively regulated by NSR1/MYR2, since the expressions of NSR1/MYR2 and ASN1 were reciprocally regulated during the light-dark cycle and ASN1 expression was down-regulated in overexpressors of NSR1/MYR2 and up-regulated in T-DNA insertion mutants of NSR1/MYR2. Therefore, the present results suggest that NSR1/MYR2 plays a role in N reutilization as a negative regulator through controlling ASN1 expression.

Precision genome editing in plants via gene targeting and piggyBac-mediated marker excision.

Precise genome engineering via homologous recombination (HR)-mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR-mediated GT is an extremely rare event, positive-negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re-integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)-tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.

A mutated cytosine deaminase gene, codA (D314A), as an efficient negative selection marker for gene targeting in rice.

Gene targeting (GT) is a powerful tool manipulating a gene of interest in a given genome specifically and precisely. To achieve efficient GT in higher plants, both positive and negative selection markers are required. In particular, a strong negative selection system is needed for enrichment of cells to eliminate those cells in which random integration of the introduced DNA has occurred in GT experiments. Currently, non-conditional negative selection marker genes are used for GT experiments in rice plants, and no conditional negative selection system is available. In this study, we describe the development of an efficient conditional negative selection system in rice plants using Escherichia coli cytosine deaminase (codA). We found that a mutant codA gene, codA(D314A), acts more efficiently than the wild-type codA for negative selection in rice plants. The codA(D314A) marker was further used as a negative selection marker for GT experiments in rice. Our conditional negative selection system effectively eliminated the cells in which random integration event(s) occurred; the enrichment factor was approximately 100-fold. This enrichment factor was similar to that found when Corynebacterium diphtheriae toxin fragment A was used. Our results suggest the codA(D314A) marker gene as a promising negative selection marker for GT of rice.

DNA replication arrest leads to enhanced homologous recombination and cell death in meristems of rice OsRecQl4 mutants.

BACKGROUND: Mammalian BLM helicase is involved in DNA replication, DNA repair and homologous recombination (HR). These DNA transactions are associated tightly with cell division and are important for maintaining genome stability. However, unlike in mammals, cell division in higher plants is restricted mainly to the meristem, thus genome maintenance at the meristem is critical. The counterpart of BLM in Arabidopsis (AtRecQ4A) has been identified and its role in HR and in the response to DNA damage has been confirmed. However, the function of AtRecQ4A in the meristem during replication stress has not yet been well elucidated. RESULTS: We isolated the BLM counterpart gene OsRecQl4 from rice and analyzed its function using a reverse genetics approach. Osrecql4 mutant plants showed hypersensitivity to DNA damaging agents and enhanced frequency of HR compared to wild-type (WT) plants. We further analyzed the effect of aphidicolin--an inhibitor of S-phase progression via its inhibitory effect on DNA polymerases--on genome stability in the root meristem in osrecql4 mutant plants and corresponding WT plants. The following effects were observed upon aphidicolin treatment: a) comet assay showed induction of DNA double-strand breaks (DSBs) in mutant plants, b) TUNEL assay showed enhanced DNA breaks at the root meristem in mutant plants, c) a recombination reporter showed enhanced HR frequency in mutant calli, d) propidium iodide (PI) staining of root tips revealed an increased incidence of cell death in the meristem of mutant plants. CONCLUSIONS: These results demonstrate that the aphidicolin-sensitive phenotype of osrecql4 mutants was in part due to induced DSBs and cell death, and that OsRecQl4 plays an important role as a caretaker, maintaining genome stability during DNA replication stress in the rice meristem.

Increase in pectin deposition by overexpression of an ERF gene in cultured cells of Arabidopsis thaliana.

Ethylene-responsive transcription factor (ERF) family genes, which are involved in regulation of metabolic pathways and/or are useful for metabolic engineering, were investigated in the cultured cells of Arabidopsis thaliana. The pectin content in the gelatinous precipitates after the ethanol precipitation of extracts derived from calli of a transgenic cell line, A17, overexpressing an ERF gene (At1g44830), increased in comparison with the control. Expression of genes involved in pectin biosynthesis was up-regulated in the A17 calli. Overexpression of the ERF gene coordinately activates the pectin biosynthetic pathway genes and increases the content of pectin. These results therefore will be useful as a genetic resource for engineering pectin biosynthesis in plants.

CDKB2 is involved in mitosis and DNA damage response in rice.

DNA damage checkpoints delay mitotic cell-cycle progression in response to DNA stress, stalling the cell cycle to allow time for repair. CDKB is a plant-specific cyclin-dependent kinase (CDK) that is required for the G2/M transition of the cell cycle. In Arabidopsis, DNA damage leads the degradation of CDKB2, and the subsequent G2 arrest gives cells time to repair damaged DNA. G2 arrest also triggers transition from the mitotic cycle to endoreduplication, leading to the presence of polyploid cells in many tissues. In contrast, in rice (Oryza sativa), polyploid cells are found only in the endosperm. It was unclear whether endoreduplication contributes to alleviating DNA damage in rice (Oryza sativa). Here, we show that DNA damage neither down-regulates Orysa;CDKB2;1 nor induces endoreduplication in rice. Furthermore, we found increased levels of Orysa;CDKB2;1 protein upon DNA damage. These results suggest that CDKB2 functions differently in Arabidopsis and rice in response to DNA damage. Arabidopsis may adopt endoreduplication as a survival strategy under genotoxic stress conditions, but rice may enhance DNA repair capacity upon genotoxic stress. In addition, polyploid cells due to endomitosis were present in CDKB2;1 knockdown rice, suggesting an important role for Orysa;CDKB2;1 during mitosis.

Constitutive expression of the GIGANTEA ortholog affects circadian rhythms and suppresses one-shot induction of flowering in Pharbitis nil, a typical short-day plant.

GIGANTEA (GI) is a key regulator of flowering time, which is closely related to the circadian clock function in Arabidopsis. Mutations in the GI gene cause photoperiod-insensitive flowering and altered circadian rhythms. We isolated the GI ortholog PnGI from Pharbitis (Ipomoea) nil, an absolute short-day (SD) plant. PnGI mRNA expression showed diurnal rhythms that peaked at dusk under SD and long-day (LD) conditions, and also showed robust circadian rhythms under continuous dark (DD) and continuous light (LL) conditions. Short irradiation with red light during the flower-inductive dark period did not change PnGI expression levels, suggesting that such a night break does not abolish flowering by affecting the expression of PnGI. In Pharbitis, although a single dusk signal is sufficient to induce expression of the ortholog of FLOWERING LOCUS T (PnFT1), PnGI mRNA expression was not reset by single lights-off signals. Constitutive expression of PnGI (PnGI-OX) in transgenic plants altered period length in leaf-movement rhythms under LL and affected circadian rhythms of PnFT mRNA expression under DD. PnGI-OX plants formed fewer flower buds than the wild type when one-shot darkness was given. In PnGI-OX plants, expression of PnFT1 was down-regulated, suggesting that PnGI functions as a suppressor of flowering, possibly in part through down-regulation of PnFT1.

Identification of genes of the plant-specific transcription-factor families cooperatively regulated by ethylene and jasmonate in Arabidopsis thaliana.

The analysis of expression patterns of transcription-factor genes will be the basis for a better understanding of their biological functions in plants. In this study, we designed and developed an oligo-DNA macroarray consisting of gene-specific probes of 60-65 nucleotides for 288 transcription-factor genes, which cover COL, DOF, ERF, and NAC family genes. To investigate transcription-factor genes that are cooperatively regulated by jasmonate and ethylene in arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants, we analyzed the expression profile of transcription-factor genes using the oligo-DNA macroarray technique in arabidopsis plants treated with methyl jasmonate and 1-aminocyclopropane-1-carboxylic acid. Then, transcript levels of candidate genes-which were selected based on the result of macroarray analysis-were evaluated by the quantitative real-time RT-PCR method. Finally, we identified an ERF family gene that is cooperatively regulated by both hormones, and designated as cooperatively regulated by ethylene and jasmonate 1 (CEJ1).