Editing of ORF138 restores fertility of Ogura cytoplasmic male sterile broccoli via mitoTALENs.

Cytoplasmic male sterility (CMS), encoded by the mitochondrial open reading frames (ORFs), has long been used to economically produce crop hybrids. However, the utilization of CMS also hinders the exploitation of sterility and fertility variation in the absence of a restorer line, which in turn narrows the genetic background and reduces biodiversity. Here, we used a mitochondrial targeted transcription activator-like effector nuclease (mitoTALENs) to knock out ORF138 from the Ogura CMS broccoli hybrid. The knockout was confirmed by the amplification and re-sequencing read mapping to the mitochondrial genome. As a result, knockout of ORF138 restored the fertility of the CMS hybrid, and simultaneously manifested a cold-sensitive male sterility. ORF138 depletion is stably inherited to the next generation, allowing for direct use in the breeding process. In addition, we proposed a highly reliable and cost-effective toolkit to accelerate the life cycle of fertile lines from CMS-derived broccoli hybrids. By applying the k-mean clustering and interaction network analysis, we identified the central gene networks involved in the fertility restoration and cold-sensitive male sterility. Our study enables mitochondrial genome editing via mitoTALENs in Brassicaceae vegetable crops and provides evidence that the sex production machinery and its temperature-responsive ability are regulated by the mitochondria.

Epistatic interactions among multiple copies of FLC genes with naturally occurring insertions correlate with flowering time variation in radish.

Brassicaceae crops, which underwent whole-genome triplication during their evolution, have multiple copies of flowering-related genes. Interactions among multiple gene copies may be involved in flowering time regulation; however, this mechanism is poorly understood. In this study, we performed comprehensive, high-throughput RNA sequencing analysis to identify candidate genes involved in the extremely late-bolting (LB) trait in radish. Then, we examined the regulatory roles and interactions of radish FLOWERING LOCUS C (RsFLC) paralogs, the main flowering repressor candidates. Seven flowering integrator genes, five vernalization genes, nine photoperiodic/circadian clock genes and eight genes from other flowering pathways were differentially expressed in the early-bolting (EB) cultivar 'Aokubinagafuto' and LB radish cultivar 'Tokinashi' under different vernalization conditions. In the LB cultivar, RsFLC1 and RsFLC2 expression levels were maintained after 40 days of cold exposure. Bolting time was significantly correlated with the expression rates of RsFLC1 and RsFLC2. Using the EB × LB F2 population, we performed association analyses of genotypes with or without 1910- and 1627-bp insertions in the first introns of RsFLC1 and RsFLC2, respectively. The insertion alleles prevented the repression of their respective FLC genes under cold conditions. Interestingly, genotypes homozygous for RsFLC2 insertion alleles maintained high RsFLC1 and RsFLC3 expression levels under cold conditions, and two-way analysis of variance revealed that RsFLC1 and RsFLC3 expression was influenced by the RsFLC2 genotype. Our results indicate that insertions in the first introns of RsFLC1 and RsFLC2 contribute to the late-flowering trait in radish via different mechanisms. The RsFLC2 insertion allele conferred a strong delay in bolting by inhibiting the repression of all three RsFLC genes, suggesting that radish flowering time is determined by epistatic interactions among multiple FLC gene copies.

Oxicam-type nonsteroidal anti-inflammatory drugs enhance Agrobacterium-mediated transient transformation in plants.

Agrobacterium-mediated transformation is a key innovation for plant breeding, and routinely used in basic researches and applied biology. However, the transformation efficiency is often the limiting factor of this technique. In this study, we discovered that oxicam-type nonsteroidal anti-inflammatory drugs, including tenoxicam (TNX), increase the efficiency of Agrobacterium-mediated transient transformation. TNX treatment increased the transformation efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana mature leaves by agroinfiltration. The increase of efficiency by TNX treatment was not observed in dde2/ein2/pad4/sid2 quadruple mutant, indicating that TNX inhibits the immune system mediated by jasmonic acid, ethylene, and salicylic acid against to Agrobacterium. We also found that TNX-treatment is applicable for the transient expression and subcellular localization analysis of fluorescent-tagged proteins in Arabidopsis leaf cells. In addition, we found that TNX increases the efficiency of Agrobacterium-mediated transient transformation of Jatropha. Given that treatment with oxicam compounds is a simple and cost effective method, our findings will provide a new option to overcome limitations associated with Agrobacterium-mediated transformation of various plant species.

Curing cytoplasmic male sterility via TALEN-mediated mitochondrial genome editing.

Sequence-specific nucleases are commonly used to modify the nuclear genome of plants. However, targeted modification of the mitochondrial genome of land plants has not yet been achieved. In plants, a type of male sterility called cytoplasmic male sterility (CMS) has been attributed to certain mitochondrial genes, but none of these genes has been validated by direct mitochondrial gene-targeted modification. Here, we knocked out CMS-associated genes (orf79 and orf125) of CMS varieties of rice and rapeseed, respectively, using transcription activator-like effector nucleases (TALENs) with mitochondria localization signals (mitoTALENs). We demonstrate that knocking out these genes cures male sterility, strongly suggesting that these genes are causes of CMS. Sequencing revealed that double-strand breaks induced by mitoTALENs were repaired by homologous recombination, and that during this process, the target genes and surrounding sequences were deleted. Our results show that mitoTALENs can be used to stably and heritably modify the mitochondrial genome in plants.

The mitochondrial genome of an asymmetrically cell-fused rapeseed, Brassica napus, containing a radish-derived cytoplasmic male sterility-associated gene.

Cytoplasmic male sterility (CMS) is an agronomically important trait whose causative genes are located in the mitochondrial genome. A CMS rapeseed, Brassica napus 'SW18', was made 25 years ago by an asymmetric (or "donor-recipient") cell fusion between B. napus 'Westar' and a CMS radish (Raphanus sativus 'Kosena'), in order to transfer the radish CMS-associated gene without disturbing the rapeseed features. Here, we determined the nucleotide sequences of the mitochondrial genomes of Kosena radish and SW18. SW18 has a recombinant mitochondrial genome, which includes the whole 222-kb genome of Westar (54 genes) and a total of 23 kb insertions of four fragments from Kosena radish (three genes: orf125, trnfM and atp1). All of the Kosena radish-derived fragments in the SW18 mitochondrial genome had sequences at their ends (ranging from 63 bp to 628 bp) that are identical to the sequences at the sites of insertion on the Westar rapeseed-derived mitochondrial genome. This suggests that these insertions were mediated by homologous recombination. These results confirm at the nucleotide level that a desired CMS-associated gene (orf125) along with a few extraneous genes from radish were successfully transferred.

CRISPR/Cas9-mediated genome editing of the fatty acid desaturase 2 gene in Brassica napus.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated genome editing system has been widely applied as a powerful tool for modifying preferable endogenous genes. This system is highly expected to be further applied for the breeding of various agronomically important plant species. Here we report the modification of a fatty acid desaturase 2 gene (FAD2), which encodes an enzyme that catalyzes the desaturation of oleic acid, in Brassica napus cv. Westar using the CRISPR/Cas9 system. Two guide RNAs were designed for BnaA.FAD2.a (FAD2_Aa). Of 22 regenerated shoots with FAD2_Aa editing vectors, three contained mutant alleles. Further analysis revealed that two of three mature plants (Aa1#13 and Aa2#2) contained the mutant alleles. The mutant fad2_Aa allele had a 4-bp deletion, which was inherited by backcross progenies (BC1) in the Aa1#13 line. Furthermore, plants with the fad2_Aa allele without transgenes were selected from the BC1 progenies and plants homozygous for fad2_Aa were then produced by self-crossing these BC1 progenies (BC1S1). Fatty acid composition analysis of their seeds revealed a statistically significant increase in the content of oleic acid compared with that in wild-type seeds. These results showed that the application of the CRISPR/Cas9 system is useful to produce desirable mutant plants with an agronomically suitable phenotype by modifying the metabolic pathway in B. napus.

The Restorer-of-fertility-like 2 pentatricopeptide repeat protein and RNase P are required for the processing of mitochondrial orf291 RNA in Arabidopsis.

Eukaryotes harbor mitochondria obtained via ancient symbiosis events. The successful evolution of energy production in mitochondria has been dependent on the control of mitochondrial gene expression by the nucleus. In flowering plants, the nuclear-encoded pentatricopeptide repeat (PPR) superfamily proteins are widely involved in mitochondrial RNA metabolism. Here, we show that an Arabidopsis nuclear-encoded RNA-binding protein, Restorer-of-fertility-like PPR protein 2 (RFL2), is required for RNA degradation of the mitochondrial orf291 transcript via endonucleolytic cleavage of the transcript in the middle of its reading frame. Both in vivo and in vitro, this RNA cleavage requires the activity of mitochondrial proteinaceous RNase P, which is possibly recruited to the site by RFL2. The site of RNase P cleavage likely forms a tRNA-like structure in the orf291 transcript. This study presents an example of functional collaboration between a PPR protein and an endonuclease in RNA cleavage. Furthermore, we show that the RFL2-binding region within the orf291 gene is hypervariable in the family Brassicaceae, possibly correlated with the rapid evolution of the RNA-recognition interfaces of the RFL proteins.

The BME3 (Blue Micropylar End 3) GATA zinc finger transcription factor is a positive regulator of Arabidopsis seed germination.

In many plant species, seed dormancy is broken by cold stratification, a pre-chilling treatment of fully imbibed seeds. Although the ecological importance of seed response to cold temperature is well appreciated, the mechanisms underlying the physiological changes during cold stratification is unknown. Here we show that the GATA zinc finger protein expressed in Arabidopsis seeds during cold stratification plays a critical role in germination. Characterization of an enhancer-trap population identified multiple lines that exhibited beta-glucuronidase (GUS) expression in the micropylar end of the seed (named Blue Micropylar End, BME lines). One of these lines, BME3, had a T-DNA insertion site in the 5' upstream region of a GATA-type zinc finger transcription factor gene (termed BME3-ZF). The BME3-ZF mRNA accumulated in seeds during cold stratification. Characterization of the BME3-ZF promoter indicated that this gene was activated specifically in the embryonic axis, which was still enclosed by the endosperm. The zinc finger gene knockout plants produced seeds exhibiting deeper dormancy, which showed reduced response to cold stratification. The ungerminated knockout seeds exhibited testa rupture, but failed to penetrate the endosperm layer. Application of gibberellic acid (GA3) rescued impaired germination of knockout seeds without cold stratification, indicating that the normal GA signal transduction pathway is present in the knockout mutants. Expression of GA20-oxidase and GA3-oxidase genes was greatly reduced in the knockout seeds, suggesting the potential involvement of the zinc finger protein in GA biosynthesis. These results suggest that the GATA zinc finger protein is a positive regulator of seed germination.

Large-scale screening of Arabidopsis enhancer-trap lines for seed germination-associated genes.

Enhancer trap is a powerful approach for identifying tissue- and stage-specific gene expression in plants and animals. For Arabidopsis research, beta-glucuronidase (GUS) enhancer-trap lines have been created and successfully used to identify tissue-specific gene expression in many plant organs. However, limited applications of these lines for seed germination research have been reported. This is probably due to the impermeability of the testa to the GUS substrate. By focusing on the stages between testa and endosperm rupture, we were able to circumvent the testa barrier to the GUS substrate and observe diverse tissue-specific gene expression during germination sensu stricto. One hundred and twenty-one positive subpools of 10 lines out of 1130 were isolated. Approximately 4500 plants from these subpools were grown in a greenhouse and one to seven individual plants exhibiting GUS expression in seeds were isolated for each subpool. This library of the Arabidopsis seed enhancer-trap lines is an efficient tool for identifying seed germination-associated genes. The individual lines from this library will be provided to the international seed biology research community. International collaboration to identify the trapped genes using genome-walking PCR and to characterize the gene functions using knockout plants will significantly enhance our understanding of the molecular mechanisms of seed germination.

Genetic characterization of a pentatricopeptide repeat protein gene, orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish.

Cytoplasmic male sterility (CMS) in plants is a maternally inherited inability to produce functional pollen, and is often associated with mitochondrial DNA abnormalities. Specific nuclear loci that suppress CMS, termed as restorers of fertility (Rf), have been identified. Previously, we identified an Rf for the CMS Kosena radish and used genetic analysis to identify the locus and create a contig covering the critical interval. To identify the Rf gene, we introduced each of the lambda and cosmid clones into the CMS Brassica napus and scored for fertility restoration. Fertility restoration was observed when one of the lambda clones was introduced into the CMS B. napus. Furthermore, introduction of a 4.7-kb BamHI/HpaI fragment of the lambda clone is enough to restore male fertility. A cDNA strand isolated from a positive fragment contained a predicted protein (ORF687) of 687 amino acids comprising 16 repeats of the 35-amino acid pentatricopeptide repeat (PPR) motif. Kosena CMS radish plants were found to express an allele of this gene possessing four substituted amino acids in the second and third repeats of the PPR suggesting that the domains formed by these repeats in ORF687 are essential for fertility restoration. Protein levels of the Kosena CMS-associated mitochondrial protein ORF125 were considerably reduced in plants in which fertility was restored, although mRNA expression was normal. Regarding the possible role for PPR-containing proteins in the regulation of the mitochondrial gene, we propose that ORF687 functions either directly or indirectly to lower the levels of ORF125, resulting in the restoration of fertility in CMS plants.