Peculiar properties of tuber starch in a potato mutant lacking the α-glucan water dikinase 1 gene GWD1 created by targeted mutagenesis using the CRISPR/dMac3-Cas9 system.

Glucose chains in starch are phosphorylated and contribute to structural stabilization. Phosphate groups contained in starch also play a role in retaining moisture. α-Glucan water dikinase 1 (GWD1) is involved in the phosphorylation of glucose chains in starch. In this study, we generated potato mutants of the GWD1 gene using the CRISPR/dMac3-Cas9 system. Observation of the phenotypes of the GWD1-deficient mutants revealed their physiological roles in tuber starch formation. The 4-allele mutants showed growth retardation and a delay in tuber formation. A significant decrease in phosphorus content was detected in the tuber starch of the gwd1 mutant. This mutant starch showed a higher amylose content than the wild-type starch, whereas its gelatinization temperature was slightly lower than that of the WT starch. The peak viscosity of the mutant starch was lower than that of the WT starch. These observations revealed that the starch of the gwd1 mutants had peculiar and unique properties compared to those of WT, sbe3 and gbss1 mutant starches. The amount of tissue-released water due to freeze-thawing treatment was determined on tubers of the gwd1 mutant and compared with those of WT and the other mutants. Significantly less water loss was found in the gwd1, sbe3 and gbss1 mutant tubers than in the WT tubers. Our results indicate that the GWD1 gene is not only important for potato growth, but also largely effective for the traits of tuber starch.

Procedure for the efficient acquisition of progeny seeds from crossed potato plants grafted onto tomato.

Potato, Solanum tuberosum L. is an important crop. However, it is difficult to breed potato cultivars by applying conventional crossing methods because potato has a tetraploid genome and is vegetatively propagated. Flower formation and tuber development occur simultaneously. Many potato cultivars hardly produce any fruits after crossing and fail to produce seeds. We report an improved procedure for obtaining progeny seeds by grafting potatoes onto tomatoes. The rate of fruit formation was more than 19% when the grafted potatoes were used for the crossing experiments, whereas crossing using the ungrafted plants showed a rate of 1.1%. This result suggests that our procedure results in the easy acquisition of null-segregant progenies by crossing mutant lines. It is also expected to improve conventional potato breeding.

Creation of a potato mutant lacking the starch branching enzyme gene StSBE3 that was generated by genome editing using the CRISPR/dMac3-Cas9 system.

The potato tuber starch trait is changed depending on the composition of amylose and amylopectin. The amount of amylopectin is determined by the activity of the starch branching enzymes SBE1, SBE2, and SBE3 in potato. SBE3, a homolog of rice BEI, is a major gene that is abundant in tubers. In this study, we created mutants of the potato SBE3 gene using CRISPR/Cas9 attached to the translation enhancer dMac3. Potato has a tetraploid genome, and a four-allele mutant of the SBE3 gene is desired. Mutations in the SBE3 gene were found in 89 of 126 transformants of potato plants. Among these mutants, 10 lines contained four mutant SBE3 genes, indicating that 8% efficiency of target mutagenesis was achieved. These mutants grew normally, similar to the wild-type plant, and yielded sufficient amounts of tubers. The potato starch in these tubers was similar to that of the rice BEI mutant. Western blot analysis revealed the defective production of SBE3 in the mutant tubers, suggesting that these transformants were loss-of-function mutants of SBE3.

A simple method to establish an efficient medium suitable for potato regeneration.

Potato (Solanum tuberosum) is one of the important crop plants, and many potato cultivars consist of a tetraploid genome with high heterozygosity. The techniques of transformation and genome editing require plant regeneration. However, no efficient regeneration method has been established except for some specific cultivars, such as 'Sayaka'. In general, it is necessary to determine the adequate concentrations of auxin and cytokinin for plant regeneration. We established an efficient method using a 24-well microplate that easily enabled determination of the concentrations of these plant growth regulators suitable for shoot regeneration. Using this method, the optimal concentrations of these factors were analyzed for two representative potato cultivars, 'Sayaka' and 'Konafubuki'. This analysis revealed there was a large difference in the optimal concentrations between them. Based on this result, a specialized medium for the efficient regeneration of 'Konafubuki' cultivars was proposed. This assay method was also applied for determination of hygromycin sensitivity of these potato cultivars, and it was observed that 'Konafubuki' was rather sensitive to hygromycin. These findings suggested that the selection of a 'Konafubuki' transformant could be achieved using a medium containing a lower amount of hygromycin than that used for 'Sayaka'.

Establishment of a conditional TALEN system using the translational enhancer dMac3 and an inducible promoter activated by glucocorticoid treatment to increase the frequency of targeted mutagenesis in plants.

Transcription activator-like effector nuclease (TALEN) is an artificial nuclease that causes DNA cleavage at the target site and induces few off-target reactions because of its high sequence specificity. Powerful and variable tools using TALENs can be used in practical applications and may facilitate the molecular breeding of many plant species. We have developed a convenient construction system for a plant TALEN vector named the Emerald Gateway TALEN system. In this study, we added new properties to this system, which led to an increase in the efficiency of targeted mutagenesis. Rice dMac3 is a translational enhancer that highly increases the efficiency of translation of the downstream ORF. We inserted dMac3 into the 5' untranslated region of the TALEN gene. In the cultured rice cells to which the TALEN gene was introduced, the frequency of targeted mutagenesis was highly increased compared with those altered using the conventional system. Next, the promoter for the TALEN gene was replaced with iPromoter, and its expression was stringently controlled by a GVG transcription factor that was activated in the presence of glucocorticoid. This conditional expression system worked effectively and led to a higher frequency of targeted mutagenesis than that by the constitutive expression system, while no mutagenesis was detected without glucocorticoid treatment. These results suggest that our system can be applied to genome editing to create the desired mutation.

Establishment of a modified CRISPR/Cas9 system with increased mutagenesis frequency using the translational enhancer dMac3 and multiple guide RNAs in potato.

CRISPR/Cas9 is a programmable nuclease composed of the Cas9 protein and a guide RNA (gRNA) molecule. To create a mutant potato, a powerful genome-editing system was required because potato has a tetraploid genome. The translational enhancer dMac3, consisting of a portion of the OsMac3 mRNA 5'-untranslated region, greatly enhanced the production of the protein encoded in the downstream ORF. To enrich the amount of Cas9, we applied the dMac3 translational enhancer to the Cas9 expression system with multiple gRNA genes. CRISPR/Cas9 systems targeting the potato granule-bound starch synthase I (GBSSI) gene examined the frequency of mutant alleles in transgenic potato plants. The efficiency of the targeted mutagenesis strongly increased when the dMac3-installed Cas9 was used. In this case, the ratio of transformants containing four mutant alleles reached approximately 25% when estimated by CAPS analysis. The mutants that exhibited targeted mutagenesis in the GBSSI gene showed characteristics of low amylose starch in their tubers. This result suggests that our system may facilitate genome-editing events in polyploid plants.

WIND1 Promotes Shoot Regeneration through Transcriptional Activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis.

Many plant species display remarkable developmental plasticity and regenerate new organs after injury. Local signals produced by wounding are thought to trigger organ regeneration but molecular mechanisms underlying this control remain largely unknown. We previously identified an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) as a central regulator of wound-induced cellular reprogramming in plants. In this study, we demonstrate that WIND1 promotes callus formation and shoot regeneration by upregulating the expression of the ENHANCER OF SHOOT REGENERATION1 (ESR1) gene, which encodes another AP2/ERF transcription factor in Arabidopsis thaliana The esr1 mutants are defective in callus formation and shoot regeneration; conversely, its overexpression promotes both of these processes, indicating that ESR1 functions as a critical driver of cellular reprogramming. Our data show that WIND1 directly binds the vascular system-specific and wound-responsive cis-element-like motifs within the ESR1 promoter and activates its expression. The expression of ESR1 is strongly reduced in WIND1-SRDX dominant repressors, and ectopic overexpression of ESR1 bypasses defects in callus formation and shoot regeneration in WIND1-SRDX plants, supporting the notion that ESR1 acts downstream of WIND1. Together, our findings uncover a key molecular pathway that links wound signaling to shoot regeneration in plants.

WIND1-based acquisition of regeneration competency in Arabidopsis and rapeseed.

Callus formation and de novo organogenesis often occur in the wounded tissues of plants. Although this regenerative capacity of plant cells has been utilized for many years, molecular basis for the wound-induced acquisition of regeneration competency is yet to be elucidated. Here we find that wounding treatment is essential for shoot regeneration from roots in the conventional tissue culture of Arabidopsis thaliana. Furthermore, we show that an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) plays a pivotal role for the acquisition of regeneration competency in the culture system. Ectopic expression of WIND1 can bypass both wounding and auxin pre-treatment and increase de novo shoot regeneration from root explants cultured on shoot-regeneration promoting media. In Brassica napus, activation of Arabidopsis WIND1 also greatly enhances de novo shoot regeneration, further corroborating the role of WIND1 in conferring cellular regenerative capacity. Our data also show that sequential activation of WIND1 and an embryonic regulator LEAFY COTYLEDON2 enhances generation of embryonic callus, suggesting that combining WIND1 with other transcription factors promote efficient and organ-specific regeneration. Our findings in the model plant and crop plant point to a possible way to efficiently induce callus formation and regeneration by utilizing transcription factors as a molecular switch.

PRC2 represses dedifferentiation of mature somatic cells in Arabidopsis.

Plant somatic cells are generally acknowledged to retain totipotency, the potential to develop into any cell type within an organism. This astonishing plasticity may contribute to a high regenerative capacity on severe damage, but how plants control this potential during normal post-embryonic development remains largely unknown(1,2). Here we show that POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a chromatin regulator that maintains gene repression through histone modification, prevents dedifferentiation of mature somatic cells in Arabidopsis thaliana roots. Loss-of-function mutants in PRC2 subunits initially develop unicellular root hairs indistinguishable from those in wild type but fail to retain the differentiated state, ultimately resulting in the generation of an unorganized cell mass and somatic embryos from a single root hair. Strikingly, mutant root hairs complete the normal endoreduplication programme, increasing their nuclear ploidy, but subsequently reinitiate mitotic division coupled with successive DNA replication. Our data show that the WOUND INDUCED DEDIFFERENTIATION3 (WIND3) and LEAFY COTYLEDON2 (LEC2) genes are among the PRC2 targets involved in this reprogramming, as their ectopic overexpression partly phenocopies the dedifferentiation phenotype of PRC2 mutants. These findings unveil the pivotal role of PRC2-mediated gene repression in preventing unscheduled reprogramming of fully differentiated plant cells.

Arabidopsis WIND1 induces callus formation in rapeseed, tomato, and tobacco.

The capacity to promote cell dedifferentiation is widespread among plant species. We have recently reported that an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) and its paralogues, WIND2-4, promote cell dedifferentiation in Arabidopsis (Arabidopsis thaliana). Phylogenetic analyses suggest that AtWIND1 orthologs are found in land plants and that the shared peptide motifs between Arabidopsis paralogues are conserved in putative orthologs in dicotyledonous and monocotyledonous plants. In this study we show that AtWIND1 chemically induced rapeseed and tomato, as well as AtWIND1 constitutively expressed tobacco, promote callus formation on phytohormone-free medium. Our results suggest that the WIND1-mediated signaling cascade to promote cell dedifferentiation might be conserved in at least several species of Brassicaceae and Solanaceae.