Pretreatment with dilute maleic acid enhances the enzymatic digestibility of sugarcane bagasse and oil palm empty fruit bunch fiber.

Lignocellulose is resistant to degradation and requires pretreatment before hydrolytic enzymes can release fermentable sugars. Sulfuric acid has been widely used for biomass pretreatment, but high amount of degradation products usually occurred when using this method. To enhance accessibility to cellulose, we studied the performances of several dilute organic acid pretreatments of sugarcane bagasse and oil palm empty fruit bunch fiber. The results revealed that pretreatment with maleic acid yields the highest xylose and glucose release among other organic acids. The effects of concentration, duration of heating and heating temperature were further studied. Dilute maleic acid 1 % (w/w) pretreatment at 180 °C was the key to its viability as a substitute for sulfuric acid. Moreover, maleic acid did not seem to highly promote the formation of either furfural or 5-HMF in the liquid hydrolysate after pretreatment.

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.

3-Amino-4-hydroxybenzoic acid production from glucose and/or xylose via recombinant Streptomyces lividans.

The aromatic compound 3-amino-4-hydroxybenzoic acid (3,4-AHBA) can be employed as a raw material for high-performance industrial plastics. The aim of this study is to produce 3,4-AHBA via a recombinant Streptomyces lividans strain containing griI and griH genes derived from Streptomyces griseus using culture medium with glucose and/or xylose, which are the main components in lignocellulosic biomass. Production of 3,4-AHBA by the recombinant S. lividans strain was successful, and the productivity was affected by the kind of sugar used as an additional carbon source. Metabolic profiles revealed that L aspartate-4-semialdehyde (ASA), a precursor of 3,4-AHBA, and coenzyme NADPH were supplied in greater amounts in xylose medium than in glucose medium. Moreover, cultivation in TSB medium with a mixed sugar (glucose/xylose) was found to be effective for 3,4-AHBA production, and optimal conditions for efficient production were designed by changing the ratio of glucose to xylose. The best productivity of 2.70 g/L was achieved using a sugar mixture of 25 g/L glucose and 25 g/L xylose, which was 1.5 times higher than the result using 50 g/L glucose alone. These results suggest that Streptomyces is a suitable candidate platform for 3,4-AHBA production from lignocellulosic biomass-derived sugars under appropriate culture conditions.

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.

Overexpression of rice OsLEA5 relieves the deterioration in seed quality caused by high-temperature stress.

Late embryogenesis abundant protein (LEA) genes are widely conserved in seed plant species and form a multigene family. While some LEAs are known to respond to environmental stresses, the function of many LEAs is unknown. OsLEA5 (Lea14A) interacts with a regulator of the endosperm storage production, FLO2, suggesting that OsLEA5 may be involved in endosperm quality control. RNAi knockdown line of OsLEA5 showed decreased seed weight. Transformant lines overexpressing OsLEA5 exhibited improved quality and seed weight of mature seeds when they were developed under high-temperature conditions, while seed quality strongly declined in wild-type plants exposed to high-temperature stress. These findings indicate that OsLEA5 contributes to suppressing the deterioration of seed quality when developed under high-temperature conditions.

Dissection of a rice OsMac1 mRNA 5' UTR to uncover regulatory elements that are responsible for its efficient translation.

The untranslated regions (UTRs) of mRNAs are involved in many posttranscriptional regulatory pathways. The rice OsMac1 mRNA has three splicing variants of the 5' UTR (UTRa, UTRb, and UTRc), which include a CU-rich region and three upstream open reading frames (uORFs). UTRc contains an additional 38-nt sequence, termed sp38, which acts as a strong translational enhancer of the downstream ORF; reporter analysis revealed translational efficiencies >15-fold higher with UTRc than with the other splice variants. Mutation analysis of UTRc demonstrated that an optimal sequence length of sp38, rather than its nucleotide sequence is essential for UTRc to promote efficient translation. In addition, the 5' 100 nucleotides of CU-rich region contribute to UTRc translational enhancement. Strikingly, three uORFs did not reveal their inhibitory potential within the full-length leader, whereas deletion of the 5' leader fragment preceding the leader region with uORFs nearly abolished translation. Computational prediction of UTRc structural motifs revealed stem-loop structures, termed SL1-SL4, and two regions, A and B, involved in putative intramolecular interactions. Our data suggest that SL4 binding to Region-A and base pairing between Region-B and the UTRc 3'end are critically required for translational enhancement. Since UTRc is not capable of internal initiation, we presume that the three-dimensional leader structures can allow translation of the leader downstream ORF, likely allowing the bypass of uORFs.

A simple method for in planta tomato transformation by inoculating floral buds with a sticky Agrobacterium tumefaciens suspension.

Tomato transformation is conventionally performed using Agrobacterium tumefaciens-infected cotyledons. Here, we propose a simple procedure for tomato transformation, by which A. tumefaciens cells were smeared onto floral buds of a tomato plant using a paintbrush. Sufficient numbers of fruits were obtained from them, although the smearing of an excess number of A. tumefaciens cells led to an adverse effect on the plant growth. Progeny plants were screened by growth on a kanamycin-containing selection medium plate. The nptII gene was detected in 10 plants among 1,599 progenies. These transformants were derived from fruits other than those obtained from the smeared buds. This suggested that A. tumefaciens cells moved to the buds located near the smeared buds and caused the transformation event. Our findings suggest that this procedure can be used for the introduction of a foreign gene into plant cells.

Characterization of novel SUMO family genes in the rice genome.

Small ubiquitin-related modifier (SUMO) is a post-translational modification factor composed of about 100 amino acid residues. Most plant species express a family of SUMO isoforms. We found three novel homologs of rice (Oryza sativa L.) SUMO genes, OsSUMO4, OsSUMO5 and OsSUMO6, in addition to the known SUMO genes OsSUMO1-OsSUMO3. Phylogenetic tree analysis revealed that rice SUMO genes have diverged considerably during their evolution. All six of these SUMO genes complemented the phenotype of the SUMO-deficient pmt3Δ mutant of fission yeast. Among the amino acid sequences of rice SUMO proteins, consensus motifs (ΨKXE/D) of the SUMO acceptor site were found in OsSUMO3, OsSUMO4, OsSUMO5 and OsSUMO6. The heat shock protein HSF7 is known to be SUMOylated in Arabidopsis thaliana. SUMOylation using a bacterial expression system revealed that the rice HSF7 homolog was modified by the six rice SUMOs, and further suggested that OsSUMO1, OsSUMO3, OsSUMO4 and OsSUMO6 are involved in its multiple SUMOylation.

AtFLL2, a member of the FLO2 gene family, affects the enlargement of leaves at the vegetative stage and facilitates the regulation of carbon metabolism and flow.

Arabidopsis thaliana FLL2, a member of the FLO2 gene family, is expressed specifically in green leaves. The fll2 mutant showed significantly large rosette leaves and reduced the chlorophyll content. The sucrose content was significantly reduced. The glucose content was higher during the vegetative growth stage but decreased during the early reproductive growth stage. The amount of assimilated starch was lower than that in the wild type plant. The expression levels of genes involved in biosynthesis of sucrose and starch were largely altered. These results suggest that, in the fll2 mutant, a small amount of photosynthetic products was used for the biosynthesis of starch, and the products were supplied to promote intracellular growth of the source organs or for transport to the sink organs. These findings suggest that FLL2 is a factor affecting the expression level of genes involved in sugar metabolism, whose mutation caused a change in the assimilated products. Abbreviations : DAS: days after sowing.

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'.

A novel FLOURY ENDOSPERM2 (FLO2)-interacting protein, is involved in maintaining fertility and seed quality in rice.

Crop plants accumulate a large amount of storage starch and storage proteins in the endosperm. Genes involved in the biosynthesis of these substances work in concert during development of the rice endosperm. The rice flo2 mutant produces aberrant seeds with reduced grain quality. FLOURRY ENDOSPERM 2 (FLO2), the causative gene of the flo2 mutant, is considered to be a regulatory protein that controls the biosynthesis of seed storage substances. FLO2 contains tetratricopeptide repeat (TPR) motifs that may mediate protein-protein interactions. In this study, we identified the protein that interacts with the TPR motif of FLO2. We generated a transformant that produced the FLAG-tagged fusion FLO2 protein in the flo2 mutant and used this in the shotgun proteomic analysis. A protein, which we named FLOC1, interacted with FLO2. In vitro pull-down assays indicated that the TPR motif was involved in this interaction. A knock-down transformant of FLOC1 showed significantly reducted fertility and generation of seeds with abnormal features. These findings suggest that FLOC1 is involved not only in seed fertility but also in seed quality. These phenotypes were also observed on the RNAi transformants of the flo2 mutant although the effect of the flo2 mutation remained. these findings imply that there is a difference in the functions of FLO2 and FLOC1 although both of appear to be involved in the control of seed quality during seed formation.

De novo transcriptome analysis reveals an unperturbed transcriptome under high cadmium conditions in the Cd-hypertolerant fern Athyrium yokoscense.

Athyrium yokoscense shows strong tolerance to cadmium exposure, even at levels that are many times greater than the toxic levels in ordinary plants. To determine the mechanism of Cd tolerance in A. yokoscense, we grew these plants under high Cd conditions and observed the tissue-specific accumulation of Cd and generation of reactive oxygen species, which is one of the major physiological responses to Cd stress. Fuchsin staining indicated the existence of a casparian strip in A. yokoscense roots, which may participate in Cd hypertolerance in A. yokoscense. Moreover, we performed RNA-seq of RNA samples from A. yokoscense plants treated with or without Cd exposure and obtained comprehensive RNA sequences as well as the Cd-responsive expression patterns of individual genes. Through de novo transcriptome assembly and gene expression analysis, we found that A. yokoscense showed normal features with no significant change in the expression levels of any transporter genes, even under high Cd exposure conditions. Our results demonstrate that A. yokoscense has an unusual mechanism that allows the invading Cd to partition into the distal roots, thus avoiding translocation of Cd into the xylem.

Construction of genomic marker sets based on the chloroplast genome of a green alga, Ulva pertusa (syn. Ulva australis), leads to simple detection of Ulva species.

In closed sea areas such as Tokyo Bay, a phenomenon known as a green tide often occurs, in which large amounts of Ulva seaweed grow abnormally and form mats along the coast. This is currently a serious environmental problem. Green tides are generated by the explosive growth of multiple types of Ulva algae. However, many Ulva species show similar characteristics to each other and are indistinguishable by appearance, making it difficult to identify the Ulva algae in green tides. In this study, we determined the entire nucleotide sequence of the chloroplast genome of Ulva pertusa (syn. Ulva australis) and identified two large inversions of gene order, suggesting the occurrence of genome inversions. We also detected structural polymorphisms among Ulva chloroplast genomes. Ulva pertusa was classified in a different clade from that containing U. lactuca and U. ohnoi, suggesting that U. pertusa is evolutionarily divergent from these species. Based on this knowledge, we constructed a genetic diagnosis system for Ulva algae. Using this approach, we established a simple method that can determine the species of Ulva algae by PCR using specific molecular markers, through which representative Ulva species such as U. lactuca, U. ohnoi and U. pertusa were easily distinguished.

Aberrant endosperm formation caused by reduced production of major allergen proteins in a rice flo2 mutant that confers low-protein accumulation in grains.

Rice flo2 mutation produces grains showing a reduced amount of storage proteins. Using Nipponbare and the flo2 mutant, we created rice transformants that showed defective production of major allergen proteins RA14 and RA33 (14-16 kDa and 33 kDa allergen proteins, respectively) by RNAi introduction. The knock-down transformant generated using Nipponbare showed greatly reduced accumulation of both allergen proteins, normal growth, and production of a sufficient amount of normal-shaped seeds. F1 seeds were obtained by crossing between the transformants containing RNAi genes to RA14 and RA33, and showed decreased accumulation of both proteins. However, a peculiar phenotype was observed in the flo2 transformants that lacked accumulation of RA14 or RA33. They showed significantly reduced fertility. A wrinkled grain feature was found on the transformant lacking accumulation of RA14. F1 seeds obtained by crossing these transformants showed significantly lower fertility. F2 seeds showed decreases in both allergen proteins but morphological abnormality with small and severely wrinkled features. These results indicated that it is hard to obtain any transformant lacking accumulation of these allergen proteins using the flo2 mutant, whereas a knock-down transformant of both allergen protein genes was obtained when a wild-type Nipponbare was used as a host. These facts strongly suggest that RA14 and RA33 have some roles in rice seeds.

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.

Effective usage of sorghum bagasse: Optimization of organosolv pretreatment using 25% 1-butanol and subsequent nanofiltration membrane separation.

We investigated the use of low concentrations of butanol (<40%, all v/v) as an organosolv pretreatment to fractionate lignocellulosic biomass into cellulose, hemicellulose, and lignin. The pretreatment conditions were optimized for sorghum bagasse by focusing on four parameters: butanol concentration, sulfuric acid concentration, pretreatment temperature, and pretreatment time. A butanol concentration of 25% or higher together with 0.5% or higher acid was effective for removing lignin while retaining most of the cellulose in the solid fraction. The highest cellulose (84.9%) and low lignin (15.3%) content were obtained after pretreatment at 200 °C for 60 min. Thus, pretreatment comprising 25% butanol, 0.5% acid, 200 °C, and 60 min process time was considered optimal. Enzymatic saccharification and fermentation by Saccharomyces cerevisiae produced 61.9 g/L ethanol from 200 g/L solid fraction obtained following pretreatment, and 10.2 g/L ethanol was obtained from the liquid fraction by xylose-utilizing S. cerevisiae following membrane nanofiltration to remove butanol.

Genotypic effects on sugar and by-products of liquid hydrolysates and on saccharification of acid-insoluble residues from wheat straw.

Wheat straw is one of the major attractive resources for low-cost raw materials for renewable energy, biofuels and biochemicals. However, like other sources of lignocellulosic biomass, straw is a heterogeneous material due to its mixed origin from different tissue and cell types. Here, to examine the genotypic effects on biorefinery usage of wheat straw, straw obtained from different wheat cultivars and experimental lines was pretreated with dilute acid. Significant differences between cultivars were observed in the concentrations of glucose and toxic by-products of the liquid hydrolysates. A higher content of xylose than glucose was found in liquid hydrolysates from wheat straw, and the xylose content appeared to be affected by both environmental and genetic factors. Analysis using chromosome substitution lines of the common wheat cultivar Chinese Spring showed that chromosomes 2A and 3A from other wheat cultivars, Hope and Timstein, significantly increased the xylose content. However, no significant relationship was observed between the liquid hydrolysate xylose content and the glucose content obtained from enzymatic saccharification of the acid-insoluble residue. These results highlight the potential of wheat breeding to improve biomass-related traits in wheat straw.

Differences in glucose yield of residues from among varieties of rice, wheat, and sorghum after dilute acid pretreatment.

Bio-refinery processes require use of the most suitable lignocellulosic biomass for enzymatic saccharification and microbial fermentation. Glucose yield from biomass solid fractions obtained after dilute sulfuric acid (1%) pretreatment (at 180 °C) was investigated using 14, 8, and 16 varieties of rice, wheat, and sorghum, respectively. Biomass solid fractions of each crop showed similar cellulose content. However, glucose yield after enzymatic hydrolysis (cellulase loading at 6.6 filter paper unit/g-biomass) was different among the varieties of each crop, indicating genotypic differences for rice, wheat, and sorghum. Nuclear magnetic resonance method revealed that the high residual level of lignin aromatic regions decreased glucose yield from solid fraction of sorghum.