Rerouting of the lignin biosynthetic pathway by inhibition of cytosolic shikimate recycling in transgenic hybrid aspen.

Lignin is a phenolic polymer deposited in the plant cell wall, and is mainly polymerized from three canonical monomers (monolignols), i.e. p-coumaryl, coniferyl and sinapyl alcohols. After polymerization, these alcohols form different lignin substructures. In dicotyledons, monolignols are biosynthesized from phenylalanine, an aromatic amino acid. Shikimate acts at two positions in the route to the lignin building blocks. It is part of the shikimate pathway that provides the precursor for the biosynthesis of phenylalanine, and is involved in the transesterification of p-coumaroyl-CoA to p-coumaroyl shikimate, one of the key steps in the biosynthesis of coniferyl and sinapyl alcohols. The shikimate residue in p-coumaroyl shikimate is released in later steps, and the resulting shikimate becomes available again for the biosynthesis of new p-coumaroyl shikimate molecules. In this study, we inhibited cytosolic shikimate recycling in transgenic hybrid aspen by accelerated phosphorylation of shikimate in the cytosol through expression of a bacterial shikimate kinase (SK). This expression elicited an increase in p-hydroxyphenyl units of lignin and, by contrast, a decrease in guaiacyl and syringyl units. Transgenic plants with high SK activity produced a lignin content comparable to that in wild-type plants, and had an increased processability via enzymatic saccharification. Although expression of many genes was altered in the transgenic plants, elevated SK activity did not exert a significant effect on the expression of the majority of genes responsible for lignin biosynthesis. The present results indicate that cytosolic shikimate recycling is crucial to the monomeric composition of lignin rather than for lignin content.

Prior secondary cell wall formation is required for gelatinous layer deposition and posture control in gravi-stimulated aspen.

Cell walls, especially secondary cell walls (SCWs), maintain cell shape and reinforce wood, but their structure and shape can be altered in response to gravity. In hardwood trees, tension wood is formed along the upper side of a bending stem and contains wood fiber cells that have a gelatinous layer (G-layer) inside the SCW. In a previous study, we generated nst/snd quadruple-knockout aspens (Populus tremula × Populus tremuloides), in which SCW formation was impaired in 99% of the wood fiber cells. In the present study, we produced nst/snd triple-knockout aspens, in which a large number of wood fibers had thinner SCWs than the wild type (WT) and some had no SCW. Because SCW layers are always formed prior to G-layer deposition, the nst/snd mutants raise interesting questions of whether the mutants can form G-layers without SCW and whether they can control their postures in response to changes in gravitational direction. The nst/snd mutants and the WT plants showed growth eccentricity and vessel frequency reduction when grown on an incline, but the triple mutants recovered their upright growth only slightly, and the quadruple mutants were unable to maintain their postures. The mutants clearly showed that the G-layers were formed in SCW-containing wood fibers but not in those lacking the SCW. Our results indicate that SCWs are essential for G-layer formation and posture control. Furthermore, each wood fiber cell may be able to recognize its cell wall developmental stage to initiate the formation of the G-layer as a response to gravistimulation.

Plasticity of functional traits and optimality of biomass allocation in elevational ecotypes of Arabidopsis halleri grown at different soil nutrient availabilities.

In mountainous areas, plant distribution is constrained by various environmental stresses. Plasticity and constancy in plant functional traits may relate to optimal strategies at respective habitats and to ecotypic differentiation along elevation. Although plant biomass allocation has been extensively studied in relation to adaptation to soil nutrient availability along elevation, its optimality is still poorly understood. We examined soil nutrient availability in the field and conducted growth analysis for two elevational ecotypes of Arabidopsis halleri grown under different nutrient availabilities. We determined plasticity in morphological and physiological traits and evaluated optimal biomass allocation using an optimality model. Our field investigation indicated that soil nitrogen (N) availability increased rather than decreased with increasing elevation. Our growth analysis revealed that lowland ecotype was more plastic in morphological variables and N concentrations, whereas the highland ecotype was more plastic in other physiological variables such as the net assimilation rate (NAR). The leaf mass ratio (LMR) in the lowland ecotype was moderately plastic at the whole range of N availabilities, whereas LMR in the highland ecotype was very plastic at higher N availabilities only. The optimality model indicated that the LMR of the lowland ecotype was nearly optimal throughout the range of studied N availabilities, whereas that of the highland ecotype was suboptimal at low N availability. These results suggest that highland ecotype is adapted only to high N availability, whereas the lowland ecotype is adapted to a relatively wide range of N availabilities as a result of natural selection in their respective habitats. We conclude that an adaptive differentiation has occurred between the two ecotypes and plasticity in the biomass allocation is directly related to its optimization in changing environments.

Discrimination of candidate subgenome-specific loci by linkage map construction with an S1 population of octoploid strawberry (Fragaria × ananassa).

BACKGROUND: The strawberry, Fragaria × ananassa, is an allo-octoploid (2n = 8x = 56) and outcrossing species. Although it is the most widely consumed berry crop in the world, its complex genome structure has hindered its genetic and genomic analysis, and thus discrimination of subgenome-specific loci among the homoeologous chromosomes is needed. In the present study, we identified candidate subgenome-specific single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) loci, and constructed a linkage map using an S1 mapping population of the cultivar 'Reikou' with an IStraw90 Axiom® SNP array and previously published SSR markers. RESULTS: The 'Reikou' linkage map consisted of 11,574 loci (11,002 SNPs and 572 SSR loci) spanning 2816.5 cM of 31 linkage groups. The 11,574 loci were located on 4738 unique positions (bin) on the linkage map. Of the mapped loci, 8999 (8588 SNPs and 411 SSR loci) showed a 1:2:1 segregation ratio of AA:AB:BB allele, which suggested the possibility of deriving loci from candidate subgenome-specific sequences. In addition, 2575 loci (2414 SNPs and 161 SSR loci) showed a 3:1 segregation of AB:BB allele, indicating they were derived from homoeologous genomic sequences. Comparative analysis of the homoeologous linkage groups revealed differences in genome structure among the subgenomes. CONCLUSIONS: Our results suggest that candidate subgenome-specific loci are randomly located across the genomes, and that there are small- to large-scale structural variations among the subgenomes. The mapped SNPs and SSR loci on the linkage map are expected to be seed points for the construction of pseudomolecules in the octoploid strawberry.

Mutant selection in the self-incompatible plant radish (Raphanus sativus L. var. sativus) using two-step TILLING.

Radish (Raphanus sativus L. var. sativus), a widely cultivated root vegetable crop, possesses a large sink organ (the root), implying that photosynthetic activity in radish can be enhanced by altering both the source and sink capacity of the plant. However, since radish is a self-incompatible plant, improved mutation-breeding strategies are needed for this crop. TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful method used for reverse genetics. In this study, we developed a new TILLING strategy involving a two-step mutant selection process for mutagenized radish plants: the first selection is performed to identify a BC1M1 line, that is, progenies of M1 plants crossed with wild-type, and the second step is performed to identify BC1M1 individuals with mutations. We focused on Rubisco as a target, since Rubisco is the most abundant plant protein and a key photosynthetic enzyme. We found that the radish genome contains six RBCS genes and one pseudogene encoding small Rubisco subunits. We screened 955 EMS-induced BC1M1 lines using our newly developed TILLING strategy and obtained six mutant lines for the six RsRBCS genes, encoding proteins with four different types of amino acid substitutions. Finally, we selected a homozygous mutant and subjected it to physiological measurements.

Development and characterization of a strawberry MAGIC population derived from crosses with six strawberry cultivars.

A strawberry Multi-parent Advanced Generation Intercrosses (MAGIC) population, derived from crosses using six strawberry cultivars was successfully developed. The population was composed of 338 individuals; genome conformation was evaluated by expressed sequence tag-derived simple short repeat (EST-SSR) markers. Cluster analysis and principal component analysis (PCA) based on EST-SSR marker polymorphisms revealed that the MAGIC population was a mosaic of the six founder cultivars and covered the genomic regions of the six founders evenly. Fruit quality related traits, including days to flowering (DTF), fruit weight (FW), fruit firmness (FF), fruit color (FC), soluble solid content (SC), and titratable acidity (TA), of the MAGIC population were evaluated over two years. All traits showed normal transgressive segregation beyond the founder cultivars and most traits, except for DTF, distributed normally. FC exhibited the highest correlation coefficient overall and was distributed normally regardless of differences in DTF, FW, FF, SC, and TA. These facts were supported by PCA using fruit quality related values as explanatory variables, suggesting that major genetic factors, which are not influenced by fluctuations in other fruit traits, could control the distribution of FC. This MAGIC population is a promising resource for genome-wide association studies and genomic selection for efficient strawberry breeding.

Efficient selection of a biallelic and nonchimeric gene-edited tree using Oxford Nanopore Technologies sequencing.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease system is a versatile and essential biotechnological tool in the life sciences that allows efficient genome editing. When generating gene-edited trees, T0-generation plants are often used for subsequent analysis because of the time that is required to obtain the desired mutants via crossing. However, T0-generation plants exhibit various unexpected mutations, which emphasizes the need to identify mutants with expected mutation patterns. The two critical checkpoints in this process are to confirm the expected mutation patterns in both alleles and to exclude somatic chimeric plants. In this study, we generated gene-edited Cryptomeria japonica plants and established a method to determine chimerism and mutation patterns using fragment analysis and Oxford Nanopore Technologies (ONT)-based amplicon sequencing. In the first screening, fragment analysis, i.e., indel detection via amplicon analysis, was used to predict indel mutation patterns in both alleles and to discriminate somatic chimeric plants in 188 candidate mutants. In the second screening, we precisely determined the mutation patterns and chimerism in the mutants using ONT-based amplicon sequencing, where confirmation of both alleles can be achieved using allele-specific markers flanking the single guide RNA target site. In the present study, a bioinformatic analysis procedure was developed and provided for the rapid and accurate determination of DNA mutation patterns using ONT-based amplicon sequencing. As ONT amplicon sequencing has a low running cost compared with other long-read analysis methods, such as PacBio, it is a powerful tool in plant genetics and biotechnology to select gene-edited plants with expected indel patterns in the T0-generation.

Effect of differences in light source environment on transcriptome of leaf lettuce (Lactuca sativa L.) to optimize cultivation conditions.

When used in closed-type plant factories, light-emitting diode (LED) illumination systems have the particular advantages of low heat emission and high luminous efficiency. The effects of illumination quality and intensity on the growth and morphogenesis of many plant species have been examined, but improvements are needed to optimize the illumination systems for better plant products with lower resource investments. In particular, new strategies are needed to reduce the wastage of plant products related to leaf senescence, and to better control the ingredients and appearance of leafy vegetables. Although the quality of light is often altered to change the characteristics of plant products, the transcriptional status underlying the physiological responses of plants to light has not been established. Herein, we performed a comprehensive gene expression analysis using RNA-sequencing to determine how red, blue, and red/blue LEDs and fluorescent light sources affect transcriptome involved in the leaf aging of leaf lettuce. The RNA-sequencing profiling revealed clear differences in the transcriptome between young and old leaves. Red LED light caused large variation between the two age classes, while a pure or mixed blue LED light spectrum induced fewer transcriptome differences between young and old leaves. Collectively, the expression levels of genes that showed homology with those of other model organisms provide a detailed physiological overview, incorporating such characteristics as the senescence, nutrient deficiency, and anthocyanin synthesis of the leaf lettuce plants. Our findings suggest that transcriptome profiles of leaf lettuce grown under different light sources provide helpful information to achieve better growth conditions for marketable and efficient green-vegetable production, with improved wastage control and efficient nutrient inputs.

A chromosome-scale draft genome sequence of horsegram (Macrotyloma uniflorum).

Horsegram (Macrotyloma uniflorum [Lam.] Verdc.) is an underutilized warm-season diploid legume (2n = 20, 22). Because of its ability to grow under water-deficient and marginal soil conditions, horsegram is a preferred choice in the era of global climate change. In recognition of its potential as a crop species, we generated and analyzed a draft genome sequence for a horsegram variety, HPK-4. Ten chromosome-scale pseudomolecules were created by aligning Illumina scaffold sequences onto a linkage map. The total length of the ten pseudomolecules was 259.2 Mbp, covering 89% of the total length of the assembled sequences. A total of 36,105 genes were predicted on the assembled sequences. Diversity analysis of 89 horsegram accessions by dd-RAD-Seq identified 277 single nucleotide polymorphisms (SNPs), suggesting narrow genetic diversity among the horsegram accessions. This is the first attempt to generate a draft genome sequence of horsegram and will provide a reference for sequence-based analysis of horsegram germplasm.

Overexpression of Plasma Membrane H+-ATPase in Guard Cells Enhances Light-Induced Stomatal Opening, Photosynthesis, and Plant Growth in Hybrid Aspen.

Stomata in the plant epidermis open in response to light and regulate CO2 uptake for photosynthesis and transpiration for uptake of water and nutrients from roots. Light-induced stomatal opening is mediated by activation of the plasma membrane (PM) H+-ATPase in guard cells. Overexpression of PM H+-ATPase in guard cells promotes light-induced stomatal opening, enhancing photosynthesis and growth in Arabidopsis thaliana. In this study, transgenic hybrid aspens overexpressing Arabidopsis PM H+-ATPase (AHA2) in guard cells under the strong guard cell promoter Arabidopsis GC1 (AtGC1) showed enhanced light-induced stomatal opening, photosynthesis, and growth. First, we confirmed that AtGC1 induces GUS expression specifically in guard cells in hybrid aspens. Thus, we produced AtGC1::AHA2 transgenic hybrid aspens and confirmed expression of AHA2 in AtGC1::AHA2 transgenic plants. In addition, AtGC1::AHA2 transgenic plants showed a higher PM H+-ATPase protein level in guard cells. Analysis using a gas exchange system revealed that transpiration and the photosynthetic rate were significantly increased in AtGC1::AHA2 transgenic aspen plants. AtGC1::AHA2 transgenic plants showed a>20% higher stem elongation rate than the wild type (WT). Therefore, overexpression of PM H+-ATPase in guard cells promotes the growth of perennial woody plants.

Identifying transcription factors that reduce wood recalcitrance and improve enzymatic degradation of xylem cell wall in Populus.

Developing an efficient deconstruction step of woody biomass for biorefinery has been drawing considerable attention since its xylem cell walls display highly recalcitrance nature. Here, we explored transcriptional factors (TFs) that reduce wood recalcitrance and improve saccharification efficiency in Populus species. First, 33 TF genes up-regulated during poplar wood formation were selected as potential regulators of xylem cell wall structure. The transgenic hybrid aspens (Populus tremula × Populus tremuloides) overexpressing each selected TF gene were screened for in vitro enzymatic saccharification. Of these, four transgenic seedlings overexpressing previously uncharacterized TF genes increased total glucan hydrolysis on average compared to control. The best performing lines overexpressing Pt × tERF123 and Pt × tZHD14 were further grown to form mature xylem in the greenhouse. Notably, the xylem cell walls exhibited significantly increased total xylan hydrolysis as well as initial hydrolysis rates of glucan. The increased saccharification of Pt × tERF123-overexpressing lines could reflect the improved balance of cell wall components, i.e., high cellulose and low xylan and lignin content, which could be caused by upregulation of cellulose synthase genes upon the expression of Pt × tERF123. Overall, we successfully identified Pt × tERF123 and Pt × tZHD14 as effective targets for reducing cell wall recalcitrance and improving the enzymatic degradation of woody plant biomass.

De novo whole-genome assembly in Chrysanthemum seticuspe, a model species of Chrysanthemums, and its application to genetic and gene discovery analysis.

Cultivated chrysanthemum (Chrysanthemum morifolium Ramat.) is one of the most economically important ornamental crops grown worldwide. It has a complex hexaploid genome (2n = 6x = 54) and large genome size. The diploid Chrysanthemum seticuspe is often used as a model of cultivated chrysanthemum, since the two species are closely related. To expand our knowledge of the cultivated chrysanthemum, we here performed de novo whole-genome assembly in C. seticuspe using the Illumina sequencing platform. XMRS10, a C. seticuspe accession developed by five generations of self-crossing from a self-compatible strain, AEV2, was used for genome sequencing. The 2.72 Gb of assembled sequences (CSE_r1.0), consisting of 354,212 scaffolds, covered 89.0% of the 3.06 Gb C. seticuspe genome estimated by k-mer analysis. The N50 length of scaffolds was 44,741 bp. For protein-encoding genes, 71,057 annotated genes were deduced (CSE_r1.1_cds). Next, based on the assembled genome sequences, we performed linkage map construction, gene discovery and comparative analyses for C. seticuspe and cultivated chrysanthemum. The generated C. seticuspe linkage map revealed skewed regions in segregation on the AEV2 genome. In gene discovery analysis, candidate flowering-related genes were newly found in CSE_r1.1_cds. Moreover, single nucleotide polymorphism identification and annotation on the C. × morifolium genome showed that the C. seticuspe genome was applicable to genetic analysis in cultivated chrysanthemums. The genome sequences assembled herein are expected to contribute to future chrysanthemum studies. In addition, our approach demonstrated the usefulness of short-read genome assembly and the importance of choosing an appropriate next genome sequencing technology based on the purpose of the post-genome analysis.

Draft genome sequence of subterranean clover, a reference for genus Trifolium.

Clovers (genus Trifolium) are widely cultivated across the world as forage legumes and make a large contribution to livestock feed production and soil improvement. Subterranean clover (T. subterraneum L.) is well suited for genomic and genetic studies as a reference species in the Trifolium genus, because it is an annual with a simple genome structure (autogamous and diploid), unlike the other economically important perennial forage clovers, red clover (T. pratense) and white clover (T. repens). This report represents the first draft genome sequence of subterranean clover. The 471.8 Mb assembled sequence covers 85.4% of the subterranean clover genome and contains 42,706 genes. Eight pseudomolecules of 401.1 Mb in length were constructed, based on a linkage map consisting of 35,341 SNPs. The comparative genomic analysis revealed that different clover chromosomes showed different degrees of conservation with other Papilionoideae species. These results provide a reference for genetic and genomic analyses in the genus Trifolium and new insights into evolutionary divergence in Papilionoideae species.