CRISPR/Cas9-mediated disruption of CjACOS5 confers no-pollen formation on sugi trees (Cryptomeria japonica D. Don).

Sugi (Cryptomeria japonica D. Don) is an economically important coniferous tree in Japan. However, abundant sugi pollen grains are dispersed and transported by the wind each spring and cause a severe pollen allergy syndrome (Japanese cedar pollinosis). The use of pollen-free sugi that cannot produce pollen has been thought as a countermeasure to Japanese cedar pollinosis. The sugi CjACOS5 gene is an ortholog of Arabidopsis ACOS5 and rice OsACOS12, which encode an acyl-CoA synthetase that is involved in the synthesis of sporopollenin in pollen walls. To generate pollen-free sugi, we mutated CjACOS5 using the CRISPR/Cas9 system. As a result of sugi transformation mediated by Agrobacterium tumefaciens harboring the CjACOS5-targeted CRISPR/Cas9 vector, 1 bp-deleted homo biallelic mutant lines were obtained. Chimeric mutant lines harboring both mutant and wild-type CjACOS5 genes were also generated. The homo biallelic mutant lines had no-pollen in male strobili, whereas chimeric mutant lines had male strobili with or without pollen grains. Our results suggest that CjACOS5 is essential for the production of pollen in sugi and that its disruption is useful for the generation of pollen-free sugi. In addition to conventional transgenic technology, genome editing technology, including CRISPR/Cas9, can confer new traits on sugi.

CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don).

Cryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

Xyloglucan for generating tensile stress to bend tree stem.

In response to environmental variation, angiosperm trees bend their stems by forming tension wood, which consists of a cellulose-rich G (gelatinous)-layer in the walls of fiber cells and generates abnormal tensile stress in the secondary xylem. We produced transgenic poplar plants overexpressing several endoglycanases to reduce each specific polysaccharide in the cell wall, as the secondary xylem consists of primary and secondary wall layers. When placed horizontally, the basal regions of stems of transgenic poplars overexpressing xyloglucanase alone could not bend upward due to low strain in the tension side of the xylem. In the wild-type plants, xyloglucan was found in the inner surface of G-layers during multiple layering. In situ xyloglucan endotransglucosylase (XET) activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, began at the inner surface layers S1 and S2 and was retained throughout G-layer development, while the incorporation of xyloglucan heptasaccharide (XXXG) for wall loosening occurred in the primary wall of the expanding zone. We propose that the xyloglucan network is reinforced by XET to form a further connection between wall-bound and secreted xyloglucans in order to withstand the tensile stress created within the cellulose G-layer microfibrils.

Stable expression of the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 in hybrid poplar cells.

The cbnA gene encoding chlorocatechol dioxygenase from the soil bacterium Ralstonia eutropha NH9 under the control of a modified cauliflower mosaic virus 35S promoter was introduced into a hybrid poplar (Populus tremula x P. tremuloides). Integration of the cbnA gene in transgenic poplar was confirmed by PCR and genomic Southern blot analysis. Expression of the cbnA gene was analyzed by Western blot analysis. Transgenic poplar calli efficiently converted 3-chlorocatechol to 2-chloro-cis,cis-muconate.

Regeneration of transgenic Cryptomeria japonica D. Don after Agrobacterium tumefaciens-mediated transformation of embryogenic tissue.

A genetic transformation procedure for Cryptomeria japonica was developed after co-cultivation of embryogenic tissues with the disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the visual reporter gene sgfp and two selectable marker genes, hpt and nptII. We were able to generate eight and three independent transgenic lines per gram of embryogenic tissue after selection on hygromycin and kanamycin medium, respectively. Transgenic plants were regenerated through somatic embryogenesis in 4 lines out of these 11 lines. Green fluorescent protein fluorescence was observed under fluorescent microscopy. Integration of the genes into the genome was confirmed by polymerase chain reaction analysis of embryogenic tissues and Southern blot analysis of regenerated plantlets.

Biosynthesis of cellulose-enriched tension wood in Populus: global analysis of transcripts and metabolites identifies biochemical and developmental regulators in secondary wall biosynthesis.

Stems and branches of angiosperm trees form tension wood (TW) when exposed to a gravitational stimulus. One of the main characteristics of TW, which distinguishes it from normal wood, is the formation of fibers with a thick inner gelatinous cell wall layer mainly composed of crystalline cellulose. Hence TW is enriched in cellulose, and deficient in lignin and hemicelluloses. An expressed sequence tag library made from TW-forming tissues in Populus tremula (L.) x tremuloides (Michx.) and data from transcript profiling using microarray and metabolite analysis were obtained during TW formation in Populus tremula (L.) in two growing seasons. The data were examined with the aim of identifying the genes responsible for the change in carbon (C) flow into various cell wall components, and the mechanisms important for the formation of the gelatinous cell wall layer (G-layer). A specific effort was made to identify carbohydrate-active enzymes with a putative function in cell wall biosynthesis. An increased C flux to cellulose was suggested by a higher abundance of sucrose synthase transcripts. However, genes related to the cellulose biosynthetic machinery were not generally affected, although the expression of secondary wall-specific CesA genes was modified in both directions. Other pathways for which the data suggested increased activity included lipid and glucosamine biosynthesis and the pectin degradation machinery. In addition, transcripts encoding fasciclin-like arabinogalactan proteins were particularly increased and found to lack true Arabidopsis orthologs. Major pathways for which the transcriptome and metabolome analysis suggested decreased activity were the pathway for C flux through guanosine 5'-diphosphate (GDP) sugars to mannans, the pentose phosphate pathway, lignin biosynthesis, and biosynthesis of cell wall matrix carbohydrates. Several differentially expressed auxin- and ethylene-related genes and transcription factors were also identified.

A transcriptional timetable of autumn senescence.

BACKGROUND: We have developed genomic tools to allow the genus Populus (aspens and cottonwoods) to be exploited as a full-featured model for investigating fundamental aspects of tree biology. We have undertaken large-scale expressed sequence tag (EST) sequencing programs and created Populus microarrays with significant gene coverage. One of the important aspects of plant biology that cannot be studied in annual plants is the gene activity involved in the induction of autumn leaf senescence. RESULTS: On the basis of 36,354 Populus ESTs, obtained from seven cDNA libraries, we have created a DNA microarray consisting of 13,490 clones, spotted in duplicate. Of these clones, 12,376 (92%) were confirmed by resequencing and all sequences were annotated and functionally classified. Here we have used the microarray to study transcript abundance in leaves of a free-growing aspen tree (Populus tremula) in northern Sweden during natural autumn senescence. Of the 13,490 spotted clones, 3,792 represented genes with significant expression in all leaf samples from the seven studied dates. CONCLUSIONS: We observed a major shift in gene expression, coinciding with massive chlorophyll degradation, that reflected a shift from photosynthetic competence to energy generation by mitochondrial respiration, oxidation of fatty acids and nutrient mobilization. Autumn senescence had much in common with senescence in annual plants; for example many proteases were induced. We also found evidence for increased transcriptional activity before the appearance of visible signs of senescence, presumably preparing the leaf for degradation of its components.

Evidence that sucrose loaded into the phloem of a poplar leaf is used directly by sucrose synthase associated with various beta-glucan synthases in the stem.

Sucrose (Suc) synthase (SuSy) is believed to function in channeling UDP-Glc from Suc to various beta-glucan synthases. We produced transgenic poplars (Populus alba) overexpressing a mutant form (S11E) of mung bean (Vigna radiata) SuSy, which appeared in part in the microsomal membranes of the stems. Expression of SuSy in these membranes enhanced the incorporation of radioactive Suc into cellulose, together with the metabolic recycling of fructose (Fru), when dual-labeled Suc was fed directly into the phloem of the leaf. This overexpression also enhanced the direct incorporation of the glucosyl moiety of Suc into the glucan backbone of xyloglucan and increased recycling of Fru, although the Fru recycling system for cellulose synthesis at the plasma membrane might differ from that for xyloglucan synthesis in the Golgi network. These findings suggest that some of the Suc loaded into the phloem of a poplar leaf is used directly by SuSys associated with xyloglucan and cellulose synthases in the stem. This may be a key function of SuSy because the high-energy bond between the Glc and Fru moieties of Suc is conserved and used for polysaccharide syntheses in this sink tissue.

The role of PopCel1 and PopCel2 in poplar leaf growth and cellulose biosynthesis.

Poplar calli transcribed two cellulase (endo-1,4-beta-glucanase) genes, PopCel1 and PopCel2, whose mRNAs were differentially located in the growing leaves of poplar during cell wall synthesis. Histochemical and RT-PCR analyses of promoter-GUS fusion gene activities in transgenic poplar demonstrated that PopCel1 promoter-derived GUS activity was localized in the petiole and leaf veins, whereas PopCel2 was confined to mesophyll cells and disappeared from the tip during the development of leaves. Autoradiography of the leaf showed that the radioactivity of [14C]sucrose incorporated into cellulose corresponded to the combination of the sucrose-induced tissue-specific patterns of PopCel1 and PopCel2. Interestingly, 2,6-dichlorobenzonitrile (DCB) not only inhibited the incorporation of the radioactivity into cellulose, but also repressed the induction of both cellulase genes. Suppression of cellulases by expression of PopCel1 antisense cDNA or co-suppression of PopCel1 mRNA by overexpression of PopCel1 sense cDNA reduced leaf growth. Therefore, we came to the conclusion that PopCel1 and PopCel2 probably function to promote leaf growth in poplar by the endohydrolysis of 1,4-beta-glucan.