SummaryStems 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.) · 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.

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.

A microarray has been created from 36,354 ESTs from Populus species and used to study autumn senescence in the leaves of the aspen tree Populus tremula.

Sucrose (Suc) synthase (SuSy) is believed to function in channeling UDP-Glc from Suc to various β-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.

SummarySuspension-cultured poplar (Populus alba) cells produce two distinct endo-1,4-b-glucanases, one of which is released in the extracellular culture medium and the other localized in their walls. Two cDNA clones, PopCel1 and PopCel2, isolated from a poplar cDNA library, encode the extracellular and the wallbound endo-1,4-b-glucanases, respectively, based upon deduced amino acid sequences. The products of these two genes contained domains conserved in endo-1,4-b-glucanase (family 9) and showed 91.5% amino acid identity. The levels of both PopCel1 and PopCel2 mRNAs increased during the lag phase of growth and decreased rapidly during the linear phase. After the levels had decreased, they were again increased by addition of sucrose to the culture medium and further enhanced by the addition of 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of sucrose. The accumulation of the mRNAs was correlated with the solubilization of cello-oligosaccharides. Cello-oligosaccharides and xyloglucan were also solubilized from the wall preparations of poplar cells incubated with enzyme preparations from the extracellular culture medium and walls. An antibody against both PopCel proteins reduced the production of cello-oligosaccharides by the extracellular enzyme by 90% and that by the wall-bound enzyme by 55%, and also prevented xyloglucan solubilization. The results show that the accumulation of poplar endo-1,4-b-glucanases is regulated indirectly by auxin in the presence of sucrose and can act on cellulose in suspension-cultured poplar cells.

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.

SummaryPoplar calli transcribed two cellulase (endo-1,4-b-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 con®ned to mesophyll cells and disappeared from the tip during the development of leaves. Autoradiography of the leaf showed that the radioactivity of [ 14 C]sucrose incorporated into cellulose corresponded to the combination of the sucrose-induced tissue-speci®c 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 cosuppression 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-b-glucan.

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.