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BACKGROUND AND AIMS: The timing of cambial reactivation plays an important role in the control of both the quantity and the quality of wood. The effect of localized heating on cambial reactivation in the main stem of a deciduous hardwood hybrid poplar (Populus sieboldii x P. grandidentata) was investigated. METHODS: Electric heating tape (20-22 °C) was wrapped at one side of the main stem of cloned hybrid poplar trees at breast height in winter. Small blocks were collected from both heated and non-heated control portions of the stem for sequential observations of cambial activity and for studies of the localization of storage starch around the cambium from dormancy to reactivation by light microscopy. KEY RESULTS: Cell division in phloem began earlier than cambial reactivation in locally heated portions of stems. Moreover, the cambial reactivation induced by localized heating occurred earlier than natural cambial reactivation. In heated stems, well-developed secondary xylem was produced that had almost the same structure as the natural xylem. When cambial reactivation was induced by heating, the buds of trees had not yet burst, indicating that there was no close temporal relationship between bud burst and cambial reactivation. In heated stems, the amount of storage starch decreased near the cambium upon reactivation of the cambium. After cambial reactivation, storage starch disappeared completely. Storage starch appeared again, near the cambium, during xylem differentiation in heated stems. CONCLUSIONS: The results suggest that, in deciduous diffuse-porous hardwood poplar growing in a temperate zone, the temperature in the stem is a limiting factor for reactivation of phloem and cambium. An increase in temperature might induce the conversion of storage starch to sucrose for the activation of cambial cell division and secondary xylem. Localized heating in poplar stems provides a useful experimental system for studies of cambial biology.

We have developed a new plant vector system for repeated transformation (called MAT for multi-autotransformation) in which a chimeric ipt gene, inserted into the transposable element Ac, is used as a selectable marker for transformation. Selectable marker genes conferring antibiotic or herbicide resistance, used to introduce economically valuable genes into crop plants, have three major problems: (i) the selective agents have negative effects on proliferation and differentiation of plant cells; (ii) there is uncertainty regarding the environmental impact of many selectable marker genes; (iii) it is difficult to perform recurrent transformations using the same selectable marker to pyramid desirable genes. The MAT vector system containing the ipt gene and the Ac element is designed to overcome these difficulties. When tobacco leaf segments were transformed and selected, subsequent excision of the modified Ac produced marker-free transgenic tobacco plants without sexual crosses or seed production. In addition, the chimeric ipt gene could be visually used as a selectable marker for transformation of hybrid aspen (Populus sieboldii x Populus grandidentata). The chimeric ipt gene, therefore, is an attractive alternative to the most widely used selectable marker genes. The MAT vector system provides a promising way to shorten breeding time for genetically engineered crops. This method could be particularly valuable for fruit and forest trees, for which long generation times are a more significant barrier to breeding and genetic analysis

Lignin is a cell wall component of vascular plants crucial for survival in terrestrial environments. While p -hydroxyphenyl lignin is minor, it is considered to be localised in the outermost part of the cell wall providing strong adhesion between cells, which determines cell shape. Transport of the lignin precursor from the cytosol to the cell wall is critical to regulate temporal and spatial lignin deposition; however, little information on the transport step is available. Here, we report transport activity of p -glucocoumaryl alcohol, a precursor of p -hydroxyphenyl lignin, in a broad-leaved tree (hybrid poplar, Populus sieboldii  ×  P . grandidentata ) and a coniferous tree (Japanese cypress, Chamaecyparis obtusa ). Membrane vesicles of both trees were prepared from differentiating xylem with vigorous lignification and used for transport assays. Several inhibition assays indicated that not ABC transporters but the proton gradient and V-ATPase are involved in p -glucocoumaryl alcohol transport depending on ATP. These results support the hypothesis that p -glucocoumaryl alcohol is loaded into the secretory vesicles and delivered to the cell wall by exocytosis. Furthermore, this transport mechanism was common in both poplar and Japanese cypress, strongly suggesting that p -glucocoumaryl alcohol transport in the differentiating xylem is conserved within woody plants.

The formation of tracheary elements was induced in calli derived from petioles of hybrid poplar (Populus sieboldii × P. grandidentata) after 10 days of culture on medium that lacked auxin but contained 1 µM brassinolide. Some differentiated cells formed broad regions of cell walls and bordered pits, which are typical features of tracheary elements of secondary xylem. Other differentiated cells resembled tracheary elements of primary xylem, with spiral or reticulate thickening of cell walls. The tracheary elements that developed in calli were formed within cell clusters. This induction system provides a new model for studies of the mechanism of differentiation of secondary xylem cells in vitro.

Key message Calli of hybrid poplar that had been exposed to desiccation in air before transfer to the induction medium differentiated into tracheary elements at higher rates than calli without air desiccation. Cells of hybrid poplar ( Populus sieboldii x P. grandidentata ) in culture can be induced to differentiate into secondary xylem-like tracheary elements that form the highly developed bordered pits and broad regions of cell walls in contrast to helical or reticulate wall thickenings in primary xylem elements. We attempted to increase the rate of differentiation of tracheary elements from calli using a combination of hormonal stimulation and partial desiccation. Calli that had been exposed to desiccation in air in a clean hood for 90 min before transfer to the induction medium differentiated into tracheary elements at higher rates than calli without air desiccation. The partial desiccation treatment had no effects on the features of the induced tracheary elements and the frequencies with which they appeared. Our results show that partial desiccation can increase, approximately threefold the rate of differentiation of secondary xylem-like tracheary elements from calli of hybrid poplar. This improvement in the rate of differentiation tracheary elements in vitro should facilitate detailed future analysis of the differentiation of secondary xylem.

Differences in the timing of cell death, differentiation and function among three different types of ray parenchyma cells in the hardwood Populus sieboldii × P. grandidentata which form uniseriate and homocellular rays were examined and clarified. Ray parenchyma cells died within 5 years, and the disappearance of nuclei from ray parenchyma cells did not occur successively from the pith side, even within individual radial cell lines of a given ray. Cell death occurred earliest in contact cells, which were connected to adjacent vessel elements through pits, in the fourth annual ring from the cambium. Cell death occurred next in intermediate cells, which were located within the same cell lines as contact cells but were not adjacent to vessel elements, in the fourth annual ring from the cambium. Finally, isolation cells, which were located within the other cell lines of a given ray, died in the fifth annual ring from the cambium. Secondary wall thickenings in contact cells and intermediate cells were initiated before those in isolation cells in the current year’s xylem. Most starch grains were localized in intermediate cells, and there were more lipid droplets in contact cells and intermediate cells than in isolation cells. In addition, the largest quantities of protein were found in contact cells. Our results indicate that the position within a ray and neighboring short-lived vessel elements might affect the timing of cell death and differentiation and, thus, the function of long-lived ray parenchyma cells in Populus sieboldii × P. grandidentata.

We previously demonstrated that overexpression of the horseradish (Armoracia rusticana) peroxidase prxC1a gene stimulated the growth rate of tobacco (Nicotiana tabacum) plants. Here, the cauliflower mosaic virus 35S::prxC1a construct was introduced into hybrid aspen (Populus sieboldii × Populus grandidentata). The growth rate of these transformed hybrid aspen plants was substantially increased under greenhouse conditions. The average stem length of transformed plants was 25% greater than that of control plants. There was no other obvious phenotypic difference between the transformed and control plants. Fast-growing transformed hybrid aspen showed high levels of expression of prxC1a and had elevated peroxidase activities toward guaiacol and ascorbate. However, there was no increase of the endogenous class I ascorbate peroxidase activities in the transformed plants by separate assay and activity staining of native polyacrylamide gel electrophoresis. Furthermore, calli derived from the transformed hybrid aspen grew faster than those from control plants and were resistant to the oxidative stress imposed by hydrogen peroxide. Therefore, enhanced peroxidase activity affects plant growth rate and oxidative stress resistance.

It is generally accepted that peroxidases catalyze the final step in the biosynthesis of lignin. In this study, to examine how expression of prxA3a, a gene for an anionic peroxidase, might be related to lignification in plant tissues, we produced transgenic tobacco plants that harbored a gene for beta-glucuronidase (GUS) fused to the prxA3a promoter. Histochemical staining for GUS activity indicated that the prxA3a promoter was active mainly in the lignifying cells of stem tissues. Further, to examine the effects of suppressing the expression of prxA3a, we transferred an antisense prxA3a gene construct into the original host, hybrid aspen ( Populus sieboldii x P. gradidentata), under the control of the original promoter of the prxA3a gene. Eleven transformed aspens were obtained and characterized, and the stable integration of the antisense construct was confirmed by PCR and Southern blotting analysis in all these lines. Assays of enzymatic activity showed that both total peroxidase activity and acidic peroxidase activity were lower in most transgenic lines than in the control plants. In addition, the reduction of peroxidase activity was associated with lower lignin content and modified lignin composition. Transgenic lines with the highest reduction of peroxidase activity displayed a higher syringyl/vanillin (S/V) ratio and a lower S+V yield, mainly because of a decreased amount of V units. Thus, our results indicate that prxA3a is involved in the lignification of xylem tissue and that the down-regulation of anionic peroxidase alters both lignin content and composition in hybrid aspen.

A bacterial strain designated PtRA-8T was isolated from the trunk surface of a Japanese aspen tree (Populus tremula var. sieboldii). Cells of strain PtRA-8T were aerobic, non-motile, non-spore forming, Gram-stain-negative rods, 1.0‒2.0 µm in width and 3.0‒10.0 µm in length. The pH range for growth was between 5.5 and 7.5, with an optimum at 6.5. The temperature range for growth was between 10 and 37 °C, with an optimum at around 25‒30 °C. Strain PtRA-8T was highly resistant to UV irradiation, similar to its Deinococcus relatives. The respiratory quinone was menaquinone MK-8. The major cellular fatty acids (> 10% of the total fatty acid content) were iso-C15:0 (17.8%), C16:0 (15.0%), iso-C17:0 (10.4%), and iso-C17:1 ω9c/C16:010-methyl (22.2%). The polar lipids consisted of four unidentified glycolipids, two unidentified aminolipids, two unidentified phospholipids, and three unidentified polar lipids. The peptidoglycan was A3β-type containing glutamic acid, glycine, alanine, and ornithine. The DNA G + C content of strain PtRA-8T was 68.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PtRA-8T was closely related to “Deinococcus radioresistens” 8AT (97.4%), Deinococcus metalli DSM 27521T (95.7%), and Deinococcus yunweiensis YIM 007T (94.5%). The DNA–DNA hybridization experiments between strain PtRA-8T and its relatives yielded relatedness values below 70%. Based on the polyphasic evidence, we concluded that strain PtRA-8T represents a novel species within the genus Deinococcus, for which the name Deinococcus populi is proposed. The type strain of D. populi is PtRA-8T (= DSM 29820T= NBRC 110763T; DPD TaxonNumber TA00271).