Peter Kitin

Antisense Down-Regulation of 4CL Expression Alters Lignification, Tree Growth, and Saccharification Potential of Field-Grown Poplar

Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplar (Populus tremula × Populus alba), we applied this strategy and examined field-grown transformants for both effects on wood biochemistry and tree productivity. The reductions in lignin contents obtained correlated well with 4CL RNA expression, with a sharp decrease in lignin amount being observed for RNA expression below approximately 50% of the nontransgenic control. Relatively small lignin reductions of approximately 10% were associated with reduced productivity, decreased wood syringyl/guaiacyl lignin monomer ratios, and a small increase in the level of incorporation of H-monomers (p-hydroxyphenyl) into cell walls. Transgenic events with less than approximately 50% 4CL RNA expression were characterized by patches of reddish-brown discolored wood that had approximately twice the extractive content of controls (largely complex polyphenolics). There was no evidence that substantially reduced lignin contents increased growth rates or saccharification potential. Our results suggest that the capacity for lignin reduction is limited; below a threshold, large changes in wood chemistry and plant metabolism were observed that adversely affected productivity and potential ethanol yield. They also underline the importance of field studies to obtain physiologically meaningful results and to support technology development with transgenic trees.

Transgenic poplars with reduced lignin show impaired xylem conductivity, growth efficiency and survival

We studied xylem anatomy and hydraulic architecture in 14 transgenic insertion events and a control line of hybrid poplar (Populus spp.) that varied in lignin content. Transgenic events had different levels of down-regulation of two genes encoding 4-coumarate:coenzyme A ligase (4CL). Two-year-old trees were characterized after growing either as free-standing trees in the field or as supported by stakes in a greenhouse. In free-standing trees, a 20 to 40% reduction in lignin content was associated with increased xylem vulnerability to embolism, shoot dieback and mortality. In staked trees, the decreased biomechanical demands on the xylem was associated with increases in the leaf area to sapwood area ratio and wood specific conductivity (k(s)), and with decreased leaf-specific conductivity (k(l)). These shifts in hydraulic architecture suggest that the bending stresses perceived during growth can affect traits important for xylem water transport. Severe 4CL-downregulation resulted in the patchy formation of discoloured, brown wood with irregular vessels in which water transport was strongly impeded. These severely 4CL-downregulated trees had significantly lower growth efficiency (biomass/leaf area). These results underscore the necessity of adequate lignification for mechanical support of the stem, water transport, tree growth and survival.

Tyloses and phenolic deposits in xylem vessels impede water transport in low-lignin transgenic poplars: a study by cryo-fluorescence microscopy

Of 14 transgenic poplar genotypes (Populus tremula × Populus alba) with antisense 4-coumarate:coenzyme A ligase that were grown in the field for 2 years, five that had substantial lignin reductions also had greatly reduced xylem-specific conductivity compared with that of control trees and those transgenic events with small reductions in lignin. For the two events with the lowest xylem lignin contents (greater than 40% reduction), we used light microscopy methods and acid fuchsin dye ascent studies to clarify what caused their reduced transport efficiency. A novel protocol involving dye stabilization and cryo-fluorescence microscopy enabled us to visualize the dye at the cellular level and to identify water-conducting pathways in the xylem. Cryo-fixed branch segments were planed in the frozen state on a sliding cryo-microtome and observed with an epifluorescence microscope equipped with a cryo-stage. We could then distinguish clearly between phenolic-occluded vessels, conductive (stain-filled) vessels, and nonconductive (water- or gas-filled) vessels. Low-lignin trees contained areas of nonconductive, brown xylem with patches of collapsed cells and patches of noncollapsed cells filled with phenolics. In contrast, phenolics and nonconductive vessels were rarely observed in normal colored wood of the low-lignin events. The results of cryo-fluorescence light microscopy were supported by observations with a confocal microscope after freeze drying of cryo-planed samples. Moreover, after extraction of the phenolics, confocal microscopy revealed that many of the vessels in the nonconductive xylem were blocked with tyloses. We conclude that reduced transport efficiency of the transgenic low-lignin xylem was largely caused by blockages from tyloses and phenolic deposits within vessels rather than by xylem collapse.

Bending characteristics of bamboo (Phyllostachys pubescens) with respect to its fiber–foam composite structure

The bending properties of split bamboo culm were compared with those of spruce and beech wood specimens. The bamboo allowed large flexural deformation since its outer layer retains the tensile stress while the softer inner layer undergoes large compressive deformation. The results suggested that the combination of the fiber-rich outer part and the compressible inner part was responsible for the flexural ductility of split bamboo. To clarify the compressible nature of the inner part of bamboo, the longitudinal surfaces of the bamboo and wood specimens were microscopically observed before and after a large longitudinal compression. Although the wood specimens showed serious and localized buckling, the inner part of the bamboo specimens showed no such visible buckling. In the latter case, the foam-like parenchyma cells absorbed the large compressive deformation by their microscopic buckling and simultaneously, the alignment of sclerenchyma fibers was maintained by the surrounding parenchyma matrix. The flexural elasticity of the bamboo was compared to that of the wood in respect of remaining strain during cyclic bending tests. No clear difference was recognized between their remaining strains. This fact indicated that the bamboo was not so flexible elastically, although its fiber–foam combination and intelligent fiber distribution improve flexural ductility.

Anatomy of the vessel network within and between tree rings of Fraxinus lanuginosa (Oleaceae)

The three-dimensional (3-D) arrangement of vessels and the vessel-to-vessel connections in the secondary xylem of the stem of the ring-porous hardwood tree Fraxinus lanuginosa were studied in series of thick transverse sections with epifluorescence microscope and confocal laser scanning microscope. Vessels were traced in sequential sections, and vessel networks were reconstructed in two segments of wood with dimensions of 2 × 1.4 × 21.2 mm(3) and 2 × 1.4 × 5.8 mm(3) (tangential × radial × axial). The arrangement of vessels and intervessel pits were visualized by scanning electron microscopy in low-density polyethylene microcasts and on exposed tangential faces of growth-ring boundaries. The vessels deviated from the stem axis in the tangential direction and, to a lesser extent, in the radial direction. Some neighboring vessels were twisted around each other. Vessels that appeared solitary in single sections were found to be sequentially contiguous with a number of other vessels, forming networks that extended in the tangential direction and across growth-ring boundaries. In the 21.2-mm wood block, all earlywood vessels at the growth-ring boundary made contact with latewood vessels in the previous tree ring. Within a growth ring however, only a single contact was observed between individual earlywood and latewood vessels. Densely arranged intervessel pits were characteristic in the regions where adjacent vessels made contact with each other. Such bordered pits were abundant in the tangential walls of vessel elements adjacent to growth-ring boundaries. Therefore, bordered pits appear to provide the pathway for the radial transport of water via the vessel network across growth-ring borders. Fiber-tracheids, observed as terminal cells in the tree rings, might also contribute to the apoplastic transfer of water across ring borders.

Anatomical features that facilitate radial flow across growth rings and from xylem to cambium in Cryptomeria japonica

BACKGROUND AND AIMS Although the lateral movement of water and gas in tree stems is an important issue for understanding tree physiology, as well as for the development of wood preservation technologies, little is known about the vascular pathways for radial flow. The aim of the current study was to understand the occurrence and the structure of anatomical features of sugi (Cryptomeria japonica) wood including the tracheid networks, and area fractions of intertracheary pits, tangential walls of ray cells and radial intercellular spaces that may be related to the radial permeability (conductivity) of the xylem. METHODS Wood structure was investigated by light microscopy and scanning electron microscopy of traditional wood anatomical preparations and by a new method of exposed tangential faces of growth-ring boundaries. KEY RESULTS Radial wall pitting and radial grain in earlywood and tangential wall pitting in latewood provide a direct connection between subsequent tangential layers of tracheids. Bordered pit pairs occur frequently between earlywood and latewood tracheids on both sides of a growth-ring boundary. In the tangential face of the xylem at the interface with the cambium, the area fraction of intertracheary pit membranes is similar to that of rays (2.8 % and 2.9 %, respectively). The intercellular spaces of rays are continuous across growth-ring boundaries. In the samples, the mean cross-sectional area of individual radial intercellular spaces was 1.2 microm(2) and their total volume was 0.06 % of that of the xylem and 2.07 % of the volume of rays. CONCLUSIONS A tracheid network can provide lateral apoplastic transport of substances in the secondary xylem of sugi. The intertracheid pits in growth-ring boundaries can be considered an important pathway, distinct from that of the rays, for transport of water across growth rings and from xylem to cambium.

Trade-offs between biomass growth and inducible biosynthesis of polyhydroxybutyrate in transgenic poplar

Polyhydroxybutyrate (PHB) is a bioplastic that can be produced in transgenic plants by the coexpression of three bacterial genes for its biosynthesis. PHB yields from plants have been constrained by the negative impacts on plant health that result from diversion of resources into PHB production; thus, we employed an ecdysone analogue-based system for induced gene expression. We characterized 49 insertion events in hybrid transgenic poplar (Populus tremula x alba) that were produced using Agrobacterium transformation and studied two high-producing events in detail. Regenerated plants contained up to 1-2% PHB (dry weight) in leaves after 6-8 weeks of induction. Strong induction was observed with 1-10 mm Intrepid and limited direct toxicity observed. Confocal fluorescence microscopy was used to visualize PHB granules in chloroplasts after chemical treatment to reduce autofluorescence. A greenhouse study indicated that there were no negative consequences of PHB production on growth unless the PHB content exceeded 1% of leaf weight; at PHB levels above 1%, growth (height, diameter and total mass) decreased by 10%-34%.

Involvement of Localized Cortical Microtubules in the Formation of a Modified Structure of Wood

Cambial cells dierentiate into secondary xylem cells through a process of expansion or elongation, cell wall thickening, cell wall sculpturing, lignification, and cell death (formation of wood). The secondary xylem cells develop modifications of the cell wall such as pits, helical thickenings, perforations and warts, through the localized deposition of cell wall materials. Recent observations have revealed that the localized appearance or disappearance of cortical microtubules is related to the localized deposition of cellulose microfibrils in secondary xylem cells. Cortical microtubules play an important role in the morphogenesis of secondary xylem cells, thereby controlling the structure of wood. Therefore, cortical microtubules provide a target for biotechnological applications to change the quality of wood.

ANALYSIS OF CAMBIUM AND DIFFERENTIATING VESSEL ELEMENTS IN KALOPANAX PICTUS USING RESIN CAST REPLICAS

A resin-casting method with subsequent scanning electron microscopy (SEM) was used to examine the three-dimensional (3-D) shapes of cells and the cell walls of cambium and differentiating xylem. Glutaraldehyde- fixed and dehydrated specimens were embedded in polystyrene and then organic material was removed by digestion with acidic solutions or enzymes. The acidic solutions used for treatment were sulphuric acid and a mixture of acetic acid and hydrogen peroxide and the enzymes used for treatment were pectinase and cellulase, with a final treatment with sodium hypochlorite. Both methods could be used for studies of the differentiation of cambial cells; however, digestion with enzymes allowed better preservation of the 3-D organisation of the tissue. Negative replicas of inner surfaces of cell walls of differentiating vessel elements revealed the sequential stages of the development of bordered pits and perforation plates. Future bordered pits at the early stages of the differentiation of cell walls were demarcated by the accumulation of organic material between adjacent pit membranes. Subsequent deposition of cell wall material resulted in formation of pit cavities and the rims of perforation plates.

EARLYWOOD VESSELS IN RING-POROUS TREES BECOME FUNCTIONAL FOR WATER TRANSPORT AFTER BUD BURST AND BEFORE THE MATURATION OF THE CURRENT-YEAR LEAVES

This paper reviews the development of xylem vessels in ring-porous dicots and the corresponding leaf phenology. Also included are our original observations on the time-course of vessel element growth, secondary wall deposition, and end wall perforation in the deciduous hardwood Kalopanax septemlobus. Different patterns of xylem growth and phenology serve different strategies of the species for adaptation to seasonal climates. Trees with ring-porous xylem form wide earlywood vessels (EWV) in spring and narrow latewood vessels in summer. The wide EWV become embolized or blocked with tyloses by the end of the growing season while the narrow vessels may remain functional for many years. The co-occurrence of wide and narrow vessels provides both efficiency and safety of the water transport as well as a potentially longer growing season. It has for a long time been assumed that EWV in ring-porous hardwoods are formed in early spring before bud burst in order to supply sap to growing leaves and shoots.However, the full time-course of development of EWV elements from initiation of growth until maturation for water transport has not been adequately studied until recently. Our observations clarify a crucial relationship between leaf maturation and the maturation of earlywood vessels for sap transport. Accumulated new evidence shows that EWV in branches and upper stem parts develop earlier than EWV lower in the stem. The first EWV elements are fully expanded with differentiated secondary walls by the time of bud burst. In lower stem parts, perforations in vessel end walls are formed after bud burst and before the new leaves have achieved full size. Therefore, the current-year EWV network becomes functional for water transport only by the time when the first new leaves are mature.