Steven L. Voelker

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.

Moving water well: comparing hydraulic efficiency in twigs and trunks of coniferous, ring‐porous, and diffuse‐porous saplings from temperate and tropical forests

*Coniferous, diffuse-porous and ring-porous trees vary in their xylem anatomy, but the functional consequences of these differences are not well understood from the scale of the conduit to the individual. *Hydraulic and anatomical measurements were made on branches and trunks from 16 species from temperate and tropical areas, representing all three wood types. Scaling of stem conductivity (K(h)) with stem diameter was used to model the hydraulic conductance of the stem network. *Ring-porous trees showed the steepest increase in K(h) with stem size. Temperate diffuse-porous trees were at the opposite extreme, and conifers and tropical diffuse-porous species were intermediate. Scaling of K(h) was influenced by differences in the allometry of conduit diameter (taper) and packing (number per wood area) with stem size. *The K(h) trends were mirrored by the modeled stem-network conductances. Ring-porous species had the greatest network conductance and this value increased isometrically with trunk basal area, indicating that conductance per unit sapwood was independent of tree size. Conductances were lowest and most size-dependent in conifers. The results indicate that differences in conduit taper and packing between functional types propagate to the network level and have an important influence on metabolic scaling concepts.

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.

Reduced wood stiffness and strength, and altered stem form, in young antisense 4CL transgenic poplars with reduced lignin contents

• Reduced lignin content in perennial crops has been sought as a means to improve biomass processability for paper and biofuels production, but it is unclear how this could affect wood properties and tree form. • Here, we studied a nontransgenic control and 14 transgenic events containing an antisense 4-coumarate:coenzyme A ligase (4CL) to discern the consequences of lignin reduction in poplar (Populus sp.). During the second year of growth, trees were grown either free-standing in a field trial or affixed to stakes in a glasshouse. • Reductions in lignin of up to 40% gave comparable losses in wood strength and stiffness. This occurred despite the fact that low-lignin trees had a similar wood density and up to three-fold more tension wood. In free-standing and staked trees, the control line had twice the height for a given diameter as did low-lignin trees. Staked trees had twice the height for a given diameter as free-standing trees in the field, but did not differ in wood stiffness. • Variation in tree morphogenesis appears to be governed by lignin x environment interactions mediated by stresses exerted on developing cells. Therefore our results underline the importance of field studies for assessing the performance of transgenic trees with modified wood properties.

Hydraulic architecture of two species differing in wood density: opposing strategies in co-occurring tropical pioneer trees

Co-occurring species often have different strategies for tolerating daily cycles of water stress. One underlying parameter that can link together the suite of traits that enables a given strategy is wood density. Here we compare hydraulic traits of two pioneer species from a tropical forest in Panama that differ in wood density: Miconia argentea and Anacardium excelsum. As hypothesized, the higher wood density of Miconia was associated with smaller diameter vessels and fibres, more water stress-resistant leaves and stems, and roughly half the capacitance of the lower wood density Anacardium. However, the scaling of hydraulic parameters such as the increases in leaf area and measures of hydraulic conductivity with stem diameter was remarkably similar between the two species. The collection of traits exhibited by Miconia allowed it to tolerate more water stress than Anacardium, which relied more heavily on its capacitance to buffer daily water potential fluctuations. This work demonstrates the importance of examining a range of hydraulic traits throughout the plant and highlights the spectrum of possible strategies for coping with daily and seasonal water stress cycles.

HISTORICAL CO2 GROWTH ENHANCEMENT DECLINES WITH AGE IN QUERCUS AND PINUS

Despite experimental evidence showing that elevated C02 levels increase growth in most plants, the isolation of a signal consistent with anthropogenically caused increases in atmospheric C02 from the dendrochronological record has shown mixed results. Our extensive sets of tree ring data from the Ozark Mountains in Missouri showed that since 1850, Quercus velutina Lam., Quercus cocc?nea Muench., and Pinus echinata Mill, trees increased in stem growth coincidently with increases in atmospheric C02. Those long-term increases in radial growth appear unrelated to historical disturbance levels for the region, to long-term changes in relevant climatic variables, or to productivity of sites sampled for the purpose of creating a time sequence of tree ring growth. It is still unclear what the potential role of nitrogen deposition might have been for tree growth. We cross-dated a large number of increment cores and aligned the ring width data by pith date for accurate age constant assessments of growth over the past 150 years. Thus, we circumvented changes in growth trend associated with differences in physiological functioning during development, as well as the need for statistical detrending that removes an unknown degree of long-term environmental signal, the so called segment length curse that applies to standard dendrochronological investigations. When the positive relationship between C02 and ring width was examined at different ages, an ontogenetic decline in the rate of growth stimulation was found. Specifically, both the pooled Quercus spp. and P. echinata were characterized by a negative exponential pattern of response over a developmental sequence through age 50. Further knowledge of an intrinsic decline in C02 sensitivity with tree age or size such as this may be important for increased accuracy in estimating terrestrial carbon stocks across successional landscapes.

Comparative hydraulic architecture of tropical tree species representing a range of successional stages and wood density

Plant hydraulic architecture (PHA) has been linked to water transport sufficiency, photosynthetic rates, growth form and attendant carbon allocation. Despite its influence on traits central to conferring an overall competitive advantage in a given environment, few studies have examined whether key aspects of PHA are indicative of successional stage, especially within mature individuals. While it is well established that wood density (WD) tends to be lower in early versus late successional tree species, and that WD can influence other aspects of PHA, the interaction of WD, successional stage and the consequent implications for PHA have not been sufficiently explored. Here, we studied differences in PHA at the scales of wood anatomy to whole-tree hydraulic conductance in species in early versus late successional Panamanian tropical forests. Although the trunk WD was indistinguishable between the successional groups, the branch WD was lower in the early successional species. Across all species, WD correlated negatively with vessel diameter and positively with vessel packing density. The ratio of branch:trunk vessel diameter, branch sap flux and whole-tree leaf-specific conductance scaled negatively with branch WD across species. Pioneer species showed greater sap flux in branches than in trunks and a greater leaf-specific hydraulic conductance, suggesting that pioneer species can move greater quantities of water at a given tension gradient. In combination with the greater water storage capacitance associated with lower WD, these results suggest these pioneer species can save on the carbon expenditure needed to build safer xylem and instead allow more carbon to be allocated to rapid growth.

Seasonal carbohydrate dynamics and growth in Douglas-fir trees experiencing chronic, fungal-mediated reduction in functional leaf area

Stored non-structural carbohydrates (NSCs) could play an important role in tree survival in the face of a changing climate and associated stress-related mortality. We explored the effects of the stomata-blocking and defoliating fungal disease called Swiss needle cast on Douglas-fir carbohydrate reserves and growth to evaluate the extent to which NSCs can be mobilized under natural conditions of low water stress and restricted carbon supply in relation to potential demands for growth. We analyzed the concentrations of starch, sucrose, glucose and fructose in foliage, twig wood and trunk sapwood of 15 co-occurring Douglas-fir trees expressing a gradient of Swiss needle cast symptom severity quantified as previous-year functional foliage mass. Growth (mean basal area increment, BAI) decreased by ∼80% and trunk NSC concentration decreased by 60% with decreasing functional foliage mass. The ratio of relative changes in NSC concentration and BAI, an index of the relative priority of storage versus growth, more than doubled with increasing disease severity. In contrast, twig and foliage NSC concentrations remained nearly constant with decreasing functional foliage mass. These results suggest that under disease-induced reductions in carbon supply, Douglas-fir trees retain NSCs (either actively or due to sequestration) at the expense of trunk radial growth. The crown retains the highest concentrations of NSC, presumably to maintain foliage growth and shoot extension in the spring, partially compensating for rapid foliage loss in the summer and fall.

Spring temperature responses of oaks are synchronous with North Atlantic conditions during the last deglaciation

Paleoclimate proxies based on the measurement of xylem cell anatomy have rarely been developed across the temperature range of a species or applied to wood predating the most recent millennium. Here we describe wood anatomy-based proxies for spring temperatures in central North America from modern bur oaks (Quercus macrocarpa Michx.). The strong coherence of temperature signals across the species range supports the use of these proxies across thousands of years of climatic change. We also used 79 subfossil oak log cross sections from northern Missouri, 14C-dated to 9.9–13.63 ka (ka is 1000 cal yr BP), to assess the frequency of oak deposition into alluvial sediments and a subset of these oaks for a wood anatomy-based reconstruction of spring paleotemperatures. Temperatures during the Younger Dryas cold period (YD) were up to 3.5°C lower than modern temperatures for that region, equivalent to or lower than those experienced at the northern edge of the modern species range. Compared to extant oaks growing a...