IAWA Journal, Vol. 27 (1), 2006: 45–54 COMPRESSION WOOD DOES NOT FORM IN THE ROOTS OF PINUS RADIATA Linda C.Y. Hsu1*, John C.F. Walker1, Brian G. Butterfield2 and Sandra L. Jackson2 SUMMARY We investigated the potential for the roots of Pinus radiata D. Don to form compression wood. Compression wood was not observed in either the tap or any lateral roots further than 300 mm from the base of the stem. This suggests that either the roots do not experience the stresses required to induce compression wood formation, or that they lack the ability to form it. Roots artificially subjected to mechanical stress also failed to develop compression wood. It is therefore unlikely that an absence of a compressive load on buried roots can account for the lack of compression wood. Application of auxin to the cambia of lateral roots was similarly ineffective at inducing the formation of compression wood. These ob- servations suggest that the buried roots of radiata pine lack the ability to develop compression wood. We also report the formation of an atypical S3 wall layer in the mechanically-stressed and auxin-treated tracheids and suggest that a reaction wood that is different to compression wood may well form in roots. Key words: Auxin, compression wood, mechanical stress, radiata pine, roots. INTRODUCTION Woody roots function to transmit the load of the stem to the soil as well as to transport water and nutrients to the canopy. In gymnosperms, tracheids, that make up the bulk of the xylem, carry out both of these tasks. Although the xylem of roots and stems undertake similar functions, root xylem appears to be exposed to less mechanical stress as the longitudinal maturation strains measured in roots are consistently smaller than those for stems (Gartner 1997; Stokes et al. 1998). Nonetheless, root xylem, like stem xylem, was responsive to differences in load as both eccentric radial growth (Fayle 1968; Timell 1986; Stokes et al. 1998; Niklas 1999) and changes in mechanical properties of the wood occurred (Stokes & Mattheck 1996; Niklas 1999). The measured alterations 1) School of Forestry, University of Canterbury, Private Bag 4800, Christchurch, New Zealand. 2) School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand. *) Current address: Crop & Food Research, Private Bag 4704, Christchurch, New Zealand [E-mail: [email protected]]. Associate Editor: Barbara Gartner Downloaded from Brill.com10/03/2021 09:42:39PM via free access 46 IAWA Journal, Vol. 27 (1), 2006 Hsu, Walker, Butterfield & Jackson — Compression wood absence in roots 47 in compressive strength and stiffness imply that tracheid morphology and/or cell wall composition/thickness have changed. In gymnosperm stems such changes occur on the compression side of an unbalanc- ed load caused by leaning of the stem or branching (Westing 1965, 1968; Boyd 1973; Wilson & Archer 1977; Siripatanadilok & Leney 1985; Yoshizawa et al. 1985; Yoshi- zawa & Idei 1987; Lee & Eom 1988). In extreme cases this compression wood was red- dish brown in colour and contained shorter tracheids that were rounded in cross section. The cylindrical shape of these cells disrupted the interlocked packing of the xylem and resulted in air spaces. In addition, the secondary cell wall was thickened even though the S3 and warty layers were absent. Cellulose microfibril distribution in the S2 layer was altered with a larger microfibril angle and larger variance in microfibril angle. Deep helical checking was also present in this layer. Finally, lignin distribution in the S2 layer was also different with more lignin in the outer portion. It is important to note that compression wood is not an all or nothing biological response. Rather it is a continuum ranging from severe (described above) to mild. Mild forms are reported to lack red coloration, have typical interlocked tracheids without air spaces with slightly perturbed cellulose microfibrils, an intact 3S layer and slightly modified lignin deposi- tion in the S2 layer. The occurrence of compression wood in gymnosperm roots is controversial. Most re- ports claim that buried roots do not form compression wood (Westing 1965; Fayle 1968; Timell 1986) except when close to the base of the stem (Hartman 1942; Fayle 1968). Nor was it possible to induce the formation of compression wood in buried roots with applied mechanical force (Westing 1965; Fayle 1968; Timell 1986) or the plant growth regulator auxin (Fayle 1968). Since these approaches successfully induced compression wood to form in stems the implication was that stems and roots were different. As a result of these observations it was concluded that roots are not capable of forming compres- sion wood (Fayle 1968). Unfortunately, the bulk of these studies used red coloration as the defining feature for the presence of compression wood. We now realise that mild forms of compression wood lack red coloration. Furthermore, there are reasons to expect milder compression wood in roots. Firstly, roots seem to experience less mechanical stress than shoots (Gartner 1997; Stokes et al. 1998) and one would expect reduced stress to cause a milder reaction. Secondly, the changes in tracheid structure that occur during compression wood formation inhibit water transport (Spicer & Gartner 1998). Given that severe compression wood is likely to inhibit water transport milder forms of compression wood may well be the only type to occur in roots. The fact that the mechanical properties of root wood respond to differential loading (Niklas 1999) shows that roots do indeed experience the biophysical stresses required to induce changes in xylem structure. Since compression wood can be present in mild forms that would have escaped detection in previous studies, we have looked at pine roots using techniques that are capable of perceiving subtle changes in this tissue. Scan- ning electron microscopy, ultraviolet (UV) epifluorescence microscopy and X-ray dif- fraction were used to distinguish differences in morphology, lignification and cellulose microfibril angle that may occur in the mild forms of compression wood. We examined several lateral and tap roots of radiata pine and also attempted to induce compression Downloaded from Brill.com10/03/2021 09:42:39PM via free access 46 IAWA Journal, Vol. 27 (1), 2006 Hsu, Walker, Butterfield & Jackson — Compression wood absence in roots 47 wood formation with both mechanical stress and the exogenous application of auxin. Radiata pine was chosen for this study because of its economic significance to many Southern Hemisphere economies. MATERIALS AND METHODS Root anatomy Roots from 8 ramets of four different clones (two trees from each clone) of 3-year- old radiata pine (Pinus radiata D. Don) were used. The trees were grown and provided by Trees and Technology Ltd, Te Teko, New Zealand. The unexposed large main tap and lateral roots were harvested from each tree. In total, 48 roots were examined. Each root was dissected at three positions, the transition zone (point where the tap root and shoot meet or the point where the lateral root met the tap root), the middle portion of the root and close to the root tip. All samples were stored in 30% ethanol. Applying mechanical stress or auxin in roots Six straight 7-year-old Pinus radiata trees from a single clone in Burnham experi- mental forest, c. 30 km south of Christchurch, New Zealand were used. The stony soil at Burnham resulted in shallow lateral roots, which were excavated in an annulus some 0.5–0.7 metres from the stem, permitting the selection of the five largest and straightest portions of rootwood. This was sufficiently distant from the stem to avoid the pos- sibility of the stem influencing the development of compression wood. The five roots of each tree were subjected to 1% α-napthalene acetic acid (NAA, Sigma Australia), 0.1% NAA, mechanical bending, and the controls for the biochemical and mechanical treatments. For NAA applications, the NAA was mixed with lanoline to make NAA con- centrations of 1% and 0.1%. A small hole (5 mm in both diameter and depth) was drilled on the upper side of each root. The holes were filled with the lanoline /NAA mixture and then bandaged with a plastic bag before the root was re-covered with soil. The drill hole of the control root was filled with pure lanoline. For the mechanical bending treatment, a saw-horse was adapted to apply a vertical tensile load to the root. Using a pulley system, one side was attached to the root while the other side carried a 28–30 kg weight in the form of four concrete blocks. One root per tree provided a control sample, which was first exposed and then re-covered with soil without applying any load. During the four-month treatment period between September and December 2001 (spring season in the Southern Hemisphere), when the speed of cambium division in- creases in plants, tree number 5 displayed the largest diameter increment for both 1% and 0.1% NAA applications. Hence, the five roots from this tree were selected for in- vestigating the formation of compression wood under SEM. The other roots were cut in disks and observed by eye for discoloration associated with compression wood. Compression wood determination To look for potential compression wood we examined tracheid anatomy, lignin distribution and cellulose microfibril angle on both the upper and lower or the auxin- treated and untreated sides of the roots. Anatomy was observed using scanning electron Downloaded from Brill.com10/03/2021 09:42:39PM via free access 48 IAWA Journal, Vol. 27 (1), 2006 Hsu, Walker, Butterfield & Jackson — Compression wood absence in roots 49 microscopy (Leica 440i; Meylan & Butterfield 1978). Adjacent samples were section- ed at 10 μm thickness with a sledge microtome and UV epifluorescence microscopy (Olympus IX 70) was used to detect differences in lignin autofluorescence.