885

Can. J. For. Res. Downloaded from www.nrcresearchpress.com by "Institute of Tibetan Plateau Research,CAS" on 05/24/16 For personal use only.

NOTE The onset of xylogenesis is not related to distance from the crown in Smith fir trees from the southeastern Tibetan Plateau Xiaoxia Li, J. Julio Camarero, Bradley Case, Eryuan Liang, and Sergio Rossi

Abstract: Young and mature trees are usually characterized by asynchronous growth resumption of xylem. Here, we test the hypothesis that the later onset of xylem growth in older trees is related to the longer distance of the stem from the developing buds, which represents the main source of hormones triggering vascular tissue differentiation. We compared the onset of xylogenesis at different heights along the stems of young and mature Smith fir (Abies georgei var. smithii (Viguie & Gaussen) W. C. Cheng & L. K. Fu)) trees in the Sygera Mountains, southeastern Tibetan Plateau. Xylem formation was monitored weekly in 2012 on anatomical sections of wood microcores. The onset of xylogenesis differed between young and mature trees, with most phases occurring 2 weeks later in mature trees. No effect of the sampling height was observed on the growth resumption. Our results suggest that the later resumption of xylogenesis in older conifer trees is not related to their longer distance from the crown. Key words: cambium, xylem formation, xylogenesis, southeastern Tibetan Plateau, Abies georgei var. smithii. Résumé : Les jeunes et les vieux arbres sont habituellement caractérisés par une reprise asynchrone de croissance du xylème. Nous avons testé l'hypothèse selon laquelle la croissance du xylème débute plus tard chez les vieux arbres parce que les bourgeons sont situés a` une plus grande distance de la tige qui est la principale source d'hormones responsables du déclenchement de la différenciation des tissus vasculaires. Nous avons comparé le début de la xylogenèse a` différentes hauteurs le long de la tige de jeunes et de vieux sapins de longue bractée (Abies georgei var. smithii (Viguie & Gaussen) W. C. Cheng & L. K. Fu) dans les montagnes Sergyemla situées dans la partie sud–est du plateau tibétain. La formation du xylème a été suivie hebdomadairement en 2012 sur des coupes anatomiques de microcarottes de bois. La xylogenèse ne débutait pas en même temps chez les jeunes arbres et les arbres matures : la plupart des phases survenaient deux semaines plus tard chez les arbres matures. La hauteur d'échantillonnage n'a eu aucun effet sur la reprise de la croissance. Nos résultats indiquent que le retard dans le début de la xylogenèse chez les vieux conifères n'est pas relié au fait qu'ils aient une plus grande couronne. [Traduit par la Rédaction] Mots-clés : cambium, formation du xylème, xylogenèse, sud–est du plateau tibétain, Abies georgei var. smithii.

Introduction As showed by increasing scientific evidence, older trees are characterized by a later onset of xylogenesis (xylem formation and differentiation) than younger trees (e.g., Deslauriers et al. 2003; Rossi et al. 2008; Li et al. 2013b). However, the factors or mechanisms driving such an age-related growth phenomenon remain elusive (Peñuelas 2005). Xylogenesis involves complex physiological processes that are affected by both external (e.g., climate) and internal (e.g., age dependence and hormones) factors (Larson 1994). Past research has shown that the polar transport of the main auxin form, indole-3-acetic acid (IAA), occurs downward from swelling and developing buds via the procambium and cambium to the root tips, which induces cambium activity and controls xylem formation (Uggla et al. 1998; Aloni 2001). However, a direct relationship between the timing of budburst and xylem formation has not always been detected. In conifers, the onset of xylem formation can occur either before or after bud break (Fahn and Werker 1990; Rossi et al. 2009; Cuny et al. 2012; Zhai et al. 2012; Antonucci et al. 2015).

Auxin is transported basipetally from the developing buds into the subjacent stem, inducing the production of xylem cells. Consequently, we raise the hypothesis that cambial activity should be increasingly delayed further down towards the lower part of stem, tracking the steady polar flow of auxin from leaves to roots. Such a hypothesis may provide a potential explanation for a later onset of xylogenesis observed in older trees, as the base of stem in older trees is more distant from the developing buds than that of younger trees (Rossi et al. 2008). Most studies have investigated cambial activity at breast height only, i.e., ca. 1.3 m (e.g., Deslauriers et al. 2003; Rossi et al. 2008; Li et al. 2013b). Although the timings of xylem formation have been investigated worldwide (e.g., Camarero et al. 2010; Gruber et al. 2010; Moser et al. 2010; Rathgeber et al. 2011; Seo et al. 2011; Huang et al. 2014), information regarding the onset of xylogenesis at different heights along the tree stem among different tree ages is still scarce (Anfodillo et al. 2012). Thus, the question remains whether later resumption of xylogenesis in older trees is related to the distance from the crown (the main source of auxin) or to age.

Received 3 March 2016. Accepted 14 April 2016. X. Li. Key Laboratory of Alpine Ecology and Biodiversity, Institute of Tibetan Plateau Research, Chinese Academy of Sciences, Beijing 100101, China. J.J. Camarero. Instituto Pirenaico de Ecología (IPE-CSIC), Avda. Montañana 1005, 50080 Zaragoza, Spain. B. Case. Department of Informatics and Enabling Technologies, Faculty of Environment, Society and Design, Lincoln University, Lincoln 7647, New Zealand. E. Liang. Key Laboratory of Alpine Ecology and Biodiversity, Institute of Tibetan Plateau Research, Chinese Academy of Sciences, Beijing 100101, China; CAS Center for Excellence in Tibetan Plateau Earth Sciences, Beijing 100101, China. S. Rossi. Département des Sciences Fondamentales, Université du Québec a` Chicoutimi, Chicoutimi, Quebec, Canada. Corresponding author: Eryuan Liang (email: [email protected]). Copyright remains with the author(s) or their institution(s). Permission for reuse (free in most cases) can be obtained from RightsLink. Can. J. For. Res. 46: 885–889 (2016) dx.doi.org/10.1139/cjfr-2016-0092

Published at www.nrcresearchpress.com/cjfr on 19 April 2016.

886

Can. J. For. Res. Vol. 46, 2016

Table 1. Onset, ending, and duration of xylogenesis and final number of xylem cells formed by young and mature Smith firs in 2012 (n = 5 trees per age class).

Can. J. For. Res. Downloaded from www.nrcresearchpress.com by "Institute of Tibetan Plateau Research,CAS" on 05/24/16 For personal use only.

Onset date

Ending date

Duration (no. of days)

Xylem cells (total no.)

Sampling height (m)

Mature

Young

Mature

Young

Mature

Young

Mature

Young

3.3 2.3 1.3 0.3

158±6 164±5 163±3 163±3

— 150±0 151±3 151±3

265±4 269±9 268±3 273±8

— 269±9 269±5 270±3

106±9 105±11 105±5 111±8

— 119±8 118±8 119±7

43±9 44±3 43±3 39±3

— 56±4 51±5 50±4

Note: The onset and ending dates of xylogenesis are given in days of the year (DOY). Values are reported as means ± standard deviation.

The objective of this study is to examine the timings of xylogenesis along the stem in young and mature Smith fir (Abies georgei var. smithii (Viguie & Gaussen) W. C. Cheng & L. K. Fu) trees in the Sygera Mountains on the southeastern Tibetan Plateau. We tested the hypothesis that the distance from the crown influences growth resumption.

Materials and methods Study site and tree selection The study site was located at 4360 m above sea level near the natural alpine treeline of Smith fir in the Sygera Mountains (29°10=–30°15=N, 93°12=–95°35=E) on the southeastern Tibetan Plateau (Liang et al. 2011). The mean annual temperature was –0.2 °C, and the mean annual precipitation was 1162 mm, with a June– August precipitation of 596 mm, as measured by an automatic weather station located close to the site in 2012. The coverage of Smith fir was less than 20% and the understory was dominated by rhododendron shrub (Rhododendron aganniphum Balf. f. et K. Ward var. schizopeplum (Balf. f. et Forrest) T. L. Ming). The selected site is located on a gentle slope with an inclination <15°, underlain by podzolic soils (see plot E1 in Liang et al. (2011)). Two age classes of Smith fir, 20–40 years old (hereafter young trees) and 70–110 years old (hereafter mature trees), were selected in April 2012 at the same study site with identical environmental conditions. Five dominant trees per age class with upright stems and homogeneous diameters were chosen. Mean heights of young and mature trees were 5.2 ± 0.7 and 8.2 ± 0.7 m, with living branches until 2.0 and 4.0 m above ground, respectively. Mean measured diameters at breast height of young and mature trees were 10.1 ± 1.0 and 23.8 ± 1.0 cm, respectively. Trees with polycormic stems, partially dead crowns, reaction wood, or evident damage were avoided. Xylem sampling and data collection Xylem formation was monitored at three (0.3, 1.3 and 2.3 m) and four (0.3, 1.3, 2.3 and 3.3 m) heights along the stems in young and mature trees on the same longitudinal line, respectively. Accordingly, the distances from the sampling positions to the crown (the lowest living branches) were ca. 2, 1, and 0 m in young trees and ca. 4, 3, 2, and 1 m in mature trees. Wood microcores (15 mm long and 2 mm in diameter) were collected weekly, from May to October 2012, with a Trephor (Rossi et al. 2006) and fixed in a formalin– ethanol–acetic acid solution. Spacing between samples at each sampling height was at least 3 cm to avoid wound reaction. Overall, 23 samples at each sampling height were extracted, resulting in a total of 805 microcores. In the laboratory, the microcores were dehydrated in a graded series of ethanol and D-limonene and embedded in paraffin (Rossi et al. 2006). Transverse sections of 9–12 ␮m thickness were cut with a rotary microtome and stained with safranin (0.5% in 95% ethanol) and astra blue (0.5% in 95% ethanol) and mounted in Euparal. Sections were observed with a light microscope under

visible and polarized lights to distinguish the developing cells. For each sample, the radial number of cells in the phase of radial enlargement and in the phase of cell wall thickening and lignification, as well as the number of mature cells, were counted along three radial files (Li et al. 2013b). In a cross section, cambial cells were characterized by thin cell walls and small radial diameters (Deslauriers et al. 2003). Cells in the radial enlargement phase contained protoplast, had thin primary walls, and a radial diameter at least twice the size of the cambial cells. Cells in the phase of cell wall thickening were determined by birefringence in the cell walls under polarized light. Tracheids were considered mature when cell walls were completely red-stained walls and empty lumina (Gricˇar et al. 2005). For each sample, the total xylem cell number was determined by counting the number of cells undergoing enlargement and cell wall thickening and the number of mature cells (Deslauriers et al. 2003). In spring, xylogenesis was considered to have started when at least one tangential row of cells was observed in the enlarging phase. In late summer, when no new cells were observed in the wall thickening or lignification phase, xylogenesis was considered to have terminated (Deslauriers et al. 2003). The duration of xylogenesis was calculated as the difference between the onset of xylem formation and the date when cell wall formation finished. Statistical analyses The onset, duration, and ending of xylogenesis and the final number of xylem cells were compared between the two age classes and among different sampling heights using generalized linear models (GLMs). Prior to GLM analyses, data were checked for normality and homoscedasticity using the Shapiro–Wilk and Levene tests, respectively.

Results The timing of xylogenesis differed between young and mature trees at each sampling height (Tables 1 and 2). In young trees, the radial enlargement of tracheids started at the end of May, which was about 2 weeks earlier than in mature trees (Fig. 2). One or two layers of enlarging tracheids were observed in young trees on 29 May (Fig. 1a), whereas the cambium in mature trees was still dormant at the same date (Fig. 1e). On 12 June, eight or nine layers of enlarging tracheids were detected in young trees (Fig. 1b) and two layers of enlarging tracheids were detected in mature trees (Fig. 1f). In late August, no enlarging tracheids were present in both young and mature trees (Figs. 1c and 1g). The wall thickening and lignification of tracheids ended at the same time, in late September, in both age classes (Figs. 1d and 1h; Tables 1 and 2). As a result, mature trees exhibited a shorter duration of xylogenesis and, consequently, produced fewer cells along the radial files than young trees (Tables 1 and 2). The number of differentiating cells showed a similar pattern of variation during the year at different sampling heights, with delayed, unimodal distributions for enlarging and wall-thickening Published by NRC Research Press

Li et al.

887

Table 2. Results from generalized linear models (F statistics and probability levels, P) testing differences in the timings of the onset, ending, and duration of xylogenesis and the final number of xylem cells among cambium samples taken at different distances from tree crowns and for two age classes (mature and young), as well as their interactions, in Smith fir trees.

Can. J. For. Res. Downloaded from www.nrcresearchpress.com by "Institute of Tibetan Plateau Research,CAS" on 05/24/16 For personal use only.

Xylogenesis Source of variation

Onset date

Ending date

Duration (no. of days)

No. of xylem cells

Tree age Distance from crown Tree age × distance from crown

78.87* 1.94 0.47

0.05 1.45 0.76

17.00* 0.38 0.35

31.15* 1.90 0.36

Note: The onset and ending dates of xylogenesis are given in days of the year (DOY). An asterisk (*) indicates P < 0.01.

Fig. 1. Types of xylem cells corresponding to different phases of xylem formation observed in (a–d) young and (e–h) mature Smith fir trees at the following dates: (a) and (e), 29 May; (b) and (f), 12 June; (c) and (g), 28 August; (d) and (h), 25 September. cz, cambium zone; ec, enlarging cells; wt, wall-thickening cells; mc, mature cells. Bars are equivalent to 100 ␮m.

cells and delayed, increasing linear growth trends for mature cells (Fig. 2). Neither the onset nor the ending of xylogenesis differed significantly among the sampling heights for each age class (Tables 1 and 2). As a result, the duration of xylogenesis was similar at different sampling heights, and consequently, no difference was detected in the final numbers of mature tracheids produced (Tables 1 and 2).

Discussion Despite the cambium maintaining its ability to divide throughout the life-span of trees, our results suggest that the resumption of xylem growth is delayed as trees get older but not necessarily as they get larger, which is consistent with previous observations for conifers growing in cold sites at high latitudes or altitudes (e.g., Deslauriers et al. 2003; Rossi et al. 2008; Li et al. 2013b). In broadleaf trees, two types of cambium reactivation have been described: basipetal and simultaneous (Lachaud et al. 1999). However, we have demonstrated for the first time here that no significant difference was found in the onset of tracheid radial enlargement among samples taken along the stem from the base to the crown in conifer trees, regardless of tree age. Anfodillo et al. (2012) also observed

that the first enlarging cells appeared synchronously along the stem in a 30-year-old Norway spruce (Picea abies (L.) Karst.) tree. These findings suggest that the resumption of xylem growth along the stem is likely not related to the effect of hormone transport from the crown in conifers. The auxin from the young shoots does not seem to be a prerequisite for the resumption of xylogenesis. For example, cambial activity was shown to resume in locally heated portions of Norway spruce and poplar stems during the dormant period before bud bursting (Gricˇar et al. 2006; Begum et al. 2007). Furthermore, no increase in the level of IAA was detected in the cambial zone of Scots pine (Pinus sylvestris L.) and Japanese larch (Larix kaempferi (Lamb.) Carr.) at the onset of cambium activity (Sundberg et al. 1991; Funada et al. 2002). In Smith fir, the onset of xylem formation occurred 15–30 days before bud break (from the end of June to early July in 2012, as we observed), suggesting that cambium activity does not require additional IAA supply from young shoots. Indeed, recent studies indicate that the auxin required for cambium reactivation might be already present in the stem (Junghans et al. 2006; Matte Risopatron et al. 2010; Li et al. 2013a). However, the role of auxin in promoting cambium differentiation into Published by NRC Research Press

888

Can. J. For. Res. Vol. 46, 2016

Can. J. For. Res. Downloaded from www.nrcresearchpress.com by "Institute of Tibetan Plateau Research,CAS" on 05/24/16 For personal use only.

Fig. 2. Number of cells in different stages of xylem formation at different heights along the stems in young and mature trees along days of the year (DOY) in 2012. Error bars indicate standard deviations (n = 5 trees per age class).

xylem cell is still unclear and remains to be further identified (Woodward and Bartel 2005; Zhang et al. 2014). In conclusion, results from this study do not provide support for the hypothesis that the onset of xylogenesis along the stem is related to the distance from the crown. Other mechanisms associated with tree ontogenetic development may influence the growth resumption of xylem. Considering the limitation of the reduced height of the sample trees at the alpine treeline, additional evidence is required to definitively confirm the results of this study.

Acknowledgements This work was supported by the National Natural Science Foundation of China (nos. 41525001 and 41130529) and the National Basic Research Program of China (no. 2012FY111400). We thank the Southeast Tibet Observation and Research Station for the Alpine Environment, Chinese Academy of Sciences, for the fieldwork and monitoring.

References Aloni, R. 2001. Foliar and axial aspects of vascular differentiation: hypotheses and evidence. J. Plant Growth Regul. 20: 22–34. doi:10.1007/s003440010001. Anfodillo, T., Deslauriers, A., Menardi, R., Tedoldi, L., Petit, G., and Rossi, S. 2012. Widening of xylem conduits in a conifer tree depends on the longer time of cell expansion downwards along the stem. J. Exp. Bot. 63: 837–845. doi:10. 1093/jxb/err309. Antonucci, S., Rossi, S., Deslauriers, A., Lombardi, F., Marchetti, M., and Tognetti, R. 2015. Synchronisms and correlations of spring phenology between apical and lateral meristems in two boreal conifers. Tree Physiol. 35: 1086–1094. doi:10. 1093/treephys/tpv077. Begum, S., Nakaba, S., Oribe, Y., Kubo, T., and Funada, R. 2007. Induction of cambial reactivation by localized heating in a deciduous hardwood Hybrid Poplar (Populus sieboldii × P. grandidentata). Ann. Bot. 100: 439–447. doi:10.1093/ aob/mcm130. Camarero, J.J., Olano, J.M., and Parras, A. 2010. Plastic bimodal xylogenesis in conifers from continental Mediterranean climates. New Phytol. 185: 471–480. doi:10.1111/j.1469-8137.2009.03073.x. Cuny, H.E., Rathgeber, C.B.K., Lebourgeois, F., Fortin, M., and Fournier, M. 2012. Life strategies in intra-annual dynamics of wood formation: example of three conifer species in a temperate forest in north–east France. Tree Physiol. 32: 612–625. doi:10.1093/treephys/tps039. Deslauriers, A., Morin, H., and Begin, Y. 2003. Cellular phenology of annual ring formation of Abies balsamea in the Quebec boreal forest (Canada). Can. J. For. Res. 33(2): 190–200. doi:10.1139/x02-178.

Fahn, A., and Werker, E. 1990. Seasonal cambial activity. In The vascular cambium. Edited by M. Iqbal. Wiley, New York. pp. 139–158. Funada, R., Kubo, T., Sugiyama, T., and Fushitani, M. 2002. Changes in levels of endogenous plant hormones in cambial regions of stems of Larix kaempferi at the onset of cambial activity in springtime. J. Wood Sci. 48: 75–80. doi:10. 1007/BF00766242. Gricˇar, J., Cˇufar, K., Oven, P., and Schmitt, U. 2005. Differentiation of terminal latewood tracheids in Silver fir trees during autumn. Ann. Bot. 95: 959–965. doi:10.1093/aob/mci112. Gricˇar, J., Zupancˇicˇ, M., Cˇufar, K., Koch, G., Schmitt, U., and Oven, P. 2006. Effect of local heating and cooling on cambial activity and cell differentiation in the stem of Norway spruce (Picea abies). Ann. Bot. 97: 943–951. doi:10.1093/aob/ mcl050. Gruber, A., Strobl, S., Veit, B., and Oberhuber, W. 2010. Impact of drought on the temporal dynamics of wood formation in Pinus sylvestris. Tree Physiol. 30: 490–501. doi:10.1093/treephys/tpq003. Huang, J.-G., Deslauriers, A., and Rossi, S. 2014. Xylem formation can be modeled statistically as a function of primary growth and cambium activity. New Phytol. 203: 831–841. doi:10.1111/nph.12859. Junghans, U., Polle, A., Düchting, P., Weiler, E., Kuhlman, B., Gruber, F., and Teichmann, T. 2006. Adaptation to high salinity in poplar involves changes in xylem anatomy and auxin physiology. Plant Cell Environ. 29: 1519–1531. doi:10.1111/j.1365-3040.2006.01529.x. Lachaud, S., Catesson, A.M., and Bonnemain, J.L. 1999. Structure and functions of the vascular cambium. C.R. Acad. Sci. III, 322: 633–650. doi:10.1016/S07644469(99)80103-6. Larson, P.R. 1994. The vascular cambium. Development and structure. SpringerVerlag, Berlin. Li, W., Ding, Q., Cui, K., and He, X. 2013a. Cambium reactivation independent of bud unfolding involves de novo IAA biosynthesis in cambium regions in Populus tomentosa Carr. Acta Physiol. Plant. 35: 1827–1836. doi:10.1007/s11738013-1220-2. Li, X., Liang, E., Gricˇar, J., Prislan, P., Rossi, S., and Cˇufar, K. 2013b. Age dependence of xylogenesis and its climatic sensitivity in Smith fir on the south– eastern Tibetan Plateau. Tree Physiol. 33: 48–56. doi:10.1093/treephys/tps113. Liang, E., Wang, Y., Eckstein, D., and Luo, T. 2011. Little change in the fir tree-line position on the southeastern Tibetan Plateau after 200 years of warming. New Phytol. 190: 760–769. doi:10.1111/j.1469-8137.2010.03623.x. Matte Risopatron, J., Sun, Y., and Jones, B. 2010. The vascular cambium: molecular control of cellular structure. Protoplasma, 247: 145–161. doi:10.1007/ s00709-010-0211-z. Moser, L., Fonti, P., Büntgen, U., Esper, J., Luterbacher, J., Franzen, J., and Frank, D. 2010. Timing and duration of European larch growing season along altitudinal gradients in the Swiss Alps. Tree Physiol. 30: 225–233. doi:10.1093/ treephys/tpp108. Peñuelas, J. 2005. Plant physiology: a big issue for trees. Nature, 437: 965–966. doi:10.1038/437965a. Rathgeber, C.B.K., Rossi, S., and Bontemps, J.-D. 2011. Cambial activity related to Published by NRC Research Press

Can. J. For. Res. Downloaded from www.nrcresearchpress.com by "Institute of Tibetan Plateau Research,CAS" on 05/24/16 For personal use only.

Li et al.

tree size in a mature silver-fir plantation. Ann. Bot. 108: 429–438. doi:10.1093/ aob/mcr168. Rossi, S., Anfodillo, T., and Menardi, R. 2006. Trephor: a new tool for sampling microcores from tree stems. IAWA J. 27: 89–97. doi:10.1163/22941932-90000139. Rossi, S., Deslauriers, A., Anfodillo, T., and Carrer, M. 2008. Age-dependent xylogenesis in timberline conifers. New Phytol. 177: 199–208. doi:10.1111/j.14698137.2007.02235.x. Rossi, S., Rathgeber, C.B.K., and Deslauriers, A. 2009. Comparing needle and shoot phenology with xylem development on three conifer species in Italy. Ann. For. Sci. 66: 206. doi:10.1051/forest/2008088. Seo, J.-W., Eckstein, D., Jalkanen, R., and Schmitt, U. 2011. Climatic control of intra- and inter-annual wood-formation dynamics of Scots pine in northern Finland. Environ. Exp. Bot. 72: 422–431. doi:10.1016/j.envexpbot.2011.01.003.

889

Sundberg, B., Little, C.H.A., Cui, K., and Sandberg, G. 1991. Level of endogenous indole-3-acetic acid in the stem of Pinus sylvestris in relation to the seasonal variation of cambial activity. Plant Cell Environ. 14: 241–246. doi:10.1111/j.13653040.1991.tb01342.x. Uggla, C., Mellerowicz, E.J., and Sundberg, B. 1998. Indole-3-acetic acid controls cambial growth in Scots pine by positional signaling. Plant Physiol. 117: 113–121. doi:10.1104/pp.117.1.113. Woodward, A.W., and Bartel, B. 2005. Auxin: Regulation, action, and interaction. Ann. Bot. 95: 707–735. doi:10.1093/aob/mci083. Zhai, L., Bergeron, Y., Huang, J.-G., and Berninger, F. 2012. Variation in intraannual wood formation, and foliage and shoot development of three major Canadian boreal tree species. Am. J. Bot. 99: 827–837. doi:10.3732/ajb.1100235. Zhang, J., Nieminen, K., Serra, J., and Helariutta, Y. 2014. The formation of wood and its control. Curr. Opin. Plant Biol. 17: 56–63. doi:10.1016/j.pbi.2013.11.003.

Published by NRC Research Press