Science of the Total Environment 493 (2014) 845–853

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Retranslocation and localization of nutrient elements in various organs of moso bamboo (Phyllostachys pubescens) Mitsutoshi Umemura ⁎, Chisato Takenaka Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

H I G H L I G H T S • • • •

The bamboo efficiently utilizes boron by the retranslocation and local accumulation. Zinc found in nodes at high concentrations may support internode elongation. The bamboo can utilize more silicon for cell wall enhancement than calcium or boron. Silicon, boron, and zinc might support the vegetative reproduction and rapid growth.

a r t i c l e

i n f o

Article history: Received 28 February 2014 Received in revised form 17 June 2014 Accepted 18 June 2014 Available online xxxx Editor: Charlotte Poschenrieder Keywords: Moso bamboo Vegetative reproduction Rapid growth Silicon Boron Zinc

a b s t r a c t Moso bamboo (Phyllostachys pubescens) is one of the major giant bamboo species growing in Japan, and the invasion of mismanaged bamboo populations into contiguous forests has been a serious problem. To understand expansion mechanisms of the bamboo, it is important to obtain some first insights into the plant's rapid growth from the viewpoints of the nutrient dynamics in bamboo organs. We have investigated seasonal changes in the concentrations of several nutrient elements in leaves of the plants from three P. pubescens forests and the distributions of those elements in both mature (culms, branches, leaves, roots, and rhizomes) and growing organs (shoots and rhizomes). Among all elements analyzed, boron (B) concentrations in leaves showed a specific seasonal variation that was synchronous across all study sites. Boron was detected at high concentrations in the younger parts of growing rhizomes and shoots, and in mature leaves. These results indicate that P. pubescens could actively utilize B for vegetative reproduction by the retranslocation and the local accumulation behaving as mobile B. Silicon (Si) was found in high concentrations in surface parts of culms and in the mature sheaths of growing rhizomes and shoots following those in mature leaves. P. pubescens, a plant known to accumulate Si, accumulated only low levels of Ca and B in the leaves, indicating that it is possible to utilize more Si for cell wall enhancement than Ca or B. In both mature culms and rhizomes, zinc (Zn) was found at much higher concentrations in the nodes with meristematic tissue than those in internodes, indicating that Zn might play a role in promoting culm and rhizome elongation. We suggest that specific and local utilization of B, Si, and Zn in P. pubescens might support the vegetative reproduction and rapid growth. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Bamboo comprises over 70 genera including over 1200 species, and occupies more than 14 million ha worldwide (Dransfield and Widjaja, 1995; Fu and Banik, 1995). Almost 80% of all bamboo species and forests are found in South and Southeast Asia countries including China, India, and Myanmar (Kleinhenz and Midmore, 2001). Bamboo is also considered as an important bioresource in these Asian countries. To use

⁎ Corresponding author at: Laboratory of Forest Environment and Resources, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 4648601, Japan. Tel./fax: +81 52 789 4055. E-mail addresses: [email protected] (M. Umemura), [email protected] (C. Takenaka).

http://dx.doi.org/10.1016/j.scitotenv.2014.06.078 0048-9697/© 2014 Elsevier B.V. All rights reserved.

bamboo sustainably, the study of nutrient cycles in bamboo forests to improve their management is of primary importance. A number of studies have been conducted on the cycles of macronutrients such as nitrogen (N), phosphorus (P), and potassium (K) in bamboo forests (Raghubanshi, 1994; Mailly et al., 1997; Shanmughavel and Francis, 1997; Li et al., 1998; Embaye et al., 2005). Bamboo species are characterized by their high growth rate at sprouting, no secondary growth of the culm due to no cambium (Uchimura, 1994), and silicon (Si) accumulation (e.g. Ueda and Ueda, 1961; Ma and Takahashi, 2002). Therefore, we hypothesized that these characteristics should be affected by the seasonal or interannual changes in nutrient concentrations in leaves, culms, and other organs, and also by the distribution of nutrients among various organs. Studies of these nutrient translocation and cycling processes have been

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conducted for Bambusa bambos (Shanmughavel and Francis, 1997), Dendrocalamus strictus (Tripathi and Singh, 1994; Tripathi et al., 1999), Yushania alpina, Arundinaria alpina (Embaye et al., 2005), Phyllostachys pubescens (Kaneko, 1995; Li et al., 1998; Wu et al., 2009), and Phyllostachys bambusoides (Ueda et al., 1961; Nishida, 1989; Kaneko, 1995). Also, the accumulation of Si has been studied for several bamboo species (Ueda et al., 1961; Nishida, 1989; Motomura and Fujii, 2000; Lux et al., 2003; Kondo, 2010). The rapid internode elongation is supported not only by uptake of nutrient elements from soil, but also by retranslocation of nutrients and photosynthesis products from culms or leaves to the growing organs. Ueda et al. (1961) reported that the concentrations of N, P, and K in leaves of P. bambusoides changed markedly during the bamboo shoot growth season and during leaf renewal. They also found seasonal increases in calcium (Ca) and Si in leaves. In addition to nutrients, the photosynthetic products stored in both rhizomes and culms are also consumed for rhizome growth in June to November, and especially during new shoot growth in April (Ueda and Uchimura, 1958; Ueda, 1963; Uchimura, 1994). Though dynamics of macronutrients and photosynthesis products in various organs related to the rapid growth of bamboo has been clarified, there have been few studies on trace elements such as zinc (Zn) and boron (B), which are thought to be related to the vegetative reproduction and high growth rate of bamboo. The physiological functions of Zn are well known in DNA and RNA metabolism, in cell division, and protein synthesis (Coleman, 1992; Vallee and Falchuk, 1993; Klug, 1999; Broadley et al., 2007; White, 2012). Especially, in regard to the effects on internode elongation in higher plants, Zn deficiency in cereals such as wheat typically results in reduced shoot elongation (Cakmak et al., 1996). Structural strength is also necessary to sustain the rapid growth of these relatively slender plants. Silicon, Ca, and B are mainly located in the cell walls (Broadley et al., 2012), where they help to provide structural support for the stems. For example, Si deposition in the epidermis and vascular tissues of stems, leaf sheaths, and hulls of rice enhances the strength and rigidity of cell walls (Ma and Takahashi, 2002). Also, a high proportion of the total Ca in plant tissue is often located in cell walls (apoplasm) (Hawkesford et al., 2012). Further, there is a close relationship between B nutrition and the functions of primary cell walls, which are important for determining cell size and shape during development of higher plants (Blevins and Lukaszewski, 1998). The transportation and metabolism of carbohydrate are also related to B, which forms complexes by ester-binding between B and sugar alcohol with cis-diol (Fujihara et al., 2002). The retranslocation of B through the phloem has been recognized in olive (Liakopoulos et al., 2009); celery and peach (Hu et al., 1997); Ricinus communis (Eichert and Goldbach, 2009); and almond, apple, and nectarine (Brown and Hu, 1996). However, the retranslocation of B in bamboo species has not yet been observed. In particular, investigating seasonal changes in elemental concentrations, including trace elements, should clarify the nutrient retranslocation and provide important clues to understanding the specific growth mechanisms of bamboo species. In this study, we focus on moso bamboo (P. pubescens), which is one of the major giant bamboo species observed in Japan. This bamboo species was introduced from China in 1746 (Suzuki, 1978) and has since been widely utilized for various human purposes. Bamboo plantations had therefore been historically well-managed. However, the recent increase in the import of bamboo products from foreign countries has resulted in the abandonment of bamboo plantations (Torii, 2003). This has caused invasion of mismanaged bamboo populations into contiguous forests, including other plantation forests (Okutomi et al., 1996; Torii and Isagi, 1997; Isagi and Torii, 1998; Torii, 1998; Katanoda, 2003; Yamamoto et al., 2004). The bamboo shows particularly high vertical shoot growth rates (e.g., N 10 m in approximately 2 months) and high horizontal rhizome growth rates (e.g., average 1–2 m year− 1, max 5.28 m year−1) (Ueda, 1960; Nishikawa et al., 2005; Matsumoto and Kondo, 2007; Kawai et al., 2008). We hypothesized that the rapid growth of P. pubescens is sustained by the special dynamics such as

recycle and accumulation of the specific elements in each organ. The purpose of this study is to obtain some first insights into the plant's rapid growth based on the above hypothesis through the observation for viewpoints of seasonal changes in elemental concentrations in leaves and elemental distribution in various organs, especially focusing on Si, Ca, B, and Zn and several other nutrient elements. 2. Materials and methods 2.1. Site characteristics This study was conducted in P. pubescens forests in which neither stem density management nor fertilization had been conducted. The three sites selected for research, Kanpachi (35°07′ N, 137°13′ E, 110 m altitude), Seto (35°11′ N, 137°07′ E, 200 m altitude), and Noguchi (35°07′ N, 137°15′ E, 160 m altitude) are all located in Aichi Prefecture, central Japan. The annual mean temperature and precipitation at Toyota, the nearest AMeDAS (Automated Meteorological Data Acquisition System) observation site, are 14.8 °C and 1451.4 mm, respectively (Japan Meteorological Agency, 2011). Mean diameter at breast height (DBH) and density of each bamboo stand were 8.2, 10.1, and 11.2 cm and 2660, 2400, and 4790 stems/ha at Kanpachi, Seto, and Noguchi, respectively (Table 1). The density of P. pubescens at each site was constant during the period of study, from June 2008 to December 2009. Forests had sparse understory vegetation consisting of evergreen and deciduous broad-leaved plants. Understory species in Kanpachi comprised Cleyera japonica Thunb., Eurya japonica Thunb., Illicium anisatum L., Osmanthus heterophyllus P. S. Green, Quercus glauca Thunb., Callicarpa mollis S. & Z., and Wisteria sp., and in Noguchi included Aucuba japonica Thunb., C. japonica Thunb., and Nandina domestica Thunb. Only sparse C. japonica Thunb. and Camellia sinensis O. K. were observed in Seto. 2.2. Sampling of mature bamboo organs, growing shoots, and growing rhizomes To investigate the elemental distributions in bamboo, mature organs including culms, leaves, branches, rhizomes, roots, and growing organs of the shoots and rhizomes were sampled. One mature bamboo greater than 4 years in age with average DBH was harvested at each site in December 2009. Culms were cut at the internodes at 0 m, 1.3 m, 5 m, 10 m, and 15 m from the culm base (there was no sample for 15 m height at Kanpachi), and the cut segments were cross-sectioned and divided into internodes and nodes that included both culm wall and diaphragm. To examine radial elemental distribution in culms, the cross-sectioned culm of 1.3 m height was separated into outer surface (around 1 mm depth), outer vascular zone (around 3 mm depth), inner vascular zone (around 3 mm depth), and stem cavity inner surface (around 1 mm depth). Leafed branches were also collected for each height (0 m, 2 m, and 4 m) from the lowest branch on the culms. Rhizomes were sampled from 5 randomly placed subplots (30 cm soil depth × 50 cm × 50 cm) at each site in December 2009. Rhizomes were washed and all developing roots were then excised. These rhizomes were divided into internode and node segments (n = 5, respectively). To obtain fine root samples, 5 soil core samples were taken from each site in December 2009, using a liner core sampler (diameter 48.4 mm × depth 300 mm) (DIK-110C, Daiki Rika Kogyo Co., Ltd., Saitama, Japan). All core samples were transported to a laboratory and kept frozen (−20 °C) until analysis was performed. In the laboratory, core samples were separated into six subsamples 5 cm in length. Bamboo roots collected from these subsamples were carefully pre-washed with tap water to remove elemental contamination derived from soil. Roots were then classified as living or dead based on the definitions established by Vogt and Persson (1991). Living roots were separated

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Table 1 Site characteristics of three Phyllostachys pubescens forests. Site

Density of bamboo stemsa (stems/ha)

Kanpachi

2660

Seto Noguchi

2400 4790

a

Mean DBH of bamboo stems (cm)

Slope angle

Slope direction

Location

Understory species

8.2

25°

Facing southeast

Base of a mountain

10.1 11.2

35° 15°

Facing east Facing northeast

Base of a mountain A mountain side

Cleyera japonica, Eurya japonica, Illicium anisatum, Osmanthus heterophyllus, Quercus glauca, Callicarpa mollis, Wisteria sp. Cleyera japonica, Camellia sinensis Aucuba japonica, Cleyera japonica, Nandina domestica

The density of bamboo stems at these sites was previously investigated in 2008.

into two diameter classes (d ≥ 2 mm or d b 2 mm). For chemical analysis, the roots within each diameter class at 5–10 cm depth, where root biomasses were the highest, were used (n = 3–4 per diameter class). Growing bamboo shoots and growing rhizomes were sampled at Kanpachi in April and July 2010, respectively. Five growing shoots attached to rhizomes and three growing rhizomes were sampled and carefully pre-washed with tap water to remove elemental contamination derived from soil. Then, the shoot samples were separated into the following 17 parts after removing the outer sheaths: rhizome node with the shoot, stem petiole attached to rhizome, lower culm base, central culm base, upper culm base, 1st node, 5th node, 10th node, 15th node, 20th node, apex of culm, young leaf blades on sheaths, mature leaf blades on sheaths, young culm sheaths, mature culm sheaths, root tips, and young roots (Fig. 1). The growing rhizome samples were separated into 6 portions after removing outer sheaths: 0–5 cm, 10 cm, 20 cm, 30 cm, and 40 cm from the apex of the rhizome and mature sheaths. The 0–5 cm and 10 cm sections corresponded to younger sheaths and apex of stele, respectively.

2.3. Sampling of bamboo leaves to observe seasonal changes in elemental concentrations We sampled current-year leaves from 5 bamboos of average DBH once per month from June 2008 to May 2009 in order to observe seasonal changes in elemental concentrations in bamboo leaves. These leaves were sampled from lower portions of branches at the same height throughout the sampling duration to avoid possible variation in elemental concentrations due to branch height.

2.4. Leaf litter sampling Five litter traps of 50 cm × 50 cm in size were placed randomly on the forest floor of each site in July 2008. Litter samples from each trap were collected once per month from August 2008 to July 2009. The litter samples were dried at 80 °C for 48 h then sorted carefully into bamboo organs including leaves, leaf sheaths, and branches, or other organic matter. For elemental analysis, we used leaf litter collected in May or June 2009, which comprised the greatest litter supply observed throughout the year at each site. 2.5. Analysis of elemental concentrations in each bamboo organ All samples of each bamboo organ were rinsed in ultrapure water twice after they were washed in deionized water using an ultrasonic bath for less than 1 min, and dried at 80 °C for 48 h. To obtain homogeneous samples, the culms, branches, and rhizomes were sliced, and the leaves, roots, growing shoots, and growing rhizomes were ground using a mill mixer (BL-229, SUN Co., Ltd., Osaka, Japan). The inner organs covered by the sheaths of growing shoots and rhizomes were not rinsed to prevent elemental leaching. We determined Si, K, Ca, P, B, and Zn concentrations using a combination method of wet digestion with nitric acid followed by gravimetric analysis for the insoluble Si and by inductively coupled plasma atomic emission spectrometry (ICP-AES; IRIS ICARP, Jarrell Ash Nippon Corp., Japan) for soluble Si, K, Ca, P, B, and Zn (Umemura and Takenaka, 2010).

3. Results 3.1. Elemental distribution in growing and mature organs

Fig. 1. Organs sampled from a growing bamboo shoot: (a) node of rhizome, (b) stem petiole, (c) lower culm base, (d) central culm base, (e) upper culm base (f) the 1st node, (g) the 5th node, (h) the 10th node, (i) the 15th node, (j) the 20th node, (k) apex of culm, (l) young leaf blades on sheaths, (m) mature leaf blades on sheaths, (n) young culm sheaths, (o) mature culm sheaths, (p) root tips, and (q) young roots.

Silicon concentrations were higher in mature sheaths in both growing shoots and growing rhizomes than in younger organs (Figs. 2A and 3A). On the other hand, K, Ca, P, B, and Zn concentrations in each organ of the growing shoots were higher in younger parts than in mature organs (Fig. 2B–F). This tendency was consistent with that observed in growing rhizomes: higher concentrations of K, Ca, P, B, and Zn were observed in the rhizome segments of 10 cm from the apex, which are corresponding to apex of the stele, than in older portions of the rhizomes (the segments of 40 cm from the apex) (Fig. 3B–F). Boron concentrations, compared to those of other elements, showed an especially remarkable gradient from the bottom of the culm base to the apex of the culm and tended to accumulate in young leaf blades on the sheath

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Fig. 2. Elemental concentrations in each organ of growing bamboo shoots sampled at Kanpachi in April 2010. The letters (a–q) labeling each organ correspond to each organ depicted in Fig. 1. Error bars represent S.D. (n = 5).

(Fig. 2C). Silicon and Zn concentrations were particularly high in rhizome nodes with bamboo shoot (Fig. 2A and F). High concentrations of Si (47.6–68.5 mg g−1), K (6.7–11.8 mg g−1), Ca (5.2–10.5 mg g−1), P (1.0–1.5 mg g−1), and B (3.4–5.1 μg g−1) were found in mature bamboo leaves among mature organs at all three sites (Tables 2–4). However, the highest concentrations of Zn were found in the nodes of the culms or rhizomes. The distribution of Zn was especially remarkable in the rhizomes: 87.4–211 μg g−1 Zn in the nodes and 6.2–11.8 μg g−1 Zn in the internodes. In a cross section of an internode at 1.3 m height, the concentrations of Si (9.2–14.6 mg g−1) in the outer surface tissues were higher than those of Si (0.04–5.0 mg g−1) in the inner tissues of the cross section. Boron was detected in specific organs such as leaves and only at very low levels in mature culms and rhizomes at all study sites. No common tendency regarding elemental concentrations were identified in branches, the diaphragms at the nodes of culms, or roots at any of the three study sites.

and tended to either remain constant or decrease slightly from September to February, and after that increased until May (Fig. 4C). Potassium concentrations in leaves tended to increase from June to early autumn, after which they continued to decrease until the following spring at all three sites (Fig. 4D). Phosphorus concentrations of leaves tended to decrease from March to May at all three sites (Fig. 4E). The concentrations of Zn did not show any seasonal change and were almost constant at all three sites (Fig. 4F). In leaf litter, the concentrations of Si (52–87 mg g−1) and Ca (9.6–14 mg g−1) were the highest (Table 5), relative to the concentrations measured in mature leaves (Fig. 4A and B). In contrast, the concentrations of P (0.17–0.19 mg g−1) and K (1.3–2.4 mg g−1) in leaf litter were much lower than those in the living leaves, harmonizing with the seasonal change. The B and Zn concentrations in leaf litter were similar to observed seasonal concentration ranges in living leaves. 4. Discussion

3.2. Seasonal changes in nutrient elements in leaves 4.1. Remobilization and distribution of K and P Seasonal changes of nutrient elements in leaves of P. pubescens at each study site are shown in Fig. 4. Silicon and Ca showed seasonal accumulation into bamboo leaves, but without clear evidence of retranslocation to other portions of the plants. Silicon concentrations tended to increase throughout the year from 11.2–13.3 mg g− 1 in June to 20.6–37.1 mg g− 1 in May at all three study sites (Fig. 4A). Calcium concentrations also tended to increase during the year at Kanpachi and Seto, from 1.6–2.3 mg g−1 in June to 4.2–4.4 mg g−1 in the next May, but those at Noguchi were almost constant throughout the year (Fig. 4B). The concentrations of B decreased from June to July

The seasonal changes of K and P concentrations in living leaves and those concentrations in leaf litter indicated that a large amount of K and P were lost from mature leaves, reaching to 87–92% and 86–92% of the maximum contents in the mature leaves, respectively (Fig. 4D and E, and Table 5). We also found higher K and P concentrations in younger organs than those in mature organs of growing rhizomes and shoots (Figs. 2 and 3). Potassium is known to accumulate in meristems and young tissues and known to function in osmotic adjustment and phloem retranslocation of organic acids such as malate and assimilates to

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Fig. 3. Elemental concentrations in each organ of growing rhizomes sampled at Kanpachi in July 2010. Rhizome segments after mature sheaths peeled were sampled at each distance from the apex. Error bars represent S.D. (n = 3).

the roots of higher plants (Cakmak et al., 1994; Hawkesford et al., 2012; White, 2012). Ueda et al. (1961) investigated seasonal changes in K concentrations of culms and rhizomes in P. bambusoides and found high K concentrations in rhizomes from July to October and in March. Ueda

(1963) also reported that assimilates were accumulated for bamboo shoot growth in spring and for rhizome growth that peaks in September. Although we did not measure seasonal changes of K concentration in rhizomes, our results on remarkable losses of K from leaves may

Table 2 Elemental concentrations in mature organs of Phyllostachys pubescens sampled at the Kanpachi study site in December 2009. Sic

Organs

K

Ca

P

mg g−1 Culm

Internodesa (n = 4) Culm wall of nodesa (n = 4) Diaphragm of nodesa (n = 4) Cross section of an internode at 1.3 m height (n = 1)

Branches (n = 3)b Leaves (n = 3)b Rhizomes

Roots

a b c

Internodes (n = 5) Nodes (n = 5) d ≥2 mm (n = 4) d b 2 mm (n = 3)

Mean S.D. Mean S.D. Mean S.D. Outer surface Outer vascular zone Inner vascular zone Culm cavity inner surface Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D.

3.55 2.13 5.51 2.24 3.53 3.95 14.6 4.78 4.95 3.77 8.62 3.83 59.7 4.8 3.45 1.63 2.24 0.84 4.67 2.69 5.70 1.20

B

Zn

μg g−1 2.02 1.10 1.29 0.08 2.77 1.40 1.37 1.15 1.96 1.74 1.77 0.27 6.67 0.87 7.15 3.32 4.63 2.38 0.42 0.37 0.26 0.13

0.174 0.107 0.180 0.044 0.173 0.029 0.188 0.126 0.136 0.131 0.163 0.030 6.02 0.97 0.153 0.048 0.204 0.103 0.769 0.473 0.966 0.336

Values are average concentrations in organs sampled every height from the culm base. Values are average concentrations in organs sampled every height from the lowest branch. Silicon values, except culm cross sections and node segments of both the culm and rhizomes, are based on Umemura and Takenaka (2014).

0.0560 0.0240 0.0974 0.0414 0.0820 0.0414 0.119 0.0365 0.0325 0.0200 0.106 0.018 1.04 0.07 0.187 0.061 0.200 0.030 0.0589 0.0464 0.0648 0.0230

n.d. – 0.52 1.0 n.d. – 10 n.d. n.d. n.d. n.d. n.d. 4.4 0.7 n.d. – n.d. – n.d. n.d. 0.80 1.4

50.9 36.1 190 177 91.3 163 23.0 36.5 66.0 23.4 50.3 22.1 32.6 3.1 6.23 2.59 165 89 22.1 15.9 35.6 15.2

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Table 3 Elemental concentrations in mature organs of Phyllostachys pubescens sampled at the Seto study site in December 2009. Sic

Organs

K

Ca

P

B

mg g−1 Culm

Internodesa (n = 5) Culm wall of nodesa (n = 5) Diaphragm of nodesa (n = 4) Cross section of an internode at 1.3 m height (n = 1)

Branches (internodes) (n = 3)b Leaves (n = 3)b Rhizomes

Roots

a b c

Internodes (n = 5) Nodes (n = 5) d ≥ 2 mm (n = 3) d b 2 mm (n = 3)

Mean S.D. Mean S.D. Mean S.D. Outer surface Outer vascular zone Inner vascular zone Culm cavity inner surface Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D.

3.10 1.80 3.41 1.73 0.280 0.477 11.1 0.573 0.669 0.103 5.27 0.75 68.5 14.4 5.39 4.97 2.47 1.48 4.14 0.46 8.43 1.54

Zn

μg g−1 2.51 1.25 1.42 0.24 1.70 0.65 1.45 2.11 3.58 1.29 2.32 0.60 8.64 3.45 4.63 1.76 3.16 0.69 0.505 0.085 0.242 0.063

0.337 0.200 0.311 0.173 0.213 0.086 0.178 0.179 0.239 0.159 0.214 0.072 10.5 2.5 0.094 0.039 0.119 0.031 0.291 0.101 0.698 0.223

0.0926 0.0180 0.157 0.038 0.145 0.038 0.166 0.0598 0.0851 0.0744 0.135 0.019 1.20 0.13 0.377 0.037 0.298 0.066 0.125 0.011 0.0827 0.0029

n.d. – n.d. – n.d. – n.d. n.d. n.d. n.d. n.d. n.d. 5.1 1.1 n.d. – n.d. – n.d. n.d. 0.93 0.84

40.4 4.0 107 53 20.8 30.5 33.3 29.7 45.8 7.05 27.0 15.9 22.7 3.9 7.78 5.53 87.4 68.9 6.73 0.68 17.0 7.2

Values are average concentrations in organs sampled at each height from the culm base. Values are average concentrations in organs sampled at each height from the lowest branch. Silicon values, except culm cross sections and node segments of both the culm and rhizomes, are based on Umemura and Takenaka (2014).

indicate the retranslocation from mature leaves to the growing organs. On the other hand, in forest ecosystems, loss of K by leaching from leaves has been observed (Titlyanova, 2007). Sakai and Tadaki (1997) reported that K+ concentrations in the throughfall were about 4 to 6 times higher in both P. pubescens and P. bambusoides stands than in the secondary forest composed of deciduous tree species indicating K+ leaching from the bamboo leaves. Therefore, it is considered that a large amount of K loss in leaves should be resulted not only from its retranslocation within the plant, but also from its measurable leaching from the leaves.

Phosphorus is an essential macronutrient for higher plants with physiological functions including pH regulation, reproductive development, ATP production, enzyme regulation, and many other metabolic processes, and is typically a highly mobile and frequently retranslocated element (White, 2012). The observed decreases in P concentration from March to May (Fig. 4E) and prior to leaf fall (Table 5) at all study sites may indicate retranslocation of this element from leaves to other organs. However, the changes in P concentration in leaves from June to the following May differed among the three study sites (Fig. 4E). Previous reports had also discussed the different trends in seasonal changes

Table 4 Elemental concentrations in mature organs of Phyllostachys pubescens sampled at the Noguchi study site in December 2009. Sic

Organs

K

Ca

P

mg g−1 Culm

Internodesa (n = 5) Culm wall of nodesa (n = 5) Diaphragm of nodesa (n = 3) Cross section of an internode at 1.3 m height (n = 1)

Branches (internodes) (n = 3)b Leaves (n = 3)b Rhizomes

Roots

a b c

Internodes (n = 5) Nodes (n = 5) d ≥ 2 mm (n = 3) d b 2 mm (n = 3)

Mean S.D. Mean S.D. Mean S.D. Outer surface Outer vascular zone Inner vascular zone Culm cavity inner surface Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D.

1.18 2.13 3.42 1.85 0.645 0.680 9.22 1.83 0.0464 0.0432 5.07 1.57 47.6 12.1 6.72 5.06 1.97 1.44 4.71 1.32 8.56 0.48

B

Zn

μg g−1 5.99 5.52 2.66 1.24 5.24 3.72 2.14 7.32 10.6 5.27 3.09 1.00 11.8 2.5 8.58 3.73 5.33 1.64 0.449 0.050 0.220 0.061

0.185 0.111 0.231 0.099 0.190 0.047 0.122 0.123 0.117 0.136 0.143 0.034 5.19 1.77 0.174 0.053 0.231 0.070 0.415 0.155 0.960 0.143

Values are average concentrations in organs sampled at each height from the culm base. Values are average concentrations in organs sampled at each height from the lowest branch. Silicon values, except culm cross sections and node segments of both the culm and rhizomes, are based on Umemura and Takenaka (2014).

0.467 0.255 0.240 0.067 0.309 0.176 0.439 0.467 1.04 0.321 0.241 0.162 1.53 0.49 0.690 0.320 0.591 0.325 0.123 0.051 0.103 0.020

n.d. – n.d. – n.d. – n.d. n.d. n.d. n.d. n.d. n.d. 3.4 1.7 n.d. – n.d. – n.d. n.d. n.d. n.d.

10.9 7.2 74.9 56.9 22.7 24.5 17.2 6.12 10.8 7.56 11.8 6.7 17.8 2.9 11.8 7.7 211 158 8.51 1.86 17.1 8.3

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Fig. 4. Seasonal change in elemental concentrations in leaves of Phyllostachys pubescens from June 2008 to May 2009 at each site: Kanpachi (solid line), Seto (dotted line), and Noguchi (dashed line). Error bars represent S.D. (n = 5). Seasonal change of Si concentration (A) was cited from Umemura and Takenaka (2014).

in leaf P concentrations. Kaneko (1995) showed that P concentration in leaves of P. pubescens continued to decrease from May to October. On the other hand, Ueda et al. (1961) indicated that the seasonal changes in the concentration of P in leaves, culms, and rhizomes of P. bambusoides were small. These observations may imply that seasonal changes in P concentrations in leaves depend on the growing environment such as P availability, however, P mass loss (86–92%) from living leaves indicates a possibility of the retranslocation of P from leaves to other organs. 4.2. Distribution and mobilization of Si, Ca, and B Silicon is known to function in enhancement of cell structural strength and tolerance to external stress (Ma and Takahashi, 2002). The Si concentration was higher in mature sheaths of both bamboo shoots and growing rhizomes (Figs. 2 and 3). Further, we found higher Si concentrations in the outer surface tissues following that in mature leaves (Tables 2–4). Previous studies also show higher Si concentrations

Table 5 Elemental concentrations of leaf litter of Phyllostachys pubescens collected from three study sites in May or June 2009 (n = 3). Sia

Site

K

Ca

P

mg g−1 Kanpachi Seto Noguchi a

Mean S.D. Mean S.D. Mean S.D.

71.1 2.5 87.1 0.9 51.5 0.5

B

Zn

μg g−1 2.34 0.03 1.32 0.01 2.40 0.08

10.4 0.1 13.7 0.2 9.56 0.05

0.189 0.013 0.170 0.001 0.181 0.001

The silicon values are based on Umemura and Takenaka (2014).

9.9 0.9 8.6 0.1 5.8 0.1

37.7 0.7 23.6 1.2 22.1 0.5

in the outer surface parts than those in the woody inner tissue of the culms in P. pubescens, P. bambusoides, and Phyllostachys nigra (Nishida and Shirai, 1985), and increase of Si with age (Nishida, 1989). Ueda and Ueda (1961) reported that the application of calcium silicate to the soil in a P. bambusoides forest increased culm hardness. Therefore, the Si accumulation into mature sheaths and the surfaces of the mature culms found in this study is considered to provide physical strength and protection of inner or immature organs. In addition, these accumulations of Si should be derived not from the retranslocation but from the root uptake, because of a simple seasonal increase in leaves. It is known that a high proportion of total Ca in plant tissue is often located in cell walls (apoplasm), and is bound to the R-COO− groups of polygalacturonic acid (pectin) in a readily exchangeable form (Hawkesford et al., 2012). Our results showed that the concentrations of Ca in both bamboo shoots and growing rhizomes were higher in younger plant parts, particularly in the culm or stele apex (Figs. 2 and 3). Since the embryonic tissue was found in the stele apex (Uchimura, 1994), the localization of Ca is considered to be related with the cell structure of growing organs. Calcium is also known as an element with low propensity for retranslocation (Rodrigues et al., 2003; White, 2012). Therefore, the accumulation of Ca into growing organs is thought to be derived not from the retranslocation from leaves but from the root uptake. In contrast, B in P. pubescens may be transported from mature leaves to growing organs. Seasonal changes in B concentrations in leaves were the most synchronous among nutrient elements measured and remained consistent at all sites. The B concentrations in leaves increased twice, from July to September and again from February to May (Fig. 4C). In addition, from September to February, the B concentrations remained nearly constant or decreased slightly. Physiological functions of B include both structural enhancement of cell walls and transportation of carbohydrates. Because B binds to cell walls strongly and stably,

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B tends to be difficult to retranslocate (e.g. Brown and Hu, 1994; Hu and Brown, 1994; Fujihara et al., 2002). However, B also functions in the transportation and metabolism of carbohydrates by forming complexes through ester bonds between B and sugar alcohol with cis-diol, polyols such as sorbitol, mannitol, and dulcitol. The mobility of these complexes then permits B retranslocation to other plant organs through the phloem (e.g. Zimmermann, 1960; Brown and Hu, 1996, 1998; Hu et al., 1997; Fujihara et al., 2002). Therefore, the seasonal decreasing or constant characteristics found in leaves may be related with the retranslocation of B. The periods of retranslocation of B are nearly coincident with the periods of rhizome growth and starch storage in various organs in bamboo. Bamboo rhizomes begin to elongate in June and exhibit their highest growth rates in September, after which elongation stops in November (Ueda, 1963). In addition, studies of P. pubescens, P. bambusoides, and Pleioblastus pubescens Nakai show that the starch contents stored in both rhizomes and 2-year-old culms tend to increase from September to March to prepare for new shoot growth in April (Ueda and Uchimura, 1958; Uchimura, 1994). These facts may imply that mobile B in bamboo leaves forms boron–polyol complexes and retranslocates to culms or rhizomes in these seasons when sugar is transported, although further study is needed to clarify existence of polyols or boron–polyol complexes which have not been found in bamboo yet. Interestingly, B was found in leaves, growing rhizomes, and growing shoots, especially in young leaf blades on sheaths, but not in mature organs such as culms, branches, and rhizomes consistently at each study site (Figs. 2C, 3C, and Tables 2–4). This particular localization of B indicates that P. pubescens effectively distributes B into the organs where demand for B is high. Boron is related with the formation of primary cell walls (Blevins and Lukaszewski, 1998). Up to 90% of cellular B was found in the cell wall fraction (Loughman and White, 1984; Loomis and Durst, 1992). Boron contributes to cell wall structural stabilization by forming cross-links with the pectin rhamnogalacturonan II domain, and these cross-links are essential to plant growth (Kobayashi et al., 1996; O'Neill et al., 2001; Iwai et al., 2002). For example, borate-ester cross-linked rhamnogalacturonan II dimer (dRG-II-B) is found in the abscission zone in the pedicel and strengthens its adhesion to retain the fruit of tobacco (Tsukahara et al., 2013). In bamboo shoot cell walls, B cross-linking two RG-II molecules is also found, and it indicates that boron may play an important role in cell wall synthesis and/or structure (Kaneko et al., 1997). From these findings, we suggest that P. pubescens is possible to actively reutilize B through the retranslocation and the local accumulation for vegetative reproduction. As described above, Si, Ca, and B are involved in cell structure, however, it is considered that Si should be an essential element in P. pubescens. The concentrations of Si and Ca (47.6–68.5 mg g−1 of Si and 5.19–10.5 mg g− 1 of Ca (Tables 2–4)) and their ratios (6.5–9.9) clearly indicate that P. pubescens is a Si-accumulating plant, defined as plants with Si content higher than 1.0% and Si/Ca ratios greater than 1.0 ratio (Ma and Takahashi, 2002). The concentration ranges of Ca in leaves (1.3–13.7 mg g−1) (Fig. 4B and Table 5) were lower than those in leaves of other temperate broad-leaved trees such as 5–20 mg g−1 (Kanaya, unpublished data). This tendency is consistent with the previous report by Nishida and Shirai (1985) which compared between bamboo species and tree species, and with the fact that Si concentrations in all gramineous plants tend to be high and Ca concentrations tend to be low, in contrast to dicot species (Ma and Takahashi, 2002). In addition, the B concentration range of 0.01–9.9 μg g− 1 in P. pubescens leaves (Fig. 4C and Table 5) was also lower than that in leaves of other temperate broad-leaved trees (10–80 μg g−1; Kanaya, unpublished data). These findings clearly indicate that P. pubescens may also be characterized as a plant with low accumulation of Ca and B. In the leaves of P. pubescens, a graminaceous species, we found higher concentrations of Si and lower B and Ca concentrations. This indicates that P. pubescens utilizes more Si for growth and maintenance of their cell walls than Ca and B.

4.3. Distribution and mobilization of Zn We found higher concentrations of Zn in nodes (culm wall segments for culm nodes) than in internodes in both mature bamboo culms and rhizomes (Tables 2–4). This distribution of Zn was especially remarkable in the mature rhizomes, even higher than the concentration in growing bamboo shoots (Fig. 2F). The nodes of bamboo species are composed of meristematic tissue from which culm sheaths and branches arise (Kleinhenz and Midmore, 2001) and are involved in internode elongation by cell division within tissues located just above each node (Uchimura, 1994). Therefore, our findings indicate that Zn is likely involved in the internode elongation of culms and rhizomes in P. pubescens. Zinc accumulation in the nodes could depend on retranslocation from mature leaves. The Zn concentrations in leaves changed little through the year at all study sites (Fig. 4F), and the Zn concentrations in leaf litter (22–38 μg g− 1, Table 5) were similar to yearly concentration ranges of viable leaves. This small seasonal change could result from both accumulation of Zn into the leaves and subsequent retranslocation from there, as Zn is classified as an element with intermediate mobility in the phloem of higher plants (White, 2012). Shoot elongation of higher plants is known to be the major physiological role of Zn, as a deficiency of Zn results in shortening of internodes. Zinc is important also for protein and peptide synthesis (Persson et al., 2009), and high Zn concentrations (600 μg g− 1 dw) have been found in the scutellum of wheat seeds (Mazzolini et al., 1985). A Zn concentration of at least 100 μg g−1 dw is required for maintenance of protein synthesis in shoot meristems (Broadley et al., 2012). Although the relationship of protein synthesis to internode elongation has not been clarified in the present study, we suggest that the accumulation of Zn in the nodes of both the rhizomes and the culms of P. pubescens plays roles in promoting culm and rhizome elongation and may be involved in the formation of sheaths and lateral branches. 5. Conclusions We investigated both seasonal change in elemental concentrations of leaves and elemental distribution in mature and growing organs of P. pubescens. We found that this plant utilizes K, P and B through retranslocation linking to the life cycle. It suggests that the species can efficiently recycle those elements probably to sustain a fast growth. Boron and Ca were found in stele apex of both growing bamboo shoots and rhizomes at high concentrations. Silicon was accumulated in mature organs such as leaves, outer surface of the culm, and sheaths of growing organs, indicating the contribution of Si to cell structural enhancement. The specific distribution of Zn, high concentration in nodes, suggested that this element relates with internode elongation. In conclusion, P. pubescens has the specific systems in accumulation or retranslocation of elements such as Si, B, and Zn, which might support the rapid growth. Conflict of interest We have no relevant matter for conflict of interest directly relevant to the content of this article. Acknowledgments We would like to thank Toshio Koike, Yukizumi Takayama, Masaru Koide, Noboru Koike, and Masao Takayama for the permission to work on a private property in Noguchi. We thank Go Koyama, Takayuki Hukami, and the officers of Toyota, Aichi for allowing us to use the study site in Kanpachi. In addition, we would like to thank the officers of the Aichi Kaisho-no-mori Center for allowing us to use the study site in Seto. We are also grateful to Drs. Kazukiyo Yamamoto, Rie Tomioka, and Takafumi Tezuka, of Nagoya University for their advice and discussions regarding our research.

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