Immunolocalization of Endogenous Indole-3-Acetic Acid and Abscisic Acid in the Shoot Internodes of Fargesia yunnanensis Bamboo during Development

The Bambusoideae subfamily includes the fastest-growing plants worldwide, as a consequence of fast internode elongation. However, few studies have evaluated the temporal and spatial distribution of endogenous hormones during internode elongation. In this paper, endogenous indole-3-acetic acid (IAA) and abscisic acid (ABA) were detected in different developmental internodes during shoot elongation by immunolocalization. Immunohistochemistry showed that IAA was mainly present in the shoot apex, leaf sheath primordia, parenchymal cells, and vascular tissues. During internode elongation and maturation, the IAA signals decreased significantly and then increased slightly, with the weakest signals observed in the rapidly elongating internode. Based on immunogold localization, most IAA signals were detected in the cytoplasm and nuclei of both parenchymal and fiber cells, and few signals were detected in cell walls in the unelongated and elongating internodes. After the completion of internode elongation, additional IAA signals were detected in the secondary walls of both parenchymal and fiber cells. Immunohistochemical localization of ABA showed that ABA signals decreased with internode elongation and maturation, with the weakest signal observed in the internodes of 3-month-old shoots. In addition, few ABA signals were detected in the shoot apex. The strongest IAA and ABA signals in unelongated internodes suggested that both hormones participated in the mediation of internode differentiation but not in the rapid elongation. Moreover, IAA was involved in secondary cell wall deposition. Phytohormones are small molecules that are derived from secondary metabolism and play key roles in shaping the plant architecture (Santner and Estelle, 2009). The presence and effect of some of these hormones have been recognized for more than a century (Jaillais and Chory, 2010). IAA is the most abundant naturally occurring auxin and is involved in many aspects of plant growth and development, such as cell division and elongation, differentiation, tropism, apical dominance, senescence, abscission, and flowering (Chudasama and Thaker, 2007; Mano and Nemoto, 2012; Teale et al., 2006; Woodward andBartel, 2005). To date, a large number of studies have focused on the role of auxin in rhizogenesis (Dong et al., 2012, 2014; Falasca et al., 2004; Gangopadhyay et al., 2010; Ludwig-Muller et al., 2005; Xuan et al., 2008), embryogenesis and development (Chen et al., 2010; Thomas et al., 2002; Zhang et al., 2015), and floral induction (Hou and Huang, 2005). ABA is a small molecule that is naturally generated in plants and was named for its propensity to accumulate in dry, mature leaves (Moore, 1989). After ABA was first identified, it quickly became accepted as one of the five major phytohormones (Peng et al., 2006). ABA has major roles in plant responses to drought or osmotic stress, cold stress, wounding, and pathogen assault (Mauch-Mani and Mauch, 2005; Verslues and Zhu, 2005; Received for publication 17 May 2016. Accepted for publication 15 Aug. 2016. This research was fully funded by the Advanced and Characteristic Key Biological Disciplines of Yunnan Province (50097505), the Science Foundation of Yunnan Province (2015FB150), and the China National Science Foundation (31560196). These authors contributed equally to this work. Corresponding author. E-mail: [email protected]. J. AMER. SOC. HORT. SCI. 141(6):563–572. 2016. 563 Yamaguchi-Shinozaki and Shinozaki, 2005). ABA also functions in seed dormancy and germination (Gubler et al., 2005). In addition, ABA has also been reported to participate in sugar signaling (Arenas-Huertero et al., 2000; Brocard-Gifford et al., 2004; Cxakir et al., 2003; Carrari et al., 2004; Finkelstein and Gibson, 2002; Rook et al., 2001; Smeekens, 2000) and to affect the distribution of sucrose and protons across the plasmalemma in the pea (Pisum sativum) mesophyll (Opaskornkul et al., 1999). Furthermore, ABA is preferentially localized in phloem cells (Peng et al., 2003; Zhang et al., 1999, 2001a, 2001b). Therefore, ABA may play some ‘‘positive’’ role during plant development by participating in the regulation of the distribution of assimilation, in addition to its well-known function as a ‘‘negative hormone’’ (Peng et al., 2006). Bamboos (Poaceae, Bambusoideae) are the fastest-growing plants worldwide, and their culms can reach their final height within relatively short periods of 2 to 4 months (Magel et al., 2005). Although some research has been conducted to assess bamboo growth (Cui et al., 2012; Peng et al., 2013), the mechanism responsible for the fast growth rate remains unknown. Plant hormones play an important role in the regulation and coordination of cell proliferation and elongation (Wang et al., 2015). However, to date, very few studies have focused on endogenous hormones in bamboos or on endogenous hormone contents in moso bamboo (Phyllostachys pubescens) (Ding, 1997;Wang et al., 2015). Most of the reported studies have been quantitative analyses of phytohormones using various analytical methods. However, even the most advanced methods must use a certain amount of material, and the concentration of phytohormones in the target site and not the whole tissue could reflect activity levels (Dong et al., 2012). Therefore, immunohistochemical localization is an important technique for evaluating endogenous hormones during the rapid growth of bamboos. In addition, information regarding the sites of IAA and ABA storage and transport, and the characteristics of their dynamic spatial changes during shoot elongation are quite limited, necessitating further investigations. The subcellular localization of endogenous IAA and its role in shoot elongation remain unclear. In this paper, the spatial distribution patterns of endogenous IAA and ABA, and their dynamic changes during shoot elongation of Fargesia yunnanensis were investigated. The correlation between phytohormones and bamboo internode elongation was also analyzed. The findings presented will provide more information regarding the regulation of endogenous IAA and ABA during shoot internode elongation. Materials and Methods PLANT MATERIALS. Fargesia yunnanensis belongs to sympodial bamboos with a long-necked patchymorph rhizome (pseudorhizomes) that grows to a height of 10 m. Shoot growth is usually initiated in September. Among the internodes assessed herein, the first (labeled as internode 1), third (labeled as internode 3), and fifth (labeled as internode 5) aboveground internodes, the apex portion of the 0.5-monthold shoot, internode 1 of the 1-month-old culms, and internode 1 of the 3-month-old culms were obtained and used as samples for the immunohistochemical localization of IAA and ABA. All samples were collected in Sept. and Oct. 2013. The characteristics of the sampled internodes are presented in Table 1. PARAFFIN AND ULTRATHIN SECTIONS. The middle portions of all chosen internodes were cut into 3-mm blocks, which were immediately prefixed in a 2% (wt/v) aqueous solution of 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide hydrochloride (Sigma-Aldrich, St. Louis, MO) at 4 C for 8 h and then postfixed in glutaraldehyde fixative containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.025 M phosphate buffer solution (PBS, pH 7.2) at 4 C. For the microscopic observation, paraffin embedding was employed. Seven-micrometer-thick paraffin sections were cut using a rotary microtome. To determine the subcellular localization of endogenous IAA, the samples were dehydrated in a graded ethanol series and then embedded in Lowicryl K4MResin (Polysciences, Warrington, PA) at –20 C. The resins were polymerized by long-wavelength ultraviolet irradiation for 48 h. Semithin sections were cut to examine the location of vascular bundles, and then ultrathin sections (60 nm) were cut with a diamond knife on an ultramicrotome and mounted on nickel grids (100 mesh) with Formvar (Zhongjingkeyi, Beijing, China). LOCALIZATION OF IAA. The tissue-specific immunolocalization of endogenous IAAwas performed as follows. After washing three times with TBST1 solution [(0.05 M pH 7.4 Tris-HCl buffer, 0.15 M NaCl) solution + 0.3% (v/v) Triton X-100 (Solarbio, Beijing, China)], the sections were incubated for 45 min in a blocking solution [5% (wt/v) bovine serum albumin (BSA; Sigma-Aldrich) and 1.5% (wt/v) glycine in TBST1 solution] and then incubated overnight at 4 Cwith anti-IAA antibodies (Agdia, Table 1. Morphological characteristics of the sampled Fargesia yunnanensis internodes by microscopy and transmission electron microscopy. Sample Position Age (mo.) Morphological characteristics Shoots (27 cm in height) Apex and internodes close to the apex 0.5 Unelongated, soft, faint yellow, and enveloped entirely by purple