Saturated humidity accelerates lateral root development in rice (Oryza sativa L.) seedlings by increasing phloem-based auxin transport

Tory Chhun 2 Yuichi Uno 1 Shin Taketa 0 Tetsushi Azuma 1 Masahiko Ichii 0 Takashi Okamoto 2 Seiji Tsurumi 2 0 Faculty of Agriculture, Kagawa University , Miki, Kagawa, 761-0795 Japan 1 Faculty of Agriculture, Kobe University , Kobe, 657-8501 Japan 2 Center for Supports to Research and Education Activities Isotope Division, Kobe University , Kobe, 657-8501 Japan - Auxin transport plays a significant role modifying plant growth and development in response to environmental signals such as light and gravity. However, the effect of humidity on auxin transport is rarely documented. It is shown here that the transport of labelled indole3-acetic acid (IAA) from the shoot to the root is accelerated in rice (Oryza sativa L. ssp. indica cv. IR8) seedlings grown under saturated humidity (SHseedlings) compared with plants grown under normal humidity (NH-seedlings). The development of lateral roots in SH-seedlings was greatly enhanced compared with NH-seedlings. Removal of the shoot from SHseedlings reduced the density of lateral roots, and the application of IAA to the cut stem restored the lateral root density, while the decapitation of NH-seedlings did not alter lateral root development. Phloem-based auxin transport appeared responsible for enhanced lateral root formation in SH-seedlings since (i) the rate of IAA transport from the shoot to the root tip was greater than 3.5 cm h21 and (ii) naphthylphthalamic acid (NPA)-induced reduction of polar auxin transport in the shoot did not influence the number of lateral roots in SH-seedlings. It is proposed that high humidity conditions accelerate the phloem-based transport of IAA from the leaf to the root, resulting in an increase in the number of lateral roots. Introduction Auxins play a pivotal part in plant growth and development, including cell enlargement and division, lateral branching of shoots and roots, vascular differentiation, gravitropism, and early embryonic development (Davies, 1995; Hobbie, 1998). The auxin indole-3-acetic acid (IAA) is unique amongst plant hormones in being directionally transported (Lomax et al., 1995). In addition to directional transport, auxin can also move through phloem (Morris and Kadir, 1972). These auxin pathways are not constant processes but change thoroughout the plant life cycle or in response to environmental stimuli such as light and gravity (Lomax et al., 1995). In shoot tissues, polar auxin transport occurs in a basipetal direction from the shoot apex to the base. In contrast, auxin transport is more complex in roots, where IAA is transported acropetally through the central cylinder and basipetally via the outer layers of root cells (Tsurumi and Ohwaki, 1978; Estelle, 1996; Marchant et al., 1999; Muday, 2001; Blilou et al., 2005; Swarup et al., 2005). Recent knowledge about auxin transport has been primarily obtained through a series of studies on auxin influx and efflux carriers using Arabidopsis mutants. Auxin is taken up by plant cells via a carrier protein termed AUX1 * To whom correspondence should be addressed. E-mail: or Abbreviations: NH, normal humidity; NPA, naphthylphthalamic acid; SH, saturated humidity. The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: (Bennett et al., 1996; Swarup et al., 2004; Yang et al., 2006) and is mobilized out of cells via auxin efflux carriers encoded by a family of PIN genes (Galweiler et al., 1998; Benkova et al., 2003; Petrasek et al., 2006) or PGPs (Geisler et al., 2005). Mutations of AUX1 and AGR1/EIR1/ PIN2/WAV6 (Chen et al., 1998; Luschnig et al., 1998; Muller et al., 1998; Utsuno et al., 1998; Marchant et al., 1999) disrupt gravity response in roots and/or lateral root formation, demonstrating that auxin transport plays a critical role in root growth and development. Auxin synthesized in young apical parts and leaves is transported to the roots through the phloem or a polar transport system (Reed et al., 1998; Marchant et al., 2002; Ljung et al., 2005). However, the respective role of the two auxin pathways for root development is still unclear. In the present study, it is shown that the saturated humidity surrounding the shoot of rice seedlings influences lateral root development by impacting phloem-based auxin transport from the shoot to the root. This represents a novel mechanism for how an environmental stimulus could modify root architecture by influencing auxin transport. Materials and methods Plant materials and growth condition Seeds of rice cultivar IR8 (Oryza sativa L. ssp. indica) were surface-sterilized in a 0.2% (v/v) Benomyl (Dupont Company, Tokyo, Japan) solution for 24 h, rinsed, and soaked in water overnight in the dark at 30 C. Germinated seeds were transferred to a floating net and grown hydroponically in a 400 ml glass cup under continuous white light at an irradiance of approximately 100 lmol m 2 s 1 for 4 d at 25 C in a growth room, where the relative humidity was kept at 60%. For saturated humidity, glass cups were placed in a closed glass chamber of 3.0 l with or without a continuous flow (1.8 l min 1) of water-saturated fresh air (Fig. 1; see Fig. 1. Growth conditions for NH-seedlings propagated at 60% relative humidity (A) versus SH-seedlings grown at 100% relative humidity in a closed glass chamber (B). Rice seedlings were grown hydroponically in a 400 ml glass cup under continuous white light at an irradiance of approximately 100 lmol m 2 s 1 for 4 d at 25 C in a room, where the relative humidity was maintained at 60%. Supplementary Fig. S1 at JXB online). Relative humidity was measured with a digital humidity meter (HN-K, Chino Corp) Transport of labelled IAA from the shoot to the root Auxin transport from the shoot to the root was performed mainly as described by Chhun et al. (2004) with a slight modification. The shoots of 4-d-old seedlings were decapitated and the cut stem of 0.5 cm in length was capped with a small plastic tip containing 3 ll of 10 mM MES buffer (pH 5.7) supplemented with 1 lM 5-[3H]IAA (specific activity 740 MBq lmol 1, American Radiolabelled Chemicals, Inc., St Louis, MO, USA). The [3H]IAA-treated seedlings were incubated for various transport periods under 60% or 100% relative humidity. Radioactivity was measured either on the whole root or after dividing the root into three or four segments. The length of root segments was approximately 1 cm while the length of the root tip segment was slightly longer than 1 cm depending on the length of the whole root. Ten whole roots or root segments were combined, and were placed into 5 ml of scintillation fluid overnight. Radioactivity was counted with a scintillation counter (model LS6500, Beckman Instruments, Fullerton, CA, USA). Basipetal transport of labelled IAA in root segments Basipetal auxin transport in roots was measured as described by Chhun et al. (2005) with a slight modification. An apical root segment of 1.6 (...truncated)