Is naphthylphthalamic acid a specific phytotropin? It elevates ethylene and alters metabolic homeostasis in tomato

Highlights

• N-1-naphthylphthalamic acid (NPA) treatment stimulates tomato hypocotyl elongation likely by elevating ethylene emission.

• NPA treatment lowers the indole-3-butyric acid and elevates zeatin level in the seedlings.

• NPA effect on tomato is opposite to Arabidopsis, indicating that NPA-mediated responses vary in a species-specific fashion.

Abstract

In higher plants, phytohormone indole-3-acetic acid is characteristically transported from the apex towards the base of the plant, termed as polar auxin transport (PAT). Among the inhibitors blocking PAT, N-1-naphthylphthalamic acid (NPA) that targets ABCB transporters is most commonly used. NPA-treated light-grown Arabidopsis seedlings show severe inhibition of hypocotyl and root elongation. In light-grown tomato seedlings, NPA inhibited root growth, but contrary to Arabidopsis stimulated hypocotyl elongation. The NPA-stimulation of hypocotyl elongation was milder in blue, red, and far-red light-grown seedlings. The NPA-treatment stimulated emission of ethylene from the seedlings. The scrubbing of ethylene by mercuric perchlorate reduced NPA-stimulated hypocotyl elongation. NPA action on hypocotyl elongation was antagonized by 1-methylcyclopropene, an inhibitor of ethylene action. NPA-treated seedlings had reduced levels of indole-3-butyric acid and higher levels of zeatin in the shoots. NPA did not alter indole-3-acetic levels in shoots. The analysis of metabolic networks indicated that NPA-treatment induced moderate shifts in the networks compared to exogenous ethylene that induced a drastic shift in metabolic networks. Our results indicate that in addition to ethylene, NPA-stimulated hypocotyl elongation in tomato may also involve zeatin and indole-3- butyric acid. Our results indicate that NPA-mediated physiological responses may vary in a species-specific fashion.

Introduction

Plant development is mediated by coordinated synthesis and distribution of several plant hormones. Among the plant hormones, the auxin is involved in almost all developmental responses throughout the lifecycle of the plants [1]. While most plant hormones function in a cell-autonomous fashion, several developmental responses mediated by the auxins such as leaf primordia initiation, tropic movement of organs are attributed to its directional transport within the tissue and/or organ. The orientation of auxin transport in an organ or tissue is mediated by a battery of transporters and associated proteins, which together imparts the specialized transport termed as polar auxin transport (PAT) [2]. It is believed that auxin being a weak acid diffuses in the cells as an uncharged molecule, wherein it is dissociated to the charged form, and its efflux is mediated from the cells by polarly localized auxin transporters.

The analysis of the mutants defective in PAT led to the identification of several genes encoding for the proteins facilitating PAT. Several physiological and molecular evidences indicate that the PAT is mediated by the distinct influx and efflux carriers presumably localized at the plasma membrane. Molecular-genetic evidence indicated that the members of the AUX/LAX family act as auxin influx carriers [3]. The auxin efflux from cells is mediated by a group of proteins belonging to PIN and ABCB family [4] and is modulated by additional proteins such as PINOID [5]. The maintenance of polar auxin transport system is essential for normal development of the plant. Thus, the mutants altered in auxin transport display several developmental abnormalities [6,7].

Prior to molecular genetic analysis of mutants, most information regarding the contribution of auxin to plant development came from the use of pharmacological inhibitors that specifically affect auxin transport. The inhibitors like 1-naphthoxyacetic acid, 2-naphthoxyacetic acid, 3-chloro-4-hydroxyphenylacetic acid inhibit auxin influx carriers [8], whereas 1-N-naphthylphthalamic acid (NPA), 2-(1-pyrenoyl)-benzoic acid, cyclopropyl propane dione, 2,3,5-triiodobenzoic acid, morphactins inhibit auxin efflux carriers [9]. Among these inhibitors, NPA is the most widely used and classified as a phytotropin, a class of chemicals that inhibit tropic responses in stems. Using NPA, several developmental responses have been attributed to the PAT, such as leaf vein patterning [10], phyllotaxy [11], lateral root development [12] and embryo development [13]. The action of NPA on auxin efflux is attributed to its interaction with several cellular components. It binds to twisted-dwarf-1 (TWD1), ABCB1, ABCB19, and it's binding to TWD1, and ABCB1 prevents their protein-protein interaction leading to reduced PAT [14]. In addition, it also binds to plasma membrane-associated aminopeptidases APM1 and APP1 [15]. While NPA does not bind to PIN proteins, it alters the intracellular cycling of PIN proteins between endosomal vesicles and the plasma membrane [16].

Auxin-induced cell expansion is one of the most extensively studied growth response. Since post-germination Arabidopsis hypocotyl growth mainly consists of cell expansion, several studies used hypocotyls as a model system to examine the role of light and phytohormones, particularly auxin and ethylene on hypocotyl elongation [17,18]. Ethylene suppresses elongation of hypocotyls in dark-grown seedlings, while it promotes elongation in light-grown seedlings [19,20]. In light, ethylene stimulates translocation of COP1 to the nucleus, where it mediates HY5 degradation contributing to hypocotyl growth in the light [21]. The auxin transport plays an important role in hypocotyl elongation of light-grown seedlings. NPA treatment strongly inhibited hypocotyl elongation in light-grown seedlings whereas it had minimal effect in etiolated Arabidopsis seedlings [22] The above light effect is likely related to auxin transport, as the rate of PAT is lower in hypocotyls of etiolated seedlings of Arabidopsis and tomato than in light-grown seedlings [23,24]. The light-dependent auxin transport is likely mediated by both phytochrome and cryptochrome as NPA effect on hypocotyl elongation is subdued in the mutants defective in above photoreceptors [22].

Relatively little information is available about the effect of NPA on cellular metabolism and other plant hormones. Emerging evidence indicates that NPA also influences other hormonal responses. Arabidopsis plants grown on NPA containing media for five days showed significantly reduced indole-3-acetic acid (IAA) levels in the leaves [25]. A likely link between auxin transport and cytokinin level is indicated as NPA strongly decreased transcripts of cytokinin oxidases, AtCKX1, and AtCKX6 [26]. Application of NPA mimicked the cytokinin induction of the off-the-medium growth of Arabidopsis root tip [27]. NPA also mimicked the effect of exogenous cytokinin in inducing root-like organogenesis in excised hypocotyls of Arabidopsis [28]. A relationship between NPA and ethylene is also indicated as NPA sensitized roots of ethylene resistant eir1 mutant to ethylene [29].

To better understand the effect of NPA on plant hormones level and the cellular metabolism, we grew tomato seedlings on the medium containing NPA. Surprisingly, NPA stimulated hypocotyl elongation in tomato seedlings rather than inhibiting it, as reported in Arabidopsis. This variance in NPA response compared to Arabidopsis was restricted to hypocotyl, as NPA inhibited root elongation akin to Arabidopsis. NPA treatment also influenced the metabolic and hormonal homeostasis of tomato seedlings, elevating ethylene synthesis, zeatin levels, and reducing indole-3-butyric acid (IBA) level. Our study indicates that a species-specific variation exists in NPA effect on hypocotyl elongation. Importantly, in addition to influencing PAT, it also has a wider range of influence encompassing modulation of hormonal levels and cellular metabolites.