Elsevier

Plant Physiology and Biochemistry

Volume 97, December 2015, Pages 20-27
Plant Physiology and Biochemistry

Research article
Identification and functional analysis of the autofluorescent substance in Limonium bicolor salt glands

https://doi.org/10.1016/j.plaphy.2015.09.007Get rights and content

Highlights

  • The autofluorescent substance was localized in the cuticle of the salt glands.

  • The primary autofluorescent substance in the salt glands was ferulic acid.

  • Mutants showing increased or decreased fluorescence intensity were screened.

  • Ferulic acid in the cuticle was directly involved in salt secretion of salt gland.

Abstract

Limonium bicolor is a typical recretohalophyte with salt glands for the secretion of excess salts into the environment. We observed that L. bicolor salt glands showed obvious blue autofluorescence under UV excitation (330–380 nm). The aim of the present study was to identify and clarify a role for this autofluorescent substance in salt secretion. Sudan IV staining showed that the autofluorescent substance was localized in the cuticle of the salt glands. Moreover, the primary autofluorescent substance was identified as ferulic acid after treatment with 0.1 M ammonium hydroxide solution, alkaline and enzymatic hydrolysis. Additional experiments using two mutants exhibiting increased (fii) and decreased (fid) salt gland fluorescence indicated that the fluorescence intensity of salt glands under UV excitation was positively correlated with the content of ferulic acid in the cuticle, strongly suggesting that the primary autofluorescent substance in the salt glands was ferulic acid. Salt gland secretion was determined using leaf discs, and the results showed that the Na+ secretion rate per single salt gland was also positively correlated with the content of ferulic acid in the cuticle, suggesting that ferulic acid in the cuticle was directly involved in salt secretion of salt gland.

Introduction

Salinity affects more than 800 million hectares of land, accounting for more than 6% of the world's total land area (Munns and Tester, 2008). Currently, soil salinity is expected to increase due to global climate changes, population growth, industrial pollution, improper fertilizer application and irrigation practices. Salinity is one of the major factors inhibiting plant growth primarily through osmotic stress and ion toxicity (Ouhibi et al., 2014). Halophytes are able to complete a life cycle in a salt concentration of at least 200 mM NaCl and tolerate salt concentrations that kill 99% of other species (Flowers and Colmer, 2008). These organisms can be divided into recretohalophytes, euhalophytes, and pseudo-halophytes (Breckle, 1995). Limonium bicolor is a typical exo-recretohalophyte that secretes excess salt onto the plant surface under salt stress. Three hypotheses exist for the salt secretion mechanism of plant salt glands. Arisz et al. (1955) proposed that the accumulation of ions in these salt glands increases osmotic potential, and these ions are excreted out of salt gland cells through a remarkable increase in hydrostatic pressure. Ziegler and Lüttge (1967) and Shimony and Fahn (1968) speculated that salt secretion is the opposite process of exocytosis. Levering and Thomson, 1971, Levering and Thomson, 1972 proposed that salt secretion is similar to the animal flow transport system. However, the mechanism of salt secretion remains uncertain.

Yuan et al. (2013) showed that the blue autofluorescence of salt glands under UV excitation (330–380 nm) is a useful method for studying the mechanism of salt gland development and salt secretion. Plant leaves generate blue, red and far-red fluorescence under UV excitation. Red fluorescence is primarily derived from chlorophyll, whereas blue fluorescence is primarily emitted via ferulic acid and other hydroxycinnamic acids bounded to plant cell walls or the lignified cell walls of sclerenchyma fibers and xylem vessels (Harris and Hartley, 1976, Boerjan et al., 2003, Talamond et al., 2015). Blue fluorescence is an intrinsic property of lignin (Lundquist et al., 1978). Lignins are primarily composed of the monolignols p-coumaryl, coniferyl and sinapyl alcohols that give rise to the p-hydroxyphenyl (H), guaiacyl (G) and sinapyl alcohol (S) units, respectively, of the lignin polymer (Voxeur et al., 2015). Lignins are deposited in secondary thickened cells, where these polymers provide strength and impermeability to the wall (Vanholme et al., 2012). Ferulic acid and p-coumaric acid have been characterized as the primary phenylpropanoids responsible for the characteristic UV-induced blue fluorescence of the surface tissues of several plant species (Lichtenthaler and Schweiger, 1998, Karabourniotis et al., 2001). In particular, ferulic acid is the main emitter of blue fluorescence, whereas in low amounts, caffeic acid and p-coumaric acid contribute little to the overall blue fluorescence emission of leaves (Lichtenthaler and Schweiger, 1998). Moreover, due to the presence of ferulic acid, the cuticles of the leaf epidermis and stomatal guard cells also emitted blue fluorescence when excited with UV radiation (Hartley and Harris, 1981, Jones et al., 2005). Furthermore, ferulic acid exhibited blue autofluorescence at a low pH (5.4) and green autofluorescence with greater intensity at a high pH (10.3) under UV light, but lignin autofluoresced blue at both pH levels (Harris and Hartley, 1976, Harris and Trethewey, 2010).

Little is known about the components responsible for the blue autofluorescence of salt glands under UV excitation (330–380 nm) and about the role for these components in salt secretion. In the present study, we identified the substance that is primarily responsible for the blue autofluorescence of salt glands in L. bicolor. The function of this fluorescent substance in salt secretion was also investigated using two fluorescent mutants of L. bicolor induced using gamma irradiation.

Section snippets

Plant materials and growth conditions

L. bicolor seeds were collected from native soil saline–alkaline land (N37°200′, E118°360′) in the Yellow River Delta in China. The seeds were sown in plastic pots (22 cm high × 20 cm diameter) containing washed sand. After germination, the seedlings were sufficiently watered with ½ Hoagland nutrient solution daily and cultured at 24 °C under 600 μmol m−2 s−1 illumination, with a 14-h-light/10-h-dark cycle in a controlled environmental growth chamber.

Approximately 30,000 seeds were treated with

Sudan IV staining confirmed that the cuticle was involved in salt gland autofluorescence

The salt glands of L. bicolor were surrounded with blue autofluorescence under UV excitation (330–380 nm) (Fig. 1Ac and Bc). Compared with the control (Fig. 1Aa and Ba), the regions of the salt glands showing autofluorescence turned red after Sudan IV staining (Fig. 1Ab and Bb; SFig. 1). The vascular bundles and internal cuticle from the outer stomatal ledge to the substomatal cavity also showed blue autofluorescence under UV excitation (330–380 nm) (SFig. 2; SFig. 3b). The regions of the

Discussion

The cuticle, one of the key adaptations in the evolution of land plants, covers the surfaces of all aerial plant organs. The leaf surface cuticle extends from the outer stomatal ledge to the epidermal cells bordering the substomatal cavity (Wullschleger and Oosterhuis, 1989), and the outermost layer of salt glands is encapsulated by the cuticle (Feng et al., 2014). Waxes, fats and fatty acids are known to be colored red by Sudan IV which is typically used to visualize the cuticle (Lulai and

Contributions

Bao-Shan Wang designed the experiments and revised the manuscript. Yunquan Deng, Zhongtao Feng and Fang Yuan performed the experiments. Jianrong Guo and Shanshan Suo conducted the HPLC analyses. Yunquan Deng drafted the manuscript.

Acknowledgments

This work was financially supported through grants from the NSFC (National Natural Science Research Foundation of China, project No. 30870158), Programs Foundation of the Ministry of Education of China (20123704130001) and Natural Science Research Foundation of Shandong Province (ZR2014CZ002).

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