Research articleIdentification and functional analysis of the autofluorescent substance in Limonium bicolor salt glands
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).
References (40)
- et al.
Feruloyl esterases as a tool for the release of phenolic compounds from agro-industrial by-products
Carbohydr. Res.
(2006) - et al.
Phenolic constituents of the cell walls of dicotyledons
Biochem. Syst. Ecol.
(1981) - et al.
Epicuticular phenolics over guard cells: exploitation for in situ stomatal counting by fluorescence microscopy and combined image analysis
Ann. Bot.
(2001) - et al.
Potential applications of ferulic acid from natural sources
Biotechnol. Rep.
(2014) - et al.
Cell wall bound ferulic acid, the major substance of the blue-green fluorescence emission of plants
J. Plant Physiol.
(1998) - et al.
Ferulic and p-coumaric acids extraction by alkaline hydrolysis of brewer's spent grain
Ind. Crop. Prod.
(2007) Unraveling the complex network of cuticular structure and function
Curr. Opin. Plant Biol.
(2006)- et al.
Salt stress mitigation by seed priming with UV-C in lettuce plants: growth, antioxidant activity and phenolic compounds
Plant Physio. Biochem.
(2014) - et al.
Parathyroid hyperplasia in uremic rats precedes down-regulation of the calcium receptor
Kidney Int.
(2001) - et al.
Lignification: different mechanisms for a versatile polymer
Curr. Opin. Plant Biol.
(2015)
The occurrence of an internal cuticle in cotton (Gossypium hirsutum L.) leaf stomates
Environ. Exp. Bot.
An efficient autofluorescence method for screening Limonium bicolor mutants for abnormal salt gland density and salt secretion
South Afr. J. Bot.
The secretion of the salt glands of Limonium latifolium Ktze
Acta botánica Neerl.
Lignin biosynthesis
Annu. Rev. Plant Biol.
How do halophytes overcome salinity
Biol. Salt Toler. Plant.
Study on pathway and characteristics of ion secretion of salt glands of Limonium bicolor
Acta Physiol. Plant.
New insights into the properties of pubescent surfaces: peach fruit as a model
Plant Physiol.
Salinity tolerance in halophytes
New Phytol.
Detection of bound ferulic acid in cell walls of the Gramineae by ultraviolet fluorescence microscopy
Nature
The distribution of ester-linked ferulic acid in the cell walls of angiosperms
Phytochem. Rev.
Cited by (68)
Analyzing the structure-activity relationship of raspberry polysaccharides using interpretable artificial neural network model
2024, International Journal of Biological MacromoleculesThe MYB transcription factor LbCPC of Limonium bicolor negatively regulates salt gland development and salt tolerance
2023, Environmental and Experimental BotanySalt tolerance in plants: Using OMICS to assess the impact of plant growth-promoting bacteria (PGPB)
2022, Mitigation of Plant Abiotic Stress by Microorganisms: Applicability and Future DirectionsThe RING zinc finger protein LbRZF1 promotes salt gland development and salt tolerance in Limonium bicolor
2024, Journal of Integrative Plant BiologyDealing with extremes: insights into development and operation of salt bladders and glands
2024, Critical Reviews in Plant Sciences
- 1
These authors are equally contributed to this article.