Physiologia Plantarum 153: 513–524. 2015
© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317
Metabolic profiling reveals altered pattern of central metabolism in navel orange plants as a result of boron deficiency Guidong Liua , Xiaochang Donga , Leichao Liua , Lishu Wua , Shu’ang Pengb and Cuncang Jianga,* a Key b
Laboratory of Horticulture Plant Biology (HZU) MOE, Microelement Research Center, Huazhong Agricultural University, Wuhan 430070, China Key Laboratory of Horticulture Plant Biology (HZU) MOE, Huazhong Agricultural University, Wuhan 430070, China
Correspondence *Corresponding author, e-mail:
[email protected] Received 17 March 2014; revised 28 May 2014 doi:10.1111/ppl.12279
We focused on the changes of metabolite profiles in navel orange plants under long-term boron (B) deficiency using a gas chromatography–mass spectrometry (GC–MS) approach. Curling of the leaves and leaf chlorosis were observed only in the upper leaves (present before start of the treatment) of B-deficient plants, while the lower leaves (grown during treatment) did not show any visible symptoms. The metabolites with up-accumulation in B-deficient leaves were mainly proline, L-ornithine, lysine, glucoheptonic acid, fucose, fumarate, oxalate, quinate, myo-inositol and allo-inositol, while the metabolites with down-accumulation in B-deficient leaves were mainly serine, asparagine, saccharic acid, citrate, succinate, shikimate and phytol. The levels of glucose and fructose were increased only in the upper leaves by B deficiency, while starch content was increased in all the leaves and in roots. The increased levels of malate, ribitol, gluconic acid and glyceric acid occurred only in the lower leaves of B-deficient plants. The increased levels of phenols only in the upper leaves indicated that the effects of B on phenol metabolism in citrus plants may be a consequence of disruptions in leaf structure. Metabolites with opposite reactions in upper and lower leaves were mainly glutamine, glycine and pyrrole-2-carboxylic acid. To our knowledge, the phenomena of allo-inositol even higher than myo-inositol occurred characterized for the first time in this species. These results suggested that the altered pattern of central metabolism may be either specific or adaptive responses of navel orange plants to B deficiency.
Introduction The essential role of boron (B) in plants was first described 90 years ago (Warington 1923). B has been shown to be essential to the structure and function of plant cell walls, where it cross-links pectic polysaccharides through borate-diol bonding of two rhamnogalacturonan II (RG-II) molecules (Kobayashi et al. 1996, O’Neill et al. 1996, Ishii et al. 1999).
Genetic studies employing the Arabidopsis thaliana mur1 mutant further indicated that borate cross-linked RG-II is essential for normal plant growth (O’Neill et al. 2001). The evidence provided by recent cell wall studies explains many problems caused by B deficiency, but there are indeed some other aspects of plant B nutrition that go beyond cell wall structure (Blevins and Lukaszewski 1998, Goldbach and Wimmer 2007). Increasing evidence suggests that B is also an essential
Abbreviations – GABA, 𝛾-aminobutyric acid; GC–MS, gas chromatography–mass spectrometry; HCA, hierarchical clustering analysis; MDA, malondialdehyde; OAA, oxaloacetic acid; PCA, principal component analysis; RGII, rhamnogalacturonan II; TCA, tricarboxylic acid.
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element in cyanobacteria (Bonilla et al. 1997), yeast (Bennett et al. 1999) and mouse (Lanoue et al. 2000). These findings highlight the broad roles of B in biology. World-wide, B deficiency is more extensive than deficiency of any other plant micronutrient (Shorrocks 1997). In agriculture, B deficiency is a major problem that impedes crop growth and generally leads to the rapid cessation of root elongation, reduced leaf expansion and reduced fertility, mainly due to reduced cell expansion (Dell and Huang 1997). Exposure of plants to B deficiency will also result in a wide range of metabolic responses (Brown et al. 2002). In contrast to the role of B in cell wall, B role in plant metabolism, including carbohydrate (Kastori et al. 1995, El-Shintinawy 1999, Stavrianakou et al. 2006), nitrogen (Ruiz et al. 1998) and phenolic metabolism (Cakmak et al. 1995, Pfeffer et al. 1998, Dordas and Brown 2005), is still a subject of considerable debate. In addition, the information about effects of B deficiency on organic acids metabolism is very limited (Tang et al. 2011). While changes in levels of several metabolites following B deficiency stress are extensively documented (Camacho-Cristóbal et al. 2004, Han et al. 2008), less well documented are the changes that occur in the metabolome overall. An unbiased and sensitive analytical technique such as gas chromatography–mass spectrometry (GC–MS) which allows the simultaneous analysis of metabolites in a complex extract can be used to understand the metabolic networks within plants (Kueger et al. 2012). Metabolites reflect the integration of gene expression, protein interaction and other different regulatory processes and are therefore closer to the phenotype than mRNA transcripts or proteins alone (Arbona et al. 2013). The metabolomics technique has recently been used to detect the changes in a number of different metabolites like organic acids, amino acids, sugars and secondary metabolites caused by mineral deficiency (Takahashi et al. 2012, Foito et al. 2013). The effect of B deficiency on metabolism has been demonstrated in many herbaceous plant species, but information on woody plants is scarce (Han et al. 2008). It has been suggested that perennials are less vulnerable to severe B deficiency than herbaceous plants (Ruuhola et al. 2011). In China, B deficiency is frequently observed in citrus orchards, and is responsible for considerable loss of productivity and quality (Han et al. 2008, Jiang et al. 2009). However, studies on investigating the relationship between the extent of changes in leaf structure induced by B deficiency and the corresponding metabolic profiling are very limited (Alves et al. 2011). In this study, we focused on the changes of metabolite profiles in navel orange plants under long-term B deficiency. The different types of 514
leaves were found to differ in their appearance and metabolite responses to the imposed B-deficient stress. This differential response is discussed in terms of the potential involvement of particular metabolites in either specific or adaptive responses of citrus to B deficiency.
Materials and methods Plant materials and growth conditions We used 8-month-old navel orange plants (Citrus sinensis cv. Newhall) grafted on Carrizo citrange [C. sinensis (L.) Osb. × Poncirus trifoliate (L.) Raf.] rootstock with uniform stem diameter (6–7 mm), height (18–22 cm) and total fresh weight (20–25 g). The selected plants all consisted of only one main shoot of the scion and had 12–15 leaves, which were not fully expanded at the beginning of the experiment. All the plants were washed with distilled water to remove surface contaminants after soaking in tap water for 2 days, followed by transplantation to black pots (one plant per pot), each containing 8-l of nutrient solution. Prior to the experiment, the black pots were immersed in 1 M HCl and washed with distilled water. Modified from Hoagland and Arnon (1950), the composition and salt contents of the basal culture solution were: 1 mM KNO3 , 1.23 mM Ca(NO3 )2 , 0.5 mM MgSO4 , 0.07 mM Na2 HPO4 , 0.16 mM NaH2 PO4 , 4.45 μM MnCl2 , 0.8 μM ZnSO4 , 0.16 μM CuSO4 , 0.18 μM Na2 MoO4 and 28.7 M iron-ethylenediaminetetraacetic acid (Fe-EDTA). The nutrient solution was used at half strength for the first 7 days with 10 μM H3 BO3 . Subsequently, the nutrient solution was enhanced to the full strength with 20 μM H3 BO3 as a sufficient control and no B added as a B deficiency treatment. Purified water was obtained by a system consisting of three units (active charcoal, ion exchanger and reverse osmosis), and had an electric conductivity lower than 0.06 μS cm−1 (B concentration
