HR-MAS NMR metabolomics of ‘Swingle’ citrumelo rootstock genetically modified to overproduce proline Caroline S. de Oliveira, a,c Eduardo F. Carlos, b Luiz G. E. Vieira, b Luciano M. Lião c and Glaucia B. Alcantara a * The accumulation of proline is a typical physiological response to abiotic stresses in higher plants. ‘Swingle’ citrumelo, an important rootstock for citrus production, has been modified with a mutated Δ 1 -pyrroline-5-carboxylate synthetase gene (VaP5CSF129A) linked to the cauliflower mosaic virus 35S promoter to induce the overproduction of free proline. This paper presents a comparative metabolomic study of nontransgenic versus transgenic ‘Swingle’ citrumelo plants with high endogenous proline. 1 H high-resolution magic angle spinning nuclear magnetic resonance spectroscopy and multivariate analysis showed significant differences in some metabolites between the nontransgenic and transgenic leaves and roots. The overproduction of proline has reduced the sucrose content in transgenic leaves, revealing a metabolic cost for these plants. In roots, the high level of free proline acts for the adjustment of cation–anion balance, causing the reduction of acetic acid content. The same sucrose level in roots indicates that they can be considered as sucrose sink. Similar behavior may be waited for fruits produced on transgenic rootstock. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: NMR; HR-MAS; citrus; genetically modified organism; proline; chemometrics Introduction Oranges and other citrus (Citrus sp.) are the most widely grown fruits in the world, with production in more than 80 countries. [1] Brazil is the principal producer and exporter of concentrated orange juice, with over 18.2 million tons produced in 2012. [2] However, production and fruit quality of citrus in Brazil are affected by the intensity of drought. Long dry spells retard the growth of the fruit and cause young fruit to drop. Fruits that can reach a mature stage are of lower quality and are deficient in juice, thus increasing the cost of the extraction process. [3] During drought periods, plants can accumulate low molecular weight compounds known as osmolytes, which are reported to be responsible for protecting the cellular structures and, hence, for raising the plant’s tolerance to hydric deficit. The osmolytes may contribute in sustenance of physiological processes because of decrease in osmotic potential, maintenance of water absorp- tion, and cellular pressure potential. [4,5] Genetic modification for increasing the production of osmolytes is a common strategy that has been used to cope with the adverse effects of abiotic stress. This strategy has been employed with several genes, such as the Arabidopsis thaliana Δ 1 -pyrroline-5-carboxylate synthetase (AtP5CS), the sacB (isolated from Bacillus subtilis), the codA (isolated from Arthrobacter globiformis), [6] and the Escherichia coli proB74. [7] In this context, proline is one of the most important osmolytes, representing an overall response of many plants exposed to environmental stresses. [4] A P5CS gene isolated from Vigna aconitifolia, modified to eliminate the proline feedback inhibition (VaP5CSF129A), [8] has been inserted in various plants in order to overproduce this amino acid. The overexpression of this gene has enhanced tolerance to water deficit, [4,9,10] salinity, [7,9] and freezing [6] in plants. The development of citrus rootstocks with incremental accu- mulation of proline was shown to be important for maintaining the productivity of plants under stress conditions. [4,11] ‘Swingle’ citrumelo (Citrus paradisi Macf. × Poncirus trifoliata (L.) Raf.) is an important rootstock for commercial citrus production. This rootstock was developed by Walter T. Swingle in 1907 [12] and has been widely used in Florida since the 1990s. It is the second most used rootstock in Brazil because of the excellent quality of oranges in trees grown on ‘Swingle’. [4,13] Also, the ‘Swingle’ citrumelo rootstock has been used because it confers resistance to diseases such as sudden death, citrus decline, gummosis (Phytophthora spp.), citrus tristeza virus, and the citrus nematode (Tylenchulus semipenetrans). [3,13] Campos and colleagues [11] showed that transgenic (T-35S) ‘Swingle’ citrumelo was better able to endure stress caused by water deficiency than nontransgenic (NT) plants because of the direct or indirect action of proline as a mediator of osmotic adjustment and protection against free radicals. * Correspondence to: Glaucia B. Alcantara, Instituto de Química, Universidade Federal de Mato Grosso do Sul, Av. Filinto Muller, 1555, CP 549, CEP 79074-460, Campo Grande, Mato Grosso do Sul, Brazil. E-mail: glaucia.alcantara@ufms.br a Instituto de Química, Universidade Federal de Mato Grosso do Sul, Av. Filinto Muller, 1555, CP 549, CEP 79074-460, Campo Grande, Mato Grosso do Sul, Brazil b Instituto Agronômico do Paraná (IAPAR), Laboratório de Biotecnologia Vegetal, CP 481, CEP 86001-970, Londrina, Paraná, Brazil c Instituto de Química, Universidade Federal de Goiás, CP 131, CEP 74001-970, Goiânia, Goiás, Brazil Magn. Reson. Chem. (2014) Copyright © 2014 John Wiley & Sons, Ltd. Research article Received: 6 December 2013 Revised: 13 March 2014 Accepted: 21 April 2014 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/mrc.4082 1<?