Nodal segments of Dalbergia nigra reveal changes in endogenous polyamine content and proteomic profiles associated with the capacity for in vitro shoot development

Mateus Santana Rodrigues1,2, Jociel Nascimento de Noronha1, Marta Rodríguez-Ruiz2, José M. Palma2, Vanildo Silveira2, Francisco J. Corpas2, Claudete Santa-Catarina1

1. LBCT / CBB / UENF, Laboratório de Biologia Celular e Tecidual. Centro de Biociências e Biotecnologia. Universidade Estadual do Norte Fluminense Darcy Ribeiro; Campos dos Goytacazes, RJ, Brazil
2. CSIC, Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture (ARNOBA). Department of Plant Stress, Development and Signaling. Estación Experimental del Zaidín; Granada, Spain

Dalbergia nigra, commonly known as Jacarandá-da-Bahia, is a Brazilian tree species of ecological significance and high economic value due its prized wood. However, it is currently threatened with extinction. Micropropagation is an effective technique for propagation and conservation of various woody species. D. nigra can be propagated through axillary bud proliferation using nodal segments from seedlings in vitro-grown. Nevertheless, shoots that develop from cotyledonary (CS), intermediate (IS), and apical (AS) nodal segments show differences in their growth patterns. The aim of this study was to analyze the proteomic profile and the endogenous content of free polyamines (PAs) in different nodal segments of in vitro-grown D. nigra seedlings, to better understand the potential relationship between shoot development, PA metabolism, and differentially accumulated proteins (DAPs). CS, IS, and AS segments from in vitro-grown seedlings were cultured on MS (Murashige and Skoog) culture medium supplemented with 2.5 µM benzyladenine to induce shoot formation. Additionally, these nodal segments were used for PA quantification, proteomic profiling, and catalase expression and enzymatic activity analysis. As a result, CS segments exhibited the highest shoot length, followed by a progressive decrease in IS and AS segments. AS showed higher endogenous levels of putrescine and total free PAs compared to CS and IS. However, CS showed a higher endogenous content of spermine than AS and IS. In the proteomic analysis comparing AS/CS, a total of 633 proteins were identified, 71 of which were DAPs, with 13 unique to AS and 11 unique to CS. In comparison between IS/CS, 628 proteins were identified, including 50 DAPs, with 9 unique to IS and 13 unique to CS. In the AS/IS comparison, 632 proteins were identified, 46 of which were DAPs, with 9 proteins unique to IS and 15 unique to AS. In our results, catalase was down-accumulated in AS/CS comparison. Additionally, gel-based enzymatic assays and Western blotting revealed higher activity and expression levels of this protein in CS, respectively. Furthermore, the Ycf2 protein, involved in chloroplast protein import, and Patellin-3, associated with intracellular lipid transport, were uniquely identified in CS in both AS/CS and IS/CS comparisons. We conclude that this study revealed the identification of proteins potentially associated with the capacity for in vitro shoot development in D. nigra, contributing to a better understanding of the micropropagation of this emblematic Brazilian species.

Agradecimentos: Financial support and fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) are gratefully acknowledged.