New role of phenylpropanoid compounds during sugarcane micropropagation in Temporary Immersion Bioreactors (TIBs)
Introduction
Plants synthesize an array of natural products based on the phenylpropane skeleton [1]. These compounds have multiple functions, which are not yet fully understood and involve a variety of ecological and physiological phenomena [2], [3]. However, some key functions have been already identified, e.g., lignification and influence on the physicochemical properties of the cell walls [4], [5], protection from UV irradiation [6], antioxidants [7], [8] and allelochemicals [9]. A crucial role for cell wall expansion and homeostasis control during plant development has recently been identified [3].
Likewise, it is known that physical wounding (such as those produced in plant culture manipulation) triggers several important changes in plant metabolism. One of the most pronounced changes occurring after wounding is the accumulation and oxidation of phenolic compounds causing tissue-browning [10]. Wounding induces an increase of the activity of the phenylalanine ammonia-lyase (PAL; EC 4.3.1.5); a key enzyme in the synthesis of phenylpropanoid compounds [11]. PAL activity is correlated with the accumulation of phenolic compounds that are oxidized by polyphenol oxidases (PPOs) and peroxidases (PODs) [12]. Polyphenol oxidase (PPO) enzymes responsible for the typical browning of plant extracts and damaged tissues caused by spontaneous polymerization and cross-linking of o-quinones. PPOs are highly expressed in sugarcane apical meristems [13].
The antisense downregulation of polyphenol oxidase resulted in an enhanced disease susceptibility to Pseudomonas syringae in tomato plants [14] and the inhibition of the phenolic acid metabolism results in a precocious cell death and altered cell morphology in leaves of transgenic tobacco plants [15]. Rasmussen and Rixon [16] suggested that the metabolic channeling of trans-cinnamic acid requires a close association of specific forms of PAL with C4H (cinnamate 4-hydroxylase) on microsomal membranes. In parallel, roles in the defense against leaf-eating insects [17] as well as the response to both abiotic and biotic factors have been documented [18], [19], [20], [21].
Micropropagation is an advanced biotechnological system for producing cloned pathogen-free plants for agriculture and forestry uses. Recently, the cultivation in liquid media using Temporary Immersion Bioreactors (TIBs) with different frequencies of immersion was shown to improve plant quality and multiplication rates [22], [23], [24]. Ziv [25] reviewed the information about the use of bioreactors as a system for plant propagation through the organogenic pathway. Metabolite concentrations in these plant cultures are much higher in comparison to undifferentiated cell cultures. By controlling the environmental parameters, it was possible to modify the metabolite content for production of highly active plant [26]. Our primary observations showed that increased amounts of secondary metabolites called phenolic compounds were produced from sugarcane plants micropropagated in TIBs. However, though phenolics production has been widely considered as an undesired trait in conventional tissue cultures [27], [28], sugarcane plants growing in TIBs displayed a remarkable vigor and health.
In this work, for the first time, a differential genomic response in plants micropropagated in TIBs was investigated using sugarcane as model specie. The results put forward a new and benefic function of plant phenolic metabolites during micropropagation.
Section snippets
Biological materials
In vitro sugarcane plants (Saccharum officinarum spp., cv. Badila.) were obtained by sterile meristem culture and micropropagated according to He et al. [29]. Cultures were maintained at 110 μM m−2 s−1 luminosity, 12-h photoperiod, at 27 °C. Vigorous and pathogen-free plants of 15 days old (after subcultures) were selected for the experiments.
Micropropagation using Temporary Immersion Bioreactors
Selected sugarcane plants (10 plants/L medium) were transferred to sterile plastic bottles (10 L capacity). TIBs contained 5 L culture medium composed of MS
Micropropagation in TIBs
In vitro sugarcane plants were transferred to TIBs (supplemented or not with 5 mg/L of ascorbic acid) for a micropropagation cycle. As expected, the TIBs+ ascorbic acid remained transparent-pale yellow after 25 days indicating that neither phenolics nor soluble o-quinones were consistently released to the medium (Fig. 1A). This result confirmed the reliability of the ascorbic acid to control browning in sugarcane tissue cultures by suppressing key enzymes of the phenylpropanoid pathway. On the
Discussion
For the first time and using sugarcane as model, the genomic characterization of plants micropropagated in TIBs was investigated by suppressing the key genes of the phenylpropanoid pathway. A set of 50 transcripts was identified as differentially expressed, which confirmed both the effectiveness of the experimental design applied and the reliability of the molecular tool used. As expected, with the inhibition of the phenylpropanoid pathway, a control of the release of the secondary metabolites
Acknowledgements
Thanks are due to Post doctoral fellowships granted to Ariel D. Arencibia and Elva R. Carmona to perform part of this research at the Key Biotechnology Laboratory, Guangxi Academic of Agricultural Sciences, Nanning, PR China. Thank to the anonymous referees for their useful critical.
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