Abstract
Carotenoids in plants play several key functions such as acting as light-harvesters, antioxidants (Lado et al., 2016) or being precursors of strigolactones, abscisic acid, volatiles and other signaling compounds (Arbona et al., 2013). Although those functions are well-known in light-exposed tissues, information in belowground organs is limited because of reduced abundance of these pigments. In order to better understand the role of carotenoids in roots, we developed a methodology to increase the abundance of these pigments in underground tissues. We took advantage of the fact that citrus roots exposed to light develop pigmentation in order to increase the carotenoid content. Therefore, here we describe a simple method to increase carotenoids in citrus roots.
Keywords: Abscisic acid, Phytohormones, Growth chamber, in vitro culture, Root detachment, Seed germination
Background
Carotenoid abundance in roots is quite limited and, therefore, understanding the role of these compounds becomes difficult. Exposure of roots to light is a simple, fast and useful tool to increase carotenoid levels in these tissues, especially when compared to other genomic approaches such as overexpressing some key genes of the carotenoid biosynthetic pathway (Cao et al., 2015).
Materials and Reagents
Equipment
Procedure
Data analysis
The aim of this protocol is to increase the levels of carotenoids in roots. To evaluate the raise of these pigments two alternative methodologies could be used: on one hand, spectrophotometric analysis could be performed by many different protocols, evaluating in this case the total carotenoid content (i.e., Wellburn, 1994). Alternatively, detailed carotenoid composition could be achieved by liquid chromatography coupled to a diode array detector (HPLC-DAD) as detailed in Manzi et al., (2016). Carotenoid quantification should be performed accordingly to the method used.
Notes
Rates of growth may differ among citrus genotypes. To avoid unwanted effects on metabolism, avoid excessive growing of roots which may lead to an early senescence. To this, you might adjust the growing time of the plants accordingly (i.e., reducing the 3 week period under light conditions). In order to obtain high levels of sample material a good option is increasing the number of individual roots rather than extending the root growing period. Consider that 20 roots provide approximately 1.5-2.0 g of fresh tissue.
Recipes
Acknowledgments
This work was supported by the Ministerio de Economia (MINECO) and Universitat Jaume I through grants No. AGL2013- 42038-R and P1IB2013-23, respectively. MM was recipient of a ‘Santiago Grisolia’ fellowship from Generalitat Valenciana (Spain). This protocol is based on the methodology used in the manuscript Manzi et al. (2016).
References
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