Induction and Control of Chromoplast-specific Carotenoid Genes by Oxidative Stress

Abstract

The differentiation of chloroplasts into chromoplasts involves a series of biochemical changes that culminate with the intense accumulation of long chain chromophore carotenoids such as lycopene, rhodoxanthin, astaxanthin, anhydroeschsoltzxanthin, capsanthin, and capsorubin. The signal pathways mediating these transformations are unknown. Chromoplast carotenoids are known to accumulate in green tissues experiencing stress conditions, and studies indicate that they provide efficient protection against oxidative stress. We tested the role of reactive oxygen species (ROS) as regulators of chromoplast carotenoid biosynthesis in vivo. The addition of ROS progenitors, such as menadione, tert-butylhydroperoxide, or paraquat and prooxidants such as diamide or buthionine sulfoximine to green pericarp discs of pepper fruits rapidly and dramatically induce the simultaneous expression of multiple carotenogenic gene mRNAS that give rise to capsanthin. Similarly, down-regulation of catalase by amitrole induces expression of carotenogenic gene mRNAs leading to the synthesis of capsanthin in excised green pericarp discs. ROS signals from plastids and mitochondria also contribute significantly to this process. Analysis of the capsanthin-capsorubin synthase promoter in combination with a ␤-glucuronidase reporter gene reveals strong activation in transformed pepper protoplasts challenged with the above ROS. Collectively these data demonstrate that ROS act as a novel class of second messengers that mediate intense carotenoid synthesis during chromoplast differentiation.Plastids are plant organelles whose diverse functions include photosynthesis, gravity perception, and biogenesis of microand macromolecules. These functions do not occur in all plastids but are associated with structurally distinct plastid types. Plastid differentiation is a highly coordinated process involving programmed, multi-phase events that are transduced by a variety of stimuli. These transducers activate numerous morphological and biochemical changes that ultimately affect plastid compartmentalization. This is particularly evident in chloroplasts undergoing the transformation to chromoplasts, an event characterized by the synthesis and accumulation of carotenoids into unique plastid substructures. These changes are accompanied by the yellow to red color shift in flowers, fruits, and roots of certain plants (1, 2). Although the molecular events controlling these alterations are largely unknown, emerging evidence suggests that reactive oxygen species (ROS) 1 may play a vital role in this process (3). In this paper we present data showing how oxidative stress affects carotenogenesis in plants.Data from several model systems indicate that ROS operate at the molecular level. Homeostatic variations in ROS levels are known to activate normal and pathological events during animal cell development (4, 5).

Phytoene Desaturase Is Localized Exclusively in the Chloroplast and Up-Regulated at the mRNA Level during Accumulation of Secondary Carotenoids in Haematococcus pluvialis (Volvocales, Chlorophyceae),

“…Because of slight variability in the extent of SC accumulation between parallels (Grü newald, 1997), we do not interpret the small difference between the maxima in PDS mRNA and protein levels as a sign for post-translational regulation events-at least the main part of up-regulation takes place at the mRNA level. Bouvier et al (1998) showed that pepper PDS mRNA increased under different stress conditions. During accumulation of capsanthin and capsorubin, Rö mer et al (1993) observed after an increase between the mature green pepper fruit and the first defined ripening stage a constant level of PDS mRNA despite the massive increase of carotenoids occurring during the further ripening stages, and this resembles our findings here.…”

Escherichia coli as a platform for functional expression of plant P450 carotene hydroxylases

“…The first class of hydroxylases is comprised of the β-ring non-heme diiron monooxygenases. Members of this class were identified from a wide range of bacterial, algal, and plant species [24][25][26]. Plant diiron β-ring hydroxylases contain four transmembrane helices and four conserved iron-binding histidine clusters.…”

Real-time in vivo visualization of oxidative stress in duckweed (Lemna minor L.)

“…It was also used as a ROS inducer in yeast cells [8], fruits of Capsicum annuum [18], glial cells [19], Pisum sativum [20], Arabidopsis thaliana cell culture [21] and Arabidopsis thaliana plants [22]. Ascorbate, on the other hand, is -besides glutathione -the plants' main low molecular weight antioxidant and can directly reduce superoxide as well as hydrogen peroxide [25].…”

“…An additional benefit of using plants in this non-invasive technique is their inherent trait of containing esterases in the apoplast [13][14][15][16]. This enabled us to observe either apoplastic or symplastic ROS formation in the test plants after exposure to 2-methyl-1,4-naphtoquinone (menadione), a commonly used ROS inducer [8,[17][18][19][20][21][22], with slight modifications in the fluorescent dye loading protocol.…”