Synthetic conversion of leaf chloroplasts into carotenoid-rich plastids reveals mechanistic basis of natural chromoplast development

Abstract

Plastids, the defining organelles of plant cells, undergo physiological and morphological changes to fulfill distinct biological functions. In particular, the differentiation of chloroplasts into chromoplasts results in an enhanced storage capacity for carotenoids with industrial and nutritional value such as beta-carotene (provitamin A). Here, we show that synthetically inducing a burst in the production of phytoene, the first committed intermediate of the carotenoid pathway, elicits an artificial chloroplast-to-chromoplast differentiation in leaves. Phytoene overproduction initially interferes with photosynthesis, acting as a metabolic threshold switch mechanism that weakens chloroplast identity. In a second stage, phytoene conversion into downstream carotenoids is required for the differentiation of chromoplasts, a process that involves a concurrent reprogramming of nuclear gene expression and plastid morphology for improved carotenoid storage. We hence demonstrate that loss of photosynthetic competence and enhanced production of carotenoids are not just consequences but requirements for chloroplasts to differentiate into chromoplasts.

Keywords: carotenoid; chromoplast; differentiation; phytoene; synthetic.

Fig. 1.

Virus-mediated production of crtB causes leaf yellowing due to carotenoid overaccumulation. (A) Lettuce at 12 d postinoculation (dpi) with a crtB-expressing Lettuce mosaic virus (LMV)-derived vector or an empty control. (B) Zucchini from plants at 14 dpi with a crtB-expressing Zucchini yellow mosaic virus (ZYMV)-derived vector or an empty control. (C) Carotenoid analysis and representative images at 14 dpi of Arabidopsis (Col) WT and double mutant plants grown under short day conditions (8 h of low light and 16 h of darkness) for 2 wk (WT and ccd1 ccd4) or 5 wk (ator atorl) and then inoculated with the indicated viral vectors. Plot shows the mean and SD of n = 3 independent samples. Carotenoid levels are represented relative to those in WT samples inoculated with the empty vector control (TuMV). (D) Representative Arabidopsis WT plants at 38 dpi.

Fig. 2.

Chromoplast-like plastids develop from chloroplasts in leaves producing crtB or a plastid-targeted version of the enzyme. TEM images of representative plastids from the indicated species and treatments are shown. (A) Plastids from N. tabacum leaves collected 10 dpi with TEVΔNIb (empty vector) or TEVΔNIb-crtB. (B) Plastids from A. thaliana (Ler) leaves inoculated with TEV (empty vector) or TEV-crtB at 15 dpi. (C) Plastids from N. benthamiana leaves agroinfiltrated with the indicated constructs and collected at 5 dpi (first four images) or 18 dpi (Right). (D) Magnification of plastids from N. benthamiana leaves agroinfiltrated with the indicated constructs and collected at 5 dpi. Grana are marked as “g,” membrane stacks with white arrows, and plastoglobules with gray arrows. (E) Gerontoplast from a N. benthamiana leaf harvested from the plant and kept in the dark for 10 d (senescent). (F) Immunoblot analysis of plastidial proteins in leaves treated as described in A and C. Coomassie blue (C-Blue) staining is shown as a loading control. (Scale bars, 1 µm.)

Fig. 3.

Leaf tissues producing crtB show a stable phenotype of high carotenoid levels and impaired photosynthesis. (A) N. benthamiana leaf 5 d after agroinfiltration (dpi) with the indicated constructs in different sections. (B) Levels of carotenoids (CRTs) and chlorophylls (CHLs) in leaf sections like those shown in A. (C) ɸPSII and NPQ in leaf sections like those shown in A. (D) Changes in CRTs, CHLs, ɸPSII, and carotenoid-to-chlorophyll ratio (CRTs/CHLs) in leaf sections at different time points after agroinfiltration with crtB. A representative leaf at 16 dpi is shown at Left. Plots show the mean and SD of n = 3 independent samples. Values are represented relative to those in GFP controls. Asterisks in D plots mark statistically significant changes relative to 0 dpi (t test, P < 0.05).

Fig. 4.

Time-course of chloroplast-to-chromoplast differentiation in leaves. N. benthamiana leaves were agroinfiltrated with the indicated constructs, and samples were collected at the indicated time points (hours after agroinfiltration). (A) Levels of (p)crtB-encoding transcripts relative to the maximum in (p)crtB samples. (B) Absolute phytoene levels and relative downstream carotenoid contents in (p)crtB samples. (C) Representative chlorophyll fluorescence images and ɸPSII values. (D) D1 (PsbA) protein contents. (E) Levels of phytoene, downstream carotenoids, and ɸPSII in (p)crtB samples relative to those at 25 hpi. Asterisks mark significant changes relative to the 25 hpi values (t test, P < 0.05). Note the logarithmic scale. In all of the plots, values correspond to mean and SD values of n = 3 independent samples.

Fig. 5.

Transformation of leaf chloroplasts into chromoplasts requires a reduction of photosynthetic capacity and production of carotenoids downstream of phytoene. (A) Carotenoid levels and ɸPSII in leaves 96 h after agroinfiltration with the indicated constructs. In all cases, plot values correspond to mean and SD values of n = 3 independent samples. Asterisks in ɸPSII plots mark statistically significant changes relative to untreated GFP controls (t test, P < 0.05). Samples infiltrated with NF at 24 hpi or treated with DCMU 24 h before agroinfiltration are indicated. (B) Representative images of agroinfiltrated leaves at 96 hpi and their corresponding chlorophyll fluorescence for ɸPSII.

Fig. 6.

Model of the chloroplast-to-chromoplast differentiation process. Plant developmental programs create organs with different degrees of photosynthetic capacity and, hence, chloroplast identity, from strong (e.g., leaves) to weak (e.g., green fruits) or absent (e.g., roots). Weakening of chloroplast identity appears to be the first phase (I) in chromoplast differentiation. In a second phase (II), developmental cues promote the expression of genes encoding PSY and other carotenoid biosynthetic enzymes. Enhanced production of carotenoids then reprograms plastid-to-nucleus communication, changes plastidial ultrastructure, and results in the differentiation of chromoplasts, which, in turn, promote biosynthesis and improve storage of carotenoids. The two phases can be synthetically engineered in leaves by overproducing phytoene using crtB. When phytoene exceeds a certain level, it interferes with the photosynthetic capacity of leaf chloroplasts. This acts as a metabolic switch that allows the formation of chromoplasts after phytoene is converted into downstream carotenoids by endogenous enzymes.