RNA interference-mediated repression of MtCCD1 in mycorrhizal roots of Medicago truncatula causes accumulation of C27 apocarotenoids, shedding light on the functional role of CCD1

Abstract

Tailoring carotenoids by plant carotenoid cleavage dioxygenases (CCDs) generates various bioactive apocarotenoids. Recombinant CCD1 has been shown to catalyze symmetrical cleavage of C(40) carotenoid substrates at 9,10 and 9',10' positions. The actual substrate(s) of the enzyme in planta, however, is still unknown. In this study, we have carried out RNA interference (RNAi)-mediated repression of a Medicago truncatula CCD1 gene in hairy roots colonized by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. As a consequence, the normal AM-mediated accumulation of apocarotenoids (C(13) cyclohexenone and C(14) mycorradicin derivatives) was differentially modified. Mycorradicin derivatives were strongly reduced to 3% to 6% of the controls, while the cyclohexenone derivatives were only reduced to 30% to 47%. Concomitantly, a yellow-orange color appeared in RNAi roots. Based on ultraviolet light spectra and mass spectrometry analyses, the new compounds are C(27) apocarotenoic acid derivatives. These metabolic alterations did not lead to major changes in molecular markers of the AM symbiosis, although a moderate shift to more degenerating arbuscules was observed in RNAi roots. The unexpected outcome of the RNAi approach suggests C(27) apocarotenoids as the major substrates of CCD1 in mycorrhizal root cells. Moreover, literature data implicate C(27) apocarotenoid cleavage as the general functional role of CCD1 in planta. A revised scheme of plant carotenoid cleavage in two consecutive steps is proposed, in which CCD1 catalyzes only the second step in the cytosol (C(27)-->C(14)+C(13)), while the first step (C(40)-->C(27)+C(13)) may be catalyzed by CCD7 and/or CCD4 inside plastids.

Figure 1.

Simplified hypothetical pathway to C₁₃ cyclohexenone and C₁₄ mycorradicin derivatives in mycorrhizal roots. The indicated cleavage specificities of CCD1 and its presumed C₄₀ carotenoid substrate have been deduced from in vitro studies. An early biosynthetic step in the MEP pathway previously targeted for gene silencing (DXS2) is highlighted. The carotenoid precursor in mycorrhizal roots is still elusive. DMAPP, Dimethylallyl diphosphate; DXP, 1-deoxy-d-xylulose 5-phosphate; DXR, 1-deoxy-d-xylulose 5-phosphate reductoisomerase; GAP, glyceraldehyde 3-phosphate; Gly, glycoside; IPP, isopentenyl diphosphate; MEP, 2-C-methyl-d-erythritol 4-phosphate; R¹, R², unknown moieties.

Figure 2.

Expression of the MtCCD1 cDNA in E. coli strains engineered for carotenoid production. MtCCD1 expression constructs in pET-28a lacking or containing a C-terminal His tag as indicated were introduced into E. coli strains containing the plasmid pAC-BETA (center) or pAC-ZEAX (right) for β-carotene or zeaxanthin formation, respectively. The streak on top represents the strain transformed with the EV control.

Figure 3.

Transcript levels of MtCCD1 and levels of mycorradicin and cyclohexenone derivatives in mycorrhizal hairy roots of M. truncatula reduced for MtCCD1 expression. Results from two extensive experiments of two different colonization periods are shown using EV control roots (white columns) or RNAi roots (gray columns). The 7-week experiment consisted of eight individual EV controls and eight individual RNAi plants, while nine EV plants and 13 RNAi plants were used for the 9-week experiment. A, MtCCD1 transcript levels were determined by real time RT-PCR. B and C, Mycorradicin (B) and cyclohexenone (C) derivatives were assigned by their characteristic UV light spectra and quantified by HPLC using external standards. Letters differing between columns indicate significant differences of means based on Kruskal-Wallis test (mycorradicin) or one-way test (cyclohexenone derivatives). fw, Fresh weight.

Figure 4.

Conspicuous alteration of root color in mycorrhizal hairy roots reduced for MtCCD1 expression. Total root systems or enlarged sectors of roots after 7 weeks of fungal colonization show faint orange-brown coloration of EV controls (left) or a much more intense yellow-orange coloration of MtCCD1 RNAi roots (right).

Figure 5.

HPLC separation of methanolic extracts from hairy roots. A, Comparison of mycorrhizal EV control roots (bottom trace) and mycorrhizal RNAi roots (top trace) with compound detection at 440 nm. Separation of native constituents revealed seven novel compounds, which appear exclusively in RNAi samples. Corresponding UV absorption maxima are shown in the inset. B, Comparison between nonmycorrhizal (NM) and mycorrhizal (M) roots of three individual RNAi root systems each. Root extraction, separation conditions, and compound terminology were as in A.

Figure 6.

HPLC analysis of methanolic extracts from mycorrhizal hairy roots after alkaline hydrolysis. Comparison of EV and RNAi roots revealed two major compounds appearing in RNAi roots termed Apo1 and Apo2. Their UV light spectra and absorption maxima are shown in the insets. Separation and detection conditions were as described for Figure 5.

Figure 7.

Mass spectral analysis of compound Apo2. A, Scheme of the proposed fragmentation of compound Apo2, whose proposed structure is shown at top left. Apo2 exhibits a m/z of 733 [M + H]⁺. Removing one or two hexose residues (increments of 162) results in fragments with m/z 571 and 409. Splitting of the aglycone next to the cyclohexene ring is proposed to yield fragment a (m/z 269). Additional fragments may be derived from fragmentation in the linear side chain of the Apo2 aglycone (m/z 391) at the positions indicated, resulting in fragments of m/z 201 (b) or m/z 187 (c). B and C, ESI-CID mass spectra of Apo2 (B, 15 eV; C, 25 eV).

Figure 8.

Assessment of mycorrhizal parameters from stained mycorrhizal roots of EV controls and RNAi plants of experiment I. A to C, Ink-staining analyses conducted and evaluated according to Trouvelot et al. (1986) using eight samples each of EV controls and RNAi roots. A, Frequency of fungal structures (F%). B, Density of fungal structures in the mycorrhizal part of the root (m%). C, Arbuscule abundance in the mycorrhizal part of the roots (a%). D, Evaluation of arbuscule morphologies from confocal images after acid-fuchsin staining of roots. Individual arbuscules were classified into one of four developmental stages as indicated using four samples each of EV controls and RNAi roots (containing 3%–6% residual MtCCD1 transcripts). One hundred arbuscules were evaluated from each root system. Statistical significance of alterations was analyzed by the Kruskal-Wallis test (developing stage) or the one-way test (other stages). * P ≤ 0.05; ** P ≤ 0.01. sd values are shown below the graph.

Figure 9.

Proposed organization of C₄₀ carotenoid formation and cleavage to apocarotenoids in a compartmentalized mycorrhizal root cell. C₄₀ carotenoids (exemplified by lactucaxanthin as the tentatively proposed precursor for AM-induced apocarotenoids) synthesized inside plastids are proposed to be cleaved by plastidial CCD7 to one C₂₇ aldehyde and one C₁₃ ketone intermediate, which both can be exported into the cytosol in an unmodified or modified state (white arrows). The C₂₇ intermediate in the cytosol is further cleaved by CCD1 to a C₁₄ and a second molecule of C₁₃, which are, together with the first C₁₃ molecule, modified to yield the oxidized C₁₃ and C₁₄ apocarotenoids (cyclohexenone and mycorradicin derivatives) found in mycorrhizal roots. For abbreviations, see Figure 1.