Expression and functional analyses of the plastid lipid-associated protein CHRC suggest its role in chromoplastogenesis and stress

Abstract

Chromoplastogenesis during flower development and fruit ripening involves the dramatic overaccumulation of carotenoids sequestered into structures containing lipids and proteins called plastid lipid-associated proteins (PAPs). CHRC, a cucumber (Cucumis sativus) PAP, has been suggested to be transcriptionally activated in carotenoid-accumulating flowers by gibberellin (GA). Mybys, a MYB-like trans-activator identified here, may represent a chromoplastogenesis-related factor: Its expression is flower specific and parallels that of ChrC during flower development; moreover, as revealed by stable ectopic and transient-expression assays, it specifically trans-activates ChrC promoter in flowers accumulating carotenoids and flavonoids. A detailed dissection of ChrC promoter revealed a GA-responsive element, gacCTCcaa, the mutation of which abolished ChrC activation by GA. This cis-element is different from the GARE motif and is involved in ChrC activation probably via negative regulation, similar to other GA-responsive systems. The GA responsiveness and MYBYS floral activation of the ChrC promoter do not overlap with respect to cis-elements. To study the functionality of CHRC, which is activated in vegetative tissues similar to other PAPs by various biotic and abiotic stresses, we employed a tomato (Lycopersicon esculentum) plant system and generated RNAi-transgenic lines with suppressed LeCHRC. Transgenic flowers accumulated approximately 30% less carotenoids per unit protein than controls, indicating an interrelationship between PAPs and flower-specific carotenoid accumulation in chromoplasts. Moreover, the transgenic LeCHRC-suppressed plants were significantly more susceptible to Botrytis cinerea infection, suggesting CHRC's involvement in plant protection under stress conditions and supporting the general, evolutionarily preserved role of PAPs.

Figure 1.

A, Nucleotide and predicted amino acid sequences of mybys. The conserved R2/R3 DNA-binding domains at the 5′ end of the mybys (GenBank DQ311672) sequence are underlined and a typical activation domain at the 3′ end is in bold. The terminal codon is marked with an asterisk. B, Temporal and spatial regulation of mybys transcript levels in cucumber tissues. Total RNA extracted from cucumber leaves (L) and corollas at different developmental stages (stages 2–4) was probed with a radiolabeled fragment of mybys specific to the 3′ end of the gene. The same RNA blot was rehybridized with radiolabeled ChrC.

Figure 2.

MYBYS transcription factor specifically activates the ChrC promoter. A, Histochemical visualization of GUS activity in petunia flowers bombarded with ChrC:GUS alone, or ChrC:GUS or 137ChrC:GUS (containing GUS driven by 3,500 or 137 bp of the ChrC promoter, respectively) cobombarded with 35S:MYBYS. In control cobombardment experiments, MYBYS was replaced by another MYB factor, PAP, which regulates the anthocyanin pathway (35S:PAP). B, Histochemical visualization of GUS activity in young green transgenic tomato flowers constitutively expressing 35S:MYBYS (line 10) following bombardment with ChrC:GUS. As a control, nontransgenic flowers (WT) were also bombarded with ChrC:GUS and histochemically analyzed (top). Accumulation of mybys in 35S:MYBYS-transgenic tomato flowers (independent transgenic lines 4, 10, and 110) is shown in the bottom section. RNA-blot analysis was performed using radiolabeled 3′ mybys as a probe. A nontransgenic line (WT) and a transgenic line with no expression of mybys (line 3) were used as controls.

Figure 3.

Identification of GA₃-responsive cis-elements in the ChrC promoter region. A, ChrC promoter fragments fused to GUS via the 35S minimal promoter (TATA) were cobombarded with 35S:GFP into stage 1 cucumber corollas grown in vitro without (W) or with GA₃ (GA). GUS expression was normalized to the GFP signal using ImageJ software. The results of five replicates ±se are presented. B, Histochemical visualization of GUS activity in stage 1 cucumber corollas grown in vitro without (W) or with GA₃ (GA) following bombardment with ChrC promoter lacking 212 bp (−290 to −78) fused to GUS (Δ212ChrC:GUS) or the 212-bp fragment of the promoter (−290 to −78) fused to GUS via an 35S minimal promoter (212ChrC:GUS).

Figure 4.

Effect of mutations on GA₃ responsiveness of the ChrC promoter. A, Mutated region of the ChrC promoter. The 6-bp sequence GTA TCT was used to replace the original sequence of the promoter, with three-base gaps between each of the four (MG1–MG4) mutations. B, ChrC promoter fragments, original and mutated, fused to GUS via an 35S minimal promoter (TATA) were cobombarded with 35S:GFP into stage 1 cucumber corollas grown in vitro without (W) or with GA₃ (GA). GUS expression was normalized to the GFP signal using ImageJ software. The results of five replicates ±se are presented.

Figure 5.

Induction of ChrC expression in cucumber leaves by biotic and abiotic stresses. A, Activation of ChrC promoter by heat shock and fungal inoculation. Cucumber leaves were cultured in vitro for 4 h at 42°C (HS) or room temperature (RT). In addition, leaves from plants infected (+) with powdery mildew Sphaerotheca fuliginea (Oidium sp.) were compared to control uninfected leaves (−). Following bombardment with ChrC:GUS, leaves were histochemically analyzed for GUS expression. B, Effect of heat shock and fungal inoculation on ChrC transcript levels. Total RNA was extracted from leaves treated as in A, and following blotting probed with radiolabeled ChrC. T0, Detached leaves prior to the in vitro culture.

Figure 6.

Comparison of CHRC homolog amino acid sequences. Multiple sequence alignment was performed with ClustalW (Thompson et al., 1994). GenBank accession numbers of the CHRC homologs are as follows: tomato LeChrC, DQ310151; C. annuum Fib, CAA50750; and cucumber CHRC, AAD05165. The transit peptide of the CHRC sequence is underlined. Identical residues in the column are marked with an asterisk (*), and conserved (:) and semiconserved (.) substitutions are indicated as well.

Figure 7.

Transgenic tomato plants with suppressed LeCHRC. A and B, Molecular analysis of transgenic tomato flowers with suppressed LeChrC generated via the RNAi approach. Total RNA from stage 2 corollas was extracted and probed with radiolabeled LeChrC. RNA-blot analysis of RNAi LeChrC-transgenic plants (independent lines 11, 13, 21, 28, 33, and 37) versus control transgenic lines with no suppression (independent lines 2, 19, and 25) and control nontransgenic (WT) tomato plants is presented. Analyses were performed with both T0 (A) and T2 (B) generation plants. C, Western-blot analyses of CHRC levels in stage 2 corollas of RNAi LeChrC-transgenic plants (independent lines 11, 13, and 37) versus control transgenic lines with no suppression (line 19) and control nontransgenic (WT) tomato plants (T2 generation). Equal loading, as revealed by Ponceau-S red staining of the membrane prior to incubation with affinity-purified polyclonal antibodies against CHRC, is shown in the bottom section.

Figure 8.

Susceptibility of transgenic tomato plants with modulated ChrC expression levels to B. cinerea infection. Transgenic tomato overexpressing ChrC (sense lines 2 and 5), control nontransgenic (WT), and RNAi LeChrC-suppressed plants (RNAi lines 11 and 37) were infected with B. cinerea. A, B. cinerea disease symptoms in leaves and stems 6 and 8 d after inoculation, respectively. B, RT-PCR analysis of ChrC/LeChrC (and actin as a control) expression in control and transgenic leaves prior to infection (−) or 3 d after infection (+) with B. cinerea.