It was suggested that enzymes of ALA synthesis are controlled by thiol-based regulation (Richter and Grimm, 2013). Dithiothreitol (DTT) is used in a standard SDS-PAGE to reduce disulfide bridges of proteins. To explore the potential of redox-active cysteine residues in FLU/GluTR, a non-reducing SDS-PA gel (without DTT) was applied to separate the total protein extract of WT, and FLUOE lines. Plant materials were harvested in the dark (23:00, 5h transferred to dark) or light (12:00). Proteins were then transferred to a membrane and probed with specific antibodies against GluTR or FLU. Under reducing condition (+DTT), FLU migrated as a 23 kDa protein, which is the predicted size of the monomeric FLU, while under non-reducing condition (-DTT), both in WT and FLUOE lines, FLU migrated as a 45 kDa protein band, which is the size of FLU dimers (Figure 3.28). The majority of GluTR migrated at the 55kDa level, which is related to the predicted size of GluTR monomers (Figure 3.28). This data indicated that FLU in plants forms a homo-dimer, probably through the formation of an intermolecular disulfide bond between two FLU proteins. The FLU homodimer was also analyzed under various growth conditions in WT, such as HL, LL, cold stress, or in etiolated and de-etiolated seedlings. However, no change in dimer formation was found for any of the examined conditions (Figure S1).
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Figure 3.28 Redox-state of FLU and GluTR in WT or FLUOE lines in the dark and in the light. Total
protein samples of WT or FLUOE lines were extracted under non-reducing conditions (-DTT) or reducing conditions (+DTT). Plants were grown under light-dark conditions (10h light/14 dark) for two weeks. Samples from light-grown seedlings were harvested in the middle of the day, and dark samples were harvested 5 hours after a transition from light to darkness. Protein samples were separated on a SDS-PA-gel. Subsequently, proteins were transferred to a membrane and probed with specific antibodies reacting with FLU or GluTR. The immune-reacting bands migrate at the predicted size of monomeric or dimeric FLU and GluTR, respectively, as indicated by arrows.
In Arabidopsis thaliana, FLU has two conserved cysteines (Cys119 and Cys292) (Figure S2). To determine which cysteine residue is required for the formation of the FLU dimer, two cysteine-substitution mutants (FLUC119S#, FLUC292S#) were generated expressing one of the site-directed FLU mutants. As a negative control, FLU(WT)# flu complementation lines expressed the WT FLU in the flu mutant. The expression of these mutant genes was under the control of the FLU promoter. Both FLUC119S# and FLUC292S# lines were able to grow under light-dark conditions (14h light/10h dark). A 20% reduced Chl content was determined in the FLUC119S lines compared to WT (Figure 3.29B). The ALA synthesis rate was decreased in the FLUC119S lines, while the other lines showed similar levels to WT (Figure 3.29C). Pchlide substantially accumulated in flu in the dark (Meskauskiene et al., 2001). Successful complementation of flu is defined when Pchlide accumulation in the dark reaches WT levels. Pchlide levels of the flu complementation lines in the dark were measured. Plant material was harvested at several time points in darkness. It was found that the Pchlide accumulation in all cysteine-substitution lines was not higher than the dark-grown WT (Figure S3). The expression of the WT FLU, FLUC119S and FLUC292S proteins in flu suppressed the Pchlide accumulation of flu in the dark. Pchlide levels of FLUC119S lines in the dark were even lower than in FLU(WT) lines (Figure S3).
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Figure 3.29 The image of single plants, Chl contents and ALA synthesis rate of WT, and FLU cysteine-substitution lines. Plants were grown under light-dark conditions for three weeks.
FLU(WT)#5 and FLU(WT)#6 are two lines expressing the WT FLU in the flu background. FLUC119S#3 and #5 are two individual lines expressing a DNA sequence coding for a serine substitution FLU at the position 119; FLUC119S#2 and #4 are two flu complementation lines expressing a DNA sequence coding for a mutant FLU with the cysteine292 substituted by a serine. The expression of all mutant genes is under the control of the FLU promoter.
Under the non-reducing condition FLU of WT and FLU(WT), and FLUC292S of FLUC292S lines migrated as a 45kDa protein band in the SDS-PA gel, which is the predicted size of the FLU-dimer, while FLUC119S of FLUC119S lines migrated at a 23kDa protein, which is the predicted size of the FLU monomer (Figure 3.30A). The mutation of cysteine119, rather than cysteine292, on FLU, disrupted the FLU homo-dimer formation in plants. The signal intensity of the FLU monomer in FLUC119S lines was significantly lower than that of the FLU-dimer in WT or other complementation lines (Figure 3.30A). The substantially lower signal intensity of the band representing the monomer of FLU than of the band representing the dimer of FLU is probably due to the antibody recognizing the FLU dimer more efficiently than the monomeric form of FLU. Under reducing condition, the steady-state level of FLU in all the complementation lines was higher than in WT, which was due to the elevated FLU transcripts in the complementation lines (Figure 3.30C).
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Figure 3.30 Immune-blotting analysis of FLU under non-reducing (A) and reducing conditions (B) and the levels of the FLU transcript in WT and flu complementation lines (C). Plants were grown under
light-dark conditions for three weeks. Protein samples were extracted under non-reducing condition (A) or reducing condition (B) and then separated by SDS-PAGE. Proteins were transferred to a membrane and probed with FLU antibody. FLU(WT)#5 and FLU(WT)#6 are two lines expressing FLU in the flu mutant background. FLUC119S#3 and #5 are two individual lines expressing a DNA sequence coding for a mutated FLU with cysteine119 substituted with a serine in flu. FLUC119S#2 and #4 are two flu complementation lines expressing a DNA sequence coding for a mutated FLU with cysteine292 substituted with a serine. The expression of all complementation lines is under the control of the original FLU promoter.