Knockdown of FIBRILLIN4 Gene Expression in Apple Decreases Plastoglobule Plastoquinone Content

Abstract

Fibrillin4 (FBN4) is a protein component of plastoglobules, which are antioxidant-rich sub-compartments attached to the chloroplast thylakoid membranes. FBN4 is required for normal plant biotic and abiotic stress resistance, including bacterial pathogens, herbicide, high light intensity, and ozone; FBN4 is also required for the accumulation of osmiophilic material inside plastoglobules. In this study, the contribution of FBN4 to plastoglobule lipid composition was examined using cultivated apple trees in which FBN4 gene expression was knocked down using RNA interference. Chloroplasts and plastoglobules were isolated from leaves of wild-type and fbn4 knock-down trees. Total lipids were extracted from chloroplasts and plastoglobules separately, and analyzed using liquid chromatography-mass spectrometry (LC–MS). Three lipids were consistently present at lower levels in the plastoglobules from fbn4 knock-down apple leaves compared to the wild-type as determined by LC-MS multiple ion monitoring. One of these species had a molecular mass and fragmentation pattern that identified it as plastoquinone, a known major component of plastoglobules. The plastoquinone level in fbn4 knock-down plastoglobules was less than 10% of that in wild-type plastoglobules. In contrast, plastoquinone was present at similar levels in the lipid extracts of whole chloroplasts from leaves of wild-type and fbn4 knock-down trees. These results suggest that the partitioning of plastoquinone between the plastoglobules and the rest of the chloroplast is disrupted in fbn4 knock-down leaves. These results indicate that FBN4 is required for high-level accumulation of plastoquinone and some other lipids in the plastoglobule. The dramatic decrease in plastoquinone content in fbn4 knock-down plastoglobules is consistent with the decreased plastoglobule osmiophilicity previously described for fbn4 knock-down plastoglobules. Failure to accumulate the antioxidant plastoquinone in the fbn4 knock-down plastoglobules might contribute to the increased stress sensitivity of fbn4 knock-down trees.

Fibrillin 5 Is Essential for Plastoquinone-9 Biosynthesis by Binding to Solanesyl Diphosphate Synthases in Arabidopsis

“…FBN5 is a phylogenetically distant relative of other fibrillins in Arabidopsis (Singh and McNellis, 2011), and it localizes to the stroma, with a lower pI and lower hydrophobicity than the other fibrillins (Lundquist et al, 2012). Therefore, FBN5 may play different roles from those previously reported for the fibrillins that localize primarily to the plastoglobules (Trebst, 1978;Yang et al, 2006;Singh et al, 2010Singh et al, , 2012Youssef et al, 2010). Surprisingly, we found that FBN5 specifically interacts with two solanesyl diphosphate synthases, SPS1 and SPS2, in the chloroplasts.…”

“…We used HPLC to determine the amounts of PQ-9, tocochromanols, carotenoids, and chlorophylls in the mutant and wild-type plants because the photosynthetic capacity of the fbn5-1 plants was reduced and FBN4 is involved in plastoquinone accumulation in the plastoglobules (Singh et al, 2012). Although fbn5-1 is seedling-lethal when plants are grown in soil, seedlings cultivated on agar medium supplemented with 1% sucrose were pale green, survived for several weeks, and produced sufficient tissue for biochemical analyses.…”

“…It has been suggested that FBN4, with its lipocalin domain, is involved in the transport of PQ-9 and other molecules from the thylakoids to the plastoglobules (Singh et al, 2010(Singh et al, , 2012. However, the precise biological functions of the fibrillins require further investigation.…”

“…These forms of plastoquinone (PQ) are likely part of the photoactive PQ pool and participate in both electron transport and antioxidant functions (Kruk and Karpinski, 2006). PQ-9 is also found in plastoglobules and the inner membrane of plastids (Tevini and Steinmüller, 1985;Eugeni Piller et al, 2012;Singh et al, 2012). These PQ-9 molecules are thought to be nonphotoactive, storage forms that are used to replenish the photoactive PQ pool (Zbierzak et al, 2010;Singh et al, 2012).…”

“…PQ-9 is also found in plastoglobules and the inner membrane of plastids (Tevini and Steinmüller, 1985;Eugeni Piller et al, 2012;Singh et al, 2012). These PQ-9 molecules are thought to be nonphotoactive, storage forms that are used to replenish the photoactive PQ pool (Zbierzak et al, 2010;Singh et al, 2012). Previous studies have shown that PQ-9 biosynthesis occurs in the membranes of the plastid envelope (Hutson and Threlfall, 1980;Soll et al, 1980Soll et al, , 1985Joyard et al, 2009).…”