Abstract
BackgroundHSP90.5 is a chloroplast localized HSP90 family molecular chaperone in Arabidopsis, and it has been implicated in plant abiotic stress resistance, photomorphogenesis and nuclear-encoded protein import into the chloroplast. However, how these processes are controlled by HSP90 is not well understood. To understand the role of HSP90.5 in chloroplast function and biogenesis, in this study, we generated transgenic Arabidopsis plants that overexpress a C-terminally FLAG-tagged HSP90.5. By characterizing three HSP90.5 cosuppression lines, we demonstrated the essential role of HSP90.5 in plant growth and chloroplast biogenesis.ResultsImmunoblotting and quantitative PCR analyses revealed three independent HSP90.5 cosuppressing transgenic lines. All three cosuppression lines displayed a certain degree of variegated phenotype in photosynthetic tissues, and the cosuppression did not affect the expression of cytosolic HSP90 isoforms. HSP90.5 cosuppression was shown to be developmentally regulated and occurred mostly at late developmental stage in adult leaves and inflorescence tissues. HSP90.5 cosuppression also caused significantly reduced rosette leaf growth, transient starch storage, but did not affect rosette leaf initiation or inflorescence production, although the fertility was reduced. Isolation of chloroplasts and size exclusion chromatography analysis indicated that the FLAG at the HSP90.5 C-terminus does not affect its proper chloroplast localization and dimerization. Finally, transmission electron microscopy indicated that chloroplast development in HSP90.5 cosuppression leaves was significantly impaired and the integrity of chloroplast is highly correlated to the expression level of HSP90.5.ConclusionWe thoroughly characterized three HSP90.5 cosuppression lines, and demonstrated that properly controlled expression of HSP90.5 is required for plant growth and development in many tissues, and especially essential for chloroplast thylakoid formation. Since the homozygote of HSP90.5 knockout mutant is embryonically lethal, this study provides transgenic lines that mimic the conditional knockout line or siRNA line of the essential HSP90.5 gene in Arabidopsis.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-643) contains supplementary material, which is available to authorized users.
Highlights
HSP90.5 is a chloroplast localized HSP90 family molecular chaperone in Arabidopsis, and it has been implicated in plant abiotic stress resistance, photomorphogenesis and nuclear-encoded protein import into the chloroplast
Some transgenic plants expressing FLAG-tagged HSP90.5 display variegated phenotype in photosynthetic tissues The initial goal of generating transgenic Arabidopsis plants that overexpress C-terminally FLAG-tagged HSP90.5 is to purify HSP90.5 complex by affinity chromatography and to determine HSP90.5 interactors via mass spectrometry
Similar to a previous study [38], no obvious phenotype was observed for the primary transgenic plants that were confirmed to contain transgenic FLAG-tagged HSP90.5 gene by PCR genotyping (Additional file 1: Figure S1 and S2A) under normal growth conditions
Summary
HSP90.5 is a chloroplast localized HSP90 family molecular chaperone in Arabidopsis, and it has been implicated in plant abiotic stress resistance, photomorphogenesis and nuclear-encoded protein import into the chloroplast. How these processes are controlled by HSP90 is not well understood. To ensure the proper protein folding and turnover of these proteins as well as newly synthesized ones from its own de novo protein synthesis machinery, a comprehensive protein quality control system exists in the chloroplast This complex system includes chaperone families of HSP40, HSP60, HSP70, HSP90 and HSP100, and proteases ClpP, FtsH, DegP, and SPP [11,12]. Malfunction of protein quality control components have been shown to impair chloroplast function and plant development [13,14,15]
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