Cosuppression of the chloroplast localized molecular chaperone HSP90.5 impairs plant development and chloroplast biogenesis in Arabidopsis

Oh SE, Yeung C, Babaei-Rad R, Zhao R

BMC Res Notes 2014;7:643

PMID: 25216779


BACKGROUND: 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. 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.

RESULTS: Immunoblotting 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.

CONCLUSION: We 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.

A highly charged region in the middle domain of plant endoplasmic reticulum (ER)-localized heat-shock protein 90 is required for resistance to tunicamycin or high calcium-induced ER stresses

Chong LP, Wang Y, Gad N, Anderson N, Shah B, Zhao R

J. Exp. Bot. 2015 Jan;66(1):113-24

PMID: 25297550


Heat-shock protein 90 (HSP90) is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes under both physiological and stress conditions. In Arabidopsis, there are seven HSP90 isoforms (HSP90.1-HSP90.7) that are localized in the cytoplasm/nucleus, mitochondrion, chloroplast, and endoplasmic reticulum (ER) where protein folding actively takes place. In this study, we analysed the sequence of ER-localized Arabidopsis HSP90.7 and the other ER GRP94 proteins from plants and animals, and identified a short, charged region that is specifically present in the middle domain of plant-derived GRP94 proteins. To understand the role of this charged region, we analysed transgenic plants that expressed a mutant protein, HSP90.7(Δ22), which had this charged region deleted. We showed that seedlings expressing HSP90.7(Δ22) had significantly enhanced sensitivity to ER stress induced by tunicamycin or a high concentration of calcium, although its general chaperone activity in preventing the model protein from heat-induced aggregation was not significantly affected. We also analysed the ATP-binding and hydrolysis activity of both wild-type and mutant HSP90.7 proteins, and found that they had slightly different ATP-binding affinities. Finally, using a yeast two-hybrid screen, we identified a small set of HSP90.7 interactors and showed that the charged region is not required for the candidate client interaction, although it may affect their binding affinity, thus providing potential targets for further investigation of HSP90.7 functions.

A tetratricopeptide repeat domain-containing protein SSR1 located in mitochondria is involved in root development and auxin polar transport in Arabidopsis

Zhang M, Wang C, Lin Q, Liu A, Wang T, Feng X, Liu J, Han H, Ma Y, Bonea D, Zhao R, Hua X

Plant J. 2015 Aug;83(4):582-599

PMID: 26072661


Auxin polar transport mediated by a group of Pin-formed (PIN) transporters plays important roles in plant root development. However, the mechanism underlying the PIN expression and targeting in response to different developmental and environmental stimuli is still not fully understood. Here, we report a previously uncharacterized gene SSR1, which encodes a mitochondrial protein with tetratricopeptide repeat (TPR) domains, and show its function in root development in Arabidopsis thaliana. In ssr1-2, a SSR1 knock-out mutant, the primary root growth was dramatically inhibited due to severely impaired cell proliferation and cell elongation. Significantly lowered level of auxin was found in ssr1-2 roots by auxin measurement and was further supported by reduced expression of DR5-driven reporter gene. As a result, the maintenance of the root stem cell niche is compromised in ssr1-2. It is further revealed that the expression level of several PIN proteins, namely, PIN1, PIN2, PIN3, PIN4 and PIN7, were markedly reduced in ssr1-2 roots. In particular, we showed that the reduced protein level of PIN2 on cell membrane in ssr1-2 is due to impaired retrograde trafficking, possibly resulting from a defect in retromer sorting system, which destines PIN2 for degradation in vacuoles. In conclusion, our results indicated that SSR1 is functioning in root development in Arabidopsis, possibly by affecting PIN protein expression and subcellular targeting.