Knockout of endoplasmic reticulum-localized molecular chaperone HSP90.7 impairs seedling development and cellular auxin homeostasis in Arabidopsis.

The Arabidopsis endoplasmic reticulum-localized heat shock protein HSP90.7 modulates tissue differentiation and stress responses; however, complete knockout lines have not been previously reported. In this study, we identified and analyzed a mutant allele, hsp90.7-1, which was unable to accumulate the HSP90.7 full-length protein and showed seedling lethality. Microscopic analyses revealed its essential role in male and female fertility, trichomes and root hair development, proper chloroplast function, and apical meristem maintenance and differentiation. Comparative transcriptome and proteome analyses also revealed the role of the protein in a multitude of cellular processes. Particularly, the auxin-responsive pathway was specifically downregulated in the hsp90.7-1 mutant seedlings. We measured a much-reduced auxin content in both root and shoot tissues. Through comprehensive histological and molecular analyses, we confirmed PIN1 and PIN5 accumulations were dependent on the HSP90 function, and the TAA-YUCCA primary auxin biosynthesis pathway was also downregulated in the mutant seedlings. This study therefore not only fulfilled a gap in understanding the essential role of HSP90 paralogs in eukaryotes but also provided a mechanistic insight on the ER-localized chaperone in regulating plant growth and development via modulating cellular auxin homeostasis.

Oxidative and salt stresses alter the 26S proteasome holoenzyme and associated protein profiles in Arabidopsis thaliana.

BACKGROUND: The 26S proteasome, canonically composed of multi-subunit 19S regulatory (RP) and 20S core (CP) particles, is crucial for cellular proteostasis. Proteasomes are re-modeled, activated, or re-localized and this regulation is critical for plants in response to environmental stresses. The proteasome holoenzyme assembly and dissociation are therefore highly dynamic in vivo. However, the stoichiometric changes of the plant proteasomes and how proteasome associated chaperones vary under common abiotic stresses have not been systematically studied. RESULTS: Here, we studied the impact of abiotic stresses on proteasome structure, activity, and interacting partners in Arabidopsis thaliana. We analyzed available RNA expression data and observed that expressions of proteasome coding genes varied substantially under stresses; however, the protein levels of a few key subunits did not change significantly within 24 h. Instead, a switch in the predominant proteasome complex, from 26S to 20S, occurs under oxidative or salt stress. Oxidative stress also reduced the cellular ATP content and the association of HSP70-family proteins to the 20S proteasome, but enhanced the activity of cellular free form CP. Salt stress, on the other hand, did not affect cellular ATP level, but caused subtle changes in proteasome subunit composition and impacted bindings of assembly chaperones. Analyses of an array of T-DNA insertional mutant lines highlighted important roles for several putative assembly chaperones in seedling establishment and stress sensitivity. We also observed that knockout of PBAC1, one of the α-ring assembly chaperones, resulted in reduced germination and tearing of the seed coat following sterilization. CONCLUSIONS: Our study revealed an evolutionarily conserved mechanism of proteasome regulation during oxidative stress, involving dynamic regulation of the holoenzyme formation and associated regulatory proteins, and we also identified a novel role of the PBAC1 proteasome assembly chaperone in seed coat development.

HSP90C interacts with PsbO1 and facilitates its thylakoid distribution from chloroplast stroma in Arabidopsis.

Arabidopsis plastidic HSP90C is an HSP90 family molecular chaperone that is required for chloroplast development and function. To understand the mechanism of action of HSP90C within the chloroplast, we conducted a yeast two-hybrid screening and revealed it interacts directly with the photosystem II extrinsic protein PsbO1, which performs a canonical function in the thylakoid lumen. To understand the biological significance of HSP90C-PsbO1 interaction, we investigated the role of HSP90C in modulating the stromal and thylakoid distribution of PsbO1GFP fusion protein. Fusion to GFP significantly delays the PsbO1 thylakoid transport and induces a variegation phenotype. Overexpression of HSP90C promotes the thylakoid distribution of PsbO1GFP and alleviates the leaf variegation. By tracking the chloroplast maturation during photomorphogenesis, we observed PsbO1GFP tends to form distinct fluorescent clusters within the stroma with delayed thylakoid membrane biogenesis, while HSP90C overexpression corrects these adverse effects. We also demonstrated that active HSP90C function is specifically required for stable accumulation of mature PsbO1GFP in thylakoid by using specific inhibitor geldanamycin. This study therefore not only identified novel HSP90C interactors, but also reports for the first time that PsbO1 enroute from the cytoplasm to thylakoid lumen is tightly regulated by the HSP90C chaperone complex in plastid stroma; whereas the proper HSP90C homeostasis is also critical for chloroplast maturation and function.

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

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.