Arabidopsis V-ATPase d2 Subunit Plays a Role in Plant Responses to Oxidative Stress

Vacuolar-type H+-ATPase (V-ATPase), a multisubunit proton pump located on the endomembrane, plays an important role in plant growth. The Arabidopsis thaliana V-ATPase d subunit (VHA-d) consists of two isoforms; AtVHA-d1 and AtVHA-d2. In this study, the function of AtVHA-d2 was investigated. Histochemical analysis revealed that the expression of AtVHA-d1 and AtVHA-d2 was generally highly overlapping in multiple tissues at different developmental stages of Arabidopsis. Subcellular localization revealed that AtVHA-d2 was mainly localized to the vacuole. AtVHA-d2 expression was significantly induced by oxidative stress. Analysis of phenotypic and H2O2 content showed that the atvha-d2 mutant was sensitive to oxidative stress. The noninvasive microtest monitoring demonstrated that the net H+ influx in the atvha-d2 roots was weaker than that in the wild-type under normal conditions. However, oxidative stress resulted in the H+ efflux in atvha-d2 roots, which was significantly different from that in the wild-type. RNA-seq combined with qPCR analysis showed that the expression of several members of the plasma membrane H+-ATPase gene (AtAHA) family in atvha-d2 was significantly different from that in the wild-type. Overall, our results indicate that AtVHA-d2 plays a role in Arabidopsis in response to oxidative stress by affecting H+ flux and AtAHA gene expression.

Arabidopsis V-ATPase d2 subunit plays a role in plant responses to oxidative stress

Background: Vacuolar-type H + -ATPase (V-ATPase) is a multisubunit proton pump located on the endomembranes, which plays an important role in plant growth. The Arabidopsis V-ATPase d subunit consists of two isoforms, AtVHA-d1 and AtVHA-d2. Results: In this study, the function of the AtVHA-d2 gene was investigated. Histochemical analysis revealed that the AtVHA-d1 and AtVHA-d2 genes were generally and highly overlapping expressed in multiple tissues at different developmental stages of Arabidopsis. Subcellular localization showed that AtVHA-d2 was mainly localized to the vacuole. The AtVHA-d2 expression was significantly induced by oxidative stress. Furthermore, the phenotypic analysis showed that the atvha-d2 mutant was sensitive to oxidative stress. The non-invasive micro-test measurement demonstrated that the net H + influx in the atvha-d2 roots was weaker than that of the wild type under normal conditions. However, oxidative stress resulted in the H + efflux in atvha-d2 roots, which was significantly different from the wild type. RNA-seq combined with qPCR analysis showed that the expression of several members of the plasma membrane H + -ATPase gene ( AtAHA ) family in atvha-d2 were significant different from wild type under normal and oxidative stress. Conclusion: Overall, our results indicate that AtVHA-d2 plays a role in Arabidopsis in response to oxidative stress by affecting H + flux and AtAHA gene expression.

Heterologous Expression of Human Norovirus GII.4 VP1 Leads to Assembly of T=4 Virus-Like Particles

ABSTRACTHuman noroviruses are a leading cause of acute gastroenteritis, yet there are still no vaccines or antivirals available. Expression of the norovirus capsid protein (VP1) in insect cells typically results in the formation of virus-like particles (VLPs) that are morphologically and antigenically comparable to native virions. Previous structural analysis of norovirus VLPs showed that the capsid has a T=3 icosahedral symmetry and is composed of 180 copies of VP1 that are folded into three quasi-equivalent subunits (A, B, and C). In this study, we determined the cryo-EM VLP structures of two GII.4 variants, termed CHDC-1974 and NSW-2012. Surprisingly, we found that greater than 95% of these GII.4 VLPs were larger than virions and 3D reconstruction showed that these VLPs exhibited T=4 icosahedral symmetry. We found that the T=4 VLPs showed several structural differences to the T=3 VLPs. The T=4 particles assemble from 240 copies of VP1 that adopt four quasi-equivalent conformations (A, B, C, and D) that form two distinct dimers, A/B and C/D. The T=4 protruding domains were elevated ∼21-Å off the capsid shell, which was ∼7-Å more than the previously studied GII.10 T=3 VLPs. A small cavity and flap-like structure at the icosahedral twofold axis disrupted the contiguous T=4 shell, a consequence of the D-subunit S-domains having smaller contact interfaces with neighboring dimers. Overall, our findings that old and new GII.4 VP1 sequences assemble T=4 VLPs might have implications for the design of potential future vaccines.IMPORTANCEThe discovery that the GII.4 VLPs have a T=4 symmetry is of significance, since this represents the first known T=4 calicivirus structure. Interestingly, the GII.4 2012 variant shares 96% amino acid identity with a current GII.4 VLP vaccine candidate sequence, which suggests that this vaccine might also have a T=4 symmetry. Our previous results with these GII.4 VLPs showed functional binding properties to antibodies and Nanobodies that were raised against T=3 (GII.10) VLPs. This suggests that the T=4 VLPs were antigenically comparable to T=3 particles, despite the obvious structural and size differences. On the other hand, these larger T=4 VLPs with novel structural features and possibly new epitopes might elicit antibodies that do not recognize equivalent epitopes on the T=3 VLPs. Further structural and binding studies using a library of GII.4-specific Nanobodies are planned in order to precisely investigate whether new epitopes are formed.