cis-prenyltransferase 3 and α/ß-hydrolase are new determinants of dolichol accumulation in Arabidopsis.

Dolichols (Dols), ubiquitous components of living organisms, are indispensable for cell survival. In plants, as well as other eukaryotes, Dols are crucial for post-translational protein glycosylation, aberration of which leads to fatal metabolic disorders in humans and male sterility in plants. Until now, the mechanisms underlying Dol accumulation remain elusive. In this study, we have analysed the natural variation of the accumulation of Dols and six other isoprenoids among more than 120 Arabidopsis thaliana accessions. Subsequently, by combining QTL and GWAS approaches, we have identified several candidate genes involved in the accumulation of Dols, polyprenols, plastoquinone and phytosterols. The role of two genes implicated in the accumulation of major Dols in Arabidopsis-the AT2G17570 gene encoding a long searched for cis-prenyltransferase (CPT3) and the AT1G52460 gene encoding an α/ß-hydrolase-is experimentally confirmed. These data will help to generate Dol-enriched plants which might serve as a remedy for Dol-deficiency in humans.

Modulation of Expression of PVYNTN RNA-Dependent RNA Polymerase (NIb) and Heat Shock Cognate Host Protein HSC70 in Susceptible and Hypersensitive Potato Cultivars.

Potato virus Y (PVY) belongs to the genus Potyvirus and is considered to be one of the most harmful and important plant pathogens. Its RNA-dependent RNA polymerase (RdRp) is known as nuclear inclusion protein b (NIb). The recent findings show that the genome of PVY replicates in the cytoplasm of the plant cell by binding the virus replication complex to the membranous structures of different organelles. In some potyviruses, NIb has been found to be localized in the nucleus and associated with the endoplasmic reticulum membranes. Moreover, NIb has been shown to interact with other host proteins that are particularly involved in promoting the virus infection cycle, such as the heat shock proteins (HSPs). HSP70 is the most conserved among the five major HSP families that are known to affect the plant-pathogen interactions. Some plant viruses can induce the production of HSP70 during the development of infection. To understand the molecular mechanisms underlying the interactive response to PVYNTN (necrotic tuber necrosis strain of PVY), the present study focused on StHSC70-8 and PVYNTN-NIb gene expression via localization of HSC70 and NIb proteins during compatible (susceptible) and incompatible (hypersensitive) potato-PVYNTN interactions. Our results demonstrate that NIb and HSC70 are involved in the response to PVYNTN infections and probably cooperate at some stages of the virus infection cycle. Enhanced deposition of HSC70 proteins during the infection cycle was associated with the dynamic induction of PVYNTN-NIb gene expression and NIb localization during susceptible infections. In hypersensitive response (HR), a significant increase in HSC70 expression was observed up to 3 days post-inoculation (dpi) in the nucleus and chloroplasts. Thereafter, between 3 and 21 dpi, the deposition of NIb decreased, which can be attributed to a reduction in the levels of both virus accumulation and PVYNTN-NIb gene expression. Therefore, we postulate that increase in the expression of both StHSC70-8 and PVYNTN-NIb induces the PVY infection during susceptible infections. In contrast, during HRs, HSC70 cooperates with PVYNTN only at the early stages of interaction and mediates the defense response signaling pathway at the later stages of infection.

Long-Chain Polyisoprenoids Are Synthesized by AtCPT1 in Arabidopsis thaliana.

Arabidopsis roots accumulate a complex mixture of dolichols composed of three families, (i.e., short-, medium- and long-chain dolichols), but until now none of the cis-prenyltransferases (CPTs) predicted in the Arabidopsis genome has been considered responsible for their synthesis. In this report, using homo- and heterologous (yeast and tobacco) models, we have characterized the AtCPT1 gene (At2g23410) which encodes a CPT responsible for the formation of long-chain dolichols, Dol-18 to -23, with Dol-21 dominating, in Arabidopsis. The content of these dolichols was significantly reduced in AtCPT1 T-DNA insertion mutant lines and highly increased in AtCPT1-overexpressing plants. Similar to the majority of eukaryotic CPTs, AtCPT1 is localized to the endoplasmic reticulum (ER). Functional complementation tests using yeast rer2Δ or srt1Δ mutants devoid of medium- or long-chain dolichols, respectively, confirmed that this enzyme synthesizes long-chain dolichols, although the dolichol chains thus formed are somewhat shorter than those synthesized in planta. Moreover, AtCPT1 acts as a homomeric CPT and does not need LEW1 for its activity. AtCPT1 is the first plant CPT producing long-chain polyisoprenoids that does not form a complex with the NgBR/NUS1 homologue.

Polyprenols Are Synthesized by a Plastidial cis-Prenyltransferase and Influence Photosynthetic Performance.

Plants accumulate a family of hydrophobic polymers known as polyprenols, yet how they are synthesized, where they reside in the cell, and what role they serve is largely unknown. Using Arabidopsis thaliana as a model, we present evidence for the involvement of a plastidial cis-prenyltransferase (AtCPT7) in polyprenol synthesis. Gene inactivation and RNAi-mediated knockdown of AtCPT7 eliminated leaf polyprenols, while its overexpression increased their content. Complementation tests in the polyprenol-deficient yeast ∆rer2 mutant and enzyme assays with recombinant AtCPT7 confirmed that the enzyme synthesizes polyprenols of ∼55 carbons in length using geranylgeranyl diphosphate (GGPP) and isopentenyl diphosphate as substrates. Immunodetection and in vivo localization of AtCPT7 fluorescent protein fusions showed that AtCPT7 resides in the stroma of mesophyll chloroplasts. The enzymatic products of AtCPT7 accumulate in thylakoid membranes, and in their absence, thylakoids adopt an increasingly "fluid membrane" state. Chlorophyll fluorescence measurements from the leaves of polyprenol-deficient plants revealed impaired photosystem II operating efficiency, and their thylakoids exhibited a decreased rate of electron transport. These results establish that (1) plastidial AtCPT7 extends the length of GGPP to ∼55 carbons, which then accumulate in thylakoid membranes; and (2) these polyprenols influence photosynthetic performance through their modulation of thylakoid membrane dynamics.

Short-chain polyisoprenoids in the yeast Saccharomyces cerevisiae - New companions of the old guys.

Dolichols are, among others, obligatory cofactors of protein glycosylation in eukaryotic cells. It is well known that yeast cells accumulate a family of dolichols with Dol-15/16 dominating while upon certain physiological conditions a second family with Dol-21 dominating is noted. In this report we identified the presence of additional short-chain length polyprenols - all-trans Pren-7 in three yeast strains (SS328, BY4741 and L5366), Pren-7 was accompanied by traces of putative Pren-6 and -8. Moreover, in two of these strains a single polyprenol mainly-cis-Pren-11 was synthesized at the stationary phase of growth. Identity of polyprenols was confirmed by HR-HPLC/MS, NMR and metabolic labeling. Additionally, simvastatin inhibited their biosynthesis.

Identification and functional analysis of Joka2, a tobacco member of the family of selective autophagy cargo receptors.

Two main mechanisms of protein turnover exist in eukaryotic cells: the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Autophagy is an emerging important constituent of many physiological and pathological processes, such as response to nutrient deficiency, programmed cell death and innate immune response. In mammalian cells the selectivity of autophagy is ensured by the presence of cargo receptors, such as p62/SQSTM1 and NBR1, responsible for sequestration of the ubiquitinated proteins. In plants no selective cargo receptors have been identified yet. The present report indicates that structural and functional homologs of p62 and NBR1 proteins exist in plants. The tobacco protein, named Joka2, has been identified in yeast two-hybrid search as a binding partner of a small coiled-coil protein, a member of UP9/LSU family of unknown function, encoded by the UP9C gene strongly and specifically induced during sulfur deficiency. The typical domains of p62 and NBR1 are conserved in Joka2. Similarly to p62, Joka2-YFP has dual localization (cytosolic speckles and the nucleus); it forms homodimers and interacts with a member of the ATG8 family. Increased expression of Joka2 and ATG8f was observed in roots of tobacco plants grown for two days in nutrient-deficient conditions. Constitutive ectopic expression of Joka2-YFP in tobacco resulted in attenuated response (manifested by lesser yellowing of the leaves) to nutrient deficiency. In conclusion, Joka2, and presumably the process of selective autophagy, might constitute an important part of plant response to environmental stresses.