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During dark adaptation, plant nuclei move centripetally toward the midplane of the leaf blade; thus, the nuclei on both the adaxial and abaxial sides become positioned at the inner periclinal walls of cells. This centripetal nuclear positioning implies that a characteristic cell polarity exists within a leaf, but little is known about the mechanism underlying this process. Here, we show that ANGUSTIFOLIA (AN) and ACTIN7 regulate centripetal nuclear positioning in Arabidopsis (

Lipid body (LB) is recognized as the cellular carbon and energy storage organelle in many organisms. LBs have been observed in the marine haptophyte alga Tisochrysis lutea that produces special lipids such as long-chain (C37-C40) ketones (alkenones) with 2–4 trans-type double bonds. In this study, we succeeded in developing a modified method to isolate LB from T. lutea. Purity of isolated LBs was confirmed by the absence of chlorophyll auto-fluorescence and no contamination of the most abundant cellular protein ribulose-1,5-bisphosphate carboxylase/oxygenase. As alkenones predominated in the LB by GC-MS analysis, the LB can be more appropriately named as “alkenone body (AB).” Extracted AB-containing proteins were analyzed by the combination of 1DE (SDS-PAGE) and MS/MS for confident protein identification and annotated using BLAST tools at National Center for Biotechnology Information. Totally 514 proteins were identified at the maximum. The homology search identified three major proteins, V-ATPase, a hypothetical protein EMIHUDRAFT_465517 found in other alkenone-producing haptophytes, and a lipid raft-associated SPFH domain-containing protein. Our data suggest that AB of T. lutera is surrounded by a lipid membrane originating from either the ER or the ER-derived four layer-envelopes chloroplast and function as the storage site of alkenones and alkenes.

The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus. The ER network was RHD3-dependently reconstituted from the cytosol and microsome fraction of tobacco (Nicotiana tabacum) cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in vitro assay system of ER tubule formation with Arabidopsis ER vesicles, in which addition of GTP caused ER sac formation from the ER vesicles. Subsequent application of a shearing force to this system triggered the formation of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence of a shearing force, Ser/Thr kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple Ser and Thr residues in the C terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the Ser cluster was deleted or when the Ser residues were replaced with Ala residues, kinase treatment had no effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither phosphorylation-dependent modulation of membrane fusion nor oligomerization has been reported for atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated oligomerization of RHD3 enhances ER membrane fusion to form the ER network.

Iron (Fe) is required by plants for basic redox reactions in photosynthesis and respiration, and for many other key enzymatic reactions in biological processes. Fe homeostatic mechanisms have evolved in plants to enable the uptake and sequestration of Fe in cells. To elucidate the network of proteins that regulate Fe homeostasis and transport, we optimized the iTRAQ-OFFGEL method to identify and quantify the number of proteins that respond to Fe deficiency in the model plant Arabidopsis. In this study, Fe deficiency was created using Fe-deficient growth conditions, excess zinc (Zn), and use of the irt1-1 mutant in which the IRT1 Fe transporter is disrupted. Using the iTRAQ-OFFGEL approach, we identified 1139 proteins, including novel Fe deficiency-responsive proteins, in microsomal fractions isolated from 3 different types of Fe-deficient shoots compared with just 233 proteins identified using conventional iTRAQ-CEX. Further analysis showed that greater numbers of low-abundance proteins could be identified using the iTRAQ-OFFGEL method and that proteins could be identified from numerous cellular compartments. The improved iTRAQ-OFFGEL method used in this study provided an efficient means for identifying greater numbers of proteins from microsomal fractions of Arabidopsis shoots. The proteome identified in this study provides new insight into the regulatory cross talk between Fe-deficient and excess Zn conditions.

Polar transport of the plant hormone auxin is controlled by PIN- and ABCB/PGP-efflux catalysts. PIN polarity is regulated by the AGC protein kinase, PINOID (PID), while ABCB activity was shown to be dependent on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Using co-immunoprecipitation (co-IP) and shotgun LC–MS/MS analysis, we identified PID as a valid partner in the interaction with TWD1. In-vitro and yeast expression analyses indicated that PID specifically modulates ABCB1-mediated auxin efflux in an action that is dependent on its kinase activity and that is reverted by quercetin binding and thus inhibition of PID autophosphorylation. Triple ABCB1/PID/TWD1 co-transfection in tobacco revealed that PID enhances ABCB1-mediated auxin efflux but blocks ABCB1 in the presence of TWD1. Phospho-proteomic analyses identified S634 as a key residue of the regulatory ABCB1 linker and a very likely target of PID phosphorylation that determines both transporter drug binding and activity. In summary, we provide evidence that PID phosphorylation has a dual, counter-active impact on ABCB1 activity that is coordinated by TWD1–PID interaction.