In higher plants, the chloroplast NAD(P)H dehydrogenase (NDH) complex mediates photosystem I (PSI) cyclic and chlororespiratory electron transport. We reported previously that NDH interacts with the PSI complex to form a supercomplex (NDH-PSI). In this study, NDH18 and FKBP16-2 (FK506 Binding Protein 16-2), detected in the NDH-PSI supercomplex by mass spectrometry, were shown to be NDH subunits by the analysis of their knockdown lines. On the basis of extensive mutant characterization, we propose a structural model for chloroplast NDH, whereby NDH is divided into four subcomplexes. The subcomplex A and membrane subcomplex are conserved in cyanobacteria, but the subcomplex B and lumen subcomplex are specific to chloroplasts. Two minor light-harvesting complex I proteins, Lhca5 and Lhca6, were required for the full-size NDH-PSI supercomplex formation. Similar to crr pgr5 double mutants that completely lack cyclic electron flow activity around PSI, the lhca6 pgr5 double mutant exhibited a severe defect in growth. Consistent with the impaired NDH activity, photosynthesis was also severely affected in mature leaves of lhca6 pgr5. We conclude that chloroplast NDH became equipped with the novel subcomplexes and became associated with PSI during the evolution of land plants, and this process may have facilitated the efficient operation of NDH.

The nuclear pore complex (NPC) facilitates nucleocytoplasmic transport, a crucial process for various cellular activities.The NPC comprises ;30 nucleoporins and is well characterized in vertebrates and yeast. However, only eight plant nucleoporins have been identified, and little information is available about the complete molecular structure of plant NPCs. In this study, an interactive proteomic approach was used to identify Arabidopsis thaliana nucleoporins. A series of five cycles of interactive proteomic analysis was performed using green fluorescent protein (GFP)-tagged nucleoporins. The identified nucleoporins were then cloned and subcellular localization analyses were performed. We found that the plant NPC contains at least 30 nucleoporins, 22 of which had not been previously annotated. Surprisingly, plant nucleoporins shared a similar domain organization to their vertebrate (human) and yeast (Saccharomyces cerevisiae) counterparts. Moreover, the plant nucleoporins exhibited higher sequence homology to vertebrate nucleoporins than to yeast nucleoporins. Plant NPCs lacked seven components (NUCLEOPORIN358 [Nup358], Nup188, Nup153, Nup45, Nup37, NUCLEAR DIVISION CYCLE1, and PORE MEMBRANE PROTEIN OF 121 kD) that were present in vertebrate NPCs. However, plants possessed a nucleoporin, Nup136/Nup1, that contained Phe-Gly repeats, and sequence analysis failed to identify a vertebrate homolog for this protein. Interestingly, Nup136-GFP showed greater mobility on the nuclear envelope than did other nucleoporins, and a Nup136/Nup1 deficiency caused various defects in plant development. These findings provide valuable new information about plant NPC structure and function.

Some subunits of chloroplast NAD(P)H dehydrogenase (NDH) are related to those of the respiratory complex I, and NDH mediates photosystem I (PSI) cyclic electron flow. Despite extensive surveys, the electron donor and its binding subunits have not been identified. Here, we identified three novel components required for NDH activity. CRRJ and CRRL are J-and J-like proteins, respectively, and are components of NDH subcomplex A. CRR31 is an Src homology 3 domain-like fold protein, and its C-terminal region may form a tertiary structure similar to that of PsaE, a ferredoxin (Fd) binding subunit of PSI, although the sequences are not conserved between CRR31 and PsaE. Although CRR31 can accumulate in thylakoids independently of NDH, its accumulation requires CRRJ, and CRRL accumulation depends on CRRJ and NDH. CRR31 was essential for the efficient operation of Fd-dependent plastoquinone reduction in vitro. The phenotype of crr31 pgr5 suggested that CRR31 is required for NDH activity in vivo. We propose that NDH functions as a PGR5-PGRL1 complexindependent Fd:plastoquinone oxidoreductase in chloroplasts and rename it the NADH dehydrogenase-like complex.

The Arabidopsis thaliana K+ channel KAT1 has been suggested to have a key role in mediating the aperture of stomata pores on the surface of plant leaves. Although the activity of KAT1 is thought to be regulated by phosphorylation, the endogenous pathway and the primary target site for this modification remained unknown. In the present study, we have demonstrated that the C-terminal region of KAT1 acts as a phosphorylation target for the Arabidopsis calcium-independent ABA (abscisic acid)-activated protein kinase SnRK2.6 (Snf1-related protein kinase 2.6). This was confirmed by LC-MS/MS (liquid chromatography tandem MS) analysis, which showed that Thr306 and Thr308 of KAT1 were modified by phosphorylation. The role of these specific residues was examined by single point mutations and measurement of KAT1 channel activities in Xenopus oocyte and yeast systems. Modification of Thr308 had minimal effect on KAT1 activity. On the other hand, modification of Thr306 reduced the K+ transport uptake activity of KAT1 in both systems, indicating that Thr306 is responsible for the functional regulation of KAT1. These results suggest that negative regulation of KAT1 activity, required for stomatal closure, probably occurs by phosphorylation of KAT1 Thr306 by the stress-activated endogenous SnRK2.6 protein kinase.

The cell nucleus communicates with the cytoplasm through a nucleocytoplasmic linker that maintains the shape of the nucleus and mediates its migration. In contrast to animal nuclei, which are moved by motor proteins (kinesins and dyneins) along the microtubule cytoskeleton, plant nuclei move rapidly and farther along an actin filament cytoskeleton. This implies that plants use a distinct nucleocytoplasmic linker for nuclear dynamics, although its molecular identity is unknown. Here, we describe a new type of nucleocytoplasmic linker consisting of a myosin motor and nuclear membrane proteins. In the Arabidopsis thaliana mutant kaku1, nuclear movement was impaired and the nuclear envelope was abnormally invaginated. The responsible gene was identified as myosin XI-i, which encodes a plant-specific myosin. Myosin XI-i is specifically localized on the nuclear membrane, where it physically interacts with the outer-nuclear-membrane proteins WIT1 and WIT2. Both WIT proteins are required for anchoring myosin XI-i to the nuclear membrane and for nuclear movement. A striking feature of plant cells is dark-induced nuclear positioning in mesophyll cells. A deficiency of either myosin XI-i or WIT proteins diminished dark-induced nuclear positioning. The unique nucleocytoplasmic linkage in plants might enable rapid nuclear positioning in response to environmental stimuli.