AtRMR1 functions as a cargo receptor for protein trafficking to the protein storage vacuole

Organellar proteins are sorted by cargo receptors on the way to their final destination. However, receptors for proteins that are destined for the protein storage vacuole (PSV) are largely unknown. In this study, we investigated the biological role that Arabidopsis thaliana receptor homology region transmembrane domain ring H2 motif protein (AtRMR) 1 plays in protein trafficking to the PSV. AtRMR1 mainly colocalized to the prevacuolar compartment of the PSV, but a minor portion also localized to the Golgi complex. The coexpression of AtRMR1 mutants that were localized to the Golgi complex strongly inhibited the trafficking of phaseolin to the PSV and caused accumulation of phaseolin in the Golgi complex or its secretion. Coimmunoprecipitation and in vitro binding assays revealed that the lumenal domain of AtRMR1 interacts with the COOH-terminal sorting signal of phaseolin at acidic pH. Furthermore, phaseolin colocalized with AtRMR1 on its way to the PSV. Based on these results, we propose that AtRMR1 functions as the sorting receptor of phaseolin for its trafficking to the PSV

Evolution of rubisco complex small subunit transit peptides from algae to plants

Chloroplasts evolved from a free-living cyanobacterium acquired by the ancestor of all photosynthetic eukaryotes, including algae and plants, through a single endosymbiotic event. During endosymbiotic conversion, the majority of genes in the endosymbiont were transferred to the host nucleus and many of the proteins encoded by these genes must therefore be transported into the chloroplast after translation in the cytosol. Chloroplast-targeted proteins contain a targeting signal, named the transit peptide (TP), at the N-terminus. However, the evolution of TPs is not well understood. In this study, TPs from RbcS (rubisco small subunit) were compared between lower and higher eukaryotes. Chlamydomonas reinhardtii RbcS (CrRbcS) TP was non-functional in Arabidopsis. However, inclusion of a critical sequence motif, FP-RK, from Arabidopsis thaliana RbcS (AtRbcS) TP allowed CrRbcS TP to deliver proteins into plant chloroplasts. The position of the FP-RK motif in CrRbcS TP was critical for function. The QMMVW sequence motif in CrRbcS TP was crucial for its transport activity in plants. CrRbcS TPs containing additional plant motifs remained functional in C. reinhardtii. These results suggest that TPs evolved by acquiring additional sequence motifs to support protein targeting to chloroplasts during evolution of land plants from algae.113Ysciescopu

SCYL2 Genes Are Involved in Clathrin-Mediated Vesicle Trafficking and Essential for Plant Growth

Protein transport between organelles is an essential process in all eukaryotic cells and is mediated by the regulation of processes such as vesicle formation, transport, docking, and fusion. In animals, SCY1-LIKE2 (SCYL2) binds to clathrin and has been shown to play roles in trans-Golgi network-mediated clathrin-coated vesicle trafficking. Here, we demonstrate that SCYL2A and SCYL2B, which are Arabidopsis (Arabidopsis thaliana) homologs of animal SCYL2, are vital for plant cell growth and root hair development. Studies of the SCYL2 isoforms using multiple single or double loss-of-function alleles show that SCYL2B is involved in root hair development and that SCYL2A and SCYL2B are essential for plant growth and development and act redundantly in those processes. Quantitative reverse transcription-polymerase chain reaction and a beta-glucuronidase-aided promoter assay show that SCYL2A and SCYL2B are differentially expressed in various tissues. We also show that SCYL2 proteins localize to the Golgi, trans-Golgi network, and prevacuolar compartment and colocalize with Clathrin Heavy Chain1 (CHC1). Furthermore, bimolecular fluorescence complementation and coimmunoprecipitation data show that SCYL2B interacts with CHC1 and two Soluble NSF Attachment Protein Receptors (SNAREs): Vesicle Transport through t-SNARE Interaction11 (VTI11) and VTI12. Finally, we present evidence that the root hair tip localization of Cellulose Synthase-Like D3 is dependent on SCYL2B. These findings suggest the role of SCYL2 genes in plant cell developmental processes via clathrin-mediated vesicle membrane trafficking.11Ysciescopu

AtCAP2 is crucial for lytic vacuole biogenesis during germination by positively regulating vacuolar protein trafficking

Protein trafficking is a fundamental mechanism of subcellular organization and contributes to organellar biogenesis. AtCAP2 is an Arabidopsis homolog of the Mesembryanthemum crystallinum calcium-dependent protein kinase 1 adaptor protein 2 (McCAP2), a member of the syntaxin superfamily. Here, we show that AtCAP2 plays an important role in the conversion to the lytic vacuole (LV) during early plant development. The AtCAP2 loss-of-function mutant atcap2-1 displayed delays in protein storage vacuole (PSV) protein degradation, PSV fusion, LV acidification, and biosynthesis of several vacuolar proteins during germination. At the mature stage, atcap2-1 plants accumulated vacuolar proteins in the pre-vacuolar compartment (PVC) instead of the LV. In wild-type plants, AtCAP2 localizes to the PVC as a peripheral membrane protein and in the PVC compartment recruits glyceraldehyde-3-phosphate dehydrogenase C2 (GAPC2) to the PVC. We propose that AtCAP2 contributes to LV biogenesis during early plant development by supporting the trafficking of specific proteins involved in the PSV-to-LV transition and LV acidification during early stages of plant development.11Ysciescopu

Physiological and Molecular Processes Associated with Long Duration of ABA Treatment

Plants need to respond to various environmental stresses such as abiotic stress for proper development and growth. The responses to abiotic stress can be biochemically demanding, resulting in a trade-off that negatively affects plant growth and development. Thus, plant stress responses must be fine-tuned depending on the stress severity and duration. Abscisic acid, a phytohormone, plays a key role in responses to abiotic stress. Here, we investigated time-dependent physiological and molecular responses to long-term ABA treatment in Arabidopsis as an approach to gain insight into the plant responses to long-term abiotic stress. Upon ABA treatment, the amount of cellular ABA increased to higher levels, reaching to a peak at 24 h after treatment (HAT), and then gradually decreased with time whereas ABA-GE was maintained at lower levels until 24 HAT and then abruptly increased to higher levels at 48 HAT followed by a gradual decline at later time points. Many genes involved in dehydration stress responses, ABA metabolism, chloroplast biogenesis, and chlorophyll degradation were strongly expressed at early time points with a peak at 24 or 48 HAT followed by gradual decreases in induction fold or even suppression at later time points. At the physiological level, long-term ABA treatment caused leaf yellowing, reduced chlorophyll levels, and inhibited chloroplast division in addition to the growth suppression whereas short-term ABA treatment did not affect chlorophyll levels. Our results indicate that the duration of ABA treatment is a crucial factor in determining the mode of ABA-mediated signaling and plant responses: active mobilization of cellular resources at early time points and suppressive responses at later time points.11Ysciescopu

Production of active Exendin-4 in Nicotiana benthamiana and its application in treatment of type-2 diabetics

GLP-1 (Glucagon-like peptide-1) is a peptide that stimulates insulin secretion from the β-cell for glycemic control of the plasma blood glucose level. Its mimetic exenatide (synthetic Exendin-4) with a longer half-life of approximately 3.3–4 h is widely used in clinical application to treat diabetes. Currently, exenatide is chemically synthesized. In this study, we report that the GLP-1 analogue recombinant Exendin-4 (Exdn-4) can be produced at a high level in Nicotiana benthamiana, with an estimated yield of 50.0 µg/g fresh biomass. For high-level expression, we generated a recombinant gene, B:GB1:ddCBD1m:8xHis : Exendin-4 (BGC : Exdn-4), for the production of Exendin-4 using various domains such as the BiP signal peptide, the GB1 domain (B1 domain of streptococcal G protein), a double cellulose binding domain 1 (CBD1), and 8 His residues (8xHis) to the N-terminus of Exendin-4. GB1 was used to increase the expression, whereas double CBD1 and 8xHis were included as affinity tags for easy purification using MCC beads and Ni(2+)-NTA resin, respectively. BGC : Exdn-4 was purified by single-step purification to near homogeneity using both Ni(2+)-NTA resin and microcrystalline cellulose (MCC) beads. Moreover, Exdn-4 without any extra residues was produced from BGC : Exdn-4 bound onto MCC beads by treating with enterokinase. Plant-produced Exdn-4 (Exendin-4) was as effective as chemically synthesized Exendin-4 in glucose-induced insulin secretion (GIIS) from mouse MIN6m9 cells a pancreatic beta cell line

Comparative transcriptomics reveals the role of altered energy metabolism in the establishment of single-cell C4 photosynthesis in Bienertia sinuspersici

Single-cell C4 photosynthesis (SCC4) in terrestrial plants without Kranz anatomy involves three steps: initial CO2 fixation in the cytosol, CO2 release in mitochondria, and a second CO2 fixation in central chloroplasts. Here, we investigated how the large number of mechanisms underlying these processes, which occur in three different compartments, are orchestrated in a coordinated manner to establish the C4 pathway in Bienertia sinuspersici, a SCC4 plant. Leaves were subjected to transcriptome analysis at three different developmental stages. Functional enrichment analysis revealed that SCC4 cycle genes are coexpressed with genes regulating cyclic electron flow and amino/organic acid metabolism, two key processes required for the production of energy molecules in C3 plants. Comparative gene expression profiling of B. sinuspersici and three other species (Suaeda aralocaspica, Amaranthus hypochondriacus, and Arabidopsis thaliana) showed that the direction of metabolic flux was determined via an alteration in energy supply in peripheral chloroplasts and mitochondria via regulation of gene expression in the direction of the C4 cycle. Based on these results, we propose that the redox homeostasis of energy molecules via energy metabolism regulation is key to the establishment of the SCC4 pathway in B. sinuspersici

Presence of a bi-directional S phase-specific transcription regulatory element in the promoter shared by testis-specific TH2A and TH2B histone genes

, 19, pp. 93–98 (1991) EMBL accession nos X59961–X5996