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Young seedlings use nutrients stored in the seeds to grow and acquire photosynthetic potential. This process, called seedling establishment, involves a developmental phase transition from heterotrophic to autotrophic growth. Some membrane-trafficking mutants of Arabidopsis (Arabidopsis thaliana), such as the katamari2 (kam2) mutant, exhibit growth arrest during seedling development, with a portion of individuals failing to develop true leaves on sucrose-free solid medium. However, the reason for this seedling arrest is unclear. In this study, we show that seedling arrest is a temporal growth arrest response that occurs not only in kam2 but also in wild-type (WT) Arabidopsis; however, the threshold for this response is lower in kam2 than in the WT. A subset of the arrested kam2 seedlings resumed growth after transfer to fresh sucrose-free medium. Growth arrest in kam2 on sucrose-free medium was restored by increasing the gel concentration of the medium or covering the surface of the medium with a perforated plastic sheet. WT Arabidopsis seedlings were also arrested when the gel concentration of sucrose-free medium was reduced. RNA sequencing revealed that transcriptomic changes associated with the rate of seedling establishment were observed as early as 4 d after sowing. Our results suggest that the growth arrest of both kam2 and WT seedlings is an adaptive stress response and is not simply caused by the lack of a carbon source in the medium. This study provides a new perspective on an environmental stress response under unfavorable conditions during the phase transition from heterotrophic to autotrophic growth in Arabidopsis. [ABSTRACT FROM AUTHOR]

The adaptor protein (AP) complexes play crucial roles in vesicle formation in post-Golgi trafficking. Land plants have five types of AP complexes (AP-1 to AP-5), each of which consists of two large subunits, one medium subunit and one small subunit. Here, we show that the Arabidopsis AP-1 complex mediates the polarized secretion and accumulation of a pectic polysaccharide called mucilage in seed coat cells. Previously, a loss-of-function mutant of AP1M2, the medium subunit of AP-1, has been shown to display deleterious growth defects because of defective cytokinesis. To investigate the function of AP-1 in interphase, we generated transgenic Arabidopsis plants expressing AP1M2-GFP (green fluorescent protein) under the control of the cytokinesis-specific KNOLLE (KN) promoter in the ap1m2 background. These transgenic plants, designated pKN lines, successfully rescued the cytokinesis defect and dwarf phenotype of ap1m2. pKN lines showed reduced mucilage extrusion from the seed coat. Furthermore, abnormal accumulation of mucilage was found in the vacuoles of the outermost integument cells of pKN lines. During seed development, the accumulation of AP1M2–GFP was greatly reduced in the integument cells of pKN lines. These results suggest that trans -Golgi network (TGN)-localized AP-1 is involved in the trafficking of mucilage components to the outer surface of seed coat cells. Our study highlights an evolutionarily conserved function of AP-1 in polarized sorting in eukaryotic cells. [ABSTRACT FROM AUTHOR]

The trans-Golgi network (TGN) is a tubular–vesicular organelle that matures from the trans cisternae of the Golgi apparatus. In plants, the TGN functions as a central hub for three trafficking pathways: the secretory pathway, the vacuolar trafficking pathway and the endocytic pathway. Here, we describe a novel TGN-localized membrane protein, CONTINUOUS VASCULAR RING (COV1), that is crucial for TGN function in Arabidopsis. The COV1 gene was originally identified from the stem vascular patterning mutant of Arabidopsis thaliana. However, the molecular function of COV1 was not identified. Fluorescently tagged COV1 proteins co-localized with the TGN marker proteins, SYNTAXIN OF PLANTS 4 (SYP4) and vacuolar-type H+-ATPase subunit a1 (VHA-a1). Consistently, COV1-localized compartments were sensitive to concanamycin A, a specific inhibitor of VHA. Intriguingly, cov1 mutants exhibited abnormal Golgi morphologies, including a reduction in the number of Golgi cisternae and a reduced association between the TGN and the Golgi apparatus. A deficiency in COV1 also resulted in a defect in vacuolar protein sorting, which was characterized by the abnormal accumulation of storage protein precursors in seeds. Moreover, we found that the development of an idioblast, the myrosin cell, was abnormally increased in cov1 leaves. Our results demonstrate that the novel TGN-localized protein COV1 is required for Golgi morphology, vacuolar trafficking and myrosin cell development. [ABSTRACT FROM AUTHOR]

Targeted genome modification technologies are key tools for functional genomics. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 system (CRISPR/Cas9) is an emerging technology for targeted genome modification. The CRISPR/Cas9 system consists of a short guide RNA (gRNA), which specifies the target genome sequence, and the Cas9 protein, which has endonuclease activity. The CRISPR/Cas9 system has been applied to model animals and flowering plants, including rice, sorghum, wheat, tobacco and Arabidopsis. Here, we report the application of CRISPR/Cas9 to targeted mutagenesis in the liverwort Marchantia polymorpha L., which has emerged as a model species for studying land plant evolution. The U6 promoter of M. polymorpha was identified and cloned to express the gRNA. The target sequence of the gRNA was designed to disrupt the gene encoding auxin response factor 1 (ARF1) in M. polymorpha. Using Agrobacterium-mediated transformation, we isolated stable mutants in the gametophyte generation of M. polymorpha. CRISPR/Cas9-based site-directed mutagenesis in vivo was achieved using either the Cauliflower mosaic virus 35S or M. polymorpha EF1α promoter to express Cas9. Isolated mutant individuals showing an auxin-resistant phenotype were not chimeric. Moreover, stable mutants were produced by asexual reproduction of T1 plants. Multiple arf1 alleles were easily established using CRIPSR/Cas9-based targeted mutagenesis. Our results provide a rapid and simple approach for molecular genetics in M. polymorpha, and raise the possibility that CRISPR/Cas9 may be applied to a wide variety of plant species. [ABSTRACT FROM AUTHOR]

Formation of clathrin-coated vesicles (CCVs) requires the scaffolding adaptor protein (AP) complexes, which are conserved across all eukaryotes. The Arabidopsis genome encodes five AP complexes (AP-1 to AP-5), and each complex consists of four subunits. In this study, we characterized the poorly defined AP-1 complex by using genetics, proteomics and live cell imaging. We showed that the AP-1 µ adaptin subunit (AP1M2) was localized to the trans-Golgi network (TGN) and interacted physically with the AP-1 subunits in Arabidopsis. During treatment with brefeldin A (BFA), the functional fluorophore-tagged AP1M2 relocated to the BFA compartment. The AP1M2 loss-of-function mutant ap1m2 displayed deleterious growth defects, which were particularly evident in the compromised cytokinesis that was revealed by the presence of cell wall stubs in multinucleate cells. Immunolocalization of the cytokinesis-specific syntaxin KNOLLE (KN) in ap1m2 showed that KN was mislocalized and aggregated around the division plane, while a secretory marker targeting to the cell plate remained unaffected. Taken together, we propose that the AP-1 complex is required for cell plate-targeted trafficking of KN in dividing plant cells, and that it has a common role in mediating plant and yeast/animal cytokinesis systems which are fundamentally different. [ABSTRACT FROM AUTHOR]

Seed storage proteins are synthesized on rough endoplasmic reticulum (ER) in a precursor form and then are transported to protein storage vacuoles (PSVs) where they are converted to their mature form. To understand the mechanisms by which storage proteins are transported, we screened Arabidopsis maigo mutants to identify those that abnormally accumulate storage protein precursors. Here we describe a new maigo mutant, maigo 4 (mag4), that abnormally accumulates the precursors of two major storage proteins, 12S globulin and 2S albumin, in dry seeds. Electron microscopy revealed that mag4 seed cells abnormally develop a large number of novel structures that exhibit a highly electron-dense core. Some of these structures were surrounded by ribosomes. Immunogold analysis suggests that the electron-dense core is an aggregate of 2S albumin precursors and that 12S globulins are localized around the core. The MAG4 gene was identified as At3g27530, and the MAG4 protein has domains homologous to those found in bovine vesicular transport factor p115. MAG4 molecules were concentrated at cis-Golgi stacks. Our findings suggest that MAG4 functions in the transport of storage protein precursors from the ER to the Golgi complex in plants. In addition, the mag4 mutant exhibits a dwarf phenotype, suggesting that MAG4 is involved in both the transport of storage proteins and in plant growth and development. [ABSTRACT FROM PUBLISHER]

Cutinase is an esterase that degrades the polyester cutin, a major component of the plant cuticle. Although cutinase activity has been detected in pollen, the genes encoding this enzyme have not been identified. Here, we report the identification and characterization of Arabidopsis CDEF1 (cuticle destructing factor 1), a novel candidate gene encoding cutinase. CDEF1 encodes a member of the GDSL lipase/esterase family of proteins, although fungal and bacterial cutinases belong to the α/β hydrolase superfamily which is different from the GDSL lipase/esterase family. According to the AtGenExpress microarray data, CDEF1 is predominantly expressed in pollen. The ectopic expression of CDEF1 driven by the 35S promoter caused fusion of organs, including leaves, stems and flowers, and increased surface permeability. Ultrastructural analysis revealed that the cuticle of the transgenic plants was often disrupted and became discontinuous. Subcellular analysis with green fluorescent protein (GFP)-tagged CDEF1 showed that the protein is secreted to the extracellular space in leaves. The recombinant CDEF1 protein has esterase activity. These results are consistent with cutinase being secreted from cells and directly degrading the polyester in the cuticle. CDEF1 promoter activity was detected in mature pollen and pollen tubes, suggesting that CDEF1 is involved in the penetration of the stigma by pollen tubes. Additionally, we found CDEF1 expression at the zone of lateral root emergence, which suggests that CDEF1 degrades cell wall components to facilitate the emergence of the lateral roots. Our findings suggest that CDEF1 is a candidate gene for the unidentified plant cutinase. [ABSTRACT FROM PUBLISHER]

Seed storage proteins are synthesized on rough endoplasmic reticulum (ER) as larger precursors and are sorted to protein storage vacuoles, where they are converted into the mature forms. We report here an Arabidopsis mutant, maigo 1 (mag1), which abnormally accumulates the precursors of two major storage proteins, 12S globulin and 2S albumin, in dry seeds. Electron microscopy revealed that mag1 seeds mis-sort storage proteins by secreting them from cells. mag1 seeds have smaller protein storage vacuoles in the seeds than do wild-type seeds. The MAG1 gene encodes a homolog of the yeast (Saccharomyces cerevisiae) protein VPS29. VPS29 is a component of a retromer complex for recycling a vacuolar sorting receptor VPS10 from the pre-vacuolar compartment to the Golgi complex. Our findings suggest that MAG1/AtVPS29 protein is involved in recycling a plant receptor for the efficient sorting of seed storage proteins. The mag1 mutant exhibits a dwarf phenotype. A plant retromer complex plays a significant role in plant growth and development. [ABSTRACT FROM AUTHOR]

Myrosin cells in Capparales plants are idioblasts that accumulate thioglucoside glucohydrolase (TGG, also called myrosinase), which hydrolyzes glucosinolates to produce toxic compounds for repelling pests. Here, we show that AtVAM3 is involved in development of myrosin cells. It has been shown that yeast VAM3 is a Qa-SNARE that is involved in vesicle transport of vacuolar proteins and vacuolar assembly. We found that two Arabidopsis atvam3 alleles, atvam3-3 and atvam3-4/ssm, accumulate large amounts of TGG1 and TGG2 that are enzymatically active. An immunogold analysis revealed that TGGs were specifically localized in the vacuole of myrosin cells in atvam3 mutants. This result indicates that TGGs are normally transported to vacuoles in these mutants and that AtVAM3 is not essential for vacuolar transport of the proteins. We developed a staining method with Coomassie brilliant blue that detects myrosin cells in whole leaves by their high TGG content. This method showed that atvam3 leaves have a larger number of myrosin cells than do wild-type leaves. Myrosin cells were scattered along leaf veins in wild-type leaves, while they were abnormally distributed in atvam3 leaves. The mutants developed a network of myrosin cells throughout the leaves: myrosin cells were not only distributed continuously along leaf veins, but were also observed independent of leaf veins. The excess of myrosin cells in atvam3 mutants might be responsible for the abnormal abundance of TGGs and the reduction of elongation of inflorescence stems and leaves in these mutants. Our results suggest that AtVAM3 has a plant-specific function in development of myrosin cells. [ABSTRACT FROM AUTHOR]

The short stem and midrib (ssm) mutants of Arabidopsis thaliana show both semi-dwarf and wavy leaf phenotypes due to defects in the elongation of the stem internodes and leaves. Moreover, these abnormalities cannot be recovered by exogenous phytohormones. ssm was originally identified as a single recessive mutant of the ecotype Columbia (Col-0), but genetic crossing experiments have revealed that this mutant phenotype is restored by another gene that is functional in the ecotype Landsberg erecta (Ler) and not in Col-0. Map-based cloning of the gene that is defective in ssm mutants has uncovered a small deletion in the sixth intron of a gene encoding a syntaxin, VAM3/SYP22, which has been implicated in vesicle transport to the vacuole. This mutation appears to cause a peptide insertion in the deduced VAM3/SYP22 polypeptide sequence due to defective splicing of the shortened sixth intron. Significantly, when compared with the wild-type Ler genome, the wild-type Col-0 genome has a single base pair deletion causing a frameshift mutation in SYP23, a gene with the highest known homology to VAM3/SYP22. These findings suggest that VAM3/SYP22 and SYP23 have overlapping functions and that the vesicle transport mediated by these syntaxins is important for shoot morphogenesis. [ABSTRACT FROM AUTHOR]

A novel vesicle, referred to as a precursor-accumulating (PAC) vesicle, mediates the transport of storage protein precursors to protein storage vacuoles in maturing pumpkin seeds. PV72, a type I integral membrane protein with three repeats of epidermal growth factor, was found on the membrane of the PAC vesicles. PV72 had an ability to bind to pro2S albumin, a storage protein precursor, in a Ca2+-dependent manner, via the C-terminal region of pro2S albumin, which was found to function as a vacuolar targeting signal. This implies that PV72 is a vacuolar sorting receptor of the storage protein. PV72 was specifically and transiently accumulated at the middle stage of seed maturation in association with the synthesis of storage proteins. Subcellular fractionation showed that PV72 was also accumulated in the microsomal fraction. A fusion protein consisting of GFP and the transmembrane domain and the cytosolic tail of PV72 was localized in Golgi complex. PV72 in the isolated PAC vesicles had a complex type of oligosaccharide, indicating that PV72 passed though the Golgi complex. These results suggest that PV72 is recycled between PAC vesicles and Golgi complex/post-Golgi compartments. PV72 appears to be responsible for recruiting pro2S albumin molecules from the Golgi complex to the PAC vesicles. [ABSTRACT FROM PUBLISHER]

Plants degrade cellular materials during senescence and under various stresses. We report that the precursors of two stress-inducible cysteine proteinases, RD21 and a vacuolar processing enzyme (VPE), are specifically accumulated in ~0.5 µm diameter × ~5 µm long bodies in Arabidopsis thaliana. Such bodies have previously been observed in Arabidopsis but their function was not known. They are surrounded with ribosomes and thus are assumed to be directly derived from the endoplasmic reticulum (ER). Therefore, we propose to call them the ER bodies. The ER bodies are observed specifically in the epidermal cells of healthy seedlings. These cells are easily wounded and stressed by the external environment. When the seedlings are stressed with a concentrated salt solution, leading to death of the epidermal cells, the ER bodies start to fuse with each other and with the vacuoles, thereby mediating the delivery of the precursors directly to the vacuoles. This regulated, direct pathway differs from the usual case in which proteinases are transported constitutively from the ER to the Golgi complex and then to vacuoles, with intervention of vesicle-transport machinery, such as a vacuolar-sorting receptor or a syntaxin of the SNARE family. Thus, the ER bodies appear to be a novel proteinase-storing system that assists in cell death under stressed conditions. [ABSTRACT FROM PUBLISHER]

We have shown the localization and mobilization of modified green fluorescent proteins (GFPs) with various signals in different compartments in a vacuolar-sorting system of tobacco BY-2 cells. In contrast to the efficient secretion of GFP from the transformed cells expressing SP-GFP composed of a signal peptide and GFP, accumulation of GFP in the vacuoles was observed in the cells expressing SP-GFP fused with the C-terminal peptide of pumpkin 2S albumin. This indicated that this peptide is sufficient for vacuolar targeting. Interestingly, the fluorescence in the vacuoles disappeared sharply at 7 d after inoculation of the cells, but it appeared again after re-inoculation into a new culture medium. When SP-GFP was fused with the region, termed PV72C, including a transmembrane domain and a cytosolic tail of a vacuolar-sorting receptor PV72, GFP-PV72C was detected in the Golgi-complex-like small particles. Prolonged culture showed that GFP-PV72C that reached the prevacuolar compartments was cleaved off the PV72C region to produce GFP, that arrived at the vacuoles to be diffused. These findings suggested that the vacuolar-sorting receptor might be recycled between the Golgi complex and prevacuolar compartments. [ABSTRACT FROM PUBLISHER]

Precursor-accumulating (PAC) vesicles were previously shown to mediate the transport of the precursor of a major storage protein (pro2S albumin) to protein-storage vacu-oles in developing pumpkin cotyledons. In this study, we characterized two homologous proteins from PAC vesicles, a 72 kDa protein (PV72) and an 82 kDa protein (PV82). PV72 and PV82 showed an ability to bind to peptides derived from both an internal propeptide and a C-terminal peptide of pro2S albumin. PV72 was predicted to be a type I integral membrane protein with epidermal growth factor (EGF)-like motifs. These results suggest that PV72 and PV82 are potential sorting receptors for 2S albumin to protein-storage vacuoles. [ABSTRACT FROM AUTHOR]

The binding protein (BiP) has been implicated in cotranslational folding of nascent polypeptides, and in the recognition and disposal of aberrant polypeptides. To elucidate the involvement of BiP in the biosynthesis of vacuolar proteins, we have characterized the protein in pumpkin cotyledons during seed maturation and seedling growth. Isolated microsomes from maturing pumpkin cotyledons contained a significant amount of BiP, protein-disulfide isomerase and calreticulin. We have purified a 70-kDa protein; sequences of the N-terminus and internal fragments of this protein exhibited a high identity to the sequence of soybean BiP. Immunoblot analysis with specific antibodies raised against the purified BiP showed that the amount of BiP in a cotyledon increased markedly at the middle stages and then decreased. The increase was accompanied by the synthesis of storage proteins and the development of the endoplasmic reticulum in the cotyledons at the middle stage of seed maturation. Most of these storage proteins degraded dramatically between 2 and 5 days after seed germination, and the degradation was also accompanied by a rapid increase in the level of BiP. Subcellular fractionation of the 4-day-old cotyledons showed a high accumulation of BiP in the endoplasmic reticulum. It is possible that BiP might be involved in the synthesis of seed storage proteins during maturation and in the synthesis of hydrolytic enzymes responsible for the degradation of the storage proteins during seed germination. [ABSTRACT FROM AUTHOR]