Chieko Saito

Involvement of the Vacuoles of the Endodermis in the Early Process of Shoot Gravitropism in Arabidopsis

The endodermal cells of the shoot are thought to be the gravity-sensing cells in Arabidopsis. The amyloplasts in the endodermis that sediment in the direction of gravity may act as statoliths. Endodermis-specific expression of SGR2 and ZIG using the SCR promoter could complement the abnormal shoot gravitropism of the sgr2 and zig mutants, respectively. The abnormalities in amyloplast sedimentation observed in both mutants recovered simultaneously. These results indicate that both genes in the endodermal cell layer are crucial for shoot gravitropism. ZIG encodes AtVTI11, which is a SNARE involved in vesicle transport to the vacuole. The fusion protein of SGR2 and green fluorescent protein localized to the vacuole and small organelles. These observations indicate that ZIG and SGR2 are involved in the formation and function of the vacuole, a notion supported by the results of subcellular analysis of the sgr2 and zig mutants with electron microscopy. These results strongly suggest that the vacuole participates in the early events of gravitropism and that SGR2 and ZIG functions are involved.

A SNARE complex containing SGR3/AtVAM3 and ZIG/VTI11 in gravity-sensing cells is important for Arabidopsis shoot gravitropism

Plants can sense the direction of gravity and change the growth orientation of their organs. The molecular mechanisms of gravity sensing and signal transduction during gravitropism are not well known. We have isolated several shoot gravitropism (sgr) mutants of Arabidopsis. The sgr3-1 mutant exhibits a reduced gravitropic response in the inflorescence stems. In the inflorescence stems of Arabidopsis, gravity is sensed in endodermal cells that contain sedimentable amyloplasts. In sgr3-1, some amyloplasts in the endodermis failed to sediment in the direction of gravity. SGR3 encodes a syntaxin, AtVAM3, which had previously been cloned as a homologue of yeast Vam3p. AtVAM3 is localized to the prevacuolar compartment and vacuole and is suggested to function in vesicle transport to the vacuole. We have also cloned another soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), ZIG/AtVTI11, a mutation that causes abnormal gravitropism. This mutant displayed an abnormal distribution of amyloplasts in the endodermal cells similar to that in sgr3-1. Endodermis-specific expression of SGR3 and ZIG by using the SCR promoter could complement the abnormal shoot gravitropism of each mutant. Protein–protein interaction between AtVAM3 and AtVTI11 in the endodermal cells was detected immunologically. The sgr3-1 mutation appeared to reduce the affinity of AtVAM3 for AtVTI11 or SYP5. These results suggest that vesicle transport to the prevacuolar compartment/vacuole in the endodermal cells, mediated by a specific SNARE complex containing AtVAM3 and AtVTI11, plays an important role in shoot gravitropism.

Short actin-based mechanism for light-directed chloroplast movement in Arabidopsis

Organelle movement is essential for proper function of living cells. In plants, these movements generally depend on actin filaments, but the underlying mechanism is unknown. Here, in Arabidopsis, we identify associations of short actin filaments along the chloroplast periphery on the plasma membrane side associated with chloroplast photorelocation and anchoring to the plasma membrane. We have termed these chloroplast-actin filaments (cp-actin filaments). Cp-actin filaments emerge from the chloroplast edge and exhibit rapid turnover. The presence of cp-actin filaments depends on an actin-binding protein, chloroplast unusual positioning1 (CHUP1), localized on the chloroplast envelope. chup1 mutant lacked cp-actin filaments but showed normal cytoplasmic actin filaments. When irradiated with blue light to induce chloroplast movement, cp-actin filaments relocalize to the leading edge of chloroplasts before and during photorelocation and are regulated by 2 phototropins, phot1 and phot2. Our findings suggest that plants evolved a unique actin-based mechanism for organelle movement.

Live-Cell Imaging Reveals the Dynamics of Two Sperm Cells during Double Fertilization in Arabidopsis thaliana

Flowering plants have evolved a unique reproductive process called double fertilization, whereby two dimorphic female gametes are fertilized by two immotile sperm cells conveyed by the pollen tube. The two sperm cells are arranged in tandem with a leading pollen tube nucleus to form the male germ unit and are placed under the same genetic controls. Genes controlling double fertilization have been identified, but whether each sperm cell is able to fertilize either female gamete is still unclear. The dynamics of individual sperm cells after their release in the female tissue remain largely unknown. In this study, we photolabeled individual isomorphic sperm cells before their release and analyzed their fate during double fertilization in Arabidopsis thaliana. We found that sperm delivery was composed of three steps. Sperm cells were projected together to the boundary between the two female gametes. After a long period of immobility, each sperm cell fused with either female gamete in no particular order, and no preference was observed for either female gamete. Our results suggest that the two sperm cells at the front and back of the male germ unit are functionally equivalent and suggest unexpected cell-cell communications required for sperm cells to coordinate double fertilization of the two female gametes.

Application of Lifeact Reveals F-Actin Dynamics in Arabidopsis thaliana and the Liverwort, Marchantia polymorpha

Actin plays fundamental roles in a wide array of plant functions, including cell division, cytoplasmic streaming, cell morphogenesis and organelle motility. Imaging the actin cytoskeleton in living cells is a powerful methodology for studying these important phenomena. Several useful probes for live imaging of filamentous actin (F-actin) have been developed, but new versatile probes are still needed. Here, we report the application of a new probe called Lifeact for visualizing F-actin in plant cells. Lifeact is a short peptide comprising 17 amino acids that was derived from yeast Abp140p. We used a Lifeact–Venus fusion protein for staining F-actin in Arabidopsis thaliana and were able to observe dynamic rearrangements of the actin meshwork in root hair cells. We also used Lifeact–Venus to visualize the actin cytoskeleton in the liverwort Marchantia polymorpha; this revealed unique and dynamic F-actin motility in liverwort cells. Our results suggest that Lifeact could be a useful tool for studying the actin cytoskeleton in a wide range of plant lineages.

Qa-SNAREs localized to the trans-Golgi network regulate multiple transport pathways and extracellular disease resistance in plants

In all eukaryotic cells, a membrane-trafficking system connects the post-Golgi organelles, such as the trans-Golgi network (TGN), endosomes, vacuoles, and the plasma membrane. This complex network plays critical roles in several higher-order functions in multicellular organisms. The TGN, one of the important organelles for protein transport in the post-Golgi network, functions as a sorting station, where cargo proteins are directed to the appropriate post-Golgi compartments. Unlike its roles in animal and yeast cells, the TGN has also been reported to function like early endosomal compartments in plant cells. However, the physiological roles of the TGN functions in plants are not understood. Here, we report a study of the SYP4 group (SYP41, SYP42, and SYP43), which represents the plant orthologs of the Tlg2/syntaxin16 Qa-SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) that localizes on the TGN in yeast and animal cells. The SYP4 group regulates the secretory and vacuolar transport pathways in the post-Golgi network and maintains the morphology of the Golgi apparatus and TGN. Consistent with a secretory role, SYP4 proteins are required for extracellular resistance responses to a fungal pathogen. We also reveal a plant cell-specific higher-order role of the SYP4 group in the protection of chloroplasts from salicylic acid-dependent biotic stress.

Arabidopsis dynamin-related proteins DRP3A and DRP3B are functionally redundant in mitochondrial fission, but have distinct roles in peroxisomal fission

Two similar Arabidopsis dynamin-related proteins, DRP3A and DRP3B, are thought to be key factors in both mitochondrial and peroxisomal fission. However, the functional and genetic relationships between DRP3A and DRP3B have not been fully investigated. In a yeast two-hybrid assay, DRP3A and DRP3B interacted with themselves and with each other. DRP3A and DRP3B localized to mitochondria and peroxisomes, and co-localized with each other in leaf epidermal cells. In two T-DNA insertion mutants, drp3a and drp3b, the mitochondria are a little longer and fewer in number than those in the wild-type cells. In the double mutant, drp3a/drp3b, mitochondria are connected to each other, resulting in massive elongation. Overexpression of either DRP3A or DRP3B in drp3a/drp3b restored the particle shape of mitochondria, suggesting that DRP3A and DRP3B are functionally redundant in mitochondrial fission. In the case of peroxisomal fission, DRP3A and DRP3B appear to have different functions: peroxisomes in drp3a were larger and fewer in number than those in the wild type, whereas peroxisomes in drp3b were as large and as numerous as those in the wild type, and peroxisomes in drp3a/drp3b were as large and as numerous as those in drp3a. Although overexpression of DRP3A in drp3a/drp3b restored the shape and number of peroxisomes, overexpression of DRP3B did not restore the phenotypes, and often caused elongation instead. These results suggest that DRP3B and DRP3A have redundant molecular functions in mitochondrial fission, whereas DRP3B has a minor role in peroxisomal fission that is distinct from that of DRP3A.

Chapter 4: functions of RAB and SNARE proteins in plant life.

In all eukaryotic cells, vesicular trafficking is crucial for maintaining cellular and organelle functions. RABs and SNAREs play key roles in vesicle/organelle identity and exchange. Budding yeast genetics and mammalian cell biochemistry were the most effective approaches for investigating molecular mechanisms underlying vesicular trafficking and remain important in exploring new horizons. The field of vesicular trafficking attracted plant biologists in the early 1990s. Today, this field continues to stimulate a wide range of research. This review starts with some history of RAB and SNARE research in yeast and mammals and introduces a widely accepted general model. Then we summarize recent reports regarding plant RABs and SNAREs, focusing on functional diversity. Finally, we discuss how plants assign new roles to conserved vesicular trafficking proteins to perform divergent, higher order plant functions. Plants have apparently evolved a unique set of plant-specific RAB and SNARE molecules that play significant roles in plant life.

Vacuolar/pre-vacuolar compartment Qa-SNAREs VAM3/SYP22 and PEP12/SYP21 have interchangeable functions in Arabidopsis

SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) mediate specific membrane fusion between transport vesicles or organelles and target membranes. VAM3/SYP22 and PEP12/SYP21 are Qa-SNAREs that act in the vacuolar transport pathway of Arabidopsis thaliana, and are localized predominantly on the vacuolar membrane and the pre-vacuolar compartment (PVC), respectively. Previous studies have shown that loss-of-function mutants of VAM3/SYP22 or PEP12/SYP21 showed male gametophytic lethality, suggesting that VAM3/SYP22 and PEP12/SYP21 possess different, non-redundant functions. We have re-evaluated the effects of mutations in these genes using T-DNA insertion mutants in the Columbia accession. We found that a mutation in VAM3/SYP22 (vam3-1) caused pleiotropic abnormalities, including semi-dwarfism and wavy leaves. In contrast, a loss-of-function mutant of PEP12/SYP21 (pep12) showed no apparent abnormal phenotype. We also found that the double vam3-1 pep12 mutant had severely reduced fertilization competence, although male and female gametophytes (vam3-1(-) pep12(-) ) maintained the ability to fertilize. Moreover, promoter swapping analysis revealed that expression of a GFP-PEP12/SYP21 fusion under the control of the VAM3/SYP22 promoter suppressed all phenotypes of the vam3-1 mutant. These results indicate that the functions of VAM3/SYP22 and PEP12/SYP21 were redundant and interchangeable.

High-curvature domains of the ER are important for the organization of ER exit sites in Saccharomyces cerevisiae.

Summary Protein export from the endoplasmic reticulum (ER) to the Golgi apparatus occurs at specialized regions known as the ER exit sites (ERES). In Saccharomyces cerevisiae, ERES appear as numerous scattered puncta throughout the ER. We examined ERES within the peripheral ER, finding that the proteins comprising the ERES localize on high-curvature ER domains where curvature-stabilizing protein Rtn1 is present. &Dgr;rtn1 &Dgr;rtn2 &Dgr;yop1 cells have fewer high-curvature ER domains, but ERES accumulate at the remaining high-curvature ER domains on the edge of expanded ER sheets. We propose that membrane curvature is a key geometric feature for the regulation of ERES localization. We also investigated a spatial relationship between ERES and Golgi cisternae. Golgi cisternae in S. cerevisiae are unstacked, dispersed, and moving in the cytoplasm with cis-cisternae positioned adjacent to ERES, whereas trans-cisternae are not. Morphological changes in the ER of &Dgr;rtn1 &Dgr;rtn2 &Dgr;yop1 cells resulted in aberrant Golgi structures, including cis- and trans-markers, and there was reduced movement at ERES between expanded ER sheets and the plasma membrane.