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.actin filament ͉ chloroplast photorelocation ͉ chloroplast unusual positioning1 (CHUP1) ͉ organelle movement ͉ phototropin

Salvia leucophylla, a shrub observed in coastal south California, produces several volatile monoterpenoids (camphor, 1,8-cineole, beta-pinene, alpha-pinene, and camphene) that potentially act as allelochemicals. The effects of these were examined using Brassica campestris as the test plant. Camphor, 1,8-cineole, and beta-pinene inhibited germination of B. campestris seeds at high concentrations, whereas alpha-pinene and camphene did not. Root growth was inhibited by all five monoterpenoids in a dose-dependent manner, but hypocotyl growth was largely unaffected. The monoterpenoids did not alter the sizes of matured cells in either hypocotyls or roots, indicating that cell expansion is relatively insensitive to these compounds. They did not decrease the mitotic index in the shoot apical region, but specifically lowered mitotic index in the root apical meristem. Moreover, morphological and biochemical analyses on the incorporation of 5-bromo-2'-deoxyuridine into DNA demonstrated that the monoterpenoids inhibit both cell-nuclear and organelle DNA synthesis in the root apical meristem. These results suggest that the monoterpenoids produced by S. leucophylla could interfere with the growth of other plants in its vicinity through inhibition of cell proliferation in the root apical meristem.

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), ZIG͞AtVTI11, 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 compartment͞vacuole in the endodermal cells, mediated by a specific SNARE complex containing AtVAM3 and AtVTI11, plays an important role in shoot gravitropism.

SummaryThe plant vacuole is a multifunctional organelle which is essential for growth and development. To visualize the dynamics of plant vacuolar membranes, g-TIP (tonoplast intrinsic protein) was fused to GFP and expressed in Arabidopsis thaliana. The marker molecule was targeted to the vacuolar membranes in most tissues, as expected. In rapidly expanding cells, some additional spherical structures were often observed within the lumen of vacuoles, which emitted strong¯uorescence. To con®rm their normal presence, we examined wild-type Arabidopsis cotyledons by transmission electron microscopy. The metal-contact rapid-freezing method revealed that the vacuolar lumen of epidermal cells contained many cytoplasmic projections, which often formed spherical structures (1±3 mm diameter) consisting of double membranes. Thus we concluded that these structures are authentic and named them`bulbs'. Threedimensional reconstruction from serial electron microscopic images demonstrates that bulbs are very intricately folded, but are continuous with the limiting vacuolar membrane. The¯uorescence intensity of bulbs is about threefold higher than that of vacuolar membrane. GFP-AtRab75c, another marker of the vacuole, did not give¯uorescent signals of bulbs in transgenic plants, but the existence of bulbs was still con®rmed by electron microscopy. These results suggest that bulbs de®ne a subregion in the continuous vacuolar membrane, where some proteins are concentrated and others segregated.

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.

We developed an adequate method for the in vivo analysis of organelle dynamics in the gravity-perceptive cell (endodermis) of the Arabidopsis thaliana inflorescence stem, revealing behavior of amyloplasts and vacuolar membranes in those cells. Amyloplasts in the endodermis showed saltatory movements even before gravistimulation by reorientation, and these movements were confirmed as microfilament dependent. From our quantitative analysis in the wild type, the gravityoriented movement of amyloplasts mainly occurred during 0 to 3 min after gravistimulation by reorientation, supporting findings from our previous physiological study. Even after microfilament disruption, the gravity-oriented movement of amyloplasts remained. By contrast, in zig/sgr4 mutants, where a SNARE molecule functioning in vacuole biogenesis has been disrupted, the movement of amyloplasts in the endodermis is severely restricted both before and after gravistimulation by reorientation. Here, we describe vacuolar membrane behavior in these cells in the wild-type, actin filament-disrupted, and zig/sgr4 mutants and discuss its putatively important features for the perception of gravity. We also discuss the data on the two kinds of movements of amyloplasts that may play an important role in gravitropism: (1) the leading edge amyloplasts and (2) the en mass movement of amyloplasts.

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. INTRODUCTIONGravitropism is an important environmental response in plants. In higher plants, shoots and roots show negative and positive gravitropism, respectively. The gravitropic response is composed of four sequential steps: gravity sensing, signal formation, signal transduction, and differential growth of the upper and lower tissues of the responding organ (Tasaka et al., 1999(Tasaka et al., , 2001. The perception of gravity and the conversion of the physical stimulus (i.e., the gravity vector) to chemical signals in the cell are particularly interesting, but they are the least characterized events in gravitropism. Physiological and cytological analyses with various plants have shown that amyloplasts, which are plastids containing starch granules, sediment in the direction of gravity and are able to move downward in response to the change of gravity stimulation (Konings, 1995;Blancaflor et al., 1998). Thus, amyloplasts are believed to act as statoliths for gravity. The columella cells of the root cap and the endodermal cells or starch sheath of the shoot, which contain sedimented amyloplasts, are thought to act as gravity-sensing cells (Sack, 1991(Sack, , 1997.In the shoot, the statolith hypothesis is supported by genetic studies. Some of the genes involved in the development or differentiation of gravity-sensing cells or amyloplasts have been cloned. In the Arabidopsis shoot, the epidermis, the cortex, the endodermis, and the stele containing vascular tissues are arranged in a radially symmetrical manner progressing from the outside to the inside. The sgr1 ( shoot gravitropism 1 ) and sgr7 mutants, whose inflorescence stems and hypocotyls both lack gravitropic ability, also lack the normal endodermal cell layer (Fukaki et al., 1996(Fukaki et al., , 1998. These two mutants are allelic to scr ( scarecrow ) and shr ( short-root ), respectively, both of which were isolated as radial pattern formation mutants in the root and enco...

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 | membrane traffic | membrane fusion I n eukaryotic cells, membrane fusion is an essential process in protein secretion and endocytosis (1, 2). Selective membrane fusion occurs with the concerted functions of several molecules, including SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), Rab GTPases, tethering factors, and Sec1p/Munc18 (SM) proteins. SNAREs are membrane-anchored proteins that contain α-helical heptad repeats and a characteristic central amino acid within the SNARE motif. The SNARE complex comprises four SNAREs, including three with a central glutamine residue in SNARE motif (Q-SNAREs: Qa, Qb, and Qc) and one with a central arginine (R-SNARE) in SNARE motif. The Q-SNAREs reside on the target membrane, and the R-SNARE resides on the transport vesicle. This complex first forms a bridge between the target organelle membrane and the vesicle, and then compresses to bring the two membranes close enough to mediate specific membrane fusion. After fusion is complete, the SNARE complex is dissociated by a NSF (N-ethylmaleimide-sensitive factor), and the SNAREs are recycled. To execute the correct membrane fusion, SNAREs must be localized on the membrane of specific organelles or transport vesicles. QaSNAREs also serve as organelle markers (3-5), by virtue of their specific localization on the membrane of target organelles.The TGN was first defined as a special organelle on the transside of the Golgi stack that is responsible for protein sorting to the plasma membrane or lysosomes (6). The TGN contains multiple sorting domains and functions as the compartment of cargo sorting. In addition, the TGN ...