An anchoring complex recruits katanin for microtubule severing at the plant cortical nucleation sites.

Microtubules are severed by katanin at distinct cellular locations to facilitate reorientation or amplification of dynamic microtubule arrays, but katanin targeting mechanisms are poorly understood. Here we show that a centrosomal microtubule-anchoring complex is used to recruit katanin in acentrosomal plant cells. The conserved protein complex of Msd1 (also known as SSX2IP) and Wdr8 is localized at microtubule nucleation sites along the microtubule lattice in interphase Arabidopsis cells. Katanin is recruited to these sites for efficient release of newly formed daughter microtubules. Our cell biological and genetic studies demonstrate that Msd1-Wdr8 acts as a specific katanin recruitment factor to cortical nucleation sites (but not to microtubule crossover sites) and stabilizes the association of daughter microtubule minus ends to their nucleation sites until they become severed by katanin. Molecular coupling of sequential anchoring and severing events by the evolutionarily conserved complex renders microtubule release under tight control of katanin activity.

Basic Proline-Rich Protein-Mediated Microtubules Are Essential for Lobe Growth and Flattened Cell Geometry.

Complex cell shapes are generated first by breaking symmetry, and subsequent polar growth. Localized bending of anticlinal walls initiates lobe formation in the epidermal pavement cells of cotyledons and leaves, but how the microtubule cytoskeleton mediates local cell growth, and how plant pavement cells benefit from adopting jigsaw puzzle-like shapes, are poorly understood. In Arabidopsis (Arabidopsis thaliana), the basic Pro-rich protein (BPP) microtubule-associated protein family comprises seven members. We analyzed lobe morphogenesis in cotyledon pavement cells of a BPP1;BPP2;BPP5 triple knockout mutant. New image analysis methods (MtCurv and BQuant) showed that anticlinal microtubule bundles were significantly reduced and cortical microtubules that fan out radially across the periclinal wall did not enrich at the convex side of developing lobes. Despite these microtubule defects, new lobes were initiated at the same frequency as in wild-type cells, but they did not expand into well-defined protrusions. Eventually, mutant cells formed nearly polygonal shapes and adopted concentric microtubule patterns. The mutant periclinal cell wall bulged outward. The radius of the calculated inscribed circle of the pavement cells, a proposed proxy for maximal stress in the cell wall, was consistently larger in the mutant cells during cotyledon development, and correlated with an increase in cell height. These bpp mutant phenotypes provide genetic and cell biological evidence that initiation and growth of lobes are distinct morphogenetic processes, and that interdigitated cell geometry effectively suppresses large outward bulging of pavement cells.

Polar vacuolar distribution is essential for accurate asymmetric division of Arabidopsis zygotes.

In most flowering plants, the asymmetric cell division of the zygote is the initial step in establishing the apical-basal axis of the mature plant. The zygote is polarized, possessing the nucleus at the apical tip and large vacuoles at the basal end. Despite their known polar localization, whether the positioning of the vacuoles and the nucleus is coordinated and what the role of the vacuole is in the asymmetric zygotic division remain elusive. In the present study, we utilized a live-cell imaging system to visualize the dynamics of vacuoles during the entire process of zygote polarization in Arabidopsis Image analysis revealed that the vacuoles formed tubular strands around the apically migrating nucleus. They gradually accumulated at the basal region and filled the space, resulting in asymmetric distribution in the mature zygote. To assess the role of vacuoles in the zygote, we screened various vacuole mutants and identified that shoot gravitropism2 (sgr2), in which the vacuolar structural change was impaired, failed to form tubular vacuoles and to polarly distribute the vacuole. In sgr2, large vacuoles occupied the apical tip and thus nuclear migration was blocked, resulting in a more symmetric zygotic division. We further observed that tubular vacuole formation and asymmetric vacuolar distribution both depended on the longitudinal array of actin filaments. Overall, our results show that vacuolar dynamics is crucial not only for the polar distribution along actin filaments but also for adequate nuclear positioning, and consequently zygote-division asymmetry.

Modification of growth anisotropy and cortical microtubule dynamics in Arabidopsis hypocotyls grown under microgravity conditions in space.

We carried out a space experiment, denoted as Aniso Tubule, to examine the effects of microgravity on the growth anisotropy and cortical microtubule dynamics in Arabidopsis hypocotyls, using lines in which microtubules are visualized by labeling tubulin or microtubule-associated proteins (MAPs) with green fluorescent protein (GFP). In all lines, GFP-tubulin6 (TUB6)-, basic proline-rich protein1 (BPP1)-GFP- and spira1-like3 (SP1L3)-GFP-expressing using a constitutive promoter, and spiral2 (SPR2)-GFP- and GFP-65 kDa MAP-1 (MAP65-1)-expressing using a native promoter, the length of hypocotyls grown under microgravity conditions in space was longer than that grown at 1 g conditions on the ground. In contrast, the diameter of hypocotyls grown under microgravity conditions was smaller than that of the hypocotyls grown at 1 g. The percentage of cells with transverse microtubules was increased under microgravity conditions, irrespective of the lines. Also, the average angle of the microtubules with respect to the transverse cell axis was decreased in hypocotyls grown under microgravity conditions. When GFP fluorescence was quantified in hypocotyls of GFP-MAP65-1 and SPR2-GFP lines, microgravity increased the levels of MAP65-1, which appears to be involved in the maintenance of transverse microtubule orientation. However, the levels of SPR2 under microgravity conditions were comparable to those at 1 g. These results suggest that the microgravity-induced increase in the levels of MAP65-1 is involved in increase in the transverse microtubules, which may lead to modification of growth anisotropy, thereby developing longer and thinner hypocotyls under microgravity conditions in space.

Regulation of organ straightening and plant posture by an actin-myosin XI cytoskeleton.

Plants are able to bend nearly every organ in response to environmental stimuli such as gravity and light(1,2). After this first phase, the responses to stimuli are restrained by an independent mechanism, or even reversed, so that the organ will stop bending and attain its desired posture. This phenomenon of organ straightening has been called autotropism(3) and autostraightening(4) and modelled as proprioception(5). However, the machinery that drives organ straightening and where it occurs are mostly unknown. Here, we show that the straightening of inflorescence stems is regulated by an actin-myosin XI cytoskeleton in specialized immature fibre cells that are parallel to the stem and encircle it in a thin band. Arabidopsis mutants defective in myosin XI (specifically XIf and XIk) or ACTIN8 exhibit hyperbending of stems in response to gravity, an effect independent of the physical properties of the shoots. The actin-myosin XI cytoskeleton enables organs to attain their new position more rapidly than would an oscillating series of diminishing overshoots in environmental stimuli. We propose that the long actin filaments in elongating fibre cells act as a bending tensile sensor to perceive the organ's posture and trigger the straightening system.

A unique HEAT repeat-containing protein SHOOT GRAVITROPISM6 is involved in vacuolar membrane dynamics in gravity-sensing cells of Arabidopsis inflorescence stem.

Plant vacuoles play critical roles in development, growth and stress responses. In mature cells, vacuolar membranes (VMs) display several types of structures, which are formed by invagination and folding of VMs into the lumenal side and can gradually move and change shape. Although such VM structures are observed in a broad range of tissue types and plant species, the molecular mechanism underlying their formation and maintenance remains unclear. Here, we report that a novel HEAT-repeat protein, SHOOT GRAVITROPISM6 (SGR6), of Arabidopsis is involved in the control of morphological changes and dynamics of VM structures in endodermal cells, which are the gravity-sensing cells in shoots. SGR6 is a membrane-associated protein that is mainly localized to the VM in stem endodermal cells. The sgr6 mutant stem exhibits a reduced gravitropic response. Higher plants utilize amyloplast sedimentation as a means to sense gravity direction. Amyloplasts are surrounded by VMs in Arabidopsis endodermal cells, and the flexible and dynamic structure of VMs is important for amyloplast sedimentation. We demonstrated that such dynamic features of VMs are gradually lost in sgr6 endodermal cells during a 30 min observation period. Histological analysis revealed that amyloplast sedimentation was impaired in sgr6. Detailed live-cell imaging analyses revealed that the VM structures in sgr6 had severe defects in morphological changes and dynamics. Our results suggest that SGR6 is a novel protein involved in the formation and/or maintenance of invaginated VM structures in gravity-sensing cells.

An atypical tubulin kinase mediates stress-induced microtubule depolymerization in Arabidopsis.

BACKGROUND: As sessile organisms, plants adapt to adverse environmental conditions by quickly adjusting cell physiology and metabolism. Transient depolymerization of interphase microtubules is triggered by various acute stresses and biotic interactions with pathogenic organisms. Although rapid remodeling of plant microtubule arrays in response to external stresses is an intriguing phenomenon, the underlying molecular mechanisms and the advantages of this response to plant performance are poorly understood. RESULTS: A domain with weak homology to the slime mold actin-fragmin kinase in the Arabidopsis mitogen-activated protein kinase phosphatase PROPYZAMIDE-HYPERSENSITIVE 1 (PHS1) is a Mn2+-dependent kinase. This atypical kinase domain phosphorylates Thr349 of α-tubulin at the longitudinal interdimer interface, thereby generating a polymerization-incompetent isoform, and effectively depolymerizes microtubule arrays when ectopically expressed in plant or animal cells. The intrinsic tubulin kinase activity is normally suppressed by the phosphatase activity of PHS1 but is unmasked immediately after osmotic stress. In the phs1 null mutant, stress-induced microtubule depolymerization does not occur. CONCLUSIONS: The rapid and reversible modification of tubulin subunits by PHS1-mediated phosphorylation enables dynamic remodeling of the plant microtubule cytoskeleton in response to external stimuli. Suppression of the potent tubulin kinase activity by the juxtaposed phosphatase domain tightly controls this stress-activated microtubule regulator.

Purification and characterization of novel microtubule-associated proteins from Arabidopsis cell suspension cultures.

Plant microtubules (MTs) play essential roles in cell division, anisotropic cell expansion, and overall organ morphology. Microtubule-associated proteins (MAPs) bind to MTs and regulate their dynamics, stability, and organization. Identifying the full set of MAPs in plants would greatly enhance our understanding of how diverse MT arrays are formed and function; however, few proteomics studies have characterized plant MAPs. Using liquid chromatography-tandem mass spectrometry, we identified hundreds of proteins from MAP-enriched preparations derived from cell suspension cultures of Arabidopsis (Arabidopsis thaliana). Previously reported MAPs, MT regulators, kinesins, dynamins, peroxisome-resident enzymes, and proteins implicated in replication, transcription, and translation were highly enriched. Dozens of proteins of unknown function were identified, among which 12 were tagged with green fluorescent protein (GFP) and examined for their ability to colocalize with MTs when transiently expressed in plant cells. Six proteins did indeed colocalize with cortical MTs in planta. We further characterized one of these MAPs, designated as BASIC PROLINE-RICH PROTEIN1 (BPP1), which belongs to a seven-member family in Arabidopsis. BPP1-GFP decorated interphase and mitotic MT arrays in transgenic Arabidopsis plants. A highly basic, conserved region was responsible for the in vivo MT association. Overexpression of BPP1-GFP stabilized MTs, caused right-handed helical growth in rapidly elongating tissues, promoted the formation of transverse MT arrays, and resulted in the outgrowth of epidermal cells in light-grown hypocotyls. Our high-quality proteome database of Arabidopsis MAP-enriched preparations is a useful resource for identifying novel MT regulators and evaluating potential MT associations of proteins known to have other cellular functions.

Amyloplast displacement is necessary for gravisensing in Arabidopsis shoots as revealed by a centrifuge microscope.

The starch-statolith hypothesis proposes that starch-filled amyloplasts act as statoliths in plant gravisensing, moving in response to the gravity vector and signaling its direction. However, recent studies suggest that amyloplasts show continuous, complex movements in Arabidopsis shoots, contradicting the idea of a so-called 'static' or 'settled' statolith. Here, we show that amyloplast movement underlies shoot gravisensing by using a custom-designed centrifuge microscope in combination with analysis of gravitropic mutants. The centrifuge microscope revealed that sedimentary movements of amyloplasts under hypergravity conditions are linearly correlated with gravitropic curvature in wild-type stems. We next analyzed the hypergravity response in the shoot gravitropism 2 (sgr2) mutant, which exhibits neither a shoot gravitropic response nor amyloplast sedimentation at 1 g. sgr2 mutants were able to sense and respond to gravity under 30 g conditions, during which the amyloplasts sedimented. These findings are consistent with amyloplast redistribution resulting from gravity-driven movements triggering shoot gravisensing. To further support this idea, we examined two additional gravitropic mutants, phosphoglucomutase (pgm) and sgr9, which show abnormal amyloplast distribution and reduced gravitropism at 1 g. We found that the correlation between hypergravity-induced amyloplast sedimentation and gravitropic curvature of these mutants was identical to that of wild-type plants. These observations suggest that Arabidopsis shoots have a gravisensing mechanism that linearly converts the number of amyloplasts that settle to the 'bottom' of the cell into gravitropic signals. Further, the restoration of the gravitropic response by hypergravity in the gravitropic mutants that we tested indicates that these lines probably have a functional gravisensing mechanism that is not triggered at 1 g.

Arabidopsis GCP3-interacting protein 1/MOZART 1 is an integral component of the γ-tubulin-containing microtubule nucleating complex.

Microtubules in eukaryotic cells are nucleated from ring-shaped complexes that contain γ-tubulin and a family of homologous γ-tubulin complex proteins (GCPs), but the subunit composition of the complexes can vary among fungi, animals and plants. Arabidopsis GCP3-interacting protein 1 (GIP1), a small protein with no homology to the GCP family, interacts with GCP3 in vitro, and is a plant homolog of vertebrate mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1), a recently identified component of the γ-tubulin complex in human cell lines. In this study, we characterized two closely related Arabidopsis GIP1s: GIP1a and GIP1b. Single mutants of gip1a and gip1b were indistinguishable from wild-type plants, but their double mutant was embryonic lethal, and showed impaired development of male gametophytes. Functional fusions of GIP1a with green fluorescent protein (GFP) were used to purify GIP1a-containing complexes from Arabidopsis plants, which contained all the subunits (except NEDD1) previously identified in the Arabidopsis γ-tubulin complexes. GIP1a and GIP1b interacted specifically with Arabidopsis GCP3 in yeast. GFP-GIP1a labeled mitotic microtubule arrays in a pattern largely consistent with, but partly distinct from, the localization of the γ-tubulin complex containing GCP2 or GCP3 in planta. In interphase cortical arrays, the labeled complexes were preferentially recruited to existing microtubules, from which new microtubules were efficiently nucleated. However, in contrast to complexes labeled with tagged GCP2 or GCP3, their recruitment to cortical areas with no microtubules was rarely observed. These results indicate that GIP1/MOZART1 is an integral component of a subset of the Arabidopsis γ-tubulin complexes.

Mitogen-activated protein kinase phosphatase PHS1 is retained in the cytoplasm by nuclear extrusion signal-dependent and independent mechanisms.

The organization of plant microtubule arrays is thought to be regulated by phosphorylation and other signaling cascades, but the molecular components involved are largely unknown. We have previously found that a dominant missense mutation (phs1-1) in a putative kinase-docking motif of an Arabidopsis PHS1 phosphatase, which belongs to the mitogen-activated protein kinase phosphatase (MKP) family, compromises the stability of cortical microtubules. We here report that suppressor screening of phs1-1 recovered several intragenic recessive mutations in PHS1. In contrast to our previous report, null alleles of PHS1 were almost indistinguishable from the wild type in morphology, but their roots skewed to the abnormal direction when grew in the presence of low doses of a microtubule-destabilizing drug. PHS1 is mainly expressed in elongating cells, where the protein was distributed in the cytoplasm, predominantly in a microsomal fraction. Recruitment of green fluorescent protein-tagged PHS1 in endomembrane aggregates after treatment with brefeldin A or in an endomembrane-organization mutant suggests that an association with endomembranes retains PHS1 in the cytoplasm. A nuclear export signal identified in the C-terminal tail also contributes to the robust cytoplasmic retention of PHS1.

Defects in dynamics and functions of actin filament in Arabidopsis caused by the dominant-negative actin fiz1-induced fragmentation of actin filament.

We isolated frizzy1 (fiz1), a novel dominant actin mutant from Arabidopsis. In the fiz1 mutant, Glu272 was substituted with lysine in the hydrophobic loop of ACT8, which is very important for the polymerization. Live imaging of actin filaments revealed that the fiz1 mutation induced fragmentation of actin filaments in a semi-dominant manner. In addition, the dynamics of Golgi stacks and mitochondria were disrupted by the fiz1 effects. From these results, it was strongly suggested that the fiz1 mutation had dominant-negative effects on actin polymerization, which causes defects in the functions of actin filaments such as organelle transport.

The gene MACCHI-BOU 4/ENHANCER OF PINOID encodes a NPH3-like protein and reveals similarities between organogenesis and phototropism at the molecular level.

Intercellular transport of the phytohormone auxin is a significant factor for plant organogenesis. To investigate molecular mechanisms by which auxin controls organogenesis, we analyzed the macchi-bou 4 (mab4) mutant identified as an enhancer of pinoid (pid). Although mab4 and pid single mutants displayed relatively mild cotyledon phenotypes, pid mab4 double mutants completely lacked cotyledons. We found that MAB4 was identical to ENHANCER OF PINOID (ENP), which has been suggested to control PIN1 polarity in cotyledon primordia. MAB4/ENP encodes a novel protein, which belongs to the NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) family thought to function as a signal transducer in phototropism and control lateral translocation of auxin. MAB4/ENP mRNA was detected in the protodermal cell layer of the embryo and the meristem L1 layer at the site of organ initiation. In the mab4 embryo, the abundance of PIN1:GFP was severely decreased at the plasma membrane in the protodermal cell layer. In addition, subcellular localization analyses indicated that MAB4/ENP resides on a subpopulation of endosomes as well as on unidentified intracellular compartments. These results indicate that MAB4/ENP is involved in polar auxin transport in organogenesis.

The auxin-regulated AP2/EREBP gene PUCHI is required for morphogenesis in the early lateral root primordium of Arabidopsis.

Organ primordia develop from founder cells into organs due to coordinated patterns of cell division. How patterned cell division is regulated during organ formation, however, is not well understood. Here, we show that the PUCHI gene, which encodes a putative APETALA2/ethylene-responsive element binding protein transcription factor, is required for the coordinated pattern of cell divisions during lateral root formation in Arabidopsis thaliana. Recessive mutations in PUCHI disturbed cell division patterns in the lateral root primordium, resulting in swelling of the proximal region of lateral roots. PUCHI expression was initially detected in all of the cells in early lateral root primordia, and later it was restricted to the proximal region of the primordia. Stable expression of PUCHI required auxin-responsive elements in its promoter region, and exogenous auxin increased the level of PUCHI mRNA accumulation. These results suggest that PUCHI acts downstream of auxin signaling and that this gene contributes to lateral root morphogenesis through affecting the pattern of cell divisions during the early stages of primordium development.

A C2H2-type zinc finger protein, SGR5, is involved in early events of gravitropism in Arabidopsis inflorescence stems.

Plants can sense the direction of gravity and change the growth orientation of their organs. To elucidate the molecular mechanisms of gravity perception and the signal transduction of gravitropism, we have characterized a number of shoot gravitropism (sgr) mutants of Arabidopsis. The sgr5-1 mutant shows reduced gravitropism in the inflorescence stem but its root and hypocotyl have normal gravitropism. SGR5 encodes a zinc finger protein with a coiled-coil motif. The SGR5-GFP fusion protein is localized in the nucleus of Arabidopsis protoplasts, suggesting that SGR5 may act as a transcription factor. Analysis of GUS expression under the control of the SGR5 promoter revealed that SGR5 is mainly expressed in the endodermis, the gravity-sensing tissue in inflorescence stems. Furthermore, the observation that endodermis-specific expression of SGR5 using the SCR promoter in the sgr5-1 mutant restores shoot gravitropism indicates that it could function in the gravity-sensing endodermal cell layer. In contrast to other sgr mutants reported previously, almost all amyloplasts in the endodermal cells of the sgr5-1 mutant sedimented in the direction of gravity. Taken together, our results suggest that SGR5 may be involved in an early event in shoot gravitropism such as gravity perception and/or a signaling process subsequent to amyloplast sedimentation as a putative transcription factor in gravity-perceptive cells.

Cortical control of plant microtubules.

The cortical microtubule array of plant cells appears in early G(1) and remodels during the progression of the cell cycle and differentiation, and in response to various stimuli. Recent studies suggest that cortical microtubules are mostly formed on pre-existing microtubules and, after detachment from the initial nucleation sites, actively interact with each other to attain distinct distribution patterns. The plus end of growing microtubules is thought to accumulate protein complexes that regulate both microtubule dynamics and interactions with cortical targets. The ROP family of small GTPases and the mitogen-activated protein kinase pathways have emerged as key players that mediate the cortical control of plant microtubules.

Amyloplasts and vacuolar membrane dynamics in the living graviperceptive cell of the Arabidopsis inflorescence stem.

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 gravity-oriented 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 gravitropism defective 2 mutants of Arabidopsis are deficient in a protein implicated in endocytosis in Caenorhabditis elegans.

The gravitropism defective 2 (grv2) mutants of Arabidopsis show reduced shoot phototropism and gravitropism. Amyloplasts in the shoot endodermal cells of grv2 do not sediment to the same degree as in wild type. The GRV2 gene encodes a 277-kD polypeptide that is 42% similar to the Caenorhabditis elegans RME-8 protein, which is required for endocytosis. We hypothesize that a defect in endocytosis may affect both the initial gravity sensing via amyloplasts sedimentation and the subsequent more general tropic growth response.

PIN-FORMED1 and PINOID regulate boundary formation and cotyledon development in Arabidopsis embryogenesis.

In dicotyledonous plants, two cotyledons are formed at bilaterally symmetric positions in the apical region of the embryo. Single mutations in the PIN-FORMED1 (PIN1) and PINOID (PID) genes, which mediate auxin-dependent organ formation, moderately disrupt the symmetric patterning of cotyledons. We report that the pin1 pid double mutant displays a striking phenotype that completely lacks cotyledons and bilateral symmetry. In the double mutant embryo, the expression domains of CUP-SHAPED COTYLEDON1 (CUC1), CUC2 and SHOOT MERISTEMLESS (STM), the functions of which are normally required to repress growth at cotyledon boundaries, expand to the periphery and overlap with a cotyledon-specific marker, FILAMENTOUS FLOWER. Elimination of CUC1, CUC2 or STM activity leads to recovery of cotyledon growth in the double mutant, suggesting that the negative regulation of these boundary genes by PIN1 and PID is sufficient for primordium growth. We also show that PID mRNA is localized mainly to the boundaries of cotyledon primordia and early expression of PID mRNA is dependent on PIN1. Our results demonstrate the redundant roles of PIN1 and PID in the establishment of bilateral symmetry, as well as in the promotion of cotyledon outgrowth, the latter of which involves the negative regulation of CUC1, CUC2 and STM genes, which are boundary-specific downstream effectors.

Role of endodermal cell vacuoles in shoot gravitropism.

In higher plants, shoots and roots show negative and positive gravitropism, respectively. Data from surgical ablation experiments and analysis of starch deficient mutants have led to the suggestion that columella cells in the root cap function as gravity perception cells. On the other hand, endodermal cells are believed to be the statocytes (that is, gravity perceiving cells) of shoots. Statocytes in shoots and roots commonly contain amyloplasts which sediment under gravity. Through genetic research with Arabidopsis shoot gravitropism mutants, sgr1/scr and sgr7/shr, it was determined that endodermal cells are essential for shoot gravitropism. Moreover, some starch biosynthesis genes and EAL1 are important for the formation and maturation of amyloplasts in shoot endodermis. Thus, amyloplasts in the shoot endodermis would function as statoliths, just as in roots. The study of the sgr2 and zig/sgr4 mutants provides new insights into the early steps of shoot gravitropism, which still remains unclear. SGR2 and ZIG/SGR4 genes encode a phospholipase-like and a v-SNARE protein, respectively. Moreover, these genes are involved in vacuolar formation or function. Thus, the vacuole must play an important role in amyloplast sedimentation because the sgr2 and zig/sgr4 mutants display abnormal amyloplast sedimentation.