Miyo Terao Morita

A Genetic Framework for the Control of Cell Division and Differentiation in the Root Meristem

Plant growth and development are sustained by meristems. Meristem activity is controlled by auxin and cytokinin, two hormones whose interactions in determining a specific developmental output are still poorly understood. By means of a comprehensive genetic and molecular analysis in Arabidopsis, we show that a primary cytokinin-response transcription factor, ARR1, activates the gene SHY2/IAA3 (SHY2), a repressor of auxin signaling that negatively regulates the PIN auxin transport facilitator genes: thereby, cytokinin causes auxin redistribution, prompting cell differentiation. Conversely, auxin mediates degradation of the SHY2 protein, sustaining PIN activities and cell division. Thus, the cell differentiation and division balance necessary for controlling root meristem size and root growth is the result of the interaction between cytokinin and auxin through a simple regulatory circuit converging on the SHY2 gene.

Directional Gravity Sensing in Gravitropism

Plants can reorient their growth direction by sensing organ tilt relative to the direction of gravity. With respect to gravity sensing in gravitropism, the classic starch statolith hypothesis, i.e., that starch-accumulating amyloplast movement along the gravity vector within gravity-sensing cells (statocytes) is the probable trigger of subsequent intracellular signaling, is widely accepted. Several lines of experimental evidence have demonstrated that starch is important but not essential for gravity sensing and have suggested that it is reasonable to regard plastids (containers of starch) as statoliths. Although the word statolith means sedimented stone, actual amyloplasts are not static but instead possess dynamic movement. Recent studies combining genetic and cell biological approaches, using Arabidopsis thaliana, have demonstrated that amyloplast movement is an intricate process involving vacuolar membrane structures and the actin cytoskeleton. This review covers current knowledge regarding gravity sensing, particularly gravity susception, and the factors modulating the function of amyloplasts for sensing the directional change of gravity. Specific emphasis is made on the remarkable differences in the cytological properties, developmental origins, tissue locations, and response of statocytes between root and shoot systems. Such an approach reveals a common theme in directional gravity-sensing mechanisms in these two disparate organs.

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.

SGR2, a Phospholipase-Like Protein, and ZIG/SGR4, a SNARE, Are Involved in the Shoot Gravitropism of Arabidopsis

In higher plants, the shoot and the root generally show negative and positive gravitropism, respectively. To elucidate the molecular mechanisms involved in gravitropism, we have isolated many shoot gravitropism mutants in Arabidopsis. The sgr2 and zig/sgr4 mutants exhibited abnormal gravitropism in both inflorescence stems and hypocotyls. These genes probably are involved in the early step(s) of the gravitropic response. The sgr2 mutants also had misshapen seed and seedlings, whereas the stem of the zig/sgr4 mutants elongated in a zigzag fashion. The SGR2 gene encodes a novel protein that may be part of a gene family represented by bovine phosphatidic acid–preferring phospholipase A1 containing a putative transmembrane domain. This gene family has been reported only in eukaryotes. The ZIG gene was found to encode AtVTI11, a protein that is homologous with yeast VTI1 and is involved in vesicle transport. Our observations suggest that the two genes may be involved in a vacuolar membrane system that affects shoot gravitropism.

Role of PIN-mediated auxin efflux in apical hook development of Arabidopsis thaliana.

The apical hook of dark-grown Arabidopsis seedlings is a simple structure that develops soon after germination to protect the meristem tissues during emergence through the soil and that opens upon exposure to light. Differential growth at the apical hook proceeds in three sequential steps that are regulated by multiple hormones, principally auxin and ethylene. We show that the progress of the apical hook through these developmental phases depends on the dynamic, asymmetric distribution of auxin, which is regulated by auxin efflux carriers of the PIN family. Several PIN proteins exhibited specific, partially overlapping spatial and temporal expression patterns, and their subcellular localization suggested auxin fluxes during hook development. Genetic manipulation of individual PIN activities interfered with different stages of hook development, implying that specific combinations of PIN genes are required for progress of the apical hook through the developmental phases. Furthermore, ethylene might modulate apical hook development by prolonging the formation phase and strongly suppressing the maintenance phase. This ethylene effect is in part mediated by regulation of PIN-dependent auxin efflux and auxin signaling.

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.

Gravity-induced PIN transcytosis for polarization of auxin fluxes in gravity-sensing root cells

Auxin is an essential plant-specific regulator of patterning processes that also controls directional growth of roots and shoots. In response to gravity stimulation, the PIN3 auxin transporter polarizes to the bottom side of gravity-sensing root cells, presumably redirecting the auxin flux toward the lower side of the root and triggering gravitropic bending. By combining live-cell imaging techniques with pharmacological and genetic approaches, we demonstrate that PIN3 polarization does not require secretion of de novo synthesized proteins or protein degradation, but instead involves rapid, transient stimulation of PIN endocytosis, presumably via a clathrin-dependent pathway. Moreover, gravity-induced PIN3 polarization requires the activity of the guanine nucleotide exchange factors for ARF GTPases (ARF-GEF) GNOM-dependent polar-targeting pathways and might involve endosome-based PIN3 translocation from one cell side to another. Our data suggest that gravity perception acts at several instances of PIN3 trafficking, ultimately leading to the polarization of PIN3, which presumably aligns auxin fluxes with gravity vector and mediates downstream root gravitropic response.

The VTI Family of SNARE Proteins Is Necessary for Plant Viability and Mediates Different Protein Transport Pathways

The Arabidopsis genome contains a family of v-SNAREs: VTI11, VTI12, and VTI13. Only VTI11 and VTI12 are expressed at appreciable levels. Although these two proteins are 60% identical, they complement different transport pathways when expressed in the yeast vti1 mutant. VTI11 was identified recently as the mutated gene in the shoot gravitropic mutant zig. Here, we show that the vti11 zig mutant has defects in vascular patterning and auxin transport. An Arabidopsis T-DNA insertion mutant, vti12, had a normal phenotype under nutrient-rich growth conditions. However, under nutrient-poor conditions, vti12 showed an accelerated senescence phenotype, suggesting that VTI12 may play a role in the plant autophagy pathway. VTI11 and VTI12 also were able to substitute for each other in their respective SNARE complexes, and a double-mutant cross between zig and vti12 was embryo lethal. These results suggest that some VTI1 protein was necessary for plant viability and that the two proteins were partially functionally redundant.

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.

Dynamic Aspects of Ion Accumulation by Vesicle Traffic Under Salt Stress in Arabidopsis

The intracellular membrane dynamics of Arabidopsis cells under high salt treatment were investigated. When Arabidopsis was treated with high levels of NaCl in hydroponic culture, root tip cells showed rapid changes in the vacuolar volume, a decrease in the number of small acid compartments, active movement of vesicles and accumulation of Na(+) both in the central vacuole and in the vesicles around the main vacuole observed with the Na(+)-dependent fluorescence of Sodium Green. Detailed observation of Arabidopsis suspension-cultured cells under high salt treatment showed a similar pattern of response to that observed in root tip cells. Immunostaining of suspension-cultured cells with antibodies against AtNHX1 clearly showed the occurrence of dotted fluorescence in the cytoplasm only under salt treatment. We also confirmed the existence of AtNHX1 in the vacuolar membrane isolated from suspension-cultured cells with immunofluorescence. Knockout of the vacuolar Q(a)-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein VAM3/SYP22 caused an increase in salt tolerance. In mutant plants, the distribution of Na(+) between roots and shoots differed from that of wild-type plants, with Na(+) accumulating more in roots and less in the shoots of the mutant plants. The role of vesicle traffic under salt stress is discussed.