The shape of the inflorescence in Arabidopsis thaliana ecotype Columbia is a raceme with individual flowers developing acropetally. The ecotype Landsberg harboring the erecta (er) mutation shows a corymb-like inflorescence, namely a compact inflorescence with a flattened arrangement of flower buds at the tip. To gain insight into inflorescence development, we previously isolated corymb-like inflorescence mutants, named corymbosa1 (crm1), and found that the corymb-like inflorescence in crm1-1 was due to reduced cell elongation of pedicels and stem internodes. Double mutants of crm1 with er and crm2, and crm1-1 crm2-1 er-105 triple mutants show an additive phenotype. crm1-1 is caused by a mutation in BIG, which is required for polar auxin transport. CRM1/BIG is expressed in inflorescence meristems, floral meristems and vascular tissues. We analyzed a collection of 12 reduced lateral root formation (rlr) mutants, which are allelic to crm1-1, and categorized the mutants into three classes, depending on the plant developmental defects. Although all 12 alleles had new stop codons, the phenotype of heterozygous crm1-1/doc1-1 and Northern blotting suggest that new crm1/big mutant alleles are hypomorphic. Auxin-responsive DR5rev::GFP expression was decreased in crm1-1 vasculature of pedicels and stem internodes. PINFORMED1 (PIN1) and CRM1/BIG are expressed in vasculature of pedicels and stem internodes. The severity of corymb-like inflorescence in crm1/big mutants correlated with increased levels of PIN1. Our results suggest that CRM1/BIG controls the elongation of the pedicels and stem internodes through auxin action.
To sequence a DNA segment inserted into a cosmid vector under the directed sequencing strategy, we established a simple and rapid method for generating nested deletions which uses the in vitro packaging system of bacteriophage T3 DNA. The principle is based on the previous finding that this system can translocate any linear double-stranded DNA up to 40 kb into the phage capsid in a time-dependent manner and the encapsulated DNA becomes DNase-resistant. For this purpose, we constructed a cosmid vector that carries two different antibiotic selection markers at both sides of the multiple cloning site, and after insertion of a DNA segment, the clone was linearized by λ-terminase at the cos site. After the packaging reaction in vitro followed by DNase treatment, the encapsulated DNA was introduced into Escherichia coli cells to give clones with unidirectional deletions by differential antibiotic selection. Restriction and sequence analyses of deletion clones demonstrated that an ordered set of clones with nested deletions, ranging from less than 1 kb to 25 kb, was created from either the end of the DNA segment. Thus, nested deletion clones that cover the entire region of a ∼40-kb cosmid insert can be obtained by a single packaging reaction, and its restriction map can be simultaneously obtained.
The SNARE complex is a key regulator of vesicular traffic, executing membrane fusion between transport vesicles or organelles and target membranes. A functional SNARE complex consists of four coiled-coil helical bundles, three of which are supplied by Q-SNAREs and another from an R-SNARE. Arabidopsis thaliana VAMP727 is an R-SNARE, with homologs only in seed plants. We have found that VAMP727 colocalizes with SYP22/ VAM3, a Q-SNARE, on a subpopulation of prevacuolar compartments/endosomes closely associated with the vacuolar membrane. Genetic and biochemical analyses, including examination of a synergistic interaction of vamp727 and syp22 mutations, histological examination of protein localization, and coimmunoprecipitation from Arabidopsis lysates indicate that VAMP727 forms a complex with SYP22, VTI11, and SYP51 and that this complex plays a crucial role in vacuolar transport, seed maturation, and vacuole biogenesis. We suggest that the VAMP727 complex mediates the membrane fusion between the prevacuolar compartment and the vacuole and that this process has evolved as an essential step for seed development.
During gravitropism, the directional signal of gravity is perceived by gravity-sensing cells called statocytes, leading to asymmetric distribution of auxin in the responding organs. To identify the genes involved in gravity signaling in statocytes, we performed transcriptome analyses of statocyte-deficient Arabidopsis thaliana mutants and found two candidates from the LAZY1 family, AtLAZY1/LAZY1-LIKE1 (LZY1) and AtDRO3/AtNGR1/LZY2 We showed that LZY1, LZY2, and a paralog AtDRO1/AtNGR2/LZY3 are redundantly involved in gravitropism of the inflorescence stem, hypocotyl, and root. Mutations of LZY genes affected early processes in gravity signal transduction without affecting amyloplast sedimentation. Statocyte-specific expression of LZY genes rescued the mutant phenotype, suggesting that LZY genes mediate gravity signaling in statocytes downstream of amyloplast displacement, leading to the generation of asymmetric auxin distribution in gravity-responding organs. We also found that lzy mutations reversed the growth angle of lateral branches and roots. Moreover, expression of the conserved C-terminal region of LZY proteins also reversed the growth direction of primary roots in the lzy mutant background. In lateral root tips of lzy multiple mutants, asymmetric distribution of PIN3 and auxin response were reversed, suggesting that LZY genes regulate the direction of polar auxin transport in response to gravity through the control of asymmetric PIN3 expression in the root cap columella.
A significant feature of plant cells is the extensive motility of organelles and the cytosol, which was originally defined as cytoplasmic streaming. We suggested previously that a three-way interaction between plant-specific motor proteins myosin XIs, actin filaments, and the endoplasmic reticulum (ER) was responsible for cytoplasmic streaming. (1) Currently, however, there are no reports of molecular components for cytoplasmic streaming other than the actin-myosin-cytoskeleton and ER-related proteins. In the present study, we found that elongated cells of inflorescence stems of Arabidopsis thaliana exhibit vigorous cytoplasmic streaming. Statistical analysis showed that the maximal velocity of plastid movements is 7.26 µm/s, which is much faster than the previously reported velocities of organelles. Surprisingly, the maximal velocity of streaming in the inflorescence stem cells was significantly reduced to 1.11 µm/s in an Arabidopsis mutant, abcb19-101, which lacks ATP BINDING CASSETTE SUBFAMILY B19 (ABCB19) that mediates the polar transport of the phytohormone auxin together with PIN-FORMED (PIN) proteins. Polar auxin transport establishes the auxin concentration gradient essential for plant development and tropisms. Deficiency of ABCB19 activity eventually caused enhanced gravitropic responses of the inflorescence stems and abnormally flexed inflorescence stems. These results suggest that ABCB19-mediated auxin transport plays a role not only in tropism regulation, but also in cytoplasmic streaming.
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.
Intracellular sedimentation of highly dense, starch-filled amyloplasts toward the gravity vector is likely a key initial step for gravity sensing in plants. However, recent live-cell imaging technology revealed that most amyloplasts continuously exhibit dynamic, saltatory movements in the endodermal cells of Arabidopsis stems. These complicated movements led to questions about what type of amyloplast movement triggers gravity sensing. Here we show that a confocal microscope equipped with optical tweezers can be a powerful tool to trap and manipulate amyloplasts noninvasively, while simultaneously observing cellular responses such as vacuolar dynamics in living cells. A near-infrared (λ=1064 nm) laser that was focused into the endodermal cells at 1 mW of laser power attracted and captured amyloplasts at the laser focus. The optical force exerted on the amyloplasts was theoretically estimated to be up to 1 pN. Interestingly, endosomes and trans-Golgi network were trapped at 30 mW but not at 1 mW, which is probably due to lower refractive indices of these organelles than that of the amyloplasts. Because amyloplasts are in close proximity to vacuolar membranes in endodermal cells, their physical interaction could be visualized in real time. The vacuolar membranes drastically stretched and deformed in response to the manipulated movements of amyloplasts by optical tweezers. Our new method provides deep insights into the biophysical properties of plant organelles in vivo and opens a new avenue for studying gravity-sensing mechanisms in plants.
Abstract Gravitropism is the plant organ bending in response to gravity, while a straightening mechanism prevents bending beyond the gravitropic set-point angle. The promotion and prevention of bending occur simultaneously around the inflorescence stem tip. How these two opposing forces work together and what part of the stem they affect are unknown. To understand the mechanical forces involved, we rotated wild type and organ-straightening-deficient mutant ( myosin xif xik ) Arabidopsis plants to a horizontal position to initiate bending. The mutant stems started to bend before the wild-type stems, which led us to hypothesize that the force preventing bending was weaker in mutant. We modeled the wild-type and mutant stems as elastic rods, and evaluated two parameters: an organ-angle-dependent gravitropic-responsive parameter ( β ) and an organ-curvature-dependent proprioceptive-responsive parameter ( γ ). Our model showed that these two parameters were lower in mutant than in wild type, implying that, unexpectedly, both promotion and prevention of bending are weak in mutant. Subsequently, finite element method simulations revealed that the compressive stress in the middle of the stem was significantly lower in wild type than in mutant. The results of this study show that myosin-XIk-and-XIf-dependent organ straightening adjusts the stress distribution to achieve a mechanically favorable shape.
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