Genetic regulation of gravitropism in higher plants.

Gravitropism is a classical subject in plant physiology. However, the molecular mechanisms that regulate gravitropism are unknown. Recently, many gravitropic mutants have been isolated from Arabidopsis thaliana and several genes for gravitropism have been cloned and characterized. These studies have shown that (1) the endodermis is essential for shoot gravitropism and (2) an auxin transport system and signaling pathway are necessary for gravitropism. Recent studies in Arabidopsis are reviewed and genetic regulation of gravitropism in this organism is discussed.

The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling.

Asymmetric cell divisions play an important role in the establishment and propagation of the cellular pattern of plant tissues. The SHORT-ROOT (SHR) gene is required for the asymmetric cell division responsible for formation of ground tissue (endodermis and cortex) as well as specification of endodermis in the Arabidopsis root. We show that SHR encodes a putative transcription factor with homology to SCARECROW (SCR). From analyses of gene expression and cell identity in genetically stable and unstable alleles of shr, we conclude that SHR functions upstream of SCR and participates in a radial signaling pathway. Consistent with a regulatory role in radial patterning, ectopic expression of SHR results in supernumerary cell divisions and abnormal cell specification in the root meristem.

Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot.

Mutation of the SCARECROW (SCR) gene results in a radial pattern defect, loss of a ground tissue layer, in the root. Analysis of the shoot phenotype of scr mutants revealed that both hypocotyl and shoot inflorescence also have a radial pattern defect, loss of a normal starch sheath layer, and consequently are unable to sense gravity in the shoot. Analogous to its expression in the endodermis of the root, SCR is expressed in the starch sheath of the hypocotyl and inflorescence stem. The SCR expression pattern in leaf bundle sheath cells and root quiescent center cells led to the identification of additional phenotypic defects in these tissues. SCR expression in a pin-formed mutant background suggested the possible origins of the starch sheath in the shoot inflorescence. Analysis of SCR expression and the mutant phenotype from the earliest stages of embryogenesis revealed a tight correlation between defective cell divisions and SCR expression in cells that contribute to ground tissue radial patterning in both embryonic root and shoot. Our data provides evidence that the same molecular mechanism regulates the radial patterning of ground tissue in both root and shoot during embryogenesis as well as postembryonically.

Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning.

To accelerate gene isolation from plants by positional cloning, vector systems suitable for both chromosome walking and genetic complementation are highly desirable. Therefore, we developed a transformation-competent artificial chromosome (TAC) vector, pYLTAC7, that can accept and maintain large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. Furthermore, it has the cis sequences required for Agrobacterium-mediated gene transfer into plants. We cloned large genomic DNA fragments of Arabidopsis thaliana into the vector and showed that most of the DNA fragments were maintained stably. Several TAC clones carrying 40- to 80-kb genomic DNA fragments were transferred back into Arabidopsis with high efficiency and shown to be inherited faithfully among the progeny. Furthermore, we demonstrated the practical utility of this vector system for positional cloning in Arabidopsis. A TAC contig was constructed in the region of the SGR1 locus, and individual clones with ca. 80-kb inserts were tested for their ability to complement the gravitropic defects of a homozygous mutant line. Successful complementation enabled the physical location of SGR1 to be delimited with high precision and confidence.

The endodermis and shoot gravitropism

Shoots and roots of higher plants exhibit negative and positive gravitropism, respectively. A variety of gravitropic mutants have recently been isolated from Arabidopsis, the characterization of which demonstrates that the molecular mechanisms of the gravitropic responses in roots, hypocotyls and inflorescence stems are different. The cytological and molecular analysis of two mutants, shoot gravitropism 1 (sgrl), which is allelic to scarecrow (scr), and sgr7, which is allelic to short-root(shr), indicate that the endodermis is the site of gravity perception in shoots. These data suggest a new model for shoot gravitropism.

Gravity perception and gravitropic response of inflorescence stems in Arabidopsis thaliana.

Shoots of higher plants exhibit negative gravitropism. However, little is known about the site of gravity perception in shoots and the molecular mechanisms of shoot gravitropic responses. Our recent analysis using shoot gravitropism 1(sgr1)/scarecrow(scr) and sgr7/short-root (shr) mutants in Arabidopsis thaliana indicated that the endodermis is essential for shoot gravitropism and strongly suggested that the endodermis functions as the gravity-sensing cell layer in dicotyledonous plant shoots. In this paper, we present our recent analysis and model of gravity perception and gravitropic response of inflorescence stems in Arabidopsis thaliana.

Genetic evidence that the endodermis is essential for shoot gravitropism in Arabidopsis thaliana.

Shoots of higher plants exhibit negative gravitropism. However, little is known about the mechanism or site of gravity perception in shoots. We have identified two loci that are essential for normal shoot gravitropism in Arabidopsis thaliana. Genetic analysis demonstrated that the shoot gravitropism mutants sgr1 and sgr7 are allelic to the radial pattern mutants, scr and shr, respectively. Characterization of the aerial phenotype of these mutants revealed that the primary defect is the absence of a normal endodermis in hypocotyls and influorescence stems. This indicates that the endodermis is essential for shoot gravitropism and strongly suggests that this cell layer functions as the gravity-sensing cell layer in dicotyledonous plant shoots. These results also demonstrate that, in addition to their previously characterized role in root radial patterning, SCR and SHR regulate the radial organization of the shoot axial organs in Arabidopsis.

The RHG gene is involved in root and hypocotyl gravitropism in Arabidopsis thaliana.

In higher plants, shoots show negative gravitropism and roots show positive gravitropism. To elucidate the molecular mechanisms of root and hypocotyl gravitropism, we segregated the second mutation from the original phyB-1 mutant line which impaired both root and hypocotyl gravitropism and characterized this novel mutation named rhg (for root and hypocotyl gravitropism). The rhg is a single recessive nuclear mutation and it is mapped on the lower part of the chromosome 1. Analyses on the gravitropic responses of the rhg mutant indicate that root and hypocotyl gravitropism are severely impaired but inflorescence stem gravitropism is not affected by the rhg mutation. In the rhg mutant seedlings, amyloplasts (statoliths for gravity-perception) were present in the presumptive statocytes of roots and hypocotyls. Phototropism by roots and hypocotyls was not impaired in the rhg mutant. These results suggest that the RHG gene product probably acts on the gravity-perception and/or the gravity-signal transduction in root and hypocotyl gravitropism. This is the first report about the genetic locus specifically involved in both root and hypocotyl gravitropism but not inflorescence stem gravitropism, supporting our hypothesis that the mechanisms of gravitropism are genetically different between hypocotyls and inflorescence stems.

Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant.

Mutations in CUC1 and CUC2 (for CUP-SHAPED COTYLEDON), which are newly identified genes of Arabidopsis, caused defects in the separation of cotyledons (embryonic organs), sepals, and stamens (floral organs) as well as in the formation of shoot apical meristems. These defects were most apparent in the double mutant. Phenotypes of the mutants suggest a common mechanism for separating adjacent organs within the same whorl in both embryos and flowers. We cloned the CUC2 gene and found that the encoded protein was homologous to the petunia NO APICAL MERISTEM (NAM) protein, which is thought to act in the development of embryos and flowers.

Mutations in the SGR4, SGR5 and SGR6 loci of Arabidopsis thaliana alter the shoot gravitropism.

Shoots of higher plants grow upward in response to gravity. To elucidate the molecular mechanism of this response, we have isolated shoot gravitropism (sgr) mutants in Arabidopsis thaliana. In this report, we describe three novel mutants, sgr4-1, sgr5-1 and sgr6-1 whose inflorescence stems showed abnormal gravitropic responses as previously reported for sgr1, sgr2 and sgr3. These new sgr mutations were recessive and occurred at three independent genetic loci. The sgr4-1 mutant showed severe defect in gravitropism of both inflorescence stem and hypocotyl but were normal in root gravitropism as were sgr1 and sgr2. The sgr5-1 and sgr6-1 mutants showed reduced gravitropism only in inflorescence stems but normal in both hypocotyls and roots as sgr3. These results support the hypothesis that some mechanisms of gravitropism are genetically different in these three organs in A. thaliana. In addition, these mutants showed normal phototropic responses, suggesting that SGR4, SGR5 and SGR6 genes are specifically involved in gravity perception and/or gravity signal transduction for the shoot gravitropic response.

How do plant shoots bend up? The initial step to elucidate the molecular mechanisms of shoot gravitropism using Arabidopsis thaliana.

In higher plants, shoots show a negative gravitropic response. To elucidate the molecular mechanisms of this phenomenon, mutational analyses using Arabidopsis thaliana are in progress. This minireview aims to present recent developments in the genetic analysis of shoot gravitropism in this organism. We focus mainly on our studies on the novel shoot gravitropic (sgr) mutants in Arabidopsis thaliana that have dramatic defects in shoot gravitropism.

Gravitropic response of inflorescence stems in Arabidopsis thaliana.

We have characterized the gravitropic response of inflorescence stems in Arabidopsis thaliana. When the inflorescence stems were placed horizontally, they curved upward about 90 degrees within 90 min in darkness at 23 degrees C, exhibiting strong negative gravitropism. Decapitated stem segments (without all flowers, flower buds, and apical apices) also showed gravitropic responses when they included the elongation zone. This result indicates that the minimum elements needed for the gravitropic response exist in the decapitated inflorescence stem segments. At least the 3-min gravistimulation time was sufficient to induce the initial curvature at 23 degrees C after a lag time of about 30 min. In the gravitropic response of inflorescence stems, (a) the gravity perception site exists through the elongating zone, (b) auxin is involved in this response, (c) the gravitropic curvature was inhibited at 4 degrees C but at least the gravity perception step could occur, and (d) two curvatures could be induced in sequence at 23 degrees C by two opposite directional horizontal gravistimulations at 4 degrees C.

SGR1, SGR2, SGR3: novel genetic loci involved in shoot gravitropism in Arabidopsis thaliana.

In higher plants shoots show a negative gravitropic response but little is known about its mechanism. To elucidate this phenomenon, we have isolated a number of mutants with abnormal shoot gravitropic responses in Arabidopsis thaliana. Here we describe mainly three mutants: sgr1-1, sgr2-1, and sgr3-1 (shoot gravitropism). Genetic analysis confirmed that these mutations were recessive and occurred at three independent loci, named SGR1, SGR2, and SGR3, respectively. In wild type, both inflorescence stems and hypocotyls show negative gravitropic responses. The sgr1-1 mutants showed no response to gravity either by inflorescence stems or by hypocotyls. The sgr2-1 mutants also showed no gravitropic response in inflorescence stems but showed a reduced gravitropic response in hypocotyls. In contrast, the sgr3-1 mutant was found to have reduced gravitropic responses in inflorescence stems but normal gravitropic responses in hypocotyls. These results suggest that some genetic components of the regulatory mechanisms for gravitropic responses are common between inflorescence stems and hypocotyls, but others are not. In addition, these sgr mutants were normal with respect to root gravitropism, and their inflorescence stems and hypocotyls could carry out phototropism. We conclude that SGR1, SGR2, and SGR3 are novel genetic loci specifically involved in the regulatory mechanisms of shoot gravitropism in A. thaliana.