Expression of theRSI-1 gene during development of roots and reproductive organs in tomato

TheRSI-1 gene is expressed in pericycle cells just prior to the first round of cell division for lateral root development in tomato. We transformed tomato plants with theRSI-1 gene promoter linked to a GUS reporter gene. GUS activity was detected not only at the sites of initiation for lateral and adventitious roots, but also at the primary root tip. Expression of the fusion gene was also regulated at various stages of tissue development: in particular, during the formation of reproductive organs such as pollen and fruit Overexpression of theRSI-1 gene in either the sense or antisense orientation resulted in arrest of fruit development and seed germination. TheRSI-1 gene product, therefore, may play a role in auxin-induced cell division in various developing tissues. Inter and intramolecular disulfide bridges between cysteines rich in the RSI-1 protein might be involved in cell-wall modifications that are essential for new cell division. These hypotheses for the role of theRSI-1 gene in lateral-root and reproductive-organ development remain to be tested.     

Transgenic white clover. Studies with the auxin-responsive promoter,GH3, in root gravitropism and lateral root development

We report an improved method for white clover (Trifolium repens) transformation usingAgrobacterium tumefaciens. High efficiencies of transgenic plant production were achieved using cotyledons of imbibed mature seed. Transgenic plants were recovered routinely from over 50% of treated cotyledons. Thebar gene and phosphinothricin selection was shown to be a more effective selection system thannptII (kanamycin selection) oraadA (spectinomycin selection). White clover was transformed with the soybean auxin responsive promoter, GH3, fused to the GUS gene (-glucuronidase) to study the involvement of auxin in root development. Analysis of 12 independent transgenic plants showed that the location and pattern of GUS expression was consistent but the levels of expression varied. The level of GH3:GUS expression in untreated plants was enhanced specifically by auxin-treatment but the pattern of expression was not altered. Expression of the GH3:GUS fusion was not enhanced by other phytohormones. A consistent GUS expression pattern was evident in untreated plants presumably in response to endogenous auxin or to differences in auxin sensitivity in various clover tissues. In untreated plants, the pattern of GH3:GUS expression was consistent with physiological responses which are regarded as being auxin-mediated. For the first time it is shown that localised spots of GH3:GUS activity occurred in root cortical tissue opposite the sites where lateral roots subsequently were initiated. Newly formed lateral roots grew towards and through these islands of GH3:GUS expression, implying the importance of auxin in controlling lateral root development. Similarly, it is demonstrated for the first time that gravistimulated roots developed a rapid (within 1 h) induction of GH3:GUS activity in tissues on the non-elongating side of the responding root and this induction occurred concurrently with root curvature. These transgenic plants could be useful tools in determining the physiological and biochemical changes that occur during auxin-mediated responses.     

The Arabidopsis dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) is regulated by auxin in both shoots and roots

The AtNRT1.1 (CHL1) gene of Arabidopsis encodes a dual-affinity nitrate transporter and contributes to both low and high affinity nitrate uptake. Localization studies have shown that CHL1 expression is preferentially targeted to nascent organs and growing regions of roots and shoots in Arabidopsis. In roots, CHL1 expression is concentrated in the tips of primary and lateral roots and is activated during lateral root initiation. In shoots, strong CHL1 expression is found in young leaves and developing flower buds. These findings suggest that CHL1 expression might be regulated by a growth signal such as the phytohormone auxin. To test this, auxin regulation of CHL1 was examined. Using transgenic Arabidopsis plants containing CHL1::GUS/GFP DNA constructs, it was found that treatment with exogenous auxin or introduction of the auxin overproducing mutations (yucca and rooty) resulted in a strong increase in CHL1::GUS/GFP signals in roots and leaves. When mature roots were treated with auxin to induce lateral root formation, CHL1::GFP signals were dramatically enhanced in dividing pericycle cells and throughout primordia development. RNA blot analysis showed that CHL1 mRNA levels in whole seedlings increase within 30 min of auxin treatment. The distribution of CHL1 expression in Arabidopsis roots and shoots was found to be similar to that of DR5::GUS, a synthetic, auxin-responsive gene. These results indicate that auxin acts as an important signal regulating CHL1 expression and contributes to the targeting of CHL1 expression to nascent organs and root tips in Arabidopsis.     

The tobacco Cel7 gene promoter is auxin-responsive and locally induced in nematode feeding sites of heterologous plants

Emerging evidence suggests that plant cell-wall-modifying enzymes induced by root-parasitic nematodes play important roles in feeding cell formation. We previously identified a tobacco endo-β-1,4-glucanase (cellulase) gene, NtCel7 , that was strongly induced in both root-knot and cyst nematode feeding cells. To characterize further the developmental and nematode-responsive regulation of NtCel7 , we isolated the NtCel7 promoter and analysed its expression over a time course of nematode infection and in response to auxin, gibberellin, ethylene and sucrose in soybean and tomato hairy roots and in Arabidopsis containing the NtCel7 promoter fused to the β-glucuronidase (GUS) reporter gene. Histochemical analyses of transgenic plant materials revealed that the NtCel7 promoter exhibited a unique organ-specific expression pattern during plant development suggestive of important roles for NtCel7 in both vegetative and reproductive growth. In all plant species tested, strong GUS expression was observed in root tips and lateral root primordia of uninfected roots with weaker expression in the root vasculature. Further analyses of transgenic Arabidopsis plants revealed expression in shoot and root meristems and the vasculature of most organs during plant development. We also determined that the NtCel7 promoter was induced by auxin, but not gibberellin, ethylene or sucrose. Moreover, strong GUS activity was observed in both cyst and root-knot nematode-induced feeding sites in transgenic roots of soybean, tomato and Arabidopsis. The conserved developmental and nematode-responsive expression of the NtCel7 promoter in heterologous plants indicates that motifs of this regulatory element play a fundamental role in regulating NtCel7 gene expression within nematode feeding sites and that this regulation may be mediated by auxin.     

Cell-cycle regulation of hydroxyproline-rich glycoprotein HRGPnt3 gene expression during the initiation of lateral root meristems

Analysis of transgenic tobacco plants containing a tobacco hydroxyproline-rich glycoprotein HRGPnt3 gene promoter-β-glucuronidase (GUS) gene fusion (HRGPnt3-uidA) showed that this promoter is active not only in the early stages of initiation of lateral roots as previously described, but also in the initiation of adventitious roots, with similar selective expression in a subset of pericycle cells. HRGPnt3 is also induced during initiation of hairy roots following transformation by Agrobacterium rhizogenes. The auxin indole acetic acid (IAA) induces an increase in the number of characteristic discrete sites of HRGP-nt3 expression. It is shown that these sites are destined to form new root primordia from pericycle cells of both adventitious and main roots. Dose-dependent induction of root meristems by auxin overcomes the limitations of this naturally stochastic process and makes lateral root initiation amenable to biochemical analysis. Quiescent pericycle cells, which are developmentally arrested in the G₂ phase of the cell cycle, rapidly progress into M phase upon mitogenic stimulation. Colchicine and nocodazole, which block completion of mitosis, inhibited the activation of the HRGPnt3 promoter but did not block auxin induction of parA, a marker for de-differentiation in leaf mesophyll cell-derived protoplasts. Hydroxyurea, which inhibits cell-cycle progression at the G₁/S-phase transition and also blocks lateral root initiation, did not inhibit HRGPnt3 induction. Thus, HRGPnt3 induction precedes completion of the first cell division during primordium formation, and is one of the initial steps in a sequential program of gene expression activated upon stimulation of cell division for the development of a new meristem during lateral root initiation.     

Residual phosphorus and long-term management strategies for an Ultisol

Due to their extensive growth potential, transgenic root systems arising from inoculation withAgrobacterium rhizogenes became popular in the last decade as model systems in domains as diverse as production of secondary metabolites, interactions with pathogens and symbionts, examination of gene importance in control of root development or in regulation of gene expression in roots. Wild-type bacterial strains have also been considered as useful tools to stimulate rooting on recalcitrant cuttings or microcuttings as they cause abundant root initiation at the site of inoculation.Root initiation and the in vitro growth characteristics of transformed roots result from the transfer of genes located on the root-inducing plasmid (Ri) to plant cells and their expression therein. Two sets of pRi genes are involved in the root induction process: therol (rootloci) genes located in the TL region and theaux genes of the TR region. Some of these genes being able to interact, the system appears also as a new tool to study the role of auxin in the process of root initiation. The distinctive phenotype of the transformed roots which are capable of hormone autonomous growth seems to be controlled mainly by therol genes. Theserol genes, i.e. the geneticloci rol A, rol B, rol C androl D correspond to open reading frames ORFs 10, 11, 12 and 15. In vitro experiments determined the functions of the Rol B and Rol C proteins but the functions of Rol A and Rol D are still unknown. Altered metabolism of developmental regulators or modified sensitivity to auxin have been suspected to mediate root induction and morphological abnormalities of transformed roots and plants.The target cells for transformation and the cells which are competent for root initiation will be characterized as well as the subsequent development of transgenic roots provided with various constructs from the whole T-DNA to singlerol genes. Results dealing with auxin contents in relation with root growth kinetics, phenotype and structure, will also be presented and discussed with the potential use of therol genes to control root biomass. F J de Bruijn Section editor     

Elevated auxin and reduced cytokinin contents in rootstocks improve their performance and grafting success

Plant grafting is an important technique for horticultural and silvicultural production. However, many rootstock plants suffer from undesirable lateral bud outgrowth, low grafting success rates or poor rooting. Here, we used a root‐predominant gene promoter (SbUGT) to drive the expression of a tryptophan‐2‐monooxygenase gene (iaaM) from Agrobacterium tumefaciens to increase auxin levels in tobacco. The transgenic plants, when used as a rootstock, displayed inhibited lateral bud outgrowth, enhanced grafting success rate and improved root initiation. However, root elongation and biomass of SbUGT::iaaM transgenic plants were reduced compared to those of wild‐type plants. In contrast, when we used this same promoter to drive CKX (a cytokinin degradation gene) expression, the transgenic tobacco plants displayed enhanced root elongation and biomass. We then made crosses between the SbUGT::CKX and SbUGT::iaaM transgenic plants. We observed that overexpression of the CKX gene neutralized the negative effects of auxin overproduction on root elongation. Also, the simultaneous expression of both the iaaM and CKX genes in rootstock did not disrupt normal growth and developmental patterns in wild‐type scions. Our results demonstrate that expression of both the iaaM and CKX genes predominantly in roots of rootstock inhibits lateral bud release from rootstock, improves grafting success rates and enhances root initiation and biomass.     

Overexpression of OsRAA1 causes pleiotropic phenotypes in transgenic rice plants, including altered leaf, flower, and root development and root response to gravity

There are very few root genes that have been described in rice as a monocotyledonous model plant so far. Here, the OsRAA1 (Oryza sativa Root Architecture Associated 1) gene has been characterized molecularly. OsRAA1 encodes a 12.0-kD protein that has 58% homology to the AtFPF1 (Flowering Promoting Factor 1) in Arabidopsis, which has not been reported as modulating root development yet. Data of in situ hybridization and OsRAA1::GUS transgenic plant showed that OsRAA1 expressed specifically in the apical meristem, the elongation zone of root tip, steles of the branch zone, and the young lateral root. Constitutive expression of OsRAA1 under the control of maize (Zea mays) ubiquitin promoter resulted in phenotypes of reduced growth of primary root, increased number of adventitious roots and helix primary root, and delayed gravitropic response of roots in seedlings of rice (Oryza sativa), which are similar to the phenotypes of the wild-type plant treated with auxin. With overexpression of OsRAA1, initiation and growth of adventitious root were more sensitive to treatment of auxin than those of the control plants, while their responses to 9-hydroxyfluorene-9-carboxylic acid in both transgenic line and wild type showed similar results. OsRAA1 constitutive expression also caused longer leaves and sterile florets at the last stage of plant development. Analysis of northern blot and GUS activity staining of OsRAA1::GUS transgenic plants demonstrated that the OsRAA1 expression was induced by auxin. At the same time, overexpression of OsRAA1 also caused endogenous indole-3-acetic acid to increase. These data suggested that OsRAA1 as a new gene functions in the development of rice root systems, which are mediated by auxin. A positive feedback regulation mechanism of OsRAA1 to indole-3-acetic acid metabolism may be involved in rice root development in nature.     

Mutations in the Diageotropica (Dgt) gene uncouple patterned cell division during lateral root initiation from proliferative cell division in the pericycle

In angiosperms, root branching requires a continuous re-initiation of new root meristems. Through some unknown mechanism, in most eudicots pericycle cells positioned against the protoxylem change identity and initiate patterned division, leading to formation of lateral root primordia that further develop into lateral roots. This process is auxin-regulated. We have observed that three mutations in the Diageotropica (Dgt) gene in tomato prevent primordium formation. Detailed analysis of one of these mutants, dgt1-1, demonstrated that the mutation does not abolish the proliferative capacity of the xylem-adjacent pericycle in the differentiated root portion. Files of shortened pericycle cells found in dgt1-1 roots were unrelated to primordium formation. Auxin application stimulated this unusual proliferation, leading to formation of a multi-layered xylem-adjacent pericycle, but did not rescue the primordium formation. In contrast to wild type, auxin could not induce any cell divisions in the pericycle of the most distal dgt1-1 root-tip portion. In wild-type roots, the Dgt gene promoter was expressed strongly in lateral root primordia starting from their initiation, and on auxin treatment was induced in the primary root meristem. Auxin level and distribution were altered in dgt1-1 root tissues, as judged by direct auxin measurements, and the tissue-specific expression of an auxin-response reporter was altered in transgenic plants. Together, our data demonstrate that the Dgt gene product, a type-A cyclophilin, is essential for morphogenesis of lateral root primordia, and that the dgt mutations uncouple patterned cell division in lateral root initiation from proliferative cell division in the pericycle.