Jennifer F. Topping

hydra Mutants of Arabidopsis Are Defective in Sterol Profiles and Auxin and Ethylene Signaling

The hydra mutants of Arabidopsis are characterized by a pleiotropic phenotype that shows defective embryonic and seedling cell patterning, morphogenesis, and root growth. We demonstrate that the HYDRA1 gene encodes a Δ8-Δ7 sterol isomerase, whereas HYDRA2 encodes a sterol C14 reductase, previously identified as the FACKEL gene product. Seedlings mutant for each gene are similarly defective in the concentrations of the three major Arabidopsis sterols. Promoter::reporter gene analysis showed misexpression of the auxin-regulated DR5 and ACS1 promoters and of the epidermal cell file–specific GL2 promoter in the mutants. The mutants exhibit enhanced responses to auxin. The phenotypes can be rescued partially by inhibition of auxin and ethylene signaling but not by exogenous sterols or brassinosteroids. We propose a model in which correct sterol profiles are required for regulated auxin and ethylene signaling through effects on membrane function.

The POLARIS Gene of Arabidopsis Encodes a Predicted Peptide Required for Correct Root Growth and Leaf Vascular Patterning

The POLARIS (PLS) gene of Arabidopsis was identified as a promoter trap transgenic line, showing β-glucuronidase fusion gene expression predominantly in the embryonic and seedling root, with low expression in aerial parts. Cloning of the PLS locus revealed that the promoter trap T-DNA had inserted into a short open reading frame (ORF). Rapid amplification of cDNA ends PCR, RNA gel blot analysis, and RNase protection assays showed that the PLS ORF is located within a short (∼500 nucleotides) auxin-inducible transcript and encodes a predicted polypeptide of 36 amino acid residues. pls mutants exhibit a short-root phenotype and reduced vascularization of leaves. pls roots are hyperresponsive to exogenous cytokinins and show increased expression of the cytokinin-inducible gene ARR5/IBC6 compared with the wild type. pls seedlings also are less responsive to the growth-inhibitory effects of exogenous auxin and show reduced expression of the auxin-inducible gene IAA1 compared with the wild type. The PLS peptide-encoding region of the cDNA partially complements the pls mutation and requires the PLS ORF ATG for activity, demonstrating the functionality of the peptide-encoding ORF. Ectopic expression of the PLS ORF reduces root growth inhibition by exogenous cytokinins and increases leaf vascularization. We propose that PLS is required for correct auxin-cytokinin homeostasis to modulate root growth and leaf vascular patterning.

Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants

As part of a gene tagging strategy to study the developmental regulation of patterns of plant gene expression, a promoterlessuidA (gus A) gene, encoding the β-glucuronidase (GUS) reporter, was introduced into populations of tobacco,Arbidopsis and potato byAgrobacterium-mediated gene transfer. The objective was to generate random functional fusions following integration of thegusA gene downstream of native gene promoters. We describe here a detailed analysis of levels and patterns ofgusA activation in diverse organs and cell types in those populations.gusA activation occurred at high frequency in all three species, and unique patterns of fusion gene expression were found in each transgenic line. The frequency ofgusA activation was differentially blased in different organs in the three species. Fusion gene activity was identified in a wide range of cell types in all organs studied, and expression patterns were stably transmissible to the T2 and T3 progeny. Developmentally-regulated and environmentally-inducible expression ofgusA is described for one transgenic line. Phenotypic variants were detected in the transgenic population. These results demonstrate the potential of T-DNA insertion as a means of creating functional tags of genes expressed in a wide spectrum of cell types, and the value of the approach as a complement to standard T-DNA insertional mutagenesis and transposon tagging for developmental studies is discussed.

Transcriptional analysis through RNA sequencing of giant cells induced by Meloidogyne graminicola in rice roots

One of the reasons for the progressive yield decline observed in aerobic rice production is the rapid build-up of populations of the rice root knot nematode Meloidogyne graminicola. These nematodes induce specialized feeding cells inside root tissue, called giant cells. By injecting effectors in and sipping metabolites out of these cells, they reprogramme normal cell development and deprive the plant of its nutrients. In this research we have studied the transcriptome of giant cells in rice, after isolation of these cells by laser-capture microdissection. The expression profiles revealed a general induction of primary metabolism inside the giant cells. Although the roots were shielded from light induction, we detected a remarkable induction of genes involved in chloroplast biogenesis and tetrapyrrole synthesis. The presence of chloroplast-like structures inside these dark-grown cells was confirmed by confocal microscopy. On the other hand, genes involved in secondary metabolism and more specifically, the majority of defence-related genes were strongly suppressed in the giant cells. In addition, significant induction of transcripts involved in epigenetic processes was detected inside these cells 7 days after infection.

Modelling and experimental analysis of hormonal crosstalk in Arabidopsis.

An important question in plant biology is how genes influence the crosstalk between hormones to regulate growth. In this study, we model POLARIS (PLS) gene function and crosstalk between auxin, ethylene and cytokinin in Arabidopsis. Experimental evidence suggests that PLS acts on or close to the ethylene receptor ETR1, and a mathematical model describing possible PLS–ethylene pathway interactions is developed, and used to make quantitative predictions about PLS–hormone interactions. Modelling correctly predicts experimental results for the effect of the pls gene mutation on endogenous cytokinin concentration. Modelling also reveals a role for PLS in auxin biosynthesis in addition to a role in auxin transport. The model reproduces available mutants, and with new experimental data provides new insights into how PLS regulates auxin concentration, by controlling the relative contribution of auxin transport and biosynthesis and by integrating auxin, ethylene and cytokinin signalling. Modelling further reveals that a bell‐shaped dose–response relationship between endogenous auxin and root length is established via PLS. This combined modelling and experimental analysis provides new insights into the integration of hormonal signals in plants.

Abscisic acid regulates root growth under osmotic stress conditions via an interacting hormonal network with cytokinin, ethylene and auxin.

Summary Understanding the mechanisms regulating root development under drought conditions is an important question for plant biology and world agriculture. We examine the effect of osmotic stress on abscisic acid (ABA), cytokinin and ethylene responses and how they mediate auxin transport, distribution and root growth through effects on PIN proteins. We integrate experimental data to construct hormonal crosstalk networks to formulate a systems view of root growth regulation by multiple hormones. Experimental analysis shows: that ABA‐dependent and ABA‐independent stress responses increase under osmotic stress, but cytokinin responses are only slightly reduced; inhibition of root growth under osmotic stress does not require ethylene signalling, but auxin can rescue root growth and meristem size; osmotic stress modulates auxin transporter levels and localization, reducing root auxin concentrations; PIN1 levels are reduced under stress in an ABA‐dependent manner, overriding ethylene effects; and the interplay among ABA, ethylene, cytokinin and auxin is tissue‐specific, as evidenced by differential responses of PIN1 and PIN2 to osmotic stress. Combining experimental analysis with network construction reveals that ABA regulates root growth under osmotic stress conditions via an interacting hormonal network with cytokinin, ethylene and auxin.

Insertional mutagenesis and promoter trapping in plants for the isolation of genes and the study of development

Insertional mutagenesis, whether by transposable elements or T-DNAs fromAgrobacterium tumefaciens, provides a powerful experimental strategy to investigate the genetic basis of plant growth, metabolism and development. The linkage of an insertion element with a mutant phenotype of interest greatly facilitates the isolation of the wild-type gene. A further refinement of this strategy is the incorporation of promoter traps or enhancer traps into the insertion elements. These can act as functional tags of regulatory elements associated with genes in the host genome, potentially can improve further the efficiency of screening for target mutant phenotypes, and may provide valuable markers of specific cell types for developmental analysis. We discuss the use of these techniques to study the molecular genetics of plant development.

Interaction of PLS and PIN and hormonal crosstalk in Arabidopsis root development.

Understanding how hormones and genes interact to coordinate plant growth is a major challenge in developmental biology. The activities of auxin, ethylene, and cytokinin depend on cellular context and exhibit either synergistic or antagonistic interactions. Here we use experimentation and network construction to elucidate the role of the interaction of the POLARIS peptide (PLS) and the auxin efflux carrier PIN proteins in the crosstalk of three hormones (auxin, ethylene, and cytokinin) in Arabidopsis root development. In ethylene hypersignaling mutants such as polaris (pls), we show experimentally that expression of both PIN1 and PIN2 significantly increases. This relationship is analyzed in the context of the crosstalk between auxin, ethylene, and cytokinin: in pls, endogenous auxin, ethylene and cytokinin concentration decreases, approximately remains unchanged and increases, respectively. Experimental data are integrated into a hormonal crosstalk network through combination with information in literature. Network construction reveals that the regulation of both PIN1 and PIN2 is predominantly via ethylene signaling. In addition, it is deduced that the relationship between cytokinin and PIN1 and PIN2 levels implies a regulatory role of cytokinin in addition to its regulation to auxin, ethylene, and PLS levels. We discuss how the network of hormones and genes coordinates plant growth by simultaneously regulating the activities of auxin, ethylene, and cytokinin signaling pathways.

Analysis of Vascular Development in the hydra Sterol Biosynthetic Mutants of Arabidopsis

Background The control of vascular tissue development in plants is influenced by diverse hormonal signals, but their interactions during this process are not well understood. Wild-type sterol profiles are essential for growth, tissue patterning and signalling processes in plant development, and are required for regulated vascular patterning. Methodology/Principal Findings Here we investigate the roles of sterols in vascular tissue development, through an analysis of the Arabidopsis mutants hydra1 and fackel/hydra2, which are defective in the enzymes sterol isomerase and sterol C-14 reductase respectively. We show that defective vascular patterning in the shoot is associated with ectopic cell divisions. Expression of the auxin-regulated AtHB8 homeobox gene is disrupted in mutant embryos and seedlings, associated with variably incomplete vascular strand formation and duplication of the longitudinal axis. Misexpression of the auxin reporter proIAA2∶GUS and mislocalization of PIN proteins occurs in the mutants. Introduction of the ethylene-insensitive ein2 mutation partially rescues defective cell division, localization of PIN proteins, and vascular strand development. Conclusions The results support a model in which sterols are required for correct auxin and ethylene crosstalk to regulate PIN localization, auxin distribution and AtHB8 expression, necessary for correct vascular development.

Rescue of defective auxin-mediated gene expression and root meristem function by inhibition of ethylene signalling in sterol biosynthesis mutants of Arabidopsis

The roles of sterols in plant development are not well understood, but evidence is emerging that they are required for cell division, polarity and patterning by mechanisms that are independent of brassinosteroids, of which they are precursors. Previous evidence shows that two sterol-defective mutants of Arabidopsis thaliana (L.) Heynh., hyd1 and fkhyd2, are defective in root development. Here we show that the HYD1 gene, like the FK gene, is transcriptionally active in both primary and lateral root meristems, though not in the shoot apical meristem. The patterns of cell division during early stages of lateral root initiation in the hyd1 and fkhyd2 mutants appear normal. Previous evidence also suggests that auxin and ethylene signalling is defective in the mutants. Here we show that the cytokinin- and ethylene-responsive ACS1::GUS reporter in the fkhyd2 mutant responds to exogenous cytokinins but not to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, indicative of normal cytokinin signalling but supporting the hypothesis that ethylene signalling is defective. The defective root meristem cell division activity and expression patterns of the auxin-regulated DR5::GUS and IAA2::GUS reporters can be rescued to a significant extent by the pharmacological or genetic inhibition of ethylene signalling, but not by treatment with aminoethoxyvinylglycine, an inhibitor of ethylene synthesis. This supports the emerging view that the hyd1 and fkhyd2 mutants exhibit an enhanced and unregulated ethylene signalling activity, which accounts for at least part of the observed mutant phenotypes, including disrupted auxin signalling. The possible relationship between ethylene signalling, membrane sterols and meristem function is discussed.