Keith Lindsey

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.

Early transcriptomic events in microdissected Arabidopsis nematode‐induced giant cells

Root-knot nematodes differentiate highly specialized feeding cells in roots (giant cells, GCs), through poorly characterized mechanisms that include extensive transcriptional changes. While global transcriptome analyses have used galls, which are complex root structures that include GCs and surrounding tissues, no global gene expression changes specific to GCs have been described. We report on the differential transcriptome of GCs versus root vascular cells, induced in Arabidopsis by Meloidogyne javanica at a very early stage of their development, 3 days after infection (d.p.i.). Laser microdissection was used to capture GCs and root vascular cells for microarray analysis, which was validated through qPCR and by a promoter-GUS fusion study. Results show that by 3 d.p.i., GCs exhibit major gene repression. Although some genes showed similar regulation in both galls and GCs, the majority had different expression patterns, confirming the molecular distinctiveness of the GCs within the gall. Most of the differentially regulated genes in GCs have no previously assigned function. Comparisons with other transcriptome analyses revealed similarities between GCs and cell suspensions differentiating into xylem cells. This suggests a molecular link between GCs and developing vascular cells, which represent putative GC stem cells. Gene expression in GCs at 3 d.p.i. was also found to be similar to crown galls induced by Agrobacterium tumefaciens, a specialized root biotroph.

The Significance of Microspore Division and Division Symmetry for Vegetative Cell-Specific Transcription and Generative Cell Differentiation.

The significance of the onset and symmetry of pollen mitosis I (PMI) for the subsequent differentiation of the vegetative and generative cells was investigated by the in vitro maturation of isolated microspores of transgenic tobacco. Free uninucleate microspores of transgenic plants harboring the vegetative cell (VC)-specific late anther tomato lat52 promoter fused to the [beta]-glucuronidase (gus) gene showed normal asymmetric cell division at PMI and activated the lat52 promoter specifically in the nascent VC during in vitro maturation. In vitro maturation in the presence of high levels of colchicine effectively blocked PMI, resulting in the formation of uninucleate pollen grains in which the lat52 promoter was activated. Furthermore, matured uninucleate pollen grains were capable of germination and pollen tube growth despite the absence of a functional generative cell (GC). Lower levels of colchicine induced symmetric division at PMI, producing two similar daughter cells in which typical GC chromatin condensation was prevented. Similar cultures of transgenic microspores harboring the lat52 promoter driving the expression of a nuclear-targeted GUS fusion protein showed that lat52 promoter activation occurred in both symmetric daughter cells. These results directly demonstrate that division asymmetry at PMI is essential for correct GC differentiation and that activation of VC-specific transcription and functional VC maturation may be uncoupled from cytokinesis at PMI. These results are discussed in relation to models proposed to account for the role and distribution of factors controlling the differing fates of the vegetative and generative cells.

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.

Extracellular ATP Functions as an Endogenous External Metabolite Regulating Plant Cell Viability

ATP is a vital molecule used by living organisms as a universal source of energy required to drive the cogwheels of intracellular biochemical reactions necessary for growth and development. Animal cells release ATP to the extracellular milieu, where it functions as the primary signaling cue at the epicenter of a diverse range of physiological processes. Although recent findings revealed that intact plant tissues release ATP as well, there is no clearly defined physiological function of extracellular ATP in plants. Here, we show that extracellular ATP is essential for maintaining plant cell viability. Its removal by the cell-impermeant traps glucose–hexokinase and apyrase triggered death in both cell cultures and whole plants. Competitive exclusion of extracellular ATP from its binding sites by treatment with β,γ-methyleneadenosine 5′-triphosphate, a nonhydrolyzable analog of ATP, also resulted in death. The death response was observed in Arabidopsis thaliana, maize (Zea mays), bean (Phaseolus vulgaris), and tobacco (Nicotiana tabacum). Significantly, we discovered that fumonisin B1 (FB1) treatment of Arabidopsis triggered the depletion of extracellular ATP that preceded cell death and that exogenous ATP rescues Arabidopsis from FB1-induced death. These observations suggest that extracellular ATP suppresses a default death pathway in plants and that some forms of pathogen-induced cell death are mediated by the depletion of extracellular ATP.

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 Profiling of the Arabidopsis Embryo

We have used laser-capture microdissection to isolate RNA from discrete tissues of globular, heart, and torpedo stage embryos of Arabidopsis (Arabidopsis thaliana). This was amplified and analyzed by DNA microarray using the Affymetrix ATH1 GeneChip, representing approximately 22,800 Arabidopsis genes. Cluster analysis showed that spatial differences in gene expression were less significant than temporal differences. Time course analysis reveals the dynamics and complexity of gene expression in both apical and basal domains of the developing embryo, with several classes of synexpressed genes identifiable. The transition from globular to heart stage is associated in particular with an up-regulation of genes involved in cell cycle control, transcriptional regulation, and energetics and metabolism. The transition from heart to torpedo stage is associated with a repression of cell cycle genes and an up-regulation of genes encoding storage proteins, and pathways of cell growth, energy, and metabolism. The torpedo stage embryo shows strong functional differentiation in the root and cotyledon, as inferred from the classes of genes expressed in these tissues. The time course of expression of the essential EMBRYO-DEFECTIVE genes shows that most are expressed at unchanging levels across all stages of embryogenesis. We show how identified genes can be used to generate cell type-specific markers and promoter activities for future application in cell biology.

Modulation of Cyclin Transcript Levels in Cultured Cells of Arabidopsis thaliana

Previous studies on the cell cycle of Arabidopsis thaliana have been hindered by the lack of synchronous cell culture systems. We have used liquid callus cultures and a cycloheximide-synchronized suspension culture of Arabidopsis to investigate changes in cyclin transcript levels in response to exogenous auxin, cytokinin, and nutrients, and during the cell cycle. CYCD1 ([delta]1) transcript was virtually undetectable in liquid-cultured callus or suspension-culture cells. CYCD2 ([delta]2) transcript levels were largely unaffected by the readdition of phytohormones or nitrate to the growth medium, and remained constant throughout the cell cycle in suspension-culture cells. CYCD3 ([delta]3) transcript levels were strongly dependent on nitrate, and were induced at the G1/S transition following phytohormone readdition. In synchronized suspension-culture cells, CYCD3 transcript accumulated during the S phase, and remained constant thereafter. These results support the hypothesis that D cyclins function as part of the cellular machinery that integrates diverse signals impinging upon commitment to cell division. In synchronized cells transcripts of the mitotic cyclins CYC1, CYC2, and CYC3 reached a maximum with peak mitotic index, but CYC3 transcript levels increased earlier than those of CYC1 or CYC2. The kinetics of accumulation of CYC transcript levels support their classification as A-type (CYC3) and B-type (CYC1 and CYC2) cyclins, respectively.

Small RNA and degradome sequencing reveal complex miRNA regulation during cotton somatic embryogenesis

MicroRNAs (miRNAs) are endogenous non-coding ~21 nucleotide RNAs that regulate gene expression at the transcriptional and post-transcriptional levels in plants and animals. They play an important role in development, abiotic stress, and pathogen responses. miRNAs with their targets have been widely studied in model plants, but limited knowledge is available on the small RNA population of cotton (Gossypium hirsutum)—an important economic crop, and global identification of related targets through degradome sequencing has not been developed previously. In this study, small RNAs and their targets were identified during cotton somatic embryogenesis (SE) through high-throughput small RNA and degradome sequencing, comparing seedling hypocotyl and embryogenic callus (EC) of G. hirsutum YZ1. A total of 36 known miRNA families were found to be differentially expressed, of which 19 miRNA families were represented by 29 precursors. Twenty-five novel miRNAs were identified. A total of 234 transcripts in EC and 322 transcripts in control (CK) were found to be the targets of 23 and 30 known miRNA families, respectively, and 16 transcripts were targeted by eight novel miRNAs. Interestingly, four trans-acting small interfering RNAs (tas3-siRNAs) were also found in degradome libraries, three of which perfectly matched their precursors. Several targets were further validated via RNA ligase-mediated rapid amplification of 5’ cDNA ends (RLM 5’-RACE). The profiling of the miRNAs and their target genes provides new information on the miRNAs network during cotton SE.

Peptides: new signalling molecules in plants

For many years, our insight into intercellular signalling in plants was based upon our knowledge of the so-called five classical plant hormones--auxin, cytokinin, ethylene, gibberellin and abscisic acid. However, biochemical and genetic studies have identified peptides that play crucial roles in plant growth and development, including defence mechanisms in response to wounding by pests, the control of cell division and expansion, and pollen self-incompatibility. Genome sequencing has revealed many predicted peptide-encoding genes and possible receptors, and a major challenge of the post-genomics era is to determine the function of these molecules.