Biomedical subjects
Publications and source records attributed to K Lindsey.
A polypeptide from tobacco has been found to be processed into multiple functional peptides, each with independent hormone-like activities. This adds to a growing set of small peptides known to function as signal molecules in plants.
The establishment of the apical-basal axis is a critical event in plant embryogenesis, evident from the earliest stages onwards. Polarity is evident in the embryo sac, egg cell, zygote, and embryo-suspensor complex. In the embryo-proper, two functionally distinct meristems form at each pole, through the localized expression of key genes. A number of mutants, notably of the model genetic organism Arabidopsis thaliana, have revealed new gene functions that are required for patterning of the apical-basal axis. There is now increasing evidence that two particular modes of signalling, via auxin and cell wall components, play important roles in co-ordinating the gene expression programmes that define determinative roles in the establishment of polarity.
Plants used by Southern African traditional healers for the treatment of menstrual pains were screened for prostaglandin-synthesis inhibitors and the ability to reduce isolated uterine muscle contraction using the cyclooxygenase and in vitro uterine bioassays respectively. Prostaglandins are synthesized from arachidonic acid and the enzyme that drives this reaction is cyclooxygenase. The excessive production of prostaglandins by the myometrium and endometrium induces uterine contractions. Inhibition of cyclooxygenase and hence of the prostaglandin biosynthetic pathway may lead to relief of menstrual pain. Ten plants used by traditional healers for menstrual pains were assayed for cyclooxygenase inhibitory activity. Several plant extracts exhibited high inhibitory activity in the assay. The highest activities were obtained with ethanolic extracts of Siphonochilus aethiopicus, Cenchrus ciliaris and Solanum mauritianum. Generally ethanolic extracts gave higher activity than the aqueous extracts. None of the ethanolic plant extracts were able to relax or reduce the contractions of the precontracted guinea pig uterus.
The mechanism by which higher plant cells divide is quite different from the animal system, and has implications for the way in which the processes of pattern formation, morphogenesis and cell differentiation are executed. The relationship between cell shape, cell division and differentiation and development will be considered from an evolutionary perspective, and the ways in which new molecular markers can inform our interpretation of these relationships will be considered.
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Development can be considered to comprise the co-ordinated regulation of patterning at different levels: patterning of cells to form tissues, patterning of tissues to form organs, and patterning of organs to generate the characteristic architecture of the organism. These processes are expected, in turn, to be mediated by the precise spatial and temporal regulation of patterns of gene expression during development, which depend on appropriate signalling mechanisms. In order to investigate molecular events of morphogenesis in plants, we have utilized a system of promoter trap insertional mutagenesis in Arabidopsis, to generate both phenotypic mutants and gene fusions that represent markers useful in studying the regulation of patterning. A screen of transgenic seedlings containing a T-DNA promoter trap has led to the identification of mutants defective in seedling shape and embryonic development, and of GUS fusion genes that are expressed in spatially restricted patterns. Mutants have been crossed with marker lines expressing cell type-specific GUS activities, to investigate their cellular organization. For example, the POLARIS marker gene is expressed in the embryonic and seedling root tip. When crossed with hydra, which lacks an embryonic root, and with emb30, which lacks both embryonic and seedling roots, it is nevertheless expressed in the correct relative position, and we hypothesize that it represents a novel marker of root positional information, independent of root morphogenesis.
A gene encoding a predicted nucleic acid helicase was identified in Arabidopsis thaliana by activation of a promoter trap. An in vivo transcriptional fusion between the helicase gene and a promoterless uidA (gusA) gene was generated, which was expressed specifically in the tapetum and vascular tissues. The tagged gene, designated HVT1 (Helicase in Vascular tissue and Tapetum), encodes a native transcript of approximately 4.4 kb, which is of very low abundance. The predicted HVT1 protein, of 1291 amino acid residues, is homologous to the Drosophila MALELESS, human RNA helicase A and bovine nuclear DNA helicase proteins, and represents the first identified member of a new subgroup within the mle group of the DEAH family. Low stringency genomic Southern blot analysis indicates that at least two structurally related genes exist in Arabidopsis. We suggest that the HVT1 protein may play a role in nucleic acid unwinding in restricted cell types during both vegetative and reproductive phases of the Arabidopsis life cycle.
To investigate mechanisms involved in establishing polar organization in Arabidopsis embryos and seedlings, we used promoter trapping to identify molecular markers (beta-glucuronidase fusion genes) expressed in spatially restricted patterns along the apical-basal axis. Three markers were identified that are expressed, respectively, in the embryonic and seedling root tip (POLARIS), cotyledons and shoot and root apices (EXORDIUM), and root cap (COLUMELLA). Each marker was crossed into the mutants hydra and emb30, which are defective in embryonic and seedling morphogenesis. All three markers were expressed in hydra mutants in patterns similar to those observed in phenotypically wild-type embryos and seedlings. In emb30 mutants, the EXORDIUM marker was expressed in cotyledons but not in the expected position of shoot and root meristems, and the marker COLUMELLA was not expressed at all, which is consistent with the view that the emb30 mutant, but not hydra, lacks shoot and root meristems. However, POLARIS was expressed in the basal part of hydra embryos lacking an embryonic root and in the basal parts of both hydra and emb30 seedlings. Expression of POLARIS is inducible by exogenous auxin and suppressed by cytokinin but is unaffected by inhibitors of polar auxin transport or cell division. We conclude that POLARIS differentiates positional aspects of polar development from structural aspects.
In the quest for plant regulatory sequences capable of driving nematode-triggered effector gene expression in feeding structures, we show that promoter tagging is a valuable tool. A large collection of transgenic Arabidopsis plants was generated. They were transformed with a beta-glucuronidase gene functioning as a promoter tag. Three T-DNA constructs, pGV1047, p delta gusBin19, and pMOG553, were used. Early responses to nematode invasion were of primary interest. Six lines exhibiting beta-glucuronidase activity in syncytia induced by the beet cyst nematode were studied. Reporter gene activation was also identified in galls induced by root knot and ectoparasitic nematodes. Time-course studies revealed that all six tags were differentially activated during the development of the feeding structure. T-DNA-flanking regions responsible for the observed responses after nematode infection were isolated and characterized for promoter activity.
Mutations in the HYDRA1 (HYD1) gene of Arabidopsis thaliana can prevent normal morphological development of embryos and seedlings. Three allelic mutants (hydra 1-1, hydra1-2 and hydra1-3) have been identified, and in each the seedling is characterized by having a variable number of cotyledons, a short and wide hypocotyl and a much reduced root system. hydra1 embryos appear to develop normally to the octant stage, but fail to establish a distinct protoderm and lack bilateral symmetry, developing multiple cotyledonary primordia of irregular size and shape. Cells of the embryo proper, but not the suspensor, exhibit abnormalities in size and shape. The hydra1 embryo fails to develop an embryonic root, but embryos and seedlings express molecular markers of apical-basal polarity. Mutant seedlings produce leaves to form a small cabbage-like habit and may occasionally produce sterile flowers, though the mutation is commonly seedling-lethal. hydra1 seedlings exhibit abnormal radial patterning, but nevertheless express at least one molecular marker of vascular cell differentiation. A model is proposed in which the HYDRA1 protein functions as an essential component of the cell expansion system.
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.
Transgenic plants of tobacco and Arabidopsis that produce genetically marked pollen, expressing the reporter gene uidA (gusA), were generated to determine whether pollen proteins can be expressed and stable in honey, a potential route by which foreign proteins might enter the wider environment. Hydrated tobacco pollen was found to lose viability rapidly in honey, while pollen in the natural dehydrated form remained viable for at least several days and in some cases several weeks, as determined by FDA staining activity and germinability. Dehydrated pollen was found to be capable of transient foreign gene expression, following microprojectile bombardment, after incubation in honey for at least 120 h. PCR amplification of transgene sequences in pollen of transgenic plants revealed that pollen DNA can remain relatively intact after 7 weeks in honey. GUS enzyme activity analysis and SDS-PAGE of pollen proteins revealed that foreign and native pollen proteins are stable in pollen incubated in honey for at least 6 weeks. We conclude that pollen may represent an ecologically important vector for transgenic protein products.
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The technique of promoter trapping has been exploited to identify markers of embryogenesis in Arabidopsis thaliana. A population of transgenic A. thaliana was generated containing the promoter trap vector pdeltagusBin19, following Agrobacterium tumefaciens-mediated transformation. This vector contains at the T-DNA left border, a promoterless gusA gene, which is activated following integration downstream of a native gene promoter that directs transgene transcription. Screening of a population of 430 independent transgenic lines revealed that 74 lines (17.2%) exhibited GUS activity in siliques, as determined by fluorimetric assay. Histochemical GUS analysis was used to identify lines that expressed GUS in embryos, and three lines that were demonstrated to contain single T-DNA inserts were analysed in detail. Each line showed a distinct pattern of GUS fusion activity. Fusion transcripts were identified, demonstrating that transcription was initiated in the genomic DNA flanking the T-DNA left border. Inverse PCR was used to clone the T-DNA flanking sequences, and for one line a corresponding cDNA was identified, demonstrating that the tagged sequences are transcribed. The markers represent the earliest embryonic genes known to be expressed in plants.
As part of a gene tagging strategy to study the developmental regulation of patterns of plant gene expression, a promoterless uidA (gusA) gene, encoding the beta-glucuronidase (GUS) reporter, was introduced into populations of tobacco, Arabidopsis and potato by Agrobacterium-mediated gene transfer. The objective was to generate random functional fusions following integration of the gusA gene downstream of native gene promoters. We describe here a detailed analysis of levels and patterns of gusA 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 of gusA activation was differentially biased 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 of gusA 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.