Stuart A. Casson

Influence of environmental factors on stomatal development

Stomata play a pivotal role in the regulation of gas exchange in flowering plants and are distributed throughout the aerial epidermis. In leaves, the pattern of stomatal distribution is highly variable between species but is regulated by a mechanism that maintains a minimum of one cell spacing between stomata. In Arabidopsis, a number of the genetic components of this mechanism have been identified and include, SDD1, EPF1 and the putative receptors TMM and the ERECTA-gene family. A mitogen-activated protein (MAP) kinase signalling cascade is believed to act downstream of these putative receptors while a number of transcription factors including SPCH, MUTE and FAMA have been identified that control consecutive steps of stomatal development. The environment also has significant effects on stomatal development. In a number of species both light intensity and CO(2) concentrations have been shown to influence the frequency at which stomata develop on leaves. Long-distance signalling mechanisms have been implicated in these environmental responses with the conditions sensed by mature leaves determining the stomatal frequency in developing leaves. Thus, changes in the environment appear to act by modulating the developmental and patterning pathways to determine stomatal frequency.

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 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.

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.

Environmental regulation of stomatal development

Stomata are microscopic structures in the epidermis of the aerial parts of flowering plants formed by two specialized guard cells flanking a central pore. The role of stomata is to optimize gas exchange (the uptake of carbon dioxide and the loss of water vapor) to suit the prevailing environmental conditions. To do this plants open and close the stomatal pores and regulates the number of stomata that develop on the epidermes. Both these responses are controlled by integrating information from environmental cues and hormonal signals. Recent work has resulted in significant advances in our understanding of the underlying pathway controlling stomatal development. Here we shall discuss how environmental cues might modulate this pathway such that gas exchange is optimized to suit the prevailing environmental conditions.

phytochrome B and PIF4 Regulate Stomatal Development in Response to Light Quantity

Stomata are pores on the surfaces of leaves that regulate gas exchange between the plant interior and the atmosphere [1]. Plants adapt to changing environmental conditions in the short term by adjusting the aperture of the stomatal pores, whereas longer-term changes are accomplished by altering the proportion of stomata that develop on the leaf surface [2, 3]. Although recent work has identified genes involved in the control of stomatal development [4], we know very little about how stomatal development is modulated by environmental signals, such as light. Here, we show that mature leaves of Arabidopsis grown at higher photon irradiances show significant increases in stomatal index (S.I.) [5] compared to those grown at lower photon irradiances. Light quantity-mediated changes in S.I. occur in red light, suggesting that phytochrome photoreceptors [6] are involved. By using a genetic approach, we demonstrate that this response is dominated by phytochrome B and also identify a role for the transcription factor, PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) [7]. In sum, we identify a photoreceptor and downstream signaling protein involved in light-mediated control of stomatal development, thereby establishing a tractable system for investigating how an environmental signal modulates stomatal development.

Land Plants Acquired Active Stomatal Control Early in Their Evolutionary History

Stomata are pores that regulate plant gas exchange [1]. They evolved more than 400 million years ago [2, 3], but the origin of their active physiological responses to endogenous and environmental cues is unclear [2-6]. Recent research suggests that the stomata of lycophytes and ferns lack pore closure responses to abscisic acid (ABA) and CO(2). This evidence led to the hypothesis that a fundamental transition from passive to active control of plant water balance occurred after the divergence of ferns 360 million years ago [7, 8]. Here we show that stomatal responses of the lycophyte Selaginella [9] to ABA and CO(2) are directly comparable to those of the flowering plant Arabidopsis [10]. Furthermore, we show that the underlying intracellular signaling pathways responsible for stomatal aperture control are similar in both basal and modern vascular plant lineages. Our evidence challenges the hypothesis that acquisition of active stomatal control of plant carbon and water balance represents a critical turning point in land plant evolution [7, 8]. Instead, we suggest that the critical evolutionary development is represented by the innovation of stomata themselves and that physiologically active stomatal control originated at least as far back as the emergence of the lycophytes (circa 420 million years ago) [11].

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.

The POLARIS Peptide of Arabidopsis Regulates Auxin Transport and Root Growth via Effects on Ethylene Signaling

The rate and plane of cell division and anisotropic cell growth are critical for plant development and are regulated by diverse mechanisms involving several hormone signaling pathways. Little is known about peptide signaling in plant growth; however, Arabidopsis thaliana POLARIS (PLS), encoding a 36–amino acid peptide, is required for correct root growth and vascular development. Mutational analysis implicates a role for the peptide in hormone responses, but the basis of PLS action is obscure. Using the Arabidopsis root as a model to study PLS action in plant development, we discovered a link between PLS, ethylene signaling, auxin homeostasis, and microtubule cytoskeleton dynamics. Mutation of PLS results in an enhanced ethylene-response phenotype, defective auxin transport and homeostasis, and altered microtubule sensitivity to inhibitors. These defects, along with the short-root phenotype, are suppressed by genetic and pharmacological inhibition of ethylene action. PLS expression is repressed by ethylene and induced by auxin. Our results suggest a mechanism whereby PLS negatively regulates ethylene responses to modulate cell division and expansion via downstream effects on microtubule cytoskeleton dynamics and auxin signaling, thereby influencing root growth and lateral root development. This mechanism involves a regulatory loop of auxin–ethylene interactions.

The turnip Mutant of Arabidopsis Reveals That LEAFY COTYLEDON1 Expression Mediates the Effects of Auxin and Sugars to Promote Embryonic Cell Identity

The transition from embryonic to vegetative growth marks an important developmental stage in the plant life cycle. The turnip (tnp) mutant was identified in a screen for modifiers of POLARIS expression, a gene required for normal root growth. Mapping and molecular characterization of tnp shows that it represents a gain-of-function mutant of LEAFY COTYLEDON1 (LEC1), due to a promoter mutation. This results in the ectopic expression of LEC1, but not of other LEC genes, in vegetative tissues. The LEC class of genes are known regulators of embryogenesis, involved in the control of embryonic cell identity by currently unknown mechanisms. Activation of the LEC-dependent pathway in tnp leads to the loss of hypocotyl epidermal cell marker expression and loss of SCARECROW expression in the endodermis, the ectopic accumulation of starch and lipids, and the up-regulation of early and late embryonic genes. tnp also shows partial deetiolation during dark growth. Penetrance of the mutant phenotype is strongly enhanced in the presence of exogenous auxin and sugars, but not by gibberellin or abscisic acid, and is antagonized by cytokinin. We propose that the role of LEC1 in embryonic cell fate control requires auxin and sucrose to promote cell division and embryonic differentiation.