Julie E. Gray

Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia.

The vanilloid receptor-1 (VR1) is a ligand-gated, non-selective cation channel expressed predominantly by sensory neurons. VR1 responds to noxious stimuli including capsaicin, the pungent component of chilli peppers, heat and extracellular acidification, and it is able to integrate simultaneous exposure to these stimuli. These findings and research linking capsaicin with nociceptive behaviours (that is, responses to painful stimuli in animals have led to VR1 being considered as important for pain sensation. Here we have disrupted the mouse VR1 gene using standard gene targeting techniques. Small diameter dorsal root ganglion neurons isolated from VR1-null mice lacked many of the capsaicin-, acid- and heat-gated responses that have been previously well characterized in small diameter dorsal root ganglion neurons from various species. Furthermore, although the VR1-null mice appeared normal in a wide range of behavioural tests, including responses to acute noxious thermal stimuli, their ability to develop carrageenan-induced thermal hyperalgesia was completely absent. We conclude that VR1 is required for inflammatory sensitization to noxious thermal stimuli but also that alternative mechanisms are sufficient for normal sensation of noxious heat.

The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1).

The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR‐based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 μM) and capsaicin (1 μM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 μM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC50 values of 5.94±0.06 (n=5) and 7.13±0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pKB of 7.40±0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide‐induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1.

Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes

The role of the cell wall hydrolase polygalacturonase (PG) during fruit ripening was investigated using novel mutant tomato lines in which expression of the PG gene has been down regulated by antisense RNA. Tomato plants were transformed with chimaeric genes designed to express anti-PG RNA constitutively. Thirteen transformed lines were obtained of which five were analysed in detail. All contained a single PG antisense gene, the expression of which led to a reduction in PG enzyme activity in ripe fruit to between 5% and 50% that of normal. One line, GR16, showed a reduction to 10% of normal PG activity. The reduction in activity segregated with the PG antisense gene in selfed progeny of GR16. Plants homozygous for the antisense gene showed a reduction of PG enzyme expression of greater than 99%. The PG antisense gene was inherited stably through two generations. In tomato fruit with a residual 1% PG enzyme activity pectin depolymerisation was inhibited, indicating that PG is involved in pectin degradation in vivo. Other ripening parameters, such as ethylene production, lycopene accumulation, polyuronide solubilisation, and invertase activity, together with pectinesterase activity were not affected by the expression of the antisense gene.

The signaling peptide EPF2 controls asymmetric cell divisions during stomatal development.

Stomata are pores in the plant epidermis that control carbon dioxide uptake and water loss. They are major regulators of global carbon and water cycles [1]. Several signaling components that regulate stomatal development have been characterized. These include a putative secretory peptide EPF1, LRR receptor components TMM and ER, and a peptidase SDD1 [2-4]. We have identified EPF2, a peptide related to EPF1 that is expressed in proliferating cells of the stomatal lineage, known as meristemoids, and in guard mother cells, the progenitors of stomata. EPF2 expression during leaf development affects stomatal density on the mature leaf. In the absence of EPF2, excessive numbers of cells enter the stomatal lineage and produce numerous small epidermal cells that express stomatal lineage reporter genes, whereas plants overexpressing EPF2 produce virtually no stomata. Results from genetic experiments indicate that EPF2 regulates a different aspect of stomatal development to EPF1 and are consistent with EPF2 acting in a pathway to regulate stomatal density that involves ER and TMM, but not SDD1. We propose that EPF2 is expressed earlier in leaf development than EPF1 and is involved in determining the number of cells that enter, and remain in, the stomatal lineage.

The Arabidopsis Cyclophilin Gene Family

Database searching has allowed the identification of a number of previously unreported single and multidomain isoform members of the Arabidopsis cyclophilin gene family. In addition to the cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, the latter contain a variety of other domains with characterized functions. Transcriptional analysis showed they are expressed throughout the plant, and different isoforms are present in all parts of the cell including the cytosol, nucleus, mitochondria, secretory pathway, and chloroplast. The abundance and diversity of cyclophilin isoforms suggests that, like their animal counterparts, plant cyclophilins are likely to be important proteins involved in a wide variety of cellular processes. As well as fulfilling the basic role of protein folding, they may also play important roles in mRNA processing, protein degradation, and signal transduction and thus may be crucial during both development and stress responsiveness.

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.

Nitric Oxide Sensing in Plants Is Mediated by Proteolytic Control of Group VII ERF Transcription Factors

Summary Nitric oxide (NO) is an important signaling compound in prokaryotes and eukaryotes. In plants, NO regulates critical developmental transitions and stress responses. Here, we identify a mechanism for NO sensing that coordinates responses throughout development based on targeted degradation of plant-specific transcriptional regulators, the group VII ethylene response factors (ERFs). We show that the N-end rule pathway of targeted proteolysis targets these proteins for destruction in the presence of NO, and we establish them as critical regulators of diverse NO-regulated processes, including seed germination, stomatal closure, and hypocotyl elongation. Furthermore, we define the molecular mechanism for NO control of germination and crosstalk with abscisic acid (ABA) signaling through ERF-regulated expression of ABSCISIC ACID INSENSITIVE5 (ABI5). Our work demonstrates how NO sensing is integrated across multiple physiological processes by direct modulation of transcription factor stability and identifies group VII ERFs as central hubs for the perception of gaseous signals in plants.

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.

Action of the Style Product of the Self-Incompatibility Gene of Nicotiana alata (S-RNase) on in Vitro-Grown Pollen Tubes.

The products of the S-locus expressed in female tissues of Nicotiana alata are ribonucleases (S-RNases). The arrest of growth of incompatible pollen tubes in styles may result from entry of the S-RNase into the pollen tube and degradation of pollen tube RNA. We investigated the action of isolated S-RNases on pollen tubes grown in vitro and found that S-RNase is taken up by the pollen without substantial alteration. The S-RNases inhibit incorporation of exogenously added radioactive amino acids into protein by the germinated pollen. The S-RNases also inhibit in vitro translation of pollen tube RNA in a wheat germ cell-free extract. We found no evidence for a specific mRNA substrate for the S-RNases, which implies that if RNase activity is involved in the control of self-incompatibility, allelic specificity is more likely to depend on the selective uptake of S-RNases into pollen tubes or their selective activation or inactivation by pollen factors, rather than cleavage of a specific substrate. Heat treating S2-RNase largely destroys its RNase activity but increases its inhibitory effect on in vitro pollen tube growth. This effect is not due to an increased uptake of S2-RNase by the pollen but is associated with a greatly enhanced accumulation of S2-RNase on the outer surface of the pollen grains.

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].