RESUMEN
Spores of the fern Ceratopteris richardii have proven to be a valuable single-cell system for studying gravity responses. The earliest cellular change directed by gravity in these cells is a trans-cell calcium current, which peaks near 10 h after the spores are induced to germinate. This current is needed for gravity-directed axis alignment, and its peak is coincident with the time period when gravity polarises the direction of subsequent nuclear migration and rhizoid growth. Transcriptomic analysis of genes expressed at the 10-h time point revealed several that encode proteins likely to be key components that either drive the current or regulate it. Notable among these is a plasma membrane (PM)-type Ca(2+) ATPase, CrACA1, whose activity pumping Ca(2+) out of cells is regulated by gravity. This report provides an initial characterisation of the structure and expression of this protein, and demonstrates its heterologous function complementing the K616 mutant of yeast, which is deficient in PM-type Ca(2+) pump activity. Gravity-induced changes in the trans-cell Ca(2+) current occur within seconds, a result consistent with the hypothesis that the force of gravity can rapidly alter the post-translational state of the channels and pumps that drive this current across spore cells. This report identifies a transporter likely to be a key driver of the current, CrACA1, and characterises the role of this protein in early germination and gravity-driven polarity fixation through analysis of expression levels, functional complementation and pharmacological treatments. These data, along with newly available transcriptomic data obtained at the 10-h time point, indicate that CrACA1 is present, functional and likely a major contributing component of the trans-cell Ca(2+) efflux. CrACA1 is not necessary for polar axis alignment, but pharmacological perturbations of it disrupt rhizoid development. These data support and help refine the post-translational modification model for gravity responses.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Membrana Celular/enzimología , Helechos/enzimología , Gravitación , Proteínas de Plantas/metabolismo , Esporas/enzimología , Secuencia de Aminoácidos , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/química , Membrana Celular/efectos de los fármacos , Pruebas de Enzimas , Inhibidores Enzimáticos/farmacología , Helechos/citología , Helechos/efectos de los fármacos , Helechos/crecimiento & desarrollo , Prueba de Complementación Genética , Datos de Secuencia Molecular , Proteínas de Plantas/química , Estructura Terciaria de Proteína , Alineación de Secuencia , Esporas/citología , Esporas/efectos de los fármacos , Esporas/crecimiento & desarrollo , Relación Estructura-ActividadRESUMEN
Annexins are a multigene family in most plant species and are suggested to play a role in a wide variety of essential cellular processes. In Arabidopsis thaliana there are eight different annexins (AnnAt1-8), which range from 29% to 83% in deduced amino acid sequence identity. As a first step toward clarifying the individual functions of these annexins, in this study we have used quantitative real time reverse transcription PCR to assess their differential expression in different tissues or after different stimuli. We determined which annexins are expressed during germination and early seedling growth by assaying annexin expression levels in dry and germinating seeds and in 7-day-old light-grown seedlings. Our results indicate that transcripts for all eight annexins are present in germinating seeds and that transcript levels for all the annexins increase by 7 days of normal growth. We assayed transcript levels in dark grown roots, cotyledons, and hypocotyls and found that the relative abundance of each annexin varied in these dark-grown tissues. We also examined the effects of red and far red light treatments on annexin expression in 5.5-day-old etiolated seedlings. Light treatments significantly altered transcript levels in hypocotyls and cotyledons for only two members of the gene family. Finally, we monitored annexin expression changes in response to a variety of abiotic stresses. We found that the expression of most of the Arabidopsis annexin genes is differentially regulated by exposure to salt, drought, and high- and low-temperature conditions, indicating a likely role for members of this gene family in stress responses.