In guard cells, the plant hormone abscisic acid (ABA) inhibits stomatal opening and induces stomatal closure through the coordinated regulation of ion transport. Despite this central role of ABA in regulating stomatal function, the signal transduction events leading to altered ion fluxes remain incompletely understood. We report that the activity of the enzyme phospholipase D (PLD) transiently increased in guard cell protoplasts at 2.5 and 25 min after ABA application. Treatment of guard cell protoplasts with phosphatidic acid (PtdOH), one of the products of PLD activity, led to an inhibition of the activity of the inward K ؉ channel. PtdOH also induced stomatal closure and inhibited stomatal opening when added to epidermal peels. Application of 1-butanol (1-buOH), a selective inhibitor of PtdOH production by PLD, inhibited the increase in PtdOH production elicited by ABA. 1-BuOH treatment also partially prevented ABA-induced stomatal closure and ABA-induced inhibition of stomatal opening. This inhibitory effect of buOH was enhanced by simultaneous application of nicotinamide, an inhibitor of cADP ribose action. These results suggest that in the guard cell, ABA activates the enzyme PLD, which leads to the production of PtdOH. This PtdOH is then involved in triggering subsequent ABA responses of the cell via a pathway operating in parallel to cADP ribose-mediated events.

The plant hormones abscisic acid (ABA) and gibberellic acid (GA) are important regulators of the dormancy and germination of seeds. In cereals, GA enhances the synthesis and secretion of enzymes (principally ␣-amylases) in the aleurone cells of the endosperm, which then mobilize the storage reserves that fuel germination. ABA inhibits this enhanced secretory activity and delays germination. Despite the central role of ABA in regulating germination, the signal transduction events leading to altered gene expression and cellular activity are essentially unknown. We report that the application of ABA to aleurone protoplasts increased the activity of the enzyme phospholipase D (PLD) 10 min after treatment. The product of PLD activity, phosphatidic acid (PPA), also increased transiently at this time. The application of PPA to aleurone protoplasts led to an ABA-like inhibition of ␣-amylase production, and induction of the ABA upregulated proteins ASI (amylase subtilisin inhibitor) and RAB (responsive to ABA). Inhibition of PLD activity by 0.1% 1-butanol during the initial 20 min of ABA treatment resulted in inhibition of ABA-regulated processes. This inhibition coincided with the timing of PLD activation by ABA and was overcome by simultaneous addition of PPA. These results suggest that ABA activates the enzyme PLD to produce PPA that is involved in triggering the subsequent ABA responses of the aleurone cell.

The organization of microtubule arrays in the plant cell cortex involves interactions with the plasma membrane, presumably through protein bridges. We have used immunochemistry and monoclonal antibody 6G5 against a candidate bridge protein, a 90-kD tubulin binding protein (p90) from tobacco BY-2 membranes, to characterize the protein and isolate the corresponding gene. Screening an Arabidopsis cDNA expression library with the antibody 6G5 produced a partial clone encoding phospholipase D (PLD), and a full-length gene was obtained by sequencing a corresponding expressed sequence tag clone. The predicted protein of 857 amino acids contains the active sites of a phospholipidmetabolizing enzyme and a Ca 2 ؉-dependent lipid binding domain and is identical to Arabidopsis PLD ␦. Two amino acid sequences obtained by Edman degradation of the tobacco p90 are identical to corresponding segments of a PLD sequence from tobacco. Moreover, immunoprecipitation using the antibody 6G5 and tobacco BY-2 protein extracts gave significant PLD activity, and PLD activity of tobacco BY-2 membrane proteins was enriched 6.7-fold by tubulin-affinity chromatography. In a cosedimentation assay, p90 bound and decorated microtubules. In immunofluorescence microscopy of intact tobacco BY-2 cells or lysed protoplasts, p90 colocalized with cortical microtubules, and taxol-induced microtubule bundling was accompanied by corresponding reorganization of p90. Labeling of p90 remained along the plasma membrane when microtubules were depolymerized, although detergent extraction abolished the labeling. Therefore, p90 is a specialized PLD that associates with membranes and microtubules, possibly conveying hormonal and environmental signals to the microtubule cytoskeleton.

The range of processes regulated by gibberellins (GAs) covers all aspects of the life history of the plant from seed germination to vegetative growth and flowering. In seeds there has been an intensive search, using the techniques of both biochemistry and cell biology, for the regulatory molecules linking GA perception to gene regulation and the events of germination. Although a GA receptor has yet to be identified, the site of perception has been localized to the plasma membrane. Calmodulin, Ca2+ and cGMP have also been identified as elements of the GA signal transduction pathway. These regulators parallel many of the signalling elements identified in the transduction of other signals such as phytochrome and ABA. Studies of GA‐regulated gene expression, principally of the α‐amylases of cereal aleurone, have identified core GA‐responsive promoter elements, such as the gibberellin response element (GARE), box‐1 and pyrimidine boxes, as well as elements that may lend specificity to GA‐regulated expression, such as the Opaque‐2‐similar element (O2S), and TRE and CRE motifs. One of the most striking features of all of these studies of the molecular basis of GA action is the interaction of GA‐dependent regulatory elements with those of other factors such as ABA. GA‐response elements also appear to be conserved between disparate GA‐response systems. For example, Myb transcription factors appear to regulate a multitude of GA‐induced genes in cereal aleurone as well as to alter GA responses when expressed in Arabidopsis. Thus the study of GA signal transduction and response systems is highlighting the conservation of regulatory elements used by plants. These common factors, used by distinct signal transduction systems, provide a molecular basis for the integration of the GA signal with other growth regulators that is the hallmark of plant growth and development.

We have previously determined that phospholipase D (PLD) is activated by abscisic acid (ABA), and this activation is required for the ABA response of the cereal aleurone cell. In this study, ABA-stimulated PLD activity was reconstituted in vitro in microsomal membranes prepared from aleurone protoplasts. The transient nature (20 min) and degree (1.5-to 2-fold) of activation in vitro were similar to that measured in vivo. Stimulation by ABA was only apparent in the membrane fraction and was associated with a fraction enriched in plasma membrane. These results suggest that an ABA receptor system and elements linking it to PLD activation are associated with the aleurone plasma membrane. The activation of PLD in vitro by ABA was dependent on the presence of GTP. Addition of GTP␥S transiently stimulated PLD in an ABA-independent manner, whereas treatment with GDP␤S or pertussis toxin blocked the PLD activation by ABA. Application of pertussis toxin to intact aleurone protoplasts inhibited the ability of ABA to activate PLD as well as antagonizing the ability of ABA to down-regulate gibberellic acid-stimulated ␣-amylase production. All of these data support the hypothesis that ABA stimulation of PLD activity occurs at the plasma membrane and is mediated by G-protein activity.