The phytohormone abscisic acid (ABA) promotes plant water conservation by decreasing the apertures of stomatal pores in the epidermis through which water loss occurs. We found that Arabidopsis thaliana plants harboring transferred DNA insertional mutations in the sole prototypical heterotrimeric GTP-binding (G) protein alpha subunit gene, GPA1, lack both ABA inhibition of guard cell inward K(+) channels and pH-independent ABA activation of anion channels. Stomatal opening in gpa1 plants is insensitive to inhibition by ABA, and the rate of water loss from gpa1 mutants is greater than that from wild-type plants. Manipulation of G protein status in guard cells may provide a mechanism for controlling plant water balance.

significant differences between axon-bearing and ax-presented (abstract 291.5) at the 29th Meeting of the 26. A. 8. Ali and A. M. Thomson,]. ~h~s i o l . (london) 507, on-lacking dendrites.Abscisic acid (ABA) stimulates stomatal closure and thus supports water conservation by plants during drought. Mass spectrometry-generated peptide sequence information was used t o clone a Vicia faba complementary DNA, AAPK, encoding a guard cell-specific ABA-activated serine-threonine protein kinase (AAPK). Expression in transformed guard cells of AAPK altered by one amino acid (lysine 43 t o alanine 43) renders stomata insensitive t o ABA-induced closure by eliminating ABA activation of plasma membrane anion channels. This information should allow cell-specific, targeted biotechnological manipulation of crop water status.

Summary• Guard cells play an important role in the physiology and development of plants. The genetic resources available for Arabidopsis thaliana make it the most favorable plant species for the study of guard cell processes, but it is not easy to isolate highly purified preparations of large numbers of guard cells from this species. Here, we describe methods for isolation of both guard cell and mesophyll cell protoplasts from A. thaliana and their use in the study of unique biochemical and cellular properties of these cell types.• Protocols developed for large-and small-scale preparation of guard cell protoplasts and mesophyll cell protoplasts are described, followed by specific examples of their use in electrophysiological, biochemical and molecular approaches such as patch clamping, enzyme assays, and reverse-transcription polymerase chain reaction.• The protocols described yield millions of highly purified, viable guard cell protoplasts and mesophyll cell protoplasts from A. thaliana. These protoplasts have been used successfully in the study of ion channel properties, assay of ABA activation in phospholipase D activity and comparisons of gene and protein expression levels.• These techniques make it possible to elucidate electrophysiological, biochemical and molecular genetic pathways of guard cell function.

No abstract