Functional Interactions of Lanthanum and Phospholipase D with the Abscisic Acid Signaling Effectors VP1 and ABI1-1 in Rice Protoplasts

Abstract

cis,trans-Abscisic acid (ABA) plays an important role in plant growth and development, regulation of seed maturation, germination, and adaptation to environmental stresses. Knowledge of ABA mechanisms of action and the interactions of components required for ABA signal transduction is far from complete. Using transient gene expression in rice protoplasts, we observed additive and inhibitory effects between maize VP1 (Viviparous-1, a transcriptional activator) and a dominant-negative mutant protein phosphatase, ABI1-1 (ABA-insensitive-1-1), from Arabidopsis. Lanthanide ions were shown to be specific agonists of ABA-inducible gene expression and to interact synergistically with ABA and overexpressed VP1. Both VP1 and lanthanum activities could be antagonized by coexpression of ABI1-1, which demonstrates the specific ABA dependence of these effectors on ABA-regulated gene expression. We obtained pharmacological evidence that phospholipase D (PLD) functions in ABA-inducible gene expression in rice. Antagonism of ABA, VP1, and lanthanum synergy by 1-butanol, a specific inhibitor of PLD, was similar to the inhibition by coexpression of ABI1-1. These results demonstrate that ABA, VP1, lanthanum, PLD, and ABI1 are all involved in ABA-regulated gene expression and are consistent with an integrated model whereby La 3؉ acts upstream of PLD.cis,trans-Abscisic acid (ABA) 1 modulates seed development, dormancy, cell division, stomatal movements, and cellular responses to environmental stresses such as drought, cold, salt, pathogen attack, and UV light (1-3). Despite its importance in plant growth and development, the mechanisms of ABA action are largely unknown, and there may be more than one ABA signal transduction pathway leading to both fast (guard cell ion fluxes) and slow (gene regulation) cellular responses (4 -6). Transient gene expression studies with LEA (late embryogenesis abundant) and drought-inducible gene promoters have defined the cis-acting elements necessary and sufficient to confer ABA-inducible transcription (7-10). Separate ABA-responsive elements and coupling elements function cooperatively and redundantly as an ABA response complex (9,11,12).Recent insight into the mechanisms of ABA-inducible transcription have come from cloning of TRAB1 (transcription factor responsible for ABA regulation), a basic leucine zipper transcription factor that binds ABA response complex promoter elements and VP1 (Viviparous-1), a transcriptional activator required for ABA-regulated gene expression during seed maturation (13-15). Other proteins interact with ABA response complexes, VP1 orthologs, and basic leucine zipper factors, but there is no direct evidence that these factors function in ABA signaling (16 -27). Regulation by ABA of TRAB1 and VP1 transactivation of ABA response complexes is not at the level of DNA binding, based on transient gene expression and in vivo footprinting assays (13,28). Consistent with this model is the finding that the bean ortholog of VP1, PvALF, functions to remodel chromatin independent of e...

Abscisic Acid Biosynthesis and Response

“…As described for guard cell signaling, ABA-induction of gene expression depends on action of Ca 2+ , IP 3 , PA and cADPR as secondary messengers (Gilroy and Jones, 1992;HeimovaaraDijkstra et al, 1995;Wu et al, 1997;Ritchie and Gilroy, 1998;Ghelis et al, 2000b;Webb et al, 2001) and may require S-type anion channel activity (Ghelis et al, 2000a). Interactions among transcription factors and their dependence on specific secondary messengers or phosphorylation states has also been analyzed in transient assays following bombardment or electroporation of genes encoding these signaling components (Hagenbeek et al, 2000;Uno et al, 2000;Gampala et al, 2001b).…”

“…Secondary messengers in ABA signaling regulating stomatal function and gene expression include inositol triphosphate (IP 3 ) and phosphatidic acid (PA), produced by phospholipase C (PLC) and phospholipase D (PLD), respectively (see following section) (Gilroy et al, 1990;Jacob et al, 1999;Ritchie and Gilroy, 1998;Gampala et al, 2001b). Arabidopsis contains 6 PLC genes; ABA induces expression of only one of these, AtPLC1 (Hirayama et al, 1995).…”

A Battery of Transcription Factors Involved in the Regulation of Secondary Cell Wall Biosynthesis in Arabidopsis

“…Arabidopsis leaf protoplasts were cotransfected with the reporter and effector constructs together with a construct containing the firefly luciferase gene driven by the CaMV 35S promoter for determination of the transfection efficiency. After a 20-h incubation, transfected protoplasts were lysed and the soluble extracts were used for analysis of GUS and luciferase activities as described (Gampala et al, 2001) or for real-time quantitative PCR analysis of the effector mRNA level. The GUS activity was normalized against the luciferase activity in each transfection, and the data were the average of three biological replicates.…”

The MYB46 Transcription Factor Is a Direct Target of SND1 and Regulates Secondary Wall Biosynthesis in Arabidopsis

“…Arabidopsis leaf protoplasts were cotransfected with the reporter and effector constructs together with a construct containing the firefly luciferase gene driven by the CaMV 35S promoter for determination of the transfection efficiency. After 20 h of incubation, transfected protoplasts were lysed, and the soluble extracts were used for analysis of GUS and luciferase activities as described (Gampala et al, 2001). The GUS activity was normalized against the luciferase activity in each transfection, and the data were the average of three biological replicates.…”