Characterization and functional analysis of ABSCISIC ACID INSENSITIVE3‐like genes from Physcomitrella patens

Abstract

SummaryAlthough the moss Physcomitrella patens is known to respond to abscisic acid (ABA) by activating gene expression, the transcriptional components involved have not been characterized. Initially, we used the ABAresponsive Em promoter from wheat linked to b-glucuronidase (GUS) to determine whether ABI3/VP1, transcriptional regulators in the ABA-signaling pathway in angiosperms, were similarly active in the ABA response of P. patens. We show by particle bombardment that ABI3 and VP1 affect Em-GUS expression in P. patens in a manner similar to angiosperms. We also show the involvement of ABI1 in the pathway, utilizing the abi1-1 mutant allele. We isolated three ABI3-like genes from P. patens. Using an Em-like ABA-responsive promoter from P. patens (PpLea1), we demonstrate that PpABI3A, only in the presence of ABA, strongly enhances PpLea1-GUS expression in P. patens. PpABI3A also enhances ABA-induced Em-GUS expression in P. patens. In barley aleurone, PpABI3A transactivates Em-GUS but to a lesser extent than VP1 and ABI3. PpABI3A:GFP is localized to the nucleus of both protonemal cells and barley aleurone, indicating that the nuclear localization signals are conserved. We show that at least a part of the inability of PpABI3A to fully complement the phenotypes of the Arabidopsis abi3-6 mutant is due to a weak interaction between PpABI3A and the bZIP transcription factor ABI5, as assayed functionally in barley aleurone and physically in the yeasttwo-hybrid assay. Our data clearly demonstrate that P. patens will be useful for comparative structural and functional studies of components in the ABA-response pathway such as ABI3.

Oil Bodies and Oleosins in Physcomitrella Possess Characteristics Representative of Early Trends in Evolution

“…Such a plant system was not previously available (Miao and Jiang, 2007), and transient expression of genes in Physcomitrella has been performed only with juvenile protonema (e.g. Marella et al, 2006), which contain few or no OBs (Fig. 1A).…”

Evolutionarily Conserved Regulatory Mechanisms of Abscisic Acid Signaling in Land Plants: Characterization of ABSCISIC ACID INSENSITIVE1-Like Type 2C Protein Phosphatase in the Liverwort Marchantia polymorpha

“…To show molecular responses to ABA in M. polymorpha, we conducted transient assays using the Em-GUS construct, which had been successfully used for studies on ABA signaling (Marella et al, 2006). Specific sequence elements such as the ABA-responsive element ABRE found in the promoter of Em are thought to function in both higher plant and moss cells (Knight et al, 1995;Kamisugi and Cuming, 2005).…”

“…Transient assays of P. patens protonema cells were carried out according to the protocol described by Marella et al (2006). In brief, 6-d-old P. patens cells were bombarded with Em-GUS as a reporter construct, Ubi-LUC as an internal control construct, and the wild-type and mutant MpABI1 genes as effecter constructs using the PDS-1000He particle delivery system (Bio-Rad).…”

“…The Em-GUS construct has been used for studies of ABA-responsive gene expression in transient assays of higher and lower plants (Marcotte et al, 1989;Marella et al, 2006). To examine the effect of MpABI1 overexpression on GUS activity, MpABI1 cDNA was fused to the downstream region of the rice (Oryza sativa) actin promoter ( Fig.…”

“…Marella et al (2006) identified PpABI3, which positively regulates ABA-induced expression of the wheat (Triticum aestivum) Em promoter fused to the GUS reporter gene (Em-GUS). This indicated that transcriptional machinery for ABA-induced gene expression is conserved in mosses.…”