We show in bryophytes that abscisic acid (ABA) pretreatment of moss (Physcomitrella patens) cells confers desiccation tolerance. In angiosperms, both ABA and the transcriptional regulator ABSCISIC ACID INSENSITIVE 3 (ABI3) are required to protect the seed during desiccation. ABA was not able to protect moss cells in stable deletion lines of ABI3 (DeltaPpabi3). Hence, moss has the same functional link between ABA, ABI3, and the desiccation tolerance phenotype that is found in angiosperms. Furthermore, we identified 22 genes that were induced during ABA pretreatment in wild-type lines. When their expression was compared with that of DeltaPpabi3 during ABA pretreatment and immediately after desiccation, a new target of ABI3 action appears to be in the recovery period.

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.

The root-knot nematode, Meloidogyne incognita, is an obligate parasite which depends entirely on the host plant for its nutrition. Root-knot nematodes induce the formation of a highly specialized feeding site consisting of several giant cells surrounded by a network of vascular tissues. Nutrients, including amino acids and sugars, are transferred apoplastically from the vascular tissues to the feeding site. Using Arabidopsis thaliana lacking the vascular-expressed amino acid permeases (AAP) AAP3 or AAP6, we demonstrate that disruption of amino acid transport can affect nematode parasitism. Nematode infestation levels are significantly reduced on the aap3 and aap6 mutants. AAP3 and AAP6 act distinctly in the transport of amino acids to the feeding site, as demonstrated by differences in their carrying capacity profiles. Furthermore, analyses of promoter: β-glucuronidase lines show different expression patterns for AAP3 and AAP6 in infected roots. In the aap3-3 mutant, part of the decrease in infestation is connected to a defect in early infection, where juveniles enter but then leave the root. Both aap3-3 and aap6-1 produce fewer females and produce more adult male nematodes. Additionally, detrimental effects are observed in the nematodes harvested from aap3-3 and aap6-1 mutants, including decreased egg hatching and infectivity and lower levels of lipid reserves. The transport of amino acids by AAP3 and AAP6 is important for nematode infection and success of the progeny.

The transcriptional regulator VIVIPA-ROUS1 (VP1) is composed of four functional domains that control different aspects of gene expression during seed development. The B2 domain is required for its role as a transcriptional activator, functioning at the site of transcription and/or for its transport into the nucleus. Previous work showed that the B2 domain was required for transactivation of the Em promoter. We demonstrate that VP1::GFP localizes to the nucleus of barley (Hordeum vulgare) aleurone cells, but when B2 is deleted, nuclear accumulation is lost. However, the B2 domain itself is not sufficient for nuclear localization of GFP::GUS. Using point mutagenesis on the putative NLS within B2, we show that the VP1::GFP still accumulates in the nucleus. Utilizing a comparative approach, through the alignment of B2 domains from various VP1/ABI3 proteins, oincluding the ABI3 orthologs from Physcomitrella patens, revealed the involvement of other conserved amino acids. Mutating VP1 at the conserved threonine on the N-terminal side of the putative NLS and at a conserved arginine-glutamine-arginine sequence on the C-terminal side prevented nuclear localization of VP1. A single amino acid change, from alanine to threonine, within this NLS found in the Arabidopsis abi3-7 mutant prevents transcription of AtEm1 and AtEm6 in vivo. We show that this same mutation in VP1 prevents transactivation of the Em-GUS reporter in barley aleurone but does not interfere with nuclear localization. Our data demonstrate that the B2 domain of VP1 is bifunctional in nature regulating both nuclear localization and transactivation.

We describe an efficient inducible system to regulate gene expression in plants based on quorum-sensing components found in Gram-negative bacteria such as Agrobacterium tumefaciens. These bacteria monitor their own population density by utilizing members of the N-acyl homoserine lactone family as inducers and a transcriptional activator as its receptor. In our study, we utilize the components from A. tumefaciens (i.e. 3-oxooctanyl-L-homoserine lactone [OOHL]) synthesized by the TraI protein and its receptor, TraR. When OOHL binds to TraR, it recognizes its specific cis-element, the tra box. We translationally fused the eukaryotic VP16 activation domain to the N terminus of TraR. In the presence of OOHL, the chimeric VP16:TraR transcriptional regulator induces reporter gene expression in moss (Physcomitrella patens), barley (Hordeum vulgare), and carrot (Daucus carota) cells, as well as in transgenic Arabidopsis (Arabidopsis thaliana) seedlings. The inducible system shows a low level of reporter gene expression in the absence of the inducer. Foliar application and a floating-leaf assay in the presence of the inducer shows a 30-and 200-fold induction, respectively. Induction by foliar application of the inducer to whole seedlings is achieved within 8 h. The VP16:TraR activator also shows specificity for binding to its cognate inducer, OOHL. Based on microarray analyses, endogenous gene expression is not significantly affected due to overexpression of the TraR protein or presence of OOHL in either wild-type or lactone-inducible transgenic plants.