Flow cytometry and surface plasmon resonance analyses demonstrate that the monoclonal antibody JIM19 interacts with a rice cell surface component involved in abscisic acid signalling in protoplasts.

Abscisic acid (ABA) is a plant hormone involved in many developmental and physiological processes, but as yet, no ABA receptor has been identified. Flow cytometry of rice protoplasts and immunoblotting of purified plasma membranes (PMs) have been used to demonstrate that the monoclonal antibody JIM19 recognizes carbohydrate epitopes of cell surface glycoproteins. Using surface plasmon resonance technology specific binding of PMs to JIM19 was observed. Such interaction was antagonized significantly by ABA, but not by the biologically inactive ABA catabolite phaseic acid. These in vitro interactions were correlated with the biological activities of JIM19, ABA and phaseic acid on activation of the ABA-inducible Em promoter using two different transient reporter gene assays, beta-glucuronidase/luciferase and quantitative flow cytometry of Aequoria green fluorescent protein. Pre-treatment with JIM19 resulted in significant inhibition of ABA-inducible gene expression. Taken together, these data suggest that JIM19 interacts with a functional PM complex involved in ABA signalling.     

Lanthanide ions are agonists of transient gene expression in rice protoplasts and act in synergy with ABA to increase Em gene expression

Previous work has shown that in rice suspension cells, NaCl at 0.4 M can induce Em gene expression and act synergistically with ABA, possibly by potentiating the ABA response pathway through a rate-limiting intermediate (R.M. Bostock and R.S. Quatrano (1992) Plant Physiol., 98, 1356–1363). Since calcium is an intermediate in ABA regulation of stomatal closure, we tested the effect of calcium changes on ABA-inducible Em gene expression in transiently transformed rice protoplasts. We show that calcium is required for ABA-inducible Em-GUS expression and can act in synergy with ABA. The trivalent ions lanthanum, gadolinium, and aluminum, which are known to interact with calcium- and other signaling pathways, can act at sub-millimolar concentrations to increase GUS reporter gene expression driven by several promoters in transiently transformed rice protoplasts. This effect is not specific for the ABA-inducible Em promoter, but is synergistic with ABA. The lanthanum synergy with ABA does not require calcium. In rice suspension cells, lanthanum alone does not induce Em gene expression, in contrast to transiently transformed protoplasts, yet can act synergistically with ABA to effectively increase the sensitivity to ABA greater than tenfold. Trivalent ions may be a useful tool to study the regulation of gene expression. The possible effects of trivalent ions on ABA signal transduction and gene expression are discussed.     

Trivalent ions activate abscisic acid-inducible promoters through an ABI1-dependent pathway in rice protoplasts

The plant hormone abscisic acid (ABA) mediates many vital processes in plant growth and development, including seed dormancy, cell division, water use efficiency, and adaptation to drought, salinity, chilling, pathogen attack, and UV light. Our understanding of ABA signal transduction is fragmentary and would benefit from specific and facile probes of the process. Protoplasts from rice (Oryza sativa L. cv IR54) embryonic suspension cultures cotransformed with effector plasmids encoding the maize (Zea mays) VIVIPAROUS1 cDNA and/or the Arabidopsis dominant negative mutant (abi1-1) ABA-insensitive cDNA demonstrated genetic interactions of VIVIPAROUS1 and abi1-1 in transactivation of the ABA-inducible HVA1 promoter from barley (Hordeum vulgare), suggesting the mechanisms of these effectors are conserved among monocots and dicots. Trivalent ions have been shown to act as an effector of gene expression in plants and animals, although the mechanism of action is unknown. We show in two complementary transient ABA-inducible gene expression assays (beta-glucuronidase and luciferase enzymatic activities and quantitative flow cytometry of green fluorescent protein) that trivalent ions specifically interact with an ABI1-dependent ABA-signaling pathway leading to gene expression. Trivalent ions mimic ABA effects on gene expression and may be a useful tool to study ABA signaling.     

The cis-regulatory element CCACGTGG is involved in ABA and water-stress responses of the maize gene rab28

The maize gene rab28 has been identified as ABA-inducible in embryos and vegetative tissues. It is also induced by water stress in young leaves. The proximal promoter region contains the conserved cis-acting element CCACGTGG (ABRE) reported for ABA induction in other plant genes. Transient expression assays in rice protoplasts indicate that a 134 bp fragment (-194 to -60 containing the ABRE) fused to a truncated cauliflower mosaic virus promoter (35S) is sufficient to confer ABA-responsiveness upon the GUS reporter gene. Gel retardation experiments indicate that nuclear proteins from tissues in which the rab28 gene is expressed can interact specifically with this 134 bp DNA fragment. Nuclear protein extracts from embryo and water-stressed leaves generate specific complexes of different electrophoretic mobility which are stable in the presence of detergent and high salt. However, by DMS footprinting the same guanine-specific contacts with the ABRE in both the embryo and leaf binding activities were detected. These results indicate that the rab28 promoter sequence CCACGTGG is a functional ABA-responsive element, and suggest that distinct regulatory factors with apparent similar affinity for the ABRE sequence may be involved in the hormone action during embryo development and in vegetative tissues subjected to osmotic stress.     

Plant natriuretic peptides are apoplastic and paracrine stress response molecules

Higher plants contain biologically active proteins that are recognized by antibodies against human atrial natriuretic peptide (ANP). We identified and isolated two Arabidopsis thaliana immunoreactive plant natriuretic peptide (PNP)-encoding genes, AtPNP-A and AtPNP-B, which are distantly related members of the expansin superfamily and have a role in the regulation of homeostasis in abiotic and biotic stresses, and have shown that AtPNP-A modulates the effects of ABA on stomata. Arabidopsis PNP (PNP-A) is mainly expressed in leaf mesophyll cells, and in protoplast assays we demonstrate that it is secreted using AtPNP-A:green fluorescent protein (GFP) reporter constructs and flow cytometry. Transient reporter assays provide evidence that AtPNP-A expression is enhanced by heat, osmotica and salt, and that AtPNP-A itself can enhance its own expression, thereby generating a response signature diagnostic for paracrine action and potentially also autocrine effects. Expression of native AtPNP-A is enhanced by osmotica and transiently by salt. Although AtPNP-A expression is induced by salt and osmotica, ABA does not significantly modulate AtPNP-A levels nor does recombinant AtPNP-A affect reporter expression of the ABA-responsive RD29A gene. Together, these results provide experimental evidence that AtPNP-A is stress responsive, secreted into the apoplastic space and can enhance its own expression. Furthermore, our findings support the idea that AtPNP-A, together with ABA, is an important component in complex plant stress responses and that, much like in animals, peptide signaling molecules can create diverse and modular signals essential for growth, development and defense under rapidly changing environmental conditions.     

Regulation of the maizerab17 gene promoter in transgenic heterologous systems

The maizerab17 gene is expressed in different plant parts in response to ABA and osmotic stress (J. Vilardellet al., Plant Mol Biol 14 (1990) 423–432). Here we demonstrate that 5 upstream sequences of therab17 gene confer the appropriate patterns of expression on the chloramphenicol acetyl transferase (CAT) reporter gene in transgenic tobacco plants, as well as in protoplasts derived from cultured rice cells. Specifically, a CAT construct containing a large 5 upstream fragment ofrab17 (–1330/+29) results in high levels of CAT activity in embryos, leaves and roots of transgenic plants subjected to water stress or ABA treatment. Transient expression assays in rice protoplasts transfected with CAT genes fused torab17 promoter deletions indicate that a 300 bp DNA fragment (–351/–102) is sufficient to confer ABA responsiveness upon the reporter gene. Furthermore, a 100 bp sequence (–219/–102) is capable of conferring ABA responsiveness upon a minimal promoter derived from the 35S CaMV promoter. Gel retardation experiments indicate that maize nuclear proteins bind to this fragment. This region of 100 bp contains a sequence (ACGTGGC) which has been identified as an abscisic acid response element in studies of other ABA-responsive plant genes.     

Cre-lox univector acceptor vectors for functional screening in protoplasts: analysis of <Emphasis Type="Italic">Arabidopsis</Emphasis> donor cDNAs encoding ABSCISIC ACID INSENSITIVE1-like protein phosphatases

The 14,200 available full length Arabidopsis thaliana cDNAs in the universal plasmid system (UPS) donor vector pUNI51 should be applied broadly and efficiently to leverage a “functional map-space” of homologous plant genes. We have engineered Cre-lox UPS host acceptor vectors (pCR701- 705) with N-terminal epitope tags in frame with the loxH site and downstream from the maize Ubiquitin promoter for use in transient protoplast expression assays and particle bombardment transformation of monocots. As an example of the utility of these vectors, we recombined them with several Arabidopsis cDNAs encoding Ser/Thr protein phosphatase type 2C (PP2Cs) known from genetic studies or predicted by hierarchical clustering meta-analysis to be involved in ABA and stress responses. Our functional results in Zea mays mesophyll protoplasts on ABA-inducible expression effects on the Late Embryogenesis Abundant promoter ProEm:GUS reporter were consistent with predictions and resulted in identification of novel activities of some PP2Cs. Deployment of these vectors can facilitate functional genomics and proteomics and identification of novel gene activities. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.