Quantitative analysis by flow cytometry of abscisic acid-inducible gene expression in transiently transformed rice protoplasts.

BACKGROUND: Quantifying plant gene expression by flow cytometry (FCM) would allow multidimensional cell-parameter analysis on a per-cell basis, thereby providing insight into the cellular mechanisms of plant gene regulation. Here we sought to establish quantitation by FCM of plant hormone (abscisic acid, ABA)-inducible green fluorescent protein (GFP) expression and to compare the method directly with traditional reporter enzyme assays. MATERIALS AND METHODS: GFP, beta-glucuronidase, and luciferase reporter genes driven by ABA-inducible or constitutive promoter constructs were expressed in transiently cotransformed rice protoplasts and reporter activities quantified by FCM (for GFP) or traditional enzyme assays. Treatments included cotransformations with specific ABA signaling effector cDNA constructs (encoding VIVIPAROUS-1, an ABA transcription factor, and ABA-INSENSITIVE1-1, a dominant-negative protein phosphatase regulator) and the ABA agonist lanthanum chloride. Dual-color FCM was also performed on GFP-expressing cells immunodecorated with an mAb recognizing a rice cell surface epitope. RESULTS: Quantitative analysis of ABA-inducible gene expression by FCM using GFP as reporter gave comparable results to traditional reporter enzyme assays, although the signal-to-noise ratio was less for FCM, which can be a limitation of the method at low promoter strengths. Multiparameter-correlated analysis of ABA-inducible GFP expression with a plasma membrane marker showed no apparent correlation between ABA sensitivity, marked by GFP, and presence of a cell surface arabinogalactan glycoprotein. CONCLUSIONS: Quantitative FCM of GFP-expressing plant cells is a rapid, robust, reproducible, and value-added method relative to traditional enzymatic reporter gene assays.

Functional interactions of lanthanum and phospholipase D with the abscisic acid signaling effectors VP1 and ABI1-1 in rice protoplasts.

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.

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.

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.

Plant response to eggs vs. Host marking pheromone as factors inhibiting oviposition byPieris brassicae.

Pieris brassicae L. butterflies secrete miriamides onto their eggs. These avenanthramide alkaloids are strong oviposition deterrents when sprayed onto a cabbage leaf. However, these compounds could not be detected in cabbage leaves from which egg batches had been removed two days after deposition and that still showed oviposition deterrency. It was concluded that the miriamides were not directly responsible for the avoidance by females of occupied leaves while searching for an oviposition site. Evidence was obtained that cabbage leaves themselves produce oviposition deterrents in response to egg batches. Fractions containing potent oviposition deterrents could be isolated from surface extracts of leaves from which previously laid egg batches had been removed. The term host marking pheromone that was used previously is not applicable in this case.