Bratislav Stankovic

Characterization of the Variation Potential in Sunflower.

A major candidate for intercellular signaling in higher plants is the stimulus-induced systemic change in membrane potential known as variation potential (VP). We investigated the mechanism of occurrence and long-distance propagation of VP in sunflower (Helianthus annuus L.) plants. Here we present evidence of the relationship among injury-induced changes in xylem tension, turgor pressure, and electrical potential. Although locally applied wounding did trigger a change in membrane potential, it evoked even faster changes in tissue deformation, apparently resulting from pressure surges rapidly transmitted through the xylem and experienced throughout the plant. Externally applied pressure mimicked flame wounding by triggering an electrical response resembling VP. Our findings suggest that VP in sunflower is not a propagating change in electrical potential and not the consequence of chemicals transmitted via the xylem, affecting ligand-modulated ion channels. Instead, VP appears to result from the surge in pressure in the xylem causing a change in activity of mechanosensitive, stretch-responsive ion channels or pumps in adjacent, living cells. The ensuing ion flux evokes local plasma membrane depolarization, which is monitored extracellularly as VP.

Molecular cloning and characterization of a tomato cDNA encoding a systemically wound-inducible bZIP DNA-binding protein

Abstract. Localized wounding of one leaf in intact tomato (Lycopersicon esculentum Mill.) plants triggers rapid systemic transcriptional responses that might be involved in defense. To better understand the mechanism(s) of intercellular signal transmission in wounded tomatoes, and to identify the array of genes systemically up-regulated by wounding, a subtractive cDNA library for wounded tomato leaves was constructed. A novel cDNA clone (designated LebZIP1) encoding a DNA-binding protein was isolated and identified. This clone appears to be encoded by a single gene, and belongs to the family of basic leucine zipper domain (bZIP) transcription factors shown to be up-regulated by cold and dark treatments. Analysis of the mRNA levels suggests that the transcript for LebZIP1 is both organ-specific and up-regulated by wounding. In wounded wild-type tomatoes, the LebZIP1 mRNA levels in distant tissue were maximally up-regulated within only 5 min following localized wounding. Exogenous abscisic acid (ABA) prevented the rapid wound-induced increase in LebZIP1 mRNA levels, while the basal levels of LebZIP1 transcripts were higher in the ABA mutants notabilis (not), sitiens (sit), and flacca ( flc), and wound-induced increases were greater in the ABA-deficient mutants. Together, these results suggest that ABA acts to curtail the wound-induced synthesis of LebZIP1 mRNA.

Electrical Signals, the Cytoskeleton, and Gene Expression: a Hypothesis on the Coherence of the Cellular Responses to Environmental Insult

When plant tissue is abiotically injured by crushing, cutting, heat-wounding, electrical stimulation, or by several other means, the injured (perceiving) tissue generates electrical signals (action potentials and variation potentials) and transmits them to distant (responding) tissue. Here they evoke apparently disparate responses, such as callose formation, closing of plasmodesmata, stoppage of cytoplasmic streaming, inhibition of ribosome movement along messenger RNA (mRNA), and ultrarapid but transient accumulation of over 100 transcripts, which are degraded without being translated. These apparently disparate responses can be reconciled by one fundamental hypothesis that assumes that “the plant does not know what hit it” and thus “expecting the worst” mounts a holistic defense response against its most potent nemesis, a putative viral invasion. We postulate that the basis for this response is calcium influx into the cytoplasm via voltage-gated channels (action potential) associated with the microtubules, or via mechano-sensitive channels (variation potential) associated with microfilaments. The calcium interacts with calcium and/or calmodulin-dependent cytoskeleton-associated protein kinases. This causes the phosphorylation of myosin, which stops cytoplasmic streaming, and of elongation factor 2F, which slows elongation and termination and causes ribosomes to pile up on polyribosomes. This decreases protein synthesis, but protects preexisting “host” transcripts from degradation. The phosphorylation signal then passes into the nucleus, where it phosphorylates RNA polymerase II, which goes into overdrive (i.e., does not stop at accuracy checkpoints), thus causing the synthesis of large amounts of mismade mRNA. The mRNA is transported into the cytoplasm, where it is scanned (checked for accuracy) by ribosomes, and found to be incorrect. This surveillance mechanism stimulates ribonuclease activity, which degrades the free (non-polysome-associated), mismade RNA, but leaves the original, “host” transcripts unscathed since they are protected by ribosomes. The ribonuclease also (and here is the crux of the matter) attacks other free mRNAs, including viral mRNAs, so these are disposed of before they can be translated. Within minutes this reaction is over, cytoplasmic steaming resumes, translation continues, ribosomes are released and so can be used to translate new (correctly made) transcripts.

Rapid systemic up-regulation of genes after heat-wounding and electrical stimulation

When one leaf of a tomato plant is electrically-stimulated or heat-wounded, proteinase inhibitor genes are rapidly up-regulated in distant leaves. The identity of the systemic wound signal(s) is not yet known, but major candidates include hormones transmitted via the phloem or the xylem, the electrically-stimulated self-propagating electrical signal in the phloem (the action potential, AP), or the heat-wound-induced surge in hydraulic pressure in the xylem evoking a local change in membrane potential in adjacent living cells (the variation potential, VP). In order to discriminate between these signals we have adopted two approaches. The first approach involves applying stimuli that evoke known signals and determining whether these signals have similar effects on the “model” transcripts for proteinase inhibitors (pin) and calmodulin (cal). Here we show that a heat wound almost invariably evokes a VP, while an electrical stimulation occasionally evokes an AP, and both of these signals induce accumulation of transcripts encoding proteinase inhibitors. The second approach involves identifying the array of genes turned on by heat-wounding. To this end, we have constructed a subtractive library for heat-wounded tissue, isolated over 800 putatively up-regulated clones, and shown that all but two of the fifty that we have analyzed by Northern hybridization are, indeed, up-regulated. Here we show the early kinetics of up-regulation of three of these transcripts in the terminal (4th) leaf in response to heat-wounding the 3rd leaf, about 5 cm away. Even though these transcripts show somewhat different time courses of induction, with one peaking at 30 min, another at 15 min, and another at 5 min after flaming of a distant leaf, they all exhibit a similar pattern, i.e., a transient period of transcript accumulation preceding a period of transcript decrease, followed by a second period of transcript accumulation.

Signalomics: Diversity and Methods of Analysis of Systemic Signals in Plants

We provide a brief definition and history of signals, pointing out how differences in body plan between plants and animals require fundamentally different signaling mechanisms, and then list the diversity of chemical and physical signals along with their pathways of transmission, providing details on molecular signals and focusing on the phloem and xylem as being the main conduits for (rapid) systemic signaling. The two major electrical (action potentials and variation potentials) as well as hydraulic signals are then described. The latter part of the chapter deals with methods of analysis of molecular signals, including accessing the phloem and identifying the array of gene products transported therein. A description is provided of the modern methods used in metabolomics and phenotyping to analyze the metabolic consequences of signal action. Conventional techniques for analyzing electrical and hydraulic signals and their ionic components using electrodes are then furnished. Finally we describe novel techniques developed recently in the animal field using fluorescence to monitor real-time changes in membrane potential, which could be adapted for plants to open up new vistas in our understanding of electrical signals in plants.

Plant Cytomics: Novel Methods to View Molecules on the Move

We provide our definition and the brief history of “cytomics” followed by an overview of general methodological approaches of optical imaging, especially fluorescence microscopy. We then go into detail on novel fluor-linking agents (nanobodies, aptamers, and aldehydes) and the array of novel fluors available. We describe many of the new techniques developed for superfast, super-resolution microscopy (photoreactivated localization microscopy, structured illumination microscopy, stimulated emission depletion microscopy, and stochastic optical reconstruction microscopy) followed by quantitative microscopy and image analysis. We then delve into unconventional methods, novel light systems, and alternatives to fluorescence (non-liner optical imaging, single-molecule light absorption, luminescent proteins). We then describe how these systems have been employed recently for proteins, nucleic acids, the cytoskeleton, and also small molecules of major interest to plants. We finish with a description of recent findings specific to plant cytomics and furnish several impressive images and other illustrations from the recent plant literature.