Abstract
Because plants lack a circulating adaptive immune system, they have evolved multicomponent defense mechanisms to protect themselves against pathogen attack. These defense mechanisms/responses are either constitutively active in the plant, or they are inducible by pathogens. Understanding of the plant response to pathogen attack has advanced rapidly in recent years. Bacterial and fungal pathogenicity factors have been isolated, and mechanisms that are utilized by the plant to recognize the pathogen and initiate a plethora of defense mechanisms have been identified. In contrast to the well-documented effects of LPS on mammalian cells, the effects of LPS on plant cells have been far less studied.
The present study focused on the involvement of lipopolysaccharides (LPS) isolated from the outer cell wall of the Gram-negative bacteria, Burkholderia cepacia (strain ASP B 2D), and yeast elicitor (YE, a cell wall preparation from Saccharomyces cerevisiae) on the molecular mechanisms and components involved in signal transduction and defense-related responses in suspension cultured cells derived from tobacco plants (Nicotiana tabacum cv. Samsun). LPS was extracted, analyzed by denaturing electrophoresis and characterized with regard to 2-keto-3-deoxyoctonate (KDO) content, carbohydrate content, and protein content. The purified LPS and YE were found to trigger defense- and resistance-related responses in the tobacco cells. These responses included a rapid influx of Ca2+ into the cytoplasm of transgenic aequorin-transformed tobacco cells, the production of reactive oxygen species (ROS) during the oxidative burst, alkalinization of the extracellular culture medium of the cells, and changes in the protein phosphorylation patterns of the cells. Time- and concentration-dependent studies for the induction of perception and signal transduction-related responses by YE and LPS indicated that 100 µg.ml-1 of either elicitor was sufficient to induce significant responses in the cells.
YE and LPS both induced a rapid transient increase in cytosolic Ca2+ levels, returning to basal levels after seconds, followed by a second, larger and long-term increase in cytosolic Ca2+. The YE-induced cytosolic Ca2+ influx was 7.5 fold higher than that of LPS. Luminol-dependent chemiluminescence measurements of hydrogen peroxide (H2O2) produced during the YE- and LPS-induced oxidative burst reactions indicated 3.5 fold higher levels of H2O2 induced by YE than that induced by LPS. Total inhibition of H2O2 production by YE- and LPS-induced cells was observed upon treatment of the cells with the H2O2-degrading enzyme, catalase. ROS production was also analyzed by the H2DCF-DA-derived fluorescence assay. The degree of ROS production by YE-treated cells was larger than that of cells treated with LPS, suggesting that YE is a more potent inducer of plant defense responses than LPS. Categorization of the origins of the oxidative bursts, induced by YE and LPS, by the addition of a ROS scavenger (NAC), inhibitors of ROS production (DPI and DDC) and a nitric oxide scavenger (PTIO) indicated that YE and LPS induced different quantities of the same ROS species. The induced ROS included O2-·, H2O2 and perhaps other ROS species as well. In addition, both YE and LPS induced a remarkable burst of nitric oxide (NO), as determined by the 97% and 95% respective inhibitions of the H2DCF-DA-derived fluorescence by the nitric oxide scavenger PTIO. Alkalinization of the extracellular culture medium of the tobacco cells was observed after treatment of the cells with YE and LPS. Both of these elicitors induced a significant increase in extracellular pH from resting pH values of 5.7 to pH 6.3 by YE, and 6.0 by LPS. Notably, the YE-induced response returned to near basal pH levels after 50 min, while the LPS-induced response showed no signs of declining and fluctuated around pH 5.9 for the duration of the experiment. YE and LPS both induced the hyperphosphorylation of two distinct proteins with approximate molecular masses of 28 kDa and 2 kDa. Changes in the pattern of the [32P]-radiolabeled proteins pp28 became visible after 20 min of YE-elicitation and 30 min of LPS-elicitation and changes in pp2 phosphorylation became visible after 20 min treatment of the cells with both elicitors.
Addition of the protein kinase inhibitor, staurosporine, to the cells followed by subsequent elicitation by YE or LPS, resulted in inhibition or abolishment of the elicitor-induced responses during the oxidative burst, extracellular alkalinization, and protein phosphorylation. In contrast, the addition of the protein phosphatase inhibitor, calyculin A, was found to mimic elicitor action in several aspects, including extracellular alkalinization, the oxidative burst and protein phosphorylation, even in the absence of elicitors or any other stimulus. Thus, a fine balance between the actions of certain protein kinases and protein phosphatases is an essential component of signal transduction during YE and LPS elicitation of tobacco cells but the identification and characterization of the staurosporine-sensitive protein kinases and their substrates are necessary to gain a better understanding of the chemosensory perception and signal transduction of the YE and LPS elicitor signals in plant cells. Moreover, the question of whether these perception and transduction mechanisms are connected with a reduced activity of a protein phosphatase, or with the increased activity of a protein kinase, or even a combination of both, remains to be elucidated.
Prof. I.A. Dubery