Alex Levine

The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea.

BACKGROUND Plants have evolved efficient mechanisms to combat pathogen attack. One of the earliest responses to attempted pathogen attack is the generation of oxidative burst that can trigger hypersensitive cell death. This is called the hypersensitive response (HR) and is considered to be a major element of plant disease resistance. The HR is thought to deprive the pathogens of a supply of food and confine them to initial infection site. Necrotrophic pathogens, such as the fungi Botrytis cinerea and Sclerotinia sclerotiorum, however, can utilize dead tissue. RESULTS Inoculation of B. cinerea induced an oxidative burst and hypersensitive cell death in Arabidopsis. The degree of B. cinerea and S. sclerotiorum pathogenicity was directly dependent on the level of generation and accumulation of superoxide or hydrogen peroxide. Plant cells exhibited markers of HR death, such as nuclear condensation and induction of the HR-specific gene HSR203J. Growth of B. cinerea was suppressed in the HR-deficient mutant dnd1, and enhanced by HR caused by simultaneous infection with an avirulent strain of the bacterium Pseudomonas syringae. HR had an opposite (inhibitory) effect on a virulent (biotrophic) strain of P. syringae. Moreover, H(2)O(2) levels during HR correlated positively with B. cinerea growth but negatively with growth of virulent P. syringae. CONCLUSIONS We show that, although hypersensitive cell death is efficient against biotrophic pathogens, it does not protect plants against infection by the necrotrophic pathogens B. cinerea and S. sclerotiorum. By contrast, B. cinerea triggers HR, which facilitates its colonization of plants. Hence, these fungi can exploit a host defense mechanism for their pathogenicity.

The Involvement of Cysteine Proteases and Protease Inhibitor Genes in the Regulation of Programmed Cell Death in Plants

Programmed cell death (PCD) is a process by which cells in many organisms die. The basic morphological and biochemical features of PCD are conserved between the animal and plant kingdoms. Cysteine proteases have emerged as key enzymes in the regulation of animal PCD. Here, we show that in soybean cells, PCD-activating oxidative stress induced a set of cysteine proteases. The activation of one or more of the cysteine proteases was instrumental in the PCD of soybean cells. Inhibition of the cysteine proteases by ectopic expression of cystatin, an endogenous cysteine protease inhibitor gene, inhibited induced cysteine protease activity and blocked PCD triggered either by an avirulent strain of Pseudomonas syringae pv glycinea or directly by oxidative stress. Similar expression of serine protease inhibitors was ineffective. A glutathione S-transferase–cystatin fusion protein was used to purify and characterize the induced proteases. Taken together, our results suggest that plant PCD can be regulated by activity poised between the cysteine proteases and the cysteine protease inhibitors. We also propose a new role for proteinase inhibitor genes as modulators of PCD in plants.

Overexpression of Arabidopsis hexokinase in tomato plants inhibits growth, reduces photosynthesis, and induces rapid senescence.

Sugars are key regulatory molecules that affect diverse processes in higher plants. Hexokinase is the first enzyme in hexose metabolism and may be a sugar sensor that mediates sugar regulation. We present evidence that hexokinase is involved in sensing endogenous levels of sugars in photosynthetic tissues and that it participates in the regulation of senescence, photosynthesis, and growth in seedlings as well as in mature plants. Transgenic tomato plants overexpressing the Arabidopsis hexokinase-encoding gene AtHXK1 were produced. Independent transgenic plants carrying single copies of AtHXK1 were characterized by growth inhibition, the degree of which was found to correlate directly to the expression and activity of AtHXK1. Reciprocal grafting experiments suggested that the inhibitory effect occurred when AtHXK1 was expressed in photosynthetic tissues. Accordingly, plants with increased AtHXK1 activity had reduced chlorophyll content in their leaves, reduced photosynthesis rates, and reduced photochemical quantum efficiency of photosystem II reaction centers compared with plants without increased AtHXK1 activity. In addition, the transgenic plants underwent rapid senescence, suggesting that hexokinase is also involved in senescence regulation. Fruit weight, starch content in young fruits, and total soluble solids in mature fruits were also reduced in the transgenic plants. The results indicate that endogenous hexokinase activity is not rate limiting for growth; rather, they support the role of hexokinase as a regulatory enzyme in photosynthetic tissues, in which it regulates photosynthesis, growth, and senescence.

Programmed cell death of the dinoflagellate Peridinium gatunense is mediated by CO2 limitation and oxidative stress

Abstract The phytoplankton assemblage in Lake Kinneret is dominated in spring by a bloom of the dinoflagellate Peridinium gatunense , which terminates sharply in summer [1]. The pH in Peridinium patches rises during the bloom to values higher than pH9 [2] and results in CO 2 limitation. Here we show that depletion of dissolved CO 2 (CO 2(dis) ) stimulated formation of reactive oxygen species (ROS) and induced cell death in both natural and cultured Peridinium populations. In contrast, addition of CO 2 prevented ROS formation. Catalase inhibited cell death in culture, implicating hydrogen peroxide (H 2 O 2 ) as the active ROS. Cell death was also blocked by a cysteine protease inhibitor, E-64, a treatment which stimulated cyst formation. Intracellular ROS accumulation induced protoplast shrinkage and DNA fragmentation prior to cell death. We propose that CO 2 limitation resulted in the generation of ROS to a level that induced programmed cell death, which resembles apoptosis in animal and plant cells. Our results also indicate that cysteine protease(s) are involved in processes that determine whether a cell is destined to die or to form a cyst.

Induction of Salt and Osmotic Stress Tolerance by Overexpression of an Intracellular Vesicle Trafficking Protein AtRab7 (AtRabG3e)

Adaptation to stress requires removal of existing molecules from various cellular compartments and replacing them with new ones. The transport of materials to and from the specific compartments involved in the recycling and deposition of macromolecules is carried out by an intracellular vesicle trafficking system. Here, we report the isolation of a vesicle trafficking-regulating gene, AtRabG3e (formerly AtRab7), from Arabidopsis. The gene was induced during programmed cell death after treatment of intact leaves with superoxide and salicylic acid or infection with necrogenic pathogens. Transgenic plants that expressed the AtRabG3e gene under the constitutive 35S promoter from cauliflower mosaic virus exhibited accelerated endocytosis in roots, leaves, and protoplasts. The transgenic plants accumulated sodium in the vacuoles and had higher amounts of sodium in the shoots. The transgenic plants also showed increased tolerance to salt and osmotic stresses and reduced accumulation of reactive oxygen species during salt stress. These results imply that vesicle trafficking plays an important role in plant adaptation to stress, beyond the housekeeping function in intracellular vesicle trafficking.

Dinoflagellate-Cyanobacterium Communication May Determine the Composition of Phytoplankton Assemblage in a Mesotrophic Lake

The reasons for annual variability in the composition of phytoplankton assemblages are poorly understood but may include competition for resources and allelopathic interactions. We show that domination by the patch-forming dinoflagellate, Peridinium gatunense, or, alternatively, a bloom of a toxic cyanobacterium, Microcystis sp., in the Sea of Galilee may be accounted for by mutual density-dependent allelopathic interactions. Over the last 11 years, the abundance of these species in the lake displayed strong negative correlation. Laboratory experiments showed reciprocal, density-dependent, but nutrient-independent, inhibition of growth. Application of spent P. gatunense medium induced sedimentation and, subsequently, massive lysis of Microcystis cells within 24 hr, and sedimentation and lysis were concomitant with a large rise in the level of McyB, which is involved in toxin biosynthesis by Microcystis. P. gatunense responded to the presence of Microcystis by a species-specific pathway that involved a biphasic oxidative burst and activation of certain protein kinases. Blocking this recognition by MAP-kinase inhibitors abolished the biphasic oxidative burst and affected the fate (death or cell division) of the P. gatunense cells. We propose that patchy growth habits may confer enhanced defense capabilities, providing ecological advantages that compensate for the aggravated limitation of resources in the patch. Cross-talk via allelochemicals may explain the phytoplankton assemblage in the Sea of Galilee.

The involvement of poly(ADP‐ribose) polymerase in the oxidative stress responses in plants

In plants many biotic and abiotic stresses can cause secondary oxidative stress. Earlier work showed that, depending on the severity of the oxidative stress, plants can activate either cell protective genes or programmed cell death (PCD). Poly(ADP‐ribose) polymerase (PARP) has been implicated as one of the enzymes in the apoptotic pathways induced by DNA damaging agents or oxidative stress. We show that in cultured soybean cells, PARP is involved in responses to mild and severe oxidative stresses, by mediating DNA repair and PCD processes, respectively. Addition of PARP inhibitors reduced the degree of cell death triggered by H2O2. Two windows of NAD consumption after H2O2 treatment were detected. Experiments with transient overexpression of Arabidopsis PARP cDNA promoted DNA repair and inhibited cell death caused by mild oxidative stress. However, following severe stress PARP overexpression increased cell death. Expression of antisense PARP produced the opposite effects: an increase in DNA nicks and inhibition of cell death at high, but not mild doses of H2O2.

Sugar-induced apoptosis in yeast cells

Sugars induce death of Saccharomyces cerevisiae within a few hours in the absence of additional nutrients to support growth; by contrast, cells incubated in water or in the presence of other nutrients without sugar remain viable for weeks. Here we show that this sugar-induced cell death (SICD) is characterized by rapid production of reactive oxygen species (ROS), RNA and DNA degradation, membrane damage, nucleus fragmentation and cell shrinkage. Addition of ascorbic acid to sugar-incubated cells prevents SICD, indicating that SICD is initiated by ROS. The lack of a protection mechanism against SICD suggests that sugars use to be the limiting nutrients for yeast and are probably depleted before all other nutrients. Being the limiting nutrient, sugars became the growth-stimulating agent, signaling the presence of sufficient nutrients for growth, but in the absence of the complementing nutrients they induce apoptotic death.

Anoxia pretreatment protects soybean cells against H2O2-induced cell death: possible involvement of peroxidases and of alternative oxidase

Anoxia followed by reoxygenation causes extensive damage to cellular components through generation of reactive oxygen intermediates. We examined cellular responses to oxidative stress after anoxia in cultured soybean or human fibroblast cells. Anoxia pretreatment protected soybean but not fibroblasts against H2O2 concentrations that induced programmed cell death in normoxic cells. H2O2 removal in anoxia‐pretreated soybean cultures was faster. Protection was associated with increased action of alternative oxidase (AOX) and peroxidases. AOX inhibitors abolished the protective effect, while induction of AOX protected normoxic cells against H2O2. We propose that during anoxia, plant cells can prepare for reoxygenation injury by up‐regulating their antioxidant capacity, and that AOX is involved in this process.

Reduced expression of the v-SNAREs AtVAMP71/AtVAMP7C gene family in Arabidopsis reduces drought tolerance by suppression of abscisic acid-dependent stomatal closure

Stomatal closure during water stress is a major plant mechanism for reducing the loss of water through leaves. The opening and closure of stomata are mediated by endomembrane trafficking. The role of the vacuolar trafficking pathway, that involves v-SNAREs of the AtVAMP71 family (formerly called AtVAMP7C) in stomatal movements, was analysed. Expression of AtVAMP711–14 genes was manipulated in Arabidopsis plants with sense or antisense constructs by transformation of the AtVAMP711 gene. Antisense plants exhibited decreased stomatal closure during drought or after treatment with abscisic acid (ABA), resulting in the rapid loss of leaf water and tissue collapse. No improvement was seen in plants overexpressing the AtVAMP711 gene, suggesting that wild-type levels of AtVAMP711 expression are sufficient. ABA treatment induced the production of reactive oxygen species (ROS) in guard cells of both wild-type and antisense plants, indicating that correct hormone sensing is maintained. ROS were detected in nuclei, chloroplasts, and vacuoles. ABA treatment caused a significant increase in ROS-containing small vacuoles and also in plastids and nuclei of neighbouring epidermal and mesophyll cells. Taken together, our results show that VAMP71 proteins play an important role in the localization of ROS, and in the regulation of stomatal closure by ABA treatment. The paper also describes a novel aspect of ROS signalling in plants: that of ROS production in small vacuoles that are dispersed in the cytoplasm.