Waterlogging‐induced adventitious root formation in cucumber is regulated by ethylene and auxin through reactive oxygen species signalling

Development of adventitious roots (ARs) at the base of the shoot is an important adaptation of plants to waterlogging stress; however, its physiological mechanisms remain unclear. Here, we investigated the regulation of AR formation under waterlogged conditions by hormones and reactive oxygen species (ROS) in Cucumis sativus L., an agriculturally and economically important crop in China. We found that ethylene, auxin, and ROS accumulated in the waterlogged cucumber plants. On the other hand, application of the ethylene receptor inhibitor 1‐methylcyclopropene (1‐MCP), the auxin transport inhibitor 1‐naphthylphthalamic acid (NPA), or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) decreased the number of ARs induced by waterlogging. Auxin enhanced the expression of ethylene biosynthesis genes, which led to ethylene entrapment in waterlogged plants. Both ethylene and auxin induced the generation of ROS. Auxin‐induced AR formation was inhibited by 1‐MCP, although ethylene‐induced AR formation was not inhibited by NPA. Both ethylene‐ and auxin‐induced AR formation were counteracted by DPI. These results indicate that auxin‐induced AR formation is dependent on ethylene, whereas ethylene‐induced AR formation is independent of auxin. They also show that ROS signals mediate both ethylene‐ and auxin‐induced AR formation in cucumber plants.     

OsNPR1 negatively regulates herbivore‐induced JA and ethylene signaling and plant resistance to a chewing herbivore in rice

NPR1 (a non‐expressor of pathogenesis‐related genes1) has been reported to play an important role in plant defense by regulating signaling pathways. However, little to nothing is known about its function in herbivore‐induced defense in monocot plants. Here, using suppressive substrate hybridization, we identified a NPR1 gene from rice, OsNPR1, and found that its expression levels were upregulated in response to infestation by the rice striped stem borer (SSB) Chilo suppressalis and rice leaf folder (LF) Cnaphalocrocis medinalis, and to mechanical wounding and treatment with jasmonic acid (JA) and salicylic acid (SA). Moreover, mechanical wounding induced the expression of OsNPR1 quickly, whereas herbivore infestation induced the gene more slowly. The antisense expression of OsNPR1 (as‐npr1), which reduced the expression of the gene by 50%, increased elicited levels of JA and ethylene (ET) as well as of expression of a lipoxygenase gene OsHI‐LOX and an ACC synthase gene OsACS2. The enhanced JA and ET signaling in as‐npr1 plants increased the levels of herbivore‐induced trypsin proteinase inhibitors (TrypPIs) and volatiles, and reduced the performance of SSB. Our results suggest that OsNPR1 is an early responding gene in herbivore‐induced defense and that plants can use it to activate a specific and appropriate defense response against invaders by modulating signaling pathways.     

Serotonin modulates Arabidopsis root growth via changes in reactive oxygen species and jasmonic acid–ethylene signaling

Serotonin (5‐hydroxytryptamine) is a bioactive indoleamine with neurotransmitter function in vertebrates, which represents an emerging signaling molecule in plants, playing key roles in the development and defense. In this study, the role of reactive oxygen species (ROS) and jasmonic acid (JA)–ethylene (Et) signaling in root developmental alterations induced by serotonin was investigated. An Arabidopsis thaliana mutant defective at the RADICAL‐INDUCED CELL DEATH1 (RCD1) locus was resistant to paraquat‐induced ROS accumulation in primary roots and showed decreased inhibition or root growth in response to serotonin. A suite of JA‐ and Et‐related mutants including coronatine insensitive1, jasmonic acid resistant1 (jar1), etr1, ein2 and ein3 showed tolerance to serotonin in the inhibition of primary root growth and ROS redistribution within the root tip when compared with wild‐type (WT) seedlings. Competence assays between serotonin and AgNO₃, a well‐known blocker of Et action, showed that primary root growth in medium supplemented with serotonin was normalized by AgNO_3, whereas roots of eto3, an Et overproducer mutant, were oversensitive to serotonin. Comparison of ROS levels in WT, etr1, jar1 and rcd1 primary root tips using the ROS‐specific probe 2′,7′‐dichlorofluorescein diacetate and confocal imaging showed that serotonin inhibition of primary root growth likely occurs independently of its conversion into melatonin. Our results provide compelling evidence that serotonin affects ROS distribution in roots, involving RCD1 and components of the JA–Et signaling pathways.     

Reactive oxygen species‐induced cell death of rat primary astrocytes through mitochondria‐mediated mechanism

Astrocytes, the most abundant glial cell population in the central nervous system (CNS), play physiological roles in neuronal activities. Oxidative insult induced by the injury to the CNS causes neural cell death through extrinsic and intrinsic pathways. This study reports that reactive oxygen species (ROS) generated by exposure to the strong oxidizing agent, hexavalent chromium (Cr(VI)) as a chemical‐induced oxidative stress model, caused astrocytes to undergo an apoptosis‐like cell death through a caspase‐3‐independent mechanism. Although activating protein‐1 (AP‐1) and NF‐κB were activated in Cr(VI)‐primed astrocytes, the inhibition of their activity failed to increase astrocytic cell survival. The results further indicated that the reduction in mitochondrial membrane potential (MMP) was accompanied by an increase in the levels of ROS in Cr(VI)‐primed astrocytes. Moreover, pretreatment of astrocytes with N‐acetylcysteine (NAC), the potent ROS scavenger, attenuated ROS production and MMP loss in Cr(VI)‐primed astrocytes, and significantly increased the survival of astrocytes, implying that the elevated ROS disrupted the mitochondrial function to result in the reduction of astrocytic cell viability. In addition, the nuclear expression of apoptosis‐inducing factor (AIF) and endonuclease G (EndoG) was observed in Cr(VI)‐primed astrocytes. Taken together, evidence shows that astrocytic cell death occurs by ROS‐induced oxidative insult through a caspase‐3‐independent apoptotic mechanism involving the loss of MMP and an increase in the nuclear levels of mitochondrial pro‐apoptosis proteins (AIF/EndoG). This mitochondria‐mediated but caspase‐3‐independent apoptotic pathway may be involved in oxidative stress‐induced astrocytic cell death in the injured CNS. J. Cell. Biochem. 107: 933–943, 2009. © 2009 Wiley‐Liss, Inc.     

Biochemical and molecular changes in rice seedlings (Oryza sativa L.) to cope with chromium stress

Chromium (Cr) is very toxic to both humans and plants. This investigation aimed to understand the physiological and molecular responses of rice seedlings to Cr stress. Cr toxicity did not significantly affect morphological features and Cr accumulation in roots and shoots in Pokkali but not in BRRI 51, although there was a reduction in chlorophyll concentration in leaves of both genotypes. These results imply that Pokkali has mechanisms to cope with Cr supplementation. We therefore performed quantitative real‐time PCR on the expression pattern of two chelator genes, OsPCS1 and OsMT1, but there were no significant changes in expression in roots and shoots of Pokkali and BRRI 51 following Cr stress. This suggests that there was no metal sequestration following heavy metal stress in roots of these genotypes. Moreover, no expression of two heavy metal transporter genes, OsHMA3 and OsNRAMP1, was induced after Cr stress in roots and shoots, suggesting that these transporter genes are not induced by Cr stress or might not be involved in Cr uptake in rice. We also performed a targeted study on the effect of Cr on Fe uptake mechanisms. Our studies showed a consistent reduction in Fe uptake, Fe reductase activity and expression of Fe‐related genes (OsFRO1 and OsIRT1) under Cr stress in both roots and leaves of Pokkali. In contrast, these parameters and genes were significantly increased in Cr‐sensitive BRRI 51 under Cr stress. The results confirm that limiting Fe uptake through the down‐regulation of Fe reductase and Fe transporter genes is the main strategy of Cr‐tolerant Pokkali to cope with Cr stress. Finally, increased CAT, POD and GR activity and elevated glutathione and proline synthesis might provide strong antioxidant defence against Cr stress in Pokkali. Taken together, our findings reveal that Cr stress tolerance in rice (Pokkali) is not related to metal sequestration but is associated with reduced Fe transport and increased antioxidant defence.     

Hexavalent chromium disrupts mitosis by stabilizing microtubules in Lens culinaris root tip cells

Hexavalent chromium [Cr(VI)] is an accumulating environmental pollutant due to anthropogenic activities, toxic for humans, animals and plants. Therefore, the effects of Cr(VI) on dividing root cells of lentil (Lens culinaris) were investigated by tubulin immunofluorescence and DNA staining. In Cr(VI)‐treated roots, cell divisions were perturbed, the chromosomes formed irregular aggregations, multinucleate cells were produced and tubulin clusters were entrapped within the nuclei. All cell cycle‐specific microtubule (MT) arrays were affected, indicating a stabilizing effect of Cr(VI) on the MTs of L. culinaris. Besides, a time‐ and concentration‐dependent gradual increase of acetylated α‐tubulin, an indicator of MT stabilization, was observed in Cr(VI)‐treated roots by both immunofluorescence and western blotting. Evidence is also provided that reactive oxygen species (ROS) caused by Cr(VI), determined with the specific marker dichlorofluorescein, may be responsible for MT stabilization. Combined treatments with Cr(VI) and oryzalin revealed that Cr(VI) overcomes the depolymerizing ability of oryzalin, as it does experimentally introduced hydrogen peroxide, further supporting its stabilizing effect. In conclusion, it is suggested that the mitotic aberrations caused by Cr(VI) in L. culinaris root cells may be the result of MT stabilization rather than depolymerization, which consequently disturbs MT dynamics and their related functions.     

Expression of a novel <Emphasis Type="Italic">OSPGYRP</Emphasis> (rice proline-, glycine- and tyrosine-rich protein) gene,which is involved in vesicle trafficking,enhanced cold tolerance in <Emphasis Type="Italic">E. coli</Emphasis>

A novel OSPGYRP gene encoding a rice proline-, glycine- and tyrosine-rich protein was isolated from cold-stress treated rice seedlings using suppression subtractive hybridization. Both amino acid sequence analysis and subcellular localization confirm that OsPGYRP is a novel protein involved in vesicle trafficking. The expression of the OSPGYRP gene was induced by cold, salt, and osmotic stress. In addition, expression of the OSPGYRP gene in E. coli increased the resistance to cold stress. These results show that OsPGYRP is a novel protein involved in vesicle trafficking and plays an important role in plant adaptation to stress. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

A rice calcium-dependent protein kinase OsCPK12 oppositely modulates salt-stress tolerance and blast disease resistance

Calcium-dependent protein kinases (CDPKs) regulate the downstream components in calcium signaling pathways. We investigated the effects of overexpression and disruption of an Oryza sativa (rice) CDPK (OsCPK12) on the plant's response to abiotic and biotic stresses. OsCPK12-overexpressing (OsCPK12-OX) plants exhibited increased tolerance to salt stress. The accumulation of hydrogen peroxide (H(2) O(2) ) in the leaves was less in OsCPK12-OX plants than in wild-type (WT) plants. Genes encoding reactive oxygen species (ROS) scavenging enzymes (OsAPx2 and OsAPx8) were more highly expressed in OsCPK12-OX plants than in WT plants, whereas the expression of the NADPH oxidase gene, OsrbohI, was decreased in OsCPK12-OX plants compared with WT plants. Conversely, a retrotransposon (Tos17) insertion mutant, oscpk12, and plants transformed with an OsCPK12 RNA interference (RNAi) construct were more sensitive to high salinity than were WT plants. The level of H(2) O(2) accumulation was greater in oscpk12 and OsCPK12 RNAi plants than in the WT. These results suggest that OsCPK12 promotes tolerance to salt stress by reducing the accumulation of ROS. We also observed that OsCPK12-OX seedlings had increased sensitivity to abscisic acid (ABA) and increased susceptibility to blast fungus, probably resulting from the repression of ROS production and/or the involvement of OsCPK12 in the ABA signaling pathway. Collectively, our results suggest that OsCPK12 functions in multiple signaling pathways, positively regulating salt tolerance and negatively modulating blast resistance.     

Targeting and signaling of Rho of plants guanosine triphosphatases require synergistic interaction between guanine nucleotide inhibitor and vesicular trafficking

Most eukaryotic cells are polarized. Common toolbox regulating cell polarization includes Rho guanosine triphosphatases (GTPases), in which spatiotemporal activation is regulated by a plethora of regulators. Rho of plants (ROPs) are the only Rho GTPases in plants. Although vesicular trafficking was hinted in the regulation of ROPs, it was unclear where vesicle‐carried ROP starts, whether it is dynamically regulated, and which components participate in vesicle‐mediated ROP targeting. In addition, although vesicle trafficking and guanine nucleotide inhibitor (GDI) pathways in Rho signaling have been extensively studied in yeast, it is unknown whether the two pathways interplay. Unclear are also cellular and developmental consequences of their interaction in multicellular organisms. Here, we show that the dynamic targeting of ROP through vesicles requires coat protein complex II and ADP‐ribosylation factor 1‐mediated post‐Golgi trafficking. Trafficking of vesicle‐carried ROPs between the plasma membrane and the trans‐Golgi network is mediated through adaptor protein 1 and sterol‐mediated endocytosis. Finally, we show that GDI and vesicle trafficking synergistically regulate cell polarization and ROP targeting, suggesting that the establishment and maintenance of cell polarity is regulated by an evolutionarily conserved mechanism.     

植物细胞活性氧种类、代谢及其信号转导

越来越明显的证据表明,植物体十分活跃的产生着活性氧并将之作为信号分子、进而控制着诸如细胞程序性死亡、非生物胁迫响应、病原体防御和系统信号等生命过程,而不仅是传统意义上的活性氧是有氧代谢的附产物。日益增多的证据显示,由脱落酸、水杨酸、茉莉酸与乙烯以及活性氧所调节的激素信号途径,在生物和非生物胁迫信号的“交谈”中起重要作用。活性氧最初被认为是动物吞噬细胞在宿主防御反应时所释放的附产物,现在的研究清楚的表明,活性氧在动物和植物细胞信号途径中均起作用。活性氧可以诱导细胞程序性死亡或坏死、可以诱导或抑制许多基因的表达,也可以激活上述级联信号。近来生物化学与遗传学研究证实过氧化氢是介导植物生物胁迫与非生物胁迫的信号分子,过氧化氢的合成与作用似乎与一氧化氮有关系。过氧化氢所调节的下游信号包括钙“动员”、蛋白磷酸化和基因表达等。     

Ethylene mediates dichromate‐induced inhibition of primary root growth by altering AUX1 expression and auxin accumulation in Arabidopsis thaliana

The hexavalent form of chromium [Cr(VI)] causes a major reduction in yield and quality of crops worldwide. The root is the first plant organ that interacts with Cr(VI) toxicity, which inhibits primary root elongation, but the underlying mechanisms of this inhibition remain elusive. In this study, we investigate the possibility that Cr(VI) reduces primary root growth of Arabidopsis by modulating the cell cycle‐related genes and that ethylene signalling contributes to this process. We show that Cr(VI)‐mediated inhibition of primary root elongation was alleviated by the ethylene perception and biosynthesis antagonists silver and cobalt, respectively. Furthermore, the ethylene signalling defective mutants (ein2‐1 and etr1‐3) were insensitive, whereas the overproducer mutant (eto1‐1) was hypersensitive to Cr(VI). We also report that high levels of Cr(VI) significantly induce the distribution and accumulation of auxin in the primary root tips, but this increase was significantly suppressed in seedlings exposed to silver or cobalt. In addition, genetic and physiological investigations show that AUXIN‐RESISTANT1 (AUX1) participates in Cr(VI)‐induced inhibition of primary root growth. Taken together, our results indicate that ethylene mediates Cr(VI)‐induced inhibition of primary root elongation by increasing auxin accumulation and polar transport by stimulating the expression of AUX1.     

Ethylene‐dependent aerenchyma formation in adventitious roots is regulated differently in rice and maize

In roots of gramineous plants, lysigenous aerenchyma is created by the death and lysis of cortical cells. Rice (Oryza sativa) constitutively forms aerenchyma under aerobic conditions, and its formation is further induced under oxygen‐deficient conditions. However, maize (Zea mays) develops aerenchyma only under oxygen‐deficient conditions. Ethylene is involved in lysigenous aerenchyma formation. Here, we investigated how ethylene‐dependent aerenchyma formation is differently regulated between rice and maize. For this purpose, in rice, we used the reduced culm number1 (rcn1) mutant, in which ethylene biosynthesis is suppressed. Ethylene is converted from 1‐aminocyclopropane‐1‐carboxylic acid (ACC) by the action of ACC oxidase (ACO). We found that OsACO5 was highly expressed in the wild type, but not in rcn1, under aerobic conditions, suggesting that OsACO5 contributes to aerenchyma formation in aerated rice roots. By contrast, the ACO genes in maize roots were weakly expressed under aerobic conditions, and thus ACC treatment did not effectively induce ethylene production or aerenchyma formation, unlike in rice. Aerenchyma formation in rice roots after the initiation of oxygen‐deficient conditions was faster and greater than that in maize. These results suggest that the difference in aerenchyma formation in rice and maize is due to their different mechanisms for regulating ethylene biosynthesis.     

Interactions between ethylene, gibberellin and abscisic acid regulate emergence and growth rate of adventitious roots in deepwater rice

Growth of adventitious roots is induced in deepwater rice (Oryza sativa L.) when plants become submerged. Ethylene which accumulates in flooded plant parts is responsible for root growth induction. Gibberellin (GA) is ineffective on its own but acts in a synergistic manner together with ethylene to promote the number of penetrating roots and the growth rate of emerged roots. Studies with the GA biosynthesis inhibitor paclobutrazol revealed that root emergence was dependent on GA activity. Abscisic acid (ABA) acted as a competitive inhibitor of GA activity. Root growth rate on the other hand was dependent on GA concentration and ABA acted as a potent inhibitor possibly of GA but also of ethylene signaling. The results indicated that root emergence and elongation are distinct phases of adventitious root growth that are regulated through different networking between ethylene, GA and ABA signaling pathways. Adventitious root emergence must be coordinated with programmed death of epidermal cells which cover root primordia. Epidermal cell death is also controlled by ethylene, GA and ABA albeit with cell-type specific cross-talk. Different interactions between the same hormones may be a means to ensure proper timing of cell death and root emergence and to adjust the growth rate of emerged adventitious roots.