Salt tolerance requires cortical microtubule reorganization in Arabidopsis

Although the results of some studies indicate that salt stress affects the organization of microtubules, it remains an open question whether microtubules play an active role in the plant's ability to withstand salt stress. In the present study, we showed that salt stress-induced wild-type Arabidopsis seedling roots display right-handed skewed growth and depolymerization of the cortical microtubules. The results of a long-term observational study showed that cortical microtubules depolymerized then reorganized themselves under salt stress. Stabilization of microtubules with paclitaxel resulted in more seedling death under salt stress, while disruption of microtubules with oryzalin or propyzamide rescued seedlings from death. Seedlings in which the cortical microtubules were reorganized did not succumb to salt stress. These results suggest that both depolymerization and reorganization of the cortical microtubules are important for the plant's ability to withstand salt stress. Depolymerizing microtubules by drugs rescues seedlings from death under salt stress. This rescue effect was abolished by removing calcium from the medium or treatment with a calcium channel inhibitor. Depolymerization of the microtubules is followed by an increase in the free cytoplasmic calcium concentration. The addition of calcium to the growth medium increased the number of seedlings in which recovery of the cortical microtubules occurred, whereas the removal of calcium decreased the number of seedlings in which recovery occurred. Therefore, depolymerization of the cortical microtubules raises intracellular calcium concentrations, while reorganization of the cortical microtubules and seedling survival may be mediated by calcium influx in salt stress.     

The plant growth-promoting fungus Penicillium simplicissimum GP17-2 induces resistance in Arabidopsis thaliana by activation of multiple defense signals

Arabidopsis thaliana grown in soil amended with barley grain inocula of Penicillium simplicissimum GP17-2 or receiving root treatment with its culture filtrate (CF) exhibited clear resistance to Pseudomonas syringae pv. tomato DC3000 (Pst). To assess the contribution of different defense pathways, Arabidopsis genotypes implicated in salicylic acid (SA) signaling expressing the NahG transgene or carrying disruption in NPR1 (npr1), jasmonic acid (JA) signaling (jar1) and ethylene (ET) signaling (ein2) were tested. All genotypes screened were protected by GP17-2 or its CF. However, the level of protection was significantly lower in NahG and npr1 plants than it was in similarly treated wild-type plants, indicating that the SA signaling pathway makes a minor contribution to the GP17-2-mediated resistance and is insufficient for a full response. Examination of local and systemic gene expression revealed that GP17-2 and its CF modulate the expression of genes involved in both the SA and JA/ET signaling pathways. Subsequent challenge of GP17-2-colonized plants with Pst was accompanied by direct activation of SA-inducible PR-2 and PR-5 genes as well as potentiated expression of the JA-inducible Vsp gene. In contrast, CF-treated plants infected with Pst exhibited elevated expression of most defense-related genes (PR-1, PR-2, PR-5, PDF1.2 and Hel) studied. Moreover, an initial elevation of SA responses was followed by late induction of JA responses during Pst infection of induced systemic resistance (ISR)-expressing plants. In conclusion, we hypothesize the involvement of multiple defense mechanisms leading to an ISR of Arabidopsis by GP17-2.     

Role of the methionine sulfoxide reductase MsrB3 in cold acclimation in Arabidopsis

Methionine residues of proteins are a major target for oxidation by reactive oxygen species (ROS), which are generated in response to a variety of stress conditions. Methionine sulfoxide (MetO) reductases are present in most organisms and play protective roles in the cellular response to oxidative stress, reducing oxidized MetO back to Met. Previously, an Arabidopsis MetO reductase, MsrB3, was identified as a cold-responsive protein. Here we report that MsrB3 functions in the process of cold acclimation, thus contributing to cold tolerance. In contrast to normal, wild-type plants, msrb3 mutant plants lost the ability to become tolerant to freezing temperatures following cold pre-treatment. Furthermore, when exposed to low temperature, msrb3 plants exhibited a larger increase in MetO and H(2)O(2) content and electrolyte leakage compared with wild-type and MsrB3 transgenic plants. It is also shown that MsrB3 is localized at the endoplasmic reticulum (ER). We propose that MsrB3 plays an important role in cold tolerance by eliminating MetO and ROS that accumulate at the ER during cold acclimation.     

The chilling injury induced by high root temperature in the leaves of rice seedlings

Root temperature is found to be a very important factor forleaves to alter the response and susceptibility to chillingstress. Severe visible damage was observed in the most activeleaves of seedlings of a japonica rice (Oryza sativa cv. Akitakomachi),e.g. the third leaf at the third-leaf stage, after the treatmentwhere only leaves but not roots were chilled (L/H). On the otherhand, no visible damage was observed after the treatment whereboth leaves and roots were chilled simultaneously (L/L). Thechilling injury induced by L/H, a novel type of chilling injury,required the light either during or after the chilling in orderto develop the visible symptoms such as leaf bleaching and tissuenecrosis. Chlorophyll fluorescence parameters measured aftervarious lengths of chilling treatments showed that significantchanges were induced before the visible injury. The effectivequantum yield and photochemical quenching of PSII dropped dramaticallywithin 24 h in both the presence and absence of a 12 h lightperiod. The maximal quantum yield and non-photochemical quenchingof PSII decreased significantly only in the presence of light.On the other hand, L/H chilling did not affect the functionof PSI, but caused a significant decrease in the electron availabilityfor PSI. These results suggest that the leaf chilling with highroot temperature destroys some component between PSII and PSIwithout the aid of light, which causes the over-reduction ofPSII in the light, and thereby the visible injury is inducedonly in the light.     

Bacterial lipopolysaccharides induce defense responses associated with programmed cell death in rice cells

PAMP (pathogen-associated molecular pattern) recognition plays an important role during the innate immune response in both plants and animals. Lipopolysaccharides (LPS) derived from Gram-negative bacteria are representative of typical PAMP molecules and have been reported to induce defense-related responses, including the suppression of the hypersensitive response, the expression of defense genes and systemic resistance in plants. However, the details regarding the precise molecular mechanisms underlying these cellular responses, such as the molecular machinery involved in the perception and transduction of LPS molecules, remain largely unknown. Furthermore, the biological activities of LPS on plants have so far been reported only in dicots and no information is thus available regarding their functions in monocots. In our current study, we report that LPS preparations for various becteria, including plant pathogens and non-pathogens, can induce defense responses in rice cells, including reactive oxygen generation and defense gene expression. In addition, global analysis of gene expression induced by two PAMPs, LPS and chitin oligosaccharide, also reveals a close correlation between the gene responses induced by these factors. This indicates that there is a convergence of signaling cascades downstream of their corresponding receptors. Furthermore, we show that the defense responses induced by LPS in the rice cells are associated with programmed cell death (PCD), which is a finding that has not been previously reported for the functional role of these molecules in plant cells. Interestingly, PCD induction by the LPS was not detected in cultured Arabidopsis thaliana cells.     

The bacterial stringent response, conserved in chloroplasts, controls plant fertilization

The chloroplast, an essential organelle for plants, performs a wide variety of metabolic processes for host cells, which include photosynthesis as well as amino acid and fatty acid biosynthesis. The organelle conserves many bacterial systems in its functions, implicating its origin from symbiosis of a photosynthetic bacterium. In bacterial cells, the stringent response acts as a global regulatory system for gene expression mediated by a small nucleotide, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), that is necessary for cell adaptation to diverse environmental stimuli such as amino acid starvation. Recent studies indicated that proteins similar to the bacterial ppGpp synthase/hydrolyase are conserved in plants, although their precise roles are not known. Here we show that the stringent response in chloroplasts is crucial for normal plant fertilization. Specifically, one of the Arabidopsis ppGpp synthase homologs, CRSH (Ca(2+)-activated RelA/SpoT homolog), exhibits calcium-dependent ppGpp synthesis activity in vitro, and is localized in chloroplasts in vivo. A knockdown mutation of CRSH in Arabidopsis results in a significant reduction in silique size and seed production, indicating that plant reproduction is under the control of chloroplast function through a ppGpp-mediated stringent response.     

Potential role of annexin AnnAt1 from Arabidopsis thaliana in pH-mediated cellular response to environmental stimuli

Plant annexins, Ca(2+)- and membrane-binding proteins, are probably implicated in the cellular response to stress resulting from acidification of cytosol. To understand how annexins can contribute to cellular ion homeostasis, we investigated the pH-induced changes in the structure and function of recombinant annexin AnnAt1 from Arabidopsis thaliana. The decrease of pH from 7.0 to 5.8 reduced the time of the formation of ion channels by AnnAt1 in artificial lipid membranes from 3.5 h to 15-20 min and increased their unitary conductance from 32 to 63 pS. These changes were accompanied by an increase in AnnAt1 hydrophobicity as revealed by hydrophobicity predictions, by an increase in fluorescence of 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS) bound to AnnAt1 and fluorescence resonance energy transfer from AnnAt1 tryptophan residues to TNS. Concomitant lipid partition of AnnAt1 at acidic pH resulted in its partial protection from proteolytic digestion. Secondary structures of AnnAt1 determined by circular dichroism and infrared spectroscopy were also affected by lowering the pH from 7.2 to 5.2. These changes were characterized by an increase in beta-sheet content at the expense of alpha-helical structures, and were accompanied by reversible formation of AnnAt1 oligomers as probed by ultracentrifugation in a sucrose gradient. A further decrease of pH from 5.2 to 4.5 or lower led to the formation of irreversible aggregates and loss of AnnAt1 ionic conductance. Our findings suggest that AnnAt1 can sense changes of the pH milieu over the pH range from 7 to 5 and respond by changes in ion channel conductance, hydrophobicity, secondary structure of the protein and formation of oligomers. Further acidification irreversibly inactivated AnnAt1. We suggest that the pH-sensitive ion channel activity of AnnAt1 may play a role in intracellular ion homeostasis.     

Blue light-dependent nuclear positioning in Arabidopsis thaliana leaf cells

The plant nucleus changes its intracellular position not only upon cell division and cell growth but also in response to environmental stimuli such as light. We found that the nucleus takes different intracellular positions depending on blue light in Arabidopsis thaliana leaf cells. Under dark conditions, nuclei in mesophyll cells were positioned at the center of the bottom of cells (dark position). Under blue light at 100 mumol m(-2) s(-1), in contrast, nuclei were located along the anticlinal walls (light position). The nuclear positioning from the dark position to the light position was fully induced within a few hours of blue light illumination, and it was a reversible response. The response was also observed in epidermal cells, which have no chloroplasts, suggesting that the nucleus has the potential actively to change its position without chloroplasts. Light-dependent nuclear positioning was induced specifically by blue light at >50 mumol m(-2) s(-1). Furthermore, the response to blue light was induced in phot1 but not in phot2 and phot1phot2 mutants. Unexpectedly, we also found that nuclei as well as chloroplasts in phot2 and phot1phot2 mutants took unusual intracellular positions under both dark and light conditions. The lack of the response and the unusual positioning of nuclei and chloroplasts in the phot2 mutant were recovered by externally introducing the PHOT2 gene into the mutant. These results indicate that phot2 mediates the blue light-dependent nuclear positioning and the proper positioning of nuclei and chloroplasts. This is the first characterization of light-dependent nuclear positioning in spermatophytes.     

Magnesium enhances aluminum-induced citrate secretion in rice bean roots (Vigna umbellata) by restoring plasma membrane H+-ATPase activity

We demonstrated that magnesium (Mg) can alleviate aluminum (Al) toxicity in rice bean [Vigna umbellata (Thunb.) Ohwi & Ohashi] more effectively than is expected from a non-specific cation response. Micromolar concentrations of Mg alleviated the inhibition of root growth by Al but not by lanthanum, and neither strontium nor barium at the micromolar level alleviates Al toxicity. Aluminum also induced citrate efflux from rice bean roots, and this response was stimulated by inclusion of 10 microM Mg in the treatment solution. The increase in the Al-induced citrate efflux by Mg paralleled the improvement in root growth, suggesting that the ameliorative effect of Mg might be related to greater citrate efflux. Vanadate (an effective H+-ATPase inhibitor) decreased the Al-induced citrate efflux, while addition of Mg partly restored the efflux. Mg addition also increased the activity of Al-reduced plasma membrane H+-ATPase, as well as helping to maintain the Mg and calcium contents in root apices. We propose that the addition of Mg to the toxic Al treatment helps maintain the tissue Mg content and the activity of the plasma membrane H+-ATPase. These changes enhanced the Al-dependent efflux of citrate which provided extra protection from Al stress.     

Elicitation of the hypersensitive response by Pseudomonas syringae

The hypersensitive response (HR), a rapid local necrosis accompanied by the production of antimicrobial compounds, is a manifestation of the primary defense mechanisms through which plants prevent colonization by potentially pathogenic microorganisms. The pathogen factors that trigger HR-associated defense mechanisms have yet to be established. Application of transposon mutagenesis and molecular cloning techniques have provided new insights into the bacterial elicitation of the HR. This review focuses on the interaction of Pseudomonas syringae with non-host plants as a model system for the bacterial induction of the HR and discusses (a) the genetics of Pseudomonas syringae pathogenicity and HR elicitation, (b) the environmental conditions controlling expression of Pseudomonas syringae pathogenicity/HR determinants, (c) the nature of the HR elicitor and (d) early plant responses to the pathogen.     

Nod Factor/Nitrate-Induced CLE Genes that Drive HAR1-Mediated Systemic Regulation of Nodulation

Host legumes control root nodule numbers by sensing externaland internal cues. A major external cue is soil nitrate, whereasa feedback regulatory system in which earlier formed nodulessuppress further nodulation through shoot–root communicationis an important internal cue. The latter is known as autoregulationof nodulation (AUT), and is believed to consist of two long-distancesignals: a root-derived signal that is generated in infectedroots and transmitted to the shoot; and a shoot-derived signalthat systemically inhibits nodulation. In Lotus japonicus, theleucine-rich repeat receptor-like kinase, HYPERNODULATION ABERRANTROOT FORMATION 1 (HAR1), mediates AUT and nitrate inhibitionof nodulation, and is hypothesized to recognize the root-derivedsignal. Here we identify L. japonicus CLE-Root Signal 1 (LjCLE-RS1)and LjCLE-RS2 as strong candidates for the root-derived signal.A hairy root transformation study shows that overexpressingLjCLE-RS1 and -RS2 inhibits nodulation systemically and, furthermore,that the systemic suppression depends on HAR1. Moreover, LjCLE-RS2expression is strongly up-regulated in roots by nitrate addition.Based on these findings, we propose a simple model for AUT andnitrate inhibition of nodulation mediated by LjCLE-RS1, -RS2peptides and the HAR1 receptor-like kinase.     

Disruption of a gene encoding C4-dicarboxylate transporter-like protein increases ozone sensitivity through deregulation of the stomatal response in Arabidopsis thaliana

To understand better the plant response to ozone, we isolated and characterized an ozone-sensitive (ozs1) mutant strain from a set of T-DNA-tagged Arabidopsis thaliana ecotype Columbia. The mutant plants show enhanced sensitivity to ozone, desiccation and sulfur dioxide, but have normal sensitivity to hydrogen peroxide, low temperature and high light levels. The T-DNA was inserted at a single locus which is linked to ozone sensitivity. Identification of the genomic sequences flanking the T-DNA insertion revealed disruption of a gene encoding a transporter-like protein of the tellurite resistance/C(4)-dicarboxylate transporter family. Plants with either of two different T-DNA insertions in this gene were also sensitive to ozone, and these plants failed to complement ozs1. Transpiration levels, stomatal conductance levels and the size of stomatal apertures were greater in ozs1 mutant plants than in the wild type. The stomatal apertures of ozs1 mutant plants responded to light fluctuations but were always larger than those of the wild-type plants under the same conditions. The stomata of the mutant and wild-type plants responded similarly to stimuli such as light, abscisic acid, high concentrations of carbon dioxide and ozone. These results suggest that OZS1 helps to close stomata, being not involved in the responses to these signals.     

Phytochrome-regulated PIL1 derepression is developmentally modulated

We define the photoresponsiveness, during seedling de-etiolation,of PHYTOCHROME-INTERACTING FACTOR 3-LIKE 1 (PIL1), initiallyidentified by microarray analysis as an early-response genethat is robustly repressed by first exposure to light. We showthat PIL1 mRNA abundance declines rapidly, with a half-timeof 15 min, to a new steady-state level, 10-fold below the initialdark level, within 45 min of first exposure to red light. Analysisof phy-null mutants indicates that multiple phytochromes, includingphyA and phyB, impose this repression. Conversely, PIL1 expressionis rapidly derepressed by subsequent far-red irradiation ofpreviously red light-exposed seedlings. However, the magnitudeof this derepression is modulated over time, in a biphasic manner,in response to increasing duration of pre-exposure to continuousred light: (i) an early phase (up to about 6 h) of relativelyrapidly increasing effectiveness of far-red reversal of repression,as declining phyA levels relieve initial very low fluence suppressionof this response; and (ii) a second phase (beyond 6 h) of graduallydeclining effectiveness of far-red reversal, to only 20% ofmaximal derepression, within 36 h of continuous red light exposure,with no evidence of circadian modulation of this responsiveness,an observation in striking contrast to a previous report forentrained, green seedlings exposed to vegetative shade. Thesedata, together with analysis of phytochrome signaling mutantsand overexpressors with aberrant de-etiolation phenotypes, suggestthat the second-phase decline in robustness of PIL1 derepressionis an indirect consequence of the global developmental transitionfrom the etiolated to the de-etiolated state, and that circadiancoupling of derepression requires entrainment.     

Genetic linkages of the circadian clock-associated genes, TOC1, CCA1 and LHY, in the photoperiodic control of flowering time in Arabidopsis thaliana

In Arabidopsis thaliana, the flowering time is regulated through the circadian clock that measures day-length and modulates the photoperiodic CO-FT output pathway in accordance with the external coincidence model. Nevertheless, the genetic linkages between the major clock-associated TOC1, CCA1 and LHY genes and the canonical CO-FT flowering pathway are less clear. By employing a set of mutants including an extremely early flowering toc1 cca1 lhy triple mutant, here we showed that CCA1 and LHY act redundantly as negative regulators of the photoperiodic flowering pathway. The partly redundant CCA1/LHY functions are largely, but not absolutely, dependent on the upstream TOC1 gene that serves as an activator. The results of examination with reference to the expression profiles of CO and FT in the mutants indicated that this clock circuitry is indeed linked to the CO-FT output pathway, if not exclusively. For this linkage, the phase control of certain flowering-associated genes, GI, CDF1 and FKF1, appears to be crucial. Furthermore, the genetic linkage between TOC1 and CCA1/LHY is compatible with the negative and positive feedback loop, which is currently believed to be a core of the circadian clock. The results of this study suggested that the circadian clock might open an exit for a photoperiodic output pathway during the daytime. In the context of the current clock model, these results will be discussed in connection with the previous finding that the same clock might open an exit for the early photomorphogenic output pathway during the night-time.