Sang-Uk Lee

Nitric oxide and S‐nitrosoglutathione function additively during plant immunity

Nitric oxide (NO) is emerging as a key regulator of diverse plant cellular processes. A major route for the transfer of NO bioactivity is S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol forming an S-nitrosothiol (SNO). Total cellular levels of protein S-nitrosylation are controlled predominantly by S-nitrosoglutathione reductase 1 (GSNOR1) which turns over the natural NO donor, S-nitrosoglutathione (GSNO). In the absence of GSNOR1 function, GSNO accumulates, leading to dysregulation of total cellular S-nitrosylation. Here we show that endogenous NO accumulation in Arabidopsis, resulting from loss-of-function mutations in NO Overexpression 1 (NOX1), led to disabled Resistance (R) gene-mediated protection, basal resistance and defence against nonadapted pathogens. In nox1 plants both salicylic acid (SA) synthesis and signalling were suppressed, reducing SA-dependent defence gene expression. Significantly, expression of a GSNOR1 transgene complemented the SNO-dependent phenotypes of paraquat resistant 2-1 (par2-1) plants but not the NO-related characters of the nox1-1 line. Furthermore, atgsnor1-3 nox1-1 double mutants supported greater bacterial titres than either of the corresponding single mutants. Our findings imply that GSNO and NO, two pivotal redox signalling molecules, exhibit additive functions and, by extension, may have distinct or overlapping molecular targets during both immunity and development.

Bacillus aryabhattai SRB02 tolerates oxidative and nitrosative stress and promotes the growth of soybean by modulating the production of phytohormones

Plant growth promoting rhizobacteria (PGPR) are diverse, naturally occurring bacteria that establish a close association with plant roots and promote the growth and immunity of plants. Established mechanisms involved in PGPR-mediated plant growth promotion include regulation of phytohormones, improved nutrient availability, and antagonistic effects on plant pathogens. In this study, we isolated a bacterium from the rhizospheric soil of a soybean field in Chungcheong buk-do, South Korea. Using 16S rRNA sequencing, the bacterium was identified as Bacillus aryabhattai strain SRB02. Here we show that this strain significantly promotes the growth of soybean. Gas chromatography—mass spectrometry analysis showed that SRB02 produced significant amounts of abscisic acid, indole acetic acid, cytokinin and different gibberellic acids in culture. SRB02-treated soybean plants showed significantly better heat stress tolerance than did untreated plants. These plants also produced consistent levels of ABA under heat stress and exhibited ABA-mediated stomatal closure. High levels of IAA, JA, GA12, GA4, and GA7, were recorded in SRB02-treated plants. These plants produced longer roots and shoots than those of control plants. B. aryabhattai SRB02 was found to be highly tolerant to oxidative stress induced by H2O2 and MV potentiated by high catalase (CAT) and superoxide dismutase (SOD) activities. SRB02 also tolerated high nitrosative stress induced by the nitric oxide donors GSNO and CysNO. Because of these attributes, B. aryabhattai SRB02 may prove to be a valuable resource for incorporation in biofertilizers and other soil amendments that seek to improve crop productivity.

Nitric Oxide Mediated Transcriptome Profiling Reveals Activation of Multiple Regulatory Pathways in Arabidopsis thaliana

Imbalance between the accumulation and removal of nitric oxide and its derivatives is a challenge faced by all plants at the cellular level, and is especially important under stress conditions. Exposure of plants to various biotic and abiotic stresses causes rapid changes in cellular redox tone potentiated by the rise in reactive nitrogen species that serve as signaling molecules in mediating defensive responses. To understand mechanisms mediated by these signaling molecules, we performed a large-scale analysis of the Arabidopsis transcriptome induced by nitrosative stress. We generated an average of 84 and 91 million reads from three replicates each of control and 1 mM S-nitrosocysteine (CysNO)-infiltrated Arabidopsis leaf samples, respectively. After alignment, more than 95% of all reads successfully mapped to the reference and 32,535 genes and 55,682 transcripts were obtained. CysNO infiltration caused differential expression of 6436 genes (3448 up-regulated and 2988 down-regulated) and 6214 transcripts (3335 up-regulated and 2879 down-regulated) 6 h post-infiltration. These differentially expressed genes were found to be involved in key physiological processes, including plant defense against various biotic and abiotic stresses, hormone signaling, and other developmental processes. After quantile normalization of the FPKM values followed by students T-test (P < 0.05) we identified 1165 DEGs (463 up-regulated and 702 down-regulated) with at least 2-folds change in expression after CysNO treatment. Expression patterns of selected genes involved in various biological pathways were verified using quantitative real-time PCR. This study provides comprehensive information about plant responses to nitrosative stress at transcript level and would prove helpful in understanding and incorporating mechanisms associated with nitrosative stress responses in plants.

Nitric Oxide Responsive Heavy Metal-Associated Gene AtHMAD1 Contributes to Development and Disease Resistance in Arabidopsis thaliana.

Exposure of plants to different biotic and abiotic stress condition instigates significant change in the cellular redox status; resulting in the elevation of reactive nitrogen species that play signaling role in mediating defense responses. Heavy metal associated (HMA) domain containing genes are required for spatio-temporal transportation of metal ions that bind with various enzymes and co-factors within the cell. To uncover the underlying mechanisms mediated by AtHMA genes, we identified 14 Arabidopsis HMA genes that were differentially expressed in response to nitrosative stress through RNA-seq analysis. Of those 14 genes, the expression of eight HMA genes was significantly increased, whereas that of six genes was significantly reduced. We further validated the RNA-seq results through quantitative real-time PCR analysis. Gene ontology analysis revealed the involvement of these genes in biological processes such as hemostasis and transport. The majority of these nitric oxide (NO)-responsive AtHMA gene products are carrier/transport proteins. AtHMAD1 (At1g51090) showed the highest fold change to S-nitrosocystein. We therefore, further investigated its role in oxidative and nitrosative mediated stress conditions and found that AtHMAD1 has antagonistic role in shoot and root growth. Characterization of AtHMAD1 through functional genomics showed that the knock out mutant athmad1 plants were resistant to virulent Pseudomonas syringae (DC3000) and showed early induction and high transcript accumulation of pathogenesis related gene. Furthermore, inoculation of athamd1 with avirulent strain of the same bacteria showed negative regulation of R-gene mediated resistance. These results were supported by hypersensitive cell death response and cell death induced electrolyte leakage. AtHMAD1 was also observed to negatively regulate systemic acquired resistance SAR as the KO mutant showed induction of SAR marker genes. Overall, these results imply that NO-responsive AtHMA domain containing genes may play an important role in plant development and immunity.

Influence of Gypsum, Popped Rice Hulls and Zeolite on Contents of Ca 2+ , Mg 2+ , Na + , K + in Reclaimed Tideland Soils in Kyehwado

The effect of application of gypsum (G), popped rice hulls (PRH), and zeolite (Z) in exchangeable cations concentrations of reclaimed tideland soil in Kyehwado was investigated for 3 years from 2004 to 2006 in a pot experiment with bermuda grass (Cynodon dactylon). Treatments with three soil conditioner and with three applications were established with three replications; G1 (1,550 kg ), G2 (3,100), and G3 (6,200) for gypsum, H1 (1,000), H2 (2,000), and H3 (3,000) for PRH, and HZ1 (200), HZ2 (400), and HZ3 (800) for co-application of zeolite with PRH at the 1,500 kg . At 60, 90, 120 days after treatment (DAT), exchangeable cations (, , , and ) were analyzed Gypsum application significantly decreased , , in the soil probably due to exchange and subsequent leaching of these cations by from the gypsum applied. Overall, concentration was gradually decreased by continuous application of soil conditioners and was in the order of 2004>2005>2006 regardless of the kinds and application rate of soil conditioners. Comparing concentrations among the soil conditioners in the same year, its concentration was in the order of gypsum concentration; i.e. concentration was in the order of gypsumPRH also showed a similar pattern to . Gypsum application significantly increased concentration and in the gypsum treated soil concentration increased with years.

Influence of Gypsum, Popped Rice Hulls and Zeolite on Contents of Cation in Reclaimed Tideland Soils in Mangyeong

Soil conditioner, such as (gypsum), popped rice hulls (PRH), and PRH with zeolite, were treated to the silt loam of Mangyeong in Saemangeum tideland reclaimed as 1550 (G1), 3100 (G2) and 6200 (G3) of gypsum kg/10 a, 1000(H1), 2000(H2), and 3000 (H3) of PRH kg/10 a, and 200 (HZ1), 400 (HZ2), 800 (HZ3) of zeolite kg/10 a added to 1500 PRH kg/10 a, respectively, each year until 2006 from 2004 for soil aggregation. Under these conditions with growing bermuda grass (Cynodon dactylon) it was analyzed cations in soil, such as , , , and , at 60, 90, and 120 days after treatment (DAT) to research how soil conditioners influenced to change those contents in soils, respectively. The change of cations in soil was almost the same things as fine sandy loam that gypsum treated decreased remarkably contents of , , in soil. The change of content in soil by continuous using soil conditioners was gradually decreased in the order of 2004>2005>2006, regardless of the sorts and levels of soil treated conditioners, and content was high in the order of gypsum content was high in the order of gypsum content in soil was increased in the order of gypsum content in soil was remarkably increased with continuous treatment of gypsum, and its level was in the order of 2004

Transcriptome profile of NO-induced Arabidopsis transcription factor genes suggests their putative regulatory role in multiple biological processes

TFs are important proteins regulating plant responses during environmental stresses. These insults typically induce changes in cellular redox tone driven in part by promoting the production of reactive nitrogen species (RNS). The main source of these RNS is nitric oxide (NO), which serves as a signalling molecule, eliciting defence and resistance responses. To understand how these signalling molecules regulate key biological processes, we performed a large scale S-nitrosocysteine (CySNO)-mediated RNA-seq analysis. The DEGs were analysed to identify potential regulatory TFs. We found a total of 673 (up- and down-regulated) TFs representing a broad range of TF families. GO-enrichment and MapMan analysis suggests that more than 98% of TFs were mapped to the Arabidopsis thaliana genome and classified into pathways like hormone signalling, protein degradation, development, biotic and abiotic stress, etc. A functional analysis of three randomly selected TFs, DDF1, RAP2.6, and AtMYB48 identified a regulatory role in plant growth and immunity. Loss-of-function mutations within DDF1 and RAP2.6 showed compromised basal defence and effector triggered immunity, suggesting their positive role in two major plant defence systems. Together, these results imply an important data representing NO-responsive TFs that will help in exploring the core mechanisms involved in biological processes in plants.

S-nitrosocysteine-responsive genes modulate diverse regulatory pathways in Oryza sativa: a transcriptome profiling study

Rice (Oryza sativa L.) is a major food crop and also a well-established genetic model. Nitric oxide (NO) and its derivatives are important signalling molecules that actively participate in various signalling pathways in response to different stresses. In this study, we performed RNA-seq mediated transcriptomic analysis of rice after treatment with the nitric oxide donor, S-nitroso-L-cysteine (CySNO), generating an average of 37.5 and 41.5 million reads from control and treated leaf samples respectively. More than 95% of the reads were successfully mapped to the O. sativa reference genome yielding a total of 33539 differentially expressed genes (DEGs, P < 0.05). Further analyses identified 825 genes with at least 2-fold change in the expression following treatment with CySNO (P < 0.01). The DEGs identified were involved in diverse molecular functions such as catalytic activity, binding, transport, and receptor activity and were mostly located in the membrane, organelles such as nucleus, Golgi apparatus and mitochondria. DEGs also contained several genes that regulate responses to abiotic stresses such as drought, heat, cold and salt stress and biotic stresses. We also found significantly similar expression patterns of CySNO-responsive DEGs of rice with the CySNO-responsive DEGs of Arabidopsis in a previous study. Expression patterns of genes involved in key biological functions were verified using quantitative real time (qRT)-PCR. The findings of this study suggest that NO regulates the transcriptional control of genes involved in a wide variety of physiological functions in rice, and that NO-mediated transcriptional networks are highly conserved across the plant kingdom. This study provides useful information regarding the transcriptional response of plants to nitrosative stress.

High-throughput Screening of Rice for Nitrosative-stress Response and the Identification of Effective pH Range for Nitric Oxide Donor S-Nitrocysteine

Nitric oxide (NO)-mediated signaling regulates growth, development, and responses to biotic and abiotic stresses in plants. It involves various NO-derivatives, such as NO radical, nitroxyl anion (NO ), peroxynitrite (ONOO ), and S-nitrosothiols (SNOs). Many of these SNOs including S-nitrosocysteine (CysNO), can modify intracellular protein thiols through trans-nitrosation reactions. Various NO donors have been used as tools to unravel the fundamental mechanisms of NO signaling. However, the effect of NO donors can be analyzed only under specific conditions. Here, we report a high-throughput screening system for identifying mutant lines exhibiting differential responses to the frequently used NO donor CysNO. To determine the effect of CysNO on seed germination and growth at various pH levels, we grew Arabidopsis thaliana seeds on MS medium with various concentrations of CysNO at a pH range of 2–5.8. The pH was adjusted using different volumes of HCl. Seeds were found to be highly sensitive to pH below 4.5 at any given CysNO concentration; therefore, the effects of the CysNO treatments could not be determined. A similar effect of pH was observed in the case of rice plants. However, when the same concentrations of CysNO were prepared using EPPS (3-[4-(2-Hydroxyethyl)-1-piperazinyl] propanesulfonic acid) buffer to maintain pH at a suitable level, significantly different responses to CysNO were observed among wild-type and mutant rice lines, indicating the importance of optimum pH conditions. Using this technique, we identified several rice Ac/Ds transposon mutant lines that showed tolerance or sensitivity to exogenous NO stress. In conclusion, pH is a critical factor for plant germination and growth. However, information about changes of pH caused CysNO solution was lacking. In this study we demonstrated the effective pH range for maximum efficiency and absorbance of CysNO using EPPS buffer system in rice. Hence, the EPPS-based buffer system is more efficient for high-throughput screening in rice against nitrosative stress induced by the nitric oxide donor S-Nitrocysteine.