Cheol Ho Hwang

Soybean Nodule-Enhanced CLE Peptides in Roots Act as Signals in GmNARK-Mediated Nodulation Suppression

The number of nodules formed in the roots of leguminous plants is systemically controlled by autoregulation of nodulation (AON). This study characterized two of the CLAVATA3/endosperm-surrounding region (CLE) genes involved in AON signal transduction. The GmRIC1 and GmRIC2 genes initiated expression solely in the roots at approximately 3 days after inoculation (DAI) with Nod factor-producing rhizobia, corresponding to the time point of AON, and the expression was up-regulated by cytokinins. Levels of GmRIC1 and GmRIC2 gene expression were much higher in the supernodulation mutant, SS2-2, than in wild-type (WT) soybeans during nodule development, even after initiation of nitrogen fixation. At 3 DAI, GmRIC2 was induced in the cells of the pericycle and the outer cortex, which undergo cell division to form nodule primordia and spreads from the central region to the whole nodule as it develops. Overexpression of GmRIC1 and GmRIC2 strongly suppressed the nodulation of WT roots as well as transgenic hairy roots in a GmNARK-dependent manner. This systemic suppression of nodulation was caused by the secretion of two CLE proteins into the extracellular space. Double grafting between WT and SS2-2 soybeans showed that signal Q is larger in SS2-2 than in WT roots during nodulation. The results of this study suggest that GmRIC1 and GmRIC2 are good candidates for root-derived signal Q in AON signal transduction.

Genetic Analysis of Genes Controlling Natural Variation of Seed Coat and Flower Colors in Soybean

Soybean exhibits natural variation in flower and seed coat colors via the deposition of various anthocyanin pigments in the respective tissues. Although pigmentation in seeds or flowers has been well dissected at molecular level in several plant species, the genes controlling natural variation in anthocyanin traits in the soybean are not completely understood. To evaluate the genetic correlation between genetic loci and genes, 8 enzyme-encoding gene families and a transcription factor were localized in a soybean genome-wide genetic map. Among the seed coat color-controlling loci, the genetic location of the gene encoding for W1 was substantiated in the context of the current soybean molecular genetic map and O was postulated to correspond to anthocyanidin reductase. Among the genetic loci that regulate flower pigmentation, the genetic locations of the genes encoding for W1, W4, and Wp were identified, W3 was mapped on soybean linkage group B2 (chromosome 14), and W2 was postulated to correspond to an MYB transcription factor. Correlation studies between the developed markers and 3 color-controlling loci provided important empirical data that should prove useful in the design of marker-assisted breeding schemes as well as future association studies involving soybean.

Distinct roles of the pepper hypersensitive induced reaction protein gene CaHIR1 in disease and osmotic stress, as determined by comparative transcriptome and proteome analyses.

A Capsicum annuum hypersensitive induced reaction protein1 (CaHIR1) was recently proposed as a positive regulator of hypersensitive cell death in plants. Overexpression of CaHIR1 in transgenic Arabidopsis plants conferred enhanced resistance against the hemi-biotrophic Pseudomonas syringae pv. tomato (Pst) and the biotrophic Hyaloperonospora parasitica. Infection by avirulent Pseudomonas strains carrying avrRpm1 or avrRpt2 caused enhanced resistance responses in transgenic plants, suggesting that CaHIR1 is involved in basal disease resistance in a race-nonspecific manner. H.parasitica exhibited low levels of asexual sporulation on CaHIR1 seedlings. In contrast, transgenic plants were susceptible not only to the necrotrophic fungal pathogen Botrytis cinerea but were also sensitive to osmotic stress caused by high salinity and drought. To identify proteins whose expression was altered by CaHIR1 overexpression in Arabidopsis leaves, a quantitative comparative proteome analysis using two-dimensional gel electrophoresis coupled with mass spectrometry was performed. Of about 400 soluble proteins, 11 proteins involved in several metabolic pathways were up- or down-regulated by CaHIR1 overexpression. Genes encoding glycine decarboxylase (At2g35370) and an unidentified protein (At2g03440), which were strongly upregulated in CaHIR1-overexpressing Arabidopsis, were also differentially induced at the transcriptional level by Pst infection. Arabidopsis carbonic anhydrase (At3g01500), highly similar to tobacco salicylic acid-binding protein 3, was up-regulated by CaHIR1 overexpression. The activity of an anti-oxidant enzyme, cooper/zinc superoxide dismutase (At2g28190), was also attenuated in transgenic Arabidopsis by CaHIR1 overexpression. Together, these results suggest that CaHIR1 overexpression in Arabidopsis mediates plant responses to biotrophic, hemi-biotrophic and necrotrophic pathogens, as well as to osmotic stress in different ways.

Nitrate inhibits soybean nodulation by regulating expression of CLE genes.

Nitrogen compounds such as nitrate act as a potential inhibitor for legume nodulation. In this study, we isolated a new CLE gene, GmNIC2, from nitrate-treated roots, which shares high sequence homology with nitrate-induced CLE gene GmNIC1. Similar to GmNIC1, the expression level of GmNIC2 was not significantly altered in roots by rhizobial inoculation and was much higher in young nodules than in roots. In addition, overexpression of GmNIC2 led to similar nodulation inhibition of transgenic hairy roots to that of GmNIC1, which occurred in GmNARK-dependent manner and at the local level. By analyzing GmNARK loss-of-function mutant, SS2-2, it was found that expression levels of GmNIC1 and GmNIC2 in the SS2-2 roots were lower than in the wild type (WT) roots in response to nitrate. In contrast to GmNIC1 and GmNIC2, expressions of GmRIC1 and GmRIC2 genes that are related to the autoregulation of nodulation (AON) were strongly suppressed both of the soybeans during all periods of nitrate treatment and even were not induced by additional inoculation with rhizobia. Taken together, the results of this study suggest that GmNIC2, as an active homologous gene located in chromosome 13, acts locally to suppress nodulation, like GmNIC1, and nitrate inhibition of nodulation is led by fine-tuned regulation of both nitrate-induced CLEs and rhizobia-induced CLEs.

Comparative Proteomic Analysis of Soybean Nodulation Using a Supernodulation Mutant, SS2-2

Legumes have the ability to form root nodules that fix atmospheric nitrogen through a symbiotic interaction with nitrogen-fixing bacteria. As a first step in dissecting the molecular process of nodulation, proteome reference maps of soybean roots and nodules were constructed. Time course analysis revealed that the transition from root to nodule was accompanied with downregulation of defense-response related proteins, including Mn-superoxide dismutase, peroxidase (Prx), PR10, and stress-induced protein, leading to the initiation of a symbiotic interaction between the two partners. Following nitrogenase biosynthesis, the host plant cooperated with the rhizobia to fix atmospheric nitrogen under microaerobic conditions via expression of leghemoglobins and antioxidant proteins. Comparative proteome analysis indicated lower expression of malate dehydrogenase (MDH), leghemoglobins and nitrogenase in the nodule development of the supernodulation mutant, SS2-2, as compared to the wild type, indicating that SS2-2 forms functionally immature nodules in higher numbers with the lower activity of nitrogen fixation.

Differentially expressed genes related to symbiotic association in a supernodulating soybean mutant and its wild-type

SUMMARY To understand the molecular basis of symbiotic association, a cDNA-AFLP technique was used to identify differentially expressed transcripts between a supernodulating soybean mutant, SS2-2, and its wild-type, Sinpaldalkong 2. As sources of cDNA-AFLP templates, trifoliates of 2-week-old plants were collected 1 week after Bradyrhizobium japonicum inoculation. A total of 147 bands out of 4000 amplicons were recognized as differentially expressed fragments, with 40 transcript-derived fragments (TDFs) in SS2-2 and 65 TDFs in Sinpaldalkong 2. Qualitative and quantitative real-time RT-PCR assays suggested that the expression patterns of genes in both genotypes were clearly differentiated. TDFs homologous to nodulin (65S2) and a putative senescence-associated protein (9S1) were up-regulated in SS2-2, whereas Sinpaldalkong 2 showed up-regulation of a receptor-like kinase (48sin1) and a kinase-like protein (17sin1). This indicates that different genes may be involved in regulation of the symbiotic programme that distinguishes SS2-2 from its wild-type. A TDF showing a change in a single base from A (Sinpaldalkong 2) to T (SS2-2) in this study was identified as a Glycine max nodule autoregulation receptor-like protein kinase precursor, previously identified by map-based cloning. These results demonstrate that cDNA-AFLP is a powerful technique to detect interesting genes without prior assumptions about the nature of the genes. The differentially expressed genes between Sinpaldalkong 2 and SS2-2 suggest that different signal transduction pathways for symbiosis may be involved in the two soybean genotypes.

Genome-Wide Expression Profiling of OsWRKY Superfamily Genes during Infection with Xanthomonas oryzae pv. oryzae Using Real-Time PCR

WRKY transcription factors (TFs) are involved in regulating a range of biological processes such as growth, development, and the responses to biotic and abiotic stresses. Genome-wide expression profiling of OsWRKY TF superfamily genes in rice after infection with Xanthomonas oryzae pv. oryzae (Xoo) was performed to elucidate the function of OsWRKY TFs in the interaction between rice and Xoo. Of the 111 OsWRKY TF genes tested, the transcription of 94 genes changed after Xoo infection. The OsWRKY TF genes were classified into eight types according to their expression profiles. Eighty-two genes in Groups I, II, III, IV, VII were up-regulated after exposure to a compatible or an incompatible race of Xoo. Examination of salicylic acid (SA)-deficient rice lines revealed that SA was involved in Xa1-mediated resistance to Xoo infection. OsWRKY TF genes involved in Xa1-mediated resistance were classified according to their SA-dependent or -independent expression. In SA-deficient rice, the expression of 12 of 57 OsWRKY TF genes involved in Xa1-mediated resistance was compromised. Of these six OsWRKY TF genes were induced by SA. OsWRKY88, an example of a gene possibly involved in SA-dependent Xa1-mediated resistance, activated defense related genes and increased resistance to Xoo. Thus, expression profiling of OsWRKY TF genes may help predict the functions of OsWRKY TF genes involved in Xa1-mediated resistance.

Transformation of Artemisia adamsii, Endemic to a Gobi Desert, with CLP, Dhn5 to Enhance Environmental Stress Tolerance

Freezing and drought tolerances in plants are very important for survival in the desert. In an effort to reduce desertifcation in Gobi, a molecular breeding of Artemisia adamsii using the CLP (chitinase like protein, antifreeze protein) and Dhn5 (dehydrin5) genes from barley is performed by introducing them into Artemisia adamsii via Agrobacteria. We had found an optimal combinatorial concentration of hormones at 0.05mg/L of NAA and 0.5mg/L of BA for growth of callus in Artemisia adamsii. In addition, the higher rate of callus induction using hypocotyl as explant was observed comparing to explants of stem and leaf. There were some variations in the level of the proteins expressed among the transgenic lines such that the lines of CLP(CS1-5, 1-7,4-4) and Dhn5(DS2-2, 2-3) lines produce the protein to higher levels. The transgenic lines showing a higher level of Dhn5 exhibited better growth than nontransgenic callus in presence of 10 and 20％ PEG. In case of the CLP tansgenic lines, both CS1-5 and CS1-7 showed a higher level of freezing tolerance determined by ion leakage test.

The barley lipid transfer protein, BLT101, enhances cold tolerance in wheat under cold stress

To understand the mechanism by which the barley plants tolerate overwintering, we analyzed the BLT101 gene expression in four different cultivars including two each of spring and winter barleys, where the BLT101 transcript level varied among the cultivars with a long-term cold acclimation. The BLT101 transcript was induced at a higher level in the crown tissues in response to low temperature treatment. The BLT101 transcript level correlated with those of the corresponding protein. BLT101-overexpressing transgenic wheat lines (BLT101ox’s) grew slowly and showed decreased expression of the genes induced by hormones (such as auxin and cytokinin), compared to non-transgenic (NT) plants. After a prolonged cold treatment, the BLT101ox leaves showed normal phenotypes, while the NT plants displayed blighted and withered leaves. A substances leakage test showed that the BLT101ox wheat retains enhanced freezing tolerance, compared to the NT plants. The transgenic lines lost less water than the NT plants, under a cold-stressed condition. Our findings suggest that tolerance to water stress and the retardation of growth in cold conditions, the essential features in overwintering barleys, involve BLT101.

Root proteome analysis of Chinese cabbage in response to Plasmodipohora brassicae Woron

Clubroot disease is one of the most wide-spread and devastating diseases in the cultivation of Chinese cabbage. To develop a protein marker for resistance to clubroot disease in Chinese cabbage, a comparative proteome analysis was performed between a sensitive line, 94SK, and a resistant line, CR Shinki DH. Three proteins of two fold or higher accumulation that are specific to each line were found 3 days after innoculation of the Plasmodiphora brassicae. They are glutamine synthetase, malate dehydrogenase/oxidoreductase and fructose-bisphosphate aldolase in the 94SK and actin, phosphoglycerate kinase, and Cu/Zn superoxide dismutase in the CR Shinki line. From the comparison of the synthesized proteins in the 94SK and the CR Shinki, CR Shinki was found to produce more ATP-binding protein for the ABC transporter while 94SK showed a higher level of pathogenesis-related protein 1 production. All of these proteomic variations may lead to the development of molecular markers to accelerate the breeding process.