Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horseradish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca 2؉ -dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5-TWCG(C/T)GTKKKKTKCG-3 (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate ␤-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM.

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Nitric oxide (NO) is known for its role in the activation of plant defense responses. To examine the involvement and mode of action of NO in plant defense responses, we introduced calmodulin-dependent mammalian neuronal nitric oxide synthase (nNOS), which controls the CaMV35S promoter, into wild-type and NahG tobacco plants. Constitutive expression of nNOS led to NO production and triggered spontaneous induction of leaf lesions. Transgenic plants accumulated high amounts of H(2)O(2), with catalase activity lower than that in the wild type. nNOS transgenic plants contained high levels of salicylic acid (SA), and they induced an array of SA-, jasmonic acid (JA)-, and/or ethylene (ET)-related genes. Consequently, NahG co-expression blocked the induction of systemic acquired resistance (SAR)-associated genes in transgenic plants, implying SA is involved in NO-mediated induction of SAR genes. The transgenic plants exhibited enhanced resistance to a spectrum of pathogens, including bacteria, fungi, and viruses. Our results suggest a highly ranked regulatory role for NO in SA-, JA-, and/or ET-dependent pathways that lead to disease resistance.

We previously isolated the OsCBT gene, which encodes a calmodulin (CaM)-binding protein, from a rice expression library constructed from fungal elicitor-treated rice suspension cells. In order to understand the function of OsCBT in rice, we isolated and characterized a T-DNA insertion mutant allele named oscbt-1. The oscbt-1 mutant exhibits reduced levels of OsCBT transcripts and no significant morphological changes compared to wild-type plant although the growth of the mutant is stunted. However, oscbt-1 mutants showed significant resistance to two major rice pathogens. The growth of the rice blast fungus Magnaporthe grisea, as well as the bacterial pathogen Xanthomonas oryzae pv. oryzae was significantly suppressed in oscbt-1 plants. Histochemical analysis indicated that the hypersensitive-response was induced in the oscbt-1 mutant in response to compatible strains of fungal pathogens. OsCBT expression was induced upon challenge with fungal elicitor. We also observed significant increase in the level of pathogenesis-related genes in the oscbt-1 mutant even under pathogen-free condition. Taken together, the results support an idea that OsCBT might act as a negative regulator on plant defense.

Ixeris dentata var. albiflora is considered as a potential therapeutic agent against mithridatism, calculous, indigestion, pneumonia, hepatitis, and tumors as well as good seasoned vegetable in Far East countries. Phytoene synthase (PSY), phytoene desaturase (PDS) ξ-carotene desaturase (ZDS), lycopene β-cyclase (LCYB), lycopene ε-cyclase (LCYE), ε-ring carotene hydroxylase (CHXB), and zeaxanthin epoxidase (ZDS) are vital enzymes in the carotenoid biosynthesis pathway. We have examined these seven genes from I. dentata that are participated in carotenoid biosynthesis utilizing an Illumina/Solexa HiSeq 2000 platform. In silico analysis of the seven deduced amino acid sequences were revealed its closest homology with other Asteracea plants. Further, we explored transcript levels and carotenoid accumulation in various organs of I. dentata using quantitative real time PCR and high-performance liquid chromatography, respectively. The highest transcript levels were noticed in the leaf for all the genes while minimal levels were noticed in the root. The maximal carotenoid accumulation was also detected in the leaf. We proposed that these genes expressions are associated with the accumulation of carotenoids. Our findings may suggest the fundamental clues to unravel the molecular insights of carotenoid biosynthesis in various organs of I. dentata.