Jin Seo Jeong

The histone deacetylase OsHDAC1 epigenetically regulates the OsNAC6 gene that controls seedling root growth in rice

We have previously isolated a rice gene encoding a histone deacetylase, OsHDAC1, and observed that its transgenic overexpression increases seedling root growth. To identify the transcriptional repression events that occur as a result of OsHDAC1 overexpression (OsHDAC1(OE)), a global profiling of root-expressed genes was performed on OsHDAC1(OE) or HDAC inhibitor-treated non-transgenic (NT) roots, in comparison with untreated NT roots. We selected 39 genes that are induced and repressed in HDAC inhibitor-treated NT and OsHDAC1(OE) roots, compared with NT roots, respectively. Interestingly, OsNAC6, a member of the NAM-ATAF-CUC (NAC) family, was identified as a key component of the OsHDAC1 regulon, and was found to be epigenetically repressed by OsHDAC1 overexpression. The root phenotype of OsNAC6 knock-out seedlings was observed to be similar to that of the OsHDAC1(OE) seedlings. Conversely, the root phenotype of the OsNAC6 overexpressors was similar to that of the OsHDAC1 knock-out seedlings. These observations indicate that OsHDAC1 negatively regulates the OsNAC6 gene that primarily mediates the alteration in the root growth of the OsHDAC1(OE) seedlings. Chromatin immunoprecipitation assays of the OsNAC6 promoter region using antibodies specific to acetylated histones H3 and H4 revealed that OsHDAC1 epigenetically represses the expression of OsNAC6 by deacetylating K9, K14 and K18 on H3 and K5, K12 and K16 on H4.

Matrix attachment region from the chicken lysozyme locus reduces variability in transgene expression and confers copy number-dependence in transgenic rice plants

Matrix-attachment regions (MARs) may function as domain boundaries and partition chromosomes into independently regulated units. In this study, BP-MAR, a 1.3-kb upstream fragment of the 5′MAR flanking the chicken lysozyme locus, was tested for its effects on integration and expression of transgenes in transgenic rice plants. Using the Agrobacterium-mediated method, we transformed rice with nine different constructs containing seven and six different promoters and coding sequences, respectively. Genomic Southern blot analyses of 357 independent transgenic lines revealed that in the presence of BP-MAR, 57% of the lines contained a single copy of the transgene, whereas in its absence, only 20% of the lines contained a single copy of the transgene. RNA gel-blot and immunoblot experiments demonstrated that in the presence of BP-MAR, transgene expression levels were similar among different lines. These data were in direct contrast to those derived from transgenes expressed in the absence of BP-MAR, which varied markedly with the chromosomal integration site . Thus, it can be concluded that BP-MAR significantly reduces the variability in transgene expression between independent transformants. Moreover, the presence of BP-MAR appears to confer a copy number-dependent increase in transgene expression, although it does not increase expression levels of individual transgenes. These data contrast with results previously obtained with various MARs that increased expression levels of transgene significantly. Therefore, we conclude that the incorporation of BP-MAR sequences into the design of transformation vectors can minimize position effects and regulate transgene expression in a copy number-dependent way.

Rice NAC proteins act as homodimers and heterodimers

Members of the NAM-ATAF-CUC (NAC) protein family are plant-specific transcription factors that contain a highly conserved N-terminal NAC-domain and diverse C-terminal regions. They have been implicated in plant development and abiotic stress responses. To identify interacters of rice NAC-domain proteins (OsNACs), we performed yeast two-hybrid screening of rice cDNA library using OsNAC5 as a bait, and the results showed that OsNAC5 interacts with other OsNACs including itself. To delineate an interacting domain, a series of deletion constructs of four OsNACs were made and transformed into yeast in various combinations. The results revealed that the conserved NAC domain of OsNACs plays a primary role in homodimer and heterodimer formation, and a part of C-terminal sequence is also necessary for the interaction. In vitro pull-down assays using recombinant OsNAC proteins verified the dimer formations, together suggesting that OsNACs may act by forming homodimers and/or heterodimers in plants.

Characterization of the stress-inducible OsNCED3 promoter in different transgenic rice organs and over three homozygous generations

To be effective in crop biotechnology applications, gene promoters need to be stably active over sequential generations in a population of single-copy transgenic lines. Most of the stress-inducible promoters characterized in plants thus far have been analyzed at early (T0, T1 or T2) generations and/or by testing only a small number of transgenic lines. In our current study, we report our analysis of OsNCED3, a stress-inducible rice promoter involved in ABA biosynthesis, in various organs and tissues of transgenic rice plants over the T2–4 homozygous generations. The transgene copy numbers in the lines harboring the OsNCED3:gfp construct were determined and six single- and two double-copy transgenic lines were analyzed for promoter activity in comparison with the Wsi18, a stress-inducible promoter previously characterized. The exogenous promoter activities were found to be significantly enhanced in the roots and leaves, whereas zero or low levels of activity were evident in grains and flowers, under drought and high-salinity conditions. The highest induction levels of gfp transcripts in the OsNCED3:gfp plants upon drought treatments were 161- and 93-fold in leaves and roots, respectively, and these levels were comparable with those of gfp transcripts in the Wsi18:gfp plants. A comparison of the promoter activities between the T2–T4 plants revealed that comparable activity levels were maintained over these three homozygous generations with no evidence of silencing. Thus, our results provide the OsNCED3 promoter that is stress-inducible in a whole rice plant except for in the aleurones and endosperm and stably active over three generations.

Analysis of the APX, PGD1 and R1G1B constitutive gene promoters in various organs over three homozygous generations of transgenic rice plants

We have previously characterized the constitutively active promoters of the APX, PGD1 and R1G1B genes in rice (Park et al. 2010 in J Exp Bot 61:2459–2467). To have potential crop biotechnology applications, gene promoters must be stably active over many generations. In our current study, we report our further detailed analysis of the APX, PGD1 and R1G1B gene promoters in various organs and tissues of transgenic rice plants for three (T3–5) homozygous generations. The copy numbers in 37 transgenic lines that harbor promoter:gfp constructs were determined and promoter activities were measured by real-time qPCR. Analysis of the 37 lines revealed that 15 contained a single copy of one of the three promoter:gfp chimeric constructs. The promoter activity levels were generally higher in multi-copy lines, whereas variations in these levels over the T3–5 generations studied were observed to be smaller in single-copy than in multi-copy lines. The three promoters were further found to be highly active in the whole plant body at both the vegetative and reproductive stages of plant growth, with the exception of the APX in the ovary and R1G1B in the pistil and filaments where zero or very low levels of activity were detected. Of note, the spatial activities of the PGD1 promoter were found to be strikingly similar to those of the ZmUbi1, a widely used constitutive promoter. Our comparison of promoter activities between T3, T4 and T5 plants revealed that the APX, PGD1 and R1G1B promoters maintained their activities at comparable levels in leaves and roots over three homozygous generations and are therefore potentially viable alternative promoters for crop biotechnology applications.

Transgenic overexpression of UIP1, an interactor of the 3′ untranslated region of the Rubisco small subunit mRNA, increases rice tolerance to drought

Gene regulation at the post-transcriptional level is a well-organized process to adjust plants in response to environmental changes. Here, we identified a novel RNA-binding protein (RBP) possessing a CBS (cystathionine-β-synthase) domain through yeast three-hybrid screening. This RBP, 3′-UTR-interacting protein 1 (UIP1), interacts with 3′ untranslated region of the Rubisco small subunit mRNA (3′ RbcS)—the major mRNA element that mediates the stress-induced mRNA decay (SMD) under drought and salt stress conditions. Six deletion constructs were made to delineate the binding domain of the UIP1 protein. Co-transformation of yeast with these constructs together with three different hybrid RNAs in various combinations showed that deletion of 51 N-terminal amino acids resulted in a loss of sequence-specific binding affinity. Further deletion at the region between 52 and 212 amino acids revealed that the CBS domain of UIP1 is necessary for binding to 3′ RbcS. Transgenic overexpression of UIP1 in rice resulted in an increase in tolerance to drought stress at the vegetative stage of growth. Under drought, high salt and low temperature conditions, the maximum photochemical efficiency of photosystem II (Fv/Fm) of UIP1 plants was higher than those of the nontransgenic plants. Interestingly, the effect of UIP1 overexpression on tolerance to stress was much more pronounced under drought than under high salt and low temperature conditions. Taken together, our results demonstrate that UIP1 interacts with 3′ untranslated region of RbcS1 mRNA and increases tolerance of transgenic overexpressors to drought stress.

Erratum to: Characterization of the stress-inducible OsNCED3

To be effective in crop biotechnology applications, gene promoters need to be stably active over sequential generations in a population of single-copy transgenic lines. Most of the stress-inducible promoters characterized in plants thus far have been analyzed at early (T0, T1 or T2) generations and/or by testing only a small number of transgenic lines. In our current study, we report our analysis of OsNCED3, a stress-inducible rice promoter involved in ABA biosynthesis, in various organs and tissues of transgenic rice plants over the T2–4 homozygous generations. The transgene copy numbers in the lines harboring the OsNCED3:gfp construct were determined and six singleand two double-copy transgenic lines were analyzed for promoter activity in comparison with the Wsi18, a stress-inducible promoter previously characterized. The exogenous promoter activities were found to be significantly enhanced in the roots and leaves, whereas zero or low levels of activity were evident in grains and flowers, under drought and high-salinity conditions. The highest induction levels of gfp transcripts in the OsNCED3:gfp plants upon drought treatments were 161and 93-fold in leaves and roots, respectively, and these levels were comparable with those of gfp transcripts in the Wsi18:gfp plants. A comparison of the promoter activities between the T2–T4 plants revealed that comparable activity levels were maintained over these three homozygous generations with no evidence of silencing. Thus, our results provide the OsNCED3 promoter that is stress-inducible in a whole rice plant except for in the aleurones and endosperm and stably active over three generations.