New Plant Design Suite 2010 Activation

New Plant Design Suite 2010 Activation

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Encouraged by the above results, we assayed the MPK6 kinase activity induced by H2O2 by measuring the levels of phosphorylation in wild-type and mpk6-3 mutant plants that were exposed to different concentrations of exogenous H2O2. Exogenous application of H2O2 promoted the kinase activity of MPK6 in a dose-dependent manner (see Supplemental Figure 1B online). The obvious promotion of MPK kinase activity was observed at 30 min after treatment with 10 μM H2O2 (see Supplemental Figure 1B online). Under the same conditions, the NO production was significantly increased (Figure 1C). Application of 2 mM H2O2 resulted in the strongest activation of MPK6 (Figure 1E; see Supplemental Figure 1B online). Consistent with the above results, application of H2O2 to wild-type plants stimulated MPK6 activity significantly in a time-dependent manner (Figure 1E). The activation of MPK6 by H2O2 was rapid, with the maximum level of phosphorylation occurring 30 min after the addition of H2O2 (Figure 1E). Activation of MPK6 was still detectable at 60 min. In contrast with MPK6 activity, MPK3 activity was weak and relatively unstimulated by H2O2 under the same experimental conditions (Figure 1E).

(F) MPK6 is required for full activation of NR and NO generation. The NR activity (top panel) and NO production (middle panel) in 35S-NIA2 and mpk6-3/35S-NIA2 transgenic plants were measured as described in Figure 2. Each value represents the mean (±sd) of three independent experiments. The NR activity and NO production in the wild type were set as 100%. Protein extract (10 μg) from 35S-NIA2 and mpk6-3/35S-NIA2 transgenic plants was separated by electrophoresis, and immunoblotting with anti-Flag antibody was performed to determine the amount of Flag-tagged NIA2 proteins (bottom panel).

NO has long been defined as a positive regulator in lateral root development when applied exogenously (Pagnussat et al., 2002; Correa-Aragunde et al., 2004). Surprisingly, unlike the NO promotion effects seen in wild-type plants, NIA2D transgenic plants showed the growth retardation phenotypes of lateral root in response to exogenously applied NO (Figures 7F and 7G). One possibility is that there is a bell-shaped curve of dose response for NO, similar to that for cytokinin in root growth (Werner et al., 2001). Indeed, bell-shaped NO response curve for lateral root growth in mpk6 mutants was observed (Figure 6B). Because the level of endogenous NO might be nearly optimal for growth in NIA2 overexpression lines, supplying the NIA2D plants with exogenous NO (15 μM SNP) may cause overly high concentrations of NO that may be inhibitory. Similarly, without SNP treatment, lateral root growth in all overexpression lines of NIA2 (NIA2A, NIA2WT, and NIA2D) was strongly stimulated compared with the wild type (Figures 7F and 7G). These data are consistent with the previous observations (Pagnussat et al., 2002; Correa-Aragunde et al., 2004). By contrast, we did not find the differences in lateral root growth between wild-type and transgenic plants after application of H2O2 (see Supplemental Figure 7 online), probably due to the consistent activation of NR in the overexpression lines. Together, these data strongly support this notion that lateral root growth may require a minimum concentration of NO to promote growth but that root growth is inhibited in NIA2D by SNP concentrations that promote elongation in the wild type.

Anthocyanins are a group of flavonoid compounds that fulfill important biological functions in protecting plants against various biotic and abiotic stresses. All of the anthocyanin biosynthetic pathway genes and numerous regulatory factors have been identified from studies of Arabidopsis (Arabidopsis thaliana), maize (Zea mays), petunia (Petunia hybrida), snapdragon (Antirrhinum majus), and other plant species (Broun, 2005; Dixon et al., 2005; Koes et al., 2005; Grotewold, 2006). Transcriptional regulation of structural genes appears to be a major mechanism by which anthocyanin biosynthesis is regulated in plants. R2R3 MYB and basic helix-loop-helix (bHLH) transcription factors as well as WD40 proteins represent the three major families of anthocyanin regulatory proteins (Paz-Ares et al., 1987; Chandler et al., 1989; Ludwig and Wessler 1990; de Vetten et al., 1997; Quattrocchio et al., 1999). They form regulatory complexes to activate expression of anthocyanin structural genes (Goff et al., 1992; Grotewold et al., 2000). In Arabidopsis, several MYB proteins, including PAP1, PAP2, MYB113, MYB114, and MYBL2 (Borevitz et al., 2000; Dubos et al., 2008; Gonzalez et al., 2008; Matsui et al., 2008), three bHLH proteins of TT8, GL3, and EGL3 (Nesi et al., 2000; Payne et al., 2000; Zhang et al., 2003), and a WD40 repeat protein of TTG1 (Walker et al., 1999) are involved in anthocyanin biosynthesis. While the R2R3 MYB proteins of PAP1, PAP2, MYB113, and MYB114 cause tissue-specific anthocyanin accumulation in Arabidopsis (Borevitz et al., 2000; Gonzalez et al., 2008), the R3-MYB protein, MYBL2, acts as an inhibitor of anthocyanin biosynthesis (Dubos et al., 2008; Matsui et al., 2008). TT8 is required for the full transcriptional activation of late anthocyanin pathway genes (Nesi et al., 2000), and is partially functionally redundant with its closest homologs, GL3 and EGL3 (Zhang et al., 2003). The WD40 protein, TTG1, is known to physically interact with the MYB and bHLH transcription factors in controlling anthocyanin biosynthesis (Zhang et al., 2003).

Many regulatory genes that control anthocyanin biosynthesis have been isolated from plant species (Paz-Ares et al., 1987; Chandler et al., 1989; Ludwig and Wessler 1990; de Vetten et al., 1997; Quattrocchio et al., 1999). Since cauliflower genes typically share high coding sequence identity with Arabidopsis genes, the available sequences of anthocyanin regulatory genes from Arabidopsis and the significant amounts of Brassica sequence information in the public domains provide direct resources for designing the gene-specific primers for some cauliflower homologs. In Arabidopsis, the R2R3 MYB family proteins of PAP1, PAP2, MYB113, and MYB114, bHLH proteins of TT8 and EGL3, and WD40 protein of TTG1 are known to regulate anthocyanin biosynthesis. To investigate whether the Pr gene represented a mutation of one of the known regulatory genes, transcript levels of these homologous genes in wild type and the Pr-D mutant were examined by quantitative reverse transcription (qRT)-PCR using gene-specific primers (Yuan et al., 2009; Supplemental Table S1). As shown in Figure 3B, PAP-like MYB family genes, i.e. BoMYB2 and BoMYB4, as well as BobHLH1, a homologous gene of Arabidopsis TT8 (Nesi et al., 2000) exhibited differential expression in both leaves and curds between wild type and the Pr-D mutant.

The activity of MYB-like genes has been suggested to be the primary cause of natural variation in anthocyanin pigmentation in plants (Quattrocchio et al., 1999; Schwinn et al., 2006). Increased expression of R2R3 MYB transcription factors was found to be responsible for anthocyanin production in a number of anthocyanin-accumulating mutants. For example, the constitutive up-regulation of PAP1, ANT1, and MdMYB10 causes anthocyanin accumulation throughout the plant in pap1-D Arabidopsis (Borevitz et al., 2000), antl tomato (Solanum lycopersicum; Mathews et al., 2003), and red-fleshed apple (Espley et al., 2007). While overexpression of PAP1 and ANT1 MYB transcription factors is due to activation-tagged insertions in their promoter sequences (Borevitz et al., 2000; Mathews et al., 2003), the high transcript level of MdMYB10 was recently found to be due to the formation of a minisatellite-like structure comprising multiple repeats of a promoter segment that generates a novel autoregulatory motif (Espley et al., 2009). Unlike these reported anthocyanin-accumulating mutants, the Pr-D mutant appears to display a different mechanism in activating Pr expression.

Moreover, the mechanism underlying the reported Th1 shift after plant sterol consumption remains unknown. One possible pathway could be that plant sterols activate APCs, which results in a strong Th1 cell activation via elevated IL-12 or IL-18 production (1, 21). A second possibility could be (as also suggested by Calpe-Berdiel et al. (16, 22)) activation of transcription factor LXR, which may play a role in innate immunity (23). Indeed, we among others have shown that plant sterols can activate LXR (24, 25). By using a cell-free ligand sensing assay (LiSA), we have earlier shown that 4-desmethylsterols (e.g. sitosterol, sitostanol) but not 4,4-dimethylsterols (e.g. α-amyrin), which have different effects on cholesterol metabolism (26), activate transcription factor LXR (24). Therefore, these two plant sterol families can be used to examine whether LXR is involved in the plant sterol-induced Th1 shift. A third explanation might relate to activation of pattern recognition receptors such as TLR2 and -4. In this respect, it is known that TLR2 activation increases T-cell activity and inhibits suppressor activity by regulatory T-cells (27). Moreover, inhibition of TLR2 resulted in lower activity of Th1 cells in mice (28). The current perception is that TLR2 responds to lipid-based conserved patterns like peptidoglycan, lipoproteins, and lipopeptides of Gram-positive bacteria (29). Because plant sterols and stanols are also fatty compounds, a novel explanation for the observed Th1 shift could relate to activation of specific TLRs.

Adding sitosterol or sitostanol to human PBMCs ex vivo increased concentrations of the Th1 cytokines IFNγ and IL-2 when compared with the same concentration of cholesterol. In fact, cholesterol did not show any effects at all. This latter finding indicates that the effect of sitosterol or sitostanol is a plant sterol-specific effect and not a sterol effect in general. On the Th2 side, no differences were seen in IL-4 and IL-10 production in the sitosterol and sitostanol conditions compared with control conditions. Breytenbach et al. (14) showed that a plant sterol/sterolin mixture induced Th1 activity in HIV patients, whereas others reported similar effects for plant sterols in mice (16, 32, 33). Calpe-Berdiel et al. (16) have shown that dietary plant sterol supplementation increased the production of Th1 cytokines IL-2 and IFNγ during acute a-septic inflammation in mice, whereas no changes in the production of IL-4 and IL-10 were observed. They suggested that activation of transcription factor LXR might be the mechanism underlying the plant sterol induced Th1 shift (16, 22). We show here, that both sitosterol and α-amyrin, which belong to respectively the 4-desmethylsterol and 4.4-dimethylsterol family, induce a similar Th1 response. We have earlier shown that only the 4-desmethylsterols like sitosterol and sitostanol can activate LXR but not 4,4-dimethylsterols (24). Therefore, it is not likely that LXR activation is involved. 2b1af7f3a8