In Planta Assessment of the Role of Thioredoxin h Proteins in the Regulation of S-Locus Receptor Kinase Signaling in Transgenic Arabidopsis

The self-incompatibility (SI) response of the Brassicaceae is mediated by allele-specific interaction between the stigma-localized S-locus receptor kinase (SRK) and its ligand, the pollen coat-localized S-locus cysteine-rich protein (SCR). Based on work in Brassica spp., the thioredoxin h-like proteins THL1 and THL2, which interact with SRK, have been proposed to function as oxidoreductases that negatively regulate SRK catalytic activity. By preventing the spontaneous activation of SRK in the absence of SCR ligand, these thioredoxins are thought to be essential for the success of cross pollinations in self-incompatible plants. However, the in planta role of thioredoxins in the regulation of SI signaling has not been conclusively demonstrated. Here, we addressed this issue using Arabidopsis thaliana plants transformed with the SRKb-SCRb gene pair isolated from self-incompatible Arabidopsis lyrata. These plants express an intense SI response, allowing us to exploit the extensive tools and resources available in A. thaliana for analysis of SI signaling. To test the hypothesis that SRK is redox regulated by thioredoxin h, we expressed a mutant form of SRKb lacking a transmembrane-localized cysteine residue thought to be essential for the SRK-thioredoxin h interaction. We also analyzed transfer DNA insertion mutants in the A. thaliana orthologs of THL1 and THL2. In neither case did we observe an effect on the pollination responses of SRKb-expressing stigmas toward incompatible or compatible pollen. Our results are consistent with the conclusion that, contrary to their proposed role, thioredoxin h proteins are not required to prevent the spontaneous activation of SRK in the A. thaliana stigma.Many flowering plants possess self-incompatibility (SI), a genetic system that promotes outcrossing by preventing self-fertilization. In the Brassicaceae family, the SI response is controlled by haplotypes of the S locus, each of which contains two genes that encode highly polymorphic proteins, the S-locus receptor kinase (SRK), which is a plasma membrane resident single-pass transmembrane Ser/Thr receptor kinase displayed at the surface of stigma epidermal cells (Stein et al., 1991; Takasaki et al., 2000), and the S-locus Cys-rich protein (SCR), which is the pollen coat-localized ligand for SRK (Schopfer et al., 1999; Kachroo et al., 2001; Takayama et al., 2001). SRK and SCR exhibit allele-specific interactions, whereby only SRK and SCR encoded by the same S-locus haplotype interact. In a self-pollination, the binding of this “self” pollen-borne SCR to the extracellular domain of SRK activates the SRK kinase, thereby triggering a cellular response in stigma epidermal cells that causes inhibition of pollen germination and tube penetration into the stigma epidermal cell wall (for review, see Tantikanjana et al., 2010).Tight regulation of SRK kinase activity and its signaling cascade is critical for productive pollen-stigma interactions because constitutive (i.e. SCR-independent) activity of the receptor is expected to result in sterile stigmas that reject both compatible and incompatible pollen. In the classical view of ligand-activated receptor kinases, the receptor occurs as catalytically inactive monomers in the absence of ligand and only becomes activated upon ligand-induced dimerization (for review, see Lemmon and Schlessinger, 2010). However, some receptor kinases in both animals (Chan et al., 2000; Ehrlich et al., 2011) and plants (Giranton et al., 2000; Wang et al., 2005, 2008; Shimizu et al., 2010; Bücherl et al., 2013) form catalytically inactive dimers or oligomers in the absence of ligand, with receptor activation presumably resulting from ligand-induced higher order oligomerization or conformational changes (Lemmon and Schlessinger, 2010). Similar to the latter receptors, SRK forms oligomers in unpollinated stigmas, i.e. in the absence of SCR (Giranton et al., 2000), at least partly via ligand-independent dimerization domains located within the SRK extracellular domain (Naithani et al., 2007). It has been proposed that these ligand-independent SRK oligomers are maintained in an inactive state by thioredoxins, the ubiquitous small oxidoreductases that reduce disulfide bridges in proteins (Buchanan and Balmer, 2005). This hypothesis is supported by the following observations: (1) two Brassica napus thioredoxins, the Thioredoxin H-Like proteins THL1 and THL2, were identified as SRK interactors in a yeast (Saccharomyces cerevisiae) two-hybrid screen that used the B. napus SRK₉₁₀ kinase domain as bait (Bower et al., 1996); (2) when purified from pistils or insect cells, the Brassica oleracea SRK₃ variant was found to exhibit constitutive autophosphorylation activity in vitro, and this activity was inhibited by Escherichia coli-expressed THL1 proteins and was restored by addition of pollen coat proteins containing self but not of pollen coat proteins containing nonself SCR (Cabrillac et al., 2001); (3) the catalytic activity of THL1 was required for its inhibition of SRK₃ autophosphorylation activity in vitro (Cabrillac et al., 2001); and (4) antisense suppression of THL1/THL2 gene expression in the stigmas of a self-compatible B. napus strain reportedly produced a low-level constitutive incompatibility (Haffani et al., 2004), as might be expected if the THL1/THL2 proteins prevent the spontaneous activation of SRK-mediated signaling in stigmas.These observations notwithstanding, the in planta role of thioredoxin h proteins as negative regulators of SRK activity has not been conclusively demonstrated. To date, this proposed function has only been evaluated in a self-compatible strain of B. napus (Haffani et al., 2004). Consequently, it is not known if the proposed inhibitory effect of these thioredoxins on SRK catalytic activity is manifested in self-incompatible stigmas and if it applies to all SRK variants, be they derived from Brassica spp. or other self-incompatible species of the Brassicaceae such as Arabidopsis lyrata.In this study, we tested the in planta role of thioredoxin h proteins in the regulation of SI signaling using a transgenic self-incompatible Arabidopsis thaliana model that we generated by transforming A. thaliana with the SRKb-SCRb gene pair isolated from the Sb haplotype of self-incompatible A. lyrata (Kusaba et al., 2001; Nasrallah et al., 2002, 2004). We had previously shown that the stigmas of A. thaliana SRKb-SCRb transformants can exhibit an SI response that is as robust as the SI response observed in naturally self-incompatible A. lyrata, demonstrating that A. thaliana, which harbors nonfunctional S-locus haplotypes (Kusaba et al., 2001; Sherman-Broyles et al., 2007; Shimizu et al., 2008; Boggs et al., 2009c), has nevertheless retained all other factors required for SI. In view of the availability in A. thaliana of a highly efficient transformation method and numerous genetic resources, the A. thaliana SRK-SCR transgenic model has enabled the use of experimental approaches that are difficult or impossible to implement in Brassica species and has thus proven to be an invaluable platform for in planta analysis of SRK and SI signaling (Liu et al., 2007; Boggs et al., 2009a, 2009b; Tantikanjana et al., 2009; Tantikanjana and Nasrallah, 2012).We therefore used this transgenic A. thaliana self-incompatible model to determine if abolishing the proposed SRK-thioredoxin h interaction or eliminating expression of the major thioredoxin h proteins expressed in stigmas would affect the outcome of self- or cross pollination. To this end, we expressed a mutant form of SRKb that lacked the Cys residue previously shown to be required for the interaction of SRK with THLs (Mazzurco et al., 2001), and we analyzed plants carrying knockout insertional mutations in thioredoxin h genes. Our results are inconsistent with the proposed role of thioredoxin h proteins as negative regulators of SRK catalytic activity and SI signaling.     

Methylation of histone H3 at lysine 4 and expression of the maltase-glucoamylase gene are reduced by dietary resistant starch

Methylated histone H3 at lysine 4 (K4) is associated with euchromatin and is involved in the transactivation of genes. However, it is unknown whether histone methylation is involved with changes in gene expression induced by nutrients. In this study, we examined whether methylations of histone H3 at K4 on maltase-glucoamylase (Mgam), which is responsible for the digestion of starch in the small intestine, as well as Mgam expression were altered by feeding rats an indigestible starch (resistant starch, RS). The mRNA and protein levels and the activities of MGAM were reduced in rats fed an RS diet compared with those fed a regular starch diet. Furthermore, we found that decreases in di- and tri-methylation of histone H3 at K4, as well as reduced acetylation of histones H3 and H4 on the Mgam gene were associated with a reduction of Mgam gene expression. These results suggest that the reductions of jejunal MGAM levels and activities caused by the RS diet are regulated at the mRNA level through a decrease in methylation of histone H3 at K4 and reduced acetylation of histones H3 and H4 on the Mgam gene.     

Why do ants shift their foraging from extrafloral nectar to aphid honeydew?

When aphids parasitize plants with extrafloral nectaries (EFNs) and aphid colony size is small, ants frequently use EFNs but hardly tend aphids. However, as the aphid colony size increases, ants stop using EFNs and strengthen their associations with aphids. Although the shift in ant behavior is important for determining the dynamics of the ant–plant–aphid interaction, it is not known why this shift occurs. Here, we test two hypotheses to explain the mechanism responsible for this behavioral shift: (1) Extrafloral nectar secretion changes in response to aphid herbivory, or (2) plants do not change extrafloral nectar secretion, but the total reward to ants from aphids will exceed that from EFNs above a certain aphid colony size. To judge which mechanism is plausible, we investigated secretion patterns of extrafloral nectar produced by plants with and without aphids, compared the amount of sugar supplied by EFNs and aphids, and examined whether extrafloral nectar or honeydew was more attractive to ants. Our results show that there was no inducible extrafloral secretion in response to aphid herbivory, but the sugar concentration in extrafloral nectar was higher than in honeydew, and more ant workers were attracted to an artificial extrafloral nectar solution than to an artificial aphid honeydew solution. These results indicate that extrafloral nectar is a more attractive reward than aphid honeydew per unit volume. However, even an aphid colony containing only two individuals can supply a greater reward to ants than EFNs. This suggests that the ant behavioral shift may be explained by the second hypothesis.     

Cholesterol-lowering effect of rice bran protein containing bile acid-binding proteins

Dietary plant protein is well known to reduce serum cholesterol levels. Rice bran is a by-product of rice milling and is a good source of protein. The present study examined whether feeding rats a high-cholesterol diet containing 10% rice bran protein (RBP) for 10 d affected cholesterol metabolism. Rats fed dietary RBP had lower serum total cholesterol levels and increased excretion of fecal steroids, such as cholesterol and bile acids, than those fed dietary casein. In vitro assays showed that RBP strongly bound to taurocholate, and inhibited the micellar solubility of cholesterol, compared with casein. Moreover, the bile acid-binding proteins of the RBP were eluted by a chromatographic column conjugated with cholic acid, and one of them was identified as hypothetical protein OsJ_13801 (NCBI accession No. EAZ29742) using MALDI-TOF mass spectrometry analysis. These results suggest that the hypocholesterolemic action of the RBP may be caused by the bile acid-binding proteins.     

A high-density linkage map with 2560 markers and its application for the localization of the male-sterile genes <Emphasis Type="Italic">ms3</Emphasis> and <Emphasis Type="Italic">ms4</Emphasis> in <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> D. Don

Cryptomeria japonica pollinosis is one of the most serious allergic diseases in Japan; this is a social problem because C. japonica is the most important Japanese forestry species. In order to reduce the amount of pollen dispersed, breeding programs using trees with male-sterile genes have been implemented. High-density linkage maps with stable ordering of markers facilitate the localization of male-sterile genes and the construction of partial linkage maps around them in order to develop markers for use in marker-assisted selection. In this study, a high-density linkage map for C. japonica with 2560 markers was constructed. The observed map length was 1266.2 cM and the mean distance between adjacent markers was 0.49 cM. Using information from this high-density map, we newly located two male-sterile genes (ms3 and ms4) on the first and fourth linkage groups, respectively, and constructed partial linkage maps around these loci. We also constructed new partial linkage maps around the ms1 and ms2 loci using additional SNP markers. The closest markers to the ms1, ms2, ms3, and ms4 male-sterile loci were estSNP04188 (1.8 cM), estSNP00695 (7.0 cM), gSNP05415 (3.1 cM), and estSNP01408 (7.0 cM) respectively. These results allowed us to develop SNP markers tightly linked to the male sterile genes for use in MAS; this will accelerate the future isolation of these genes by map-based cloning approaches.     

Normal Lung Quantification in Usual Interstitial Pneumonia Pattern: The Impact of Threshold-based Volumetric CT Analysis for the Staging of Idiopathic Pulmonary Fibrosis

BackgroundAlthough several computer-aided computed tomography (CT) analysis methods have been reported to objectively assess the disease severity and progression of idiopathic pulmonary fibrosis (IPF), it is unclear which method is most practical. A universal severity classification system has not yet been adopted for IPF.ObjectiveThe purpose of this study was to test the correlation between quantitative-CT indices and lung physiology variables and to determine the ability of such indices to predict disease severity in IPF.MethodsA total of 27 IPF patients showing radiological UIP pattern on high-resolution (HR) CT were retrospectively enrolled. Staging of IPF was performed according to two classification systems: the Japanese and GAP (gender, age, and physiology) staging systems. CT images were assessed using a commercially available CT imaging analysis workstation, and the whole-lung mean CT value (MCT), the normally attenuated lung volume as defined from −950 HU to −701 Hounsfield unit (NL), the volume of the whole lung (WL), and the percentage of NL to WL (NL%), were calculated.ResultsCT indices (MCT, WL, and NL) closely correlated with lung physiology variables. Among them, NL strongly correlated with forced vital capacity (FVC) (r = 0.92, P <0.0001). NL% showed a large area under the receiver operating characteristic curve for detecting patients in the moderate or advanced stages of IPF. Multivariable logistic regression analyses showed that NL% is significantly more useful than the percentages of predicted FVC and predicted diffusing capacity of the lungs for carbon monoxide (Japanese stage II/III/IV [odds ratio, 0.73; 95% confidence intervals (CI), 0.48 to 0.92; P < 0.01]; III/IV [odds ratio. 0.80; 95% CI 0.59 to 0.96; P < 0.01]; GAP stage II/III [odds ratio, 0.79; 95% CI, 0.56 to 0.97; P < 0.05]).ConclusionThe measurement of NL% by threshold-based volumetric CT analysis may help improve IPF staging.