Gibberellins (GAs) are phytohormones that regulate growth and development throughout the life cycle of plants. RSG (REPRESSION OF SHOOT GROWTH) is a tobacco (Nicotiana tabacum) transcriptional ...activator with a basic leucine zipper domain that regulates the endogenous amount of GAs by control of GA biosynthetic enzymes. Negative feedback contributes to homeostasis of the GA levels. Previous studies suggested that RSG is directly or indirectly involved in the GA negative feedback of NtGA20ox1 encoding GA 20-oxidase. Using transgenic tobacco plants, we have identified a cis-acting region that is responsible for the feedback regulation of NtGA20ox1. This region contains an RSG-binding sequence. A mutation in the RSG-binding sequence abolished negative feedback of NtGA20ox1 in transgenic plants. Chromatin immunoprecipitation (ChIP) assays showed that RSG binds to the NtGA20ox1 promoter in vivo in response to a decrease in GA levels, and that this binding is abolished within 3 h after GA treatment. Furthermore, decreases in GA levels promote modifications of active histone marks in the promoter of NtGA20ox1. Our results suggest that RSG plays a role in the homeostasis of GAs through direct binding to the NtGA20ox1 promoter.
Breeding barley with a high content of dietary fiber, namely β-glucan, is required because β-glucan is attracting attention for its health functionalities such as suppression of postprandial blood ...glucose elevation. We analyzed the relationship between the β-glucan content and the starch synthase IIIa (ssIIIa) marker type, which is located near the region affecting the high amylose content amo1 mutation. The ssIIIa marker types were detected, and the β-glucan content, SKCS grain hardness, steely grain rate, thousand-grain weight, and hollow grain rate were measured for each of the 94 waxy lines (F5) derived from a cross between ‘Hokuriku 54’ (later ‘Yukihanarokujo’) and the amo1 waxy variety ‘YonRkei3755’, and for each of the 165 waxy lines (F5) derived from a cross between ‘YonRkei3755’ and the waxy variety ‘Haneumamochi’. As a result, there were significant differences in the β-glucan content, SKCS grain hardness, and steely grain rate depending on the ssIIIa marker type in each post-interbreeding line. The β-glucan content was about 1.6 times higher, the SKCS grain hardness was 23 points higher, and the steely grain rate was 7% higher in the ‘YonRkei3755’ ssIIIa type than that of the wild type. The β-glucan content was highly correlated with the SKCS grain hardness of r = 0.75 to 0.84 in the breeding line derived from both crosses. These results suggest that selection based on the ssIIIa marker type and/or the SKCS grain hardness are effective in breeding amo1 varieties with a high content of β-glucan.
Wintering ability is an important trait in barley, particularly in northern Japan. Although fructan is known to be related to wintering ability in wheat and temperate forage grasses, its contribution ...to wintering ability in barley remains unclear. To clarify the role of fructan with respect to wintering ability in barley, we analyzed the relationships among wintering ability, shoot fructan content, and the allelic variation of five fructan metabolic enzyme genes, 1-FEH, 6-FEH, 1-FFT, 1-SST, and 6-SFT, in 105 Japanese barley cultivars and breeding lines. The cultivars and breeding lines developed in the colder northern region of Japan exhibited a superior wintering ability and greater fructan content accumulated prior to snow cover than those bred in the warmer southern region. In our sequence analyses of the five fructan metabolic enzyme genes, 6 to 10 genotypes were found in each of the genes in the tested barley cultivars and lines. Cultivars and lines with the same combination of fructan metabolic enzyme gene genotypes were grouped, and the average wintering ability and fructan content values of the different groups were compared. A significant correlation was detected between fructan content and wintering ability, specifically, groups with a higher fructan content prior to snow cover, especially in stem tissue, exhibited a superior wintering ability. The group with the highest wintering ability possessed a unique 6-SFT genotype. The cultivars and breeding lines were classified into three groups with a high, medium, and low wintering ability based on the 1-FEH genotypes. We developed derived cleaved amplified polymorphic sequence (dCAPS) markers that identify the 6-SFT and 1-FEH genotypes observed in the group with the highest wintering ability and in the groups with a high, medium, and low wintering ability, respectively.
A Ca
2+
-dependent protein kinase not only phosphorylates a substrate but also acts as a scaffold that promotes the interaction between a phosphorylation product and its binding protein.
A molecular ...mechanism to ensure signaling specificity is a scaffold. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (
Nicotiana tabacum
) transcription factor that is involved in gibberellin feedback regulation. The 14-3-3 proteins negatively regulate RSG by sequestering it in the cytoplasm in response to gibberellins. The
N. tabacum
Ca
2+
-dependent protein kinase NtCDPK1 was identified as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of RSG. CDPKs are unique sensor responders of Ca
2+
that are only found in plants and some protozoans. Here, we report a scaffolding function of CDPK. 14-3-3 proteins bound to NtCDPK1 by a new mode. Autophosphorylation of NtCDPK1 was necessary for the formation of the binding between NtCDPK1 and 14-3-3 but not for its maintenance. NtCDPK1 formed a heterotrimer with RSG and 14-3-3. Furthermore, we found that NtCDPK1 transfers 14-3-3 to RSG after phosphorylation of RSG and that RSG dissociates from NtCDPK1 as a complex with 14-3-3. These results suggest that NtCDPK1 is an interesting scaffolding kinase that increases the specificity and efficiency of signaling by coupling catalysis with scaffolding on the same protein.
Summary
Physicochemical properties of storage starch largely determine rice grain quality and food characteristics. Therefore, modification of starch property is effective to fine‐tune cooked rice ...textures. To obtain new resources with modified starch property as breeding materials, we screened a mutant population of a
japonica
cultivar Nipponbare and found two independent mutant lines,
altered gelatinization
(
age
)
1
and
age2
, with moderate changes in starch gelatinization property. A combination of conventional genetic analyses and the latest mapping method, MutMapPlus, revealed that both of these lines harbour novel independent mutant alleles of
starch branching enzyme
II
b
(
BEII
b
) gene. In
age1
, amino acid substitution of Met‐723 to Lys completely abolished
BEII
b enzyme activity without significant reduction in its protein level. A transposon insertion in an intron of
BEII
b
gene reduced
BEII
b protein level and activity in
age2
. Production of a series of the mutant lines by combining
age
alleles and
indica
‐type
starch synthase
II
a
allele established stepwise alteration of the physicochemical properties of starch including apparent amylose content, thermal property, digestibility by α‐amylase and branched structures of amylopectin. Consistent with the alteration of starch properties, the results of a sensory evaluation test demonstrated that warm cooked rice of the mutants showed a variety of textures without marked reduction in overall palatability. These results suggest that a series of the mutant lines are capable of manipulation of cooked rice textures.
14-3-3 pproteins play essential roles in diverse cellular processes through the direct binding to target proteins. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription ...factor that is involved in gibberellin (GA) feedback regulation. The 14-3-3 proteins bind to RSG depending on the RSG phosphorylation of Ser-114 and negatively regulate RSG by sequestering it in the cytoplasm in response to GAs. The Ca
2+
-dependent protein kinase NtCDPK1 was identified as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of RSG. 14-3-3 weakly binds to NtCDPK1 by a new mode. The autophosphorylation of NtCDPK1 was necessary for the formation of the binding between NtCDPK1 and 14-3-3 but not for its maintenance. In this study, we showed that 14-3-3 binding to NtCDPK1 does not require the autophosphorylation when RSG was bound to NtCDPK1. These data suggest that 14-3-3 binds to an unphosphorylated motif in NtCDPK1 exposed by a conformational change in NtCDPK1 but not to a phosphate group generated by autophosphorylation of NtCDPK1.
Allelic variations of partial sequence of fructan: fructan 1-fructosyltransferase (1-FFT) gene among 43 Japanese barley cultivars and 5 foreign barley cultivars were investigated. Alignment analysis ...of the 540 bp region of 1-FFT gene revealed that there were 15 single nucleotide polymorphisms (SNPs) including four SNPs with amino acid substitution. The barley cultivars were classified into five groups by phylogenetic analysis. Most of the cultivars with superior snow resistance fell into the same group.
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