Chikako Honda

Thioredoxin h is one of the major proteins in rice phloem sap

Sieve tubes play important roles in the transfer of nutrients as well as signals. Hundreds of proteins were found in pure phloem sap collected from rice (Oryza sativa L. cv. Kantou) plants through the cut ends of insect stylets. These proteins may be involved in nutrient transfer and signal transduction. To characterize the nature of these proteins, the partial amino-acid sequence of a 13kDa protein, named RPP13-1, that was abundant in the pure phloem sap was determined. A cDNA clone of 687 bp, containing an open reading frame of 122 amino acids, was isolated using corresponding oligonucleotides as a probe. The deduced amino-acid sequence was very similar to that of the ubiquitous thiol redox protein, thioredoxin. The consensus sequences of thioredoxins are highly conserved. No putative signal peptide was identified. Antiserum against wheat thioredoxin h cross-reacted with RPP13-1 in the phloem sap of rice plants. RPP131 produced in Escherichia coli was reactive to antiserum against wheat thioredoxin h. Both E. coli-produced RPP13-1 and the phloem sap proteins catalyzed the reduction of the disulfide bonds of insulin in the presence of dithiothreitol. These results indicate that an active thioredoxin is a major protein translocating in rice sieve tubes.

An apple B-box protein, MdCOL11, is involved in UV-B- and temperature-induced anthocyanin biosynthesis

AbstractMain conclusionOur studies showed that an apple B-box protein, MdCOL11, the homolog of AtBBX22, is involved in UV-B- and temperature-induced anthocyanin biosynthesis in apple peel. Anthocyanin is responsible for the red pigmentation in apple peel and a R2R3 MYB gene, MdMYBA/1/10, a homolog of MdMYBA, controls its accumulation. Arabidopsis PAP1 is under the control of a series of upstream factors involved in light signal transduction and photomorphogenesis, such as ELONGATED HYPOCOTYL 5 (HY5) and B-box family (BBX) proteins. In this study, we identified and characterized the homolog of Arabidopsis BBX22 in apple, designated as MdCOL11. Overexpression of MdCOL11 in Arabidopsis enhanced the accumulation of anthocyanin. In apples, MdCOL11 was differentially expressed in all tissues, with the highest expression in petals and the lowest expression in the xylem. Transcripts of MdCOL11 noticeably accumulated at the ripening stage, concomitant with increases in the expressions of anthocyanin biosynthesis-related genes. In an in vitro treatment experiment, MdCOL11 was upregulated in an ultra-violet (UV)-B- and temperature-dependent manner, together with the inductions of anthocyanin biosynthesis-related genes and anthocyanin accumulation in apple peel. Furthermore, a dual-luciferase assay indicated that (1) MdCOL11 regulated the expression of MdMYBA and (2) MdCOL11 was a target of MdHY5. Taken together, our results suggest that MdCOL11 is involved in MdHY5-mediated signal transduction and regulates anthocyanin accumulation in apple peel, which sheds new light on anthocyanin accumulation in apples.

UDP-sugar biosynthetic pathway: contribution to cyanidin 3-galactoside biosynthesis in apple skin

UDP-galactose:flavonoid 3-O-galactosyltransferase (UFGalT) is responsible for cyanidin 3-galactoside (cy3-gal) synthesis from cyanidin (cy) and UDP-galactose (UDP-gal) which are, respectively, catalyzed by anthocyanidin synthase (ANS) and UDP-glucose 4-epimerase (UGE). To clarify the contribution of UDP-galactose pathway to cy3-gal accumulation in apple skin, we analyzed the contents of UDP-gal and UDP-glucose (UDP-glu), cy, and, cy3-gal contents along with UGE activity. We confirmed that transcript levels for apple ANS and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) coincided with anthocyanin accumulation in three apple cultivars differing in their skin colors. During fruit development, changes in level of cy coincided with that of cy3-gal, whereas UDP-gal and UGE activity showed no similar trend with cy3-gal. Significant correlation was not observed between the changes in UGE activity and UDP-sugar contents. The effect of temperature and UV-B radiation (different environmental conditions) on the accumulation of UDP-sugars, cy and cy3-gal, and UGE activity were also investigated in a pale-red cultivar. High temperature tended to depress the accumulation of both UDP-sugars and cy concomitant with the decrease in cy3-gal content irrespective of UV-B radiation. Although there was no high inhibition of both cy and UDP-sugars at low-temperature without UV-B, cy3-gal accumulation was highly depressed. UGE activity was highest at low temperature with UV-B, but not much different under other conditions. Most of the parameters under different environmental conditions were significantly correlated with each other. Based on these results, contribution of UDP-sugar biosynthetic pathway to anthocyanin biosynthesis under different environmental conditions as well as during fruit development is discussed.

Epigenetic regulation of MdMYB1 is associated with paper bagging-induced red pigmentation of apples

Paper-bagging treatment can transform non-transcribed MdMYB1 - 2 and MdMYB1 - 3 alleles into transcribed alleles through epigenetic regulations, resulting in the red pigmentation of a normally non-red apple cultivar ‘Mutsu.’ Anthocyanin biosynthesis in apples is regulated by MdMYB1/A/10, an R2R3-Type MYB gene. ‘Mutsu,’ a triploid apple cultivar harboring non-transcribed MdMYB1-2 and MdMYB1-3 alleles, retains green skin color under field conditions. However, it can show red/pink pigmentation under natural or artificial ultraviolet-B (UV-B) light exposure after paper-bagging and bag removal treatment. In the present study, we found that in ‘Mutsu,’ paper bagging-induced red pigmentation was due to the activation of non-transcribed MdMYB1-2/-3 alleles, which triggered the expression of downstream anthocyanin biosynthesis genes in a UV-B-dependent manner. By monitoring the epigenetic changes during UV-B-induced pigmentation, no significant differences in DNA methylation and histone modifications in the 5′ upstream region of MdMYB1-2/-3 were recorded between the UV-B-treated fruit skin (red) and the fruit skin treated only by white light (green). In contrast, bag treatment lowered the DNA methylation in this region of MdMYB1-2/-3 alleles. Similarly, higher levels of histone H3 acetylation and trimethylation of H3 tail at lysine 4, and lower level of trimethylation of H3 tail at lysine 27 were observed in the 5′ upstream region of MdMYB1-2/-3 in the skin of the fruit immediately after bag removal. These results suggest that bagging treatment can induce epigenetic changes, facilitating the binding of trans factor(s) to MdMYB1-2/-3 alleles, resulting in the activation of these MYBs after bag removal.AbstractMain conclusionPaper-bagging treatment can transform non-transcribedMdMYB1-2andMdMYB1-3alleles into transcribed alleles through epigenetic regulations, resulting in the red pigmentation of a normally non-red apple cultivar ‘Mutsu.’ Anthocyanin biosynthesis in apples is regulated by MdMYB1/A/10, an R2R3-Type MYB gene. ‘Mutsu,’ a triploid apple cultivar harboring non-transcribed MdMYB1-2 and MdMYB1-3 alleles, retains green skin color under field conditions. However, it can show red/pink pigmentation under natural or artificial ultraviolet-B (UV-B) light exposure after paper-bagging and bag removal treatment. In the present study, we found that in ‘Mutsu,’ paper bagging-induced red pigmentation was due to the activation of non-transcribed MdMYB1-2/-3 alleles, which triggered the expression of downstream anthocyanin biosynthesis genes in a UV-B-dependent manner. By monitoring the epigenetic changes during UV-B-induced pigmentation, no significant differences in DNA methylation and histone modifications in the 5′ upstream region of MdMYB1-2/-3 were recorded between the UV-B-treated fruit skin (red) and the fruit skin treated only by white light (green). In contrast, bag treatment lowered the DNA methylation in this region of MdMYB1-2/-3 alleles. Similarly, higher levels of histone H3 acetylation and trimethylation of H3 tail at lysine 4, and lower level of trimethylation of H3 tail at lysine 27 were observed in the 5′ upstream region of MdMYB1-2/-3 in the skin of the fruit immediately after bag removal. These results suggest that bagging treatment can induce epigenetic changes, facilitating the binding of trans factor(s) to MdMYB1-2/-3 alleles, resulting in the activation of these MYBs after bag removal.

Sulfate Uptake in Arabidopsis thaliana

Abstract Sulfate uptake of Arabidopsis thaliana seedlings were characterized. Sulfate uptake obeyed single Michaelis‐Mentens kinetics. When plants were grown in the medium containing 1,500 mM sulfate (control), Km was 20.0±3.60 mM and the Vmax ranged from 21 to 54 nmol g‐1 fresh weight hr‐1. While Km of plants grown in the sulfate‐deficient (‐S) medium containing 10 mM sulfate was not significantly changed, Vmax of plants grown in the ‐S medium increased by two fold. In the presence of selenate, an analogue to sulfate, Vmax remained unchanged, indicating that selenate is a competitive inhibitor for sulfate uptake. Metabolic inhibitors and high pH decreased sulfate uptake, suggesting that sulfate uptake system requires a H+ gradient generated by a H+‐ATPase. Transfer of seedlings from the control to the ‐S medium increased the rate of sulfate uptake and vice versa, suggesting that sulfate uptake in A. thaliana was regulated by external sulfate concentrations. These characteristics of sulfate uptake in A. ...

Transformation of kiwifruit using the ipt gene alters tree architecture

To manipulate the architecture of woody plants by controlling endogenous cytokinin levels, the isopentenyl transferase gene (ipt) from Agrobacterimtumefaciens was introduced to kiwifruit using stable transformation. Consequently, eight transgenic lines were obtained. Transgenic shoots harboring the ipt gene were recalcitrant to rooting under tissue-culture conditions; thus, their in vitro-cultivated shoots were directly grafted onto potted wild-type kiwifruit seedlings to evaluate their morphological features, and three lines (tmr2-4, tmr2-G, tmr3-C) were successfully grafted. The grafted transgenic plants had dwarfing and branching phenotypes, both of which are typical features of cytokinin overproduction. In addition, the number of buds increased and internode length was shorter in the grafted transgenic plants. The content of a precursor, trans-zeatin riboside, and an active cytokinin, trans-zeatin, increased in one transgenic line, in which the level of ipt gene expression was high, indicating that morphological changes were related to expression levels of the ipt gene and cytokinin content. Possibilities for potential utilization of the ipt gene in manipulating tree shape are discussed.

Genetic and physiological approaches toward understanding the mechanisms underlying the sulfur-regulated expression of β -conglycinin genes

Accumulation of the β subunit of β -conglycinin, a major seed storage protein of soybean, is known to be upregulated by sulfate deficiency and repressed by exogenous application of methionine in the in vitro culture of immature cotyledons. Accumulation of the β subunit mRNA increased in seeds of soybean plants as the concentration of sulfate in the media was decreased. The promoter of the β subunit was capable of upregulation, suggesting that the regulation mainly occurs at the level of transcription. The level of free sulfate in seeds of soybean grown under sulfur deficiency was dramatically reduced. Immature soybean seeds cultured in vitro in a sulfate deficient medium accumulated an elevated level of the β subunit. These results suggest that the level of sulfate concentration in seeds is a main trigger for the induction of the β subunit accumulation in plants exposed to sulfur deficiency. Possible application of genetical approaches for better understanding of the regulation mechanism is discussed.

Current trends and age-based differences of unintentional injury in Japanese children

Unintentional injury in children is a worldwide public health problem, as it increases the health burden and is a leading cause of death among children. It is important to understand the differences between different age groups of children in regard to unintentional injury, in order to effectively implement child safety education. The present study aimed to determine the current trends of unintentional injury in children, and to identify the differences between different age groups of children with regard to unintentional injury. We identified 1,521 children who attended an 18-month health checkup (18-month group), and 1,368 children who attended a 36-month health checkup (36-month group), between January 1, 2014 and December 31, 2014. The rate of hospital visits associated with unintentional injury was 10.6% (161/1,521) in the 18-month group, and 13.1% (180/1,368) in the 36-month group. In both groups, present/past illness was associated with hospital visits, and in the 36-month group, hospital visits were more common in boys than in girls. The number of unintentional injuries that occurred outdoors was higher in the 36-month group than in the 18-month group. Unintentional injuries resulting from accidental ingestion and falls were more common in the 18-month group, while unintentional injuries resulting from turning over were more common in the 36-month group. In conclusion, the number of hospital visits for unintentional injury might be higher, and the number of preventive actions taken by mothers might be lower, among children attending the 36-month health checkup than among those attending the 18-month health checkup.

Effects of seleno-methionine on accumulation of seed storage proteins of soybean

It is known that accumulation of the β subunit of β-conglycinin, a major seed storage protein, was repressed and glycinin, another major seed storage protein, was facilitated by exogenous application of methionine (Met). When 0.25 mM seleno-Met (Se-Met) was added to the culture medium, the β subunit accumulation decreased, while glycinin accumulation increased after 7 days of culture. Levels of free Met in immature cotyledons cultured with or without Se-Met were similar. These results suggest that Se-Met affect protein profiles in a similar way to Met.