Saneyuki Kawabata

Chalcone synthase family genes have redundant roles in anthocyanin biosynthesis and in response to blue/UV-A light in turnip (Brassica rapa; Brassicaceae)

PREMISE OF THE STUDY The epidermis of Brassica rapa (turnip) cv. Tsuda contains light-induced anthocyanins, visible signs of activity of chalcone synthase (CHS), a key anthocyanin biosynthetic enzyme, which is encoded by the CHS gene family. To elucidate the regulation of this light-induced pigmentation, we isolated Brassica rapa CHS1-CHS6 (BrCHS1-CHS6) and characterized their cis-elements and expression patterns. METHODS Epidermises of light-exposed swollen hypocotyls (ESHS) were harvested to analyze transcription levels of BrCHS genes by real-time PCR. Different promoters for the genes were inserted into tobacco to examine pCHS-GUS activity by histochemistry. Yeast-one-hybridization was used to detect binding activity of BrCHS motifs to transcription factors. KEY RESULTS Transcript levels of BrCHS1, -4, and -5 and anthocyanin-biosynthesis-related genes F3H, DFR, and ANS were high, while those of BrCHS2, -3, and -6 were almost undetectable in pigmented ESHS. However, in leaves, CHS5, F3H, and ANS expression was higher than in nonpigmented ESHS, but transcription of DFR was not detected. In the analysis of BrCHS1 and BrCHS3 promoter activity, GUS activity was strong in pigmented flowers of BrPCHS1-GUS-transformed tobacco plants, but nearly absent in BrPCHS3-GUS-transformed plants. Transcript levels of regulators, BrMYB75 and BrTT8, were strongly associated with the anthocyanin content and were light-induced. Coregulated cis-elements were found in promoters of BrCHS1,-4, and -5, and BrMYB75 and BrTT8 had high binding activities to the BrCHS Unit 1 motif. CONCLUSIONS The chalcone synthase gene family encodes a redundant set of light-responsive, tissue-specific genes that are expressed at different levels and are involved in flavonoid biosynthesis in Tsuda turnip.

Effect of high temperature on protein expression in strawberry plants

Strawberry plants (Fragaria×ananassa Duch.) cvs. Nyoho and Toyonoka were exposed to temperatures of 20, 33, and 42 °C for 4 h, and protein patterns in leaves and flowers was analyzed by 2-dimensional polyacrylamide gel electrophoresis and immunoblotting. In leaves and flowers of both cultivars, the content of most proteins decreased, but a few new proteins appeared in response to heat stress. These heat shock proteins (Hsps) were detected in the range of 19 – 29 kDa in leaves, and 16 – 26 kDa in flowers. The intensity of a 43 kDa protein spot increased in response to heat stress in Nyoho flowers, but not in Toyonoka flowers. The peaHsp17.7 antibody recognized one band at approximately 26 kDa in leaves, and two bands at approximately 16 and 17 kDa in flowers of both cultivars. These results show that the effects of heat stress on Hsp synthesis in strawberry plants differ between plant organs and between cultivars.

BrMYB4, a Suppressor of Genes for Phenylpropanoid and Anthocyanin Biosynthesis, is Down-Regulated by UV-B but not by Pigment-Inducing Sunlight in Turnip cv. Tsuda

The regulation of light-dependent anthocyanin biosynthesis in Brassica rapa subsp. rapa cv. Tsuda turnip was investigated using an ethyl methanesulfonate (EMS)-induced mutant R30 with light-independent pigmentation. TILLING (targeting induced local lesions in genomes) and subsequent analysis showed that a stop codon was inserted in the R2R3-MYB transcription factor gene BrMYB4 and that the encoded protein (BrMYB4mu) had lost its C-terminal region. In R30, anthocyanin accumulated in the below-ground portion of the storage root of 2-month-old plants. In 4-day-old seedlings and 2-month-old plants, expression of BrMYB4 was similar between R30 and the wild type (WT), but the expression of the cinnamate 4-hydroxylase gene (BrC4H) was markedly enhanced in R30 in the dark. In turnip seedlings, BrMYB4 expression was suppressed by UV-B irradiation in the WT, but this negative regulation was absent in R30. Concomitantly, BrC4H was repressed by UV-B irradiation in the WT, but stayed at high levels in R30. A gel-shift assay revealed that BrMYB4 could directly bind to the promoter region of BrC4H, but BrMYB4mu could not. The BrMYB4-enhanced green fluorescent protein (eGFP) protein could enter the nucleus in the presence of BrSAD2 (an importin β-like protein) nuclear transporter, but BrMYB4mu-eGFP could not. These results showed that BrMYB4 functions as a negative transcriptional regulator of BrC4H and mediates UV-B-dependent phenylpropanoid biosynthesis, while BrMYB4mu has lost this function. In the storage roots, the expression of anthocyanin biosynthesis genes was enhanced in R30 in the dark and in sunlight in both the WT and R30. However, in the WT, anthocyanin-inducing sunlight did not suppress BrMYB4 expression. Therefore, sunlight-induced anthocyanin biosynthesis does not seem to be regulated by BrMYB4.

Transient expression of a foreign gene by direct incorporation of DNA into intact plant tissue through vacuum infiltration.

We previously established a method to induce transient expression of foreign genes in intact plant tissue to detect the subcellular localization of proteins. Here, we have inserted a putative bZIP protein HY5 gene (SeqID: EU386772), isolated from the seedlings of turnips Brassica rapa L. subsp. rapa ‘Tsuda,’ and a receptor-like kinase gene AtRLK (SeqID: AY531551.1), isolated from Arabidopsis, into the plasmid pA7-GFP. We accomplished the direct incorporation of DNA into onion epidermal tissue by vacuum infiltration. By detecting GFP, which was fused with AtRLK or putative BrHY5, we determined that BrHY5 is located in the nucleus and AtRLK is located in the plasma membrane. This approach can be thus used to study the transient expression of foreign genes in intact tissue.

Development of simple sequence repeat (SSR) markers that are polymorphic between cultivars in Brassica rapa subsp. rapa

Simple sequence repeats (SSRs) markers were developed through data mining of 3,803 expressed sequence tags (ESTs) previously published. A total of 144 di- to penta-type SSRs were identified and they were screened for polymorphism between two turnip cultivars, ‘Tsuda’ and ‘Yurugi Akamaru’. Out of 90 EST-SSRs for which polymerase chain reaction (PCR) primers were designed, 85 could be successfully amplified, 13 showed polymorphism and eight were regarded as valid markers based on Hardy-Weinberg equilibrium (HWE) in the F 2 population developed from a cross between the two cultivars. Additionally, 1551 SSR markers reported in public database were also subjected to polymorphism survey. Among these SSR markers, 66 showed polymorphism between the two cultivars, while 28 showed HWE with Ho in a range of 0.4 to 0.6. A linkage map was constructed using these eight EST-SST markers and 28 published SSR markers in the F 2 population. Key words: EST-SSR, linkage map, polymorphism, single sequence repeats, turnip.

Relationship between the velvet-like texture of flower petals and light reflection from epidermal cell surfaces

Texture such as velvet lustre contributes to the ornamental character of a flower, along with shape and colour. This study aims to clarify the relationship between the formation of the velvet lustre texture and the optical characteristics of light reflection from irradiated surfaces of velvety and non-velvety petals from 30 cultivars or varieties of ornamental plants representing 19 species from various families. The angle of incident light from the petal surface was set at 90°, 60° or 30°, then light reflection from the petal surfaces was observed using a digital microscope. The observed reflected light was composed of “exterior” reflected light (ERL), which is observed as sparkling white spots on the surface of the epidermal cells, and “interior” reflected light (IRL), which is reflected from inside the petal and determines the base colour of the petals. Velvety petals had two common characteristics: conical-papillate or domed epidermal cells and a dark colour. As the angle between the petal and the incident light decreased, the ERL spots took on a belt-like shape, and total ERL intensity became stronger. We concluded that the velvety texture is derived from characteristic ERL rays coupled with dark IRL. The long sloping surface of the epidermal cells contributes to the higher ERL intensity as petals are observed from more horizontal angles, causing characteristic reverse shading effects on velvety petals.

Construction and genetic analysis of anthocyanin-deficient mutants induced by T-DNA insertion in ‘Tsuda’ turnip (Brassica rapa)

We previously showed that anthocyanin biosynthesis is specifically induced either by UV-A or co-irradiation with blue and UV-B, but not by monochromatic blue or UV-B light in the epidermis of the storage root of ‘Tsuda’ turnip (Brassica rapa L. subsp. rapa). To gain further molecular insights into the light signal transduction pathway of anthocyanin accumulation, over 10,000 germinated seeds of Tsuda turnip were sonicated and transformed using an improved Agrobacterium-mediated vacuum infiltration method. We obtained 17 anthocyanin-rich and 60 anthocyanin-deficient mutant lines from the mutant library. PCR identification, GFP assay and GUS staining showed that six anthocyanin-deficient mutants were generated by T-DNA insertion. Real-time qRT-PCR results demonstrated that expression of structural and regulatory genes of anthocyanin synthesis in the anthocyanin-deficient lines significantly decreased compared with wild type, which coincided with their anthocyanin levels in the epidermis of storage roots. In a genetic analysis of the F2 population of the mutants (g56w, g83w, g142w, and g143w) backcrossed with the wild type, the phenotypic proportions between the wild type and mutants were 3:1 following Mendelian segregation. Therefore, we speculated that the mutated trait of each mutant is controlled by a single recessive gene. This study provides a series of stable, homozygous and valuable mutant resources for elucidating the mechanisms of UV-A and blue + UV-B induced anthocyanin biosynthesis in higher plants.