Ultraviolet A-specific induction of anthocyanin biosynthesis in the swollen hypocotyls of turnip (Brassica rapa)

Ultraviolet A (UV-A)-mediated regulation of anthocyanin biosynthesis was investigated in swollen hypocotyls of the red turnip 'Tsuda'. The shaded swollen hypocotyls which contained negligible anthocyanin were exposed to artificial light sources including low fluence UV-B, UV-A, blue, red, far-red, red plus UV-A, far-red plus UV-A, and blue plus red. Among these lights, only UV-A induced anthocyanin biosynthesis and co-irradiation of red or far-red with UV-A did not affect the extent of UV-A-induced anthocyanin accumulation. The expression of phenylalanine ammonia lyase (PAL; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), flavanone 3-hydroxylase (F3H; EC 1.14.11.9), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), and anthocyanidin synthase (ANS; EC 1.14.11.19) genes was increased with time during a 24 h exposure to UV-A. In contrast, irradiation with red, blue, UV-B, and a combination of blue with red failed to induce CHS expression. Microarray analysis showed that only a few genes, including CHS and F3H, were induced significantly by UV-A, while a separate set of many genes was induced by low fluence UV-B. The UV-A-specific induction of anthocyanin biosynthesis and the unique gene expression profile upon UV-A irradiation as compared with blue and UV-B demonstrated that the observed induction of anthocyanin biosynthesis in red turnips was mediated by a distinct UV-A-specific photoreceptor, but not by phytochromes, UV-A/blue photoreceptors, or UV-B photoreceptors.     

Molecular genetic analysis of chalcone synthase in Lycopersicon esculentum and an anthocyanin-deficient mutant

Summary Twelve loci have previously been identified in tomato (Lycopersicon esculentum) that control the intensity and distribution of anthocyanin pigmentation; these are useful genetic markers because they encode phenotypes that are readily visualized in the hypocotyls of emerging seedlings. In order to obtain molecular probes for tomato anthocyanin biosynthesis genes, we isolated two cDNAs which encode chalcone synthase (CHS), one of the key enzymes in anthocyanin biosynthesis, from a tomato hypocotyl cDNA library. By comparing their nucleic acid sequences, we determined that the two CHS cDNAs have an overall similarity of 76% at the nucleotide level and 88% at the amino acid level. We identified hybridization conditions that would distinguish the two clones and by Northern analysis showed that 1.5 kb mRNA species corresponding to each cDNA were expressed in cotyledons, hypocotyls and leaves of wild-type seedlings. Hybridization of the cDNAs at low stringency to genomic blots indicated that in tomato, CHS genes comprise a family of at least three individual members. The two genes that encode the CHS cDNAs were then placed onto the tomato genetic map at unique loci by restriction fragment length polymorphism mapping. We also assayed the activity of CHS and another enzyme in the anthocyanin pathway, flavone 3-hydroxylase, in hypocotyl extracts of wild-type tomato and a number of anthocyanin-deficient mutants. Five mutants had reduced CHS activity when compared to the wildtype controls. Of these, three were also reduce in flavone 3-hydroxylase activity, suggesting a regulatory role for these loci. The other two mutants were preferentially reduced in CHS activity, suggesting a more specific role for these loci in CHS expression.     

BBX16, a B‐box protein,positively regulates light‐induced anthocyanin accumulation by activating MYB10 in red pear

The red coloration of pear (Pyrus pyrifolia) results from anthocyanin accumulation in the fruit peel. Light is required for anthocyanin biosynthesis in pear. A pear homolog of Arabidopsis thaliana BBX22, PpBBX16, was differentially expressed after fruits were removed from bags and may be involved in anthocyanin biosynthesis. Here, the expression and function of PpBBX16 were analysed. PpBBX16's expression was highly induced by white‐light irradiation, as was anthocyanin accumulation. PpBBX16's ectopic expression in Arabidopsis increased anthocyanin biosynthesis in the hypocotyls and tops of flower stalks. PpBBX16 was localized in the nucleus and showed trans‐activity in yeast cells. Although PpBBX16 could not directly bind to the promoter of PpMYB10 or PpCHS in yeast one‐hybrid assays, the complex of PpBBX16/PpHY5 strongly trans‐activated anthocyanin pathway genes in tobacco. PpBBX16's overexpression in pear calli enhanced the red coloration during light treatments. Additionally, PpBBX16's transient overexpression in pear peel increased anthocyanin accumulation, while virus‐induced gene silencing of PpBBX16 decreased anthocyanin accumulation. The expression patterns of pear BBX family members were analysed, and six additional BBX genes, which were differentially expressed during light‐induced anthocyanin biosynthesis, were identified. Thus, PpBBX16 is a positive regulator of light‐induced anthocyanin accumulation, but it could not directly induce the expression of the anthocyanin biosynthesis‐related genes by itself but needed PpHY5 to gain full function. Our work uncovered regulatory modes for PpBBX16 and suggested the potential functions of other pear BBX genes in the regulation of anthocyanin accumulation, thereby providing target genes for further studies on anthocyanin biosynthesis.     

Effects of Temperature on the Pattern of Anthocyanin Accumulation in Seedlings of Polygonum cuspidatum

Polygonum cuspidatum seedling. Anthocyanin accumulated first in the lower part of hypocotyls and then the site of accumulation gradually extended toward the upper part of hypocotyls when seedlings were irradiated with white light (WL) at 25 C. Etiolated seedlings accumulated anthocyanin only in the upper parts (hook and cotyledons) when the seedlings were irradiated with WL at 5 C. De-etiolated seedlings that had been pre-irradiated with WL for 1 day at 25 C accumulated anthocyanin both in upper and lower parts of the seedlings when the seedlings were irradiated with WL at 5 C. Spectral sensitivity was dependent on the temperature during irradiation. Red light (R), blue light (B), and near ultra-violet light (NUV) induced the accumulation of anthocyanin at 5 C but only NUV was effective in inducing the accumulation of anthocyanin at 25 C. Dichlorophenyl dimethylurea (DCMU) inhibited WL-induced anthocyanin accumulation but did not NUV-induced anthocyanin accumulation at 25 C. However, sucrose promoted NUV action at 25 C, indicating that photosynthesis can promote NUV-induced anthocyanin accumulation. Distribution of phytochrome in etiolated seedlings, that was examined by spectrophotometry, was similar to the distribution of anthocyanin at 5 C. Furthermore, phytochrome remained after 48 hr irradiation with WL at 5 C although phytochrome was rapidly degraded at 25 C. Received 12 July 1999/ Accepted in revised form 24 December 1999     

Sonication assisted <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation enhances the transformation efficiency in flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

A sonication-assisted, Agrobacterium-mediated, co-cultivation technique was used in an attempt to increase the transformation efficiency of flax. Hypocotyls and cotyledons excised from about 10-day-old flax seedlings grown in vitro were placed into a 10 mM MgSO₄ solution, and inoculated with an A. tumefaciens vector bearing the mgfp5-ER gene driven by the CaMV 35S promoter. The explants were subjected to pulses of ultrasound delivered by a sonicator apparatus (35 kHz) for 0–150 s and co-cultivated for 2 h at 27°C. The dried hypocotyls and cotyledons were grown on a selective MS medium to promote shoot regeneration. An electron microscopic study showed that the sonication treatment resulted in thousands of microwounds on and below the surface of the explants. A stereo microscope Leica MZ 12 equipped with a GFP adaptor was used to assess the infection and transformation of plant tissues in real time. After only 48 h and for at least 30 days after bacteria elimination, signs of transgene expression could be seen as a bright fluorescence. Our results show that treatment with ultrasound facilitates an enhanced uptake of plasmid DNA into the cells of flax hypocotyls and cotyledons and that its efficiency depends on the duration of the treatment and the frequency used. SAAT could be a promising tool for enhancing transformation efficiency in flax.     

Photocontrol of anthocyanin formation in turnip seedlings

Summary As measured by in vivo spectrophotometry the phytochrome content in etiolated turnip seedlings was higher in cotyledons than in hypocotyls; in the latter, it is confined to the apical part. During early growth in darkness the amount increased in both tissues to a maximum, reached about 40 hours after sowing; the levels then gradually declined. Separation of seedlings into hypocotyl and cotyledons increased the rate of phytochrome loss in the former, but not in the latter.Following 5 minutes of red light P _frdecayed very rapidly in darkness; after 1.5 hours all of the phytochrome was present as P _r, which was presumably not converted initially. In continuous red light the total phytochrome was reduced to below the detection level within 3 hours. Seedling age markedly affected the loss of phytochrome following red light; more was destroyed in older than in younger hypocotyls and apparent new synthesis occurred only in young seedlings. The capacity to synthesise phytochrome differed in cotyledons and hypocotyl. In cotyledons, synthesis occurred following shots of red light varying from 10 seconds, to 6×I minute, but the amount of newly formed phytochrome was not related to the amount destroyed: after 5 hours of continuous red light no new synthesis occurred. In hypocotyls, the amount of phytochrome synthesised was related to the amount previously destroyed, and the phytochrome content after 24 hours of darkness was similar following all red light treatments of 1 minute or longer: new synthesis occurred following 5 hours of continuous red light.In far-red light phytochrome decayed very slowly, approaching the limit of detection after 48 hours. In cotyledons some loss was already observed after 5 hours of far-red and, in hypocotyls, after about 10 hours.These results are discussed in relation to the possible role of phytochrome as the pigment mediating anthocyanin synthesis in prolonged far-red light.     

Betacyanin biosynthesis in the isolated hypocotyls bei Acetabularia mediterranea

Summary Isolated hypocotyls synthesize betacyanin after light exposure in Amaranthus caudatus L. Pigment synthesizing capacity is reduced in the hypocotyls with increased incubation of seedlings in dark after 24h. External feeding of precursors of betacyanin L-tyrosine and DOPA enhances pigment synthesis in the isolated hypocotyls to equal that of intact hypocotyls. Cotyledons are probably the source of precursors while both cotyledons and hypocotyls are the sites of betacyanin synthesis. Betacyanin synthesizing capacity is progressively lost from the base of the hypocotyl and precursors could not induce pigment synthesis in these regions.     

Photoregulation of Phenylalanine Ammonia Lyase is not Correlated with Anthocyanin Induction in Photomorphogenetic Mutants of Tomato(Lycopersicon esculentum)

Photoregulation of phenylalanine ammonia lyase (PAL)(EC 4.3.1.5 [EC] )was analyzed in wild type (WT) and mutants: phytochrome dencient-awrea(au), high pigment exhibiting exaggerated phytochrome response(hp) and the double mutant (au.hp) of tomato (Lycopersicon esculentum(Mill.) cv. Ailsa Craig). Red light, acting via phytochrome,stimulates PAL activity in cotyledons and hypocotyls of tomatoseedlings. The time course of photoinduction of PAL in cotyledonsof the mutants (au and au.hp) and WT seedlings has a peak ofactivity at 4 h, after which the activity falls sharply, exceptin hp seedlings where activity is maintained at a high level.In hypocotyls, photoinduction of PAL also shows an initial rise,reaching a maximum at 3 h, followed by a sharp decline in themutants (au and au.hp) and WT seedlings. However in hp seedlingsphotoinduction of PAL is about 3 fold that in WT. The photoinductionof PAL appears to be dependent on de novo synthesis of proteinand nucleic acids. The use of a PAL specific inhibitor a-aminooxyß-phenylpropionic acid indicated that PAL is an essentialcomponent of the anthocyanin biosyn-thetic pathway in the tomatoseedlings. However, a comparison of anthocyanin biosynthesis[Adamse et al. (1989) Photochem. Photobiol. 50: 107] and PALphotoinduction data revealed that phytochrome mediated inductionof PAL and anthocyanin in the tomato seedlings are not correlated.While au and au.hp mutant seedlings show a similar increasein PAL level as in the WT, there is little formation of anthocyaninin these mutant seedlings. The results indicate that, in contrastto the photoregulation of anthocyanin synthesis which is dependenton the presence of the labile phytochrome (IP) pool in tomatoseedlings, the photoinduction of PAL is mediated via a smallpool of phytochrome in au mutant: stable phytochrome (sP) ora residual /P pool. (Received August 6, 1991; Accepted September 27, 1991)     

酚类化合物代谢中受UV-C辐照调控的转录因子的筛选

SlPAL5基因是酚类化合物代谢的关键基因。UV-C辐照可以有效提高番茄果实中酚类化合物的含量。因此研究调控SlPAL5基因表达的转录因子,对于进一步阐明UV-C诱导番茄果实酚类化合物合成的调控机制具有重要意义。文中通过构建番茄酵母单杂交文库,利用酵母单杂交技术筛选调控酚类化合物合成关键基因SlPAL5表达的转录因子。通过测序和Blast同源性分析得到转录因子SlERF7,并证实SlERF7可以与SlPAL5的启动子相互作用。另外,UV-C辐照可以显著提高SlERF7的表达水平。结果表明受UV-C辐照诱导的SlERF7可能参与了SlPAL5的转录调控,为研究UV-C诱导番茄果实酚类化合物合成的调控机制提供了基础。     

Distribution of phenylalanine ammonia-lyase in etiolated and far-red irradiated radish seedlings

Summary In dark-grown Raphanus seedlings, most of the PAL activity is found in roots where it increases sigmoidally during organ development. In hypocotyls, the dark increase of enzyme activity is linear with time. In cotyledons and hooks, dark activity is very low and remains constant. After onset of continuous far-red irradiation, an activity increase is observed in all parts of the seedling. In cotyledons and hooks, the increase is followed by a decrease. This is comparable to light-induced PAL activity described in other materials. In roots and hypocotyls, the initial increase is not followed by a decrease. In dark-grown roots and hypocotyls PAL activity is correlated to fresh weight augmentation. In no part of the seedling could a correlation be found between light-induced PAL activity and anthocyanin formation.     

Wirkungsspektren der Anthocyansynthese in Gewebekulturen und Keimlingen von Haplopappus gracilis

Summary The biosynthesis of anthocyanin in tissue cultures and intact seedlings of Haplopappus gracilis is a light-dependent reaction which can be induced by blue light only. Anthocyanin appeared in all organs of the seedling.Wounding of the plant led to an increase in the content of anthocyanin due to increased anthocyanin synthesis in the cotyledons.The action spectra of anthocyanin formation in tissue cultures and intact seedlings have two peaks, one at 438 nm and the other at 372 nm. The limit of activity in the direction of longer wavelengths lies between 474 and 493 nm. Red light of short and long wavelength is ineffective in the induction of pigment synthesis. The photoreceptor of the light reaction is supposed to be a yellow pigment (flavoprotein or carotinoid). In contrast to the intact plants, isolated cotyledons and wounded seedlings are able to form anthocyanin not only in the blue region but also during irradiation with red light of high intensity. The action spectrum of anthocyanin synthesis in the isolated cotyledons has a marked maximum at about 440 nm and a second one at about 660 nm. A little activity can be observed throughout the visible spectrum. The pigment synthesis induced by red light can be completely suppressed by DCMU, an inhibitor of photosynthesis. This indicates that in the case of the activity in the red light caused by wounding chlorophyll serves as photoreceptor.The anthocyanin synthesis in tissue cultures and seedlings could not be influenced by low energy radiation in the red or in the far red region, even after induction of anthocyanin synthesis by blue light of high intensity. Therefore it seems that the phytochrome system is not involved in anthocyanin synthesis in Haplopappus gracilis.     

Comparative effects of gibberellic acid and N6-benzyladenine on dry matter partitioning and osmotic and water potentials in seedling organs of dwarf watermelon

Apical applications of 0.2 μg N⁶-benzyladenine (BA), a synthetic cytokinin, or 5 μg of gibberellic acid (GA₃) significantly enhanced hypocotyl elongation in intact dwarf watermelon seedlings over a 48-h period. Accompanying the increase in hypocotyl length was marked expansion of cotyledons in BA-treated seedlings and inhibition of root growth by both compounds. A study on dry matter partitioning indicated that both growth regulators caused a preferential accumulation of dry matter in hypocotyls at the expense of the roots; however, GA₃ elicited a more rapid and greater change than did BA. In comparison to untreated seedlings, BA decreased total translocation of metabolites out of the cotyledons. Water potentials of cotyledons and hypocotyls were determined by allowing organs to equilibrate for 2 h in serial concentrations of polyethylene glycol 4000. Osmotic potentials were determined by thermocouple psychrometry. During periods of rapid growth in cotyledons and hypocotyls of BA-treated seedlings and in hypocotyls of GA-treated seedlings, the osmotic potential increased and the turgor pressure decreased in relation to untreated seedlings, indicating that cell wall extensibility was being increased. Osmotic potentials were lower in hypocotyls of GA-treated than in those of BA-treated seedlings, even though growth rates were higher in GA-treated seedlings, indicating that the latter treatment was generating more osmotically active solutes in hypocotyls.