Flavonoid accumulation patterns of transparent testa mutants of arabidopsis

Flavonoids have been implicated in the regulation of auxin movements in Arabidopsis. To understand when and where flavonoids may be acting to control auxin movement, the flavonoid accumulation pattern was examined in young seedlings and mature tissues of wild-type Arabidopsis. Using a variety of biochemical and visualization techniques, flavonoid accumulation in mature plants was localized in cauline leaves, pollen, stigmata, and floral primordia, and in the stems of young, actively growing inflorescences. In young Landsberg erecta seedlings, aglycone flavonols accumulated developmentally in three regions, the cotyledonary node, the hypocotyl-root transition zone, and the root tip. Aglycone flavonols accumulated at the hypocotyl-root transition zone in a developmental and tissue-specific manner with kaempferol in the epidermis and quercetin in the cortex. Quercetin localized subcellularly in the nuclear region, plasma membrane, and endomembrane system, whereas kaempferol localized in the nuclear region and plasma membrane. The flavonoid accumulation pattern was also examined in transparent testa mutants blocked at different steps in the flavonoid biosynthesis pathway. The transparent testa mutants were shown to have precursor accumulation patterns similar to those of end product flavonoids in wild-type Landsberg erecta, suggesting that synthesis and end product accumulation occur in the same cells.     

Chemotaxonomic analysis of Astragalus caprinus (Fabaceae) based on the flavonic patterns

In previous work, 14 flavonol glycosides were characterised in the leaves of Astragalus caprinus, an endemic Fabaceae, widely distributed in North West Africa. These compounds included quercetin and kaempferol derivatives, glycosylated by two, three or four sugars with an aliphatic or aromatic acyl moiety. In this paper, we describe the flavonoid patterns of 404 individual plants investigated by HPLC and subjecting the resulting data to cluster analysis. This analysis highlighted four chemotypes associated with a high accumulation of a kaempferol triglycoside, a kaempferol tetraglycoside with its acylated derivatives, two quercetin tetraglycoside derivatives and methylated diglycoside derivatives, respectively. A discriminant analysis validated the chemotaxonomic value of the four chemical polymorphisms. These chemotypes were more or less abundant according to geographical sites which corresponded to different climatic conditions.     

Increased auxin efflux in the IAA-overproducing sur1 mutant of Arabidopsis thaliana: A mechanism of reducing auxin levels?

With the aim of investigating the mechanisms that maintain auxin homeostasis in plants, we have monitored the net uptake and metabolism of exogenously supplied indole-3-acetic acid (IAA) and naphthalene-1-acetic acid (NAA) in seedlings of wild type and the IAA-overproducing mutant sur1 of Arabidopsis thaliana . Tritiated IAA and NAA entered the seedling tissues within minutes and were mostly accumulated as metabolites, probably amino acid and sugar conjugates. The mutant seedlings were marked by a strong increase of [³H]IAA metabolism and a reduction of the accumulation levels of both free [³H]IAA and [³H]NAA. The same characteristics were observed in wild-type seedlings grown on 5 μ M picloram. We measured [³H]NAA uptake in the presence of high concentrations of unlabeled NAA or the auxin efflux carrier inhibitor naphthylphthalamic acid (NPA). This abolished the difference in free [³H]NAA accumulation between the mutant or picloram-treated seedlings and wild-type seedlings. These data indicated that active auxin efflux carriers were present in Arabidopsis seedling tissues. Picloram-treated seedlings and seedlings of the IAA-overproducing mutant sur1 displayed increased auxin efflux carrier activity as well as elevated conjugation of IAA. There is previous evidence to suggest that conjugation is a means to remove excess IAA in plant cells. Here, we discuss the possibility of efflux constituting an additional mechanism for regulating free IAA levels in the face of an excess auxin supply.     

Expression of chalcone synthase and chalcone isomerase proteins in Arabidopsis seedlings

Antibodies have been developed against the first two enzymes of flavonoid biosynthesis in Arabidopsis thaliana. Chalcone synthase (CHS) and chalcone isomerase (CHI) were overexpressed and purified from Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. The recombinant proteins were then used to immunize chickens and the resulting IgY fraction was purified from egg yolks. Immunoblots of crude protein extracts from Arabidopsis seedlings carrying wild-type and null alleles for CHS and CHI showed that the resulting antibody preparations provide useful tools for characterizing expression of the flavonoid pathway at the protein level. An initial analysis of expression patterns in seedlings shows that CHS and CHI proteins are present at high levels during a brief period of early seedling germination that just precedes the transient accumulation of flavonoid end-products.     

A gain-of-function phenotype conferred by over-expression of functional subunits of the COP9 signalosome in Arabidopsis

The COP9 signalosome is a conserved cellular regulator present in diverse organisms. To understand the structural and functional relationship of the COP9 signalosome with its subunits, we expressed in wild-type and mutant Arabidopsis backgrounds two orthologues of subunit 1, rice FUS6 (rFUS6) and human GPS1, and Arabidopsis subunit 8 (COP9). In Arabidopsis, rFUS6 can functionally replace Arabidopsis endogenous FUS6 to form the COP9 signalosome complex and rescue the null fus6-1 mutant phenotype. Moreover, light-grown rFUS6 over-expression seedlings displayed longer hypocotyls and reduced anthocyanin accumulation in comparison to wild-type seedlings, which is opposite to the fus6/cop11 mutant phenotype. The long-hypocotyl phenotype was also observed in transgenic seedlings over-expressing Arabidopsis COP9. This finding indicates that over-expression of a functional subunit 1 or subunit 8 of the COP9 signalosome confers a gain-of-function phenotype relative to the complex. Human GPS1, when expressed in the fus6-1 null mutant of Arabidopsis, can assemble into a chimeric COP9 signalosome at low efficiency, demonstrating the structural conservation of the complexes between human and Arabidopsis. This low-abundancy chimeric complex is insufficient to fully rescue the mutant but is able to attenuate the mutant severity.     

Sucrose control of phytochrome A signaling in Arabidopsis.

The expression of the Arabidopsis plastocyanin (PC) gene is developmentally controlled and regulated by light. During seedling development, PC gene expression is transiently induced, and this induction can be repressed by sucrose. In transgenic seedlings carrying a PC promoter-luciferase fusion gene, the luciferase-induced in vivo luminescence was similarly repressed by sucrose. From a mutagenized population of such transgenic seedlings, we selected for mutant seedlings that displayed a high luminescence level when grown on a medium with 3% sucrose. This screening of mutants resulted in the isolation of several sucrose-uncoupled (sun) mutants showing reduced repression of luminescence by sucrose. Analysis of the sun mutants revealed that the accumulation of PC and chlorophyll a/b binding protein (CAB) mRNA was also sucrose uncoupled, although the extent of uncoupling varied. The effect of sucrose on far-red light high-irradiance responses was studied in wild-type, sun1, sun6, and sun7 seedlings. In wild-type seedlings, sucrose repressed the far-red light-induced cotyledon opening and inhibition of hypocotyl elongation. sun7 seedlings showed reduced repression of these responses. Sucrose also repressed the far-red light-induced block of greening in wild-type seedlings, and both sun6 and sun7 were affected in this response. The results provide evidence for a close interaction between sucrose and light signaling pathways. Moreover, the sun6 and sun7 mutants genetically identify separate branches of phytochrome A-dependent signal transduction pathways.     

Fusarium graminearum gene deletion mutants map1 and tri5 reveal similarities and differences in the pathogenicity requirements to cause disease on Arabidopsis and wheat floral tissue

The Ascomycete pathogen Fusarium graminearum can infect all cereal species and lower grain yield, quality and safety. The fungus can also cause disease on Arabidopsis thaliana. In this study, the disease-causing ability of two F. graminearum mutants was analysed to further explore the parallels between the wheat (Triticum aestivum) and Arabidopsis floral pathosystems. Wild-type F. graminearum (strain PH-1) and two isogenic transformants lacking either the mitogen-activated protein kinase MAP1 gene or the trichodiene synthase TRI5 gene were individually spray- or point-inoculated onto Arabidopsis and wheat floral tissue. Disease development was quantitatively assessed both macroscopically and microscopically and deoxynivalenol (DON) mycotoxin concentrations determined by enzyme-linked immunosorbent assay (ELISA). Wild-type strain inoculations caused high levels of disease in both plant species and significant DON production. The map1 mutant caused minimal disease and DON accumulation in both hosts. The tri5 mutant, which is unable to produce DON, exhibited reduced pathogenicity on wheat ears, causing only discrete eye-shaped lesions on spikelets which failed to infect the rachis. By contrast, the tri5 mutant retained full pathogenicity on Arabidopsis floral tissue. This study reveals that DON mycotoxin production is not required for F. graminearum to colonize Arabidopsis floral tissue.     

Sinapic acid ester metabolism in wild type and a sinapoylglucose-accumulating mutant of arabidopsis.

Sinapoylmalate is one of the major phenylpropanoid metabolites that is accumulated in the vegetative tissue of Arabidopsis thaliana. A thin-layer chromatography-based mutant screen identified two allelic mutant lines that accumulated sinapoylglucose in their leaves in place of sinapoylmalate. Both mutations were found to be recessive and segregated as single Mendelian genes. These mutants define a new locus called SNG1 for sinapoylglucose accumulator. Plants that are homozygous for the sng1 mutation accumulate normal levels of malate in their leaves but lack detectable levels of the final enzyme in sinapate ester biosynthesis, sinapoylglucose:malate sinapoyltransferase. A study of wild-type and sng1 seedlings found that sinapic acid ester biosynthesis in Arabidopsis is developmentally regulated and that the accumulation of sinapate esters is delayed in sng1 mutant seedlings.     

Regulation of auxin transport by aminopeptidases and endogenous flavonoids

 The 1-N-naphthylphthalamic acid (NPA)-binding protein is a putative negative regulator of polar auxin transport that has been shown to block auxin efflux from both whole plant tissues and microsomal membrane vesicles. We previously showed that NPA is hydrolyzed by plasma-membrane amidohydrolases that co-localize with tyrosine, proline, and tryptophan-specific aminopeptidases (APs) in the cotyledonary node, hypocotyl-root transition zone and root distal elongation zone of Arabidopsisthaliana (L.) Heynh. seedlings. Moreover, amino acyl-β-naphthylamide (aa-NA) conjugates resembling NPA in structure have NPA-like inhibitory activity on growth, suggesting a possible role of APs in NPA action. Here we report that the same aa-NA conjugates and the AP inhibitor bestatin also block auxin efflux from seedling tissue. Bestatin and, to a lesser extent, some aa-NA conjugates were more effective inhibitors of low-affinity specific [³H]NPA-binding than were the flavonoids quercetin and kaempferol but had no effect on high-affinity binding. Since the APs are inhibited by flavonoids, we compared the localization of endogenous flavonoids and APs in seedling tissue. A correlation between AP and flavonoid localization was found in 5- to 6-d-old seedlings. Evidence that these flavonoids regulate auxin accumulation in vivo was obtained using the flavonoid-deficient mutant, tt4. In whole-seedling [¹⁴C]indole-3-acetic acid transport studies, the pattern of auxin distribution in the tt4 mutant was shown to be altered. The defect appeared to be in auxin accumulation, as a considerable amount of auxin escaped from the roots. Treatment of the tt4 mutant with the missing intermediate naringenin restored normal auxin distribution and accumulation by the root. These results implicate APs and endogenous flavonoids in the regulation of auxin efflux. Received: 2 December 1999 / Accepted: 16 January 2000     

Higher Activity of an Aldehyde Oxidase in the Auxin-Overproducing superroot1 Mutant of Arabidopsis thaliana

Aldehyde oxidase (AO; EC 1.2.3.1) activity was measured in seedlings of wild type or an auxin-overproducing mutant, superroot1 (sur1), of Arabidopsis thaliana. Activity staining for AO after native polyacrylamide gel electrophoresis separation of seedling extracts revealed that there were three major bands with AO activity (AO1–3) in wild-type and mutant seedlings. One of them (AO1) had a higher substrate preference for indole-3-aldehyde. This AO activity was significantly higher in sur1 mutant seedlings than in the wild type. The difference in activity was most apparent 7 d after germination, the same time required for the appearance of the remarkable sur1 phenotype, which includes epinastic cotyledons, elongated hypocotyls, and enhanced root development. Higher activity was observed in the root and hypocotyl region of the mutant seedlings. We also assayed the indole-3-acetaldehyde oxidase activity in extracts by high-performance liquid chromatography detection of indole-3-acetic acid (IAA). The activity was about 5 times higher in the extract of the sur1 seedlings, indicating that AO1 also has a substrate preference for abscisic aldehyde. Treatment of the wild-type seedlings with picloram or IAA caused no significant increase in AO1 activity. This result suggested that the higher activity of AO1 in sur1 mutant seedlings was not induced by IAA accumulation and, thus, strongly supports the possible role of AO1 in IAA biosynthesis in Arabidopsis seedlings.     

Phytochrome B affects responsiveness to gibberellins in Arabidopsis.

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. Arabidopsis thaliana seedlings lacking one of the phytochromes, phyB, have elongated hypocotyls and other tissues, suggesting that they may have an alteration in hormone physiology. We have studied the possibility that phyB mutations affect seedling gibberellin (GA) perception and metabolism by testing the responsiveness of wild-type and phyB seedlings to exogenous GAs. The phyB mutant elongates more than the wild type in response to the same exogenous concentrations of GA3 or GA4, showing that the mutation causes an increase in responsiveness to GAs. Among GAs that we were able to detect, we found no significant difference in endogenous levels between wild-type and phyB mutant seedlings. However, GA4 levels were below our limit of detectability, and the concentration of that active GA could have varied between wild-type and phyB mutant seedlings. These results suggest that, although GAs are required for hypocotyl cell elongation, phyB does not act primarily by changing total seedling GA levels but rather by decreasing seedling responsiveness to GAs.     

The gluconeogenic enzyme phosphoenolpyruvate carboxykinase in Arabidopsis is essential for seedling establishment

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate in the gluconeogenic production of sugars from storage oil in germinating oilseeds. Here, we present the results of analysis on PEPCK antisense Arabidopsis plants with a range of enzyme activities from 20% to 80% of wild-type levels. There is a direct correlation between enzyme activity and seedling establishment during early post-germinative growth, thus demonstrating the absolute requirement of PEPCK and gluconeogenesis in this process. Soluble sugar levels in the 35S-PCK1 antisense seedlings are reduced and seedling establishment can be rescued with an exogenous supply of sucrose. We observed an increase in the respiration of acetyl coenzyme A units released from fatty acid beta-oxidation and a corresponding decrease in the production of sugars with decreasing enzyme activity in 2-d-old antisense seedlings. The 35S-PCK1 antisense lines have a more extreme phenotype when compared with Arabidopsis mutants disrupted in the glyoxylate cycle. We conclude that the 35S-PCK1 antisense seedlings are compromised in the ability to use both storage lipid and storage protein through gluconeogenesis to produce soluble sugars.     

RSF1, an Arabidopsis locus implicated in phytochrome A signaling

In Arabidopsis, phytochrome A (phyA) is the major photoreceptor both for high irradiance responses to far-red light and broad spectrum very low fluence responses, but little is known of its signaling pathway(s). rsf1 was isolated as a recessive mutant with reduced sensitivity to far-red inhibition of hypocotyl elongation. At the seedling stage rsf1 mutants are affected, to various degrees, in all described phyA-mediated responses. However, in adult rsf1 plants, the photoperiodic flowering response is normal. The rsf1 mutant has wild-type levels of phyA suggesting that RSF1 is required for phyA signaling rather than phyA stability or biosynthesis. RSF1 thus appears to be a major phyA signaling component in seedlings, but not in adult, Arabidopsis plants.     

Response of selected antioxidants and pigments in tissues of Rosa hybrida and Fuchsia hybrida to supplemental UV-A exposure

The effect of supplemental UV-A (320–400 nm) radiation on tissue absorption at 355 nm, levels of various antioxidants (ascorbate, glutathione, carotenoids and flavonoids) and of antioxidant scavenging capacity were investigated with leaves and petals of Rosa hybrida , cv. Honesty and with leaves, petals and sepals of Fuchsia hybrida , cv. Dollarprinzessin. Supplemental UV-A did not result in visible changes in plant morphology of either species. In leaves it induced small increases in levels of chlorophylls a and b , the carotenoids antheraxanthin, lutein and β-carotene, and high increases in the flavonols quercetin and kaempferol. Petals hardly responded, while the coloured sepals of fuchsia showed an increase in quercetin derivatives. HPLC of unhydrolysed flavonoids showed that individual quercetin derivatives in leaves of both species and kaempferol derivatives in rose leaves increased 2-fold. Some kaempferol derivatives in fuchsia leaves were more than 2-fold enhanced or were newly induced by supplemental UV-A. Increases in l-ascorbic acid levels in fuchsia leaves, and decreases in rose leaves as result of supplemental UV-A were observed, but differences appeared statistically not significant, while l-ascorbate levels remained unchanged in the other tissues investigated. Anthocyanins and reduced glutathione levels were unaffected in all tissues. The combined UV-A induced increases in concentrations of these antioxidant species, did not lead to significant increases in antioxidant capacity of tissues, measured as Trolox equivalents in 50%-ethanol extracts. Light absorption at 355 nm of leaf extracts was significantly increased upon UV-A exposure. Our results indicate that the major protection towards UV-A exposure, in particular in the leaves, will originate from absorption of irradiation, and not from scavenging reactive oxygen species.     

Phenolics during early development of <Emphasis Type="Italic">Betula pubescens</Emphasis> seedlings: inhibition of phenylalanine ammonia lyase

We studied phenolic metabolism and plant growth in birch seedlings at the beginning of their development by inhibiting phenylalanine ammonia lyase (PAL), which is the first committed step in phenylpropanoid metabolism. Betula pubescens (Ehrh.) seeds were germinated in inhibitor-free media and the seedlings were transferred to hydroponic culture at the cotyledon stage. They were 6 days old at the start of the experiment, which lasted for 3 weeks. PAL activity was inhibited by three different concentrations of 2-aminoindane-2-phosphonic acid monohydrate (AIP) in the growing media. At the end of 3 weeks, phenolics in all plant parts (roots, stem, cotyledons, first, second and third true leaves) were determined. AIP inhibited strongly the accumulation of phenolic acids, salidroside, rhododendrins, ellagitannins and their precursors, flavan-3-ols, and soluble condensed tannins. The accumulation of lignin and flavonol glycoside derivatives was moderately inhibited. The accumulation of flavonol glycosides, such as quercetin glycosides and kaempferol glycosides, was not generally inhibited, even in leaves that emerged during the experiment, while the accumulation of insoluble condensed tannins was inhibited only slightly and not in all plant parts. This suggests that flavonol glycosides, which may have a UV-B protective role, and insoluble condensed tannins, which may have structural functions, are prioritized in seedling development. Inhibition of PAL with AIP decreased seedling growth and possible reasons for this are discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.