Regulation of S-Like Ribonuclease Levels in Arabidopsis. Antisense Inhibition of RNS1 or RNS2 Elevates Anthocyanin Accumulation1

The S-like ribonucleases (RNases) RNS1 and RNS2 of Arabidopsis are members of the widespread T2 ribonuclease family, whose members also include the S-RNases, involved in gametophytic self-incompatibility in plants. Both RNS1 and RNS2 mRNAs have been shown previously to be induced by inorganic phosphate (Pi) starvation. In our study we examined this regulation at the protein level and determined the effects of diminishing RNS1 and RNS2 expression using antisense techniques. The Pi-starvation control of RNS1 and RNS2 was confirmed using antibodies specific for each protein. These specific antibodies also demonstrated that RNS1 is secreted, whereas RNS2 is intracellular. By introducing antisense constructs, mRNA accumulation was inhibited by up to 90% for RNS1 and up to 65% for RNS2. These plants contained abnormally high levels of anthocyanins, the production of which is often associated with several forms of stress, including Pi starvation. This effect demonstrates that diminishing the amounts of either RNS1 or RNS2 leads to effects that cannot be compensated for by the actions of other RNases, even though Arabidopsis contains a large number of different RNase activities. These results, together with the differential localization of the proteins, imply that RNS1 and RNS2 have distinct functions in the plant.

Activation of the Maize Anthocyanin Gene a2 Is Mediated by an Element Conserved in Many Anthocyanin Promoters1

Two transcription factors, C1 (a Myb-domain protein) and B (a basic-helix-loop-helix protein), mediate transcriptional activation of the anthocyanin-biosynthetic genes of maize (Zea mays). To begin to assess the mechanism of activation, the sequences required for C1- and B-mediated induction have been determined for the a2 promoter, which encodes an anthocyanin-biosynthetic enzyme. Analysis of a series of 7- to 13-base-pair substitutions revealed two regions crucial for activation. One region, centered at −99, contained a C1-binding site that abolished C1 binding. The other crucial region was adjacent, centered at −91. C1 binding was not detected at this site, and mutation of this site did not prevent C1 binding at −99. An oligonucleotide dimer containing these two crucial elements was sufficient for C1 and B activation of a heterologous promoter. These data suggest that activation of the anthocyanin genes involves C1 and another factor binding at closely adjacent sites. Mutating a previously postulated anthocyanin consensus sequence within a2 did not significantly reduce activation by C1 and B. However, sequence comparisons of the crucial a2 regions with sequences important for C1- and B-mediated activation in two other anthocyanin promoters led to a revised consensus element shared by these promoters.

Reduction of Light-Induced Anthocyanin Accumulation in Inoculated Sorghum Mesocotyls1 : Implications for a Compensatory Role in the Defense Response

Sorghum (Sorghum bicolor L. Moench) accumulates the anthocyanin cyanidin 3-dimalonyl glucoside in etiolated mesocotyls in response to light. Inoculation with the nonpathogenic fungus Cochliobolus heterostrophus drastically reduced the light-induced accumulation of anthocyanin by repressing the transcription of the anthocyanin biosynthesis genes encoding flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase. In contrast to these repression effects, fungal inoculation resulted in the synthesis of the four known 3-deoxyanthocyanidin phytoalexins and a corresponding activation of genes encoding the key branch-point enzymes in the phenylpropanoid pathway, phenylalanine ammonia-lyase and chalcone synthase. In addition, a gene encoding the pathogenesis-related protein PR-10 was strongly induced in response to inoculation. The accumulation of phytoalexins leveled off by 48 h after inoculation and was accompanied by a more rapid increase in the rate of anthocyanin accumulation. The results suggest that the plant represses less essential metabolic activities such as anthocyanin synthesis as a means of compensating for the immediate biochemical and physiological needs for the defense response.

Anion Channels and the Stimulation of Anthocyanin Accumulation by Blue Light in Arabidopsis Seedlings1

Activation of anion channels by blue light begins within seconds of irradiation in seedlings and is related to the ensuing growth inhibition. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) is a potent, selective, and reversible blocker of these anion channels in Arabidopsis thaliana. Here we show that 20 μm NPPB blocked 72% of the blue-light-induced accumulation of anthocyanin pigments in seedlings. Feeding biosynthetic intermediates to wild-type and tt5 seedlings provided evidence that NPPB prevented blue light from up-regulating one or more steps between and including phenylalanine ammonia lyase and chalcone isomerase. NPPB was found to have no significant effect on the blue-light-induced increase in transcript levels of PAL1, CHS, CHI, or DFR, which are genes that encode anthocyanin-biosynthetic enzymes. Immunoblots revealed that NPPB also did not inhibit the accumulation of the chalcone synthase, chalcone isomerase, or flavanone-3-hydroxylase proteins. This is in contrast to the reduced anthocyanin accumulation displayed by a mutant lacking the HY4 blue-light receptor, as hy4 displayed reduced expression of the above enzymes. Taken together, the data indicate that blue light acting through HY4 leads to an increase in the amount of biosynthetic enzymes, but blue light must also act through a separate, anion-channel-dependent system to create a fully functional biosynthetic pathway.

Major recent and independent changes in levels and patterns of expression have occurred at the b gene, a regulatory locus in maize

The b locus encodes a transcription factor that regulates the expression of genes that produce purple anthocyanin pigment. Different b alleles are expressed in distinct tissues, causing tissue-specific anthocyanin production. Understanding how phenotypic diversity is produced and maintained at the b locus should provide models for how other regulatory genes, including those that influence morphological traits and development, evolve. We have investigated how different levels and patterns of pigmentation have evolved by determining the phenotypic and evolutionary relationships between 18 alleles that represent the diversity of b alleles in Zea mays. Although most of these alleles have few phenotypic differences, five alleles have very distinct tissue-specific patterns of pigmentation. Superimposing the phenotypes on the molecular phylogeny reveals that the alleles with strong and distinctive patterns of expression are closely related to alleles with weak expression, implying that the distinctive patterns have arisen recently. We have identified apparent insertions in three of the five phenotypically distinct alleles, and the fourth has unique upstream restriction fragment length polymorphisms relative to closely related alleles. The insertion in B-Peru has been shown to be responsible for its unique expression and, in the other two alleles, the presence of the insertion correlates with the phenotype. These results suggest that major changes in gene expression are probably the result of large-scale changes in DNA sequence and/or structure most likely mediated by transposable elements.

Expression Analysis of a Ripening-Specific, Auxin-Repressed Endo-1,4-β-Glucanase Gene in Strawberry

A cDNA (Cel1) encoding an endo-1,4-β-glucanase (EGase) was isolated from ripe fruit of strawberry (Fragaria × ananassa). The deduced protein of 496 amino acids contains a presumptive signal sequence, a common feature of cell wall-localized EGases, and one potential N-glycosylation site. Southern- blot analysis of genomic DNA from F. × ananassa, an octoploid species, and that from the diploid species Fragaria vesca indicated that the Cel1 gene is a member of a divergent multigene family. In fruit, Cel1 mRNA was first detected at the white stage of development, and at the onset of ripening, coincident with anthocyanin accumulation, Cel1 mRNA abundance increased dramatically and remained high throughout ripening and subsequent fruit deterioration. In all other tissues examined, Cel1 expression was invariably absent. Antibodies raised to Cel1 protein detected a protein of 62 kD only in ripening fruit. Upon deachenation of young white fruit to remove the source of endogenous auxins, ripening, as visualized by anthocyanin accumulation, and Cel1 mRNA accumulation were both accelerated. Conversely, auxin treatment of white fruit repressed accumulation of both Cel1 mRNA and ripening. These results indicate that strawberry Cel1 is a ripening-specific and auxin-repressed EGase, which is regulated during ripening by a decline in auxin levels originating from the achenes.

Genetic Interactions between Phytochrome A, Phytochrome B, and Cryptochrome 1 during Arabidopsis Development1

Single, double, and triple null combinations of Arabidopsis mutants lacking the photoreceptors phytochrome (phy) A (phyA-201), phyB (phyB-5), and cryptochrome (cry) 1 (hy4-2.23n) were examined for de-etiolation responses in high-fluence red, far-red, blue, and broad-spectrum white light. Cotyledon unhooking, unfolding, and expansion, hypocotyl growth, and the accumulation of chlorophylls and anthocyanin in 5-d-old seedlings were measured under each light condition and in the dark. phyA was the major photoreceptor/effector for most far-red-light responses, although phyB and cry1 modulated anthocyanin accumulation in a phyA-dependent manner. phyB was the major photoreceptor in red light, although cry1 acted as a phyA/phyB-dependent modulator of chlorophyll accumulation under these conditions. All three photoreceptors contributed to most blue light deetiolation responses, either redundantly or additively; however, phyB acted as a modulator of cotyledon expansion dependent on the presence of cry1. As reported previously, flowering time in long days was promoted by phyA and inhibited by phyB, with each suppressing the other's effect. In addition to the effector/modulator relationships described above, measurements of hypocotyls from blue-light-grown seedlings demonstrated phytochrome activity in blue light and cry1 activity in a phyAphyB mutant background.

A Fruit-Specific Putative Dihydroflavonol 4-Reductase Gene Is Differentially Expressed in Strawberry during the Ripening Process1

A cDNA clone encoding a putative dihydroflavonol 4-reductase gene has been isolated from a strawberry (Fragaria × ananassa cv Chandler) DNA subtractive library. Northern analysis showed that the corresponding gene is predominantly expressed in fruit, where it is first detected during elongation (green stages) and then declines and sharply increases when the initial fruit ripening events occur, at the time of initiation of anthocyanin accumulation. The transcript can be induced in unripe green fruit by removing the achenes, and this induction can be partially inhibited by treatment of de-achened fruit with naphthylacetic acid, indicating that the expression of this gene is under hormonal control. We propose that the putative dihydroflavonol 4-reductase gene in strawberry plays a main role in the biosynthesis of anthocyanin during color development at the late stages of fruit ripening; during the first stages the expression of this gene could be related to the accumulation of condensed tannins.

A Ds insertion alters the nuclear localization of the maize transcriptional activator R.

The R-sc gene of maize is a member of the R gene family of transcriptional activators that regulate anthocyanin biosynthesis. A derivative of R-sc, r-m9 conditions a reduced level of aleurone pigmentation due to the presence of a 2.1-kb Ds insertion near the 3' end of the coding region. Excision of Ds from r-m9 leaves a 7-bp insertion in the darker but still mutant v24 derivative. Both the 7-bp insertion in v24 and the 2.1-kb Ds in r-m9 are predicted to truncate their respective R proteins proximal to the carboxyl terminus, which was shown previously to contain one of three nuclear localization sequences. We find that the reduced expression of r-m9 and v24 are not due to mRNA or protein instability, but most likely reflect the inefficient localization of truncated R proteins to the nucleus. To our knowledge this is the first example of a transposable element insertion that alters gene expression by affecting nuclear localization. In addition, our data indicate that the carboxyl terminus of the R protein is far more important than previously suspected and illustrates the utility of natural mutations for defining functional domains in proteins.