The phytochrome A-specific signaling intermediate SPA1 interacts directly with COP1, a constitutive repressor of light signaling in Arabidopsis.

SPA1 is a phytochrome A (phyA)-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings. It contains a WD-repeat domain that shows high sequence similarity to the WD-repeat region of the constitutive repressor of light signaling, COP1. Here, using yeast two-hybrid and in vitro interaction assays, we show that SPA1 strongly and selectively binds to COP1. Domain mapping studies indicate that the putative coiled-coil domain of SPA1 is necessary and sufficient for binding to COP1. Conversely, similar deletion analyses of the COP1 protein suggest that SPA1 interacts with the presumed coiled-coil domain of COP1. To further investigate SPA1 function in the phyA signaling pathway, we tested whether SPA1, like COP1, mediates changes in gene expression in response to light. We show that spa1 mutations increase the photoresponsiveness of certain light-regulated genes within 2 h of light treatment. Taken together, the results suggest that SPA1 may function to link the phytochrome A-specific branch of the light signaling pathway to COP1. Hence, our data provide molecular support for the hypothesis that COP1 is a convergence point for upstream signaling pathways dedicated to individual photoreceptors.

Light-induced growth promotion by SPA1 counteracts phytochrome-mediated growth inhibition during de-etiolation.

Previous evidence has suggested that SPA1 is a signal transduction component that appears to require phytochrome A for function in seedling photomorphogenesis. Using digital image analysis, we examined the time course of growth inhibition induced by red light in spa1 mutants to test the interpretation that SPA1 functions early in a phyA-specific signaling pathway. By comparing wild-type and mutant responses, we found that SPA1 caused an increase in hypocotyl growth rate after approximately 2 h of continuous red light, whereas the onset of phyA-mediated inhibition was detected within several minutes. Thus, SPA1-dependent growth promotion began after phyA started to inhibit growth. The action of SPA1 persisted for approximately 2 d of red light, a period well beyond the time when the phyA photoreceptor and its influence on growth have both decayed to undetectable levels. Also, SPA1 promoted growth for many hours in the complete absence of a light stimulus when red-light-grown seedlings were shifted to darkness. We propose that SPA1 functions in a light-induced mechanism that promotes growth and thereby counteracts growth inhibition mediated by phyA and phyB. Our finding that spa1 seedlings do not display growth promotion in response to end-of-day pulses of far-red light, even in a phyA-null background, supports this interpretation. Combined, these results lead us to the view that the rate of hypocotyl elongation in light is determined by at least two independent, opposing processes; an inhibition of growth by the phytochromes and a promotion of growth by light-activated SPA1.

Signaling from the embryo conditions Vp1-mediated repression of alpha-amylase genes in the aleurone of developing maize seeds.

The VP1 transcription factor functions as both a repressor and an activator of gene expression in the developing aleurone. Vp1 activation of the anthocyanin pathway exhibits strict cell autonomy in aleurone. In contrast, Vp1-mediated repression of hydrolase genes in aleurone cells during seed development is determined by a combination of cell autonomous and cell non-autonomous signals. To analyze signaling between the embryo and aleurone during seed development, a T-B3La chromosome translocation was used to create seed that has non-concordant embryo and endosperm genotypes. We show that de-repression of an Amy-GUS reporter gene in developing vp1 mutant aleurone cells strongly depends on the presence of a viviparous embryo. Genetic ablation of the developing embryo in vp1 mutant and Vp1 seeds through the introduction of an early embryo mutation caused a similar enhancement of Amy-GUS expression in the aleurone, suggesting that the quiescent embryo present in normal seed is a critical source of inhibitory signals. Analysis of an ABA deficient vp1 vp5 double mutant indicates that ABA synthesized in the embyro interacts additively with Vp1 to prevent precocious induction of alpha-amylase genes in the aleurone of the developing seed. A lack of ABA synthesis, however, does not account for the strongly synergistic interaction between a viviparous vp1 embryo and mutant aleurone suggesting that a quiescent embyro is a source of other inhibitory signals.

SPA1, a WD-repeat protein specific to phytochrome A signal transduction.

The five members of the phytochrome photoreceptor family of Arabidopsis thaliana control morphogenesis differentially in response to light. Genetic analysis has identified a signaling pathway that is specifically activated by phytochrome A. A component in this pathway, SPA1 (for "suppressor of phyA-105"), functions in repression of photomorphogenesis and is required for normal photosensory specificity of phytochrome A. Molecular cloning of the SPA1 gene indicates that SPA1 is a WD (tryptophan-aspartic acid)-repeat protein that also shares sequence similarity with protein kinases. SPA1 can localize to the nucleus, suggesting a possible function in phytochrome A-specific regulation of gene expression.

SPA1: a new genetic locus involved in phytochrome A-specific signal transduction.

To identify mutants potentially defective in signaling intermediates specific to phytochrome A (phyA), we screened for extragenic mutations that suppress the morphological phenotype exhibited by a weak phyA mutant (phyA-105) of Arabidopsis. A new recessive mutant, designated spa1 (for suppressor of phyA-105), was isolated and mapped to the bottom of chromosome 2. spa1 phyA-105 double mutants exhibit restoration of several responses to limiting fluence rates of continuous far-red light that are absent in the parental phyA-105 mutant, such as deetiolation, anthocyanin accumulation, and a far-red light-induced inability of seedlings to green upon subsequent transfer to continuous white light. spa1 mutations do not cause a phenotype in darkness, indicating that the suppression phenotype is light dependent. Enhanced photoresponsiveness was observed in spa1 seedlings in a wild-type PHYA background as well as in the mutant phyA-105 background but not in a mutant phyA null background. These results indicate that phyA is necessary in a non-allele-specific fashion for the expression of the spa1 mutant phenotype and that phyB to phyE are not sufficient for this effect. Taken together, the data suggest that spa1 mutations specifically amplify phyA signaling and therefore that the SPA1 locus encodes a component that acts negatively early in the phyA-specific signaling pathway.

RED1 is necessary for phytochrome B-mediated red light-specific signal transduction in Arabidopsis.

Seedlings of a transgenic Arabidopsis line (ABO) that overexpresses phytochrome B (phyB) display enhanced deetiolation specifically in red light. To identify genetic loci necessary for phytochrome signal transduction in red light, we chemically mutagenized ABO seeds and screened M2 seedlings for revertants of the enhanced deetiolation response. One recessive, red light-specific extragenic revertant, designated red1, was isolated. The mutant phenotype was expressed in the original ABO background as well as in the nontransgenic Nossen (No-0) progenitor background. red1 is also deficient in several other aspects of red light-induced responses known to be mediated by phyB, such as inhibition of petiole elongation and the shade avoidance response. red1 was mapped to the bottom of chromosome 4 at a position distinct from all known photoreceptor loci. Together with complementation analysis, the data show that red1 is a novel photomorphogenic mutant. The evidence suggests that red1 represents a putative phytochrome signal transduction mutant potentially specific to the phyB pathway.

Integrated control of seed maturation and germination programs by activator and repressor functions of Viviparous-1 of maize.

The Viviparous-1 (VP1) transcriptional activator of maize is required for abscisic acid induction of maturation-specific genes late in seed development leading to acquisition of desiccation tolerance and arrest in embryo growth. Here, we show that VP1 also inhibits induction of the germination-specific alpha-amylase genes in aleurone cells of the developing seed and thereby appears to be involved in preventing precocious hydrolyzation of storage compounds accumulating in the endosperm. In developing seeds of the somatically instable vp1-m2 mutant, hydrolase activity was derepressed specifically in endosperm sectors underlying vp1 mutant aleurone. A barley alpha-amylase promoter-GUS reporter construct (Amy-GUS) was induced in developing vp1 mutant aleurone cells but not in wild-type aleurone cells. Moreover, transient expression of recombinant VP1 and vp1 mutant aleurone cells strongly inhibited expression of Amy-GUS and thus effectively complemented this aspect of the mutant phenotype. VP1 specifically repressed induction of Amy-GUS by the hormone gibberellic acid in aleurone of germinating barley seeds. Deletion of the acidic transcriptional activation domain of VP1 did not affect the inhibitory activity, indicating that VP1 has a discrete repressor function. Hence, physically combining activator and repressor functions in one protein may provide a mechanism to integrate the control of two normally consecutive developmental programs, seed maturation and seed germination.