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Stomata govern the gaseous exchange between the leaf and the external atmosphere, and their function is essential for photosynthesis and the global carbon and oxygen cycles. Rhythmic stomata movements in daily dark/light cycles prevent water loss at night and allow CO 2 uptake during the day. How the actors involved are transcriptionally regulated and how this might contribute to rhythmicity is largely unknown. Here, we show that morning stomata opening depends on the previous night period. The transcription factors PHYTOCHROME-INTERACTING FACTORS (PIFs) accumulate at the end of the night and directly induce the guard cell-specific K + channel KAT1. Remarkably, PIFs and KAT1 are required for blue light-induced stomata opening. Together, our data establish a molecular framework for daily rhythmic stomatal movements under well-watered conditions, whereby PIFs are required for accumulation of KAT1 at night, which upon activation by blue light in the morning leads to the K + intake driving stomata opening., (© 2024. The Author(s).)

The red phytochrome and blue cryptochrome plant photoreceptors play essential roles in promoting genome-wide changes in nuclear and chloroplastic gene expression for photomorphogenesis, plastid development, and greening. While their importance in anterograde signalling has been long recognized, the molecular mechanisms involved remain under active investigation. More recently, the intertwining of the light signalling cascades with the retrograde signals for the optimization of chloroplast functions has been acknowledged. Advances in the field support the participation of phytochromes, cryptochromes, and key light-modulated transcription factors, including HY5 and the PIFs, in the regulation of chloroplastic biochemical pathways that produce retrograde signals, including the tetrapyrroles and the chloroplastic MEP-isoprenoids. Interestingly, in a feedback loop, the photoreceptors and their signalling components are targets themselves of these retrograde signals, aimed at optimizing photomorphogenesis to the status of the chloroplasts, with GUN proteins functioning at the convergence points. High light and shade are also conditions where the photoreceptors tune growth responses to chloroplast functions. Interestingly, photoreceptors and retrograde signals also converge in the modulation of dual-localized proteins (chloroplastic/nuclear) including WHIRLY and HEMERA/pTAC12, whose functions are required for the optimization of photosynthetic activities in changing environments and are proposed to act themselves as retrograde signals., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

As a source of both energy and environmental information, monitoring of incoming light is crucial for plants to optimize growth throughout development 1 . Concordantly, the light signalling pathways in plants are highly integrated with numerous other regulatory pathways 2,3 . One of these signal integrators is the basic leucine zipper domain (bZIP) transcription factor LONG HYPOCOTYL 5 (HY5), which has a key role as a positive regulator of light signalling in plants 4,5 . Although HY5 is thought to act as a DNA-binding transcriptional regulator 6,7 , the lack of any apparent transactivation domain 8 makes it unclear how HY5 is able to accomplish its many functions. Here we describe the identification of three B-box containing proteins (BBX20, BBX21 and BBX22) as essential partners for HY5-dependent modulation of hypocotyl elongation, anthocyanin accumulation and transcriptional regulation. The bbx20 bbx21 bbx22 (bbx202122) triple mutant mimics the phenotypes of hy5 in the light and its ability to suppress the cop1 mutant phenotype in darkness. Furthermore, 84% of genes that exhibit differential expression in bbx202122 are also regulated by HY5, and we provide evidence that HY5 requires the B-box proteins for transcriptional regulation. Finally, expression of a truncated dark-stable version of HY5 (HY5(ΔN77)) together with BBX21 mutated in its VP motif strongly promoted de-etiolation in dark-grown seedlings, demonstrating the functional interdependence of these factors. In sum, this work clarifies long-standing questions regarding HY5 action and provides an example of how a master regulator might gain both specificity and dynamicity through the obligate dependence of cofactors.

To promote photomorphogenesis, including plastid development and metabolism, the phytochrome (phy) and the cryptochrome (cry) photoreceptors orchestrate genome-wide changes in gene expression in response to Red (R)- and Blue (B)-light cues. While phys and crys have a clear role in modulating photosynthesis, their role in the coordination of the nuclear genome and the plastome, essential for functional chloroplasts, remains underexplored. Using publicly available genome datasets for WT and phyABCDE or cry1cry2 Arabidopsis seedlings, grown, respectively, under R- or B-light, we bioinformatically analyzed the influence of light inputs and photoreceptors in the control of nuclear genes with a function in the chloroplast, and evaluated the role of phyB in the modulation of plastome-encoded genes. We show gene co-induction by R-phys and B-crys for genes with a chloroplastic function, and also apparent photoreceptor-driven preferential responses. Evidence from phyB in Arabidopsis together with published evidence from CRY2 in tomato also supports the participation of both photoreceptor families in the global modulation of the plastome genes. To begin addressing how these light-sensors orchestrate changes in an organellar genome, we evaluated their effect over genes with potential functions in plastid gene-expression regulation based on their TAIR annotation. Results indicate that both crys and phys modulate 'plastome-regulatory genes' with enrichment in the contribution of crys to all processes and of phys to post-transcription and transcription. Furthermore, we identified a new role for HY5 as a relevant light-signaling component in photoreceptor-based anterograde signaling leading to plastome gene regulation., (© 2020 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

The plastidial methylerythritol phosphate (MEP) pathway is an essential route for plants as the source of precursors for all plastidial isoprenoids, many of which are of medical and biotechnological importance. The MEP pathway is highly sensitive to environmental cues as many of these compounds are linked to photosynthesis and growth and light is one of the main regulatory factors. However, the mechanisms coordinating the MEP pathway with light cues are not fully understood. Here we demonstrate that by a differential direct transcriptional modulation, via the key-master integrators of light signal transduction HY5 and PIFs which target the genes that encode the rate-controlling DXS1, DXR and HDR enzymes, light imposes a direct, rapid and potentially multi-faceted response that leads to unique protein dynamics of this pathway, resulting in a significant difference in the protein levels. For DXS1, PIF1/HY5 act as a direct activation/suppression module. In contrast, DXR accumulation in response to light results from HY5 induction with minor contribution of de-repression by PIF1. Finally, HDR transcription increases in the light exclusively by suppression of the PIFs repression. This is an example of how light signaling components can differentially multi-target the initial steps of a pathway whose products branch downstream to all chloroplastic isoprenoids. These findings demonstrate the diversity and flexibility of light signaling components that optimize key biochemical pathways essential for plant growth., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

In plants, light receptors play a pivotal role in photoperiod sensing, enabling them to track seasonal progression. Photoperiod sensing arises from an interaction between the plant's endogenous circadian oscillator and external light cues. Here, we characterize the role of phytochrome A (phyA) in photoperiod sensing. Our metaanalysis of functional genomic datasets identified phyA as a principal regulator of morning-activated genes, specifically in short photoperiods. We demonstrate that PHYA expression is under the direct control of the PHYTOCHROME INTERACTING FACTOR transcription factors, PIF4 and PIF5. As a result, phyA protein accumulates during the night, especially in short photoperiods. At dawn, phyA activation by light results in a burst of gene expression, with consequences for physiological processes such as anthocyanin accumulation. The combination of complex regulation of PHYA transcript and the unique molecular properties of phyA protein make this pathway a sensitive detector of both dawn and photoperiod., Competing Interests: The authors declare no conflict of interest.

Plants coordinate their growth and development with the environment through integration of circadian clock and photosensory pathways. In Arabidopsis thaliana, rhythmic hypocotyl elongation in short days (SD) is enhanced at dawn by the basic-helix-loop-helix (bHLH) transcription factors PHYTOCHROME-INTERACTING FACTORS (PIFs) directly inducing expression of growth-related genes [1-6]. PIFs accumulate progressively during the night and are targeted for degradation by active phytochromes in the light, when growth is reduced. Although PIF proteins are also detected during the day hours [7-10], their growth-promoting activity is inhibited through unknown mechanisms. Recently, the core clock components and transcriptional repressors PSEUDO-RESPONSE REGULATORS PRR9/7/5 [11, 12], negative regulators of hypocotyl elongation [13, 14], were described to associate to G boxes [15], the DNA motifs recognized by the PIFs [16, 17], suggesting that PRR and PIF function might converge antagonistically to regulate growth. Here we report that PRR9/7/5 and PIFs physically interact and bind to the same promoter region of pre-dawn-phased, growth-related genes, and we identify the transcription factor CDF5 [18, 19] as target of this interplay. In SD, CDF5 expression is sequentially repressed from morning to dusk by PRRs and induced pre-dawn by PIFs. Consequently, CDF5 accumulates specifically at dawn, when it induces cell elongation. Our findings provide a framework for recent TIMING OF CAB EXPRESSION 1 (TOC1/PRR1) data [5, 20] and reveal that the long described circadian morning-to-midnight waves of the PRR transcriptional repressors (PRR9, PRR7, PRR5, and TOC1) [21] jointly gate PIF activity to dawn to prevent overgrowth through sequential regulation of common PIF-PRR target genes such as CDF5., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Carotenoids are photosynthetic pigments essential for the protection against excess light. During deetiolation, their production is regulated by a dynamic repression-activation module formed by PHYTOCHROME-INTERACTING FACTOR1 (PIF1) and LONG HYPOCOTYL5 (HY5). These transcription factors directly and oppositely control the expression of the gene encoding PHYTOENE SYNTHASE (PSY), the first and main rate-determining enzyme of the carotenoid pathway. Antagonistic modules also regulate the responses of deetiolated plants to vegetation proximity and shade (i.e. to the perception of far-red light-enriched light filtered through or reflected from neighboring plants). These responses, aimed to adapt to eventual shading from plant competitors, include a reduced accumulation of carotenoids. Here, we show that PIF1 and related photolabile PIFs (but not photostable PIF7) promote the shade-triggered decrease in carotenoid accumulation. While HY5 does not appear to be required for this process, other known PIF antagonists were found to modulate the expression of the Arabidopsis (Arabidopsis thaliana) PSY gene and the biosynthesis of carotenoids early after exposure to shade. In particular, PHYTOCHROME-RAPIDLY REGULATED1, a transcriptional cofactor that prevents the binding of true transcription factors to their target promoters, was found to interact with PIF1 and hence directly induce PSY expression. By contrast, a change in the levels of the transcriptional cofactor LONG HYPOCOTYL IN FAR RED1, which also binds to PIF1 and other PIFs to regulate shade-related elongation responses, did not impact PSY expression or carotenoid accumulation. Our data suggest that the fine-regulation of carotenoid biosynthesis in response to shade relies on specific modules of antagonistic transcriptional factors and cofactors., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

The ability to interpret daily and seasonal alterations in light and temperature signals is essential for plant survival. This is particularly important during seedling establishment when the phytochrome photoreceptors activate photosynthetic pigment production for photoautotrophic growth. Phytochromes accomplish this partly through the suppression of phytochrome interacting factors (PIFs), negative regulators of chlorophyll and carotenoid biosynthesis. While the bZIP transcription factor long hypocotyl 5 (HY5), a potent PIF antagonist, promotes photosynthetic pigment accumulation in response to light. Here we demonstrate that by directly targeting a common promoter cis-element (G-box), HY5 and PIFs form a dynamic activation-suppression transcriptional module responsive to light and temperature cues. This antagonistic regulatory module provides a simple, direct mechanism through which environmental change can redirect transcriptional control of genes required for photosynthesis and photoprotection. In the regulation of photopigment biosynthesis genes, HY5 and PIFs do not operate alone, but with the circadian clock. However, sudden changes in light or temperature conditions can trigger changes in HY5 and PIFs abundance that adjust the expression of common target genes to optimise photosynthetic performance and growth.

Light and temperature are arguably two of the most important signals regulating the growth and development of plants. In addition to their direct energetic effects on plant growth, light and temperature provide vital immediate and predictive cues for plants to ensure optimal development both spatially and temporally. While the majority of research to date has focused on the contribution of either light or temperature signals in isolation, it is becoming apparent that an understanding of how the two interact is essential to appreciate fully the complex and elegant ways in which plants utilize these environmental cues. This review will outline the diverse mechanisms by which light and temperature signals are integrated and will consider why such interconnected systems (as opposed to entirely separate light and temperature pathways) may be evolutionarily favourable., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Plastids provide plants with metabolic pathways that are unique among eukaryotes, including the methylerythritol 4-phosphate pathway for the production of isoprenoids essential for photosynthesis and plant growth. Here, we show that the first enzyme of the pathway, deoxyxylulose 5-phosphate synthase (DXS), interacts with the J-protein J20 in Arabidopsis thaliana. J-proteins typically act as adaptors that provide substrate specificity to heat shock protein 70 (Hsp70), a molecular chaperone. Immunoprecipitation experiments showed that J20 and DXS are found together in vivo and confirmed the presence of Hsp70 chaperones in DXS complexes. Mutants defective in J20 activity accumulated significantly increased levels of DXS protein (but no transcripts) and displayed reduced levels of DXS enzyme activity, indicating that loss of J20 function causes posttranscriptional accumulation of DXS in an inactive form. Furthermore, J20 promotes degradation of DXS following a heat shock. Together, our data indicate that J20 might identify unfolded or misfolded (damaged) forms of DXS and target them to the Hsp70 system for proper folding under normal conditions or degradation upon stress.

RNA-binding proteins (RNPs) participate in diverse processes of mRNA metabolism, and phosphorylation changes their binding properties. In spinach chloroplasts, 24RNP and 28RNP are associated with polynucleotide posphorylase forming a complex on charge of pre-mRNA 3'-end maturation. Here, we tested the hypothesis that the phosphorylation status of 24RNP and 28RNP, present in a spinach chloroplast mRNA 3'-UTR processing extract (CPE), controls the transition between petD precursor stabilization, 3'-UTR processing, and RNA degradation in vitro. The CPE processed or stabilized petD precursor depending on the ATP concentration present in an in vitro 3'-UTR processing (IVP) assay. These effects were also observed when ATP was pre-incubated and removed before the IVP assay. Moreover, a dephosphorylated (DP)-CPE degraded petD precursor and recovered 3'-UTR processing or stabilization activities in an ATP concentration dependent manner. To determine the role 24/28RNP plays in regulating these processes a 24/28RNP-depleted (Δ24/28)CPE was generated. The Δ24/28CPE degraded the petD precursor, but when it was reconstituted with recombinant non-phosphorylated (NP)-24RNP or NP-28RNP, the precursor was stabilized, whereas when Δ24/28CPE was reconstituted with phosphorylated (P)-24RNP or P-28RNP, it recovered 3'-UTR processing, indicating that 24RNP or 28RNP is needed to stabilize the precursor, have a redundant role, and their phosphorylation status regulates the transition between precursor stabilization and 3'-UTR processing. A DP-Δ24/28CPE reconstituted or not with NP-24/28RNP degraded petD precursor. Pre-incubation of DP-Δ24/28CPE with NP-24/28RNP plus 0.03 mM ATP recovered 3'-UTR processing activity, and its reconstitution with P-24/28RNP stabilized the precursor. However, pre-incubation of DP-Δ24/28CPE with 0.03 mM ATP, and further reconstitution with NP-24/28RNP or P-24/28RNP produced precursor stability instead of RNA degradation, and RNA processing instead of precursor stability, respectively. Moreover, in vitro phosphorylation of CPE showed that 24RNP, 28RNP, and other proteins may be phosphorylated. Altogether, these results reveal that phosphorylation of 24RNP, 28RNP, and other unidentified CPE proteins mediates the in vitro interplay between petD precursor stability, 3'-UTR processing, and degradation, and support the idea that protein phosphorylation plays an important role in regulating mRNA metabolism in chloroplast., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Phytochrome is a red (R)/far-red (FR) light-sensing photoreceptor that regulates various aspects of plant development. Among the members of the phytochrome family, phytochrome A (phyA) exclusively mediates atypical phytochrome responses, such as the FR high irradiance response (FR-HIR), which is elicited under prolonged FR. A proteasome-based degradation pathway rapidly eliminates active Pfr (the FR-absorbing form of phyA) under R. To elucidate the structural basis for the phyA-specific properties, we systematically constructed 16 chimeric phytochromes in which each of four parts of the phytochrome molecule, namely, the N-terminal extension plus the Per/Arnt/Sim domain (N-PAS), the cGMP phosphodiesterase/adenyl cyclase/FhlA domain (GAF), the phytochrome domain (PHY), and the entire C-terminal half, was occupied by either the phyA or phytochrome B sequence. These phytochromes were expressed in transgenic Arabidopsis thaliana to examine their physiological activities. Consequently, the phyA N-PAS sequence was shown to be necessary and sufficient to promote nuclear accumulation under FR, whereas the phyA sequence in PHY was additionally required to exhibit FR-HIR. Furthermore, the phyA sequence in PHY alone substantially increased the light sensitivity to R. In addition, the GAF phyA sequence was important for rapid Pfr degradation. In summary, distinct structural modules, each of which confers different properties to phyA, are assembled on the phyA molecule.

Phytochrome regulates various physiological and developmental processes throughout the life cycle of plants. Among the members of the phytochrome family, phytochrome A (phyA) exclusively mediates the far-red light high irradiance response (FR-HIR), which is elicited by continuous far-red light. In FR-HIR, nuclear accumulation of phyA, which precedes physiological responses, is proposed to be required for the response. In contrast to FR, red light induces rapid degradation of phyA to suppress undesirable long-term photomorphogenic responses of phyA. In the present study, we compared biological activities between phyA derivatives to which either a nuclear localization (NLS) or export (NES) signal sequence was attached. Those derivatives were expressed under the control of the PHYA promoter in the Arabidopsis phyA mutant. Detailed microscopic observation revealed that the phyA-green fluorescent protein (GFP) without a signal sequence is localized exclusively in the cytoplasm in darkness. Rapid nuclear entry was observed after exposure to both red and far-red light. Interestingly, both phyA-GFP-NLS and phyA-GFP-NES were rapidly degraded under continuous red light. Furthermore, a proteasome inhibitor delayed degradation equally under these two conditions. Therefore, similar mechanisms for phyA degradation may exist in the cytoplasm and nucleus. As expected from previous reports, phyA-GFP-NLS, but not phyA-GFP-NES, mediated different aspects of FR-HIR, such as inhibition of hypocotyl elongation and rapid induction of gene expression, confirming that phyA nuclear localization is required for FR-HIR. In addition, a detailed time course analysis of phyA-GFP and phyA-GFP-NLS responses revealed that they were almost indistinguishable, raising the question of the physiological relevance of phyA cytoplasmic retention in darkness.

Carotenoids are key for plants to optimize carbon fixing using the energy of sunlight. They contribute to light harvesting but also channel energy away from chlorophylls to protect the photosynthetic apparatus from excess light. Phytochrome-mediated light signals are major cues regulating carotenoid biosynthesis in plants, but we still lack fundamental knowledge on the components of this signaling pathway. Here we show that phytochrome-interacting factor 1 (PIF1) and other transcription factors of the phytochrome-interacting factor (PIF) family down-regulate the accumulation of carotenoids by specifically repressing the gene encoding phytoene synthase (PSY), the main rate-determining enzyme of the pathway. Both in vitro and in vivo evidence demonstrate that PIF1 directly binds to the promoter of the PSY gene, and that this binding results in repression of PSY expression. Light-triggered degradation of PIFs after interaction with photoactivated phytochromes during deetiolation results in a rapid derepression of PSY gene expression and a burst in the production of carotenoids in coordination with chlorophyll biosynthesis and chloroplast development for an optimal transition to photosynthetic metabolism. Our results also suggest a role for PIF1 and other PIFs in transducing light signals to regulate PSY gene expression and carotenoid accumulation during daily cycles of light and dark in mature plants.

In previous time-resolved microarray-based expression profiling, we identified 32 genes encoding putative transcription factors, signaling components, and unknown proteins that are rapidly and robustly induced by phytochrome (phy)-mediated light signals. Postulating that they are the most likely to be direct targets of phy signaling and to function in the primary phy regulatory circuitry, we examined the impact of targeted mutations in these genes on the phy-induced seedling deetiolation process in Arabidopsis thaliana. Using light-imposed concomitant inhibition of hypocotyl and stimulation of cotyledon growth as diagnostic criteria for normal deetiolation, we identified three major mutant response categories. Seven (22%) lines displayed statistically significant, reciprocal, aberrant photoresponsiveness in the two organs, suggesting disruption of normal deetiolation; 13 (41%) lines displayed significant defects either unidirectionally in both organs or in hypocotyls only, suggesting global effects not directly related to photomorphogenic signaling; and 12 (37%) lines displayed no significant difference in photoresponsiveness from the wild type. Potential reasons for the high proportion of rapidly light-responsive genes apparently unnecessary for the deetiolation phenotype are discussed. One of the seven disrupted genes displaying a significant mutant phenotype, the basic helix-loop-helix factor-encoding PHYTOCHROME-INTERACTING FACTOR3-LIKE1 gene, was found to be necessary for rapid light-induced expression of the photomorphogenesis- and circadian-related PSEUDO-RESPONSE REGULATOR9 gene, indicating a regulatory function in the early phy-induced transcriptional network.

Previous genetic analysis identified a component, RED1, that is required for normal de-etiolation of Arabidopsis thaliana (L.) Heynh. seedlings in continuous red light (Rc). red1 mutant seedlings exhibit elongated hypocotyls and reduced cotyledon size specifically in Rc and not in continuous far-red light (FRc). Here, we show that red1 is allelic to sur2 and atr4, and is defective in the cytochrome P450 CYP83B1, an enzyme required for normal auxin homeostasis. Two alleles of atr4, like red1, exhibit increased hypocotyl elongation and reduced cotyledon expansion in Rc but not in FRc. We further show that CYP83B1 transcript levels are elevated specifically in Rc-grown seedlings when compared with seedlings grown in darkness or FRc. Hence, the Rc-specific phenotype of the red1 mutant may indicate that Rc-induction of the CYP83B1 transcript is necessary for normal seedling de-etiolation in the wild type., (Copyright 2004 Springer-Verlag)

The basic/helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that bind as dimers to specific DNA target sites and that have been well characterized in nonplant eukaryotes as important regulatory components in diverse biological processes. Based on evidence that the bHLH protein PIF3 is a direct phytochrome reaction partner in the photoreceptor's signaling network, we have undertaken a comprehensive computational analysis of the Arabidopsis genome sequence databases to define the scope and features of the bHLH family. Using a set of criteria derived from a previously defined consensus motif, we identified 147 bHLH protein-encoding genes, making this one of the largest transcription factor families in Arabidopsis. Phylogenetic analysis of the bHLH domain sequences permits classification of these genes into 21 subfamilies. The evolutionary and potential functional relationships implied by this analysis are supported by other criteria, including the chromosomal distribution of these genes relative to duplicated genome segments, the conservation of variant exon/intron structural patterns, and the predicted DNA binding activities within subfamilies. Considerable diversity in DNA binding site specificity among family members is predicted, and marked divergence in protein sequence outside of the conserved bHLH domain is observed. Together with the established propensity of bHLH factors to engage in varying degrees of homodimerization and heterodimerization, these observations suggest that the Arabidopsis bHLH proteins have the potential to participate in an extensive set of combinatorial interactions, endowing them with the capacity to be involved in the regulation of a multiplicity of transcriptional programs. We provide evidence from yeast two-hybrid and in vitro binding assays that two related phytochrome-interacting members in the Arabidopsis family, PIF3 and PIF4, can form both homodimers and heterodimers and that all three dimeric configurations can bind specifically to the G-box DNA sequence motif CACGTG. These data are consistent, in principle, with the operation of this combinatorial mechanism in Arabidopsis.

Prenylation is necessary for association of the petunia calmodulin CaM53 with the plasma membrane. To determine whether post-prenylation processing of the protein was also required for plasma membrane targeting, we studied the subcellular localization of a GFP-labelled CaM53 reporter in yeast and plant cells. Blocking of carboxyl-methylation of prenylated proteins either by a specific inhibitor or in mutant yeast cells resulted in localization of green fluorescence to what appears to be the endomembrane system, in contrast with the plasma membrane localization observed in control cells. We show that a prenyl-cysteine methyltransferase (PCM) activity that carboxyl-methylates prenylated CaM53 also exists in plant cells, and that it is required for efficient plasma membrane targeting. We also report an Arabidopsis gene with homology to PCM and demonstrate that it encodes a protein with PCM activity that localizes to the endomembrane system of plant cells, similar to prenylated but unmethylated CaM53. Together, our data suggest that, following prenylation, CaM53 is probably associated with the endomembrane system, where a PCM activity methylates the prenylated protein prior to targeting it to its final destination in the plasma membrane.

The Arabidopsis MADS box transcription factor APETALA1 (AP1) was identified as a substrate for farnesyltransferase and shown to be farnesylated efficiently both in vitro and in vivo. AP1 regulates the transition from inflorescence shoot to floral meristems and the development of sepals and petals. AP1 fused to green fluorescent protein (GFP) retained transcription factor activity and directed the expected terminal flower phenotype when ectopically expressed in transgenic Arabidopsis. However, ap1mS, a farnesyl cysteine-acceptor mutant of AP1, as well as the GFP-ap1mS fusion protein failed to direct the development of compound terminal flowers but instead induced novel phenotypes when ectopically expressed in Arabidopsis. Similarly, compound terminal flowers did not develop in era1-2 transformants that ectopically expressed AP1. Together, the results demonstrate that AP1 is a target of farnesyltransferase and suggest that farnesylation alters the function and perhaps specificity of the transcription factor.