Genomic overview of INA-induced NPR1 targeting and transcriptional cascades in Arabidopsis.

The phytohormone salicylic acid (SA) triggers transcriptional reprogramming that leads to SA-induced immunity in plants. NPR1 is an SA receptor and master transcriptional regulator in SA-triggered transcriptional reprogramming. Despite the indispensable role of NPR1, genome-wide direct targets of NPR1 specific to SA signaling have not been identified. Here, we report INA (functional SA analog)-specific genome-wide targets of Arabidopsis NPR1 in plants expressing GFP-fused NPR1 under its native promoter. Analyses of NPR1-dependently expressed direct NPR1 targets revealed that NPR1 primarily activates genes encoding transcription factors upon INA treatment, triggering transcriptional cascades required for INA-induced transcriptional reprogramming and immunity. We identified genome-wide targets of a histone acetyltransferase, HAC1, including hundreds of co-targets shared with NPR1, and showed that NPR1 and HAC1 regulate INA-induced histone acetylation and expression of a subset of the co-targets. Genomic NPR1 targeting was principally mediated by TGACG-motif binding protein (TGA) transcription factors. Furthermore, a group of NPR1 targets mostly encoding transcriptional regulators was already bound to NPR1 in the basal state and showed more rapid and robust induction than other NPR1 targets upon SA signaling. Thus, our study unveils genome-wide NPR1 targeting, its role in transcriptional reprogramming, and the cooperativity between NPR1, HAC1, and TGAs in INA-induced immunity.

Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in Arabidopsis.

Shoot regeneration can be achieved in vitro through a two-step process involving the acquisition of pluripotency on callus-induction media (CIM) and the formation of shoots on shoot-induction media. Although the induction of root-meristem genes in callus has been noted recently, the mechanisms underlying their induction and their roles in de novo shoot regeneration remain unanswered. Here, we show that the histone acetyltransferase HAG1/AtGCN5 is essential for de novo shoot regeneration. In developing callus, it catalyzes histone acetylation at several root-meristem gene loci including WOX5, WOX14, SCR, PLT1, and PLT2, providing an epigenetic platform for their transcriptional activation. In turn, we demonstrate that the transcription factors encoded by these loci act as key potency factors conferring regeneration potential to callus and establishing competence for de novo shoot regeneration. Thus, our study uncovers key epigenetic and potency factors regulating plant-cell pluripotency. These factors might be useful in reprogramming lineage-specified plant cells to pluripotency.

De Novo Shoot Regeneration Controlled by HEN1 and TCP3/4 in Arabidopsis.

Plants have the ability to regenerate whole plant body parts, including shoots and roots, in vitro from callus derived from a variety of tissues. However, the underlying mechanisms for this de novo organogenesis, which is based on the totipotency of callus cells, are poorly understood. Here, we report that a microRNA (miRNA)-mediated posttranscriptional regulation plays an important role in de novo shoot regeneration. We found that mutations in HUA ENHANCER 1 (HEN1), a gene encoding a small RNA methyltransferase, cause cytokinin-related defects in de novo shoot regeneration. A hen1 mutation caused a large reduction in the miRNA319 (miR319) level and a subsequent increase in its known target (TCP3 and TCP4) transcript levels. TCP transcription factors redundantly inhibited shoot regeneration and directly activated the expression of a negative regulator of cytokinin response ARABIDOPSIS THALIANA RESPONSE REGULATOR 16 (ARR16). A tcp4 mutation at least partly rescued the shoot-regeneration defect and derepression of ARR16 in hen1. These findings demonstrate that the miR319-TCP3/4-ARR16 axis controls de novo shoot regeneration by modulating cytokinin responses.

Salicylic acid-induced transcriptional reprogramming by the HAC-NPR1-TGA histone acetyltransferase complex in Arabidopsis.

Plant immunity depends on massive expression of pathogenesis-related genes (PRs) whose transcription is de-repressed by pathogen-induced signals. Salicylic acid (SA) acts as a major signaling molecule in plant immunity and systemic acquired resistance triggered by bacterial or viral pathogens. SA signal results in the activation of the master immune regulator, Nonexpressor of pathogenesis-related genes 1 (NPR1), which is thought to be recruited by transcription factors such as TGAs to numerous downstream PRs. Despite its key role in SA-triggered immunity, the biochemical nature of the transcriptional coactivator function of NPR1 and the massive transcriptional reprogramming induced by it remain obscure. Here we demonstrate that the CBP/p300-family histone acetyltransferases, HACs and NPR1 are both essential to develop SA-triggered immunity and PR induction. Indeed HACs and NPR1 form a coactivator complex and are recruited to PR chromatin through TGAs upon SA signal, and finally the HAC-NPR1-TGA complex activates PR transcription by histone acetylation-mediated epigenetic reprogramming. Thus, our study reveals a molecular mechanism of NPR1-mediated transcriptional reprogramming and a key epigenetic aspect of the central immune system in plants.

Epigenetic control of juvenile-to-adult phase transition by the Arabidopsis SAGA-like complex.

During growth and development, plants undergo a series of phase transitions from the juvenile-to-adult vegetative phase to the reproductive phase. In Arabidopsis, vegetative phase transitions and flowering are regulated by SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) factors. SPL mRNAs are post-transcriptionally regulated by miR156 in an age-dependent manner; however, the role of other mechanisms in this process is not known. In this study, we demonstrate that the HAG1/GCN5- and PRZ1/ADA2b-containing SAGA-like histone acetyltransferase (HAT) complex directly controls the transcription of SPLs and determines the time for juvenile-to-adult phase transition. Thus, epigenetic control by the SAGA-like HAT complex determines the transcriptional output of SPLs, which might be a prerequisite for the subsequent post-transcriptional regulation by miR156. Importantly, this epigenetic control mechanism is also crucial for miR156-independent induction of SPLs and acceleration of phase transition by light and photoperiod or during post-embryonic growth.

Repression of flowering under a noninductive photoperiod by the HDA9-AGL19-FT module in Arabidopsis.

Posttranslational acetylation of histones is reversibly regulated by histone deacetylases (HDACs). Despite the evident significance of HDACs in Arabidopsis development, the biological roles and underlying molecular mechanisms of many HDACs are yet to be elucidated. By a reverse-genetic approach, we isolated an hda9 mutant and performed phenotypic analyses on it. In order to address the role of HDA9 in flowering, genetic, molecular, and biochemical approaches were employed. hda9 flowered early under noninductive short-day (SD) conditions and had increased expression of the floral integrator FLOWERING LOCUS T (FT) and the floral activator AGAMOUS-LIKE 19 (AGL19) compared with the wild-type. The hda9 mutation increased histone acetylation and RNA polymerase II occupancy at AGL19 but not at FT during active transcription, and the HDA9 protein directly targeted AGL19. AGL19 expression was higher under SD than under inductive long-day (LD) conditions, and an AGL19 overexpression caused a strong up-regulation of FT. A genetic analysis showed that an agl19 mutation is epistatic to the hda9 mutation, masking both the early flowering and the increased FT expression of hda9. Taken together, our data indicate that HDA9 prevents precocious flowering under SD conditions by curbing the hyperactivation of AGL19, an upstream activator of FT, through resetting the local chromatin environment.

Growth habit determination by the balance of histone methylation activities in Arabidopsis.

In Arabidopsis, the rapid-flowering summer-annual versus the vernalization-requiring winter-annual growth habit is determined by natural variation in FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). However, the biochemical basis of how FRI confers a winter-annual habit remains elusive. Here, we show that FRI elevates FLC expression by enhancement of histone methyltransferase (HMT) activity. EARLY FLOWERING IN SHORT DAYS (EFS), which is essential for FRI function, is demonstrated to be a novel dual substrate (histone H3 lysine 4 (H3K4) and H3K36)-specific HMT. FRI is recruited into FLC chromatin through EFS and in turn enhances EFS activity and engages additional HMTs. At FLC, the HMT activity of EFS is balanced by the H3K4/H3K36- and H3K4-specific histone demethylase (HDM) activities of autonomous-pathway components, RELATIVE OF EARLY FLOWERING 6 and FLOWERING LOCUS D, respectively. Loss of HDM activity in summer annuals results in dominant HMT activity, leading to conversion to a winter-annual habit in the absence of FRI. Thus, our study provides a model of how growth habit is determined through the balance of the H3K4/H3K36-specific HMT and HDM activities.

HDA19 is required for the repression of salicylic acid biosynthesis and salicylic acid-mediated defense responses in Arabidopsis.

To cope with a lifetime of exposure to a variety of pathogens, plants have developed exquisite and refined defense mechanisms that vary depending on the type of attacking pathogen. Defense-associated transcriptional reprogramming is a central part of plant defense mechanisms. Chromatin modification has recently been shown to be another layer of regulation for plant defense mechanisms. Here, we show that the RPD3/HDA1-class histone deacetylase HDA19 is involved in the repression of salicylic acid (SA)-mediated defense responses in Arabidopsis. Loss of HDA19 activity increased SA content and increased the expression of a group of genes required for accumulation of SA as well as pathogenesis related (PR) genes, resulting in enhanced resistance to Pseudomonas syringae. We found that HDA19 directly associates with and deacetylates histones at the PR1 and PR2 promoters. Thus, our study shows that HDA19, by modifying chromatin to a repressive state, ensures low basal expression of defense genes, such as PR1, under unchallenged conditions, as well as their proper induction without overstimulation during defense responses to pathogen attacks. Thus, the role of HDA19 might be critical in preventing unnecessary activation and self-destructive overstimulation of defense responses, allowing successful growth and development.

Negative evidence on the transgenerational inheritance of defense priming in Arabidopsis thaliana.

Defense priming allows plants to enhance their immune responses to subsequent pathogen challenges. Recent reports suggested that acquired resistances in parental generation can be inherited into descendants. Although epigenetic mechanisms are plausible tools enabling the transmission of information or phenotypic traits induced by environmental cues across generations, the mechanism for the transgenerational inheritance of defense priming in plants has yet to be elucidated. With the initial aim to elucidate an epigenetic mechanism for the defense priming in plants, we reassessed the transgenerational inheritance of plant defense, however, could not observe any evidence supporting it. By using the same dipping method with previous reports, Arabidopsis was exposed repeatedly to Pseudomonas syringae pv tomato DC3000 (Pst DC3000) during vegetative or reproductive stages. Irrespective of the developmental stages of parental plants that received pathogen infection, the descendants did not exhibit primed resistance phenotypes, defense marker gene (PR1) expression, or elevated histone acetylation within PR1 chromatin. In assays using the pressure-infiltration method for infection, we obtained the same results as above. Thus, our results suggest that the previous observations on the transgenerational inheritance of defense priming in plants should be more extensively and carefully reassessed. [BMB Reports 2022; 55(7): 342-347].

Identification of regulators required for the reactivation of FLOWERING LOCUS C during Arabidopsis reproduction.

FLOWERING LOCUS C (FLC) is a central floral repressor for the determination of flowering time in Arabidopsis. FLC expression is reactivated upon fertilization and regulated during seed development to ensure the appropriate floral behavior; however, the molecular mechanism for this process is largely unknown. Here, we report the identification of crucial regulators for FLC reactivation during embryogenesis by analyzing FLC::GUS and endogenous FLC expression. We newly define that the full reactivation of FLC requires a FRIGIDA (FRI)-containing protein complex throughout embryogenesis. Mutations in EARLY FLOWERING 7 (ELF7) and VERNALIZATION INDEPENDENCE4 (VIP4) showed severe defects in the reactivation of FLC transcription, suggesting that both of the genes, Arabidopsis homologs of the members of the yeast RNA polymerase II-associated factor 1 (Paf1) complex, are indispensable for FLC reactivation. actin-related protein 6 (arp6), arabidopsis trithorax 1 (atx1), arabidopsis trithorax-related 7 (atxr7), and atx1 atxr7 double mutants also caused the downregulation of FLC during seed development, but the defects were less severe than those in mutants for the FRI- and Paf1-complexes. These results suggest that the ARP6-containing Swr1-complex and FLC-specific histone methyltransferases, ATX1 and ATXR7, have relatively partial roles in FLC reactivation. In contrast to the roles of the histone modifiers, factors in the DNA methylation pathway and biogenesis of small RNAs are not involved in FLC regulation during reproduction. Taken together, our results demonstrate that adjustment by select FLC activators is critical for the re-establishment of an FLC expression state after fertilization to ensure competence for optimal flowering in the next generation.

Resetting and regulation of Flowering Locus C expression during Arabidopsis reproductive development.

The epigenetic regulation of the floral repressor Flowering Locus C (FLC) is one of the critical factors that determine flowering time in Arabidopsis thaliana. Although many FLC regulators, and their effects on FLC chromatin, have been extensively studied, the epigenetic resetting of FLC has not yet been thoroughly characterized. Here, we investigate the FLC expression during gametogenesis and embryogenesis using FLC::GUS transgenic plants and RNA analysis. Regardless of the epigenetic state in adult plants, FLC expression disappeared in gametophytes. Subsequently, FLC expression was reactivated after fertilization in embryos, but not in the endosperm. Both parental alleles contributed equally to the expression of FLC in embryos. Surprisingly, the reactivation of FLC in early embryos was independent of FRIGIDA (FRI) and SUPPRESSOR OF FRIGIDA 4 (SUF4) activities. Instead, FRI, SUF4 and autonomous-pathway genes determined the level of FLC expression only in late embryogenesis. Many FLC regulators exhibited expression patterns similar to that of FLC, indicating potential roles in FLC reprogramming. An FVE mutation caused ectopic expression of FLC in the endosperm. A mutation in PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 caused defects in FLC reactivation in early embryogenesis, and maintenance of full FLC expression in late embryogenesis. We also show that the polycomb group complex components, Fertilization-Independent endosperm and MEDEA, which mediate epigenetic regulation in seeds, are not relevant for FLC reprogramming. Based on our results, we propose that FLC reprogramming is composed of three phases: (i) repression in gametogenesis, (ii) reactivation in early embryogenesis and (iii) maintenance in late embryogenesis.

Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1.

Many aspects of plant growth and development are dependent on the flow of the hormone auxin down the plant from the growing shoot tip where it is synthesized. The direction of auxin transport in stems is believed to result from the basal localization within cells of the PIN1 membrane protein, which controls the efflux of the auxin anion. Mutations in two genes homologous to those encoding the P-glycoprotein ABC transporters that are especially abundant in multidrug-resistant tumour cells in animals were recently shown to block polar auxin transport in the hypocotyls of Arabidopsis seedlings. Here we show that the mdr mutants display faster and greater gravitropism and enhanced phototropism instead of the impaired curvature development expected in mutants lacking polar auxin transport. We find that these phenotypes result from a disruption of the normal accumulation of PIN1 protein along the basal end of hypocotyl cells associated with basipetal auxin flow. Lateral auxin conductance becomes relatively larger as a result, enhancing the growth differentials responsible for tropic responses.

Control of seed germination by light-induced histone arginine demethylation activity.

For optimal survival, various environmental and endogenous factors should be monitored to determine the appropriate timing for seed germination. Light is a major environmental factor affecting seed germination, which is perceived by phytochromes. The light-dependent activation of phytochrome B (PHYB) modulates abscisic acid and gibberellic acid signaling and metabolism. Thus far, several negative regulators of seed germination that act when PHYB is inactive have been reported. However, neither positive regulators of seed germination downstream of PHYB nor a direct mechanism for regulation of the hormone levels has been elucidated. Here, we show that the histone arginine demethylases, JMJ20 and JMJ22, act redundantly as positive regulators of seed germination. When PHYB is inactive, JMJ20/JMJ22 are directly repressed by the zinc-finger protein SOMNUS. However, upon PHYB activation, JMJ20/JMJ22 are derepressed, resulting in increased gibberellic acid levels through the removal of repressive histone arginine methylations at GA3ox1/GA3ox2, which in turn promotes seed germination.

The RNA binding protein ELF9 directly reduces SUPPRESSOR OF OVEREXPRESSION OF CO1 transcript levels in arabidopsis, possibly via nonsense-mediated mRNA decay.

SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) is regulated by a complex transcriptional regulatory network that allows for the integration of multiple floral regulatory inputs from photoperiods, gibberellin, and FLOWERING LOCUS C. However, the posttranscriptional regulation of SOC1 has not been explored. Here, we report that EARLY FLOWERING9 (ELF9), an Arabidopsis thaliana RNA binding protein, directly targets the SOC1 transcript and reduces SOC1 mRNA levels, possibly through a nonsense-mediated mRNA decay (NMD) mechanism, which leads to the degradation of abnormal transcripts with premature translation termination codons (PTCs). The fully spliced SOC1 transcript is upregulated in elf9 mutants as well as in mutants of NMD core components. Furthermore, a partially spliced SOC1 transcript containing a PTC is upregulated more significantly than the fully spliced transcript in elf9 in an ecotype-dependent manner. A Myc-tagged ELF9 protein (MycELF9) directly binds to the partially spliced SOC1 transcript. Previously known NMD target transcripts of Arabidopsis are also upregulated in elf9 and recognized directly by MycELF9. SOC1 transcript levels are also increased by the inhibition of translational activity of the ribosome. Thus, the SOC1 transcript is one of the direct targets of ELF9, which appears to be involved in NMD-dependent mRNA quality control in Arabidopsis.

Temporal and spatial expression patterns of nine Arabidopsis genes encoding Jumonji C-domain proteins.

Diverse posttranslational modifications of histones, such as acetylation and methylation, play important roles in controlling gene expression. Histone methylation in particular is involved in a broad range of biological processes, including heterochromatin formation, X-chromosome inactivation, genomic imprinting, and transcriptional regulation. Recently, it has been demonstrated that proteins containing the Jumonji (Jmj) C domain can demethylate histones. In Arabidopsis, twenty-one genes encode JmjC domain-containing proteins, which can be clustered into five clades. To address the biological roles of the Arabidopsis genes encoding JmjC-domain proteins, we analyzed the temporal and spatial expression patterns of nine genes. RT-PCR analyses indicate all nine Arabidopsis thaliana Jmj (AtJmj) genes studied are actively expressed in various tissues. Furthermore, studies of transgenic plants harboring AtJmj::beta-glucuronidase fusion constructs reveal that these nine AtJmj genes are expressed in a developmentally and spatially regulated manner.

Repression of FLOWERING LOCUS T chromatin by functionally redundant histone H3 lysine 4 demethylases in Arabidopsis.

FLOWERING LOCUS T (FT) plays a key role as a mobile floral induction signal that initiates the floral transition. Therefore, precise control of FT expression is critical for the reproductive success of flowering plants. Coexistence of bivalent histone H3 lysine 27 trimethylation (H3K27me3) and H3K4me3 marks at the FT locus and the role of H3K27me3 as a strong FT repression mechanism in Arabidopsis have been reported. However, the role of an active mark, H3K4me3, in FT regulation has not been addressed, nor have the components affecting this mark been identified. Mutations in Arabidopsis thaliana Jumonji4 (AtJmj4) and EARLY FLOWERING6 (ELF6), two Arabidopsis genes encoding Jumonji (Jmj) family proteins, caused FT-dependent, additive early flowering correlated with increased expression of FT mRNA and increased H3K4me3 levels within FT chromatin. Purified recombinant AtJmj4 protein possesses specific demethylase activity for mono-, di-, and trimethylated H3K4. Tagged AtJmj4 and ELF6 proteins associate directly with the FT transcription initiation region, a region where the H3K4me3 levels were increased most significantly in the mutants. Thus, our study demonstrates the roles of AtJmj4 and ELF6 as H3K4 demethylases directly repressing FT chromatin and preventing precocious flowering in Arabidopsis.

Arabidopsis homologs of components of the SWR1 complex regulate flowering and plant development.

The SWR1 complex (SWR1C) in yeast catalyzes the replacement of nucleosomal H2A with the H2AZ variant, which ensures full activation of underlying genes. We compared the phenotype of mutants in the homologs of SWR1C components in Arabidopsis thaliana. Mutations in Arabidopsis SWC6 (AtSWC6), SUPPRESSOR OF FRIGIDA 3 (SUF3) and PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 (PIE1), homologs of SWC6, ARP6 and SWR1, respectively, caused similar developmental defects, including leaf serration, weak apical dominance, flowers with extra petals and early flowering by reduction in expression of FLOWERING LOCUS C (FLC), a strong floral repressor. Chromatin immunoprecipitation assays showed that AtSWC6 and SUF3 bind to the proximal region of the FLC promoter, and protoplast transfection assays showed that AtSWC6 colocalizes with SUF3. Protein interaction analyses suggested the formation of a complex between PIE1, SUF3, AtSWC6 and AtSWC2. In addition, H2AZ, a substrate of SWR1C, interacts with both PIE1 and AtSWC2. Finally, knockdown of the H2AZ genes by RNA interference or artificial microRNA caused a phenotype similar to that of atswc6 or suf3. Our results strongly support the presence of an SWR1C-like complex in Arabidopsis that ensures proper development, including floral repression through full activation of FLC.

Role of plant CBP/p300-like genes in the regulation of flowering time.

CREB-binding protein (CBP) and its homolog p300 possess histone acetyltransferase activity and function as key transcriptional co-activators in the regulation of gene expression that controls differentiation and development in animals. However, the role of CBP/p300-like genes in plants has not yet been elucidated. Here, we show that Arabidopsis CBP/p300-like genes promote flowering by affecting the expression of a major floral repressor FLOWERING LOCUS C (FLC). Although animal CBP and p300 generally function as co-activators, Arabidopsis CBP/p300-like proteins are required for the negative regulation of FLC. This CBP/p300-mediated FLC repression may involve reversible protein acetylation independent of histone modification within FLC chromatin.

Multiple heme oxygenase family members contribute to the biosynthesis of the phytochrome chromophore in Arabidopsis.

The oxidative cleavage of heme by heme oxygenases (HOs) to form biliverdin IXalpha (BV) is the committed step in the biosynthesis of the phytochrome (phy) chromophore and thus essential for proper photomorphogenesis in plants. Arabidopsis (Arabidopsis thaliana) contains four possible HO genes (HY1, HO2-4). Genetic analysis of the HY1 locus showed previously that it is the major source of BV with hy1 mutant plants displaying long hypocotyls and decreased chlorophyll accumulation consistent with a substantial deficiency in photochemically active phys. More recent analysis of HO2 suggested that it also plays a role in phy assembly and photomorphogenesis but the ho2 mutant phenotype is more subtle than that of hy1 mutants. Here, we define the functions of HO3 and HO4 in Arabidopsis. Like HY1, the HO3 and HO4 proteins have the capacity to synthesize BV from heme. Through a phenotypic analysis of T-DNA insertion mutants affecting HO3 and HO4 in combination with mutants affecting HY1 or HO2, we demonstrate that both of the encoded proteins also have roles in photomorphogenesis, especially in the absence of HY1. Disruption of HO3 and HO4 in the hy1 background further desensitizes seedlings to red and far-red light and accelerates flowering time, with the triple mutant strongly resembling seedlings deficient in the synthesis of multiple phy apoproteins. The hy1/ho3/ho4 mutant can be rescued phenotypically and for the accumulation of holo-phy by feeding seedlings BV. Taken together, we conclude that multiple members of the Arabidopsis HO family are important for synthesizing the bilin chromophore used to assemble photochemically active phys.

Genetic and molecular analysis of phytochromes from the filamentous fungus Neurospora crassa.

Phytochromes (Phys) comprise a superfamily of red-/far-red-light-sensing proteins. Whereas higher-plant Phys that control numerous growth and developmental processes have been well described, the biochemical characteristics and functions of the microbial forms are largely unknown. Here, we describe analyses of the expression, regulation, and activities of two Phys in the filamentous fungus Neurospora crassa. In addition to containing the signature N-terminal domain predicted to covalently associate with a bilin chromophore, PHY-1 and PHY-2 contain C-terminal histidine kinase and response regulator motifs, implying that they function as hybrid two-component sensor kinases activated by light. A bacterially expressed N-terminal fragment of PHY-2 covalently bound either biliverdin or phycocyanobilin in vitro, with the resulting holoprotein displaying red-/far-red-light photochromic absorption spectra and a photocycle in vitro. cDNA analysis of phy-1 and phy-2 revealed two splice isoforms for each gene. The levels of the phy transcripts are not regulated by light, but the abundance of the phy-1 mRNAs is under the control of the circadian clock. Phosphorylated and unphosphorylated forms of PHY-1 were detected; both species were found exclusively in the cytoplasm, with their relative abundances unaffected by light. Strains containing deletions of phy-1 and phy-2, either singly or in tandem, were not compromised in any known photoresponses in Neurospora, leaving their function(s) unclear.