Cab Gene Expression Research Articles

Correction: Transcriptome analysis of tomato (Solanum lycopersicum L.) shoots reveals a crosstalk between auxin and strigolactone.

[This corrects the article DOI: 10.1371/journal.pone.0201124.].

Transcriptome analysis of tomato (Solanum lycopersicum L.) shoots reveals a crosstalk between auxin and strigolactone.

Auxin and strigolactone (SL) are two important phytohormones involved in shoot branching and morphology. Tomato (Solanum lycopersicum L.), a member of the Solanaceae family, is one of the most popular food crops with high economic value in the world. To seek a better understanding of the responses to exogenous hormones, transcriptome analyses of the tomato shoots treated with exogenous auxin and SL, separately or together, were performed. A total of 2326, 260 and 1379 differential expressed genes (DEGs) were identified under the IAA, GR24 and IAA+GR24 treatments, respectively. Network analysis pointed out two enriched interaction clusters, including “ethylene biosynthesis” and “photosynthesis”. Several ethylene biosynthesis and metabolism-related genes were up-regulated under both IAA and IAA+GR24 treatments, suggesting their involvement in the regulation of ethylene biosynthesis. Besides, auxin-SLs-triggered the expression of several CAB genes may lead to systemic increases in the induction of photosynthesis. Several auxin-activated metabolic pathways could be reduced by the GR24 treatment, indicated that the crosstalk between auxin and SLs may be involved in the metabolic regulation of tomato. Further analysis showed that SLs affect the responses of tomato shoots to auxin by inducing the expression of a series of auxin downstream genes. On the other hand, auxin regulated the biosynthesis of SLs by affecting the genes in the “Carotenoid biosynthesis” pathway. Our data will give us an opportunity to reveal the crosstalk between auxin and SLs in the shoots of tomato.

Melatonin is involved in skotomorphogenesis by regulating brassinosteroid biosynthesis in rice plants.

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis catalyzing the conversion of serotonin into N-acetylserotonin. In plants, SNAT is encoded by 2 isogenes of which SNAT1 is constitutively expressed and its overexpression confers increased yield in rice. However, the role of SNAT2 remains to be clarified. In contrast to SNAT1, the diurnal rhythm of SNAT2 mRNA expression peaks at night. In this study, transgenic rice plants in which SNAT2 expression were suppressed by RNAi technology showed a decrease in melatonin and a dwarf phenotype with erect leaves, reminiscent of brassinosteroids (BR)-deficient mutants. Of note, the dwarf phenotype was dependent on the presence of dark, suggesting that melatonin is involved in dark growth (skotomorphogenesis). In support of this suggestion, SNAT2 RNAi lines exhibited photomorphogenic phenotypes such as inhibition of internodes and increased expression of light-inducible CAB genes in the dark. The causative gene for the melatonin-mediated BR biosynthetic gene was DWARF4, a rate-limiting BR biosynthetic gene. Exogenous melatonin treatment induced several BR biosynthetic genes, including DWARF4, D11, and RAVL1. As expected from the erect leaves, the SNAT2 RNAi lines produced less BR than the wild type. Our results show for the first time that melatonin is a positive regulator of dark growth or shade outgrowth by regulating BR biosynthesis in plants.

Light Restriction Delays Leaf Senescence in Winter Oilseed Rape (Brassica napus L.)

Oilseed rape (Brassica napus L.) is a crop with a complex aerial architecture that can cause self-shading leading to a vertical light gradient over the foliage. Mutual shading between neighboring plants at a high sowing density also results in an alteration of photosynthetically active radiation (PAR) absorption by lower leaves. The aim of this study was to analyze the impact that light restriction on lower leaves has on shoot architecture, biomass production and allocation, nitrogen (N) fluxes, and progression of sequential senescence. Field-grown plants were collected at the end of the vegetative rest period and grown in hydroponic conditions until pod maturity. A shading treatment corresponding to a 43.4 % reduction of PAR was applied at the early flowering stage. N uptake and fluxes of N allocation and remobilization were determined by supplying K15NO3 in the nutrient solution. Photosynthesis and expression of SAG12 and Cab genes (indicators of leaf senescence progression) were also analyzed on different leaf ranks. The results showed that shading enhanced leaf development on the main stem and ramifications to optimize light capture. The expression pattern of the SAG12/Cab molecular indicator suggested a delay in leaf senescence that allowed leaf life span to be extended resulting in a more efficient leaf compound remobilization, with lower N residual contents in fallen leaves under shading. N uptake increased and N remobilization fluxes were enhanced from source organs (leaves and stem) toward sink organs (flowers). Profuse branching and late senescing varieties would be of interest for further selection programs under high sowing densities.

Light-dependent regulation of chlorophyll b biosynthesis in chlorophyllide a oxygenase overexpressing tobacco plants

Chlorophyllide a oxygenase (CAO) that converts chlorophyllide a to chlorophyllide b was overexpressed in tobacco to increase chlorophyll (Chl) b biosynthesis and alter the Chl a/b ratio. Transgenic plants along with their wild-type cultivars were grown in low and high light intensities. In low light there was 20% increase in chlorophyll b contents in transgenic plants, which resulted in 16% reduction in the Chl a/b ratio. In high light, total Chl contents were 31% higher in transgenic plants than those of wild type. The increase in Chl a was 19% and that of Chl b was 72% leading to 31% decline of Chl a/b ratio. The increase in Chl b contents was accompanied by enhanced CAO expression that was highly pronounced in low light. As compared to low light, in high light Lhcb1 and Chl a/b transcripts abundance was significantly increased in transgenic plants suggesting a close relationship between Chl b synthesis and cab gene expression. However, there was a small increase in expression of LHCII proteins, which did not correspond to 72% increase in Chl b content in transgenic line, implying that LHCPII has the ability to bind more Chl b molecules.

Evidence for type II phytochrome-induced rapid signalling leading to cab::luciferase gene expression in tobacco cotyledons.

We studied the activation of cab gene expression by phytochrome-induced intercellular signalling and report insights into the mechanism of induction and outspread of a plant internal signal. By micro-beam irradiation techniques and use of a photon-imaging charge-coupled device (CCD) camera system we monitored cab::luciferase reporter gene expression in cotyledons of transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) plants. We found that (i) the photoreceptor triggering intercellular signalling and reporter-gene expression is type II but not type I phytochrome, (ii) phytochrome in its far-red-absorbing (Pfr) form is necessary for the induction but not for the outspread of the signalling, (iii) red/far-red reversibility is restricted to the red-irradiated cells, and (iv) the phytochrome-induced signal spreads rapidly throughout the cotyledon and reaches its target cells within minutes.

SRL1: a new locus specific to the phyB‐signaling pathway in Arabidopsis

As part of an effort to isolate new Arabidopsis mutants specifically defective in responsiveness to red light, we identified srl1 (short hypocotyl in red light) by screening an EMS-mutagenized M2 population derived from a phytochrome B (phyB)-overexpressor line (ABO). The srl1 mutant shows enhanced responsiveness to continuous red but not far-red light, in both wild-type and ABO backgrounds, consistent with involvement in the phyB-signaling pathway but not that of phyA. The hypersensitive phenotype of srl1 is not due to overexpression of endogenous phyA or phyB, and the locus maps to the center of chromosome 2, distinct from any other known photomorphogenic mutants. srl1 seedlings display enhancement of several phyB-mediated responses, including shorter hypocotyls, more expanded cotyledons, shorter petioles and modestly higher levels of CAB gene expression under red light than the wild type. Double mutant analyses show that the hypersensitive phenotype of srl1 is completely phyB-dependent. The data suggest, therefore, that SRL1 may encode a negatively acting component specific to the phyB-signaling pathway.

Cab gene expression in bleached leaves of carotenoid-deficient maize.

The chloroplast photo-oxidation and the expression of the Cab gene Lhcb1, encoding the Lhcb1 light-harvesting chlorophyll a/b-protein of PS II, have been studied in leaf cells of maize treated with the two bleaching herbicides norflurazon and amitrole and of the two carotenoid-free mutants vp9 and vp2 grown under high photodamaging light. Both herbicides and mutations caused severe photo-oxidation of organelles. However, the plastids of norflurazon-treated and vp2 leaves were totally devoid of thylakoids and did not contain any chlorophyll, while the organelles of amitrole-treated and vp9 leaves still had a few altered and photosynthetically unfunctional membranes and very small quantities of chlorophylls. Despite the dramatic photodamage undergone by the plastids over several days, the cells of amitrole-treated and vp9 leaves maintained a certain expression of the Lhcb1 gene which, on the contrary, was completely blocked in the cells of norflurazon-treated and vp2 leaves. The experimental results, obtained by integrating biochemical and molecular analyses with ultrastructural observations, show that the maintainance of Cab gene expression does not strictly depend on intact and functional chloroplasts. The transcription of these genes, still maintained in cells with greatly photo-oxidized organelles, seems to be inversely related to the degree of thylakoid demolition, which can affect the last steps of chlorophyll biosynthesis.

The expression of light-regulated genes in the high-pigment-1 mutant of tomato

Three light-regulated genes, chlorophyll a/b-binding protein (CAB), ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, and chalcone synthase (CHS), are demonstrated to be up-regulated in the high-pigment-1 (hp-1) mutant of tomato (Lycopersicon esculentum Mill. ) compared with wild type (WT). However, the pattern of up-regulation of the three genes depends on the light conditions, stage of development, and tissue studied. Compared with WT, the hp-1 mutant showed higher CAB gene expression in the dark after a single red-light pulse and in the pericarp of immature fruits. However, in vegetative tissues of light-grown seedlings and adult plants, CAB mRNA accumulation did not differ between WT and the hp-1 mutant. The ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit mRNA accumulated to a higher level in the hp-1 mutant than WT under all light conditions and tissues studied, whereas CHS gene expression was up-regulated in de-etiolated vegetative hp-1-mutant tissues only. The CAB and CHS genes were shown to be phytochrome regulated and both phytochrome A and B1 play a role in CAB gene expression. These observations support the hypothesis that the HP-1 protein plays a general repressive role in phytochrome signal transduction.

Transcription of Arabidopsis and wheat Cab genes in single tobacco transgenic seedlings exhibits independent rhythms in a developmentally regulated fashion.

Transcription of Cab genes has been previously shown to be regulated by a circadian oscillator coupled to the red light-absorbing plant photoreceptor phytochrome in various plant species. In addition, it has recently been suggested that rhythmic expression of the Cab genes could also be affected by a phytochrome-independent circadian oscillator in a developmentally regulated fashion. This study has shown that a red light-insensitive oscillator and a phytochrome-coupled circadian clock indeed coregulate the oscillating expression of individual Cab genes at the level of transcription at an early developmental stage. The study involved analysing the expression patterns of transgenes, containing short fragments of the Arabidopsis thaliana Cab2 or the wheat Cab-1 promoter fused to the firefly luciferase reporter gene, by a video-imaging system in single, etiolated tobacco seedlings. Germination and red/far-red light treatments applied between 12 and 36 h after sowing lead to the appearance of two independent circadian rhythms. These rhythms coexist, both exhibiting period lengths close to 25 h but phased differently. However, repeated red-light treatments given 60 h or later after sowing synchronize these free-running rhythms and induce a single new circadian oscillation. These data indicate that both oscillators regulate the expression of the Cab genes studied at the level of transcription and that the cis-acting element(s) of the wheat Cab-1 and A. thaliana Cab2 genes mediating these responses are located on short, 250 bp promoter regions. Furthermore, these red-light induced rhythms are also inducible by far-red light treatments alone. Therefore, in tobacco, the phytochrome-coupled oscillator is regulated, at least partially, by the very low fluence response of phytochrome A.

Phytochrome‐induced intercellular signalling activates cab::luciferase gene expression

SummaryThe phytochrome‐induced expression pattern of the chlorophyll a/b binding protein (cab) gene was studied using a cab2::luciferase reporter transgene in the tobacco cotyledon. The role of developmentally regulated competence, cooperativity among cells and signal propagation was investigated by red‐light microbeam irradiation of distinct areas of the cotyledon. Even with a minimal fluence, the response was not restricted to the irradiated cells. Following irradiation of the cotyledon base, the luciferase activity revealed a robust propagation of activating signals to areas that had not received a light stimulus. The acropetal outspread formed distinct expression patterns depending on the site of the irradiation. The combination of imaging luciferase activity in living seedlings and microbeam microscopy provides significant experimental evidence of how cellular light perception and intercellular signalling contribute to the cab gene expression pattern.

A chlorate-resistant mutant defective in the regulation of nitrate reductase gene expression in Arabidopsis defines a new HY locus.

Light acts both directly as a signal and indirectly through photosynthesis to regulate the expression of genes encoding nitrate reductase (NR). Here, we report the isolation and characterization of a novel chlorate-resistant mutant that is defective in the regulation of NR gene expression. The response of NR2, but not NR1 or the gene encoding nitrate reductase (NiR), to light signals was impaired in this Arabidopsis mutant, designated cr88. In addition to NR2, the light regulation of the genes encoding the chlorophyll a/b binding protein (CAB) and the small subunit of ribulose bisphosphate carboxylase (RBCS) was also impaired in this mutant. These results suggest that the pathway through which light regulates the expression of NR2, CAB, and RBCS genes is different from those that regulate the expression of NR1 and NiR. An examination of the deetiolation process under different light spectrum showed that cr88 is defective in red light-mediated deetiolation. Complementation tests with various long hypocotyl (hy) mutants indicated that CR88 identifies a new HY locus. The possible functions of CR88 are discussed.

1. Phototransduction and Circadian Clock Pathways Regulating Gene Transcription in Higher Plants

Publisher Summary Plants serve as one of the best model systems for the investigation of the integration of the phototransduction and circadian clock pathways regulating gene transcription. The use of combined biochemical, molecular, and genetic approaches provide information on each of the three components of the circadian system involved in the regulation of plant gene transcription. The three basic components are: (1) input signal transduction pathways from the environmental cues (primarily light-dark signals), which entrain the activity of the circadian oscillator and determine the phase of the free-running rhythm; (2) a circadian oscillator that generates the rhythm; and (3) output signal transduction pathways for the temporal regulation of specific biological processes. This chapter discusses the identity and function of plant photoreceptors, the input signal transduction pathways from the photoreceptors, the analysis of the circadian oscillator by genetic screens for mutants with altered regulation of CAB expression, and the characterization of the output signal transduction pathways starting from the identification of circadian clock- and light-responsive cis-acting CAB promoter elements. Expression of the plant CAB gene family, encoding the chlorophyll a and b binding proteins of the light-harvesting complex of photosystem II, is one of the most extensively investigated plant gene families whose expression is circadian clock-regulated at the level of transcription. Analysis of CAB gene expression has revealed that a network of regulatory pathways controls CAB transcription. The potential for the combinatorial interaction of a multiplicity of inputs to CAB regulation provides a means of generating complex expression patterns.

Light intensity regulation of cab gene transcription is signaled by the redox state of the plastoquinone pool.

The eukaryotic green alga Dunaliella tertiolecta acclimates to decreased growth irradiance by increasing cellular levels of light-harvesting chlorophyll protein complex apoproteins associated with photosystem II (LHCIIs), whereas increased growth irradiance elicits the opposite response. Nuclear run-on transcription assays and measurements of cab mRNA stability established that light intensity-dependent changes in LHCII are controlled at the level of transcription. cab gene transcription in high-intensity light was partially enhanced by reducing plastoquinone with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), whereas it was repressed in low-intensity light by partially inhibiting the oxidation of plastoquinol with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Uncouplers of photosynthetic electron transport and inhibition of water splitting had no effect on LHCII levels. These results strongly implicate the redox state of the plastoquinone pool in the chloroplast as a photon-sensing system that is coupled to the light-intensity regulation of nuclear-encoded cab gene transcription. The accumulation of cellular chlorophyll at low-intensity light can be blocked with cytoplasmically directed phosphatase inhibitors, such as okadaic acid, microcystin L-R, and tautomycin. Gel mobility-shift assays revealed that cells grown in high-intensity light contained proteins that bind to the promoter region of a cab gene carrying sequences homologous to higher plant light-responsive elements. On the basis of these experimental results, we propose a model for a light intensity signaling system where cab gene expression is reversibly repressed by a phosphorylated factor coupled to the redox status of plastoquinone through a chloroplast protein kinase.

Circadian regulation of the Cat3 catalase gene in maize (Zea mays L.): entrainment of the circadian rhythm of Cat3 by different light treatments

The Cat3 maize catalase gene is a non‐photosynthetic, non‐chloroplast‐associated gene that exhibits a transcriptionally regulated circadian rhythm. Cat3 transcript accumulation was examined in leaves of maize seedlings grown under different light regimes. Plants grown in a 12 h light/12 h dark photoperiod (LD) show a distinct diurnal pattern with maximal Cat3 transcript accumulation late in the light period (CT 10, circadian time) and low to undetectable levels late in the dark and early light period (CT 22‐02). The Cat3 gene is expressed at high and constant levels without any diurnal fluctuations in leaves of maize seedlings grown post‐imbibition in constant dark (DD) or constant light (LL). The circadian rhythm of Cat3 is inducible by transferring DD‐grown plants and/ or LL‐grown plants to a 12 h (light/dark LD) or 12 h (dark/light DL) cycle. Entrainment of the rhythm requires a cyclic light signal consisting of one DL‐transition (‘light on’‐signal) followed by one LD transition (‘light off’‐signal). When etiolated maize seedlings were treated with single light pulses of white light and different monochromatic light and transferred to constant darkness (DD) a similar induction pattern of the Cat3 circadian rhythm was observed. Cat3 mRNA accumulates to peak levels 4 h after the light pulse; the next peak appeared 24–28 h later. Induction by far red‐light (FR) exhibits characteristics of a very low fluence phytochrome response. Also blue light and ultraviolet light pulses cause induction of the Cat3 circadian rhythm, but do not influence the periodicity of Cat3 expression. In order to demonstrate differences of the Cat3 circadian regulation from photosynthetic genes, the maize cab gene expression was used for comparison.

Chapter 31 Molecular Methods for Isolation of Signal Transduction Pathway Mutants

Publisher Summary This chapter reviews the methodology for Arabidopsis transformation, mutagenesis, and positive and negative genetic selections based on selectable marker gene cassettes. Several criteria need to be met for performing these types of molecular genetic studies in higher plants. The plant system chosen must be easily transformable and a good genetic organism. The signal transduction or developmental pathway under analysis should have well-defined downstream events that are manifested by tightly regulated transcriptional activation or repression. To isolate mutants that overexpressed CAB in the dark, 98,000 M2 seeds are screened. From these, 375 putative mutants are selected as hygromycin resistant. After rescreening for the expression of both reporter genes in the next few generations, eight mutants consistently shows longer hypocotyls on hygromycin-containing media and have higher GUS (β-glucuronidase) activity than the wild type in the dark. The experiments described in the chapter have identified at least seven new regulatory genes that control CAB gene expression under a variety of conditions.

The Role of Plastids in the Expression of Nuclear Genes for Thylakoid Proteins Studied with Chimeric [beta]-Glucuronidase Gene Fusions.

We have analyzed plastid and nuclear gene expression in tobacco seedlings using the carotenoid biosynthesis inhibitor nor-flurazon. mRNA levels for three nuclear-encoded chlorophyll-binding proteins of photosystem I and photosystem II (CAB I and II and the CP 24 apoprotein) are no longer detectable in photobleached seedlings, whereas those for other components of the thylakoid membrane (the 33- and 23-kD polypeptides and Rieske Fe/S polypeptide) accumulate to some extent. Transgenic tobacco seedlings with promoter fusions from genes for thylakoid membrane proteins exhibit a similar expression behavior: a CAB-[beta]-glucuronidase (GUS) gene fusion is not expressed in herbicide-treated seedlings, whereas PC-, FNR-, PSAF-, and ATPC-promoter fusions are expressed, although at reduced levels. All identified segments in nuclear promoters analyzed that have been shown to respond to light also respond to photodamage to the plastids. Thus, the regulatory signal pathways either merge prior to gene regulation or interact with closely neighboring cis elements. These results indicate that plastids control nuclear gene expression via different and gene-specific cis-regulatory elements and that CAB gene expression is different from the expression of the other genes tested. Finally, a plastid-directing import sequence from the maize Waxy gene is capable of directing the GUS protein into the photodamaged organelle. Therefore, plastid import seems to be functional in photobleached organelles.

Phytochrome A and Phytochrome B Have Overlapping but Distinct Functions in Arabidopsis Development.

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. In Arabidopsis thaliana, there are genes encoding at least five phytochromes, and it is of interest to learn if the different phytochromes have overlapping or distinct functions. To address this question for two of the phytochromes in Arabidopsis, we have compared light responses of the wild type with those of a phyA null mutant, a phyB null mutant, and a phyA phyB double mutant. We have found that both phyA and phyB mutants have a deficiency in germination, the phyA mutant in far-red light and the phyB mutant in the dark. Furthermore, the germination defect caused by the phyA mutation in far- red light could be suppressed by a phyB mutation, suggesting that phytochrome B (PHYB) can have an inhibitory as well as a stimulatory effect on germination. In red light, the phyA phyB double mutant, but neither single mutant, had poorly developed cotyledons, as well as reduced red-light induction of CAB gene expression and potentiation of chlorophyll induction. The phyA mutant was deficient in sensing a flowering response inductive photoperiod, suggesting that PHYA participates in sensing daylength. In contrast, the phyB mutant flowered earlier than the wild type (and the phyA mutant) under all photoperiods tested, but responded to an inductive photoperiod. Thus, PHYA and PHYB appear to have complementary functions in controlling germination, seedling development, and flowering. We discuss the implications of these results for possible mechanisms of PHYA and PHYB signal transduction.

Blue-Light Regulation of the Arabidopsis thaliana Cab1 Gene.

The steady-state level of Cab RNA in etiolated Arabidopsis thaliana increases as a result of a single pulse of blue light. The threshold for the response is at or below 10[deg] [mu]mol m-2 and begins within 1 h of irradiation. The response is not prevented by far-red treatment, and the blue-light source used does not elicit and observable very low fluence phytochrome response for RbcS RNA. The time course for blue-light-induced transcript accumulation differs from that of red, the blue beginning more quickly. Transcripts derived from the Cab1 (AB140; Lhcb1*3) member of the gene family are responsible in part for the blue-light-induced accumulation. This is the same member of the gene family that is responsible for phytochrome-induced Cab gene expression (G.A. Karlin-Neumann, L. Sun, E.M. Tobin [1988] Plant Physiol 88: 1323-1331). The mutant hy4, which lacks blue-light-induced suppression of hypocotyl elongation, retains the ability of Cab RNA to respond to blue light.

Dark and Circadian Regulation of mRNA Accumulation in the Short-Day Plant Pharbitis nil.

The developmental transition of the meristem from vegetative to reproductive growth is controlled by the cyclic alternation of light and darkness in photoperiodic plants. Photoperiod is perceived in the leaves or cotyledons, where a flower-inducing signal is produced and transmitted to the apex. To begin to understand the molecular basis of the photoperiodic induction of flowering, we investigated changes in gene expression at the level of mRNA abundance that occur in association with dark induction of flowering in the short-day species Pharbitis nil. Several cDNAs were isolated that corresponded to mRNAs whose abundance is altered after the transition to darkness. The pattern of increase in mRNA levels corresponding to one cDNA clone, PN1, showed a dark-induced maximum at 8 h of darkness, whereas a second clone, PN9, showed a dark-induced accumulation of mRNA with peak levels at 12 to 16 h of darkness. When plants were held in continuous darkness, both PN1 and PN9 exhibited rhythmic patterns of mRNA accumulation with an approximate circadian periodicity, suggesting that their expression is under the control of an endogenous clock. The observed pattern of expression of PN1 and PN9 in cotyledon tissue was unusual in that darkness rather than light promoted mRNA accumulation, which is a temporal pattern of expression distinct from that of several other Pharbitis genes, including Cab, PsaG, and actin, whose mRNAs were most prevalent or equally prevalent in the light. Brief illumination of an inductive dark period by a red light night break strongly inhibited the accumulation of both PN1 and PN9 mRNA. The expression of both PN1 and PN9 was spatially regulated in that mRNA transcripts were detected in the cotyledons and stems, but not the roots, of photoperiodically competent seedlings. Both PN1 and PN9 appeared to be present as single-copy genes in the Pharbitis genome. Sequence analysis has not determined the identity of these genes. Overall, the accumulation of mRNAs corresponding to both PN1 and PN9 closely paralleled the process of photoperiodic floral induction in P. nil, but a clear involvement with this process cannot be established from our findings because of the difficulty of separating photoperiodic events from other light-regulated processes, especially those involved in photosynthesis, such as Cab gene expression. These results identify the products of circadian-regulated genes in photoreceptive tissue of P. nil and support the concept that circadian-regulated gene expression interacting with darkness may be involved in the regulation of photoperiodically controlled physiological processes, including flower induction.