Nitric Oxide Represses the Arabidopsis Floral Transition

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Science  24 Sep 2004:
Vol. 305, Issue 5692, pp. 1968-1971
DOI: 10.1126/science.1098837


The correct timing of flowering is essential for plants to maximize reproductive success and is controlled by environmental and endogenous signals. We report that nitric oxide (NO) repressed the floral transition in Arabidopsis thaliana. Plants treated with NO, as well as a mutant overproducing NO (nox1), flowered late, whereas a mutant producing less NO (nos1) flowered early. NO suppressed CONSTANS and GIGANTEA gene expression and enhanced FLOWERING LOCUS C expression, which indicated that NO regulates the photoperiod and autonomous pathways. Because NO is induced by environmental stimuli and constitutively produced, it may integrate both external and internal cues into the floral decision.

The life of flowering plants is divided into two distinct phases, an initial vegetative phase during which meristems produce leaves and a subsequent reproductive phase during which meristems produce flowers. Genetic studies of the timing of flowering in Arabidopsis have revealed four major pathways (1). The photoperiod and vernalization pathways integrate external signals into the floral decision, whereas the autonomous and gibberellin (GA) pathways act independently of environmental cues (2).

NO plays a pivotal role in animals and has emerged as a key growth regulator in plants (3, 4). NO promotes leaf expansion, inhibits maturation and senescence, stimulates light-dependent germination, and promotes de-etiolation (5, 6). Excess endogenous NO reduces growth and delays development in tobacco plants (7). NO production is induced by biotic and abiotic stimuli, such as drought, salt stress, and pathogen infection (4). In addition, substantial NO is emitted from plants into the atmosphere. Conversely, atmospheric NO, a major greenhouse pollutant produced by combustion of fossil fuels, can affect plants. Thus, NO has a central role in coordinating plant growth and development with environmental conditions. However, little is known about the molecular mechanisms underlying the function of NO in plants.

Treatment of Arabidopsis seedlings with an NO donor, sodium nitroprusside (SNP), enhanced vegetative growth and delayed flowering (Fig. 1). SNP increased shoot growth by ∼65% at low concentrations (≤100 μM), although it inhibited growth at high concentrations (Fig. 1, A and B; fig S1A). The optimal SNP concentration for promoting shoot growth was ∼100 μM. A similar promotive effect of NO on chlorophyll content was also found (8). SNP delayed flowering in a dose-dependent manner, as measured by the increase in rosette leaf number and days to bolting—swift upward growth at the transition to flowering (Fig. 1, A, C, and D; fig. S1A). A standard indicator for flowering time is the number of leaves produced on the primary shoot before the first flower is initiated; plants that flower late form more leaves (9).

Fig. 1.

Exogenous NO promotes vegetative growth but inhibits reproductive development. (A) The effects of an NO donor SNP on plant growth and development. Arabidopsis seedlings were grown in petri dishes containing SNP during long days (16-hour light/8-hour dark) for 5 weeks (10). (B) The effect of SNP concentration on shoot growth. (C and D) The effect of SNP on flowering times. Fresh weight per shoot (B), the rosette leaf number (C), and days to bolting (D) from experiments as in (A) and fig. S1A plotted as a function of the concentrations of SNP that were applied, respectively. Data from four separate experiments are presented (mean ± SD; n = 150 seedlings).

Exogenously applied NO may not replicate the function of endogenous NO and may have side effects in plants. Thus, analysis of genetic mutants with altered endogenous NO levels was conducted to determine the in vivo relevance of NO. An NO-hypersensitive screen for NO overproducer (nox) mutants in Arabidopsis was performed (10). NO inhibition of root growth was used as a phenotype for the initial screen (fig. S1B). Subsequently, NO production was measured with an NO-sensitive dye, 4,5-diaminofluorescein diacetate (11, 12). Six nox1 alleles were isolated (Fig. 2A; fig. S2) that contained high levels of NO in roots (8) and leaves (table S1) compared with wild type (WT) (Fig. 2B). The nox1 mutant, which refers to nox1-1 unless otherwise specified, showed the most root-growth hypersensitivity to SNP of all the mutants isolated. Mutants with altered NO biosynthesis or signaling have not yet been isolated via genetic screens, so nox1 could provide a powerful tool for dissecting NO function. Despite recent identification of two types of NO synthase (NOS), pathogen-inducible iNOS and constitutive AtNOS, the sources of NO in plants remain to be fully elucidated (4, 11, 12).

Fig. 2.

Endogenous NO represses the floral transition. (A) The root growth phenotype in nox1 mutant. (B) The endogenous NO levels in nox1 and WT. Leaves were stained with DAF-2DA. Fluorescence was analyzed with excitation 490 nm and emission 515 nm (top) with the same exposure times (10). White-light images are shown at the bottom. (C) The levels of l-Arg and NO in WT, nox1, and cue1-5. Plants grown under 12-hour light/12-hour dark cycles were harvested 6 hours after dawn (10). The absolute levels of l-Arg and NO were 51.8 and 0.45 nmol per gram of fresh rosette leaves in WT, respectively. Values are normalized to those of WT. Each data point represents nine independent measurements (r.u., relative unit). (D) The nox1 mutant flowers late. WT and nox1 were grown in soil under 12-hour light/12-hour dark cycles and were photographed after 60 days of growth. (E and F) Flowering times of nox1 and cue1-5 mutants. The rosette leaf number (E) and days to flowering (F) from experiments as in (D) were scored (mean ± SD; n ≥ 25 plants). (G) The NO synthase 1 (nos1) mutant that produces fewer NO flowers early under long days. (H) The nos1 mutant flowers earlier than WT under SNP treatments. Experiments similar to that in (G) were analyzed (mean ± SD; n = 20 to 30 plants).

NOX1 was identified as either very close or identical to CUE1 by map-based cloning (fig. S3A). The morphological phenotype of nox1 was almost identical to that described for chlorophyll a/b binding protein (CAB) underexpressed 1 mutant (cue1), including small plant size and pale green leaves with a reticulate pattern (13). CUE1 encodes a chloroplast phosphoenolpyruvate/phosphate translocator (14). Several lines of molecular genetic evidence demonstrated that NOX1 is CUE1 (fig. S3). The cue1 mutants were hypersensitive to SNP and displayed an elevated level of endogenous NO. The cue1 could not complement nox1 phenotypes. In all six nox1 alleles, the CUE1 gene was deleted. Because NO is synthesized from the conversion of l-arginine (l-Arg) to l-citrulline (l-Cit) by NOS (3), free l-Arg and l-Cit are several-fold higher in cue1 mutants than in WT plants (14), and nox1 overproduces NO, we reasoned that disruption of the CUE1 gene would increase the endogenous l-Arg concentration and thus would promote its conversion to NO. nox1 and the cue1-5 mutant harboring a single amino acid mutation (14) indeed exhibited larger amounts of l-Arg, l-Cit, and NO than WT (Fig. 2C; fig. S4), indicating that NOS-based NO production occurs in Arabidopsis.

Soil-grown nox1 and cue1 mutants showed a late-flowering phenotype (Fig. 2, D to F; table S1). This phenotype is not photoperiod-dependent, as nox1 flowered late under all photoperiods. However, the phenotypic severity was influenced by photoperiods, with 18%, 61%, and 17% increases in the rosette leaf number seen under the light/dark (hours) cycles of 16/8, 12/12, and 8/16, respectively (table S1). This observation is consistent with the light-dependent phenotypes seen in cue1 mutants (13).

In addition, we determined whether down-regulation of endogenous NO promotes the floral transition. The mutant Atnos1 (nos1) that contains a reduced amount of NO (11) flowered earlier than WT (Fig. 2G), but still displayed sensitivity to SNP (Fig. 2H). The NO content of nos1 plants was about 18% that of WT (8). The positive correlation between endogenous NO and the number of leaves produced indicates that NO may have a specific role in controlling the floral transition.

To test which components in the floral pathways are affected by NO, we analyzed expression of the floral meristem identity gene LEAFY (LFY). LFY is an important determinant in flower initiation, and its expression increases gradually before flowering commences (15). SNP suppressed LFY expression in a dose-dependent manner (Fig. 3, A and C). LFY expression was low in nox1, but was high in nos1 plants compared with WT (Fig. 3, B and C). The negative correlation between LFY expression and endogenous NO suggests that NO repression of the floral transition is mediated, at least in part, by LFY.

Fig. 3.

NO affects the expression of genes that control the floral transition. (A, D and G) The effect of NO on the expression of LFY, FLC, and CO, respectively. Seedlings were grown in media containing SNP under long days. Leaves were collected 8 hours after dawn. The LFY and CO mRNA abundance was analyzed by using reverse transcription–polymerase chain reaction (PCR) and FLC mRNA by Northern blot (10). Ubiquitin mRNA (UBQ10) was used as a loading control. Similar results were seen for plants grown in 12-hour light/12-hour dark cycles. (B, E, and H) The expression levels of LFY, FLC, and CO, respectively, in WT, nox1, and nos1 plants. Materials were prepared, and mRNA was analyzed as described in (A). (C, F, and I) Analysis of the effects of NO on LFY, FLC, and CO expression, respectively. The relative mRNA abundance was normalized to the UBQ levels. The maximum value was arbitrarily set to 1 (mean ± SEM; n = 3).

Genetic epistasis studies have placed the genes that regulate the floral transition into four major pathways in Arabidopsis, all of which converge on the target LFY (2). The nox1 mutants flowered late and showed a dwarf phenotype, resembling those of GA-deficient mutants (16). GA promoted flowering in nox1 and WT plants but could not reverse the nox1 dwarf phenotype (8), which suggests that NO may function in parallel with GA. Because nox1 and mutants of the autonomous pathway flower late on both long and short days (1), we reasoned that nox1 might affect this pathway. The vernalization and autonomous pathways converge on a floral repressor, FLOWERING LOCUS C (FLC), and late-flowering mutants of the autonomous pathway always have elevated FLC expression (17). Treatment with low concentrations (≤50 μM) of SNP decreased FLC expression, whereas high concentrations (>50 μM) increased FLC expression (Fig. 3, D and F). FLC expression was high in nox1 and slightly reduced in nos1 compared with WT (Fig. 3, E and F). The late-flowering phenotype observed in WT plants treated with high doses of SNP or in nox1 may result from up-regulation of FLC expression. However, the late-flowering phenotype in plants treated with low doses of SNP may be caused by components independent of FLC. Nonetheless, the flowering phenotypes observed in nox1, WT, and nos1 are consistent with the expression level of FLC, which suggests that endogenous NO may down-regulate the autonomous pathway, which results in late flowering.

Because previous studies have indicated the light-dependent nature of NO effects in plants, and cue1 mutants show various defects in light perception and photomorphogenesis (6, 13), we investigated whether NO regulates the photoperiod pathway. Arabidopsis is a facultative, long-day plant; long days promote flowering (9). CONSTANS (CO) is the most genetically downstream component of this pathway identified so far that promotes floral induction, and it acts as a link between the circadian clock and the control of flowering (18, 19). SNP suppressed CO expression in a dose-dependent manner (Fig. 3, G and I). The CO expression was high in nos1 but low in nox1 plants compared with WT (Fig. 3, H and I). Consistently, CO and FLC expression were down- and up-regulated, respectively, in cue1-5 plants (fig. S5). CO expression displays a diurnal rhythm (18); thus, the repression of CO by NO could be due to a reduction of amplitude, a phase shift, or both.

To quantify this effect of NO, we determined CO mRNA expression over a 12-hour light/12-hour dark cycle. The overall CO mRNA abundance was lower in nox1 and higher in nos1 than in WT, although the phase of CO mRNA level was not greatly disturbed (Fig. 4, A and B; fig. S6), consistent with previous studies on gigantea (gi) and early flowering 3 (elf3), mutants of the photoperiod pathway (20, 21). A gi lesion down-regulates CO expression, which results in late flowering, whereas an elf3 lesion up-regulates CO expression, which leads to early flowering (18). To test whether NO directly affects CO expression, we analyzed GIGANTEA (GI), which is upstream of CO (21). The circadian amplitudes of GI mRNA abundance, similar to those of CO, were lower in nox1 than WT (Fig. 4, C and D), which suggests that NO acts upstream of CO. Additionally, we analyzed the flowering phenotypes of elf3 and CO overexpressing plants (35S::CO) (22) in response to NO. NO suppression of flowering was largely abolished in elf3 mutants and 35S::CO plants (Fig. 4F), which further supports the role of NO in the photoperiod pathway.

Fig. 4.

NO suppresses the photoperiod pathway. (A and C) The mRNA abundance of CO and GI over a 24-hour time course. Plants were grown under 12-hour light/12-hour dark cycles for 10 days. Seedlings were collected every 4 hours for CO and every 3 hours for GI starting at dawn. The black and white bars at the top represent objective lights off and on, respectively. (B and D) Quantification of CO and GI mRNA abundance in (A) and (C), respectively (mean ± SEM; n = 5). (E) Circadian rhythms of cotyledon movements. Seedlings were grown under 12-hour light/12-hour dark cycles for 5 days, and cotyledon movements were recorded under constant dim light (10). (F) Analysis of the flowering time of elf3 and 35S::CO in response to SNP treatments (mean ± SD; n = 150 seedlings).

The expression of CO is regulated by the circadian clock and photoperiod, and the circadian rhythm of CAB expression can represent the circadian output in plants (2). The amplitudes of CAB mRNA abundance after switching from 12-hour light/12-hour dark cycles to continuous light for 3 days were greatly reduced in nox1, as well as in cue1 mutants (13), whereas the circadian phases were not altered (8), similar to the expression of CO and GI. The amplitudes of another circadian output, cotyledon leaf movements, were also reduced by 43.8 ± 4.9% in nox1 compared with WT, and no alteration of the circadian period was observed (Fig. 4E; fig S7). To analyze whether NO affects the circadian clock itself, we examined components of circadian clock central oscillators, TIMING OF CAB EXPRESSION (TOC1) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) (23, 24). Neither the amplitude nor the circadian period of TOC1 and CCA1 expression was altered in nox1 (8). Therefore, it is more likely that NO may affect circadian outputs rather than these central oscillators. Additionally, NO content in leaves in the middle of the night was 70.6% of that in the middle of the day in a 12-hour light/12-hour dark cycle for WT plants and 58.0% for nox1 (table S2). This result suggests that the endogenous NO levels display a diurnal rhythm, consistent with a previous study (7). Although it is unclear whether NO production is dependent on light or the circadian clock, the alteration of NO homeostasis in nox1 may cause its flowering phenotype.

We have provided pharmacological, physiological, molecular, and genetic data demonstrating that NO represses the photoperiod and autonomous floral pathways. These results are largely consistent with previous observations of delayed development in tobacco where NO is overproduced and of its inhibitory effects on maturation and senescence (5, 7). Given that NO is induced by environmental changes, as well as constitutively produced, external and internal cues may converge on the regulation of endogenous NO status, which then relays these signals to the transcription regulatory network that controls the floral transition, which provides a unique layer of floral regulation. Oscillating NO levels, in addition to CCA1 and TOC, may provide a mechanism to measure day/night switches, acting downstream of light perception to regulate certain circadian outputs and thus to control the floral transition, although the precise action of NO remains to be determined. Because several genes affected by NO (CAB, CO, and GI) are also regulated by light, the primary effect of NO may be on light signaling, and the effect on flowering may be secondary. Thus, identification of the direct target of NO will further our understanding of the molecular function of NO in plants.

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Materials and Methods

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Figs. S1 to S7

Tables S1 and S2

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