The interaction of light quality and irradiance with gibberellins, cytokinins and auxin in regulating growth of Helianthus annuus hypocotyls


L. V. Kurepin. Fax: 1 403 289 9311; e-mail:


A reduced red to far-red (R/FR) light ratio and low photosynthetically active radiation (PAR) irradiance are both strong signals for inducing etiolation growth of plant stems. Under natural field conditions, plants can be exposed to either a reduced R/FR ratio or lower PAR, or to a combination of both. We used Helianthus annuus L., the sunflower, to study the effect of reduced R/FR ratio, low PAR or their combination on hypocotyl elongation. To accomplish this, we attempted to uncouple light quality from light irradiance as factors controlling hypocotyl elongation. We measured alterations in the levels of endogenous gibberellins (GAs), cytokinins (CKs) and the auxin indole-3-acetic acid (IAA), and the effect of exogenous hormones on hypocotyl growth. As expected, both reduced R/FR ratio and lower PAR can significantly promote sunflower hypocotyl elongation when given separately. However, providing the reduced R/FR ratio at a low PAR resulted in the greatest hypocotyl growth, and this was accompanied by significantly higher levels of endogenous IAA, GA1, GA8, GA20 and of a wide range of CKs. Providing a reduced R/FR ratio under normal PAR also significantly increased growth and again gave significantly higher levels of endogenous IAA, GAs and CKs. However, only under the de-etiolating influence of a normal R/FR ratio did lowering PAR significantly increase levels of GA1, GA8 and GA20. We thus conclude that light quality (e.g. the R/FR ratio) is the most important component of shade for controlling hypocotyl growth and elevated growth hormone content.


Stems and hypocotyls elongate in response to a modified light quality [reduced red to far-red (R/FR) ratio] and a lowered irradiance of photosynthetically active radiation (PAR), the two components of vegetative shade (Smith 2000; Franklin & Whitelam 2005; Vandenbussche et al. 2005). A reduced R/FR ratio is believed to be the more important component (Ballare, Scopel & Sanchez 1990; Smith & Whitelam 1997; Smith 2000), although low PAR is also a significant factor (Ballare, Scopel & Sanchez 1991; Ballare 1999; Kurepin et al. 2006a).

The increase in shoot elongation growth seen under reduced R/FR ratio or low PAR appears to be mediated by at least two classes of plant growth-promoting phytohormones, gibberellins (GAs) and auxin [indole-3-acetic acid (IAA)]. A low PAR coupled with a normal R/FR ratio induces stem elongation and coincidentally raises the level of growth-active GAs in a range of plant species, including Pisum sativum (Gawronska et al. 1995), Brassica napus (Potter, Rood & Zanewich 1999) and Stellaria longipes (Kurepin et al. 2006b). A reduced R/FR ratio coupled with a normal PAR increases growth and raises levels of endogenous GAs in shoots of bean plants (Beall, Yeung & Pharis 1996). End-of-day far-red (FR) light enrichment has also been shown to increase endogenous IAA levels in the third internode of P. sativum seedlings (Behringer & Davies 1992). Finally, Arabidopsis hypocotyl elongation under reduced R/FR ratio coupled with low PAR is IAA dependent (Steindler et al. 1999). In addition, a low PAR coupled with a normal R/FR ratio induces auxin activity (IAA-mediated gene expression) in rosette leaves of Arabidopsis (Vandenbussche et al. 2003).

The role of cytokinins (CKs) in mediating the effects of light on plant growth, including stem elongation, is complex and appears to involve interplay with other hormone groups (Thomas, Hare & van Staden 1997). Thus, while the link between light and CKs is not as well documented as for IAA or GA, Fankhauser (2002) cites reports which imply that CKs do play an important role in light-mediated stem elongation responses. In terms of light irradiance or absence of light (dark), work on Arabidopsis has shown that applied CKs inhibit hypocotyl elongation in the dark (Su & Howell 1995) and that this inhibition is mediated by an increase in ethylene production (Cary, Liu & Howell 1995). In contrast, CK application usually has no observable effect on leaf and petiole elongation in light-grown Arabidopsis seedlings (Su & Howell 1995). That said, applied CK can promote hypocotyl elongation of Arabidopsis in light if either ethylene action or IAA transport is blocked (Smets et al. 2005).

With respect to light quality, treatments with R light or CKs have been shown to have similar inhibiting effects on seedling growth, yet several studies have concluded that they act independently and/or additively (see Thomas et al. 1997). For example, it has been suggested that the effects of R and CK converge at the level of a CK response regulator (ARR4). This regulator apparently stabilizes the Pfr form of phyB (Sweere et al. 2001). Specifically, Arabidopsis plants over-expressing a CK response regulator, ARR4, have been shown by Sweere et al. (2001) to be more sensitive to narrow-band R at fluence rates between 0.1 and 10 µmol m−2 s−1. However, To et al. (2004) observed that double and higher-order Type A arr T-DNA insertion mutants of Arabidopsis showed an altered response to R and, in particular, there was a stronger inhibition of hypocotyl elongation in response to R treatments.

Yet, despite the previous evidence that CKs have an important role in light quality responses, mutants with loss of function for CK receptors (i.e. AHK2, AHK3, CRE1/AHK4), including triple mutants (ahk, ahk3 and cre1), show no difference from the wild type in hypocotyl elongation under a range of light conditions (Riefler et al. 2006). The light conditions tested include light, dark, continuous R (3.4 µmol m−2 s−1) and continuous FR (3.4 µmol m−2 s−1) treatments. Given these details, the putative roles of CKs in the response of hypocotyl growth to light remain unclear, particularly in terms of the influence of FR or of a reduced R/FR ratio on endogenous CK levels, or on activity of shoot-applied CKs.

In the present study, we utilized seedlings of sunflower (Helianthus annuus L.) with rapidly elongating hypocotyls in an attempt to uncouple the effect of light quality (R/FR ratio) from the effect of PAR irradiance. We did this in order to examine the respective roles of each component of shade light in promoting shoot growth (etiolation) via changes in endogenous GA, IAA and CK levels.


Plants and experimental system

Sunflower seeds (6946; Pioneer Seeds, Johnston, IA, USA) were soaked in the dark for 24 h under flowing water at 37 °C. Germinated seeds were planted in a soil mix [2.0 parts of peat moss, 1.0 part Perlite, 1.0 part Vermiculate and 0.25 parts Terragreen (a crushed baked clay medium from Professional Gardener, Calgary, Alberta, Canada)]. The plants were grown in growth chambers (Conviron, Manitoba, Canada) equipped with fluorescent (Sylvania cool white 160 W; Sylvania, Mississauga, ON, Canada) and incandescent lights (Philips 60 W; Philips, Markham, ON, Canada). Temperature was maintained at 20 °C during the16 h of light and at 16 °C during the 8 h dark period. The sunflower seedlings were watered each day with 0.25% strength Hoagland's solution (Hoagland & Arnon 1950).

The seedlings were collected on day 7 following planting and were photographed, with the hypocotyls being measured for length (cotyledon to root) and average width with Scion Image for Windows (Scion Corporation, Frederick, MD, USA). Hypocotyl tissue was then quickly frozen (see subsequent discussion) for use in hormone analysis.

Combinations of fluorescent and incandescent light sources were used to alter the R/FR ratios, and changing the distance between the lights and the cotyledons was used to alter PAR. Values for both the R/FR ratio and PAR combinations were measured with a Li-Cor LI-1800/22 quantum sensor (Li-Cor Inc., Lincoln, NE, USA) and were given in Table 1. For the narrow-band light experiments, light-emitting diode (LED) units (Quantum Inc., Barneveld, WI, USA) producing a narrow bandwidth of R light peaking at 670 nm and FR light peaking at 735 nm were utilized.

Table 1.  Light quality [red to far-red (R/FR) ratio] and irradiance [photosynthetically active radiation (PAR)] conditions used for experimental treatments
 Low PAR (138 µmol m−2 s−1)Normal PAR (510 µmol m−2 s−1)
  1. R/FR values represent mean ± SE values. R/FR ratios and PAR irradiances were measured with a Li-Cor LI-1800/22 quantum sensor (Li-Cor Inc., Lincoln, Nebraska, USA).

Low R/FR ratio0.83 ± 0.040.85 ± 0.02
Normal R/FR ratio1.37 ± 0.041.42 ± 0.04
High R/FR ratio4.69 ± 0.034.94 ± 0.05

Analysis of GAs, IAA and abscisic acid (ABA)

Hypocotyl tissue was immediately frozen in liquid N2 at the time of harvest, and was then freeze dried. One-half to one gram dry weight (DW) of each sample was ground with liquid N2 and washed sea sand (Fisher Scientific, Fair Lawn, NJ, USA), then extracted in 80% MeOH (H2O : MeOH = 20:80, v/v) and purified using the same methods detailed in Kurepin et al. (2006b). Briefly, to the aqueous MeOH we added 266 ng of [13C6] IAA, 200 ng of [2H6] ABA (a gift from Drs L. Rivier and M. Saugy, University of Lausanne, Switzerland) and 33 ng of each of [2H2] GA1, GA8 and GA20 as quantitative internal standards. After purification with a C18 preparative column (C18-PC), the dried eluate was dissolved in 1 mL of 10% MeOH with 1% acetic acid and injected into a reversed-phase high-performance liquid chromatography (HPLC) column as described in Kurepin et al. (2006b). The C18 HPLC fractions containing GAs, IAA and ABA were dried in vacuo and methylated by ethereal CH2N2 for 20 min. For analysis of GAs, the methylated sample was then trimethylsilylated by BSTFA with 1% TMCS (Hedden 1987; Gaskin & MacMillan 1991).

The identification and quantification of IAA, ABA and GAs was carried out using a gas chromatograph connected to a mass spectrometer [gas chromatography-mass spectrometry(GC-MS)] using the -selected ion monitoring (-SIM) mode as described in Kurepin et al. (2006b). For identification of the endogenous GAs, we compared the gas chromatograph (GC) retention time (Rt) of the putative endogenous GA and its [2H2] GA internal standard. Relative intensities of the molecular ion (M+) pairs were compared for each endogenous GA and its [2H2] internal standard. The same approach was taken for identification of IAA and ABA utilizing the [13C6]-IAA and [2H6]-ABA internal standards. Because the stable isotope-labelled internal standards were added in known amounts at the extraction stage, we could quantify levels of endogenous GAs, IAA and ABA based on stable isotope dilution using equations from Gaskin & MacMillan (1991) as adapted by D.W. Pearce (see Jacobsen et al. 2002) from Gaskin & MacMillan (1991).

Extraction and purification of CKs

CKs were extracted from a separate set of tissue samples and separated under conditions established by Emery et al. (1998) and Ferguson et al. (2005) designed to prevent enzyme activity that could cause CK nucleotide degradation and CK isomerization. Then, frozen tissue samples of known fresh weight were homogenized (Ultra-Turrax T8; IKA-Werke GmbH, Staufen, Germany) over ice in cold (−20 °C) modified Bieleski extraction buffer [CH3OH : H2O : HCOOH (15:4:1, v/v/v)] at 20 mL g −1 DW and were extracted according to Dobrev & Kaminek (2002). One hundred nanograms of each deuterated CK, [2H6]iP, [2H6][9R]iP, trans-[2H5]Z, [2H3]DZ, trans-[2H5][9R]Z, [2H3][9R]DHZ, [2H6][9R-MP]iP and [2H6][9R-MP]DHZ (OlChemIm Ltd, Olomouc, Czech Republic) were added as quantitative internal standards. As deuterated standards of cis-[9R]Z and cis-[9RMP]Z are not available, the levels of these two compounds were quantified based on the recovery of the deuterated standards of the corresponding trans compounds. Pooled extract supernatants were dried in vacuo at 40 °C with residues reconstituted in 5 mL of 1.0 M HCOOH for purification on an Oasis MCX column (Waters, Mississauga, Canada) as described in Dobrev & Kaminek (2002). Eluted nucleotides were converted to nucleosides for quantification, and resultant nucleosides were further purified on a reversed-phase C18 column (AccuBOND ODS; Fisher Scientific, Mississauga, Canada) as described in Emery, Ma & Atkins (2000).

Liquid chromatography-tandem mass spectrometry conditions for CK analysis

These purified CK fractions were separated and analyzed by a Waters 2680 Alliance HPLC system (Waters, Milford, MA USA) linked to a Quattro-LC triple quadrupole mass spectrometer (MS) (Micromass, Altrincham, UK). The MS was equipped with a Z-electrospray ionization (ESI) source. Positive-ion mode was used for all analyses [LC-(+)ESI-MS/MS]. A 20 µL aliquot was injected on a Genesis C18 reversed-phase column (4 µm, 150 × 2.1 mm; Jones Chromatography, Foster City, CA USA), and the CKs were eluted with an increasing gradient of acetonitrile (A) mixed with 0.1% formic acid in 20 mM ammonium acetate (v/v) at a pH adjusted to 4.0 (B) at a flow rate of 0.2 mL min−1. The initial conditions were 8% A and 92% B, changing linearly after 5 min to 15% A and 85% B for 2 min, followed by 100% A for 2 min, then linearly returning back to initial conditions for 2 min. The HPLC effluent was introduced into the electrospray source (source block temperature 80 °C, desolvation temperature 250 °C) using conditions specific for each CK where quantification was obtained by multiple reaction monitoring (MRM) of the mother (parent) ion and the appropriate daughter (product) ion as in Prinsen et al. (1995).

Statistical analysis

Each experiment was repeated at least three times, and endogenous phytohormones were measured in tissue from three different experiments. Data analysis was accomplished using the analysis of variance (anova) test on SPSS software version 13 (SPSS Inc., Chicago, IL, USA).


Uncoupling light quality from light irradiance

Based on our own measurements in the field, in southern Alberta, Canada, natural daylight conditions are the following: full sunlight gives a normal R/FR ratio of 1.1 to 1.3 when coupled with a normal PAR of 1300–1500 µmol m−2 s−1; a natural plant canopy shade can give a low R/FR ratio of 0.2 to 0.9 when coupled with a low PAR of 50–200 µmol m−2 s−1; a natural shade from neighbouring vegetation gives a low R/FR ratio of 0.6 to 0.9 when coupled with lower then normal PAR. Light at the end of the day is characterized by a reduced R/FR ratio and a lower PAR, while light on a cloudy day will have a normal R/FR ratio of 1.1 to 1.3 coupled with a relatively low PAR of 200–300 µmol m−2 s−1.

By comparison, when plants are grown in growth chambers or growth rooms equipped with regular fluorescent lighting only, they will usually be subjected to abnormally high R/FR ratios (ca 4.6 or greater), coupled with either a low or normal PAR irradiance.

In our experimental system, the hypocotyls of young sunflower plants elongated almost threefold under simulated canopy shade light (e.g. a low PAR and a low R/FR ratio), relative to simulated sunlight (e.g. a normal PAR and a normal R/FR ratio, Fig. 1). Under simulated neighbouring shade (low R/FR ratio and higher PAR), hypocotyls elongated almost twofold relative to simulated sunlight (Fig. 1). Reduction in R/FR ratio resulted in increased elongation under both PARs, although significance for all three R/FR ratios was gained only for low PAR. For normal PAR, significant growth increases occurred only at the low R/FR ratio, relative to growth at a normal R/FR ratio (Fig. 1).

Figure 1.

Hypocotyl length (mm) of 7-day-old sunflower seedlings grown under varying red to far-red (R/FR) ratios and light irradiances: low photosynthetically active radiation (PAR) (open bars) and normal PAR (solid bars). The error bars indicate one SE of the mean, and mean values with the same letter do not differ significantly at P = 0.05 based on Tukey's multiple comparison test.

The absolute increase in hypocotyl elongation induced by reducing the R/FR ratio under low and normal PAR was similar, for example, 28–32 mm. However, the proportional growth increase in response to low PAR was higher (ca twofold) under normal and high R/FR ratios (where plants were de-etiolated) than under a low R/FR ratio (ca 1.5-fold). Here, we should reiterate that under canopy shade, both reduced R/FR ratio and low PAR are considered causal for the resultant increase in shoot etiolation (Smith 2000).

Endogenous IAA, ABA, GA and CK levels

Lowering the PAR irradiance had no consistent or significant effect on endogenous IAA content of the sunflower hypocotyls (Fig. 2). However, reducing the R/FR ratio from normal to low significantly increased IAA levels (e.g. 1.5- to 2.0-fold) under both low and normal PAR irradiances. Effects of the six light treatments on endogenous ABA levels were quite variable and showed no consistent trends (data not shown).

Figure 2.

Indole-3-acetic acid (IAA) levels in hypocotyls harvested from 7-day-old sunflower seedlings grown under varying red to far-red (R/FR) ratios and light irradiances: low photosynthetically active radiation (PAR) (open bars) and normal PAR (solid bars). The error bars indicate one SE of the mean, and mean values with the same letter do not differ significantly at P = 0.05 based on Tukey's multiple comparison test. DW, dry weight.

In sunflower shoots, only the early 13-hydroxylation pathway of GA metabolism appeared to be functioning, and GA1 was the only growth-active GA present in sunflower shoots (Pearce, Reid & Pharis 1991 and our findings herein). Thus, we examined the endogenous levels of several GAs from this pathway, GA19 → GA20 → GA1 → GA8. Low R/FR ratio resulted in a three- to ninefold higher endogenous GA1 levels at both PAR irradiance levels when compared to normal or high R/FR ratios ( Fig. 3c). In contrast, PAR irradiance was not a significant variable for endogenous GA1 levels under the reduced R/FR ratio, whereas it was significant under a normal R/FR ratio (based on Student's t-test) (Fig. 3c). The endogenous levels of GA8, the inactive catabolite of GA1 (Fig. 3d), mirrored changes seen for levels of GA1 under all six light treatments.

Figure 3.

GA19 (a), GA20 (b), GA1 (c) and GA8 (d) levels in hypocotyls harvested from 7-day-old sunflower seedlings grown under varying red to far-red (R/FR) ratios and light irradiances: low photosynthetically active radiation (PAR) (open bars) and normal PAR (solid bars). The error bars indicate one SE of the mean, and mean values with the same letter do not differ significantly at P = 0.05 based on Tukey's multiple comparison test. However, when Student's t-test was utilized, GA1 and GA8 levels were significantly increased by low PAR at the normal R/FR ratio. GA, gibberellin; DW, dry weight.

The levels of GA20, the immediate precursor of GA1, also showed significant increases in response to lowering the R/FR ratio (Fig. 3b). There was also an increase in GA20 in response to low PAR, but only under normal and high R/FR ratios. This suggests that there is a more rapid turnover (metabolism) of GA biosynthesis under both the low R/FR ratio and low PAR. The significantly elevated levels of GA20 (which is C-3 deoxy) under lower PAR irradiances may imply increased biosynthesis of GA20 from the C20 GA, GA19 (Fig. 3a). Conversely, the very reduced levels of all three GAs under the normal and abnormally high R/FR ratios, when PAR is at a higher irradiance, strongly imply a reduced conversion of the C20 GA, GA19, to the C19 GA, GA20. This apparent ‘blockage’ in formation of GA20, however, appears to be corrected by the low R/FR ratio, at both PAR irradiances, and by low PAR at the two higher R/FR ratios, as previously noted, and in Fig. 3c.

The levels of individual CKs are presented in Table 2, and total CKs are shown in Fig. 4. The total CK levels of the hypocotyl were thus 8- to 10-fold higher when plants were grown under the lowest R/FR ratio, relative to the two higher R/FR ratios, for both PAR irradiances. The effect of varying PAR irradiance on increasing total CK levels was not significant (NS) (Fig. 4). However, this tendency becomes significant for some light treatments when one examines individual CKs and CK groups (see Table 2, low PAR versus normal PAR). Specifically early pathway CKs, the nucleotides ([9RMP]Z, [9RMP]DZ and [9RMP]iP) and isopentenyl adenosine (iPA) were all significantly increased in hypocotyl tissue under the higher PAR irradiance when the young sunflower plants were also exposed to normal and high R/FR ratios (Table 2). To summarize, reducing the R/FR ratio significantly elevates total CK levels under both low and normal PAR (Table 2), although the PAR effect (of increased CKs with increased irradiance) is significant only for nucleotide CKs and iPA, and only when the R/FR ratio is normal or high (Table 2). Hence, for GAs and IAA, the light quality signal appears to be the major controlling factor in elevating hypocotyl CK levels for young seedlings of sunflower plants.

Table 2.  Levels of endogenous cytokinins (CKs) (trans-ZR, cis-ZR, iPA, iP, DZR and CKs in nucleotide and free base form) expressed as picomole per gram dry weight (DW) in hypocotyls of 7-day-old sunflower seedlings grown under varying red to far-red (R/FR) ratios and light irradiances
Low PARLow R/FR ratioNormal R/FR ratioHigh R/FR ratio
Free base CKs3950 ± 725177 ± 58.6117 ± 12.1
Nucleotide CKs949 ± 726126 ± 21.6175 ± 41.4
trans-ZR934 ± 25489.4 ± 10.274.3 ± 10.2
cis-ZR3140 ± 52553.2 ± 15.213.4 ± 2.8
iPA596 ± 38.9143 ± 6.32189 ± 27.4
iP68.6 ± 48.13.79 ± 0.432.82 ± 0.16
DZR165 ± 31.87.31 ± 1.234.17 ± 0.96
Normal PARLow R/FR ratioNormal R/FR ratioHigh R/FR ratio
  1. Means are the average of two replicate tissue samples, each harvested from a separate experiment. The (±) indicates one SE of the mean.

  2. PAR, photosynthetically active radiation.

Free base CKs3257 ± 438200 ± 54.1130 ± 47.1
Nucleotide CKs204 ± 42.3241 ± 51.2287 ± 33.1
trans-ZR689 ± 116117 ± 18.7117 ± 10.9
cis-ZR1955 ± 16037.6 ± 6.619.1 ± 3.75
iPA680 ± 63.6274 ± 37.7270 ± 9.39
iP26.9 ± 4.754.27 ± 0.082.72 ± 0.14
DZR111 ± 17.58.83 ± 1.637.73 ± 1.21
Figure 4.

Total cytokinin (CK) levels (trans-ZR + cis-ZR + iPA + iP + DZR + CKs in nucleotide and free base form – see Table 2 for individual values) in hypocotyls harvested from 7-day-old sunflower seedlings grown under varying red to far-red (R/FR) ratios and light irradiances: low photosynthetically active radiation (PAR) (open bars) and normal PAR (solid bars). The error bars indicate one SE of the mean, and mean values with the same letter do not differ significantly at P = 0.05 based on Tukey's multiple comparison test. DW, dry weight.

Uncoupling FR from R light under a very low PAR irradiance

In an attempt to better understand the role of light quality in plant canopy shade, we examined growth and endogenous phytohormone levels of sunflower hypocotyls harvested from young plants maintained for a prolonged period at a very low PAR (3 µmol m−12 s−1) under either FR or R narrow-band light. The data is presented in Fig. 5, where all dark data were set to 1, and the R and FR data were calculated relative to the dark (i.e. ‘1’). As expected, hypocotyls from 7-day-old sunflower seedlings etiolated more in the dark and less under FR light (both relative to R light). Under these treatments, endogenous IAA levels were similar, or tended to be decreased (NS) under FR light. The hypocotyls of plants grown under FR light had the highest GA1 levels. Consistent with these trends, the de-etiolated hypocotyls of the R light-treated plants showed a 4+-fold increase in GA20 levels, relative to GA1, which remained low under all FR, R and dark treatments. This strongly suggests an R light-mediated block of GA20 → GA1 metabolism when seedlings are grown under a narrow-band R light treatment.

Figure 5.

Relative (to dark, which was set to ‘1’) hypocotyl lengths and endogenous hormone levels between red (blank bars) and far-red (FR) (solid bars) light treatments at very low photosynthetically active radiation (PAR) of 3 µmol m−2 s−1. The error bars indicate one SE of the mean. IAA, indole-3-acetic acid, GA, gibberellin; CK, cytokinin; FB, free base; NT, nucleotide.

The total CK levels (Fig. 5) were similar under narrow-band R light or in dark, but increased threefold under FR light (the total free base/riboside and nucleotide CKs showed the same trends). The levels of several individual CKs (trans-zeatin riboside, cis-zeatin riboside, dihydrozeatin riboside, iPA and isopentenyl adenine) were consistently at least threefold higher under FR light than under R light or in darkness. Interestingly, we found detectable levels of trans-Z and cis-Z under this very low (3 µmol m−2 s−1) irradiance R and FR light, as well as in the dark. These unique CKs were, however, not detectable in any light quality treatment at the higher PAR irradiances of 138 and 510 µmol m−2 s−1. Even more surprising was the fact that the trans-Z levels were threefold higher and cis-Z levels were up to 30-fold higher in the dark treatment, than under narrow-band FR or R light (just the opposite of what is seen for the other individual CKs, including iP –Fig. 5). This may mean that the free base pathway (iP → t-Z ↔ cis-Z) is inhibited under the R and FR light treatments, as well as under the normal PAR irradiance –Table 2).

Application of phytohormones to sunflower seedlings under varying R/FR ratios and a low PAR of 138 µmol m−2 s−1

We also examined the effects of exogenous application of several plant growth regulators on the growth of the hypocotyls under low PAR coupled with three light quality treatments. As expected, the use of the early-stage GA biosynthesis inhibitor 2-chloroethyltrimethylammonium chloride (CCC) significantly inhibited hypocotyl growth and reduced levels of endogenous GAs, including GA1 (data not shown). Applied IAA inhibited hypocotyl elongation (and coincidentally reduced endogenous GA1– data not shown) and application of the CK, 6N-BA, had no effect on hypocotyl elongation (data not shown). Finally, application of GA3 significantly increased hypocotyl growth under all light treatments, and this increase was significantly higher for hypocotyls grown under high R/FR ratio, where endogenous GA levels are low, than for hypocotyls grown under low R/FR ratio, where endogenous GA levels are high (data not shown).


Both main components of vegetative canopy shade, a low R/FR ratio and reduced PAR irradiation, promoted elongation of sunflower hypocotyls, as was previously shown for other plant systems (reviewed in Smith 2000; Franklin & Whitelam 2005; Vandenbussche et al. 2005) and more recently for S. longipes (Kurepin et al. 2006a). The low R/FR ratio treatment, when coupled with either of reduced or normal PAR irradiances, promoted hypocotyl elongation equally well, relative to the very much reduced hypocotyl growth seen for seedlings exposed to a normal R/FR ratio (Fig. 1). Response trends, for example, a twofold increase in length (ca 60 mm), relative to the effects of varying low R/FR ratio alone (at normal PAR), or lowering PAR alone (at normal or high R/FR ratios), lead us to conclude that simultaneously reducing the R/FR ratio to 0.8 and lowering the PAR are equally important for canopy shade-induced etiolation of sunflower hypocotyls.

That said, it is the reduction in light quality, not irradiance, that appears to be most important for inducingsignificant increases in the three main classes of plant growth-promoting phytohormones, GAs (and specifically the growth-active GA1), CKs and IAA.

Thus, in these young sunflower seedlings, the reduced R/FR ratio significantly increased endogenous levels of GA20, GA1 and GA8, but not of GA19, the immediate precursor of GA20. Beall et al. (1996) also reported appreciable and significant increases in endogenous GA levels (all of GA19, GA20, and GA1) in internodes of bean plants grown under a low PAR of 147–169 µmol m−2 s−1, which had been enriched in FR irradiation, thereby yielding a 0.5 R/FR ratio.

For our sunflower seedlings, a low level of PAR irradiance, as the shade component, significantly increased GA20 levels, but only under the normal R/FR or very high R/FR ratio treatments. A similar pattern of increase for GA1 and GA8 was seen, but only under the normal R/FR ratio was significance gained (Fig. 3c,d). While the higher endogenous GA20 and GA1 levels fit well with results obtained for P. sativum (Gawronska et al. 1995), B. napus (Potter et al. 1999) and S. longipes (Kurepin et al. 2006b), the inability of a low PAR irradiance at the lowest R/FR to increase endogenous GA1, GA8 or GA20 levels (Fig. 3b–d) in sunflower hypocotyls is puzzling, because this combination (low PAR coupled with low R/FR ratio) gave appreciable and significant enhancement of hypocotyl elongation (Fig. 1).

Very little is known about the effects of light quality on CK profiles or concentrations. We can now report that, as in the case of GAs, endogenous CK levels were markedly increased in response to a low R/FR ratio, irrespective of PAR irradiance (Table 2). It is also clear that FR narrow-band light can increase CK levels above that seen for either R or darkness. However, these positive correlations of elevated CKs with FR-enriched light, or with narrow-band FR light, are not proof that CKs control light-mediated hypocotyl elongation. Further, applications of a CK (6N-BA) have no significant effect on elongation of our sunflower hypocotyls under either R/FR ratio. Tong, Kasemir & Mohr (1983) have also reported that for intact mustard seedlings, application of CKs did not affect light quality-mediated growth increases.

In terms of R light effects, both CK and R light have been thought of as factors that lead to decreased hypocotyl elongation, and do so in an additive or independent manner (Thomas et al. 1997). Subsequently, Sweere et al. (2001) proposed that their effects converged at the level of phy B Pfr, whereby a type A CK response regulator, ARR4, was thought to stabilize Pfr. To et al. (2004) questioned this model, observing that Type A arr mutants enhanced, rather than inhibited, hypocotyl sensitivity to R light. Instead, To et al. (2004) proposed that if interactions with phytochrome play a significant role, it may be that the activity of the ARRs is regulated by phytochromes rather than the ARRs regulating phytochrome activity, as originally proposed by Sweere et al. (2001). The To et al. (2004) explanation may be more consistent with the patterns we observed, which show that light quality is the critical factor for increased CK accumulation. That is, either a low R/FR ratio or the use of FR narrow-band light alone gives significantly elevated CK concentrations in the hypocotyls.

Yet, because there was no correlation between CK application and hypocotyl elongation, this would suggest that there is no direct role for this class of hormone as an important controlling factor in the elongation of the hypocotyl in response to light quality or light irradiance changes. Such a conclusion is in agreement with the results of Riefler et al. (2006), who assessed the hypocotyl growth of an Arabidopsis T-DNA mutant with all known CK receptors knocked out (i.e. ahk2 ahk3 cre1 triple mutant) under a range of light conditions. They reported that hypocotyl elongation of this triple mutant was similar to the wild type among several different light treatments including light, dark, continuous R and continuous FR. Presumably, this mutant is unable to perceive CKs. If correct, this would imply that any changes in CKs that we observed in the present study are not directly relevant to the hypocotyl elongation obtained by the various light treatments. However, the increase in endogenous CK under FR light or low R/FR ratio can still be vital for stem elongation. Further experiments involving CK biosynthesis mutants may be essential for our understanding of the role that CKs may play in elongation under FR enrichment. In addition, the increase in CK levels observed under FR enrichment can play an indirect role in stem elongation by interacting and influencing the endogenous levels and/or sensitivity of other plant hormones.

Endogenous IAA levels almost double in response to a reduced R/FR ratio, irrespective of PAR (both relative to normal R/FR). In P. sativum seedlings, end-of-day FR light increased endogenous IAA levels in the third internode (Behringer & Davies 1992). In Arabidopsis, low R/FR ratio-mediated hypocotyl elongation is IAA dependent (Steindler et al. 1999), and low PAR-mediated shoot etiolation induces auxin activity (IAA-mediated gene expression) (Vandenbussche et al. 2003). Further, Morelli & Ruberti (2000) proposed a model, whereby a low R/FR ratio changes the distribution of IAA (from vertical to lateral) in Arabidopsis hypocotyls. Unfortunately, the endogenous IAA levels were not assessed in either of the Arabidopsis studies discussed earlier.

In contrast, narrow-band FR (at the very low PAR of 3.5 µmol m−2 s−1) in our study showed a modest (but significant) reduction in endogenous IAA levels, relative to narrow-band R or to dark (Fig. 5). Hence, for IAA at least, one cannot generalize between a low R/FR ratio administered via white light (under relatively high PAR –Fig. 2) and narrow-band FR at exceptionally low PAR.

To conclude, a reduction in both R/FR ratio (light quality change) and light irradiance can significantly promote elongation of sunflower hypocotyls. Further, this light quality (especially) or light irradiance-induced elongation is positively and significantly correlated with coincidental increases of plant hormones, including IAA and certain GAs. Endogenous CK levels also increase, but the causal involvement of this CK increase in hypocotyl elongation seems doubtful.

Thus, in this rather unique system (the elongating hypocotyl of a ‘sun’ plant), a low R/FR ratio appears to be the more potent signal for yielding increased endogenous levels of the growth-active GA1 (and its precursor GAs), and of IAA and CKs. In contrast, a lowering of PAR irradiance, although effective in promoting hypocotyl growth, increases levels of the growth-active GA1 only under a normal R/FR ratio (Fig. 3c). Similarly, its precursor, GA20, is also appreciably and significantly elevated by low PAR irradiance, but again, only under the higher R/FR ratios. Hence, low light irradiance may be able to exert its influence on GA metabolism only when seedlings are subjected to the de-etiolating influence of higher R/FR ratios.

The difference in magnitude of the increased endogenous GA response to our reduced R/FR ratio versus our lower PAR irradiance can possibly be explained by the nature of these two light signals. The low PAR signal is quite common in nature, but usually occurs without being coupled to a low R/FR ratio (e.g. it most often occurs in response to cloud cover and adjacent non-canopy shade). In contrast, the low R/FR ratio signal in nature is restricted to natural canopy shade and neighbouring plant shade, or end-of-the-day irradiance, where it is usually coupled with lower than normal PAR. Therefore, it seems reasonable that various plant organs have developed two light-induced mechanisms to control shoot growth via changes in plant hormones. For the hypocotyl system, one is low PAR mediated and appears to involve subtle increases in growth-active GA levels, but only under a normal R/FR. The other is low R/FR ratio mediated and involves massive increases in GA levels. Thus, under conditions of natural canopy shade, that is, low R/FR ratio and low PAR, the low R/FR ratio signal may override the low PAR signal in controlling endogenous GA and IAA levels.


We would like to thank Ms B. Smith and Mr K. Girard for excellent greenhouse assistance and Dr R. Zhang for his advice and help with GC-MS analysis. This work was funded by Natural Sciences and Engineering Research Council (NSERC) (Canada) grants to R.P. Pharis, D.M. Reid and R.J.N Emery.