JXB Advance Access originally published online on May 8, 2007
Journal of Experimental Botany 2007 58(8):2145-2157; doi:10.1093/jxb/erm068
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RESEARCH PAPER |
Uncoupling light quality from light irradiance effects in Helianthus annuus shoots: putative roles for plant hormones in leaf and internode growth
1Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4
2Biology Department, Trent University, Peterborough, Ontario, Canada K9J 7B8
* To whom correspondence should be addressed. E-mail: leon{at}phytophys.com
Received 15 August 2006; Revised 6 March 2007 Accepted 8 March 2007
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Abstract |
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An attempt has been made to uncouple the effects of the two primary components of shade light, a reduced red to far-red (R/FR) ratio and low photosynthetically active radiation (PAR), on the elongation of the youngest internode of sunflower (Helianthus annuus) seedlings. Maximal internode growth (length and biomass) was induced by a shade light having a reduced R/FR ratio (0.85) under the low PAR of 157 µmol m–2 s–1. Reducing the R/FR ratio under normal PAR (421 µmol m–2 s–1) gave similar growth trends, albeit with a reduced magnitude of the response. Leaf area growth showed a rather different pattern, with maximal growth occurring at the higher (normal) PAR of 421 µmol m–2 s–1), but with variable effects being seen with changes in light quality. Reducing the R/FR ratio (by enrichment with FR) gave significant increases in gibberellin A1 (GA1) and indole-3-acetic acid (IAA) contents in both internodes and leaves. By contrast, a lower PAR irradiance had no significant effect on GA1 and IAA levels in internodes or leaves, but did increase the levels of other GAs, including two precursors of GA1. Interestingly, both leaf and internode hormone content (GAs, IAA) are positively and significantly correlated with growth of the internode, as are leaf levels of abscisic acid (ABA). However, changes in these three hormones bear little relationship to leaf growth. By implication, then, the leaf may be the major source of GAs and IAA, at least, for the rapidly elongating internode. Several other hormones were also assessed in leaves for plants grown under varying R/FR ratios and PARs. Leaf ethylene production was not influenced by changes in R/FR ratio, but was significantly reduced under the normal (higher) PAR, the irradiance treatment which increased leaf growth. Levels of the growth-active free base and riboside cytokinins were significantly increased in leaves under a reduced R/FR ratio, but only at the higher (normal) PAR irradiance; other light quality treatments evoked no significant changes. Taken in toto, these results indicate that both components of shade light can influence the levels of a wide range of endogenous hormones in internodes and leaves while evoking increased internode elongation and biomass accumulation. However, it is light quality changes (FR enrichment) which are most closely tied to increased hormone content, and especially with increased GA and IAA levels. Finally, the increases seen in internode and leaf GA content with a reduced R/FR ratio are consistent with FR enrichment inducing an overall increase in sunflower seedling GA biosynthesis.
Key words: Internode, leaf, light irradiance, light quality, PAR, phytohormone, R/FR ratio
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Introduction |
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The internodes and leaves of plants grown in the shade of canopy or taller neighbouring plants often exhibit what is called the shade avoidance syndrome (Smith, 2000). Vegetative shade conditions are characterized by a low red to far-red (R/FR) ratio and low photosynthetically active radiation (PAR) (Smith and Whitelam, 1997; Smith, 2000). A low R/FR ratio is considered to be the more important signalling factor (Ballare et al., 1990; Smith and Whitelam, 1997; Smith, 2000), although low PAR is also considered to be an important signal in shade avoidance (Ballare et al., 1991; Ballare, 1999; Franklin and Whitelam, 2005). Both low R/FR ratio and low PAR can induce the primary morphological trait associated with internode growth in shade, which is stem elongation (Ballare, 1999; Smith, 2000). Leaves of plants that develop under a canopy or in proximity to shade from neighbouring plants will usually have reduced leaf growth as well as elongated internode growth (Smith, 2000).
The internode elongation seen in plants grown under a low R/FR ratio and/or low PAR appears to be mediated by changes in endogenous plant hormone levels (Beall et al., 1996; Vandenbussche et al., 2005; Kurepin et al., 2006a, b). Gibberellins (GAs) are potent promoters of stem elongation and two growth-active GAs, GA1 and GA4, have been shown to mediate a variety of light responses including stem growth (see review by Sponsel and Hedden, 2005). An increase in endogenous gibberellins A1, A20, and A19 in the internodes of bean plants under a low R/FR ratio (with normal PAR) was reported previously (Beall et al., 1996) and stem elongation induced by low PAR was associated with an increase in GA levels in Pisum sativum (Gawronska et al., 1995), Brassica napus (Potter et al., 1999), and Stellaria longipes (Kurepin et al., 2006b) plants. Gibberellins have also been shown to be causal for leaf growth of winter wheat (Triticum aestivum L.; Appleford and Lenton, 1991), ‘Himalaya’ barley (Hordeum vulgare L.; Smith et al., 1996), and a dwarf mutant of rice (Oryza sativa L. cv. Tan-ginbozu; Matsukura et al., 1998). Mutants of Arabidopsis that are deficient in endogenous GAs, or have impaired GA signalling, show reduced stem elongation and also have smaller leaves, relative to wild-type (WT) plants (Sun, 2004).
Indole-3-acetic acid (IAA) is the predominant auxin in most plants, with higher levels in young, growing tissues, i.e. shoot tips, young buds and leaves, young fruits, and immature seeds (Bartel, 1997). Auxins are implicated in stem elongation as well as in phototropism, gravitropism, vascular tissue differentiation, and cell expansion (Cleland, 2004; Aloni, 2004). IAA was shown to be an important factor in the low R/FR ratio-mediated increase in hypocotyl elongation in Arabidopsis (Steindler et al., 1999) and an increase in auxin activity (IAA-mediated gene expression) was detected in rosette leaves of Arabidopsis plants grown under low PAR (Vandenbussche et al., 2003). Auxin has also been shown to be important in the initiation of new leaves in tomato (Lycopersicon esculentum; Reinhardt et al., 2000), in the cell division phase of leaf expansion in Arabidopsis (Ljung et al., 2001), and in long-term inhibition of leaf blade expansion in beans (Phaseolus vulgaris) and Arabidopsis (Keller et al., 2004).
The relationship between CKs and the effects of light on plant growth, including leaf and internode growth, is complex, although applied CKs, low levels of PAR irradiance, or light with a reduced R/FR ratio are each known to elicit similar morphogenic and biochemical responses in a wide range of plant species (Thomas et al., 1997). However, whether endogenous CKs mediate photomorphogenic processes is still a matter of debate (Halliday and Fankhauser, 2003).
Very little is known about a possible role for ABA in leaf growth under shade light, although Weatherwax et al. (1996) reported that a FR light pulse (given after a R light pulse) increased ABA levels (based on radioimmunoassay) in leaves of Lemna gibba plants. By contrast, incubation of leaf discs on ABA solutions can decrease leaf expansion (Van Volkenburgh and Davies, 1983). Finally, work with the ABA-deficient maize mutant vp5 has shown that ABA can function in both a promotional and inhibitory manner in regulating shoot development under drought stress, depending on the stage of seedling development (Saab et al., 1990; Sharp and LeNoble, 2002). Thus, it should be emphasized that any role of ABA in controlling growth of the intact plant is still a matter of debate (Dodd and Davies, 2004).
For ethylene there are many research papers linking both applied and evolved ethylene to low R/FR ratio- and low PAR-mediated growth responses, although few of these have examined leaf area growth. For example, Sorghum bicolor seedlings containing a null mutation in the gene encoding phytochrome B (phyB-1) exhibited a constitutive phenotype similar to plants grown in shade and these mutants overproduced ethylene under a low R/FR ratio (Finlayson et al., 1998, 1999). Another study, using Nicotiana tabacum plants, noted that a low concentration of applied ethylene increased growth under light with a reduced R/FR ratio (Pierik et al., 2003). Further, ethylene-insensitive transgenic N. tabacum plants failed to elongate to the same extent as WT plants in response to a low R/FR ratio (Pierik et al., 2003). Finally, low PAR was shown to increase ethylene evolution in A. thaliana rosette-stage plants (Vandenbussche et al., 2003).
Here, changes in the levels of several endogenous leaf and internode hormones under a range of light quality and irradiance treatments are assessed and the likelihood that leaf-derived hormones may be causal not only for leaf growth per se, but also for growth of the internode subtending the primary leaves of young sunflower seedlings is discussed.
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Materials and methods |
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Plants and experimental system
Sunflower seeds (6946, Pioneer Seeds, USA) were imbibed in the dark for 24 h under flowing distilled water at 22 °C. Germinated seeds (24 h) were planted in soil mix (peat moss:perlite:vermiculate:fritted clay, 2:1:1:0.25 by vol.). The growth chambers (Conviron, Manitoba) were equipped with fluorescent (Sylvania cool white 160 W) and incandescent lights (Philips 60 W). Temperature was maintained at 20 °C during the 16 h light period and at 16 °C during the 8 h dark period and the seedlings were watered each day with 25% strength Hoagland's solution (Hoagland and Arnon, 1950). The varied PAR irradiance and R/FR ratio treatments began immediately after planting the seeds.
The first (youngest) internode and the expanding leaf pair at the node above the sunflower seedlings was collected and photographed on day 17 following planting (before the appearance of the second internode and second pair of leaves). These photographs were then used to obtain internode length and width, and leaf area, length, and width parameters with a Scion Image for Windows (Scion Corporation, USA). Data were analysed with ANOVA and Spearman's Rank Correlation tests on SPSS software version 13. Each experiment was done at three different times with 10 plants being subsampled for growth measurements for each replicate experiment.
Combinations of fluorescent and incandescent light sources were used to alter the R/FR ratios. Varying the distance between lights and the soil of the pot was used to alter PAR. For the highest R/FR ratio only fluorescent light sources were used, therefore yielding mostly R and blue (B) light irradiation. For the normal R/FR ratio treatment, incandescent light sources were added, thereby giving irradiation from all three wavelength bands (R, B, and FR). For the low R/FR ratio the number of incandescent light sources was increased and the number of fluorescent light sources was decreased, thereby achieving reduced R and B light irradiations and increasing FR light irradiation (i.e. mimicking canopy shade light). The irradiance values of the R/FR ratio and PAR combinations are given in Table 1. Both R/FR ratio and PAR values were measured with a Li-Cor LI-1800/22 (Li-Cor, Inc., Lincoln, Nebraska, USA) spectroradiometer. To determine R/FR ratio values, the wavelengths of 665±5 nm for red light and 730±5 for far-red light were utilized. The R/FR ratio and PAR irradiance values are given in Table 1.
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Analysis of GAs, IAA, and ABA
Internode and leaf tissue was collected and immediately frozen in liquid N2, then freeze-dried in a Freezemobile 12EL (Virtis Inc., Cardiner, NY, USA). One gram dry weight (DW) of each tissue sample was ground with liquid N2 and washed sea sand (Fisher Scientific, New Jersey 07410, USA), then extracted in 80% MeOH (H2O:MeOH=20:80, v/v). Following this, 266 ng [13C6] IAA (gift from Dr J Cohen, available from Cambridge Isotope Laboratories, Inc.), 200 ng [2H6] ABA (a gift from Drs L Rivier and M Saugy, University of Lausanne, Switzerland; added only to leaf samples) and 33 ng each of [2H2] GA1, GA8, GA19, and GA20 (deuterated GAs were obtained from Professor LN Mander, Research School of Chemistry, Australian National University, Canberra, Australia) were added to the aqueous MeOH extraction solvent as internal standards. The 80% MeOH extract was filtered through Whatman No. 2 filter paper (55 mm, Whatman International Ltd, Maidstone, England) and then purified with a C18 preparative column (C18-PC) made of a syringe barrel (inside diameter 2 cm) filled with 3 g of C18 preparative reversed-phase material (Waters Ltd) (Koshioka et al., 1983). The 80% MeOH eluate from this column was dried in vacuo at 35 °C.
The dry sample was dissolved in 1 ml of 10% MeOH with 1% acetic acid and injected into the HPLC using the method described initially by Koshioka et al. (1983). The HPLC (Waters Ltd) apparatus consisted of two pumps (model M-45), an automated gradient controller (model 680), and a Rheodyne injector (model 7125). The solvents were, pump A: 10% MeOH in 1% acetic acid (H2O:MeOH:acetic acid, 89:10:1, by vol.), pump B: 100% MeOH. A reversed phase C18 Radial-PAK (µ Bondapak column (8 mmx10 cm) was used with a manually implemented 10–73% gradient programme at a flow rate of 2 ml min–1, i.e. 0–10 min (pump A, 100%; pump B, 0%), 10–50 min (pump A, 30%; pump B, 70%), 50–80 min (pump A, 0%; pump B, 100%), 80–90 min (pump A, 100%; pump B, 0%). The HPLC fractions (4 ml) were dried in vacuo at 35 °C. Fractions from the C18 HPLC which were expected to contain the plant hormones were transferred with 100% MeOH to 2 ml glass vials, dried in vacuo, and methylated by ethereal CH2N2 for 20 min. For analysis of GAs the methylated sample was trimethylsilylated by N,O,-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) (Hedden, 1987; Gaskin and MacMillan, 1991).
The identification and quantification of IAA and GAs was carried out using a gas chromatograph connected to a mass spectrometer (GC-MS) using the selected ion monitoring (SIM) mode. The derivatized sample was injected into a capillary column installed in an Agilent 6890 GC with a capillary direct interface to the Agilent 5973 mass selective detector (MSD). The dimensions of the capillary column were 0.25 µm film thickness, 0.25 mm internal diameter, 30 m DB-1701 (Model No.: J&W122-0732, J&W Scientific, Inc.). The GC temperature programme was: 1 min at 60 °C, followed by an increase to 240 °C at 25 °C min–1, then an increase at 5 °C min–1 to 280 °C where it remained constant for 15 min before returning to 60 °C. The interface temperature was maintained at 280 °C. The dwell time was 100 ms and data was processed using HP G1034C MS ChemStation Software.
For identification of the endogenous GAs, a comparison of GC-retention times (Rts) of the GA and [2H2]-GA was used, as well as a comparison of the relative intensities of the molecular ion (M+) pairs (except for GA19/2H2-GA19, where the ion pair at m/z 434/436 was used. The relative intensities of at least two other characteristic m/z ion pairs for each endogenous GA and its deutero standard were also compared. The same approach was taken for identification of IAA and ABA utilizing [13C6]-IAA and [2H6] ABA as the internal standard. All stable isotope-labelled internal standards were added at the extraction stage with appropriate purification and chromatography being accomplished (see above) prior to GC-MS-SIM. Quantification was accomplished by reference to the stable isotope-labelled internal standard using equations for isotope dilution analysis, adapted by DW Pearce (Jacobsen et al., 2002) from Gaskin and MacMillan (1991). As noted above, each experiment was repeated three times, with the youngest internode from c. 40–120 individual plants and the youngest leaves from c. 10–20 individual plants being extracted for each replicate experiment in order to assess the endogenous hormone levels.
Extraction and purification of cytokinins from leaf tissue
Endogenous CKs were extracted from leaf tissue samples harvested from different plants than those utilized for GAs, IAA, and ABA) and purified under conditions established by Emery et al. (1998) and Ferguson et al. (2005) to prevent enzyme activity that could cause CK nucleotide degradation and CK isomerization. Thus, frozen tissue samples of known FW 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, by vol.) at 20 ml g–1 estimated DW and were extracted according to Dobrev and Kaminek (2002). One hundred ng each of 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. Since deuterated standards of cis-[9R]Z and cis-[9RMP]Z were not available, the levels of these two compounds were estimated based on the recovery of the deuterated standards of the corresponding trans-CKs. Pooled extract supernatants were dried in vacuo at 40 °C with residues reconstituted in 5 ml 1.0 M HCOOH for purification on an Oasis MCX column (Waters, Mississauga, Canada) as described in Dobrev and Kaminek (2002). Eluted nucleotides were converted to nucleosides for quantification and the resultant nucleosides were further purified on a reversed-phase C18 column (AccuBOND ODS; Fisher Scientific, Mississauga, Canada) as described in Emery et al. (2000).
LC-MS/MS conditions for cytokinin analysis from leaf tissue
Purified CK fractions were separated and analysed by a Waters 2680 Alliance HPLC system (Waters, Milford, USA) linked to a Quattro-LC triple quadrupole MS (Micromass, Altrincham, UK). The MS was equipped with a Z-electrospray ionization source (ESI). 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, 150x2.1 mm; Jones Chromatography, Foster City, 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 the 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).
Measurement of ethylene evolution from leaves
Ethylene evolution by 17-d-old leaves was measured by incubating c. 0.1 g tissue in a 3 ml syringe (1.5 ml volume) for 15 min. A 1 ml gas sample was collected and injected into a Photovac 10Splus GC (Photovac Inc., Markham, Ontario) with a photoionization detector and a 40/60 Carbopack B column (Supelco Canada, Oakville, Ontario).
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Results |
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Reducing the normal R/FR ratio to a low R/FR ratio of 0.85 significantly increased internode elongation under both low and normal PAR irradiances (Fig. 1A). By contrast, an increase in R to yield the abnormally high R/FR ratios of 4.5–4.7, gave a significant reduction in internode length, relative to the internode elongation seen under the normal R/FR ratio (Fig. 1A). This decrease in elongation occurred under both low and normal PAR irradiances (Fig. 1A). When the internode elongation that occurs in response to low PAR is compared between the three R/FR ratios (Fig. 1A), there is a notable (and significant) trend for greater elongation as the R/FR ratio decreases (Fig. 1A).
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0.05.Coincidental with the decrease of internode elongation, the width of internodes increased significantly under the abnormally high R/FR ratio (relative to the normal R/FR ratio) and this increase in width occurred under both low and normal PARs (Fig. 1B).
Internode fresh weight (FW) and dry weight (DW) under low PAR (Fig. 1C, D) followed the trends observed for internode elongation (Fig. 1A), i.e. an increase in FW or DW was associated with a decrease in R/FR ratio. Under the low PAR, all comparisons between R/FR ratios were significant. However, under normal PAR the FW and DW increases were significant only when the abnormally high R/FR ratio was compared with the normal R/FR ratio. Thus, plants grown under an abnormally high R/FR ratio showed significant reductions in both fresh and dry internode weights at both PAR irradiances.
The endogenous growth-active GA in sunflower hypocotyls was shown previously to be GA1 (Pearce et al., 1991). In the first internode of the young sunflower plant GA1 is also present and it increased significantly (2–3-fold) in response to the low R/FR ratio treatment under both PAR irradiances (Fig. 2A). Similar trends were observed for GA8, the inactive catabolite of GA1 (Fig. 2B), as well as for GA20, the precursor of GA1 (Fig. 2C). However, there was no significant PAR irradiance effect on levels of endogenous GA1 in these young internodes at any of the three R/FR ratio treatments (Fig. 2A). By contrast, low PAR significantly enhanced levels of both GA8 and GA20 at the reduced R/FR ratio. Also, levels of GA19 were significantly promoted at all R/FR ratios by low PAR irradiance (Fig. 2D).
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Endogenous IAA levels were significantly elevated in the sunflower internodes in response to a reduced R/FR ratio and significantly lowered in response to the abnormally high R/FR ratio (both relative to the normal R/FR ratio). These responses occurred under both low and normal PAR irradiances (Fig. 3). Thus, endogenous IAA levels in sunflower internodes showed the same trend as seen above for GA1 levels (Fig. 2A). However, there was no significant effect of PAR irradiance on IAA content of the internode tissue.
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Leaf size and plant hormone levels in leaves were also assessed at day 17 following germination. At this time, the first pair of sunflower leaves has just surpassed 50% of their final growth and the second pair of leaves and second internode were undeveloped, with only the epicotyl being visible.
Under the low PAR irradiance (157 µmol m–2 s–1), a reduction from a normal R/FR ratio of 1.36 to a low R/FR ratio of 0.85 significantly promoted leaf area growth (Fig. 4A). Interestingly, an increase from the normal R/FR ratio to an abnormally high R/FR ratio of 4.52 under these low PAR conditions gave the same result (Fig. 4A). However, it should be emphasized that total light irradiance remained constant under all R/FR ratio treatments. For plants grown under the low R/FR ratio at a low PAR irradiance, it was the increased leaf length (width remained constant) that was responsible for the resultant (significant) leaf area growth increase (compare Fig. 4B with 4C). In contrast, the increase in leaf area for plants grown under the high R/FR ratio at the low PAR irradiance was due to an increased leaf width (length remained constant; compare Fig. 4C with 4B). Leaf biomass (DW) for plants grown under the low PAR (Fig. 7A) roughly mimicked the trends seen for leaf area (Fig. 4A), as did leaf FW (data not shown).
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When plants were grown at the higher (normal) PAR irradiance (421 µmol m–2 s–1) leaf area growth was maximal under both a reduced (0.87) or normal (1.42) R/FR ratio. However, when the R/FR ratio was increased to an abnormally high 4.73 under the normal PAR irradiance, leaf areas were significantly diminished (Fig. 4A) due to significant reductions in both length and width (Fig. 4B, C).
At the low PAR irradiance, the levels of endogenous GA1 found in these young sunflower leaves increased significantly when plants were grown under a low R/FR ratio, relative to our normal or very high R/FR ratios (Fig. 5A). However, as R/FR ratio was reduced these steadily increasing and significant trends in leaf GA1 content (Fig. 5A) bore no obvious relationship to leaf area growth (Fig. 4A). Interestingly, although, these leaf GA1 content trends across the three R/FR treatments were correlated with the trends seen for internode growth (Fig. 1A) and internode GA1 content (Fig. 2). Thus, leaf GA1 trends with FR-enrichment were positively correlated (P=0.01) with internode length (Fig. 1A) and also with internode GA1 content under each of low and normal PAR irradiances (Fig. 2). Similar trends also occurred across R/FR treatments under the low PAR irradiance for leaf GA8, GA20, and GA19 levels (Fig. 5B, C, D). But, as seen for GA1 content, trends for these other leaf GAs were not correlated with leaf growth. Rather they were positively correlated (at P=0.01 based on Spearman's Rank Correlation test) with internode growth and internode GA content, for example compare Fig. 5 with Fig. 2.
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Sunflower seedlings grown under a low PAR irradiance showed a positive effect (P=0.01 based on Spearman's Rank Correlation test) of a reduced R/FR ratio on endogenous IAA levels in the growing leaf (Fig. 6A). Under normal PAR there was a similar trend of higher IAA levels with a reduction in R/FR ratio (Fig. 6A). This trend in IAA parallels the similar significant trends seen for leaf GAs, although neither data set was correlated with leaf growth per se. Instead, the trends for increasing leaf IAA content across R/FR ratio treatments are positively correlated (P=0.01 based on Spearman's Rank Correlation test) with the growth trends seen for internode length and internode DW (Fig. 1).
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Leaf CKs were also measured and a significant increase in total CKs of the leaves was observed in the low R/FR ratio treatment under normal PAR, for which CK levels were double those seen for all other treatments (Table 2). This increase in total CKs was comprised mainly of 3–8-fold increases in free base (FB) and riboside (R) CKs (Table 2). It is these CKs which are presumed to be ‘growth-active’ (Sakakibara, 2006). In particular, two ribosides, [9R]DHZ, [9R]iP, showed markedly higher levels. [9R]DHZ is considered active and resistant to breakdown by cytokinin oxidase/dehydrogenase and [9R]iP is its precursor. By contrast, nucleotide (NT) CK concentrations remained relatively constant among all treatments.
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Endogenous leaf ABA content was also measured under each of the low and normal PAR irradiance levels (Fig. 6B). The ABA content increased significantly as R/FR ratio decreased (high
normallow) under both PAR irradiance levels. Thus, as seen for leaf IAA and GA1 levels, there is a significant trend for FR-enrichment (decreasing R/FR ratio treatments) to elevate leaf ABA levels. By contrast, low PAR irradiance significantly reduced leaf ABA levels at all R/FR ratios. Interestingly, trends in leaf area and leaf length growth, at the normal to the low R/FR ratio (Fig. 4A, B) were positively correlated (P=0.01 based on Spearman's Rank Correlation test) with leaf ABA content (Fig. 6B). In addition, leaf ABA increases (at both PAR irradiances) across all three decreasing R/FR ratios were also positively correlated (P=0.01 based on Spearman's Rank Correlation test) with internode elongation growth at both irradiances (Fig. 1A). Thus, elevated leaf ABA levels were associated with increased growth for both leaves and internodes. Leaf biomass changes (Fig. 7A) showed an unusual pattern at the higher (normal) PAR irradiance, one which was not correlated with ABA content. Rather, leaf biomass changes were closely correlated with the ratios of ABA:GA1 content (Fig. 7B) and ABA:IAA content (Fig. 7C). For example the light treatment (normal R/FR ratio) that yielded significant 1.3–1.6-fold-increases in the ABA:GA1 and ABA:IAA ratios also gave similar (significant) 1.6-fold increase in leaf DW accumulation. Ethylene evolution by sunflower leaves (Fig. 6C) increased significantly under reduced PAR irradiance. Elevated ethylene production was thus correlated with very reduced leaf areas (Fig. 4A) across all R/FR ratio treatments, although significance occurred only at the low and normal R/FR ratios (Fig. 6C).
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Discussion |
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In this experimental system, sunflower internodes elongated significantly in response to each of low PAR irradiance and a reduced R/FR ratio. This is the expected response for a ‘sun’ plant (Ballare, 1999; Smith, 2000; Kurepin et al., 2007a) and the effect of lowering PAR irradiance was maximal when given under the reduced R/FR ratio treatment. Also, as expected, internodes decreased in length significantly in response to an abnormally high R/FR ratio treatment under each of the low and normal PAR irradiances. Thus, for plants growing under low PAR, a change from a normal to a low R/FR ratio gave significantly increased internode elongation accompanied by a significant DW increase, but with no reduction in internode width (Fig. 1B). Internode DW trends (Fig. 1D), as the R/FR ratio is decreased, are thus very similar to internode length trends (Fig. 1A). Hence, the light quality component of shade not only etiolates the internode, it makes the internode a stronger sink for photoassimilate allocation, and it does this under both reduced and normal PAR irradiances. Of even more interest, when one expands the light quality option to include an abnormally high R/FR ratio, the above trends are extended, with most growth responses (decrease in elongation) to enrichment with R light being significant (Fig. 1A–D).
Leaf area decreased appreciably and significantly in response to the reduced PAR component of shade, due to significant decreases in both leaf length and width (Fig. 2A–C). There were also significant decreases in leaf DW (Fig. 7A). Similar results have been reported previously (reviewed by Ballare, 1999; Smith, 2000; Franklin and Whitelam, 2005; Vandenbussche et al., 2005). However, changes in the R/FR component of shade gave ‘mixed’ responses in leaf growth, which appeared to be dependent on PAR irradiance. Thus, under normal PAR there was a significant trend for the abnormally high R/FR ratio to reduce both leaf length and width, and thus area (Fig. 2A–C). However, under low PAR, deviation from a normal R/FR ratio in either direction significantly increased leaf area (Fig. 2A), due either to increased leaf length (Fig. 2B) or to increased leaf width (Fig. 2C). These leaf area and DW responses are thus appreciably different from those seen for internodes.
Associated with the reduced R/FR ratio treatment (under either low or normal PAR irradiances) were significantly increased levels of growth-active GA1 in these rapidly growing internodes (as reported for elongating bean stems by Beall et al., 1996, and for elongating hypocotyls by Kurepin et al., 2007a). IAA levels also increased significantly as the R/FR ratio was reduced, similar to previously reported data for sunflower hypocotyls (Kurepin et al., 2007a). These significant increases in GA1 and IAA in response to a reduced R/FR ratio imply phytochrome mediation and both increases have, essentially, parallel slopes (Figs 2A, 3). Gibberellin A1 and IAA may thus be acting in concert as causal growth-effectors of the internode elongation that is induced by FR enrichment.
In addition, the levels of GA20 and GA19, two biosynthetic precursors of GA1, are also elevated in the internodes by reduced R/FR ratio treatments (especially under low PAR), as is the level of GA8, the inactive catabolite of GA1 (Fig. 2). This suggests that the combination of a low R/FR ratio with low PAR is enhancing overall GA biosynthesis in the young sunflower seedling, i.e. it is increasing the metabolic flow of GA19GA20GA1GA8. An analogous situation has recently been shown for shoots of Stellaria longipes grown under low PAR (Kurepin et al., 2006b) and for hypocotyls of sunflower seedlings grown under low R/FR ratio (Kurepin et al., 2007a). Also, a rapid metabolism of tritiated GA20GA1GA8 was associated with inherently rapid growth in maize hybrids (Rood et al., 1983).
In contrast to modifying PAR, changing the R/FR ratio appears to be a more potent factor in affecting internode GA level (Fig. 2). Even so, reducing PAR irradiance at the low R/FR ratio significantly increased levels of endogenous GA8, GA19, and GA20 in the internodes, although not GA1 (Fig. 2). In Pisum sativum (Gawronska et al., 1995), Stellaria longipes (Kurepin et al., 2006b), and Brassica napus (Potter et al., 1999), the increased stem elongation that was induced by low PAR irradiance was also significantly correlated with an increase in endogenous GA levels.
Leaf GA levels were appreciably affected by FR-enrichment (use of the low R/FR ratio treatment). Thus, a low R/FR ratio, when coupled with either low or normal PARs, significantly increased levels of endogenous growth-active GA1, its inactive catabolite, GA8, and its immediate precursor, GA20, in leaves, all relative to levels of these GAs found under the normal R/FR ratio treatment. However, these GA increases were positively correlated with leaf elongation (and the resultant leaf area increase) only at the low PAR irradiance under the low R/FR ratio. Even so, it seems reasonable to conclude that plants grown under ‘canopy shade’ do utilize endogenous GAs to promote leaf elongation, thereby increasing leaf area expansion. Interestingly, elevated levels of endogenous leaf GAs showed positive and significant correlations with increased internode growth across the range of R/FR ratios at both levels of PAR irradiance (compare Fig. 5 with Fig. 1). Hence, the primary leaf pair may well be the source of growth-active GA1 and its precursor GA20 for both the increased internode growth seen under canopy shade and also for growth of the sunflower internode under normal R/FR ratio and normal PAR irradiance. The transport of GAs in phloem and xylem tissues is well known (Chin and Lockhart, 1965; Proebsting et al., 1992; Davies, 2004).
Endogenous levels of IAA in the sunflower internodes were not significantly affected by reducing the PAR to 157 µmol m–2 s–1, although reducing the R/FR ratio (from abnormally high to normal and from normal to low) gave highly significant increases in internode IAA content, increases which paralleled the elongation response (compare Fig. 1A with Fig. 3). Thus, it seems likely that one cannot generalize between species or genera in allocating the effects of reduced light (PAR) irradiance, relative to a reduction in R/FR ratio, on modifications in levels of endogenous plant growth hormones. That said, we cannot rule out the possibility that PAR irradiances that are associated with full canopy shading (i.e. below 150 µmol m–2 s–1), where elongation is even greater, may influence the internode's endogenous hormone content differently.
Endogenous levels of leaf IAA also increased with FR-enrichment (e.g. as the R/FR ratio decreased from the normal R/FR ratio to a low R/FR ratio) under both PAR irradiances. However, this increase in leaf IAA content was significant only at the normal PAR irradiance (where the leaf area growth increase was not significant). Thus, shade-induced trends in leaf IAA content are not significantly correlated with leaf growth. However, shade light- (reduced R/FR ratio) induced increases in leaf IAA are positively correlated with elongation growth of the subtending internode under both normal and low PAR irradiances (Spearman Rank Correlation of P=0.01; compare Fig. 6A with Fig. 1). As for GAs, then, there is an implication of movement of IAA from the primary leaves down to the rapidly growing internode. In Arabidopsis seedlings, low R/FR ratio-mediated hypocotyl elongation has also been shown to be auxin dependent (Steindler et al., 1999) and treatment with NPA (a putative inhibitor of IAA transport) significantly reduced the low R/FR ratio-induced hypocotyl elongation of wild-type Arabidopsis seedlings (Steindler et al., 1999).
Vandenbussche et al. (2003) has also shown that low PAR will increase auxin activity (IAA-mediated gene expression) in A. thaliana rosette seedlings, although light quality effects on auxin activity were not examined.
There was a substantially enhanced (maximal) elongation of sunflower internodes when both components of ‘shade light’ were increased and this elongation was also associated with a significantly increased DW of the internodes. This suggests that both major components of shade light will likely be required for successful competition by a ‘sun’ plant when it grows in association with taller neighbouring vegetation.
To date no other studies that report on CK profiles in leaves exposed to changing R/FR ratios have been seen and there have only been a few reports where CKs at different light irradiances were examined (Hammerton et al., 1998; Bukhov et al., 1999; Pons et al., 2001). In these latter studies, detection was either non-specific for CK form, or only a few of the CKs were identified and quantified. Only one of our light quality treatments, a reduced R/FR ratio at the normal PAR, yielded significant changes in leaf CK concentrations (Table 2). Levels of individual CKs, specifically iPA, DZR, t-ZR, c-ZR, and c-Z were appreciably elevated under this treatment. A similar trend in growth (increases under a reduced R/FR ratio) was also seen for leaf area, length and width (Fig. 4A, B, C). However, since there were no CK changes at other R/FR ratios or for different PAR irradiances (Table 2), it seems premature to assign any causal role for CKs in leaf growth. Although it is possible that CK levels in internodes do increase in response to low R/FR ratio, as was shown for sunflower hypocotyls subjected to the similar levels of R/FR ratios and PARs (Kurepin et al., 2007a).
When the R/FR ratio was decreased from highnormallow, ABA levels were significantly increased in leaves under both PAR irradiance levels (Fig. 6B). These leaf ABA contents at the higher PAR irradiance were positively correlated with increased leaf elongation and even leaf width increases (and thus with leaf area) as FR enrichment increased (compare Fig. 6B with Fig. 4A, B, C).
Changes in leaf ABA content across R/FR ratios were also positively and significantly correlated with concomitant changes in internode length and internode biomass (compare Fig. 6B with Fig. 1A and D). Such trends (increased ABA being associated with the growth induced by changes in light quality) have been reported in the literature. For example, a FR light pulse given after an R light pulse increased ABA levels (compared with the R light pulse alone) in L. gibba plants (Weatherwax et al., 1996).
While changes in leaf ABA content (Fig. 6B) show no obvious correlation with leaf DW trends as light quality changes (compare Fig. 6B with Fig.7A at the higher, normal PAR irradiance), there are good correlations between leaf biomass changes (Fig. 7A) and the ratios of ABA:GA1 content (Fig. 7B) and ABA:IAA content (Fig. 7C). Thus, the allometric distribution of photoassimilate within the plant may be influenced not only by levels of a specific hormone, but also by its interactions with other hormones. It has been reported that photoassimilate accumulation (sink strength) can be enhanced in cotyledons, seeds, and other tissues by exogenously applied ABA, whereas applied IAA and GA do not show this effect (Brenner, 1987).
Ethylene evolution by sunflower leaves was significantly enhanced under low PAR when the plants were also grown under the low and normal R/FR ratios. Similar results (but only under a normal R/FR ratio) were previously obtained for A. thaliana rosette-stage seedlings (Vandenbussche et al., 2003). Because leaf expansion was appreciably and significantly inhibited by low PAR coupled with low and normal R/FR ratios, the increased ethylene evolution that was seen under this light regime was associated with decreased leaf expansion. Alonso et al. (1999) have reported that leaves of ethylene-insensitive ein2-1 Arabidopsis seedlings were notably larger than leaves of WT seedlings. In sunflower plants, low concentrations of applied ethylene gas can promote leaf expansion, whereas higher concentrations inhibit it (Lee and Reid, 1997). Hence, it seems possible that low light irradiance levels negatively influence sunflower leaf area expansion via increased ethylene production. Although the change in R/FR ratio did not result in significantly altered ethylene levels in sunflower leaves, in internodes its levels decreased as the R/FR ratio was lowered (Kurepin et al., 2007b).
To conclude, growth of sunflower internodes and leaves is greatly influenced by both decreases in R/FR ratio and in PAR irradiance. For internode growth, reductions in both types of light appear to ‘signal’ significantly increased internode elongation. However, only lowering the R/FR ratio significantly increases the levels of endogenous GAs and IAA in internodes. Although canopy shade light (reduced R/FR ratio) did promote leaf length and leaf area growth, this increased growth only occurred at the low PAR irradiance. By contrast, high PAR invariably promoted leaf area and biomass growth across all three R/FR ratios. While the levels of several endogenous hormones, GA1, IAA, and ABA, increased significantly in leaves in response to reducing the R/FR ratio, these increases in hormone levels were not always translated into increased leaf growth. Rather, changes in the leaf content of these three hormones were best correlated with the enhanced growth of the subtending internode.
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We would like to thank Ms Linda J Walton for the assistance with ethylene analysis, Mr Ian Reeves for the assistance with LC-MS/MS analysis, Dr Ruichuan Zhang for the assistance with GC-MS analysis, and Ms Bonnie Smith and Mr Ken Girard for excellent greenhouse assistance. This work was funded by NSERC (Canada) grants to DMR, RJNE and RPP.
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