Fluorescence investigations of phytochrome (phy) in rice (Oryza sativa L. cv. Nipponbare) mutants deficient in phyA, phyB and phyA plus phyB were performed. Total content of the pigment (P_tot) and its spectroscopic and photochemical characteristics were determined in different parts of the dark-grown and far-red light (FR)-grown coleoptiles. Spectroscopically, phyA in the phyB mutant was identical to phyA in the wild-type (WT) and the extent of the conversion from Pr to lumi-R at 85 K was the same for phyA in both lines and varied similarly, depending on the part of the coleoptile used. The latter finding proved that phyA in rice is heterogeneous and comprises two phyA populations, phyA′ and phyA″. Functional properties of phyA were also determined. In the dark the phyB mutant had a higher content of phyA, inactive protochlorophyllide (Pchlide⁶³³) and active protochlorophyllide (Pchlide⁶⁵⁵) than WT and its coleoptile was longer, indicating that phyB may affect the development of WT seedlings in the dark. Constant FR drastically reduced the content of phyA, Pchlide⁶³³ and Pchlide⁶⁵⁵ and brought about coleoptile shortening and appearance of the first leaf, whereas pulsed FR of equal fluence was less effective. This suggested that the reactions were primarily of the high irradiance responses type, which are likely to be mediated by phyA′. The effects on protochlorophyllide biosynthesis and growth responses type were more pronounced in the phyB mutant than in the WT seedlings, which can be connected with the higher phyA′ content in the phyB mutant and/or phyB interference with its action in WT seedlings. In the phyA mutant induction of Pchlide⁶³³ and Pchlide⁶⁵⁵ biosynthesis was observed under constant FR, indicating that phyC may be responsible for this effect.

INTRODUCTION

Autotrophic plants adapt to environmental light conditions with the aid of a sophisticated photosensory apparatus comprising a number of photoreceptor systems, of which the phytochrome (phy) system is the most systematically investigated. Phy mediates a wide spectrum of photoresponses, ranging from induction of seed germination and seedling establishment to regulation of flowering and senescence. On the basis of light requirements these photoresponses were categorized into the irreversible very low fluence responses (VLFR) and FR-induced high irradiance responses (HIR), “classical” red (R)-induced/FR-reversible low fluence responses (LFR) and responses to ratios of R to FR (1).

The complexity of the phy functions implied that more than one phy molecular species may be involved in the photoresponses. This concept was supported by the following direct experimental observations of phy heterogeneity: labile and stable phy pools (2), immunologically distinct phy populations (3) and phy species with different fluorescence and photochemical properties (4,5). With the advancement of genetic and molecular biological techniques and the discovery of phytochrome mutants, the phy heterogeneity gained rational interpretation: a small family of phytochromes—products of phy genes—was discovered with phyA and phyB being the major types (6). phyA is light labile (type I), whereas phyB and minor phytochromes (phyC and phyE in Arabidopsis species) are light stable (type II) (1,7). phyB was shown to control the photoreversible LFR and responses to R:FR ratios, whereas phyA was shown to control irreversible VLFR and HIR. However, a number of data indicate that phyA may also mediate the LFR reactions characteristic of phyB (8–11). The minor phytochromes (phyC and phyE) perform functions that are redundant or specific to those of phyA and phyB.

The functional roles of the different members of the phy family have been characterized primarily in dicots because of the availability of their phy mutants (1). The recent discovery of phy mutants in rice, on the other hand, opened an avenue for the investigation of the individual functions of monocot phytochromes (10–12). The phy family in rice has only three members (phyA, phyB and phyC) and this makes it more advantageous to investigate functions of the individual phytochromes. An analysis of isolated null mutants of the three phytochromes showed that phyA is responsible for the inhibition of coleoptile elongation with a pulse of light (VLFR). It is involved in the inhibition of mesocotyl elongation and the induction of gravitropic response under continuous FR. A defect of phyA in rice causes changes restricted to the de-etiolation process and does not have an effect in light-grown plants. Thus, phyA operates via the VLFR and HIR modes in de-etiolation. On the other hand rice-etiolated seedlings of the wild-type (WT) and phyB and phyBphyC mutants show the R/FR photoreversibility of the induction of CAB gene expression, suggesting that phyA is involved not only in the VLFR mode but also in the R/FR reversible LFR mode, phyB is not a sole photoreceptor for R, as shown by the inhibition of the coleoptile elongation in the phyB mutant. Finally, phyC has minor roles in photomorphogenesis: no difference between WT and the phyC mutant was observed in inhibition of coleoptile or mesocotyl elongation under R or FR.

The complexity of the phyA-mediated photoresponses suggests in turn that this may be also connected with the polymorphism of this pigment. With the use of fluorescence spectroscopy and photochemistry of phy in phy mutants and transgenic plants it was found that there are two phyA populations, phyA′ and phyA″ (13–15). The most pronounced phenomenological distinction between phyA′ and phyA″ was their different photochemical activity at cryogenic temperatures (i.e. their ability to undergo the initial photoconversion from Pr to lumi-R [Pr→lumi-R]): the extent of the photoconversion at 85 K was high for phyA′ (γ₁= 0.5) and low for phyA″ (γ₁≤ 0.05). It was shown also that phyA′ had the emission (absorption) maxima (λ_max) at 687 (673) nm, whereas the λ_max for phyA″ was 682–683 (668) nm, phyA′ is quantitatively the major species but is rapidly lost in constant R (Rc) light. Its concentration is highest in apical parts of seedlings and root tips. In contrast phyA″ is a minor species that is more stable in the light and its concentration does not change significantly with tissue type. Thus, phyA′ and phyA″; belong to different phenomenological types, designated Pr′ and Pr″, respectively. Because phyB has characteristics close to those of phyA″, it belongs to the same phenomenological type (Pr″). The two phyA pools are products of the same gene and differ in posttranslational modification (possibly phosphorylation at the 6-kDa N-terminal domain) and/or in intra-cellular distribution of the pigment, phyA′ is soluble, whereas phyA″ is likely to be membrane and/or protein bound.

Experiments on phy mutants and transgenic plants with altered phyA′/phyA″ content and modified photoresponses revealed that the two phyA species have different functions (14,15). As judged by the regulation of active and inactive protochlorophyllide accumulation, growth responses and autoregulation of phyA synthesis, the light-labile phyA′ is responsible for de-etiolation via the HIR mode. It may be also active in the VLFR. phyA″, on the contrary, could participate in R-induced responses and interfere with the action of phyA′. The latter activity, in particular, is suggested by the fact that overexpression in transgenic tobacco of Δ7–69-truncated oat phyA, which is represented by phyA″, inhibits the effect of FR on the active protochlorophyllide (Pchlide⁶⁵⁵) biosynthesis and cotyledon unfolding but increases the effect of R on cotyledon unfolding, compared with WT. It is believed that, in general, the relatively light-stable phyA″ might be functional in green plants. The specificity of the phyA′ and phyA″ functions might be connected with their differential regulation by Rc and FRc. Both illuminations bring about considerable lowering of the phy content in peas. However, the effect under R was due primarily to the destruction of phyA′, whereas under FR, down-regulation of phyA synthesis dominated over destruction, with the phyA′/phyA″ ratio remaining relatively constant. This supports the notion that phyA′ is dispensable under Rc and is active under FRc, whereas the opposite is the case for phyA″. Thus, this complex interrelation between the phyA′ and phyA″ functions (including the antagonistic interactions) suggests that the maintenance of the optimal ratio between phyA′ and phyA″ could be part of the mechanism of the fine-tuning of the phyA activity.

The fact that phyA is represented in vivo by the two isoforms with properties of the Pr′ and Pr″ phenomenological pools was rigorously shown for the dicots because of the availability of their phy mutants (16–18). The two Pr types were also found in monocots (i.e. oat, wheat, barley, rice and maize (19–21)), which, similar to dicots, could be attributed to the presence of phyA′ and phyA″, on the basis of their spectroscopic and photochemical properties and their abundance in and distribution among plant species and tissues under different physiological conditions. However, the lack of phyA and phyB mutants of monocots did not allow us to present a rigorous proof that phyA in these plants also exists in the two isoforms. The isolation of the phy mutants of rice (10–12) opened this perspective and in this work we have shown that this holds true for the monocots as well.

MATERIALS AND METHODS

We used low-temperature fluorescence spectroscopy of phytochrome in etiolated tissues as a major experimental approach (16–21). The procedure of the rice seedlings growth, sample preparation and spectral measurements were essentially the same as those reported elsewhere (21). Briefly, coleoptiles of 5 day old rice seedlings (WT Oryza sativa L. cv. Nipponbare and the phyA-, phyB- and phyAphyB-deficient mutants isolated from the same background) were taken for the experiments. The seedlings were grown in complete darkness (D) or under FRc or pulsed (FRp; 7 min light and 53 min D) (λ_a≥ 720 nm) of equal fluences on floating meshes in a tray filled with tap water at 27 °C. The light source was a 100 W tungsten lamp with a combination of KS-19 and FS-7 filters (thickness, 3 mm; Optical Glass Plant, Krasnogorsk, Russia) at a fluence rate of ca 0.5 μmol m⁻² s⁻¹ for FRc and 5.0 μmol m⁻² s⁻¹ for FRp. Coleoptile segments 3–5 mm long taken from the tip, middle or base were harvested for the measurement. Three to six segments were glued to a Plexiglas sample holder by means of a water-glycerol mixture (50:50) and frozen at 85 K in a cryostat consisting of a brass sample holder and a transparent Dewar flask. All the manipulations were performed under green safe-light.

A laboratory-designed spectrofluorometer based on double-grating monochromators (DFS-24 and DFS-12, LOMO, St. Petersburg, Russia) that are commonly used for Raman spectra measurements was used for spectral measurements. The spectra of phytochrome in seedlings were measured at 85 K in the interval of 650–740 nm under excitation with low-intensity photochemically inactive light at λ_e= 632.8 nm (light source, He-Ne laser, 5 mW [LGN-207B, Ryazan′, Russia]; monochromator, MDR-2 [LOMO]; and grey filters, NS-3 and NS-9 [Optical Glass Plant] to reduce the fluence rate of the excitation light) and the spectra of protochlorophyllide were measured in the interval of 600–700 nm under excitation at λ_e= 450 nm (light source, 500 W Xenon arc lamp [DKsSh-500, Electric Lamp Factory, Saransk, Russia]; and monochromator, DFS-12). The slit width of the excitation and emission monochromators was 2 nm. In the case of phytochrome, two spectral measurements were made, one from the sample in the dark-adapted state when all the pigment is in the initial Pr form (F₀ state) and the second from the same sample after saturating illumination for 10 min with full light (without the grey filters) of the same laser. This illumination partially converted the Pr form into the first photoproduct lumi-R, which is stable at a low temperature (85 K) (F₁ state, photoequilibrium Pr↔lumi-R). The emission spectra of protochlorophyllides were measured for the same sample after the registration of the phytochrome spectra.

As it was shown elsewhere (19), the spectra of etiolated tissue are represented by the individual spectra of the phytochromes and also by the spectrum of the background fluorescence. In this work, to get individual spectra of phyA, the spectrum of the phyA mutant was subtracted from that of WT and the spectrum of the phyAphyB double mutant was subtracted from the spectrum of the phyB mutant (see below) (Fig. 1). The total content of phyA (P_tot) was evaluated (in relative U) as a ratio of the fluorescence intensity in the phyA spectrum in the maximum at ca 685 nm to the intensity of the background light at ca 660 nm which is proportional to the mass of the plant tissue under the excitation light beam; Sineshchekov (19) discusses the fluorescence technique and its precision for determining the phy content in plant tissues.

Raw (a,b), difference (c,d) and normalized (e,f) low-temperature (85 K) fluorescence emission spectra (λ_e= 632.8 nm) of phytochrome in the tips of rice coleoptiles: in the initial etiolated state, when all the pigment is in its Pr form, state 0 (curve 1), and after saturating red (λ_a= 632.8 nm) illumination at 85 K converting part of Pr into the first photoproduct stable at low temperature (Pr↔lumi-R photoequilibrium), state 1 (curve 2); wild-type (a,c,e); phyB mutant (b,d,f). The spectra of etiolated coleoptiles of the rice phyA and phyAphyB mutants (curve 3) are also given in (a) and (b), respectively. The spectra in (c) and (d) belonging to phyA are obtained by subtraction of curve 3 from curves 1 and 2 in (a) and (b), respectively, after their normalization at 660 nm, where the emission of phytochrome is neglegible. The spectra in (e,f) are the average phyA spectra obtained as in (c,d) of 3–6 different samples after their normalization in the maximum (in state 0) to 1. The spectra were not corrected for the spectral sensitivity of the fluorimeter. Bars = SD.

Photochemical properties of phy in its Pr form were judged on the basis of the extent of the Pr→lumi-R photoconversion at 85 K (defined as γ₁). The latter was evaluated from the decay in Pr fluorescence from its level in the initial phyA spectrum (F₀, state 0) to that in the spectrum of the same sample after saturating R illumination at 85 K to reach a photoequilibrium between Pr and lumi-R (F₁, state 1) (i.e.γ₁= [F₀ - F₁]/F₀) (Fig. 1). The experimental γ₁ values allowed determination of the proportion of the two phenomenological phytochrome species in the sample, using individual γ₁ values of the rice phy species determined earlier (i.e.γ₁= 0.49 ± 0.03 for Pr′ and γ₁= 0 for Pr′) (19). P_tot (in relative U) and the percentage of each Pr species thus provided the content of Pr′ and Pr″ in the tissues. From this one could determine the content of phyA′ and phyA″, taking into consideration the fact that Pr′ is represented entirely by phyA′, whereas Pr″ comprises both minor phyA″ and phyB. Because our determination of the phy spectra excluded the input of phyB in the resulting difference spectra, the content of the Pr species based on these spectra directly reflected the content of the phyA isoforms, as follows: Pr′= phyA′ and Pr″= phyA″. The content of the active and inactive protochlorophyllides was also estimated from their individual spectra after deconvolution of the experimental spectra described elsewhere (22). Three to six independent measurements of different samples were performed and the precision of the determination of the experimental parameters for P_tot, γ₁, phyA′ and phyA″, which depends on the noise of the spectra registration (ca 3–5%), was within 10–15% of the parameter values. Position of the emission maximum (λ_max) and half-band width (Δλ) were determined with an accuracy of ± 1 nm.

RESULTS

Dark-grown seedlings

Raw low-temperature fluorescence emission spectra (85 K; λ_e= 632.8 nm) of phy in etiolated coleoptiles of WT and the phyB mutant in its initial state (Pr) and in the state of the Pr↔lumi-R equilibrium are presented in Fig. 1a,b (curves 1 and 2, respectively). The spectra of the phyA and phyAphyB mutants are also given in Fig. 1 (curve 3). The spectra of the etiolated coleoptiles of WT rice and its phyB mutant have a pronounced band at 685 nm; the band for the spectra of phyA mutant is very weak and is almost completely lacking for the spectra of the phyAphyB mutant (Fig. 1). On the basis of these findings it is evident that this band belongs primarily to phyA and evaluations based on these spectra have shown that the content of phyB does not exceed ca 5%, that phyA comprises up to 95% of the total phy in WT and that the input of phyC is negligible. As is seen from the real (difference) emission spectra of phyA in WT and the phyB mutant (Fig. 1c,d), especially after their normalization in the maximum and averaging for several samples (Fig. 1e,f), the spectroscopic characteristics of phyA in WT seedlings are identical to those of the phyB mutant (λ_max= 685 nm and Δλ= 24 nm for both spectra) (Table 1).

Table 1. Characteristics of phytochrome in different parts of coleoptiles of wild-type rice and its phyB mutant grown under different light conditions.*

Rice genotype	Part of coleoptile	Light conditions	Fluorescence maximum; half-band width (λ_max; Δλ)	Extent of Pr→lumi-R conversion, γ₁	[P_tot] (rU)	Pr′/Pr″ (%)	[Pr′]/[Pr″] (rU)
Wild-type	Tip	D	685; 24	0.39 ± 0.03	1.65 ± 0.26	78/22	1.28/0.36
	FRp	685; 24	0.38 ± 0.03	1.88	± 0.2	77/23	1.44/0.44
		FRc			0.07 ± 0.01
	Middle	D	685; 24	0.39 ± 0.06	0.80 ± 0.15	78/22	0.62/0.17
	Base	D	685; 24	0.33 ± 0.02	1.16 ± 0.14	66/34	0.77/0.39
phyB	Tip	D	685; 24	0.38 ± 0.03	2.63 ± 0.06	76/24	2.0/0.63
		FRp	685; 24	0.41 ± 0.01	2.21 ± 0.13	83/17	1.85/0.36
		FRc			0.11 ± 0.02
	Middle	D	685; 24	0.43 ± 0.02	1.56 ± 0.1	86/14	1.35/0.20
	Base	D	685; 27	0.32 ± 0.01	0.83 ± 0.05	64/36	0.53/0.30

*D = darkness; FR = far-red light (λ_a≥ 720 nm); FRc = constant FR; FRp = pulsed FR; rU = relative units.

Saturating R illumination of the sample at 85 K brings about a decrease in the phyA fluorescence intensity in the Pr form because of its partial conversion into the first photoproduct (lumi-R), which is stable at low temperatures (Fig. 1). The resulting spectrum of Pr plus lumi-R is represented by curve 2 in Figure 1c–f. The extent of the Pr→lumi-R conversion at 85 K (γ₁) characterizing the photochemical activity of phyA proves to be virtually the same in WT and phyB (Figs. 1 and 2, Table 1), which suggests that the state of phyA does not change as a result of the phyB mutation. At the same time, γ₁ varies depending on the part of the coleoptile used in both lines: it is higher for the upper part of the coleoptile (0.39 for WT and 0.38 for phyB) and lower at the base of the coleoptile (0.33 for WT and 0.32 for phyB) (Table 1).

Total phyA content (a), extent of the Pr→lumi-R photoconversion at 85 K under red illumination (λ_a= 632.8 nm) (b) and proportion of the phyA phenomenological pools Pr′ and Pr″ (c) and their concentration (d) in the tips of etiolated coleoptiles of the wild-type rice and its phyB mutant.

The total phyA content is 1.5–2-fold higher in the upper and middle parts of the etiolated coleoptiles of the phyB mutant, compared with the content in WT seedlings (Fig. 2, Table 1). In the lower parts of coleoptiles the total phyA content is the same as or even somewhat lower (ca 1.3-fold) than that in WT seedlings. The fact that the extent of the Pr→lumi-R conversion (γ₁) in the phyB mutant varies according to the part of the coleoptile in the same way as WT strongly suggests heterogeneity of the phyA population in rice. Evaluations of the phyA′ and phyA″ content in the phyB mutant show that, in the dark-grown phyB mutant plants, the proportion phyA′/phyA″ is virtually the same as that in WT seedlings (Fig. 2, Table 1), although their content is somewhat higher because of the higher P_tot in the coleoptile tips and middle parts of the phyB mutant.

On the basis of the emission spectra of protochlorophyllide in the coleoptile tips of WT and phy mutants of rice (Fig. 3), the concentration of the inactive and active protochlorophyllide forms was estimated to be 1.06 and 0.44 relative U, respectively, in WT (Table 2). In the phyA mutant the content of the inactive form was close to that in WT seedlings (0.86 relative U), whereas the concentration of the active form was considerably lower (0.15 relative U). The phyB mutant revealed the highest concentration of the protochlorophyllide in both forms (1.9 relative U in the inactive form and 0.62 relative U in the active form) (Table 2).

Raw low-temperature (85 K) fluorescence emission spectra (λ_e= 450 nm) of the active (Pchlide⁶⁵⁵) and inactive (Pchlide⁶³³) protochlorophyllide in coleoptiles of wild-type rice (a,d,g) and its phyA (b,e,h) and phyB (c,f,i) mutants grown in darkness (a-c) and under pulsed (d-f) and constant (g-i) far-red illumination (λ_a≥ 720 nm) of equal fluences. The spectra were not corrected for the spectral sensitivity of the fluorimeter.

Table 2. Active (Pchlide⁶⁵⁵) and inactive (Pchlide⁶³³) protochlorophyllides in coleoptiles of wild-type rice and its phytochrome mutants.*

Rice genotype	Light conditions	[Pchlide⁶³³] (rU)	[Pchlide⁶⁵⁵] (rU)
Wild-type	D	1.06 ± 0.17	0.44 ± 0.14
	FRp	0.97 ± 0.02	0.30 ± 0.05
	FRc	0.40 ± 0.05	—
phyA	D	0.86 ± 0.11	0.15 ± 0.05
	FRp	0.88 ± 0.03	0.15 ± 0.03
	FRc	0.64 ± 0.09	0.30 ± 0.08
phyB	D	1.90 ± 0.16	0.62 ± 0.07
	FRp	1.12 ± 0.22	0.32 ± 0.08
	FRc	0.30 ± 0.06	—

*D = darkness; FR = far-red light (λ_a≥ 720 nm); FRc = constant FR; FRp = pulsed FR; rU = relative units.

Etiolated seedlings of WT and the mutants had a phenotype characteristic of monocots (i.e. the first leaf was entirely covered by the coleoptile) (Fig. 4). The seedlings had a long coleoptile and a very short mesocotyl. The seedlings of the phyB mutant were, however, ca 20% longer than the seedlings of the other lines.

Five-day-old seedlings of wild-type rice (a) and its phyA (b) and phyB (c) mutants grown in darkness (left panel) and under pulsed and constant far-red light (λ_a≥ 720 nm) of equal fluences (middle and right panels, respectively). The blue arrow indicates the point of division of the coleoptile and the mesocotyl and the rose arrow indicates the spot where the first leaf pierces through the tip of the coleoptile.

Seedlings grown under FR illumination

WT and phyB mutant seedlings grown under FRp reveal the same properties as those of the dark-grown seedlings. There were no changes in the emission spectra of phytochrome in WT and the phyB mutant; both had a maximum at 685 nm and a half-band width of 24 nm (Table 1, spectra not shown). The content of phyA (P_tot) and of phyA′ and phyA″ under FRp closely resembled that in the dark-grown WT and phyB plants (Table 1). The total phy content in WT was lower than that in the phyB mutant (1.88 relative U vs. 2.21 relative U), which confirms data on the etiolated seedlings of these lines (see above). The value of γ₁ was 0.38 in WT and 0.41 in the mutant; the proportion of the two phyA pools was 77/23% in the WT seedlings and 83/17% in the phyB mutant. As a result, the content of the two phyA pools was 1.44/0.44 relative U in the WT and 1.86/0.36 relative U in the phyB mutant.

However, FRc illumination of the same fluence as FRp produced a dramatic (>20-fold) decrease in P_tot both in WT and the phyB mutant (Table 1). The decrease of the phytochrome content was so profound that its exact spectroscopic and photochemical properties and the proportion of the two phyA pools could not be determined. Thus, FRc of the same fluence as FRp turns out to be much more effective than FRp in the autoregulation of phyA synthesis and destruction, suggesting that this photoresponse belongs primarily to the HIR type.

Under FRp the Pchlide⁶³³ and Pchlide⁶⁵⁵ contents were, respectively, 0.97 and 0.30 relative U in WT; 0.88 and 0.15 relative U in the phyB mutant; and 1.12 and 0.32 relative U in the phyA mutant (Table 2). FRc produced a steep decrease in the concentration of the two protochlorophyllide forms in WT seedlings and the phyB mutant (Fig. 3, Table 2). The Pchlide⁶³³ concentration was 0.4 in WT seedlings and 0.3 in the phyB mutant but practically no Pchlide⁶⁵⁵ was detected in the samples. In the phyA mutant the concentration of Pchlide⁶³³ was 0.64 relative U and the concentration of Pchlide⁶⁵⁵ was 0.30 relative U. Thus, FRc has a negative effect in WT and the phyB mutant, whereas, as expected, there is no such effect in the phyA mutant.

Under FRp the coleoptile tip was pierced by the leaf in WT and the phyB mutant (Fig. 4, middle panel). However, the coleoptile was much longer than the mesocotyl. FRc produced a more profound effect: a short coleoptile with a long first leaf emerged from it in both lines (Fig. 4, right panel). As expected the seedlings of the phyA mutant had a phenotype of the dark-grown seedlings both under FRp and FRc.

DISCUSSION

Evidence for the existence of the two phyA types in monocot rice

The above fluorescence and photochemical analysis of the phyA state in WT and phy mutants of rice has firmly proved for the first time that the monocot phyA comprises phyA′ and phyA″ isoforms similar to those observed on phyA in dicots. This follows from the estimations of the phyA and phyB input in the phy fluorescence of etiolated rice coleoptiles and the proportions of Pr′ and Pr″ contained in them. phyB, which is of the Pr″ type, in principle could have accounted for the appearance of the Pr″ species in rice. However, in this work and earlier studies (19,21) the proportion of the minor Pr″ species in rice was found to be relatively high (up to 20% in the upper coleoptile and up to 40% in the lower coleoptile in WT seedlings), which is much higher than the proportion of phyB (type II phy) in etiolated tissues (7).

However, the major argument for the heterogeneity of phyA is that the key phenomenological parameter that distinguishes between phyA′ and phyA″ in dicots—the extent of the Pr→lumi-R conversion at 85 K (γ₁)—varies in the different parts of the coleoptiles of the phyB mutant and practically coincides with that in the respective parts of the WT coleoptiles (Fig. 2, Table 1). Evaluations of the Pr′/Pr″ ratio in WT and the phyB mutant give almost the same results (Table 1). Thus, the lack of phyB in the phyB mutant does not significantly affect the Pr′/Pr″ balance, which strongly suggests that there are two populations of phyA with the properties of Pr′(phyA′) and Pr″ (phyA″) and supports the conclusion that the input of phyB in the fluorescence of phy in WT rice coleoptiles is low. Because the properties and relative content of the two phyA species in rice (WT seedlings and the phyB mutant) are very close to those of the two phenomenological phy pools (Pr′ and Pr′) in the WT monocots (i.e. oat, wheat, barley and maize) (19–21), we believe it likely that the two phyA populations are present in all monocots.

The view that, along with the major phyA′, there exists the minor phyA″ in rice is in line with our finding (23) that rice phyA expressed in transgenic yeast and reconstituted with phytochromobilin (PφB) is represented by the species similar to phyA″. This suggests that the minor phyA″ is a product of the same gene as that of the major phyA′ and that plant-specific posttranslational modification lacking in yeast (probably phosphorylation) is needed to form phyA′. Of interest is the comparison of the properties of phy in rice and in the pea (the classic dicot model). In experiments involving phy mutants of pea the position of the emission maximum was practically the same (685–687 nm), which almost completely belonged to phyA (95%) in WT seedlings, and the proportion of phyA′/phyA″ was close to that in the rice coleoptiles (88/12% and 51/49% in the upper and lower epicotyl, respectively, for the phyB [/v-5] mutant of pea) (18). However, the total phy content was higher in the pea than in rice (6.0 vs. 2.5 relative U).

Effects of FR and phyA functions

In dark-grown rice seedlings the total phyA content in the coleoptile is higher in the phyB mutant, compared with WT. In this connection it should be mentioned that there are data that R preillumination of seeds may affect the physiology of growing seedlings. In particular, Alconada Magliano and Casal (24) observed that R illumination of Arabidopsis seeds induces an inhibitory effect on hypocotyl elongation in seedlings grown in the dark. These observations are in line with the notion that a lower phyA concentration in WT, compared with the phyB mutant, might be the consequence of the inhibitory function of phyB in the dark-grown rice seedlings due to preillumination of seeds during their maturation. This is supported by the fact that the WT coleoptile is shorter than that of the phyB mutant. The proportion of the two phyA pools remains the same in WT and the phyB mutant despite differences in the total phy content. Thus, if phyB indeed influences the accumulation of phyA in WT, this affects the two phyA pools in the same way. This hypothesis suggests that the effect is localized upstream of the posttranslational modification of phyA, which is believed to be the reason for the difference between the two phyA pools. It should be noted that the lack of phyB in the phyB mutant has positively effects in general—the mutant seedlings are slightly longer (by ca 20%) and have higher protochlorophyllide content and their seeds germinate earlier, compared with WT (M. Takano, unpublished).

FRp does not significantly affect the total phyA content in WT and phyB mutants, nor does it affect the phyA′/phyA″ proportion and the difference in P_tot between WT and phyB mutant, as was observed in the dark-grown seedlings. Thus, we may conclude that the input of the VLFR in the autoregulation of the phyA content is minor. A completely different picture is observed under FRc. In WT and the phyB mutant, a considerable decrease (ca 95%) in the content of phyA is observed. The lack of the differences in this effect between WT and the phyB mutant suggests that phyB, as expected, is not involved in this process. Because the FRc illumination is a key feature of the HIR (1), we may attribute this negative autoregulation of phyA to this photoresponse type. Earlier, it was found that the effect of FRc on the phyA concentration in wheat (20) and the pea (25) is mediated by phyA′ and we may hypothesize that this phyA species plays the same role in rice.

In the dark the levels of active and inactive protochlorophyllide in the phyB mutant were ca one-third greater than those in WT, which correlates with the higher phyA content in this mutant. During exposure to FRp there were no significant changes in levels of both forms of protochlorophyllide in WT, whereas considerable decreases were observed for Pchlide⁶³³ (40%) and for Pchlide⁶⁵⁵ (ca 100%) in the phyB mutant. This suggests that the inhibition of the Pchlide biosynthesis under FR in the phyB mutant may have the VLFR component (especially for the Pchlide⁶⁵⁵ biosynthesis). The lack or small effect of FRp in WT seedlings suggests that phyB interferes with the action of phyA and/or that there is an increased effect of phyA activity, because of its higher content in the phyB mutant.

In seedlings grown under FRc the effect of phyA is much higher—the content of Pchlide⁶³³ decreases by 3-fold in WT seedlings and by 7-fold in the phyB mutant. In both lines Pchlide⁶⁵⁵ was almost completely absent (Fig. 3, Table 2). The latter findings agree with the data on barley, Arabidopsis, tomato and rice (21,22,26–30), which indicate that phyA under FRc negatively affects Pchlide⁶⁵⁵ biosynthesis and that this effect belongs to the HIR type. At the same time, the fact that phyA in the phyB mutant is more effective than that in WT, as judged by the Pchlide⁶³³ concentration, supports the assumption that phyB may inhibit the action of phyA.

An interesting picture was observed with regard to the phyA mutant. The level of Pchlide⁶³³ in the mutant grown in the dark was similar to that in WT and did not change under FRp. The level of Pchlide⁶⁵⁵ was ca 2-fold lower in the mutant grown in the dark and under FRp its concentration did not significantly change. However, during exposure to FRc the concentration of Pchlide⁶³³ slightly decreased, whereas the concentration of Pchlide⁶⁵⁵ increased by 100% (Fig. 3, Table 2). The positive effect of FRc on the concentration of Pchlide⁶⁵⁵ in the phyA mutant points to a possible participation of phyC in this process, which is in agreement with earlier observations of positive effects of FR on the light-dependent CAB genes in the phyA mutant, which were not found in the double phyAphyC mutant (10,11).

As expected, the phyA mutant had a phenotype of the etiolated plant both in the dark and under the two light regimens (FRp and FRc), supporting the common notion that phyB is inactive under FR. This fact also agrees with earlier reports that the input of phyC in the regulation of the growth responses under these conditions is minor (10,11). The phyB mutant, on the other hand, had the same phenotype as WT seedlings under all light regimens, although it was ca 20% longer. The phyB mutant and WT both mildly responded to FRp by shortening the coleoptile and sprouting the first leaf; the effect was much more pronounced under FRc. The difference in the extent of the effects under FRp and FRc indicate that the response under FR is rather HIR than VLFR.

It should be noted in this connection that the HIR are usually observed in dicots, whereas it is a subject of debate for monocots (31). Shlumukov et al. (32) have produced transgenic wheat overexpressing an oat PHYA gene that revealed an increased phyA content both in dark- and light-grown plants (20,32). The transgenic line showed additional inhibition of coleoptile extension, compared with WT, when it was grown under FRc but not FRp; this could be attributed to HIR. In addition, this line acquired HIR responses during FRc (anthocyanin accumulation and leaf unrolling) that were not observed in WT seedlings. Because phyA′ was virtually the only species overexpressed in transgenic wheat, the acquired HIR photoresponses were attributed to phyA′ (20). The fact that phyA′ dominates in the etiolated seedlings of WT rice and its phyB mutant suggests that this form of phyA also participates in the de-etiolation processes under FRc in rice. The role of phyA″ remains to be elucidated.

Acknowledgements—

This work was partially supported by the Russian Foundation for the Fundamental Investigations (grant 05-04-49549 to V.S.). V.S. is grateful to Prof. P. Nick and Dr. M. Riemann for helpful advice and fruitful discussions.

REFERENCES

1 Smith, H. (1997) Photomorphogenesis. Plant Cell Environ. 20, 657–844.
10.1046/j.1365-3040.1997.d01-108.x
Google Scholar
2 Brockman, J. and E. Schäfer (1982) Analysis of Pfr destruction in Amarantus caudatus L.—Evidence for two pools of phytochrome. Photochem. Photobiol. 35, 555–558.
10.1111/j.1751-1097.1982.tb02608.x
Web of Science® Google Scholar
3 Tokuhisa, J. G., S. M. Daniels and P. H. Quail (1985) Phytochrome in green tissues: spectral and immunochemical evidence for two distinct molecular species of phytochrome in light-grown Avena saliva L. Planta 164, 321–332.
10.1007/BF00402943
CAS PubMed Web of Science® Google Scholar
4 Sineshchekov, V. A. and A. V. Sineshchekov (1987) Fluorescence and absorption investigations of phytochrome in the cells of etiolated seedlings. Plant Physiol. (Russ.) 34, 730–741.
Google Scholar
5 Sineshchekov, V. A. and A. V. Sineshchekov (1989) Fluorescence of phytochrome in the cells of dark-grown plants and its connection with the phototransformation of the pigment. Photochem. Photobiol. 49, 325–330.
10.1111/j.1751-1097.1989.tb04114.x
CAS Web of Science® Google Scholar
6 Sharrock, R. A. and P. H. Quail (1989) Novel phytochrome sequences in Arabidopsis thaliana: Structure, evolution, and differential expression of a plant regulatory photoreceptor family. Genes Dev. 3, 1745–1757.
10.1101/gad.3.11.1745
CAS PubMed Web of Science® Google Scholar
7 Furuya, M. (1993) Phytochromes: Their molecular species, gene families, and functions. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 617–645.
10.1146/annurev.pp.44.060193.003153
CAS Web of Science® Google Scholar
8 Boylan, M. and P. H. Quail (1991) Phytochrome A overexpression inhibits hypocotyl elongation intransgenic Arabidopsis. Proc. Natl. Acad. Sci. U S A 88, 10806–10810.
10.1073/pnas.88.23.10806
CAS PubMed Web of Science® Google Scholar
9 Long, C. and M. Iino (2001) Light-dependent osmoregulation in pea protoplasts. Photoreceptors, tissues specificity, ion regulation, and physiological implications. Plant Physiol. 125, 1854–1869.
10.1104/pp.125.4.1854
CAS PubMed Web of Science® Google Scholar
10 Takano, M., H. Kanegae, T. Shinomura, A. Miyao, H. Hirochika and M. Furuya (2001) Isolation and characterization of rice phytochrome A mutants. Plant Cell 13, 521–534.
10.1105/tpc.13.3.521
CAS PubMed Web of Science® Google Scholar
11 Takano, M., X. Xie, N. Inagaki and T. Shinomura (2005) Distinct functions of phytochromes on the photomorphogenesis in rice. In Light Sensing in Plants (Edited by M. Wada, K. Shimazaki and M. Iino), pp. 111–117. Springer-Verlag, Tokyo , Berlin, Heidelberg, New York .
10.1007/4-431-27092-2_12
Web of Science® Google Scholar
12 Takano, M., N. Inagaki, X. Xie, N. Yuzurihara, F. Hihara, T. Ishizuka, M. Yano, M. Nishimura, A. Miyao, H. Hirochika and T. Shinomura (2005) Distinct and cooperative functions of phytochromes A, B, and C in the control of deetiolation and flowering in rice. Plant Cell 17, 3311–3325.
10.1105/tpc.105.035899
CAS PubMed Web of Science® Google Scholar
13 Sineshchekov, V. A. (1995) Photobiophysics and photobiochemistry of the heterogeneous phytochrome system. Biochim. Biophys. Acta 1228, 125–164.
10.1016/0005-2728(94)00173-3
Web of Science® Google Scholar
14 Sineshchekov, V. A. (2004) Phytochrome A: Functional diversity and polymorphism. Photochem. Photobiol. Sci. 3, 596–607.
10.1039/b315430k
CAS PubMed Web of Science® Google Scholar
15 Sineshchekov, V. A. (2005) Polymorphysm of phytochrome A and its functional implications. In: Light Sensing in Plants (Edited by M. Wada, K. Shimazaki and M. Iino), pp. 95–102. Yamada Science Foundation and Springer-Verlag, Tokyo .
10.1007/4-431-27092-2_10
Web of Science® Google Scholar
16 Sineshchekov, V. A. (1995) Evidence for the existence of two phytochrome A populations. J. Photochem. Photobiol. B. 28, 53–55.
10.1016/1011-1344(94)07094-5
CAS Web of Science® Google Scholar
17 Sineshchekov, V. A., O. B. Ogorodnikova, P. F. Devlin and G. C. Whitelam (1998) Fluorescence spectroscopy and photochemistry of phytochromes A and B in wild-type, mutant and transgenic strainsof Arabidopsis thaliana. J. Photochem. Photobiol. B. 42, 133–142.
10.1016/S1011-1344(97)00133-4
CAS PubMed Web of Science® Google Scholar
18 Sineshchekov, V. A., O. B. Ogorodnikova and J. L. Weller (1999) Fluorescence and photochemical properties of phytochromes A and B in etiolated pea seedlings. J. Photochem. Photobiol. B. 49, 204–211.
10.1016/S1011-1344(99)00061-5
CAS Web of Science® Google Scholar
19 Sineshchekov, V. A. (1994) Two spectroscopically and photochemically distinguishable phytochromes in etiolated seedlings of monocots and dicots. Photochem. Photobiol. 59, 77–86.
10.1111/j.1751-1097.1994.tb05004.x
CAS Web of Science® Google Scholar
20 Sineshchekov, V., L. Koppel, L. Shlumukov, F. Barro, P. Barcelo, P. Lazzeri and H. Smith (2001) Fluorescence and photochemical properties of phytochromes in wild-type wheat and a transgenic line over-expressing an oat phytochrome A (PHYA) gene: Functional implications. Plant Cell Environ. 24, 1289–1297.
10.1046/j.1365-3040.2001.00780.x
Web of Science® Google Scholar
21 Sineshchekov, V. A., A. V. Loskovich, M. Riemann and P. Nick (2004) The jasmonate-free mutant hebiba is affected in the response of phyA′/phyA″ pools and protochlorophyllide biosynthesis to far-red light. Photochem. Photobiol. Sci. 3, 1058–1062.
10.1039/b406795a
PubMed Web of Science® Google Scholar
22 Sineshchekov, V., O. Belyaeva and A. Sudnitsin (2004) Phytochrome A positively regulates biosynthesis of the active protochlorophyllide in dicots under far-red light. J. Photochem. Photobiol. B. 74, 47–54.
10.1016/j.jphotobiol.2004.02.001
CAS PubMed Web of Science® Google Scholar
23 Sineshchekov, V., L. Hennig, T. Lamparter, J. Hughes, W. Gärtner and E. Schäfer (2001) Recombinant phytochrome A in yeast differs by its spectroscopic and photochemical properties from the major phyA′ and is close to the minor phyA″: Evidence for post-translational modification of the pigment in plants. Photochem. Photobiol. 73, 692–696.
10.1562/0031-8655(2001)0730692RPAIYD2.0.CO2
CAS PubMed Web of Science® Google Scholar
24 Alconada Magliano, T. and J. J. Casal (2004) Pre-germination seed-phytochromc signals control stem extension in dark-grown Arabidopsis seedlings. Photochem. Photobiol. Sci. 3, 612–616.
10.1039/b315511k
PubMed Web of Science® Google Scholar
25 Sineshchekov, V. A. and J. L. Weller (2004) Two modes of the light-induced phytochrome A decline—With and without changes in the relative content of its native pools (phyA′ and phyA″): Evidence from in-situ fluorescence investigations of wild-type and mutant phyA-3D pea. J. Photochem. Photobiol. B. 75, 127–135.
10.1016/j.jphotobiol.2004.05.010
PubMed Web of Science® Google Scholar
26 Apel, K., H.-J. Santel, T. E. Redlinger and H. Falk (1980) The protochlorophyllide holochrome of barley. Isolation and characterization of the NADPH: Protochlorophylide oxidoreductase. Eur. J. Biochem. 111, 251–258.
10.1111/j.1432-1033.1980.tb06100.x
CAS PubMed Web of Science® Google Scholar
27 Apel, K. (1981) The protochlorophillide holochrome of barley (Hordeum vulgare, L.). Phytochrome induced decrease of translatable mRNA coding for the NADPH:protochlorophyllide oxidoreductase. Eur. J. Biochem. 120, 89–93.
10.1111/j.1432-1033.1981.tb05673.x
CAS PubMed Web of Science® Google Scholar
28 Batschauer, A. and K. Apel (1984) An inverse control by phytochrome of the expression of two nuclear genes in barley (Hordeum vulgare L.). Eur. J. Biochem. 143, 593–597.
10.1111/j.1432-1033.1984.tb08411.x
CAS PubMed Web of Science® Google Scholar
29 Barnes, S. A., N. K. Nishizawa, R. B. Quaggio, G. C. Whitelam and N.-H. Chua (1996) Far-red light blocks greening of Arabidopsis seedlings via phytochrome A-mediated change in plastid development. Plant Cell. 8, 601–615.
10.1105/tpc.8.4.601
CAS PubMed Web of Science® Google Scholar
30 Runge, S., U. Sperling, G. Frick, K. Apel and G. A. Armstrong (1996) Distinct roles for light-dependent NADPH:protochlorophillide oxidoreductases A and B during greening in higher plants. Plant J. 9, 513–523.
10.1046/j.1365-313X.1996.09040513.x
CAS PubMed Web of Science® Google Scholar
31 Casal, J. J., R. C. Clough and R. D. Vierstra (1996) High-irradiance responses induced by far-red light in grass seedlings of the wild-type or overexpressing phytochrome A. Planta 200, 132–137.
10.1007/BF00196660
CAS Web of Science® Google Scholar
32 Shlumukov, L., F. Barro, P. Barcelo, P. Lazzeri and H. Smith (2001) Establishment of far-red high irradiance responses in wheat through transgenic expression of an oat phytochrome A gene. Plant Cell Environ. 24, 703–712.
10.1046/j.1365-3040.2001.00718.x
CAS Web of Science® Google Scholar

Citing Literature

Volume82, Issue4

July 2006

Pages 1116-1122

This article also appears in:

Photosensory Biology

Two Native Pools of Phytochrome A in Monocots: Evidence from Fluorescence Investigations of Phytochrome Mutants of Rice

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

Dark-grown seedlings

Seedlings grown under FR illumination

DISCUSSION

Evidence for the existence of the two phyA types in monocot rice

Effects of FR and phyA functions

Acknowledgements—

REFERENCES

Citing Literature

Figures

References

Information

About Wiley Online Library

Help & Support

Opportunities

Connect with Wiley

Two Native Pools of Phytochrome A in Monocots: Evidence from Fluorescence Investigations of Phytochrome Mutants of Rice

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

Dark-grown seedlings

Seedlings grown under FR illumination

DISCUSSION

Evidence for the existence of the two phyA types in monocot rice

Effects of FR and phyA functions

Acknowledgements—

REFERENCES

Citing Literature

Figures

References

Related

Information