Phytochrome Levels

To test whether phytochrome A was selectively affected in the

ftm1-1

mutant, phyA and phyB apoprotein levels were examined by immunoblotting. Figure 4.7 shows clearly that PHY A is undetectable in crude protein extracts from etiolated ft,1

rz1-1

seedlings. In contrast, the PHYB detected by monoclonal antibody rnA T5, which is lacking in

tv

mutants

(Figure

3.10), is present at normal level in fun1-1 .

An examination of phytochrome levels in etiolated

ftm1-1

seedlings by

in vivo

difference spectrophotometry revealed the

ftm1-1

mutant to possess only about 2.5% of the

photoreversible phytochrome present in the wild type

(Table 4.2).

This indicates that phyA accounts for at least 97.5% of the spectrally active phytochrome in etiolated pea seedlings. After 4 h R treatment, phytochrome in WT seedlings was depleted to about 15% of the dark level

(Table 4.2)

while the phytochrome level in

ftm1-1

seedlings remained similar (about 2.5%). On the basis of the results in

Table 4.2,

phy A therefore makes up about 80% of the phytochrome pool present after 4 h R. Although this almost certainly overestimates the proportion of phy A in the stable pool, since phytochrome in WT seedlings can be depleted below the level present after 4 h R (see

Figures 3.17, 7.11),

it does indicate the presence of a substantial amount of phyA in the stable pool, which is in keeping with the obvious effects of phyA deficiency on the mature plant phenotype

(Figure 4.3).

The small pool of photoreversible phytochrome remaining in

ftm1

plants (about 2.5% of etiolated WT level) must therefore consist mostly of other, light-stable phytochromes. Effects of the phytochrome B-deficient

tv-5

mutant on the level of spectrally active phytochrome are undetectable

(Table 4.2),

suggesting that the contribution from phyB is small, and implying the presence of a third phytochrome. However, this may be difficult to demonstrate by

in vivo

spectrophotometry, since the phytochrome content in standard samples of

fun1

tissue is close to the detection limit of the system. Also, leakiness of the

ftm1-1

and

tv-5

mutations can not currently be ruled out.

4.2.6. R/FR Reversibility of De-Etiolation in the fun1 Mutant

It was shown in

Chapter 3

that the de-etiolation response of pea seedlings to intermittent R pulses consists of two components; a FR-reversible component which is lacking in phyB­ deficient

tv

mutants and is therefore mediated by phyB, and a non-FR-reversible

than WT, and that the response of the mutant retains a similar degree of FR-reversibility to WT. However, the response to terminal FR pulses is substantially reduced in the

ftm1 -1

mutant, indicating that this component is controlled by phyA. This result also suggests that phyA and phyB together control most if not all of the response to intermittent R pulses. PhyB-deficient pea mutants also retain a normal response to continuous R at fluence rates below about 4 x J0-4 �J,mol m-2 sec-1, and it is likely that this response is also

controlled by phyA, although this has yet to be directly tested. 4.2.7. Phenotype of Mature fun1 Plants

Mutant

ftm1

seedlings were virtually indistinguishable from WT when grown for up to 12 d under continuous cool-white fluorescent light (Figure 4.6B). However, as mentioned above, mature

fun1

plants grown under standard glasshouse LD conditions exhibited a striking phenotype

(Figure 4.3), with a reduction in internode length of up to 50% relative to WT

(Table 4.3). The onset of this dwarf phenotype was monitored in more detail by measuring

lengths of individual internodes in

ftm1

plants grown in the glasshouse. Under these

.

conditions, internodes in the ft

m1

mutant were slightly longer than WT until about node 6, beyond which a dwarfing effect of

fttn1

gradually became apparent (Figure 4.9B). The reduction in internode length in

fun1

plants coincided with a thickening (Figure 4.9A) and paling of the stem, which in later internodes often took on a yellowish, succulent

appearance, and showed prominent transverse banding (Figure 4.9C). This commenced at about node 10 and became particularly severe several internodes below the node of flower intiation. The severity of the stem phenotype appeared to gradually diminish once regular flower development was established, and above about node 24 the stems of

fun1

plants regained a relatively normal appearance.

Whereas WT plants do not show any lateral branching when grown under standard glasshouse LD conditions,

fim1

plants branched strongly both from basal and aerial nodes

(Figure 4.10). Under these conditions WT plants flower at about node 16. Flowers develop

on relatively short peduncles

(Table

4.3) and set strongly, with plants producing 5-6 flowering nodes and 20-30 seeds before apical arrest

(Table

4.3). However, in ft

m1

plants, flower initials did not appear until about node 19 and did not develop until about node 24

(Table 4.3, Figure 4.9C), with the first developed flower opening much further behind the

apical bud (lower FLR) and on a much longer peduncle than in WT plants (Table 4.3). Perhaps most striking was the large delay in senescence of

ftm1

plants, which produced

buckled appearance to leaflets

(Figure

4.11A) suggestive of a greater rate of expansion of interveinal photosynthetic tissue relative to vascular tissue. Finally,

fun1

leaves developed to a greater degree of complexity, with some leaves developing 4 pairs of leaflets and thus exceeding the maximum of 3 pairs seen in WT plants

(Figure

4.11B). 4.2.7.

Photoperiod Responses in the ftm1 Mutant

The phenotypic syndrome exhibited by

fim1

mutant plants in LD is very similar to that shown by WT plants grown in SO, which also show delayed flower initiation, retarded flower and fruit development, longer peduncles, delayed transition to more complex leaf pattern, and delayed onset of apical senescence (Murfet 1982). I therefore considered that

fim1

plants might be unable to detect the difference between long and short day conditions. To test this, WT and

furll-1

plants were grown under SO (8 h) and standard phytotron LD conditions (8 h extended with 16 h weak incandescent light). A photoperiod extension with light establishing an intermediate photoequilibrium has previously been found to be the most effective for the promotion of flowering in pea (Reid and Murfet 1977) and various other LOP species (e.g. Evans 1976, Downs and Thomas 1982, Carr-Smith et a!. 1989), while the low fluence rate used in the extension excludes a significant contribution to total PAR.

Figure 4.12 shows that ftm1

plants are in fact very similar in appearance to WT plants grown in SO. WT plants elongate and flower earlier in response to a photoperiod

extension, whereas

fim1

plants are essentially unresponsive to the extension, showing the same growth habit in LD and SO. The data in

Figure

4.13 confirm the lack of response of

fim1

plants to the photoperiod extension and show that it is manifest in a number of different characters, including time to first open flower, and number of reproductive nodes. These results indicate that phyA is the primary phytochrome responsible for the

detection of a FR-rich photoperiod extension in pea.

Since

fim1

seedlings are specifically insensitive to FR, it was considered that the inability of

fim1

plants to detect a photoperiod extension might be dependent on the spectral quality of tha t extension. However,

fim1

plants were similarly unresponsive to 16-h extensions with weak fluorescent light, which contained essentially no FR

(Figure

pea, phy A is active in the detection of low-fluence-rate photo period extensions at both high and low R:FR, and that under neither regime are other phytochromes able to compensate for the loss of phyA. The apparent FR specificity seen for phyA-induced de­ etiolation in pea seedlings therefore does not necessarily exist for other photoresponses.

4.2.8 Isolation of a ftm1 lv Double Mutant

In order to further define the roles of phytochromes A and B, a fwll tv double mutant was isolated in the F2 of the cross AF140

(fu n l - 1 ) x AF280 (lv-5). Initially, the tv segregates in

this cross were identified by their increased elongation and reduced leaf expansion under WL. Several slightly shorter

lv segregates were identified as putative double mutants.

Plants were then transferred to the glasshouse apron where the double mutants were more clearly distinguished. Identity of double mutants was confirmed by selection from tv families segregating fuu1 in the M3 generation, and by backcrossing.

Grown under standard WL conditions, double mutant plants showed the characteristic appearance of the

tv

single mutant, with pale elongated internodes and reduced leaf development, but were slightly shorter and had poorer leaf development than the single

tv

mutant

(Figure 4.15A).

The difference between

tv

and

ftml tv

plants became more pronounced after transfer of plants to the glasshouse where they received a natural photoperiod extended to 18 h with mixed fluorescent/incandescent light

(Figure 4.15B).

Under these conditions, the phenotype of the

ftml tv

double mutant was strikingly

different from WT and from ei ther single mutant. Both

funl

and

tv

single mutants grown in the glasshouse achieve essentially full de-etiolation, whereas the double mutant

retained an appearance reminiscent of dark-grown plants, with pale stem, elongated petioles and poor leaflet development. Although the presence of the

tv

mutation somewhat alleviated the dwarfing effect of ftml on internodes,

fw!l tv

double mutant internodes showed a paling and thickening considerably more severe than the

ftml

single mutant

(Figure 4.16).

For several nodes below NFI, the internodes of

funl tv

plants

appeared totally lacking in chlorophyll and had a fattened, distorted appearance which in more severe cases extended to splitting and twisting of the stem

(Figure 4.16).

Double mutant stems appeared succulent and were extremely brittle, due apparently to poor development of vascular tissue. Pedicels of double mutant flowers were also longer than WT, although neither the

funl

nor the

tv

mutant alone had a visible effect on pedicel length. In addition, in many cases the peduncles of

fwzl lv

plants hacfsmall outgrowths

the double mutant was extremely low, with few plants yielding more than 5 seeds under conditions where WT (and

lv

) plants yielded around 30 and

fun1

plants more than 70 seeds

(Table 4.3).

4.2.9 Spectral Sensitivity of the fun1 tv

Double Mutant

The etiolated phenotype of

ftm1

mutants under FR has shown that phyA is the only phytochrome with a substantial role in mediation of responses to FR in etiolated pea seedlings. However, continuous R induces substantial de-etiolation in the

lv

mutants, suggesting that at least one phytochrome in addition to phyB is active in mediating the seedling response to R. The results from R pulse experiments

(Figure

4.8) indicate that phyA can also mediate responses to R, and thus suggest that phyA may also be active under continuous R. The spectral sensitivity of the

ftm1 lv

double mutant was therefore examined.

Figures

4.17 A and 4.18 show that the

fttn1 lv

double mutant grown in continuous R had much longer internodes, and a greatly reduced leaflet area and ra�e of node

expansion relative to WT plants or either single mutant. The appearance of the

fun1 lv

double mutant under R is very similar to that of a dark-grown WT plant

(Figure

4.18, also cf

Figures

4.17 A and 4.2A), with the exception that

ftm1 lv

apical buds and leaflets do show some Chl accumulation

(Figure

4.17 A). Whether this represents some degree of induction of Chl synthesis or merely reflects the light-dependent conversion of

protochlorophyllide to Chl is not known. I n any event, this result clearly demonstrates that both phyA and phyB are active under R, that either phy alone can to a large extent compensate for the absence of the other, and that phyA and phyB together mediate virtually all of the effects of R on stem elongation and leaf development.

However, the fact that the

ftm1, /v

and

[11nl lv

double mutants grown in WL are all substantially shorter than etiolated WT plants

(Figure

4.15A) indicates that another photoreceptor in addition to phyA and phyB plays an important role in

photomorphogenesis under WL. Furthermore, the near-complete insensitivity of the double mutant to R and FR strongly suggests that B is the active waveband. Both the

ftm1

and

lv

single mutants have only a small effect on internode elongation under B

(Figures

4.6 and 3.7), and

Figures

4.17B and 4.18 show that the

ftm1 lv

mutant also retains a

substantial inhibition of stem elongation in response to B. This contrasts with the

...

that the responses of the m utant seedlings to B are closely mirrored in their responses to WL

(Figure 4.15A) suggests that the B receptor has an important role under high-fluence­

rate WL, particularly for the control of stem elongation over the early internodes.

The results in Figure 4.18 also have some other interesting implications. The de-etiolating effects of R on stem inhibition and leaf development appear to be co-ordinated, insofar as phytochrome deficiency affects both processes to a similar extent. Under B, the de­ etiolating effects of phyB are also co-ordinated in a similar fashion, with phyB deficiency causing a small increase in internode length and a small decrease in leaflet area

(Figure 4.18).

In contrast, the loss of phy A strongly decreases leaflet area in seedlings grown under B, but does not cause a corresponding increase in stem elongation. In fact, the loss of phyA causes a small

inhibition

of stem elongation relative to WT

(Figure 4.6, 4.18)

particularly over the early internodes, which is more apparent in the absence of phyB

(Figure 4.18).

This result implies that phyA is actually promoting stem elongation under B, either directly, or through interaction with a B photoreceptor.

In document Control of development by Phytochrome in the garden pea (Pisum sativum L ) (Page 69-74)