Influence of deletion of both PsbS and PPH1 proteins on light stress
resistance in Arabidopsis thaliana.
Thi Thu Huong Khuonga,b,c,d, Christophe Robagliab,c,d, Stefano Caffarrib,c,d
a
Vietnam Forestry University, CFB, Xuan Mai, Chuong My, Hanoi, Vietnam
b
Aix Marseille Univ, BVME, Plant Genetic and Biophysic Laboratory, 13288, Marseille, France
c
CEA, DSV, Institute of Environmental Biology and Biotechnologies, 13288, Marseille, France
d
CNRS, UMR 7265, Biologie Végétale et Microbiologie Environnementales, 13288, Marseille, France
Abstract
Non Photochemical Quenching (NPQ) is considered a short-term regulation
important to maintain efficient photosynthesis and to avoid photooxydative damages by
dissipation of excess energy as heat in high. The activation of NPQ requires the
protonation of the PsbS protein. “State transitions” are a second important
photosynthetic regulation to respond to changes in light quality. In state transitions,
action of the STN7 kinase and PPH1 phosphatase: phosphorylation/dephotphorylation
proteins of LHCII promote its mobilization from PSII to PSI and reverse. In mutants of
Arabidopsis inactivated for the function of PPH1 protein, thylakoids membrane is
blocked in the so-called “State II” enriched in PSI-LHCII complexes. In this report, we
produced the pph1/npq4 double mutant and characterized some photosynthetic, growth
and reproduction properties in high and low light conditions. Results indicate that in
high light the pph1/npq4 double mutant showed a resistance to high light stress similar
or slightly lower than that of the single npq4 mutant. And in low light condition, the
pph1/npq4 mutant shows a significant increase of growth and flowering as compared to
single mutants and wild type plants.
Key words: Non-photochemical quenching, PsbS protein, PPH1 protein, plant growth,
state transitions.
Introduction
Light is therefore indispensable for survival, but plants need to cope with different
environmental situations where light quantity and quality can be not optimal for
photosynthesis. If absorbed energy is more than the quantity exploitable by plant
metabolism, this can lead to a variety of harmful consequence for plant and in particular
to the production of reactive oxygen species (ROS) that can impair photosynthesis and
diminish plant growth. To avoid ROS formation, plants activate a mechanism, called
Non Photochemical Quenching (NPQ), to dissipate excess energy as heat. NPQ is
considered a short term regulation important for maintaining efficient photosynthesis
and avoid photooxydative damages in high or fluctuating light. PsbS plays the key role
for NPQ activation [5,7].
A second important photosynthetic regulation to respond to changes in light intensity
and quality is called “state transitions”[1,8]. State transitions are known as a mechanism
by which excitation energy is redistributed between PSI and PSII when plants are
1
2
exposed to lights that preferentially excite either PSI or PSII. The STN7 kinase proteins
in plants [2], and the plant PPH1 phosphatase protein [10,12] have a fundamental role in
state transitions. Under illumination conditions that are favorable for PSII excitation
(red/blue light), the plastoquinone pool (PQ) becomes more reduced, the STN7 kinase is
activated and part of phosphorylated LHCII migrates to PSI (State II condition). On the
contrary, in light conditions that preferentially excite PSI (far red light), PQ is oxidized,
the LHCII kinase is inactivated, the dephosphorylation is promoted by the PPH1
phosphatase and LHCII returns to PSII (State I). The PPH1 phosphatase is a thylakoidassociated phosphatase of 38 kDa, also called TAP38, recently shown to be responsible
for the dephosphorylation of the LHCII protein [10, 12]. In Arabidopsis plants
inactivated for the PPH1 function, P-LHCII is not dephosphorylated and thylakoid is
blocked in the so-called “State II” and enriched in PSI-LHCII complexes [10,12]. In the
context of the study of photosynthetic regulation by energy quenching (NPQ) and state
transitions, we produced the pph1/npq4 double mutant by crossing npq4 and pph1
Arabidopsis mutants. In this report, we present results on photosynthetic properties and
on growth and reproduction in low light and high light and of the pph1/npq4 double
mutant in comparison with single mutants and wild type plants.
Materials and methods
Plant growth conditions and high light stresses
Low light growth experiments, excluded the one in Figure 3, were performed in a
growth chamber with homogenous illumination by fluorescent lamps. Low light (LL) ~
20 µmol m-2 s-1; normal light (NL) ~120 µmol m-2 s-1; long day (16 hr light/8 hr dark).
High light stresses were performed using cool-white LED lights for 8h at 1500 µmol m2 -1
s .
Creation of the pph1/npq4 double mutant: To produce the pph1/npq4 double mutant, we
crossed pollen of pph1.3 with ovule of npq4.1. Double mutant were screened for psbS
mutation by NPQ phenotype (video imaging of chlorophyll fluorescence and
PAM2000); for pph1 mutation by low temperature fluorescence emission spectra.
Plant growth, flower/silique production: Plant growth was determined as dry weight of
the rosette of plants grown 17 days in normal light and then 20, 33, 37 days in low light
for test 1, test 2 and test 3, respectively (Figure 4). Siliques and flowers were counted on
plants grown 17 days in normal light then 24, 37 days in low light, indicated as test 1
and test 2 in Figure 5.
Fluorescence analysis. Fluorescence measurements were performed using a PAM2000.
The maximum PSII quantum yield and the actual PSII quantum yield during a light
period were measured accordingly to the equations: Fv/Fm = (Fm-Fo)/Fm and ΦPSII =
(Fm’ – Ft)/Fm’ [3]. Fm is the maximum fluorescence yield and Fo is the minimal
fluorescence yield of dark adapted plants; Fm’ is maximum fluorescence yield of light
adapted plants and Ft is the steady state fluorescence yield under actinic light. NPQ was
calculated as (Fm-Fm’)/Fm’.
Results and discussions
Screening to isolate the pph1/npq4 double mutant
The double pph1/npq4 mutant was screened on the F1, F2, F3 seedling using the typical
phenotypes of the single parental mutants: NPQ decrease of the npq4 mutant [7] and an
elevated PSI fluorescence emission at 77K of the pph1 mutant as compared to wild type
after a preliminary exposure to PSI light to induce State I [10,11] as in Figure 1 and 2.
2
WT
NPQ
1.8
1.6
npq4
pph1npq4
1.4
pph1
1.2
1
0.8
0.6
0.4
0.2
0
0
50
100
150
200
250
300
350
Temps (sec)
Figure 1. NPQ of chlorophyll fluorescence of wild type (wt) and mutants
Fluorescence
1.6
1.4
wt
1.2
npq4
pph1/npq4
1
pph1
0.8
0.6
0.4
0.2
0
660
680
700
720
740
760
780
800
Wave (nm)
Figure 2. Fluorescence emission spectra at 77K of leaves from wild type (wt) and mutants
Response of mutants lacking PsbS and PPH1 to light stress
In high light
Plants have been treated for 8h at 1500 µmol m-2 s-1. Fv/Fm measured before and after
stress. The results, observed in figure 3, indicate that Fv/Fm was the same for all
genotypes before stress, accordingly to previous publication on single mutants [7,10] ).
After stress, the maximum quantum yield of PSII photochemistry decreased
significantly in all genotypes, with the less photoinhibited PSII in the wild type, then
pph1, npq4 and finally pph1/npq4. Again, photoinhibition susceptibility of PSII was
higher in mutants lacking PsbS.
3
4
B
0.9
WT
0.8
npq4
0.7
pph1/npq4
Fv/Fm
0.6
pph1
0.5
0.4
0.3
*
0.2
*
0.1
0
Before stress
8h HL
Figure 3. Fv/Fm measured before and after treatment by high light. The Fv/Fm decrease of
npq4 and pph1/npq4 compared with wild type is statistically significant with P<4% (noted with *
in the figure)
This is consistent with the fact that a reduced NPQ in mutants lacking PsbS leads to
overexcitation of PSII resulting in damage at high light intensities.
Pph1 mutation has clearly a smaller impact on PSII photoinhibition under high light. It
is not easy to conclude if an effect exists, since the differences between pph1 mutant vs
wild type and pph1/npq4 vs npq4 have little statistical significance in our tests.
However, the fact that at 1500 µmol m-2 s-1 (Figure 3), pph1 has a lower Fv/Fm than
wild type, as well as pph1/npq4 as compared with the npq4 single mutant, suggest that
pph1 mutation reduces in some way photoprotection under high light. This is an
interesting point, since state transitions are considered a regulative mechanism active
under moderate light [9].
The small variation of Fv/Fm in pph1 plants as compared with wild type plants supports
however the propositions that NPQ is a mechanism to prevent photoinhibition at high
light more important than state transitions [11, 13,14].
In low and normal light
The actual quantum yield of PSII (ΦPSII) reflects the overall efficiency of PSII reaction
center in the ligh. Results (Table 1) show that ΦPSII increases in all mutants as compared
with wild type. The increases are relatively small (1.7%, 1.6%, 1.8% for npq4,
pph1/npq4 and pph1, respectively), but they are statistically significant (P < 0.001% for
T-test). Comparison between ΦPSII of pph1/npq4 and single mutants did not show a
significant difference.
For plant growing in normal light, ΦPSII of all mutants were similar to that of wild type
plants (Table 1) with no statistical significant difference between them. This indicates
that the absence of PsbS and PPH1 does not cause any negative effect on the
photosynthetic process under moderate continuous light.
Table 1. PSII yield of plants in LL and NL.
Light
WT
npq4
pph1/npq4
pph1
LL
0.758±0.001 0.771±0.001 0.770±0.000 0.772±0.001
NL
0.757±0.002 0.759±0.003 0.760±0.003 0.762±0.004
Plant lacking PsbS and PPH1 shows enhanced growth and flower production in
low light condition
In order to have some physiological characterisation of the pph1 mutant and investigate
the possibility to improve plant performances in low light, we created the pph1/npq4
double mutant and compared growth of the double mutant, single mutants and wild type
plants. In our low light condition of ~20 µmol m-2 s-1, photosynthetic performances
(growth, flower + fruit production) improved both in pph1 (as previously shown for
growth in [10] and in npq4 mutants. Interestingly, a cumulative effect of the two
mutations seems present in the pph1/npq4 double mutant (Figure 4 and 5). Indeed, in
the absence of PsbS, growth increases of 60%, 10% and 14%, in test 1, 2, 3
respectively, in the absence of PPH1 of 28 %, 19 %, 9 %, while in the pph1/npq4
double mutant of 63%, 25%, and 26% (Figure 4). A similar result was found for silique
and flower number in our second biological repetition performed for this analysis
(Figure 5).
A
80
Dry weight (mg)
70
60
50
40
wt
npq4
pph1/npq4
pph1
30
#
#
Test 2
Test 3
#
#
20
10
0
Test 1
Figure 4. Growth of wild type, double and single mutant plants.
Growth differences between wild type and mutants are all statistically significant with P<5% for T-test.
Statistically significant differences between the pph1/npq4 double mutant and the single mutants are
indicated with the # symbol above the single mutant.
We found a statistical significant difference between mutants and wild type PSII yield at
low light intensity, which could explain the improved growth of mutants, but we were
not able to see a higher PSII yield for the double mutant as compared with single
mutants. This could be explained by technical issues to discriminate small variations in
fluorescence parameters, thus making difficult to see a difference between the double
mutant and single mutants. However, a little difference in PSII yield (which is an
instantaneous measurement) is amplified after several days of growth in low light.
Indeed growth cumulates the effect of an improved photosynthesis and is a better
indicator than yield of PSII to discriminate photochemical efficiencies of different
genotypes in our low light conditions. As alternative explanation, the increased growth
in the pph1/npq4 double mutant is not only dependent on ΦPSII, but a positive effect
from association of both mutations would lead to an improved growth that does not
depend on the addition of ΦPSII increases of single mutants, which is not evident.
5
6
120.00
Silique and Flower number
#
100.00
80.00
wt
npq4
pph1/npq4
pph1
*
*
#
*
* *
60.00
40.00
20.00
0.00
Test 1
Test 2
Figure 5. Silique + flower number of wild type and mutant plants in low light condition.
Statistically significant differences between mutants and wild type are indicated with (*). In test 2, the
silique + flower number increase of the pph1/npq4 double mutant is statistically significant as compared
with single mutants values (P< 5%; indicated with the # symbol).
Plant growth under normal light
Differently to low light, the photosynthetic properties tested under normal nonfluctuating light as PSII yield (Table 1) and growth (Figure 6) of mutants were similar
to those of wild type plants, suggesting that the absence of both PsbS and PPH1 proteins
does not cause a negative effect on plants in this condition.
300
Dry weight (mg)
250
200
150
wt
npq4
pph1/npq4
pph1
100
50
0
Figure 6. The dry weight of the double mutant, single mutants and wild type plants grown 38
days in normal light.
Conclusions
These results are in line with the proposition the plants have evolved several
photosynthetic regulations to optimise plant fitness under natural conditions rather than
plant growth [9]. Thus, under particular controlled condition, it might be possible to
improve plant performances by acting on photosynthesis at a molecular level.
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Ảnh hưởng của sự thiếu hụt hai protein PsbS và PPH1 đến khả năng
chống chịu với điều kiện ánh sáng bất lợi ở cây mô hình Arabidopsis
thaliana.
Tóm tắt
NPQ là cơ chế rất quan trọng để duy trì quang hợp trong điều kiện ánh sáng cao.
Protein PsbS đóng vai trò chìa khóa để hoạt hóa NPQ. Sự di chuyển trạng thái là cơ chế
điều chỉnh thứ hai phản ứng lại sự thay đổi của chất lượng ánh sáng. Trong sự di chuyển
trạng thái, nhờ hoạt động của cặp protein phosphorylation/dephotphorylation
STN7/PPH1 dẫn đến sự di chuyển của một phần LHCII từ PSII đến PSI và ngược lại.
Đột biến bất hoạt gen pph1 dẫn đến màng thylakoid bị chặn lại ở “trạng thái II”, giàu
phức hợp PSI-LHCII. Chúng tôi đã tạo ra cây Arabidopsis thaliana đột biến kép
pph1/npq4, rồi tiến hành xác định các đặc điểm quang hợp, sinh trưởng và năng suất
dưới điều kiện ánh sáng cao và thấp. Kết quả thu được chỉ ra rằng đột biến kép thể hiện
sự tăng cường sinh trưởng và sự hình thành hoa/quả so với các đột biến đơn và cây đối
chứng.
Từ khóa: di chuyển trạng thái, NPQ, protein PsbS, protein PPH1, sinh trưởng,