adaptation of dry valley lake phytoplankton to the...
TRANSCRIPT
Adaptation of Dry Valley Lake Phytoplankton to the Polar Night
C. raudensis transplantNatural Phytoplankton
Communities
Ecophysiological Models
During Summer-Winter Transition
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Sample Summary
Date Sample # PAR Transpl
ELB 17m
ELB WLB
6m 10m 18m 20m 10m 13m 15m 20m
T0 2.55 X X X X
T1 2.68 X X X X X X X X X
T2 2.4 X X X X X X X X X
T3 0.82 X
T4 0.78 X X X X X X X X X
T5 0.32 X X X X X X X X X
T6 0.46 X X X X
Data Summary
1. C. raudensis transplant
1. Chlorophyll, total protein
2. Protein – RubisCO (RbcL), PS II (PsbA)
3. Gene Copy # - rbcL, psbA
4. Gene Expression – rbcL, psbA
2. Environmental Samples (EB, WB)
1. 18S rRNA phylogeny/distribution
2. Gene copy #/gene expression Form IA/B & Form ID rbcL
3. rbcL phylogeny
PSII PSIISummer
Winter
POLAR NIGHT ADAPTATION
Adaptation to the Polar Night
O2
O2
PSIIPSI
ATP
H+
Fd
NADPH
e- pool “ENERGY IN”
CO 2ATP
NADPH
SUGARS
“ENERGY OUT”
Balancing the Energy Budget
PsbA
RbcL
2.5 2.7 2.4 0.82 0.78 0.32
0 1 2 3 4 5
A
B
C
From the Laboratory to the Field:
Protein Expression
PAR
Weeks of incubation
RbcL
PsbA
LHCII
PSII
0 10 20 30 40
1.75
2.00
2.25
2.50
2.75
3.00
Ch
l a
/b
A
From the Laboratory to the Field:
RbcL & PsbA Protein Levels
0 5 10 15 20 25 30 35 40
0.0
0.2
0.4
0.6
0.8
1.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
pm
ol/
mg
pro
tein
Incubation Time (days)
RbcL
PsbA
PA
R
From the Laboratory to the Field:
psbA & rbcL Gene Expression
0 5 10 15 20 25 30 35 40
0
10
20
30
40
50
60
70
80
90
100
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
10
8 r
bc
L/L
Incubation Time (days)
PA
R
-5 0 5 10 15 20 25 30 35 40
200
400
600
800
1000
1200
1400
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
10
8 p
sb
A m
RN
A/L
Incubation Time (days)
PA
R
rbcL psbA
From the Laboratory to the Field:
psbA & rbcL Gene Expression
0 5 10 15 20 25 30 35 40
0
10
20
30
40
50
60
70
80
90
100
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
10
8 r
bc
L/L
Incubation Time (days)
PA
R
-5 0 5 10 15 20 25 30 35 40
200
400
600
800
1000
1200
1400
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
10
8 p
sb
A m
RN
A/L
Incubation Time (days)
PA
R
rbcL psbA
y = 229.19x + 87.566
R2 = 0.8789
0
100
200
300
400
500
600
700
800
900
0 1 2 3
PAR (µmol photons/m2 s)
10
8 p
sbA
mR
NA
/L
PSII PSII
From the Laboratory to the Field:
Adaptation to the Polar Night
CO2CO2
↓ psbA / no Δ PsbA/LHCII
↓ rbcL / ↓ RbcL
POLAR NIGHT
ADAPTATION
Remaining Experiments: Transplant
• 18 rRNA real time PCR
– Ribosomes
– Comparison with environmental samples
WB vs. EB 18S rDNA
• 1059 clones screened (sequencing/RFLP)
• 46 unique phylotypes
• 22 uncultured representatives (< 10% total clones)
Top 5 nearest neighbors (~75% total clones):
Geminigera cryophila (cryptophyte)
Nannochloropsis sp. (stramenopile)
Chlamydomonas ICE-W (chlorophyte)*
Isochrysis litoralis (prymnesiophyte)
Chrysophyceae sp. (chrysophyte)*
18S rDNA Clone Distribution
3.35%
6.32%
2.23%
13.01%
5.2%11.15%
30.48%
28.25%
1.06%4.61%1.42%
8.16%
2.13%
26.24%
38.65%
17.73%
ELB WLB
StramenopileCryptomonadChlorophyteChrysophytePrymnesiophyte
CiliateChanoflagellidAlveolate
0 10 20 30 40 50 60 70 80
20
15
10
5
0 10 20 30 40 50 60 70 80
20
15
10
5
Number of Clones (% total)
ELB
Depth
(m
)
Number of Clones (% total)
WLB
Strameno
Prymnesioph
Chrysoph
Chlorophy
Crypto
18S rDNA Clone Distribution:
WB vs. EB RubisCO
• Real Time PCR (rbcL)
– Gene copy number (DNA)
– Gene expression (cDNA)
• Two major forms
– Form IA/B – cyanobacteria, plants, green algae
– Form ID – cryptophytes, chrysophytes, prymnesiophytes
Real Time PCR: rbcL Form ID
Form ID gene number in East Bonney
0
10
20
30
40
50
60
70
80
90
0 2 4 6
Time point
10
8 G
ene n
um
ber/
L
6m
10m
18m
20m
Gene Copy # (DNA)
ELB ELB
Form ID gene number in West Bonney
0
50
100
150
200
250
0 1 2 3 4 5
Time point
10
8 G
en
e n
um
be
r/L
10m
13m
15m
20m
WLB WLB
Real Time PCR: rbcL Form ID
Form ID gene number in East Bonney
0
10
20
30
40
50
60
70
80
90
0 2 4 6
Time point
10
8 G
ene n
um
ber/
L
6m
10m
18m
20m
Form ID gene expression in East Bonney
0
1
2
3
4
5
6
7
0 2 4 6
Time point
10
8 m
RN
A/L
6m
10m
18m
20m
Gene Copy # (DNA) Gene Expression (cDNA)
ELB ELB
Form ID gene number in West Bonney
0
50
100
150
200
250
0 1 2 3 4 5
Time point
10
8 G
en
e n
um
be
r/L
10m
13m
15m
20m
Form ID gene expression in East Bonney
0
0.5
1
1.5
2
2.5
0 2 4 6
Time point
10
8 m
RN
A/L
10m
13m
15m
20m
WLB WLB
RbcL Form 1D Phylogenetics
Nannochloropsis sp. JL2/4-1Nannochloropsis sp. An1/12-10
Nannochloropsis sp.MDL11-16Nannochloropsis limnetica
ELB13-T1 WK9Nannochloropsis maritima
Vischeria helveticaEustigmatos magnus
Pinguiococcus pyrenoidosusOphiocytium parvulumTribonema regulare
Bumilleria exilisHeterothrix debilis
Cryptomonas marssoniiProteomonas sulcata
Plagioselmis sp.Uncultured Microbe FJ233412.1
ELB13-T1 WK10Geminigera cryophila
Uncultured Microbe FJ233400.1Uncultured Microbe FJ233375.1
Dinophysis triposTeleaulax sp.
Calcidiscus leptoporusUmbilicosphaera sibogae
Cruciplacolithus neohelisPleurochrysis carterae
Coccolithus pelagicusChrysochromulina parva
Gephyrocapsa oceanicaIsochrysis sp. SAG 927-2
Isochrysis galbanaWLB13-T1 WK1
ELB13-T1 WK6
100
100
100
96
55
73
92
83
100
88
97
95
82
60
100
99
57
100
100
83
57
76
57
95
59
90
76
58
61
100
21
0.02
Stramenopiles
Cryptophytes
Primnesiophyte
Conclusions
• EB & WB are dominated by 4-5 algal groups with 1-2 representatives
• Algae with form 1D RubisCO dominate both lakes at mid-depths
• Form 1D rbcL abundance/expression during Polar Night differs between WB vs. EB
Remaining Experiments: Environment
• Finish 18S clone libraries
• 18S rDNA & rRNA real time PCR
– Normalize 18S rDNA libraries (rDNA)
– Correlate with transplant exp (rRNA)
• Finish rbcL form IA/B real time PCR
• rbcL form ID & IA/B clone libraries
– Phylogenetics
– Distribution of DNA vs. cDNA (RFLP)
RbcL Expression:
Transplant vs. Environment
0 5 10 15 20 25 30 35 40 45 50
0
10
20
30
40
50
60
70
80
90
100
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0
1
2
3
4
5
6
7
8
10
8 C
. ra
ue
ns
is r
bc
L/L
Incubation Time (days)
PA
R
10
8 rb
cL
fo
rm I
D @
10
m /
L