Adaptation of Dry Valley Lake Phytoplankton to the …mus.montana.edu/priscu/images/presentations/Kiss Polar...RbcL Form 1D Phylogenetics Nannochloropsis sp. JL2/4-1 Nannochloropsis

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