Plant nanobionics approach to augment photosynthesis and ... - Nature

122 downloads 0 Views 4MB Size Report
Mar 16, 2014 - photosynthesis and biochemical sensing. Juan Pablo Giraldo1, Markita P. Landry1, Sean M. Faltermeier1, Thomas P. McNicholas1,. Nicole M.
ARTICLES PUBLISHED ONLINE: 16 MARCH 2014 | DOI: 10.1038/NMAT3890

Plant nanobionics approach to augment photosynthesis and biochemical sensing Juan Pablo Giraldo1, Markita P. Landry1, Sean M. Faltermeier1, Thomas P. McNicholas1, Nicole M. Iverson1, Ardemis A. Boghossian1,2, Nigel F. Reuel1, Andrew J. Hilmer1, Fatih Sen1,3, Jacqueline A. Brew1 and Michael S. Strano1* The interface between plant organelles and non-biological nanostructures has the potential to impart organelles with new and enhanced functions. Here, we show that single-walled carbon nanotubes (SWNTs) passively transport and irreversibly localize within the lipid envelope of extracted plant chloroplasts, promote over three times higher photosynthetic activity than that of controls, and enhance maximum electron transport rates. The SWNT–chloroplast assemblies also enable higher rates of leaf electron transport in vivo through a mechanism consistent with augmented photoabsorption. Concentrations of reactive oxygen species inside extracted chloroplasts are significantly suppressed by delivering poly(acrylic acid)–nanoceria or SWNT–nanoceria complexes. Moreover, we show that SWNTs enable near-infrared fluorescence monitoring of nitric oxide both ex vivo and in vivo, thus demonstrating that a plant can be augmented to function as a photonic chemical sensor. Nanobionics engineering of plant function may contribute to the development of biomimetic materials for light-harvesting and biochemical detection with regenerative properties and enhanced efficiency.

C

hloroplasts are the ultimate source of chemical energy in food supplies and carbon-based fuels on the planet. By capturing atmospheric CO2 , these plant organelles convert light energy into three major forms of sugars that fuel plant growth: maltose, triose phosphate, and glucose1 . Whereas photosystems interfaced with nanomaterials are extensively studied, nanoengineering chloroplast photosynthesis for enhancing solar energy harnessing remains unexplored2 . One major deterrent in using chloroplast photosynthetic power as an alternative energy source is that these organelles are no longer independently living organisms. However, isolated chloroplasts from the algae Vaucheria litorea in symbiotic association with the sea slug Elysia chlorotica remarkably remain functional for at least 9 months3,4 . Land plant chloroplast photosystem activity declines within a day after extraction5,6 , and ex vivo sugar output has been reported for only a few hours1,7 . Although chloroplasts have mechanisms in place to self-repair photodamaged proteins8 , as well as a double-stranded circular DNA with a subset of protein-encoding genes involved in photosynthesis9 , and ribosomal units for protein synthesis and assembly10 , little is known about engineering these plant organelles for long-term, stable photosynthesis ex vivo. Another limitation of chloroplast photosynthesis is that absorbed light is constrained to the visible range of the spectrum, allowing access to only roughly 50% of the incident solar energy radiation11 . Furthermore, less than 10% of full sunlight saturates the capacity of the photosynthetic apparatus12 . Photosynthetic organisms evolved for reproductive success, including shading competitors, not solely for solar energy conversion efficiency. Thus, improving photosynthetic efficiency may require extending the range of solar light absorption13 , particularly in the near-infrared spectral range, which is able to penetrate deeper into living organisms. The high stability and unique chemical and physical traits of nanomaterials have the potential to enable chloroplast-based

photocatalytic complexes both ex vivo and in vivo with enhanced and new functional properties. SWNTs embedded within chloroplasts have the potential to enhance the light reactions of photosynthesis with their distinctive optical and electronic properties. Under bright sunlight, chloroplast photosystems capture more photons than they can convert into electron flow14 . However, under non-saturating light conditions, maximizing solar energy capture is crucial15 . SWNTs absorb light over a broad range of wavelengths in the ultraviolet, visible and near-infrared spectra not captured by the chloroplast antenna pigments. The electronic bandgap of semiconducting SWNTs allows them to convert this absorbed solar energy into excitons16 that could transfer electrons to the photosynthetic machinery. Also, SWNT-based nanosensors can monitor single-molecule dynamics17 of free radicals within chloroplasts for optimizing photosynthetic environmental conditions (light and CO2 ). However, nanoengineering photosynthesis requires the delivery of nanomaterials through the chloroplast outer envelope. Nanoparticle transport through lipid bilayers has been described to be energy dependent, requiring endocytosis pathways18 that have not been reported in isolated chloroplasts. So far, nanomaterial uptake mechanisms through cell membranes are controversial19 and poorly understood in organelles such as chloroplasts. Cerium oxide nanoparticles (nanoceria) also have been demonstrated to catalyse the quenching of reative oxygen species (ROS) in retinal cells, significantly reducing their intracellular concentrations20 . Chloroplasts have natural biochemical pathways to scavenge ROS and mechanisms for photosystem protein selfrepair8 . However, most enzymes involved cannot be synthesized ex vivo because they lack the polypeptide precursors imported from the plant cell cytosol21 . Technologies that localize nanoceria at the sites of ROS generation can exploit the oxygen vacancies in the CeO2 lattice structure to effectively trap free radicals before

1 Department

of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA, 2 Department of Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA, 3 Department of Biochemistry, Dumlupinar University, Kutahya 43020, Turkey. *e-mail: [email protected] 400

NATURE MATERIALS | VOL 13 | APRIL 2014 | www.nature.com/naturematerials

© 2014 Macmillan Publishers Limited. All rights reserved

NATURE MATERIALS DOI: 10.1038/NMAT3890 a

NIR fluorescence (a.u.)

12,000

ARTICLES b

Chloroplast

(9,4)

ss(AT)15–SWNT ss(AT)15–SWNT + chloroplast

10,000 8,000

(8,6) (12,1) (8,7) (10,5)

6,000 4,000

t=0s

t = 50 s

t = 75 s

t = 100 s

t = 150 s

t = 200 s

(11,3)

2,000 0 1,100

1,200

1,300

1,400

1,500

1,600

Wavelength (nm)

c

e

d B ight Bri ght-fie ght h -fi -fie fieeld d

g

f

NIR fluorescence (a.u.)

1,000

SWNTs + chloroplast

5 µm

2,000 1,200 800 400 0

NIR fluorescence (a.u.)

s AT) ss ss( A 15 AT S NTs Tss T 15–SW

0

400

800

1,200

Time (s)

2,000

Buffer Chloroplast

1,600 1,200 800 400 0

PVA VA–SW –SWNTs –SW SW WNTs NTs NT T

0

400

800

1,200

Time (s) NIR fluorescence (a.u.)

Chi C Ch hitos osan–S osan– an– n SWN n– SWNTss SW SWNTs

Buffer Chloroplast

1,600

2,000

Buffer Chloroplast

1,600 1,200 800 400 0

0

400

800

1,200

Lip LLi iipid– d–SWN d– WNT Tss

NIR fluorescence (a.u.)

Time (s) 2,000

Buffer Chloroplast

1,600 1,200 800 400 0

0

400

800

1,200

Time (s)

Figure 1 | Mechanism of SWNT trapping by chloroplast lipid bilayers. a, Chloroplast autofluorescence was masked from near-infrared images by a long-pass 1,100 nm filter. b, Near-infrared photo still indicating rapid penetration of ss(AT)15 –SWNTs through the lipid bilayers of isolated chloroplasts. c, SWNT transport through chloroplast double membrane envelope via kinetic trapping by lipid exchange. d–f, Bright-field (×100; d) and near-infrared (×100; e) images of isolated chloroplasts indicating uptake of SWNTs coated in ss(AT)15 DNA and chitosan, but not of PVA- and lipid-coated SWNTs (×100; f). g, Change in average SWNT fluorescence in cross-sections of chloroplasts versus external buffer solution. Laser excitation 785 nm at 75 µW.

they damage nearby pigments, reaction centres and photosynthetic proteins. We have recently shown that cerium leakage, from dextran nanoceria particles not able to penetrate the chloroplast outer envelope, promotes only minor scavenging of photogenerated ROS (ref. 2). Thus, we predicted far more effective catalytic ROS

scavenging and extended chloroplast photosynthetic activity by assembling nanoceria within the photosynthetic machinery. In this work, we develop a concept that is heretofore unexplored in the literature. We examine whether and how nanomaterials can interface specifically with plant organelles ex vivo and in vivo to

NATURE MATERIALS | VOL 13 | APRIL 2014 | www.nature.com/naturematerials

© 2014 Macmillan Publishers Limited. All rights reserved

401

NATURE MATERIALS DOI: 10.1038/NMAT3890

2

−1 0

500

1 2 0

−2

−1 0

500

1 2

0

−2

x (µm)

c 100 80 60 40 20 0

2

−2

24 °C 4 °C Light

4 °C 24 °C Dark

16

0 x (µm)

Liposomes + ss(AT)15–SWNTs Liposomes + buffer

2

0

0

1,000

−1 0

500

1 2 −2

0

2

0

x (µm)

d

Liposomes + ss(AT)15–SWNTs Liposomes + buffer

0.20 0.15

12 8

−2 0 2 2 0 −2 y (µm) x (µm)

−2 y (µm)

−2

200

Z=0

1,000

Gp

Percentage of chloroplasts with SWNTs

b

0 x (µm)

0

Z=0

1,000

−10

y (µm)

x (µm)

400

−5

0

−2 0 2 2 0 −2

600

0

G peak intensity

2

200

800

5

G peak intensity

500

1

−10

G peak intensity

0

G peak intensity

−1

400

−5

0

Z=0

1,000

−2 y (µm)

−2 0 2 2 0 −2 y (µm) x (µm)

Laurdan fluorescence (a.u.)

z (µm)

Z=0

−2

z (µm)

0

−2 0 2 2 0 −2 y (µm) x (µm)

200

−10

600

0

1,000 G peak intensity

200

400

−5

800

5

10 G peak intensity

400

−5

600

0

Lipid–SWNTs −35.2 ± 2.6 mV

1,000

10 G peak intensity

600

0

1,000 800

5

G peak intensity

800

5

10

Chitosan–SWNTs 48.5 ± 1.0 mV

z (µm)

1,000

10

−10

PVA–SWNTs −6.4 ± 3.4 mV

y (µm)

ss(AT)15–SWNTs −44.6 ± 1.9 mV

y (µm)

a

z (µm)

ARTICLES

0.10 0.05

4

0.00

0 400

500

600

Wavelength (nm)

0

5

10 15 20 25 30 Time (min)

Figure 2 | The ss(AT)15 –SWNT lipid exchange with the chloroplasts’ outer envelope via a passive uptake mechanism is dependent on zeta potential. a, Confocal Raman spectroscopy 3D maps localized ss(AT)15 and chitosan SWNTs inside chloroplasts whereas relatively neutral PVA and lipid SWNTs were not present. Chloroplasts were approximately 5 µm in diameter and centred at Z = 0. Values correspond to SWNT G-band intensity (1, 580 cm−1 ) under a laser excitation of 658 nm at 145 µW. b, Average percentage of chloroplasts with SWNTs is not influenced by light or temperature conditions. c,d, ss(AT)15 –SWNTs quench laurdan fluorescence in DGDG and MGDG liposomes (c), but do not modify laurdan generalized polarization (Gp), an indicator of membrane fluidity (d). Error bars represent s.d. (n = 3).

enable new or enhanced functions. We reason that the assembly of nanoparticle complexes within the chloroplast photosynthetic machinery has the potential to enhance solar energy conversion through augmented light reactions of photosynthesis and ROS scavenging while imparting new sensing capabilities to living plants. This effort is divided into objectives that attempt to understand a newfound mechanism of transport and spontaneous assembly of nanomaterials inside chloroplasts; increase the photosynthetic activity in chloroplasts through specific nanoparticle assemblies within photosynthetic machinery; enhance the ability of the chloroplast to scavenge ROS using cerium and carbon-based nanoparticles transported to optimal sites of ROS generation; and enable real-time monitoring of free-radical species and environmental pollutants using in vivo and ex vivo embedded nanosensors. These objectives constitute a plant nanobionics approach to generating new constructs for bionanotechnology research, which may contribute to biomimetic energy generation and the creation of new plant biochemical detectors.

Nanoparticle spontaneous assembly within chloroplasts We used single-particle tracking of near-infrared fluorescent semiconducting SWNTs to investigate their interaction with 402

isolated plant chloroplasts from spinach leaves. SWNTs do not photobleach and fluoresce in the near-infrared region above 1,100 nm, where chloroplast autofluorescence is minimal (Fig. 1a). SWNT fluorescence of chiralities (9,4), (8,6), (12,1), (11,3), (8,7) and (10,5) was quantified inside chloroplasts using a laser excitation (785 nm) that is off-resonance to photosynthetic pigments. Surprisingly, SWNTs suspended in strongly cationic or anionic coatings (that is, high magnitude of the zeta potential) were found to traverse and localize rapidly within the chloroplast outer envelope and not just adsorb to the exterior. The process is observed to occur within seconds of nanoparticle interaction with the inner and outer lipid bilayer (Fig. 1b). This process is irreversible, and in the case of ss(AT)15 DNA- and chitosan-coated SWNTs dosed at 2.5 mg l−1 concentration, leaves no free nanotubes suspended in the buffer solution (Supplementary Video). Not all SWNT types are transported through lipid bilayers. Both near-infrared SWNT fluorescent images (Fig. 1d–g) and confocal three-dimensional (3D) mapping of the characteristic SWNT Raman G-band (1, 580 cm−1 ) (Fig. 2a) indicate that whereas ss(AT)15 and chitosan SWNTs are embedded within chloroplasts, polyvinyl alcohol (PVA)- and lipid-coated SWNTs do not interact with the lipid bilayer. Thus, we confirmed modelling NATURE MATERIALS | VOL 13 | APRIL 2014 | www.nature.com/naturematerials

© 2014 Macmillan Publishers Limited. All rights reserved

NATURE MATERIALS DOI: 10.1038/NMAT3890 SWNT NIR fluorescence

Overlay

a

e

Lamina 1 cm

b

NIR fluorescence (a.u.)

Autofluorescence/ Bright-field

ARTICLES 4 × 104

Control leaf SWNT leaf SWNT

3 × 104 2 × 104 1 × 104 0 900

1,000

1,100

1,200

1,300

Wavelength (nm)

Vein

f Intensity (a.u.)

16 μm

6,000

c

Control leaf SWNT leaf SWNT

4,000 G

G′

2,000

Crosssection

0

0

500 1,000 1,500 2,000 2,500 3,000

g

d

Chloroplasts

5 μm

Indexed chlorophyll content (SPAD)

Wavenumber (cm−1)

16 μm

30 20 10 Control leaf SWNT leaf

0 0

5

10

15

20

Time (days)

i

j

50 nm

100 nm

Chloroplast autofluorescence

Poly(acrylic) acid nanoceria

h

SWNT

5 μm

5 μm

Nanoceria

5 μm

SWNT–nanoceria

Figure 3 | Nanoparticle transport inside isolated chloroplasts and leaves. a, CRi Maestro images of ss(AT)15 –SWNTs within the leaf lamina of A. thaliana. b–d, Co-localization of ss(AT)15 –SWNTs near leaf veins (×20; b), in parenchyma cells (×20; c) and chloroplasts in vivo (×63; d). e, Near-infrared fluorescence signal of SWNTs in leaves relative to SWNTs in solution. f, Raman spectroscopy showed broadening of G and G’ SWNT peaks in leaves. g, Temporal patterns of chlorophyll content indicated similar lifespans for leaves with SWNTs and controls. Error bars represent s.d. (n = 4). h, Confocal images of chloroplasts assembled with PAA–NC: chlorophyll (green) co-localized with PAA–NC labelled with Alexa Fluor 405 (red). i, TEM images of the SWNT–NC complex. j, Chloroplast TEM cross-section after incubation in SWNT–NC suspension. Nanoparticles localized both in the chloroplasts’ thylakoid membranes (red arrows) and the stroma (yellow arrows). Elemental analysis by ICP-MS of chloroplasts with the SWNT–NC complex detected the presence of cerium at 95 ± 0.1 ppm.

studies identifying SWNT surface patterning and charge as key traits determining penetration through lipid membranes19,22 . A highly negative or positive SWNT zeta potential favours nanotube adsorption to the chloroplast lipid membrane (Fig. 2a). The ss(AT)15 and chitosan SWNTs, having zeta potentials of −44.6 ± 1.9 mV and 48.5 ± 1.0 mV, respectively, are transported inside chloroplasts but not PVA-coated SWNTs with more neutral values, −6.4 ± 3.4 mV. However, lipid–SWNTs with a negative zeta potential, −35.2 ± 2.6 mV, are unable to move through

the chloroplast outer envelope, confirming membrane trapping inhibition once SWNTs are coated with lipids. SWNT movement through chloroplast membranes occurs via passive mechanisms. Neither variation in temperature from 4◦ to 24 ◦ C nor light conditions influenced chloroplast ss(AT)15 – SWNT uptake (Fig. 2b). Previous studies of protein transport inside chloroplasts have used temperature as an indicator of metabolic activity and light conditions as a proxy for ATP generation23 . Together these results suggest that SWNTs are transported through

NATURE MATERIALS | VOL 13 | APRIL 2014 | www.nature.com/naturematerials

© 2014 Macmillan Publishers Limited. All rights reserved

403

8 4 0 0

1

2

3

4

5

*

20 10

30

5 mg l−1

Time (h)

Chloroplasts–SWNT

0.2 0.1 0.0

400

600

800

1,000

1,200

60 50 40 30 20 10

1,400

0

1

2

Wavelength (nm)

Superoxide concentration (µM)

50

h

No nanoparticles SWNT-NC 0.7 mg l−1 SWNT-NC 1.4 mg l−1

PAA-NC 4 µM PAA-NC 8 µM

40

SWNT-NC 2.8 mg

PAA-NC 17 µM

NIR fluorescence (a.u)

g

l−1

30 20 10

30,000 25,000

5

1

2

3

4

5

600 400 200 0

0 1,100

6

(1 1 ,3)

1,200

1,300

1

2

1,400

4,000 (11,3) 2,000 0 1,100

1,200

1

2

3 1

2

1,300

1,400

1,500

Wavelength (nm)

l

3

6

(8,7) (8,6) (10,5) (12,1)

6,000

1,500

k

5

+NO

Wavelength (nm)

Time (h)

3 4 Time (h)

SWNT leaf

8,000

15,000 10,000

0

i

SWNT chloroplasts +NO

(8,7) (8,6) (10,5) (12,1)

20,000

No nanoparticles PAA-NC 6 µM PAA-NC 12 µM PAA-NC 24 µM SWNT-NC 0.25 mg l−1 SWNT-NC 0.5 mg l−1 SWNT-NC 1.25 mg l−1

800

6

5,000

0 0

j

3 4 Time (h)

*

Photosynthetic active radiation (µmol m−2 s−1)

1,000

0

NIR fluorescence (a.u.)

SWNT

0.3

m-SWNT 0.7 mg l−1 m-SWNT 1.4 mg l−1 m-SWNT 2.8 mg l−1 SWNT 0.7 mg l−1 SWNT 1.4 mg l−1 SWNT 2.8 mg l−1 No nanoparticles

70

Chloroplasts

Intensity (a.u.)

Absorbance (a.u)

0.4

f

DCF fluorescence (a.u)

e

Reduced DCPIP (µM)

d

**

10 0

2.5 mg l−1

**

20

*

0

6

40

900−1,200

12

30

Control leaf SWNT leaf 5 mg l−1 * *

50

600−900

No nanoparticles PAA-NC 4 µM PAA-NC 8 µM PAA-NC 17 µM SWNT-NC 0.7 mg l−1 SWNT-NC 1.4 mg l−1 SWNT-NC 2.8 mg l−1

16

*

200−300

20

40

60

100−200

Maximum electron transport rate (µmol m−2 s−1)

Reduced DCPIP (µM)

24

c

Control chloroplasts SWNT chloroplasts Control leaf SWNT leaf

50

0−100

b

32 28

Electron transport rate (µmol m−2 s−1)

a

300−600

NATURE MATERIALS DOI: 10.1038/NMAT3890

ARTICLES

200

1

150

3

2

100

16 µm

0

100

200

300

400

500

Time (s)

Figure 4 | SWNT and nanoceria plant nanobionics. a, Enhanced photosynthetic activity of isolated chloroplasts with SWNT–NC was shown by electron transfer to DCPIP. b, Higher maximum electron-transport rates in extracted chloroplasts and leaves were quantified by the yield of chlorophyll fluorescence (P