Download Using chlorophyll fluorescence to rapidly discriminate C3 from C4

Using chlorophyll fluorescence to rapidly discriminate C 3 from C 4
photosynthesis in plants
RG White1
1. CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia
[email protected]
chlorophyll fluorescence, C 3 photosynthesis, C 4 photosynthesis
There is considerable interest in augmenting C 3 plant species with components of C 4
photosynthesis, to increase productivity of major crop species, particularly rice (Oryza sativa L.)
[1,2], which is the largest grain crop in the world. The driving force behind this interest is production
of more grain per unit of phosynthetic capacity, since C 4 phosynthesis is almost twice as efficient,
per unit of solar radiation, at fixing atmospheric CO 2 than the C 3 pathway [2]. This means that C 4
plants are also more water use efficient, since for the same amount of transpired water, more
carbon is fixed into photosynthates and hence, into edible plant product.
Introduction of C 4 traits into a C 3 plant such as rice requires not only considerable modification of
C 3 anatomy and biochemistry, but also a method for detecting the shift to C 4 traits. Genetic screens
will require examination of hundreds of hybrids and mutant lines displaying intermediate C 3 -C 4
characteristics in a quick and efficient way. There are several anatomical and physiological traits
that vary between C 3 and C 4 plants, a few of which can be assessed quickly.
Easily measured traits that distinguish C 3 and C 4 photosythesis include the distance between
longitudinal veins in the leaves, with C 4 species characterised by small intervein spacing such that
mesophyll cells are never more than one cell diameter distant from the bundle sheath [2]. This is
useful if the standard intervein distance has been well characterised in a particular species, but it is
possible that in new genetic lines there may be a less tight correlation between intervein distance
assessed quickly at low magnification, and C 4 metabolic characteristics.
Other rapid screening protocols include assessing changes in carbon metabolism and chloroplast
fluorescence under varying CO 2 conditions. A further method is described below, which relies on
the very different ratios of photosystem I and II in mesophyll and bundle sheath chloroplasts in C 4
plants. Earlier workers have used fluorescence differences to distinguish mesophyll and bundle
sheath photosystems in isolated chloroplasts often at low temperatures, or have used fixed and
sectioned material [3]. Here, we outline a rapid protocol to assess photosynthetic apparatus and
leaf anatomy using fluorescence microscopy of fresh leaf tissues.
Fluorescence illumination with red excitation and collection of chloroplast autofluorescence in two
wavebands in the long red spectrum distinguishes C 4 bundle sheath chloroplasts which have very
low levels of photosystem II, from chloroplasts in the C 3 bundle sheath or in the mesophyll of either
C 4 or C 3 plants, which contain a much larger proportion of photosystem II. Either confocal laser
scanning microscopy or fluorescence microscopy, with a compound microscope or a fluorescence
dissecting microscope, can be used for the assessment.
Fresh leaf tissues of the C 4 plant sorghum (Sorghum bicolor L.) or the C 3 plant rice were crosssectioned by hand, mounted in water and immediately observed on a Leica SP2 confocal
microscope. After collecting representative images showing chloroplast and cell wall fluorescence,
as shown in Fig. 1 (A-B, C-E), emission was collected in 10 nm bands at 5 nm intervals from 650 to
800 nm, and the emission calculated relative to emission at 720 nm. In sorghum, the characteristic
depletion of emission at 685 nm indicating a low proportion of photosystem II was readily detected
in bundle sheath chloroplasts, whereas the peak in emission from mesophyll chloroplasts was
typical for those containing a larger proportion of photosystem II. Rice chloroplasts showed
characteristics of C 3 photosynthetic apparatus throughout the leaves.
Fluorescence analysis using a confocal microscope is moderately time-consuming, and we are
transferring this technique to fluorescence dissecting and compound microscopes. Before
implementing fluorescence analysis as a screening technique, we are confirming the robustness of
fluorescence discrimination between C 3 and C 4 chloroplasts in a range of environmental conditions.
References
[1] Sage TL, Sage RF (2009) The functional anatomy of rice leaves: implications for refixation of
photorespiratory CO 2 and efforts to engineer C 4 photosynthesis into rice. Plant & Cell Physiology
50, 756–772.
[2] Furbank RT et al. (2009) C 4 rice: a challenge for plant phenomics. Functional Plant Biology 36,
845–856.
[3] Pfündel E, Neubohn B (1999) Assessing photosystem I and II distribution in leaves from C 4
plants using confocal laser scanning microscopy. Plant, Cell & Environment 22, 1569–1577.
[4] Thanks to Susanne von Caemmerer and Bob Furbank for useful discussions.
B
MES
D
E
BS
MES
C
relative fluorescence emission
BS
F
relative fluorescence emission
A
Figure 1. Confocal laser scanning microscope image of fresh hand sections of Sorghum (A,B) and rice (D,E)
showing chlorophyll fluorescence only (A,D) or chlorophyll and cell wall fluorescence (B,E). Fresh handsections of leaves were mounted in water and imaged using excitation of 633 nm and emission from 650-720
nm (pseudocoloured red) and 720-800 nm (pseudocoloured blue) for chlorophyl fluorescence, or with 405 nm
excitation and 420-480 nm emission for cell wall autofluorescence (pseudocoloured green in B,E). In
mesophyll chloroplasts with both photosystems I and II, the combined fluorescence is magenta, whereas in
bundle sheath chloroplasts of Sorghum, the fluorescence is purple since the contribution of PSII is greatly
reduced. In rice, PSI and PSII are found in similar proportions throughout the leaf. BS = bundle sheath
chloroplasts; MES = mesophyll chloroplasts. Scale bar = 20 µm. In C,F, the emission spectrum from either
bundle sheath chloroplasts (blue squares) or mesophyll chloroplasts (magenta squares) is shown relative to
emission at 720 nm (mean of 10 BS or MES areas).