IC4R005-Phenomics-2009-11089458

Project Title

• C4 rice: a challenge for plant phenomics

The Background of This Project

• There is now strong evidence that yield potential in rice (Oryza sativa L.) is becoming limited by ‘source’ capacity, i.e. photosynthetic capacity or efficiency, and hence the ability to fill the large number of grain ‘sinks’ produced in modern varieties. One solution to this problem is to introduce a more efficient, higher capacity photosynthetic mechanism to rice, theC4 pathway.Amajor challenge is identifying and engineering the genes necessary to installC4 photosynthesis in rice. Recently, an international research consortium was established to achieve this aim. Central to the aims of this project is phenotyping large populations of rice and sorghum (Sorghum bicolor L.) mutants for ‘C4-ness’ to identifyC3 plants that have acquired C4 characteristics or revertant C4 plants that have lost them. This paper describes a variety of plant phenomics approaches to identify these plants and the genes responsible, based on the detailed physiological knowledge of C4 photosynthesis.

Research Findings

• Genetic modification of rice to have C4 traits (functioning as a C3–C4 intermediate or C4 plant) is a challenging goal, requiring both anatomical and biochemical specialisation and compartmentation within chlorenchyma cells (Fig. 1). In C4 plants, spatial separation of the capture of atmospheric CO2 from its delivery to Rubisco is required. It has been suggested that evolution of C4 has occurred multiple times by a stepwise progression of structural and biochemical changes that were induced by CO2-limiting conditions (Sage 2004 and references therein). The occurrence of ‘intermediates’ between C3 and C4 plants has provided a basis for suggesting how C4 may have evolved from C3, to intermediates that reduce photorespiration without a C4 cycle, to intermediates having a partially functioning C4 cycle, to full development of C4 (Fig. 2).

Fig. 1. A simplified scheme of the C4 pathway (adapted from von Caemmerer 2000).

Fig. 2. A simplified scheme of the C3–C4 intermediate photosynthetic pathway (adapted from von Caemmerer 2000).

• A useful high throughput diagnostic tool that can be employed both to discover C4 anatomical revertants and C4-like rice anatomical mutants is to screen for vein spacing, easily achieved with a hand-held microscope and in more detail using laser confocal microscopy (Figs 3, 4). Figure 3a, b illustrate a high throughput technique to estimate vein spacing in intact C3 and C4 leaves using low magnification light micrographs of live rice and maize leaves, respectively, imaged from above the epidermis. Figure 3c, d shows higher resolution measurements of fluorescent dyes in transverse sections of rice and maize leaves imaged using epifluorescence microscopy, which clearly illustrate the number of cells separating the vascular bundles by outlining cells.
• Laser confocal microscopy of fresh leaf sections coupled to image analysis tools can also provide a comprehensive dataset on cell size, chloroplast number and chloroplast function (Fig. 4). In Fig. 4, a transverse section of a mature maize leaf is shown, imaged using laser confocal scanning microscopy.

Fig. 3. Panels (a) and (b) are light micrographs of young fully expanded intact rice and maize leaves, respectively, imaged through the adaxial epidermis. Panels (c) and (d) show fluorescence micrographs of transverse hand sections of leaves of similar age (rice, panel c; maize, panel d) after fixation and clearing in lactic acid and staining with acridine orange to visualise chloroplasts. Red/orange fluorescence of chloroplasts and green autofluorescence of cell wall components such as lignin and suberin allow visualisation of photosynthetic cells, inter-veinal distance and cell numbers. Scale bar in panel (a) applies to all images.

Fig. 4. (a) False colour image of the fluorescence emission of maize leaf cells in hand cut transverse sections of fresh fully expanded leaves of young vegetative stage plants. Fluorescence was collected by laser confocal microscopy detected at 650–720nm (pink) overlayed with fluorescence collected at 720–800 nm (purple). (b) Emission spectrum of chlorophyll fluorescence from mesophyll and bundle sheath cells of the same maize leaf section.

• Fig. 5 show calculated values for G* and G based on the temperature dependencies of Rubisco kinetic parameters determined by Bernacchi et al. (2002).

Fig. 5. Modelled CO2 compensation point of a C3 leaf in the presence (G) and in the absence of mitochondrial respiration (G*).

• The response of CO2 assimilation, and PSII yield measured by chlorophyll fluorescence to CO2 concentration at 2% and at air levels of O2 in C3 and C4 leaves is used in Fig. 6 to illustrate these points. In sorghum, a C4 plant, the response of CO2 assimilation to CO2 concentration (Fig. 6a) is largely independent of O2 concentration. fPSII, or PSII ‘yield’ calculated from chlorophyll fluorescence (Genty et al. 1989) (which is proportional to JO2 with measurements at a given PPFD) is slightly reduced under low O2, which reflects the presence of other electron sinks, such as Mehler reaction (Fig. 6b). In the C3 plant wheat (Triticum aestivum L.) (Fig. 6c), CO2 assimilation responds strongly to O2 concentration at lower CO2 concentrations due to inhibition of photorespiration. Conversely, however, low O2 concentration reduces fPSII in wheat due to the removal of photorespiration as an electron sink. These differences in response of C3 and C4 photosynthesis to CO2 and O2 can be used to test for altered C4 function in plants, which are identified either from high throughput screens, or rice that has been transformed for potential gain of C4 function.

Fig. 6. Response of CO2 assimilation (a, c) and fPSII, PSII yield (b, d) to intercellular CO2 concentration in sorghum and wheat at 21% and 2% O2 (closed and open symbols respectively).

Labs working on this Project

• CSIRO Plant Industry and High Resolution Plant Phenomics Centre, GPO Box 1600, Canberra, ACT 2601, Australia.
• Research School of Biology, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia.
• International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines.
• School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA.

Corresponding Author

• robert.furbank@csiro.au