The role of leaf plasmodesmata in C4 photosynthesis
Creators: name: Danila, Florence
Publisher: name: The Australian National University
Year: 2019

description: In a C4 leaf, numerous plasmodesmata in the wall interface between the mesophyll and bundle sheath cells provide symplastic transport for exchange of C4 acids and metabolites during photosynthesis. Despite their importance in understanding the function and efficiency of C4 photosynthesis, quantification studies of the plasmodesmata that exist between these two photosynthetic cells are very few. The main reason for this is the difficulty involved in capturing the plasmodesmata that occur as clusters or pit field units particularly in leaves. In this work, this problem was overcame through the development of a new and more robust method to accurately quantify plasmodesmata in leaves without resorting to serial sectioning (Chapter 3). This was achieved by combining scanning electron microscopy and three-dimensional laser confocal fluorescence microscopy (e.i. PEA-CLARITY). This new method allowed empirical quantification of plasmodesmata per cell interface area, and consequently, estimation of plasmodesmata flux when these values are combined with photosynthetic measurements (Chapter 3). The new plasmodesmata quantification method provided the opportunity to examine a diversity of grass species within the non-C4 BEP clade and PACMAD clade including representative species from C3, C3-C4 intermediate, and all the C4 biochemical subtypes, nicotinamide adenine dinucleotide phosphate - malic enzyme (NADP-ME), nicotinamide adenine dinucleotide - malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PCK) (Chapter 4). Results showed that C3 species, regardless of clade, have fewer plasmodesmata than C4 species. Panicum milioides, a C3-C4 intermediate species, had plasmodesmata density within the C4 species range. Within the C4 species, there was a substantial variation of plasmodesmata densities at the mesophyll-bundle sheath cell interfaces among the subtypes, where NAD-ME subtype had the greatest value, NADP-ME had the least, and PCK had the largest variability. The greater plasmodesmata density in C4 leaves was a result of having larger pit fields and an increased number of plasmodesmata per pit field area. Conversely, plasmodesmata densities at the mesophyll-mesophyll cell interfaces were similar in C3 and C4 species. Other C4 evolution-related anatomical traits such as bundle sheath cell size, interveinal distance, and bundle sheath surface area per leaf area (Sb) were also measured. Results revealed that Sb and, to a lesser extent, interveinal distance, but not bundle sheath cell size, were statistically different between C3 species and C4 species. In addition to plasmodesmata density survey, the new method developed here was also applied to investigate the response of plasmodesmata formation in C4 grasses to changes in photosynthetic capacity. Results showed that when C4 plants were subjected to different growth light environments (Chapter 5) or when the initial carbon fixation enzyme, phosphoenolpyruvate carboxylase (PEPC) was downregulated reducing photosynthetic capacity in these plants (Chapter 6), plasmodesmata connection between the mesophyll and bundle sheath cells were altered. Low growth irradiance caused fewer plasmodesmata connection to form between mesophyll and bundle sheath cells in leaves of both Setaria viridis and Zea mays compared to the high light-grown plants, albeit manifested at different plant ages (Chapter 5). On the other hand, an increased number of pit fields in the mesophyll-bundle sheath cell interface of the leaf resulted from impaired C4-associated PEPC gene expression in S. viridis (Chapter 6). From these results it can be concluded that increased plasmodesmata density between the mesophyll and bundle sheath cells is an essential anatomical feature of C4 photosynthesis and is responsive to changes in photosynthetic capacity in C4 plants.