Abstract
Temporal and spatial patterns of photosynthetic enzyme expression and structural maturation of chlorenchyma cells along longitudinal developmental gradients were characterized in young leaves of two single cell C4 species, Bienertia sinuspersici and Suaeda aralocaspica Both species partition photosynthetic functions between distinct intracellular domains. In the C4-C domain, C4 acids are formed in the C4 cycle during capture of atmospheric CO2 by phosphoenolpyruvate carboxylase. In the C4-D domain, CO2 released in the C4 cycle via mitochondrial NAD-malic enzyme is refixed by Rubisco. Despite striking differences in origin and intracellular positioning of domains, these species show strong convergence in C4 developmental patterns. Both progress through a gradual developmental transition towards full C4 photosynthesis, with an associated increase in levels of photosynthetic enzymes. Analysis of longitudinal sections showed undeveloped domains at the leaf base, with Rubisco rbcL mRNA and protein contained within all chloroplasts. The two domains were first distinguishable in chlorenchyma cells at the leaf mid-regions, but still contained structurally similar chloroplasts with equivalent amounts of rbcL mRNA and protein; while mitochondria had become confined to just one domain (proto-C4-D). The C4 state was fully formed towards the leaf tips, Rubisco transcripts and protein were compartmentalized specifically to structurally distinct chloroplasts in the C4-D domains indicating selective regulation of Rubisco expression may occur by control of transcription or stability of rbcL mRNA. Determination of CO2 compensation points showed young leaves were not functionally C4, consistent with cytological observations of the developmental progression from C3 default to intermediate to C4 photosynthesis.
Highlights
Young leaves from each species were fixed at 4°C in 2% (v/v) paraformaldehyde and 1.25% (v/v) glutaraldehyde in 0.05 M PIPES buffer, pH 7.2
The samples were dehydrated with a graded ethanol series and embedded in London Resin White (LR White, Electron Microscopy Sciences, Fort Washington, PA, USA) acrylic resin
The slides were washed with TBST+ BSA and treated for 1 h with protein A-gold (10 nm particles diluted 1:100 with TBST + BSA)
Summary
Young leaves from each species were fixed at 4°C in 2% (v/v) paraformaldehyde and 1.25% (v/v) glutaraldehyde in 0.05 M PIPES buffer, pH 7.2. Longitudinal sections (0.8-1 μm thick), were placed onto gelatin coated slides and blocked for 1 h with TBST + BSA (10 mM Tris-HCl, 150 mM NaCl, 0.1% v/v Tween 20, 1% w/v bovine serum albumin, pH 7.2). The slides were incubated for 3 h with the preimmune serum diluted in TBST + BSA (1:100), anti-Rubisco (1:500 dilution), or anti-PEPC (1:200 dilution). The slides were washed with TBST+ BSA and treated for 1 h with protein A-gold (10 nm particles diluted 1:100 with TBST + BSA). The sections were incubated for 3 h with the preimmune serum diluted in TBST + BSA anti-Rubisco (1:50). After washing with TBST + BSA, the sections were incubated for 1 h with Protein A-gold (10 nm) diluted 1:100 with TBST + BSA. Images were collected using a FEI Tecnai G2 equipped with Eagle FP 5271/82 4K HR200KV digital camera transmission electron microscope
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