Reporter-based forward genetic screen to identify bundle sheath anatomy mutants in A. thaliana.

The evolution of C4 photosynthesis proceeded stepwise with each small step increasing the fitness of the plant. An important pre-condition for the introduction of a functional C4 cycle is the photosynthetic activation of the C3 bundle sheath by increasing its volume and organelle number. Therefore, to engineer C4 photosynthesis into existing C3 crops, information about genes that control the bundle sheath cell size and organelle content is needed. However, very little information is known about the genes that could be manipulated to create a more C4 -like bundle sheath. To this end, an ethylmethanesulfonate (EMS)-based forward genetic screen was established in the Brassicaceae C3  species Arabidopsis thaliana. To ensure a high-throughput primary screen, the bundle sheath cells of A. thaliana were labeled using a luciferase (LUC68) or by a chloroplast-targeted green fluorescent protein (sGFP) reporter using a bundle sheath specific promoter. The signal strengths of the reporter genes were used as a proxy to search for mutants with altered bundle sheath anatomy. Here, we show that our genetic screen predominantly identified mutants that were primarily affected in the architecture of the vascular bundle, and led to an increase in bundle sheath volume. By using a mapping-by-sequencing approach the genomic segments that contained mutated candidate genes were identified.

C4 anatomy can evolve via a single developmental change.

C4 photosynthesis is a complex trait that boosts productivity in warm environments. Paradoxically, it evolved independently in numerous plant lineages, despite requiring specialised leaf anatomy. The anatomical modifications underlying C4 evolution have previously been evaluated through interspecific comparisons, which capture numerous changes besides those needed for C4 functionality. Here, we quantify the anatomical changes accompanying the transition between non-C4 and C4 phenotypes by sampling widely across the continuum of leaf anatomical traits in the grass Alloteropsis semialata. Within this species, the only trait that is shared among and specific to C4 individuals is an increase in vein density, driven specifically by minor vein development that yields multiple secondary effects facilitating C4 function. For species with the necessary anatomical preconditions, developmental proliferation of veins can therefore be sufficient to produce a functional C4 leaf anatomy, creating an evolutionary entry point to complex C4 syndromes that can become more specialised.

Light Microscopy, Transmission Electron Microscopy, and Immunohistochemistry Protocols for Studying Photorespiration.

High-resolution images obtained from plant tissues processed for light microscopy, transmission electron microscopy, and immunohistochemistry have provided crucial links between plant subcellular structure and physiology during photorespiration as well as the impact of photorespiration on plant evolution and development. This chapter presents established protocols to guide researchers in the preparation of plant tissues for high-resolution imaging with a light and transmission electron microscope and detection of proteins using immunohistochemistry. Discussion of concepts and theory behind each step in the process from tissue preservation to staining of resin-embedded tissues is included to enhance the understanding of all steps in the procedure. We also include a brief protocol for quantification of cellular parameters from high-resolution images to help researchers rigorously test hypotheses.

Why are there no C4 forests?

C4 photosynthesis is absent from the arborescent life form, with the exception of seven Hawaiian Euphorbia species and a few desert shrubs that become arborescent with age. As a consequence, wherever C3 trees can establish, their height advantage enables them to outcompete low stature C4 vegetation. Had C4 photosynthesis been able to evolve in an arborescent life form, forest cover (by C4 trees) could have been much more extensive than today, with significant consequences for the biosphere. Here, we address why there are so few C4 trees. Physiological explanations associated with low light performance of C4 photosynthesis are not supported, because C4 shade-tolerant species exhibit similar performance as shade-tolerant C3 species in terms of quantum yield, steady-state photosynthetic and use of sunflecks. Hence, hypothetical C4 trees could occur in the regeneration niche of forests. Constraints associated with the evolutionary history of the C4 lineages are more plausible. Most C4 species are grasses and sedges, which lack meristems needed for arborescence, while most C4 eudicots are highly specialized for harsh (arid, saline, hot) or disturbed habitats where arborescence may be maladapted. Most C4 eudicot clades are also young, and have not had sufficient time to radiate beyond the extreme environments where C4 evolution is favored. In the case of the Hawaiian Euphorbia species, they belong to one of the oldest and most diverse C4 lineages, which primed this group to evolve arborescence in a low-competition environment that appeared on the remote Hawaiian Islands.

Mesophyll cells of C4 plants have fewer chloroplasts than those of closely related C3 plants.

The evolution of C(4) photosynthesis from C(3) ancestors eliminates ribulose bisphosphate carboxylation in the mesophyll (M) cell chloroplast while activating phosphoenolpyruvate (PEP) carboxylation in the cytosol. These changes may lead to fewer chloroplasts and different chloroplast positioning within M cells. To evaluate these possibilities, we compared chloroplast number, size and position in M cells of closely related C(3), C(3) -C(4) intermediate and C(4) species from 12 lineages of C(4) evolution. All C(3) species had more chloroplasts per M cell area than their C(4) relatives in high-light growth conditions. C(3) species also had higher chloroplast coverage of the M cell periphery than C(4) species, particularly opposite intercellular air spaces. In M cells from 10 of the 12 C(4) lineages, a greater fraction of the chloroplast envelope was pulled away from the plasmalemma in the C(4) species than their C(3) relatives. C(3) -C(4) intermediate species generally exhibited similar patterns as their C(3) relatives. We interpret these results to reflect adaptive shifts that facilitate efficient C(4) function by enhancing diffusive access to the site of primary carbon fixation in the cytosol. Fewer chloroplasts in C(4) M cells would also reduce shading of the bundle sheath chloroplasts, which also generate energy required by C(4) photosynthesis.

Comparative studies of C3 and C4 Atriplex hybrids in the genomics era: physiological assessments.

We crossed the C3 species Atriplex prostrata with the C4 species Atriplex rosea to produce F1 and F2 hybrids. All hybrids exhibited C3-like δ(13)C values, and had reduced rates of net CO2 assimilation compared with A. prostrata. The activities of the major C4 cycle enzymes PEP carboxylase, NAD-malic enzyme, and pyruvate-Pi dikinase in the hybrids were at most 36% of the C4 values. These results demonstrate the C4 metabolic cycle was disrupted in the hybrids. Photosynthetic CO2 compensation points (Г) of the hybrids were generally midway between the C3 and C4 values, and in most hybrids were accompanied by low, C3-like activities in one or more of the major C4 cycle enzymes. This supports the possibility that most hybrids use a photorespiratory glycine shuttle to concentrate CO2 into the bundle sheath cells. One hybrid exhibited a C4-like Г of 4 µmol mol(-1), indicating engagement of a C4 metabolic cycle. Consistently, this hybrid had elevated activities of all measured C4 cycle enzymes relative to the C3 parent; however, C3-like carbon isotope ratios indicate the low Г is mainly due to a photorespiratory glycine shuttle. The anatomy of the hybrids resembled that of C3-C4 intermediate species using a glycine shuttle to concentrate CO2 in the bundle sheath, and is further evidence that this physiology is the predominant, default condition of the F2 hybrids. Progeny of these hybrids should further segregate C3 and C4 traits and in doing so assist in the discovery of C4 genes using high-throughput methods of the genomics era.

Initial events during the evolution of C4 photosynthesis in C3 species of Flaveria.

The evolution of C4 photosynthesis in many taxa involves the establishment of a two-celled photorespiratory CO2 pump, termed C2 photosynthesis. How C3 species evolved C2 metabolism is critical to understanding the initial phases of C4 plant evolution. To evaluate early events in C4 evolution, we compared leaf anatomy, ultrastructure, and gas-exchange responses of closely related C3 and C2 species of Flaveria, a model genus for C4 evolution. We hypothesized that Flaveria pringlei and Flaveria robusta, two C3 species that are most closely related to the C2 Flaveria species, would show rudimentary characteristics of C2 physiology. Compared with less-related C3 species, bundle sheath (BS) cells of F. pringlei and F. robusta had more mitochondria and chloroplasts, larger mitochondria, and proportionally more of these organelles located along the inner cell periphery. These patterns were similar, although generally less in magnitude, than those observed in the C2 species Flaveria angustifolia and Flaveria sonorensis. In F. pringlei and F. robusta, the CO2 compensation point of photosynthesis was slightly lower than in the less-related C3 species, indicating an increase in photosynthetic efficiency. This could occur because of enhanced refixation of photorespired CO2 by the centripetally positioned organelles in the BS cells. If the phylogenetic positions of F. pringlei and F. robusta reflect ancestral states, these results support a hypothesis that increased numbers of centripetally located organelles initiated a metabolic scavenging of photorespired CO2 within the BS. This could have facilitated the formation of a glycine shuttle between mesophyll and BS cells that characterizes C2 photosynthesis.

High temperature stress and its effect on pollen development and morphological components of harvest index in the C3 model grass Brachypodium distachyon.

The effect of high temperatures on harvest index (HI) and morphological components that contribute to HI was investigated in two lines (Bd21 and Bd21-3) of Brachypodium distachyon, a C3 grass recognized as a tractable plant, to address critical issues associated with enhancing cereal crop yields in the presence of global climate change. The results demonstrated that temperatures ≥32 °C eliminated HI. Reductions in yield at 32 °C were due primarily to declines in pollen viability, retention of pollen in anthers, and pollen germination, while abortion of microspores by the uninucleate stage that was correlated with abnormal tapetal development resulted in yield failure at 36 °C. Increasing temperatures from 24 to 32 °C resulted in reductions in tiller numbers but had no impact on axillary branch numbers per tiller. Grain developed at 24 and 28 °C primarily in tiller spikes, although spikes on axillary branches also formed grain. Grain quantity decreased in tiller spikes but increased in axillary branch spikes as temperatures rose from 24 to 28 °C. Differential patterns of axillary branching and floret development within spikelets between Bd21 and Bd21-3 resulted in higher grain yield in axillary branches of Bd21-3 at 28 °C. The response of male reproductive development and tiller branching patterns in B. distachyon to increasing temperatures mirrors that in other cereal crops, providing support for the use of this C3 grass in assessing the molecular control of HI in the presence of global warming.