Molecular phylogenies disprove a hypothesized C4 reversion in Eragrostis walteri (Poaceae)

Background and Aims The main assemblage of the grass subfamily Chloridoideae is the largest known clade of C4 plant species, with the notable exception of Eragrostis walteri Pilg., whose leaf anatomy has been described as typical of C3 plants. Eragrostis walteri is therefore classically hypothesized to represent an exceptional example of evolutionary reversion from C4 to C3 photosynthesis. Here this hypothesis is tested by verifying the photosynthetic type of E. walteri and its classification. Methods Carbon isotope analyses were used to determine the photosynthetic pathway of several E. walteri accessions, and phylogenetic analyses of plastid rbcL and ndhF and nuclear internal transcribed spacer DNA sequences were used to establish the phylogenetic position of the species. Results Carbon isotope analyses confirmed that E. walteri is a C3 plant. However, phylogenetic analyses demonstrate that this species has been misclassified, showing that E. walteri is positioned outside Chloridoideae in Arundinoideae, a subfamily comprised entirely of C3 species. Conclusions The long-standing hypothesis of C4 to C3 reversion in E. walteri is rejected, and the classification of this species needs to be re-evaluate

Ecophysiological traits of grasses: resolving the effects of photosynthetic pathway and phylogeny

C4 photosynthesis is an important example of convergent evolution in plants, having arisen in eudicots, monocots and diatoms. Comparisons between such diverse groups are confounded by phylogenetic and ecological differences, so that only broad generalisations can be made about the role of C4 photosynthesis in
determining ecophysiological traits. However, 60% of C4 species occur in the grasses (Poaceae) and molecular phylogenetic techniques confirm that there are between 8 and 17 independent origins of C4 photosynthesis in the Poaceae. In a screening experiment, we compared leaf physiology and growth traits across several major
independent C3 & C4 groups within the Poaceae, asking 1) which traits differ consistently between photosynthetic
types and 2) which traits differ consistently between clades within each photosynthetic type

Water relations traits of C4 grasses depend on phylogenetic lineage, photosynthetic pathway, and habitat water availability

The repeated evolution of C4 photosynthesis in independent lineages has resulted in distinct biogeographical distributions in different phylogenetic lineages and the variants of C4 photosynthesis. However, most previous studies have only considered C3/C4 differences without considering phylogeny, C4 subtype, or habitat characteristics. We hypothesized that independent lineages of C4 grasses have structural and physiological traits that adapt them to environments with differing water availability. We measured 40 traits of 33 species from two major C4 grass lineages in a common glasshouse environment. Chloridoideae species were shorter, with narrower and longer leaves, smaller but denser stomata, and faster curling leaves than Panicoideae species, but overall differences in leaf hydraulic and gas exchange traits between the two lineages were weak. Chloridoideae species had two different ways to reach higher drought resistance potential than Panicoideae; NAD-ME species used water saving, whereas PCK species used osmotic adjustment. These patterns could be explained by the interactions of lineage×C4 subtype and lineage×habitat water availability in affected traits. Specifically, phylogeny tended to have a stronger influence on structural traits, and C4 subtype had more important effects on physiological traits. Although hydraulic traits did not differ consistently between lineages, they showed strong covariation and relationships with leaf structure. Thus, phylogenetic lineage, photosynthetic pathway, and adaptation to habitat water availability act together to influence the leaf water relations traits of C4 grasses. This work expands our understanding of ecophysiology in major C4 grass lineages, with implications for explaining their regional and global distributions in relation to climate

Reduced plant water status under sub-ambient pCO2 limits plant productivity in the wild progenitors of C3 and C4 cereals.

BACKGROUND AND AIMS: The reduction of plant productivity by low atmospheric CO2 partial pressure (pCO2) during the last glacial period is proposed as a limiting factor for the establishment of agriculture. Supporting this hypothesis, previous work has shown that glacial pCO2 limits biomass in the wild progenitors of C3 and C4 founder crops, in part due to the direct effects of glacial pCO2 on photosynthesis. Here, we investigate the indirect role of pCO2 mediated via water status, hypothesizing that faster soil water depletion at glacial (18 Pa) compared to post-glacial (27 Pa) pCO2, due to greater stomatal conductance, feeds back to limit photosynthesis during drying cycles. METHODS: We grew four wild progenitors of C3 and C4 crops at glacial and post-glacial pCO2 and investigated physiological changes in gas exchange, canopy transpiration, soil water content and water potential between regular watering events. Growth parameters including leaf area were measured. KEY RESULTS: Initial transpiration rates were higher at glacial pCO2 due to greater stomatal conductance. However, stomatal conductance declined more rapidly over the soil drying cycle in glacial pCO2 and was associated with decreased intercellular pCO2 and lower photosynthesis. Soil water content was similar between pCO2 levels as larger leaf areas at post-glacial pCO2 offset the slower depletion of water. Instead the feedback could be linked to reduced plant water status. Particularly in the C4 plants, soil-leaf water potential gradients were greater at 18 Pa compared with 27 Pa pCO2, suggesting an increased ratio of leaf evaporative demand to supply. CONCLUSIONS: Reduced plant water status appeared to cause a negative feedback on stomatal aperture in plants at glacial pCO2, thereby reducing photosynthesis. The effects were stronger in C4 species, providing a mechanism for reduced biomass at 18 Pa. These results have added significance when set against the drier climate of the glacial period

Molecular Dating, Evolutionary Rates, and the Age of the Grasses

Many questions in evolutionary biology require an estimate of divergence times but, for groups with a sparse fossil record, such estimates rely heavily on molecular dating methods. The accuracy of these methods depends on both an adequate underlying model and the appropriate implementation of fossil evidence as calibration points. We explore the effect of these in Poaceae (grasses), a diverse plant lineage with a very limited fossil record, focusing particularly on dating the early divergences in the group. We show that molecular dating based on a data set of plastid markers is strongly dependent on the model assumptions. In particular, an acceleration of evolutionary rates at the base of Poaceae followed by a deceleration in the descendants strongly biases methods that assume an autocorrelation of rates. This problem can be circumvented by using markers that have lower rate variation, and we show that phylogenetic markers extracted from complete nuclear genomes can be a useful complement to the more commonly used plastid markers. However, estimates of divergence times remain strongly affected by different implementations of fossil calibration points. Analyses calibrated with only macrofossils lead to estimates for the age of core Poaceae ∼51-55 Ma, but the inclusion of microfossil evidence pushes this age to 74-82 Ma and leads to lower estimated evolutionary rates in grasses. These results emphasize the importance of considering markers from multiple genomes and alternative fossil placements when addressing evolutionary issues that depend on ages estimated for important group

Towards an integrative model of C4 photosynthetic subtypes: insights from comparative transcriptome analysis of NAD-ME, NADP-ME, and PEP-CK C-4 species

C4 photosynthesis affords higher photosynthetic carbon conversion efficiency than C3 photosynthesis and it therefore represents an attractive target for engineering efforts aiming to improve crop productivity. To this end, blueprints are required that reflect C4 metabolism as closely as possible. Such blueprints have been derived from comparative transcriptome analyses of C3 species with related C4 species belonging to the NAD-malic enzyme (NAD-ME) and NADP-ME subgroups of C4 photosynthesis. However, a comparison between C3 and the phosphoenolpyruvate carboxykinase (PEP-CK) subtype of C4 photosynthesis is still missing. An integrative analysis of all three C4 subtypes has also not been possible to date, since no comparison has been available for closely related C3 and PEP-CK C4 species. To generate the data, the guinea grass Megathyrsus maximus, which represents a PEP-CK species, was analysed in comparison with a closely related C3 sister species, Dichanthelium clandestinum, and with publicly available sets of RNA-Seq data from C4 species belonging to the NAD-ME and NADP-ME subgroups. The data indicate that the core C4 cycle of the PEP-CK grass M. maximus is quite similar to that of NAD-ME species with only a few exceptions, such as the subcellular location of transfer acid production and the degree and pattern of up-regulation of genes encoding C4 enzymes. One additional mitochondrial transporter protein was associated with the core cycle. The broad comparison identified sucrose and starch synthesis, as well as the prevention of leakage of C4 cycle intermediates to other metabolic pathways, as critical components of C4 metabolism. Estimation of intercellular transport fluxes indicated that flux between cells is increased by at least two orders of magnitude in C4 species compared with C3 species. In contrast to NAD-ME and NADP-ME species, the transcription of photosynthetic electron transfer proteins was unchanged in PEP-CK. In summary, the PEP-CK blueprint of M. maximus appears to be simpler than those of NAD-ME and NADP-ME plants

Gene Duplication and Dosage Effects During The Early Emergence of C4 Photosynthesis in The Grass Genus <i>Alloteropsis</i>

The importance of gene duplication for evolutionary diversification has been mainly discussed in terms of genetic redundancy allowing neofunctionalization. In the case of C4 photosynthesis, which evolved via the co-option of multiple enzymes to boost carbon fixation in tropical conditions, the importance of genetic redundancy has not been consistently supported by genomic studies. Here, we test for a different role for gene duplication in the early evolution of C4 photosynthesis, via dosage effects creating rapid step changes in expression levels. Using genome-wide data for accessions of the grass genus Alloteropsis that recently diversified into different photosynthetic types, we estimate gene copy numbers and demonstrate that recurrent duplications in two important families of C4 genes coincided with increases in transcript abundance along the phylogeny, in some cases via a pure dosage effect. While increased gene copy number during the initial emergence of C4 photosynthesis probably offered a rapid route to enhanced expression, we also find losses of duplicates following the acquisition of genes encoding better-suited isoforms. The dosage effect of gene duplication might therefore act as a transient process during the evolution of a C4 biochemistry, rendered obsolete by the fixation of regulatory mutations increasing expression levels

Bundle sheath chloroplast volume can house sufficient Rubisco to avoid limiting C4 photosynthesis during chilling

C4 leaves confine Rubisco to bundle sheath cells. Thus, the size of bundle sheath compartments and the total volume of chloroplasts within them limit the space available for Rubisco. Rubisco activity limits photosynthesis at low temperatures. C3 plants counter this limitation by increasing leaf Rubisco content, yet few C4 species do the same. Because C3 plants usually outperform C4 plants in chilling environments, it has been suggested that there is insufficient chloroplast volume available in the bundle sheath of C4 leaves to allow such an increase in Rubisco at low temperatures. We investigated this potential limitation by measuring bundle sheath and mesophyll compartment volumes and chloroplast contents, as well as leaf thickness and inter-veinal distance, in three C4 Andropogoneae grasses: two crops (Zea mays and Saccharum officinarum) and a wild, chilling-tolerant grass (Miscanthus × giganteus). A wild C4 Paniceae grass (Alloteropsis semialata) was also included. Despite significant structural differences between species, there was no evidence of increased bundle sheath chloroplast volume per leaf area available to the chilling-tolerant species, relative to the chilling-sensitive ones. Maximal theoretical photosynthetic capacity of the leaf far exceeded the photosynthetic rates achieved even at low temperatures. C4 bundle sheath cells therefore have the chloroplast volume to house sufficient Rubisco to avoid limiting C4 photosynthesis during chilling

Were Fertile Crescent crop progenitors higher yielding than other wild species that were never domesticated?

During the origin of agriculture in the Fertile Crescent, the broad spectrum of wild plant species exploited by hunter-gatherers narrowed dramatically. The mechanisms responsible for this specialization and the associated domestication of plants are intensely debated. We investigated why some species were domesticated rather than others, and which traits they shared. We tested whether the progenitors of cereal and pulse crops, grown individually, produced a higher yield and less chaff than other wild grasses and legumes, thereby maximizing the return per seed planted and minimizing processing time. We compared harvest traits of species originating from the Fertile Crescent, including those for which there is archaeological evidence of deliberate collection. Unexpectedly, wild crop progenitors in both families had neither higher grain yield nor, in grasses, less chaff, although they did have larger seeds. Moreover, small-seeded grasses actually returned a higher yield relative to the mass of seeds sown. However, cereal progenitors had threefold fewer seeds per plant, representing a major difference in how seeds are packaged on plants. These data suggest that there was no intrinsic yield advantage to adopting large-seeded progenitor species as crops. Explaining why Neolithic agriculture was founded on these species, therefore, remains an important unresolved challenge

Differential freezing resistance and photoprotection in C3 and C4 eudicots and grasses

Globally, C4 plants dominate hot, open environments, but this general pattern is underpinned by important differences in the biogeography of C4 lineages. In particular, the species richness of C4 Poaceae (grasses) increases strongly with increasing temperature, whereas that of the major C4 eudicot group Chenopodiaceae correlates positively with aridity. Freezing tolerance is a crucial determinant of biogeographical relationships with temperature and is mediated by photodamage and cellular disruption by desiccation, but little is known about differences between C4 families. This study hypothesized that there is a greater risk of freezing damage via these mechanisms in C4 Poaceae than Chenopodiaceae, that freezing protection differs between the taxonomic groups, and that freezing tolerance of species is linked to arid habitat preference. Chlorophyll fluorescence, water relations, and freezing injury were compared in four C3 and six C4 species of Poaceae and Chenopodiaceae from the same Mongolian flora. Contrary to expectations, freezing-induced leaf mortality and photodamage were lower in Poaceae than Chenopodiaceae species, and unrelated to photosynthetic pathway. The freezing resistance of Poaceae species resulted from constitutive protection and cold acclimation and an ability to protect the photosynthetic apparatus from photodamage. Freezing protection was associated with low osmotic potential and low tissue elasticity, and freezing damage was accompanied by electrolyte leakage, consistent with cell-membrane disruption by ice. Both Chenopodiaceae and Poaceae had the potential to develop cold acclimation and withstand freezing during the growing season, which conflicted with the hypothesis. Instead, freezing tolerance was more closely associated with life history and ecological preference in these Mongolian species