C4 Grasses Research Articles

Schizachyrium scoparium (C4) better tolerates drought than Andropogon gerardii (C4) via constant CO2 supply for photosynthesis during water stress.

Climate change is dramatically altering global precipitation patterns across terrestrial ecosystems, making it critically important that we understand both how and why plant species vary in their drought sensitivities. Andropogon gerardii and Schizachyrium scoparium, both C4 grasses, provide a model system for understanding the physiological mechanisms that determine how species of a single functional type can differ in drought responses, an issue remains a critical gap in our ability to model and predict the impacts of drought on grassland ecosystems. Despite its greater lability of foliar water content, previous experiments have demonstrated that S. scoparium maintains higher photosynthetic capacity during droughts. It is therefore likely that the ability of S. scoparium to withstand drought instead derives from a greater metabolic resistance to drought. Here, we tested the following hypotheses: (H1) A. gerardii is more vulnerable to drought than S. scoparium at both the population and organismal levels, (H2) A. gerardii is less stomatally flexible than S. scoparium, and (H3) A. gerardii is more metabolically limited than S. scoparium. Our results indicate that it is actually stomatal limitations of CO2 supply that limit A. gerardii photosynthesis during drought. Schizachyrium scoparium was more drought-resistant than A. gerardii based on long-term field data, organismal biomass production and physiological gas exchange measurements. While both S. scoparium and A. gerardii avoided metabolic limitation of photosynthesis, CO2 supply of A. gerardii was greatly reduced during late-stage drought stress. That two common, co-occurring C4 species possess such different responses to drought highlights the physiological variability inherent within plant functional groups and underscores the need for more studies of C4 drought tolerance.

Plant diversity and functional identity drive grassland rhizobacterial community responses after 15 years of CO2 and nitrogen enrichment

Abstract Improved understanding of bacterial community responses to multiple environmental filters over long time periods is a fundamental step to develop mechanistic explanations of plant–bacterial interactions as environmental change progresses. This is the first study to examine responses of grassland root‐associated bacterial communities to 15 years of experimental manipulations of plant species richness, functional group and factorial enrichment of atmospheric CO2 (eCO2) and soil nitrogen (+N). Across the experiment, plant species richness was the strongest predictor of rhizobacterial community composition, followed by +N, with no observed effect of eCO2. Monocultures of C3 and C4 grasses and legumes all exhibited dissimilar rhizobacterial communities within and among those groups. Functional responses were also dependent on plant functional group, where N2‐fixation genes, NO3−‐reducing genes and P‐solubilizing predicted gene abundances increased under resource‐enriched conditions for grasses, but generally declined for legumes. In diverse plots with 16 plant species, the interaction of eCO2+N altered rhizobacterial composition, while +N increased the predicted abundance of nitrogenase‐encoding genes, and eCO2+N increased the predicted abundance of bacterial P‐solubilizing genes. Synthesis: Our findings suggest that rhizobacterial community structure and function will be affected by important global environmental change factors such as eCO2, but these responses are primarily contingent on plant species richness and the selective influence of different plant functional groups.

Priority effects and competitive exclusion by C4 grasses on longleaf pine savanna restoration sites

On grassland and savanna restoration sites, planted native C4 grasses can competitively exclude other co‐introduced plant functional groups, with negative implications for ecosystem functioning and restoration success. Previous studies have suggested that C4 grass competitive exclusion does not occur in longleaf pine savannas of the North American Coastal Plain; however these studies were primarily conducted on old growth xeric sites. We designed an experiment to test competitive interactions between C4 grasses and forbs introduced by seed to mesic savanna restoration sites. Research installations were established on three former bahiagrass (Paspalum notatum) pastures in Florida, within which six different seed treatments were applied: (1) C4 grasses and forbs together in years 1 and 2, (2) C4 grasses alone in year 1 and forbs alone in year 2, (3) forbs alone in year 1 and C4 grasses alone in year 2, (4) C4 grasses alone in both years, (5) forbs alone in both years, and (6) an unplanted control. All plots were sampled 1 year after the second seeding event. We found that C4 grasses competitively excluded forbs, reducing cover of forbs arriving on the site at the same time, as well as those arriving in the second year. The highest forb species richness and cover was found in the three treatments that did not contain C4 grasses in year 1, and different C4 grass species exhibited different community associations. These findings suggest that restoration seed mixes for longleaf pine savannas should account for the competitive interactions of C4 grasses.

Severe and mild drought cause distinct phylogenetically linked shifts in the blue grama (Bouteloua gracilis) rhizobiome

Plants rely on a diverse rhizobiome to regulate nutrient acquisition and plant health. With increasing severity and frequency of droughts worldwide due to climate change, untangling the relationships between plants and their rhizobiomes is vital to maintaining agricultural productivity and protecting ecosystem diversity. While some plant physiological responses to drought are generally conserved, patterns of root exudation (release of small metabolites shown to influence microbes) and the consequential effects on the plant rhizobiome can differ widely across plant species under drought. To address this knowledge gap, we conducted a greenhouse study using blue grama (Bouteloua gracilis), a drought-tolerant C4 grass native to shortgrass prairie across North American plains, as a model organism to study the effect of increasing drought severity (ambient, mild drought, severe drought) on root exudation and the rhizobiome. Our previous results demonstrated physiological effects of increasing drought severity including an increase in belowground carbon allocation through root exudation and shifts in root exudate composition concurrent with the gradient of drought severity. This work is focused on the rhizobiome community structure using targeted sequencing and found that mild and severe drought resulted in unique shifts in the bacterial + archaeal and fungal communities relative to ambient, non-droughted controls. Specifically, using the change in relative abundance between ambient and drought conditions for each ZOTU as a surrogate for population-scale drought tolerance (e.g., as a response trait), we found that rhizobiome response to drought was non-randomly distributed across the phylogenies of both communities, suggesting that Planctomycetota, Thermoproteota (formerly Thaumarchaeota), and the Glomeromycota were the primary clades driving these changes. Correlation analyses indicated weak correlations between droughted community composition and a select few root exudate compounds previously implicated in plant drought responses including pyruvic acid, D-glucose, and myoinositol. This study demonstrates the variable impacts of drought severity on the composition of the blue grama rhizobiome and provides a platform for hypothesis generation for targeted functional studies of specific taxa involved in plant-microbe drought responses.

Can rewilding with giant tortoises increase woody habitat and limit fire across Madagascar's grasslands?

Societal Impact StatementTo better understand the impact of human settlement on vegetation distribution and disturbance regimes in Madagascar, the 2021 Malagasy Grassy Biomes Workshop called for an examination of the role of extinct megafauna in determining the historic distribution of habitat mosaics across regions that are today dominated by vast treeless grasslands. A systematic literature review facilitated the reconstruction and evaluation of giant tortoise habitat, feeding habitats, diets and Aldabrachelys tortoise impacts on disturbance regimes. In conclusion, reintroduction of mixed‐feeding Aldabrachelys (the diets of which were often dominated by C3 woody material, but never by C4 grasses, across its range), will likely impact plant species composition, select a tree‐rich habitat mosaic, reduce fire frequency, re‐establish lost ecological functions and improve landscape productivity at a range of scales, including rangeland palatability, benefitting both biodiversity and human wellbeing.SummaryGrasslands with little tree cover today comprise 80% of Madagascar's habitat. Determining their extent at human settlement can guide ecological restoration and enhance human well‐being, so the 2021 Malagasy Grassy Biomes Workshop identified the role of extinct megafauna in determining habitat as a critical knowledge gap. Using a systematic literature review, combined with extracted datasets, we address this, examining anticipated habitat selection by giant tortoises following reintroduction to Madagascar (where the Aldabran giant tortoise, Aldabrachelys gigantea, provides ecological functions lost when A. abrupta and A. grandidieri went extinct). When comparing current and historical tortoise selection of habitat across the Mascarenes and Aldabra with contemporary Malagasy habitat, areas in Madagascar where giant tortoises historically ranged, today have a significantly different habitat composition to the forested habitat that supported giant tortoises on other islands. Dietary 13C isotope ratios show that Malagasy Aldabrachelys and Mascarene tortoises were mixed feeders, with diets often dominated by C3 woody intake, but never by C4 grasses. Across systems, giant tortoises required and selected, tree‐rich habitat mosaics, different to current pastoralist fire‐selected Malagasy grasslands characterized by sparse tree cover. Furthermore, Aldabran Aldabrachelys tortoise turf, restricted to small areas (large tracts of unshaded vegetation present physiological challenges to Aldabrachelys' survival), is compositionally different to Malagasy and African obligate C4 grazing lawns. Ecological, palaeoecological, geomorphological and molecular evidence support a lost Malagasy habitat mosaic where hippo and tortoise diets were C3‐dominated, because they inhabited closed‐canopy systems, with abutting open‐canopy areas harbouring endemic‐rich, C4 grassy understories and limited grasslands. The review suggests that rewilding with A. gigantea will help restore ecological functions, productivity and landscape‐scale degradation lost through cattle‐based pastoralism, re‐establish tree‐rich habitat mosaics, and mitigate against frequent bushfires, benefiting biodiversity and humans at multiple scales.

A Review on Biology, Status, and Medicinal Importance of Echinochloa colona L.

Abstract Echinochloa colona L., commonly known as jungle rice, is a pervasive weed that poses significant challenges to agricultural productivity worldwide. This review explores the comprehensive aspects of the weed, including its biology, ecological status, and intriguing medicinal properties. As a C4 grass species, E. colona L. adapts well to diverse environmental conditions, leading to its wider spread distribution and adverse impact on crop yields. The review begins by delving into the weeds’ morphological and physiological characteristics, shedding light on its life cycle, growth habits, and reproductive mechanisms. The ecological status selection provides insights into E. colona L. global distribution, its impact on different crops, and the mechanisms that contribute to its resilience and adaptability. In addition to its role as a notorious weed, E. colona L. harbors medicinal properties that have been conventionally recognized in various cultures. The review explores the ethnobotanical uses of E. colona L., emphasizing its potential therapeutic benefits. Phytochemical analyses reveal the presence of bioactive compounds with antioxidants and exploration of its medicinal applications. Despite its weed status, understanding the ecological and biological aspects of E. colona L. is crucial for developing effective management strategies. Furthermore, recognizing the medicinal potential of this weed may offer alternative perspectives, fostering research into sustainable uses that could benefit both agriculture and human health. This comprehensive review provides a valuable resource for researchers, agronomists, and pharmacologists, guiding future studies aimed at harnessing the full potential of E. colona L.

How does the neighborhood of annual or perennial species affect the first one hundred days of the establishment of grasses?

Pastures that include annual and perennial grasses can play a strategic role in increasing the sustainability of temperate livestock systems. However, the impacts of the competition between annual and perennial genetically improved forage species on pastures has scarcely been studied. We evaluated the early dynamics of tillering, carbon partitioning and both intra- and interspecific competition in – or between – an annual (Bromus willdenowii; prairie grass; Bw) and a perennial (Dactylis glomerata; orchard grass; Dg) C3 grass species. We sowed 24 0.18 m2 microplots in May 2008. Each microplot constituted an experimental unit and contained five sowing lines spaced 0.175 m apart. On three of the lines, we sowed different combinations of the mentioned grasses, while on the two lines between them, we sowed red clover (Trifolium pratense). We randomly applied a factorial array of eight treatments—2 species (Dg vs. Bw) × 2 types of competition (intra vs. interspecific) × 2 levels of winter nitrogen fertilization (0 vs. 170 kg of N ha-1)—designed in three complete random blocks. Fertilization did not have any significant effect on any species. The evaluation period lasted 109 days after sowing (das), separated into three phases (0-53 das, 59-90 das and 90-109 das). The variables recorded were aerial biomass (leaves, sheets and senescent material), root biomass and tiller density per species. As expected, annual Bw inhibited the growth of perennial Dg at the latest stage of establishment but surprisingly promoted it at the earliest stage. This work suggested that a low initial density of an annual species should optimize the initial facilitating effect of the annual species on the perennial species with its subsequent inhibitory effect.

Expression profiling of selected photosynthetic genes from nine halophytic C4 grasses thriving in extreme Kutch desert of India

Expression profiling of selected photosynthetic genes from nine halophytic C4 grasses thriving in extreme Kutch desert of India

Functional Trait Responses of C4 Bunchgrasses to Fire Return Intervals in the Semi-Arid Savanna of South Africa

C4 grasses coevolved with fires, employing specialized adaptive traits to recover from recurrent fires of varying regimes, thereby maintaining plant diversity and plant population stability. However, the knowledge of how C4 bunchgrasses recover from varying fire return intervals (FRIs) is limited. Biomass, tillering, flowering, and growth-related traits of Digitaria eriantha, Themeda triandra, Sporobolus fimbriatus, and Cymbopogon plurinodis were assessed in 0- (unburned), 1-, 2-, and 4-year FRIs, each applied in two 0.5 ± 0.01 ha plots from 1980–2022 at the University of Fort Hare research farm, South Africa. FRIs and grass species interacted significantly on biomass production, crown size, tiller production, and reproductive tillers, with responses varying interspecifically depending on the FRI. Cymbopogon plurinodis attained higher total biomass in 1-year FRI, whereas T. triandra produced relatively low biomass in all FRIs compared to 0-year FRI. Nonetheless, T. triandra attained nearly two to three-fold more tillers per plant and three to five-fold more reproductive tillers in 2- and 4-year FRIs compared to other FRIs. Similarly, S. fimbriatus had two-fold more reproductive tillers in 2-year FRIs compared to 0- and 1-year FRIs. We deduce that C4 bunchgrasses respond differentially under recurrent fires depending on the fire return interval, with 2- and 4-year FRIs promoting vegetative and sexual regeneration by enhancing tillering and flowering.

Holocene and late Pleistocene bioclimatic change inferred from δ13C of organic carbon in buried alluvial soils in the tallgrass prairie, northeastern Kansas

The tallgrass prairie of the Central Plains is one of the largest North American biomes, yet little is known about the history of late-Quaternary bioclimatic change in the region because settings that are favorable for the preservation of traditional paleoenvironmental proxies, such as pollen and plant macrofossils, are rare. However, the valleys of large streams contain thick soil-stratigraphic sequences that span the Holocene and terminal Pleistocene and are archives of other paleoenvironmental proxies. To better understand late-Quaternary bioclimatic change in the tallgrass prairie, we analyzed the stable carbon isotope (δ13C) composition of soil organic matter (SOM) preserved in buried soils in floodplain, terrace, and alluvial fan deposits in the Big Blue River valley of northeast Kansas. A combination of eight cores and three outcrops at six localities were investigated, and 27 radiocarbon ages provide a robust numerical chronology. Based on our analysis, the tallgrass prairie has experienced significant bioclimatic changes over the past 27,000 years. From the LGM to the beginning of the late Holocene (ca. 25,000–4500 cal yr BP), there is an overall increase in C4 plant contributions to SOM, suggesting general warming for that period. However, warming and C4 grass expansion occurred in a non-linear fashion, with minor variations in the abundance of C4 vs C3 plants reflecting either small fluctuations in temperatures during general warming trends or other environmental factors, such as landscape disturbance, periods of drought, or the seasonal distribution of precipitation. The δ13C record also suggests that a major warming event occurred in the region between ca. 3000–1300 cal yr BP, when the contribution of C4 biomass to SOM exceeded 95 %. This climatic event does not appear to be an anomaly, as multiple paleoenvironmental proxies indicate a brief but exceedingly warm and dry interval occurred from 3300 to 2800 cal yr BP.

Geologically calibrated mammalian tree and its correlation with global events, including the emergence of humans.

A robust timetree for Mammalia was constructed using the time calibration function of BEAST v1.10.4 and MEGA 11. The analysis involved the application of times of the most recent common ancestors, including a total of 19 mammalian fossil calibration ages following Benton etal. (Palaeontologia Electronica, 2015, 1-106) for their minimum ages. Additionally, fossil calibration ages for Gorilla, Pan, and a geologic event calibration age for otters were incorporated. Using these calibration ages, I constructed a geologically calibrated tree that estimates the age of the Homo and Pan splitting to be 5.69 Ma. The tree carries several significant implications. First, after the initial rifting at 120 Ma, the Atlantic Ocean expanded by over 500 km around Chron 34 (84 Ma), and vicariant speciation between Afrotheria (Africa) and Xenarthra (South America) appears to have commenced around 70 Ma. Additionally, ordinal level differentiations began immediately following the K-Pg boundary (66.0 Ma), supporting previous hypothesis that mammalian radiation rapidly filled ecological niches left vacant by non-avian dinosaurs. I constructed a diagram depicting the relationship between base substitution rate and age using an additional function in BEAST v1.10.4. The diagram reveals an exponential increase in the base substitution rate approaching recent times. This increased base substitution rate during the Neogene period may have contributed to the expansion of biodiversity, including the extensive adaptive radiation that led to the evolution of Homo sapiens. One significant driving factor behind this radiation could be attributed to the emergence and proliferation of C4 grasses since 20 Ma. These grasses have played a role in increasing carbon fixation, reducing atmospheric CO2 concentration, inducing global cooling, and initiating Quaternary glacial-interglacial cycles, thereby causing significant climatic changes.

Sustained growth and phosphorus efficiency under iron excess in Paspalum densum, a C4 grass for mining site revegetation

Iron (Fe) is a strong phosphorus (P) chelator in Fe-rich environments. In Fe mining sites, the P deficiency might limit plant development. This study aims to understand how the interaction between the Fe excess and P deficit affects the P-use efficiency and photosynthesis in Paspalum densum, a C4 tropical native grass indicated for use in revegetation of iron mining areas. The plants were exposed to four treatments in Hoagland's nutrient solution: standard (100 % P and 0.019 mM Fe); Fe excess (100 % P and 7 mM Fe); P deficit (1 % P and 0.019 mM Fe); and the combination of P deficit and Fe excess (1 % P and 7 mM Fe). The interaction between Fe excess and P deficiency changed the P allocation in the root and root apoplast. In roots, P deficit caused a higher Fe concentration, and, in the leaf, lower levels of P were found in plants exposed to the Fe excess. The stomatal limitation was the main cause of net photosynthesis decline due to Fe excess in both P doses, however without affecting plant biomass or promoting oxidative damage. The iron excess increased the P-use efficiency (PUE) at the leaf level in P-deficient plants. The photosynthetic PUE and whole biomass PUE were higher due to P deficiency. Therefore, in an environment of Fe toxicity and P deficit, as in areas impacted by Fe mining, the adjustments in P allocation and use efficiency coupled with photosynthetic responses contribute to the P. densum performance during revegetation.

N-Alkane 13C/12C indicates differential metabolic controls of fatty lipid chain extension in C3 and C4 grasses.

Fundamental differences in metabolic control of fatty acids chain extension are reflected in the contrasting carbon isotopic composition profiles of C3 and C4 grasses.

δ13C of bulk organic matter and cellulose reveal post-photosynthetic fractionation during ontogeny in C4 grass leaves.

The 13C isotope composition (δ13C) of leaf dry matter is a useful tool for physiological and ecological studies. However, how post-photosynthetic fractionation associated with respiration and carbon export influences δ13C remains uncertain. We investigated the effects of post-photosynthetic fractionation on δ13C of mature leaves of Cleistogenes squarrosa, a perennial C4 grass, in controlled experiments with different levels of vapour pressure deficit and nitrogen supply. With increasing leaf age class, the 12C/13C fractionation of leaf organic matter relative to the δ13C of atmosphere CO2 (ΔDM) increased while that of cellulose (Δcel) was almost constant. The divergence between ΔDM and Δcel increased with leaf age class, with a maximum value of 1.6‰, indicating the accumulation of post-photosynthetic fractionation. Applying a new mass balance model that accounts for respiration and export of photosynthates, we found an apparent 12C/13C fractionation associated with carbon export of -0.5‰ to -1.0‰. Different ΔDM among leaves, pseudostems, daughter tillers, and roots indicate that post-photosynthetic fractionation happens at the whole-plant level. Compared with ΔDM of old leaves, ΔDM of young leaves and Δcel are more reliable proxies for predicting physiological parameters due to the lower sensitivity to post-photosynthetic fractionation and the similar sensitivity in responses to environmental changes.

Global distribution, climatic preferences and photosynthesis-related traits of C4 eudicots and how they differ from those of C4 grasses.

C₄ is one of three known photosynthetic processes of carbon fixation in flowering plants. It evolved independently more than 61 times in multiple angiosperm lineages and consists of a series of anatomical and biochemical modifications to the ancestral C3 pathway increasing plant productivity under warm and light-rich conditions. The C4 lineages of eudicots belong to seven orders and 15 families, are phylogenetically less constrained than those of monocots and entail an enormous structural and ecological diversity. Eudicot C4 lineages likely evolved the C4 syndrome along different evolutionary paths. Therefore, a better understanding of this diversity is key to understanding the evolution of this complex trait as a whole. By compiling 1207 recognised C4 eudicots species described in the literature and presenting trait data among these species, we identify global centres of species richness and of high phylogenetic diversity. Furthermore, we discuss climatic preferences in the context of plant functional traits. We identify two hotspots of C4 eudicot diversity: arid regions of Mexico/Southern United States and Australia, which show a similarly high number of different C4 eudicot genera but differ in the number of C4 lineages that evolved insitu. Further eudicot C4 hotspots with many different families and genera are in South Africa, West Africa, Patagonia, Central Asia and the Mediterranean. In general, C4 eudicots are diverse in deserts and xeric shrublands, tropical and subtropical grasslands, savannas and shrublands. We found C4 eudicots to occur in areas with less annual precipitation than C4 grasses which can be explained by frequently associated adaptations to drought stress such as among others succulence and salt tolerance. The data indicate that C4 eudicot lineages utilising the NAD-ME decarboxylating enzyme grow in drier areas than those using the NADP-ME decarboxylating enzyme indicating biochemical restrictions of the later system in higher temperatures. We conclude that in most eudicot lineages, C4 evolved in ancestrally already drought-adapted clades and enabled these to further spread in these habitats and colonise even drier areas.

Paleobiogeoclimatic reconstruction of the Monte Cristo cave, Minas Gerais, Brazil, through phytolith analysis

This study aims to contribute to the understanding of possible paleoclimatic variations during the Quaternary in the Serra do Espinhaço Mountain Range, Minas Gerais, Brazil, based on phytolith analysis. Two soil profiles were collected from the Monte Cristo cave, which is formed in quartzite, the first being an Organosol outside the cave and the second consisting of mostly sandy material from inside the cave, in layers varying between light and dark tones. The results showed that both profiles present a high stock of phytoliths. The phytoliths are well preserved, with a predominance of Spheroid echinate and Spheroid ornate morphotypes, in addition to the presence of Bulliform flabellate, Cross, Bilobate, Rondel, and Trapezoid. The tree density index (D/P) is between 0.19 and 0.36, indicating open vegetation. The values of the Bi% index (32.8–53%) indicate moderate water stress, which is similar to previous studies in nearby regions. The Ic (climatic) index showed variations in plant adaptation at lower temperatures, with a predominance of C3 grasses, while the Pa/P index (palm tree density) indicated the presence of palm trees along both profiles. The ages obtained using 14C-AMS in the Organosol were from 4440/4230 to 2740/2430 years cal BP, and the ages of the sandy material inside the cave were from 1000/920 to 660/550 years cal BP. The phytoliths found inside the cave may be an indication that this material has an allochthonous origin, probably transported by flooding in periods under different hydrogeomorphological conditions, which is one of the hypotheses that will continue to be investigated. The vegetation type remained stable during the analyzed period, being characteristic of rupestrian fields with the presence of trees in more humid environments.

Plasmodesmal connectivity in C4 Gynandropsis gynandra is induced by light and dependent on photosynthesis.

In leaves of C4 plants, the reactions of photosynthesis become restricted between two compartments. Typically, this allows accumulation of C4 acids in mesophyll (M) cells and subsequent decarboxylation in the bundle sheath (BS). In C4 grasses, proliferation of plasmodesmata between these cell types is thought to increase cell-to-cell connectivity to allow efficient metabolite movement. However, it is not known whether C4 dicotyledons also show this enhanced plasmodesmal connectivity and so whether this is a general requirement for C4 photosynthesis is not clear. How M and BS cells in C4 leaves become highly connected is also not known. We investigated these questions using 3D- and 2D-electron microscopy on the C4 dicotyledon Gynandropsis gynandra as well as phylogenetically close C3 relatives. The M-BS interface of C4 G. gynandra showed higher plasmodesmal frequency compared with closely related C3 species. Formation of these plasmodesmata was induced by light. Pharmacological agents that perturbed photosynthesis reduced the number of plasmodesmata, but this inhibitory effect could be reversed by the provision of exogenous sucrose. We conclude that enhanced formation of plasmodesmata between M and BS cells is wired to the induction of photosynthesis in C4 G. gynandra.

GOLDEN2-like1 is sufficient but not necessary for chloroplast biogenesis in mesophyll cells of C4 grasses.

Chloroplasts are the site of photosynthesis. In land plants, chloroplast biogenesis is regulated by a family of transcription factors named GOLDEN2-like (GLK). In C4 grasses, it has been hypothesized that genome duplication events led to the sub-functionalization of GLK paralogs (GLK1 and GLK2) to control chloroplast biogenesis in two distinct cell types: mesophyll and bundle sheath cells. Although previous characterization of golden2 (g2) mutants in maize has demonstrated a role for GLK2 paralogs in regulating chloroplast biogenesis in bundle sheath cells, the function of GLK1 has remained elusive. Here we show that, contrary to expectations, GLK1 is not required for chloroplast biogenesis in mesophyll cells of maize. Comparisons between maize and Setaria viridis, which represent two independent C4 origins within the Poales, further show that the role of GLK paralogs in controlling chloroplast biogenesis in mesophyll and bundle sheath cells differs between species. Despite these differences, complementation analysis revealed that GLK1 and GLK2 genes from maize are both sufficient to restore functional chloroplast development in mesophyll andbundle sheath cells of S. viridis mutants. Collectively our results suggest an evolutionary trajectory inC4 grasses whereby both orthologs retained the ability to induce chloroplast biogenesis but GLK2 adopted a more prominent developmental role, particularly in relation to chloroplast activation in bundle sheath cells.

Allometries of cell and tissue anatomy and photosynthetic rate across leaves of C3 and C4 grasses.

Allometric relationships among the dimensions of leaves and their cells hold across diverse eudicotyledons, but have remained untested in the leaves of grasses. We hypothesised that geometric (proportional) allometries of cell sizes across tissues and of leaf dimensions would arise due to the coordination of cell development and that of cell functions such as water, nutrient and energy transport, and that cell sizes across tissues would be associated with light-saturated photosynthetic rate. We tested predictions across 27 globally distributed C3 and C4 grass species grown in a common garden. We found positive relationships among average cell sizes within and across tissues, and of cell sizes with leaf dimensions. Grass leaf anatomical allometries were similar to those of eudicots, with exceptions consistent with the fewer cell layers and narrower form of grass leaves, and the specialised roles of epidermis and bundle sheath in storage and leaf movement. Across species, mean cell sizes in each tissue were associated with light-saturated photosynthetic rate per leaf mass, supporting the functional coordination of cell sizes. These findings highlight the generality of evolutionary allometries within the grass lineage and their interlinkage with coordinated development and function.

Epicuticular wax accumulation and regulation of wax pathway gene expression during bioenergy Sorghum stem development.

Bioenergy sorghum is a drought-tolerant high-biomass C4 grass targeted for production on annual cropland marginal for food crops due primarily to abiotic constraints. To better understand the overall contribution of stem wax to bioenergy sorghum's resilience, the current study characterized sorghum stem cuticular wax loads, composition, morphometrics, wax pathway gene expression and regulation using vegetative phase Wray, R07020, and TX08001 genotypes. Wax loads on sorghum stems (~103-215 µg/cm2) were much higher than Arabidopsis stem and leaf wax loads. Wax on developing sorghum stem internodes was enriched in C28/30 primary alcohols (~65%) while stem wax on fully developed stems was enriched in C28/30 aldehydes (~80%). Scanning Electron Microscopy showed minimal wax on internodes prior to the onset of elongation and that wax tubules first appear associated with cork-silica cell complexes when internode cell elongation is complete. Sorghum homologs of genes involved in wax biosynthesis/transport were differentially expressed in the stem epidermis. Expression of many wax pathway genes (i.e., SbKCS6, SbCER3-1, SbWSD1, SbABCG12, SbABCG11) is low in immature apical internodes then increases at the onset of stem wax accumulation. SbCER4 is expressed relatively early in stem development consistent with accumulation of C28/30 primary alcohols on developing apical internodes. High expression of two SbCER3 homologs in fully elongated internodes is consistent with a role in production of C28/30 aldehydes. Gene regulatory network analysis aided the identification of sorghum homologs of transcription factors that regulate wax biosynthesis (i.e., SbSHN1, SbWRI1/3, SbMYB94/96/30/60, MYS1) and other transcription factors that could regulate and specify expression of the wax pathway in epidermal cells during cuticle development.