Biology subjects

Cousins, A. B.

Publications and source records attributed to Cousins, A. B..

13 recordsLinked to original sources

bioRxiv2026

Heritability of leaf stable carbon isotope signature in a diversity panel of the C4 plant Sorghum bicolor

A strategy to improve agricultural water productivity is to increase water use efficiency (WUE) at the level of plant transpiration through genetic selection. This requires detectable genetic variability in WUE and the ability to phenotype and select plants with higher WUE within a population. A proxy for phenotyping leaf level WUE by measuring carbon isotope signature ({delta}13Cleaf) has been supported by theory and data in C4 species. However, the functional relationship of {delta}13Cleaf and WUE in C4 species can be driven by genetics and environment. Therefore, a wide survey of existing natural variation is needed to quantify the heritability and identify various genetic factors that influence {delta}13Cleaf and WUE. In this study a genome-wide association panel was used to quantify the heritability of {delta}13Cleaf. We measured {delta}13Cleaf across a population of 360 genetically diverse lines of the C4 species Sorghum bicolor with single nucleotide polymorphic (SNP) markers determined from whole-genome resequencing. This analysis was conducted on two independent field environments where heritability of {delta}13Cleaf was evident and was driven by small genetic effects from loci that were consistently identified across environments. Candidate genes are presented that offer insights on future targets to manipulate and explore the functional relationship between {delta}13Cleaf and WUEi in C4 plants.

plant biology↗

bioRxiv2026

The Role of Phosphoenolpyruvate Carboxylase-Protein Kinase in C4 Photosynthesis: Insights from Zea mays Mutant Analysis

Understanding the regulation of enzyme activity involved in photosynthesis is essential for engineering enhanced carbon fixation in crops. In C4 plants, the enzyme phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is one of the most abundant leaf enzymes and plays an essential role in photosynthetic carbon dioxide (CO2) fixation. The enzyme also plays a key role in central metabolism (e.g., providing intermediates to the citric acid cycle) and therefore must be highly regulated to coordinate its activity. The regulation of PEPC activity can occur allosterically by glucose 6-phosphate activation and malate inhibition, which is in part influenced by reversible phosphorylation. A specific light-dependent phosphorylation of PEPC at an N-terminal serine residue by the PEPC-protein kinase (PEPC-PK) can regulate its sensitivity to this allosteric regulation. However, the impact of this PEPC phosphorylation has not been tested in a C4 crop. Therefore, we created PEPC-PK mutant lines in Zea mays to assess the impact of PEPC phosphorylation on its allosteric regulation, photosynthesis, and growth. While the maximum PEPC activity was unchanged, PEPC in the PEPC-PK mutant plants was not phosphorylated under light and was more sensitive to malate inhibition. However, gas exchange, electron transport, and field biomass analyses showed no differences in the PEPC-PK mutant plants. These results demonstrate that in Z. mays PEPC phosphorylation affects enzyme sensitivity to malate in vitro but does not substantially alert photosynthetic performance or growth under field conditions suggesting additional regulation of PEPC activity in planta.

plant biology↗

bioRxiv2025

Plant cells tolerate high rates of organellar mistranslation

Bacteria can trigger protein mistranslation to survive stress conditions 1. Mitochondria and plastids evolved from bacteria and therefore also use prokaryotic-type expression machineries to synthesize proteins. However, fungi and animal mitochondria are highly sensitive to mistranslation, which for instance manifests in lethal mitochondrial cardiomyopathy disorder 2. The response in plant cells is unknown. Glutaminyl-transferRNAs (Gln-tRNAGln) of bacteria, mitochondria, and plastids are synthesized indirectly 3,4. Initially, tRNAGln gets charged with glutamate. Subsequently, Gln is produced through trans-amidation by the aminoacyl-tRNA amido-transferase complex GatCAB. Consequentially, affected GatCAB activity results in pools of misloaded Glu-tRNAGln. Here we show that Arabidopsis mutants with decreased GatCAB level provide global insights into organellar mistranslation in plants. Proteomics revealed Gln-to-Glu misincorporation in plastid- and mitochondrially-expressed protein complexes with only modest abundance changes in mutant plants. Plastids appear more lenient to mistranslation as they exhibit much higher Gln-to-Glu misincorporation. Through efficient compensatory mechanisms, mutant plants display surprisingly subtle phenotypes. However, their acclimation to temperature stress differs. Interestingly, wild-type plants under similar stress also have altered Gln-to- Glu misincorporation. Our study shows that the response towards organellar mistranslation varies among eukaryotes. In plants, this knowledge can be used to improve stress tolerance.

plant biology↗

bioRxiv2025

A spontaneous mutation in a key C4 pathway gene significantly alters leaf δ13C uncoupling its relationship with WUE and photosynthetic performance in Zea mays

Increases in global temperature and drought are negatively impacting the yields of major crops. Therefore, targeted improvements to intrinsic water use efficiency (WUEi) are needed to reduce the water required for agricultural production. While it is very time-consuming to directly measure WUEi, stable carbon isotope ratios ({delta}13C) are a reliable high throughput proxy trait for quantifying WUEi in C3 species. While genetic studies have improved our understanding of the relationship between WUEi and {delta}13C in C4 species, the knowledge needed to implement {delta}13C in breeding schemes is incomplete. Using a Zea mays line with an extremely negative {delta}13C value, a quantitative genetics approach was used to identify a large deletion in carbonic anhydrase1 (cah1). Carbonic anhydrase is the first enzymatic step of the C4 photosynthetic pathway and is known to affect {delta}13C. Surprisingly, the line with the mutant allele has significantly higher carbonic anhydrase activity with a concurrent reduction in {delta}13C, opposite of what would be expected based on C4 carbon isotope fractionation theory. These observed decouple {delta}13C and WUEi, which calls for further investigation into carbon isotope discrimination in C4 species.

plant biology↗

bioRxiv2023

Differences in stomatal sensitivity to COC2 and light influences variation in water use efficiency and leaf carbon isotope composition in two genotypes of the C4 plant Zea mays.

The ratio of net CO2 uptake (Anet) and stomatal conductance (gs) is an intrinsic measurement of leaf water use efficiency (WUEi) however its measurement can be challenging for large phenotypic screens. Measurements of leaf carbon isotope composition ({delta}13Cleaf) may be a scalable tool to approximate WUEi for screening because it in part reflects the competing influences of Anet and gs on the CO2 partial pressure (pCO2) inside the leaf over time. However, in C4 photosynthesis the CO2 concentrating mechanism complicates the relationship between {delta}13Cleaf and WUEi. Despite this complicated relationship, several studies have shown genetic variation in {delta}13Cleaf across C4 plants. Yet there has not been a clear demonstration of whether Anet or gs are the causal mechanisms controlling WUEi and {delta}13Cleaf. Our approach was to characterize leaf photosynthetic traits of two Zea mays recombinant inbred lines (Z007E0067 and Z007E0150) which consistently differ for {delta}13Cleaf even though they have minimal confounding genetic differences. We demonstrate that these two genotypes contrasted in WUEi driven by differences in the speed of stomatal responses to changes in pCO2 and light that lead to unproductive leaf water loss. These findings provide support that differences in {delta}13Cleaf in closely related genotypes do reflect greater WUEi and further suggests that differences in stomatal kinetic response to changing environmental conditions is a key target to improve WUEi.

plant biology↗

bioRxiv2023

Probing the in-situ volumes of Arabidopsis leaf plastids using 3D confocal and scanning electron microscopy

Leaf plastids harbor a plethora of biochemical reactions including photosynthesis, one of the most important metabolic pathways on earth. Scientists are eager to unveil the physiological processes within the organelle but also their interconnection with the rest of the plant cell. An increasingly important feature of this venture is to use experimental data in the design of metabolic models. A remaining obstacle has been the limited in situ volume information of plastids and other cell organelles. To fill this gap for chloroplasts, we established three microscopy protocols delivering in situ volumes based on: 1) chlorophyll fluorescence emerging from the thylakoid membrane, 2) a CFP marker embedded in the envelope, and 3) calculations from serial block-face scanning electron microscopy (SBFSEM). The obtained data were corroborated by comparing wild-type data with two mutant lines affected in the plastid division machinery known to produce small and large mesophyll chloroplasts, respectively. Furthermore, we also determined the volume of the much smaller guard cell plastids. Interestingly, their volume is not governed by the same components of the division machinery which defines mesophyll plastid size. Based on our three approaches the average volume of a mature Col-0 wild-type mesophyll chloroplasts is 93 {micro}m3. Wild-type guard cell plastids are approximately 18 {micro}m3. Lastly, our comparative analysis shows that the chlorophyll fluorescence analysis can accurately determine chloroplast volumes, providing an important tool to research groups without access to transgenic marker lines expressing genetically encoded fluorescence proteins or costly SBFSEM equipment. Significance statement -sentence summaryThis work describes and compares three different strategies to obtain accurate volumes of leaf plastids from Arabidopsis, the most widely used model plant. We hope our contribution will support quantitative metabolic flux modeling and spark other projects aimed at a more metric-driven plant cell biology.

plant biology↗

bioRxiv2023

Altered cell wall hydroxycinnamate composition impacts leaf and canopy-level CO2-uptake and water-use in rice

Cell wall properties can play a major role in determining photosynthetic carbon-uptake and water-use through impacts on mesophyll conductance (CO2 diffusion from substomatal cavities into photosynthetic mesophyll cells) and leaf hydraulic conductance (water movement from xylem, through leaf tissue to stomata). Consequently, modification of cell wall properties is proposed as a major path for improving photosynthesis and crop water-use efficiency. We tested this using two independent transgenic rice lines that overexpress the rice OsAT10 gene (a "BAHD" CoA acyltransferase) which altered cell wall hydroxycinnamic acid content (greater para-coumaric acid and lower ferulic acid). Plants were grown under high and low water-levels and traits related to leaf anatomy, cell wall composition, gas exchange and hydraulics, plant biomass, and canopy-level water-use were measured. Alteration of hydroxycinnamic acid content led to significant decreases in mesophyll cell wall thickness (-14%), and increased mesophyll conductance (+120%) and photosynthesis (+22%). However, concomitant increases in stomatal conductance negated increased photosynthesis, resulting in no change in intrinsic water-use efficiency (ratio of photosynthesis/stomatal conductance). The leaf hydraulic conductance was also unchanged; however, the transgenics showed small, but significant increase in above-ground biomass (+12.5%), and canopy-level water-use efficiency (+8.8%; ratio of above-ground biomass/ water-used) and performed better under low water-level. Our results demonstrate that changes in cell wall composition, specifically hydroxycinnamic acid content, can increase mesophyll conductance and photosynthesis in C3 cereal crops like rice. However, attempts to improve photosynthetic water-use efficiency will need to enhance mesophyll conductance and photosynthesis whilst maintaining or decreasing stomatal conductance.

plant biology↗

bioRxiv2021

Mesophyll conductance response to short-term changes in pCO2 is related to leaf anatomy and biochemistry in diverse C4 grasses

O_LIMesophyll CO2 conductance (gm) in C3 species responds to short-term (minutes) changes in environment potentially due to changes in some leaf anatomical and biochemical properties and due to measurement artifacts. Compared to C3 species, there is less information about gm responses to short-term changes in environment conditions like pCO2 across diverse C4 species and the potential determinants of these responses. C_LIO_LIUsing 16 diverse C4 grasses we investigated the response of gm to short-term changes in CO2 and how this response related to the leaf anatomical and biochemical traits. C_LIO_LIFor all the measured C4-grasses gm increased as CO2 decreased; however, the percent change in gm varied (+13% to +250%) and significantly related to percent changes in leaf transpiration efficiency (TEi). The percent increase in gm was highest in grasses with thinner mesophyll cell walls and greater leaf nitrogen, activities of phosphoenolpyruvate carboxylase (PEPC), Rubisco and carbonic anhydrase, and a higher affinity of PEPC for bicarbonate. C_LIO_LIOur study demonstrates that CO2 response of gm varies greatly across diverse C4 grasses and identifies the key leaf anatomical and biochemical traits related to this variation. These findings have implications for improving C4 photosynthetic models, and in attempts to improve TEi through manipulation of gm. C_LI

plant biology↗

bioRxiv2020

Leaf cell wall properties and stomatal density influence oxygen isotope enrichment of leaf water

Oxygen isotopic composition ({Delta}18OLW) of leaf water can help improve our understanding of how anatomy interacts with physiology to influence leaf water transport. Leaf water isotope models of {Delta}18OLW such as the Peclet effect model have been developed to predict {Delta}18OLW, and it incorporates transpiration rate (E) and the mixing length between unenriched xylem water and enriched mesophyll water, which can occur in the mesophyll (Lm) or veins (Lv). Here we used two cell wall composition mutants grown under two light intensities and RH to evaluate the effect of cell wall composition on {Delta}18OLW. In maize (Zea mays), the compromised ultrastructure of the suberin lamellae in the bundle sheath of the ALIPHATIC SUBERIN FERULOYL TRANSFERASE mutant (Zmasft) reduced barriers to apoplastic water movement, resulting in higher E and Lv and, consequently, lower {Delta}18OLW. In cellulose synthase-like F6 (Cslf6) mutants and wildtype of rice (Oryza sativa), the difference in {Delta}18OLW in plants grown under high and low growth light intensity co-varied with their differences in stomatal density. These results show that cell wall composition and stomatal density influence {Delta}18OLW by altering the Peclet effect and that stable isotopes can facilitate the development of a physiologically and anatomically explicit water transport model.

plant biology↗

bioRxiv2020

The maize Aliphatic Suberin Feruloyl Transferase genes affect leaf water movement but are dispensable for bundle sheath CO2 concentration

C4 grasses often outperform C3 species under hot, arid conditions due to superior water and nitrogen use efficiencies and lower rates of photorespiration. A method of concentrating CO2 around the site of carbon fixation in the bundle sheath (BS) is required to realize these gains. In NADP-malic enzyme (NADP-ME)-type C4 grasses such as maize, suberin deposition in the BS cell wall is hypothesized to act as a diffusion barrier to CO2 escape and O2 entry from surrounding mesophyll cells. Suberin is a heteropolyester comprised of acyl-lipid-derived aliphatic and phenylpropanoid-derived aromatic components. To disrupt BS suberization, we mutated two paralogously duplicated, unlinked maize orthologues of Arabidopsis thaliana ALIPHATIC SUBERIN FERULOYL TRANSFERASE, ZmAsft1 and ZmAsft2, using closely linked Dissociation transposons. Loss-of-function double mutants revealed a 97% reduction in suberin-specific omega-hydroxy fatty acids without a stoichiometric decrease in ferulic acid. However, BS suberin lamellae were deficient in electron opaque material, and cohesion between the suberin lamellae and polysaccharide cell walls was attenuated in double mutants. There were no other morphological phenotypes under ambient conditions. Furthermore, there was no significant effect on net CO2 assimilation at any intercellular CO2 concentration, and no effect on 13C isotope discrimination relative to wild type. Thus, ZmAsft expression is not required to establish a functional CO2 concentrating mechanism in in maize. Double mutant leaves exhibit elevated cell wall elasticity, transpirational, and stomatal conductance relative to WT. Thus, the ZmAsft genes are dispensable for gas exchange barrier function but may be involved in regulation of leaf water movement. One-sentence SummaryDouble mutants of two paralogously duplicated maize Aliphatic Suberin Feruloyl Transferase (ZmAsft) genes exhibit reduced aliphatic suberin content, cell wall cohesion defects, and elevated leaf transpiration, but no changes in CO2 assimilation relative to wild type.

plant biology↗

bioRxiv2020

A role for neutral variation in the evolution of C4 photosynthesis

Convergent trait evolution is a recurrent phenomenon in all domains of the tree of life. While some convergent traits are caused by simple sequence changes, many are associated with extensive changes to the sequence and regulation of large cohorts of genes. It is unknown how organisms traverse this expansive genotype space to assemble such complex convergent phenotypes. C4 photosynthesis is a paradigm of large-scale phenotypic convergence. Conceptual and mathematical models propose that C4 photosynthesis evolved from ancestral C3 photosynthesis through sequential adaptive changes. These adaptive changes could have been rapidly assembled if modifications to the activity and abundance of enzymes of the C4 cycle was neutral in C3 plants. This neutrality would enable populations of C3 plants to maintain genotypes with expression levels of C4 enzymes analogous to those in C4 species and thus enable rapid assembly of a functional C4 cycle from naturally occurring genotypes given shared environmental selection. Here we show that there is substantial natural variation in expression of genes encoding C4 cycle enzymes between natural accessions of the C3 plant Arabidopsis thaliana. We further show through targeted transgenic experiments in the C3 crop Oryza sativa, that high expression of the majority of C4 cycle enzymes in rice is neutral with respect to growth, development, biomass and photosynthesis. Thus, substantial variation in the abundance and activity of C4 cycle enzymes is permissible within the limits of operation of C3 photosynthesis and the emergence of component parts of this complex convergent trait can be facilitated by neutral variation.

plant biology↗

bioRxiv2020

The Genetic Architecture of Leaf Stable Carbon Isotope Composition in Zea mays and the Effect of Transpiration Efficiency on Elemental Accumulation

With increased demand on freshwater resources for agriculture, it is imperative that more water-use efficient crops are developed. Leaf stable carbon isotope composition, {delta}13C, is a proxy for transpiration efficiency and a possible tool for breeders, but the underlying mechanisms effecting {delta}13C in C4 plants are not known. It has been suggested that differences in specific leaf area, which potentially reflects variation in internal CO2 diffusion, can impact leaf {delta}13C. However, at this point the relationship has not been tested in maize. Furthermore, although it is known that water movement is important for elemental uptake, it is not clear how manipulation of transpiration for increased water-use efficiency may impact nutrient accumulation. Here we characterize the underlying genetic architecture of leaf {delta}13C and test its relationship to specific leaf area and the ionome in four biparental populations of maize. Five significant QTL for leaf {delta}13C were identified, including both novel QTL as well as some that were identified previously in maize kernels. One of the QTL regions contains an Erecta-like gene, the ortholog of which has been shown to regulate transpiration efficiency and leaf {delta}13C in Arabidopsis. Our data does not support a relationship between {delta}13C and specific leaf area, and of the 19 elements analyzed, only a weak correlation between molybdenum and {delta}13C was detected. Together these data begin to build a genetic understanding of leaf {delta}13C in maize and suggest the potential to improve plant water use without significantly influencing elemental homeostasis. Article SummaryQuantitative genetics approaches were used to investigate the genetic architecture of leaf stable carbon isotope discrimination ({delta}13C) in maize. Developing a better understanding of leaf {delta}13C could facilitate its use in breeding for reduced transpirational water loss. Several genomic regions were identified that contribute to the variation observed in leaf {delta}13C. Furthermore, contrary to what has been observed in other species, leaf {delta}13C was not correlated with specific leaf area. Finally, a leaf ionomic analysis indicates that a reduction in transpiration, and thus mass flow, would not result in a decrease in nutrient accumulation.

plant biology↗

bioRxiv2017

Trait components of whole plant water use efficiency are defined by unique, environmentally responsive genetic signatures in the model C4 grass Setaria

Plant growth and water use are interrelated processes influenced by the genetic control of both plant morphological and biochemical characteristics. Improving plant water use efficiency (WUE) to sustain growth in different environments is an important breeding objective that can improve crop yields and enhance agricultural sustainability. However, genetic improvements of WUE using traditional methods have proven difficult due to low throughput and environmental heterogeneity encountered in field settings. To overcome these limitations the study presented here utilizes a high-throughput phenotyping platform to quantify plant size and water use of an interspecific Setaria italica x Setaria viridis recombinant inbred line population at daily intervals in both well-watered and water-limited conditions. Our findings indicate that measurements of plant size and water use in this system are strongly correlated; therefore, a linear modeling approach was used to partition this relationship into predicted values of plant size given water use and deviations from this relationship at the genotype level. The resulting traits describing plant size, water use and WUE were all heritable and responsive to soil water availability, allowing for a genetic dissection of the components of plant WUE under different watering treatments. Linkage mapping identified major loci underlying two different pleiotropic components of WUE. This study indicates that alleles controlling WUE derived from both wild and domesticated accessions of the model C4 species Setaria can be utilized to predictably modulate trait values given a specified precipitation regime.

plant biology↗