Plant Direct

Recent studies have demonstrated the presence of kynurenine (KYN) and kynurenic acid (KYNA) in several plant species, but the metabolic function of these metabolites remains undefined. We hypothesized that KYN and KYNA are metabolites of auxin and play a role in plant morphogenesis. To test our hypothesis, we developed a plant tissue-culture-based bioassay using Hypericum perforatum (St. Johns wort; SJW), a model system for auxin and indoleamine metabolism and pharmacological inhibitors (PF-04859989, RO-61-8048, and KMO inhibitor II, JM6) of human kynurenine pathways enzymes. SJW is an interesting model system because explants root in the absence of plant growth regulators but supplementation of the culture media with 10 M IAA induces a callus response without de novo root organogenesis. Supplementation of the culture media with 10 M KYN increased root number and internodal length relative to basal media. We used a previously validated high-resolution mass spectrometry analytical method to quantify KYN, KYNA, and 3-hydroxyanthranilic acid (3-HAA). KYN, KYNA and 3-HAA were quantified in roots and shoots of SJW grown on basal media. Supplementation of the culture media with 10 M KYN increased the concentration of KYN, KYNA and 3-HAA in roots and shoots. Treatment with 10 M IAA increased KYN and 3-HAA concentration in shoots. Three pharmaceutical candidates that are kynurenine pathway inhibitors in humans were taken up into the tissues from the culture media and increased KYN content as compared to basal control. Together, these data propose a role for KYN in IAA metabolism, shoot and root organogenesis. HighlightsO_LIKynurenine metabolites are detected and accumulate in H. perforatum tissue culture C_LIO_LIIAA redirects metabolism towards accumulation of KYN and 3-HAA in shoots C_LIO_LIExogenous KYN promotes KYNA accumulation C_LIO_LIPharmacological inhibition alters kynurenine pathway metabolite profiles in a tissue-specific manner C_LIO_LIKynurenine and IAA differentially regulate root development C_LI

O_LIRationale: Xylem morphology in annual monocots is important for water use strategies in many agronomically important species. C_LIO_LIMethods: We assess how xylem perforation plates affect water use strategies in maize (Zea mays L.) through in silico modeling, empirical studies under water deficit in controlled environments, and in the field. C_LIO_LIKey Result: Significant genotypic variation for the prominence and frequency of perforation plates was observed in maize germplasm. Perforation plate phenotypes had high heritability, were associated with several QTL, and were pleiotropic across leaves, aerial nodal roots, and subterranean nodal roots. Perforation plate phenotypes did not affect vulnerability to cavitation, but modeling predicted that they should affect axial water transport, which was supported by in situ measurements of root segments. Metaxylem vessel length was correlated with the rate of root elongation, root depth, and deep-water utilization in mesocosms. Under drought stress in the field, variation in xylem vessel length was associated with leaf roll, leaf temperature, transpiration, photosynthesis, and grain yield. C_LIO_LIMain Conclusion: Phenotypic variation for xylem perforation plate phenotypes in maize directly affects axial water conductance and is part of a pleiotropic syndrome with greater root elongation and deeper rooting that improves adaptation to water deficit stress. C_LI

Epicuticular wax blooms are associated with improved drought resistance in many species, including Sorghum bicolor. While the role of wax in drought resistance is well known, we report new insights into how light and drought dynamically influence wax production. We investigated how wax quantity and composition are modulated over time and in response to different environmental stressors, as well as the molecular and genetic mechanisms involved in such. Gas chromatography-mass spectrometry and photographic results showed that sorghum leaf sheath wax load and composition were altered in mature plants grown under drought and simulated shade, though this phenomenon appears to vary by sorghum cultivar. We combined an in vitro wax induction protocol with GC-MS and RNA-seq measurements to identify a draft signaling pathway for wax bloom induction in sorghum. We also explored the potential of spectrophotometry to aid in monitoring wax bloom dynamics. Spec-trophotometric analysis showed primary differences in reflectance between bloom-rich and bloomless tissue surfaces in the 230-500nm range of the spectrum, corresponding to the blue color channel of photographic data. Our smartphone-based system detected significant differences in wax production between control and shade treatment groups, demonstrating its potential for candidate screening. Overall, our data suggest that wax extrusion can be rapidly modulated in response to light, occurring within days compared to the months required for the changes observed under greenhouse drought/simulated shade conditions. These results highlight the dynamic nature of wax modulation in response to varying environmental stimuli, especially light and water availability. Significance StatementAgricultural crops require significant freshwater for irrigation, making food security vulnerable to drought. Epicuticular wax blooms are associated with drought tolerance in many plants, including Sorghum bicolor. We investigated how environmental factors like light and drought influence wax production in sorghum. Wax production, composition, and gene expression were compared between sorghum exposed to different environmental stressors, reavealing dynamic modulation of wax production in response to environmental stress as well as signaling genes potentially involved in regulating wax production. These findings broaden our understanding of wax-related drought tolerance mechanisms, providing a foundation for future efforts to enginner crops with improved climate resilience.

Plant root systems provide critical functions to enable plant survival. From anchoring the plant in the soil to finding and acquiring water and nutrients, these organs are essential for plant productivity. Despite a variety of root functions, research typically focuses on defining only one function. In this study, we explore a trade-off hypothesis, that the optimization of one root function (i.e. anchorage) may negatively impact another root function (i.e. nitrogen uptake). Previous work has demonstrated that larger roots are stronger, but may also have a diminished capacity for nutrient acquisition due to a reduced surface area to volume ratio. Using maize brace roots that had entered the soil, we show here that larger roots are both stronger and take up more nitrogen. Despite this general relationship, there are subtle trade-offs between mechanics and uptake that occur when assessing individual genotypes. These trade-offs represent an opportunity to optimize one root function without compromising other root functions. Together these data demonstrate that our original trade-off hypothesis was incorrect for maize brace roots, and that larger roots are both stronger and take up more nitrogen.

The Arabidopsis thaliana genome contains twenty genes that are analogous to mammalian ionotropic glutamate receptors. There are sixteen mammalian glutamate receptors, which are best known for their roles in neuroplasticity, learning, and memory. The large number of glutamate receptors in A. thaliana suggests they play important roles in the plants growth and development, possibly serving to regulate function like they do in non-excitable mammalian tissues. A specific glutamate receptor, GLR3.3, is highly expressed in root tissue of plants, and has been found to promote stronger, more coordinated curvature development during the process of gravitropism. Gravitropism is the ability of a plant to change its orientation to that of the gravity vector when displaced from its gravitational set point angle (GSPA). A previous association study identified six candidate genes which were correlated with the same phenotypic characteristics of gravitropism as GLR3.3. Utilizing real time RT-PCR (qRT-PCR) expression profiles were created for each candidate gene, including GLR3.3. A qRT-PCR method was developed to provide a more quantitative and sensitive way for measuring gene expression than traditional PCR methods. Furthermore, MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines were followed to ensure data robustness. Expression profiles that were similar to GLR3.3 were hypothesized to be good candidates as cell signaling components of this novel pathway. This is the beginning of a process that will identify a GLR-dependent pathway, the role of this novel pathway in the gravitropic response, and the influence of GLRs in plant physiology.

In a screen for mutations that alter cell morphology and adhesion as a marker for cell wall composition alterations, an Arabidopsis seedling with root hairs that fail to elongate was identified. Root hairs are rounded and bubble-like, and the mutation was named Bubbles or bbl1. No other visible whole plant phenotypes were observed. Using backcrosses with wild type, and genomic sequencing of pooled F2 individuals with the Bubbles phenotype, 14 candidates for the mutant allele were identified. T-DNA alleles of one of these candidates showed a similar but partial phenotype to bbl1, and indicate that bbl1 is an allele of AT2G39110 previously identified as PBL38 (AvrPphB SUSCEPTIBLE1-LIKE38), a member of receptor kinase subfamily VII. PBL38 has been associated with the response to pathogens. bbl1 is a point mutation that causes a Glutamic acid to Lysine change at position 140, and is complimented by a C-terminal GFP fusion to the coding region of AT2G39110, indicating that the BBL1 gene (PBL38) is involved in correct root hair formation. AT2G39110 is expressed only in roots early in seedling maturation, and the GFP fusion protein localizes to the cell surface, consistent with the prediction that the gene encodes a receptor-like protein kinase. In bbl1 root hairs, the actin cytoskeleton does not form, while in bbl1 roots and other tissues normal actin cytoskeleton is observed.

Improving nitrogen use efficiency in rice would provide economic and environmental benefits, but little is known about root morphological and anatomical responses to low nitrogen. In this study, two sets of rice genotypes, one set from the RDP1 panel, and one set of recombinant inbred lines, were used to characterize responses to gradual nitrogen depletion by plant uptake and movement of nitrogen to deeper soil strata as a result of leaching, so that more nitrogen was available at depth in aerobic soil mesocosms in a greenhouse. There was significant genetic variation in shoot biomass reductions in response to low nitrogen. The root to shoot biomass ratio was increased by low nitrogen in both sets of genotypes. Relative investment in nodal root number was accentuated with low nitrogen, and shoot biomass was correlated with numbers and lengths of nodal and large lateral roots. There was genetic variation for nodal root number and length in both sets of genotypes. Anatomical responses to low nitrogen were assessed in nodal roots of the RILs, where root cross-sectional area, stele area, and metaxylem vessel number were reduced by low nitrogen, and root diameter was reduced in the RDP1 genotypes. There were significant interactions of nitrogen with genotype for stele area and percent aerenchyma in the RILs. Genetic variation for low nitrogen responses may be useful for selection of rice lines with greater nitrogen acquisition under nitrogen-leaching conditions.

Auxin is a hormone that is required for hypocotyl elongation during seedling development. In response to auxin rapid changes in transcript and protein abundance occur in hypocotyls and some auxin responsive gene expression is linked to hypocotyl growth. To functionally validate proteomic studies, a reverse genetics screen was performed on mutants in auxin-regulated proteins to identify novel regulators of plant growth. This uncovered a long hypocotyl mutant, which we called slim shady, in an annotated insertion line in IMMUNOREGULATORY RNA-BINDING PROTEIN (IRR). Overexpression of the IRR gene failed to rescue the slim shady phenotype and characterization of a second T-DNA allele of IRR found that it had a wild-type hypocotyl length. The slim shady mutant has an elevated expression of numerous genes associated with the brassinosteroid-auxin-phytochrome (BAP) regulatory module compared to wild-type, including transcription factors that regulate brassinosteroid, auxin and phytochrome pathways. Additionally, slim shady seedlings fail to exhibit a strong transcriptional response to auxin. Using whole genome sequence and transcriptomics data for SALK_015201C we determined that a novel single nucleotide polymorphism in PHYTOCHROME B was responsible for the slim shady phenotype. This is predicted to convert induce a frameshift and premature stop codon at leucine 1125, within the histidine kinase-related domain of the carboxy terminus of PHYB, which is required for phytochrome signaling and function. Genetic complementation analyses with phyb-9 confirmed that slim shady is a mutant allele of PHYB. This study advances our understanding of the molecular mechanisms in seedling development, by furthering our understanding of how light signaling is linked to auxin dependent cell elongation. Furthermore, this study highlights the importance of confirming the genetic identity of research material before attributing phenotypes to known mutations sourced from T-DNA stocks.

Expansins are vital proteins that facilitate cell wall loosening, playing a crucial role in plant growth and development. This study investigates the structural and functional characteristics of the alpha-expansin precursor (GenBank ID: AAL79710.1) in Oryza sativa (Japanese rice). Through bioinformatics analyses, including ProtParam, CELLO, and conserved domain identification, we identified key biochemical properties, such as a molecular weight of approximately 28 kDa, a basic isoelectric point (pI 9.40), and significant levels of alanine and glycine. The CELLO analysis predicted the proteins localization primarily in the extracellular space, consistent with its role in modifying the cell wall. Homology searches revealed high similarity to expansin-A29 proteins in related species, while phylogenetic analysis indicated a close evolutionary relationship among monocots. Structural modeling predicted a well-folded protein, though refinement is necessary to address certain discrepancies highlighted in the QMEANDisCo analysis. Our findings underscore the evolutionary conservation of alpha-expansins and their integral role in plant physiology, particularly in cell wall dynamics and stress responses. This research enhances our understanding of alpha-expansins in rice and lays the groundwork for future studies aimed at manipulating these proteins to improve crop resilience and yield under changing environmental conditions.

Plant-specific membrane receptor kinases with structurally diverse extracellular domains regulate key processes in plant growth, development, immunity and symbiosis. Structural studies of these glycoproteins are often hampered by the limited quantities in which they can be obtained. Here, we describe the LRR crystallization screen, which has enabled the successful crystallization and structure determination of multiple receptor kinase ectodomains, including ligand-and co-receptor-bound complexes. As an example, we report the 1.5 [A] resolution crystal structure of the leucine-rich repeat (LRR) domain of STRUBBELIG-RECEPTOR FAMILY 6 (SRF6) from Arabidopsis thaliana. The SRF6 ectodomain contains seven LRRs and a disulfide-bond-stabilised N-terminal capping domain but lacks the canonical C-terminal cap and the N-glycosylation pattern typically observed in other family members. Previously reported protein-protein interactions between the SRF6 and SRF7 ectodomains and the receptor kinases BRI1, BRL1, BRL3, SERK3 and BIR1-3 could not be confirmed by quantitative isothermal titration calorimetry and grating-coupled interferometry assays, suggesting that these structurally conserved LRR receptor kinases may have signalling functions outside the brassinosteroid pathway. SynopsisA crystallisation screen that has enabled the structural analysis of various extracellular domains of plant membrane receptor kinases is described together.

Filamentous structures were observed in purified extracts from chrysanthemum, gerbera, sunflower and zinnia. When purified filament proteins were subjected to SDS-PAGE, the major protein associated with filaments from all three species has an apparent molecular mass of {approx}25 kDa. Protein bands from chrysanthemum, gerbera, and zinnia were subjected to N-terminal protein sequencing while proteins from sunflower were sequenced by CID MS/MS. All of the sequences shared highest similarity to the kunitz trypsin inhibitor family. The sequencing results indicated that the proteins lacked the signal sequences. We tested the gerbera filament protein for glycosylation and found that it was a glycoprotein. Together these results indicate that the filaments are composed of mature KTI protein. This is the first report of a KTI assembling into filaments and the first report of a filament forming Asteraceae enzyme.

Screening a transposon-mutagenized soybean population led to the discovery of a recessively inherited chlorotic phenotype. This "vir1" phenotype results in smaller stature, weaker stems, and a smaller root system with smaller nodules. Genome sequencing identified 15 candidate genes with mutations likely to result in a loss of function. Amplicon sequencing of a segregating population was then used to narrow the list to a single candidate mutation, a single-base change in Glyma.07G102300 that disrupts splicing of the second intron. Single cell transcriptomic profiling indicates that this gene is expressed primarily in mesophyll cells and RNA sequencing data indicates it is upregulated in germinating seedlings by cold stress. Previous studies have shown that mutations to Os05g34040, the rice homolog of Glyma.07G102300, produced a chlorotic phenotype that was more pronounced in cool temperatures. Growing soybean vir1 mutants at lower temperatures also resulted in a more severe phenotype. In addition, transgenic expression of wild type Glyma.07G102300 in the knockout mutant of the Arabidopsis homolog At4930720 rescues the chlorotic phenotype, further supporting the hypothesis that the mutation in Glyma.07G102300 is causal of the vir1 phenotype.

Sorghum is a crop that has become more relevant in recent years due to its uses and properties (biofuel, gluten-free flours) as well as its versatility to grow in unfavorable environmental conditions. Salinity is one of the main abiotic stresses affecting crop production and yield worldwide. The aim of this work was to study the response of sorghum seedlings to soil salinity in two genotypes with known performance to cope with water stress. Physiological parameters related to plant water status as well as the sodium content in the plant were analyzed. We studied the possible role of pricklet and microhairs in the response to salt conditions. Our results showed a differential response to salinity, probably denoting different mechanisms that involve internal water redistribution (in 200 mM NaCl) and a specific replacement of silicon by sodium (when the NaCl reaches 300 mM). The main result was that sodium was absent in all analyzed hairs and leaf surface. Surprisingly, we detected the presence of silicon inside the pricklet at 300 mM NaCl after 24 hours, but not in the microhair. NIPs aquaporins could be involucrate in silicon transport. Our novel results provide further evidence regarding the role of silicon in the response to salt stress. HighlightGrain sorghum has a different strategy to deal with salinity stress depending on the salt concentration, that involves the leaves pricklets and the migration of silicon.

I.Almond (Prunus dulcis [Mill.] D.A.Webb) exhibits an age-related disorder called non-infectious bud-failure (BF) affecting vegetative bud development and nut yield. The underlying cause of BF remains unknown but is hypothesized to be associated with heritable epigenetic mechanisms. To address this disorder and its epigenetic components, we utilized a monozygotic twin study model profiling genome-wide DNA methylation and gene expression in two sets of twin almonds discordant for BF-exhibition. Analysis of DNA methylation patterns show that BF-exhibition and methylation, namely hypomethylation, are not independent phenomena. Transcriptomic data generated from the twin pairs also shows genome-wide differential gene expression associated with BF-exhibition. After identifying differentially methylated regions (DMRs) in each twin pair, a comparison revealed 170 shared DMRs between the two twin pairs. These DMRs and the associated genetic components may play a role in BF-exhibition. A subset of 52 shared DMRs are in close proximity to genes involved in meristem maintenance, cell cycle regulation, and response to heat stress. Annotation of specific genes included involvement in processes like cell wall development, calcium ion signaling, and DNA methylation. Results of this work support the hypothesis that BF-exhibition is associated with hypomethylation in almond, and identified DMRs and differentially expressed genes can serve as potential biomarkers to assess BF-potential in almond germplasm. Our results contribute to an understanding of the contribution of epigenetic disorders in agricultural performance and biological fitness of perennials. II. SignificanceThis study examines epigenetic components underlying noninfectious bud failure, an aging-related disorder affecting almond. Results from this work contribute to our understanding of the implications of DNA methylation on agricultural production, namely perennial fruit and nut production, due to effects on growth, development, and reproduction. Describing the methylome of discordant, monozygotic twin almonds enables the study of genomic features underlying noninfectious bud failure in this economically important crop.

Radial expansion is a classic response of roots to mechanical impedance that has generally been assumed to aid penetration. We analysed the response of maize nodal roots to impedance to test the hypothesis that radial expansion is not related to the ability of roots to cross a compacted soil layer. Genotypes varied in their ability to cross the compacted layer, and those with a steeper approach to the compacted layer or less radial expansion in the compacted layer were more likely to cross the layer and achieve greater depth. Root radial expansion was due to cortical cell size expansion, while cortical cell file number remained constant. Genotypes and nodal root classes that exhibited radial expansion upon encountering the compacted soil layer also thickened in response to exogenous ethylene in hydroponic culture, i.e. radial expansion in response to ethylene was correlated with the thickening response to impedance in soil. We propose that ethylene insensitive roots, i.e. those that do not thicken and are able to overcome impedance, have a competitive advantage under mechanically impeded conditions as they can maintain their elongation rates. We suggest that prolonged exposure to ethylene could function as a stop signal for axial root growth.

Small signaling peptides (SSPs) are critical regulators of plant growth, development, and responses to biotic and abiotic stress, yet their role in the C4 grass Sorghum bicolor is largely uncharacterized. To help fill this knowledge gap, 219 S. bicolor genes that encode SSPs were identified based on SSP sequences previously identified in Arabidopsis thaliana, Oryza sativa, Zea mays, Triticum aestivum, and Brachypodium distachyon. The 219 sorghum genes were assigned to 19 gene families, analyzed for the presence of motifs, and aligned with genes that encode SSPs in other plants using phylogenetic analysis. Expression of the 219 SSP encoding genes in sorghum organs, during stem development, and in stem tissues and cell types revealed distinct spatial, temporal and developmental patterns of expression. Genes associated with the SbCEP and SbRGF families were preferentially expressed in roots, whereas SbEPF genes were expressed in stems and panicles. The expression of genes during bioenergy sorghum stem growth and development was investigated because stems account for [~]80% of harvested biomass and serve as conduits for water and nutrient transport between leaves and roots. During stem development, 28 SSP encoding sorghum genes in several families (CLE, EPF, CEP, GASS, PSY, ES, PSK, CAPE, POE) were expressed at higher levels in zones of cell proliferation. For example, the TDIF homologs SbCLE41 and SbCLE42 were expressed at high levels in nascent stem nodes where they may regulate cambial activity and vascular bundle cell differentiation. A different set of 15 genes in the CIF, POE, CAPE, PSY, CEP, RALF, and CLE families were expressed at higher levels in zones of stem tissue differentiation highlighted by elevated expression of 5 SbRALFs in the stem nodal plexus. Cell type specific expression of many SSP encoding sorghum genes was also observed in fully elongated internodes indicating gene expression is regulated with high spatial resolution. Overall, the results provide a foundation of information for analysis of SSP functions in sorghum that can be integrated with knowledge of sorghum gene regulatory networks to modulate traits important for production of sorghum crops.

3-Deoxy-D-manno-oct-2-ulosonic acid (KDO) is an essential component of rhamnogalacturonan II (RG-II), a complex pectic polysaccharide required for plant growth and development. While most steps of the KDO biosynthetic pathway have been characterized in plants, KDO-8-phosphatase (KDO8Pase), the phosphatase responsible for converting KDO 8-phosphate (KDO8P) to KDO, remained unidentified. To identify this missing component, we performed gene co-expression analysis and identified At5g57440 (GPP2) as the primary candidate in Arabidopsis (Arabidopsis thaliana L.). Recombinant GPP2 protein exhibited KDO8P-specific phosphohydrolase activity in vitro. A GFP-tagged GPP2 protein was predominantly localized to mitochondria, consistent with the compartmentation of the subsequent step in KDO biosynthesis. Null mutants of GPP2 exhibited significant growth retardation under boron-limited conditions, in which expression of GPP2 and other KDO biosynthetic genes was up-regulated. The growth retardation was also observed in liquid culture in normal media, a condition that induces rapid growth and thus likely increases metabolic demand for KDO. Despite this growth defect, the KDO content per unit cell wall in gpp2 remained equivalent to that in wild-type plants. These results are consistent with the identification of GPP2 as the elusive plant KDO8Pase and suggest a model where KDO availability becomes the rate-limiting factor for cell wall production. Our findings complete the plant KDO biosynthetic pathway and provide new insights into the physiological significance of RG-II in cell wall biosynthesis. Significance statementThis study identifies the previously unknown plant KDO-8-phosphatase, thereby completing the biosynthetic pathway for KDO in rhamnogalacturonan II. Our findings demonstrate that KDO synthesis is up-regulated under boron deficiency, and its supply becomes a rate-limiting factor for cell wall formation.

Drought stress significantly impairs growth, and microbial interactions in sorghum. This study explores the transcriptional and microbial shifts in sorghum under drought, revealing key adaptations to water deficit. LC-MS (Liquid Chromatography-Mass Spectrometry) analyses revealed that drought stress induced abscisic acid while significantly reducing jasmonic acid levels in sorghum roots, likely due to resource conservation strategies during drought. Transcriptional reprogramming highlighted the upregulation of genes in the roots involved in mineral homeostasis (Ferritin 1, Iron dehydrogenase, Nitrate transporter 1), hormone signaling (Ethylene-insensitive protein 3, Gibberellin 2-oxidase), and osmotic regulation (Aquaporin, Dehydrin), underlining key adaptive responses to maintain nutrient uptake, redox status, and cellular turgor. In Fe-supplemented plants, increased Fe in roots correlated with increased Ferritin 1 expression, improved plant health, and reduced Fenton reaction rate and H O levels. This suggests that ferritin helps minimize oxidative stress under drought in sorghum. Drought reduced root-associated bacterial diversity and richness while enriching drought tolerance-associated genera, such as Burkholderia, Caballeronia and Paraburkholderia, known for promoting plant growth through auxin production, phosphate solubilization, and siderophore-mediated iron acquisition. In contrast, fungal diversity and richness remained unchanged, dominated by Talaromyces, which showed a statistically non-significant increase under drought. Random forest models could not identify functional predictors for fungi but revealed a shift in bacterial functional groups under drought, with enrichment in phototrophy, methylotrophy, and nitrate reduction, traits emphasizing microbial roles in nutrient cycling and drought adaptation of sorghum. This study provides insights into the role of ferritin and potential bacterial bioinoculants that could enhance sorghum resilience to drought. Future research should validate these findings to integrate them into breeding programs and biofertilizer formulation for drought-tolerant sorghum and climate-resilient agriculture.

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.

Leaf development is crucial to establish the photosynthetic competency of plants. It is a process that requires coordinated changes in cell number and differentiation, transcriptomes, metabolomes and physiology. However, despite the importance of leaf formation for our major crops, early developmental processes for rice have not been comprehensively described. Here we detail the temporal developmental trajectory of early rice leaf development and connect morphological changes to metabolism. In particular, a developmental index based on the patterning of epidermal differentiation visualised by electron microscopy enabled high resolution staging of early growth for single primordium metabolite profiling. These data demonstrate that a switch in the constellation of tricarboxylic acid (TCA) cycle metabolites defines a narrow window towards the end of the P3 stage of leaf development. Taken in the context of other data in the literature, our results substantiate that this phase of rice leaf growth, equivalent to a change of primordium length from around 5 to 7.5 mm, defines a major shift in rice leaf determination towards a photosynthetically defined structure. We speculate that efforts to engineer rice leaf structure should focus on the developmental window prior to these determining events. HighlightRice leaves undergo a shift in fundamental metabolism during a very early and narrow developmental window which co-incides with them acquiring the ability to capture light for photosynthesis