Dynamics of mRNA fate during light stress and recovery: from transcription to stability and translation.

Transcript stability is an important determinant of its abundance and, consequently, translational output. Transcript destabilisation can be rapid and is well suited for modulating the cellular response. However, it is unclear the extent to which RNA stability is altered under changing environmental conditions in plants. We previously hypothesised that recovery-induced transcript destabilisation facilitated a phenomenon of rapid recovery gene downregulation (RRGD) in Arabidopsis thaliana (Arabidopsis) following light stress, based on mathematical calculations to account for ongoing transcription. Here, we test this hypothesis and investigate processes regulating transcript abundance and fate by quantifying changes in transcription, stability and translation before, during and after light stress. We adapt syringe infiltration to apply a transcriptional inhibitor to soil-grown plants in combination with stress treatments. Compared with measurements in juvenile plants and cell culture, we find reduced stability across a range of transcripts encoding proteins involved in RNA binding and processing. We also observe light-induced destabilisation of transcripts, followed by their stabilisation during recovery. We propose that this destabilisation facilitates RRGD, possibly in combination with transcriptional shut-off that was confirmed for HSP101, ROF1 and GOLS1. We also show that translation remains highly dynamic over the course of light stress and recovery, with a bias towards transcript-specific increases in ribosome association, independent of changes in total transcript abundance, after 30 min of light stress. Taken together, we provide evidence for the combinatorial regulation of transcription and stability that occurs to coordinate translation during light stress and recovery in Arabidopsis.

Reducing PHYTOENE SYNTHASE activity fine-tunes the abundance of a cis-carotene-derived signal that regulates the PIF3/HY5 module and plastid biogenesis.

PHYTOENE SYNTHASE (PSY) is a rate-limiting enzyme catalysing the first committed step of carotenoid biosynthesis, and changes in PSY gene expression and/or protein activity alter carotenoid composition and plastid differentiation in plants. Four genetic variants of PSY (psy-4, psy-90, psy-130, and psy-145) were identified using a forward genetics approach that rescued leaf virescence phenotypes and plastid abnormalities displayed by the Arabidopsis CAROTENOID ISOMERASE (CRTISO) mutant ccr2 (carotenoid and chloroplast regulation 2) when grown under a shorter photoperiod. The four non-lethal mutations affected alternative splicing, enzyme-substrate interactions, and PSY:ORANGE multi-enzyme complex binding, constituting the dynamic post-transcriptional fine-tuning of PSY levels and activity without changing localization to the stroma and protothylakoid membranes. psy genetic variants did not alter total xanthophyll or ß-carotene accumulation in ccr2, yet they reduced specific acyclic linear cis-carotenes linked to the biosynthesis of a currently unidentified apocarotenoid signal regulating plastid biogenesis, chlorophyll biosynthesis, and photomorphogenic regulation. ccr2 psy variants modulated the PHYTOCHROME-INTERACTING FACTOR 3/ELONGATED HYPOCOTYL 5 (PIF3/HY5) ratio, and displayed a normal prolamellar body formation in etioplasts and chlorophyll accumulation during seedling photomorphogenesis. Thus, suppressing PSY activity and impairing PSY:ORANGE protein interactions revealed how cis-carotene abundance can be fine-tuned through holoenzyme-metabolon interactions to control plastid development.

Deregulation of ζ-carotene desaturase in Arabidopsis and tomato exposes a unique carotenoid-derived redundant regulation of floral meristem identity and function.

A level of redundancy and interplay among the transcriptional regulators of floral development safeguards a plant's reproductive success and ensures crop production. In the present study, an additional layer of complexity in the regulation of floral meristem (FM) identity and flower development is elucidated linking carotenoid biosynthesis and metabolism to the regulation of determinate flowering. The accumulation and subsequent cleavage of a diverse array of ζ-carotenes in the chloroplast biogenesis 5 (clb5) mutant of Arabidopsis results in the reprogramming of meristematic gene regulatory networks establishing FM identity mirroring that of the FM identity master regulator, APETALA1 (AP1). The immediate transition to floral development in clb5 requires long photoperiods in a GIGANTEA-independent manner, whereas AP1 is essential for the floral organ development of clb5. The elucidation of this link between carotenoid metabolism and floral development translates to tomato exposing a regulation of FM identity redundant to and initiated by AP1 and proposed to be dependent on the E class floral initiation and organ identity regulator, SEPALLATA3 (SEP3).

Enhancing crop yields through improvements in the efficiency of photosynthesis and respiration.

The rate with which crop yields per hectare increase each year is plateauing at the same time that human population growth and other factors increase food demand. Increasing yield potential ( Y p ) of crops is vital to address these challenges. In this review, we explore a component of Y p that has yet to be optimised - that being improvements in the efficiency with which light energy is converted into biomass ( ε c ) via modifications to CO2 fixed per unit quantum of light (α), efficiency of respiratory ATP production ( ε prod ) and efficiency of ATP use ( ε use ). For α, targets include changes in photoprotective machinery, ribulose bisphosphate carboxylase/oxygenase kinetics and photorespiratory pathways. There is also potential for ε prod to be increased via targeted changes to the expression of the alternative oxidase and mitochondrial uncoupling pathways. Similarly, there are possibilities to improve ε use via changes to the ATP costs of phloem loading, nutrient uptake, futile cycles and/or protein/membrane turnover. Recently developed high-throughput measurements of respiration can serve as a proxy for the cumulative energy cost of these processes. There are thus exciting opportunities to use our growing knowledge of factors influencing the efficiency of photosynthesis and respiration to create a step-change in yield potential of globally important crops.

Potential abiotic stress targets for modern genetic manipulation.

Research into crop yield and resilience has underpinned global food security, evident in yields tripling in the past 5 decades. The challenges that global agriculture now faces are not just to feed 10+ billion people within a generation, but to do so under a harsher, more variable, and less predictable climate, and in many cases with less water, more expensive inputs, and declining soil quality. The challenges of climate change are not simply to breed for a "hotter drier climate," but to enable resilience to floods and droughts and frosts and heat waves, possibly even within a single growing season. How well we prepare for the coming decades of climate variability will depend on our ability to modify current practices, innovate with novel breeding methods, and communicate and work with farming communities to ensure viability and profitability. Here we define how future climates will impact farming systems and growing seasons, thereby identifying the traits and practices needed and including exemplars being implemented and developed. Critically, this review will also consider societal perspectives and public engagement about emerging technologies for climate resilience, with participatory approaches presented as the best approach.

Detection and analysis of novel and known plant volatile apocarotenoids.

As the climate becomes increasingly unpredictable due to global warming, plants will encounter a greater challenge to adapt to their hostile environment (e.g., drought, heat, pollution). Volatile apocarotenoids (VAs) are an integral part of this necessary adaptation. VAs are involved in diverse plant life processes such as defense against biotic or abiotic stresses and regulate various aspects of plant development. The discovery of new VAs will help enhance abiotic and biotic stress tolerance, optimize biomass and crop yield, improve root development to better search for nutrients and promote symbiotic associations. This chapter describes an optimized method, HeadSpace Solid-Phase MicroExtraction (HS-SPME) coupled to Gas Chromatography-Mass Spectrometry (GC/MS), for the sensitive, reproducible, accurate, and high-throughput detection and quantification of novel and known VAs. Further optimization of this method can be performed by (1) adapting optimal growth conditions for your plants, (2) identifying the correct SPME fiber coating chemistry for the VAs of interest, (3) adapting optimal sample HS-SPME extraction temperature and time, and the desorption time in the GC inlet, (4) identifying the correct GC column and applying the optimal GC/MS parameters for good chromatographic baseline separation of the VAs, mass spectral matching and retention index (RI) validation, and (5) performing suitable quantification and statistical analyses. With this optimized and validated analytical technique, we detected and quantified 28 VAs; 20 of these were identified for the first time in Arabidopsis.

Enzymes degraded under high light maintain proteostasis by transcriptional regulation in Arabidopsis.

Photoinhibitory high light stress in Arabidopsis leads to increases in markers of protein degradation and transcriptional up-regulation of proteases and proteolytic machinery, but proteostasis is largely maintained. We find significant increases in the in vivo degradation rate for specific molecular chaperones, nitrate reductase, glyceraldehyde-3 phosphate dehydrogenase, and phosphoglycerate kinase and other plastid, mitochondrial, peroxisomal, and cytosolic enzymes involved in redox shuttles. Coupled analysis of protein degradation rates, mRNA levels, and protein abundance reveal that 57% of the nuclear-encoded enzymes with higher degradation rates also had high light­induced transcriptional responses to maintain proteostasis. In contrast, plastid-encoded proteins with enhanced degradation rates showed decreased transcript abundances and must maintain protein abundance by other processes. This analysis reveals a light-induced transcriptional program for nuclear-encoded genes, beyond the regulation of the photosystem II (PSII) D1 subunit and the function of PSII, to replace key protein degradation targets in plants and ensure proteostasis under high light stress.

Elucidating the role of SWEET13 in phloem loading of the C4 grass Setaria viridis.

Photosynthetic efficiency and sink demand are tightly correlated with rates of phloem loading, where maintaining low cytosolic sugar concentrations is paramount to prevent the downregulation of photosynthesis. Sugars Will Eventually be Exported Transporters (SWEETs) are thought to have a pivotal role in the apoplastic phloem loading of C4 grasses. SWEETs have not been well studied in C4 species, and their investigation is complicated by photosynthesis taking place across two cell types and, therefore, photoassimilate export can occur from either one. SWEET13 homologues in C4 grasses have been proposed to facilitate apoplastic phloem loading. Here, we provide evidence for this hypothesis using the C4 grass Setaria viridis. Expression analyses on the leaf gradient of C4 species Setaria and Sorghum bicolor show abundant transcript levels for SWEET13 homologues. Carbohydrate profiling along the Setaria leaf shows total sugar content to be significantly higher in the mature leaf tip compared with the younger tissue at the base. We present the first known immunolocalization results for SvSWEET13a and SvSWEET13b using novel isoform-specific antisera. These results show localization to the bundle sheath and phloem parenchyma cells of both minor and major veins. We further present the first transport kinetics study of C4 monocot SWEETs by using a Xenopus laevis oocyte heterologous expression system. We demonstrate that SvSWEET13a and SvSWEET13b are high-capacity transporters of glucose and sucrose, with a higher apparent Vmax for sucrose, compared with glucose, typical of clade III SWEETs. Collectively, these results provide evidence for an apoplastic phloem loading pathway in Setaria and possibly other C4 species.

Autophagy mutants show delayed chloroplast development during de-etiolation in carbon limiting conditions.

Autophagy is a conserved catabolic process that plays an essential role under nutrient starvation conditions and influences different developmental processes. We observed that seedlings of autophagy mutants (atg2, atg5, atg7, and atg9) germinated in the dark showed delayed chloroplast development following illumination. The delayed chloroplast development was characterized by a decrease in photosynthetic and chlorophyll biosynthetic proteins, lower chlorophyll content, reduced chloroplast size, and increased levels of proteins involved in lipid biosynthesis. Confirming the biological impact of these differences, photosynthetic performance was impaired in autophagy mutants 12 h post-illumination. We observed that while gene expression for photosynthetic machinery during de-etiolation was largely unaffected in atg mutants, several genes involved in photosystem assembly were transcriptionally downregulated. We also investigated if the delayed chloroplast development could be explained by lower lipid import to the chloroplast or lower triglyceride (TAG) turnover. We observed that the limitations in the chloroplast lipid import imposed by trigalactosyldiacylglycerol1 are unlikely to explain the delay in chloroplast development. However, we found that lower TAG mobility in the triacylglycerol lipase mutant sugardependent1 significantly affected de-etiolation. Moreover, we showed that lower levels of carbon resources exacerbated the slow greening phenotype whereas higher levels of carbon resources had an opposite effect. This work suggests a lack of autophagy machinery limits chloroplast development during de-etiolation, and this is exacerbated by limited lipid turnover (lipophagy) that physically or energetically restrains chloroplast development.

Addressing Research Bottlenecks to Crop Productivity.

Asymmetry of investment in crop research leads to knowledge gaps and lost opportunities to accelerate genetic gain through identifying new sources and combinations of traits and alleles. On the basis of consultation with scientists from most major seed companies, we identified several research areas with three common features: (i) relatively underrepresented in the literature; (ii) high probability of boosting productivity in a wide range of crops and environments; and (iii) could be researched in 'precompetitive' space, leveraging previous knowledge, and thereby improving models that guide crop breeding and management decisions. Areas identified included research into hormones, recombination, respiration, roots, and source-sink, which, along with new opportunities in phenomics, genomics, and bioinformatics, make it more feasible to explore crop genetic resources and improve breeding strategies.

Deconvoluting apocarotenoid-mediated retrograde signaling networks regulating plastid translation and leaf development.

Signals originating within plastids modulate organelle differentiation by transcriptionally regulating nuclear-encoded genes. These retrograde signals are also integral regulators of plant development, including leaf morphology. The clb5 mutant displays severe leaf morphology defects due to Apocarotenoid Signal 1 (ACS1) accumulation in the developmentally arrested plastid. Transcriptomic analysis of clb5 validates that ACS1 accumulation deregulates hundreds of nuclear genes, including the suppression of most genes encoding plastid ribosomal proteins. Herein, we order the molecular events causing the leaf phenotype associated with the accumulation of ACS1, which includes two consecutive retrograde signaling cascades. Firstly, ACS1 originating in the plastid drives inhibition of plastid translation (IPT) via nuclear transcriptome remodeling of chlororibosomal proteins, requiring light as an essential component. Subsequently, IPT results in leaf morphological defects via a GUN1-dependent pathway shared with seedlings undergoing chemical IPT treatments and is restricted to an early window of the leaf development. Collectively, this work advances our understanding of the complexity within plastid retrograde signaling exemplified by sequential signal exchange and consequences that in a particular temporal and spatial context contribute to the modulation of leaf development.

A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development.

Carotenoids are a core plastid component and yet their regulatory function during plastid biogenesis remains enigmatic. A unique carotenoid biosynthesis mutant, carotenoid chloroplast regulation 2 (ccr2), that has no prolamellar body (PLB) and normal PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR) levels, was used to demonstrate a regulatory function for carotenoids and their derivatives under varied dark-light regimes. A forward genetics approach revealed how an epistatic interaction between a ζ-carotene isomerase mutant (ziso-155) and ccr2 blocked the biosynthesis of specific cis-carotenes and restored PLB formation in etioplasts. We attributed this to a novel apocarotenoid retrograde signal, as chemical inhibition of carotenoid cleavage dioxygenase activity restored PLB formation in ccr2 etioplasts during skotomorphogenesis. The apocarotenoid acted in parallel to the repressor of photomorphogenesis, DEETIOLATED1 (DET1), to transcriptionally regulate PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (POR), PHYTOCHROME INTERACTING FACTOR3 (PIF3) and ELONGATED HYPOCOTYL5 (HY5). The unknown apocarotenoid signal restored POR protein levels and PLB formation in det1, thereby controlling plastid development.

Prospects for Carotenoid Biofortification Targeting Retention and Catabolism.

Due to the ongoing prevalence of vitamin A deficiency (VAD) in developing countries there has been a large effort towards increasing the carotenoid content of staple foods via biofortification. Common strategies used for carotenoid biofortification include altering flux through the biosynthesis pathway to direct synthesis to a specific product, generally ß-carotene, or via increasing the expression of genes early in the carotenoid biosynthesis pathway. Recently, carotenoid biofortification strategies are turning towards increasing the retention of carotenoids in plant tissues either via altering sequestration within the cell or via downregulating enzymes known to cause degradation of carotenoids. To date, little attention has focused on increasing the stability of carotenoids, which may be a promising method of increasing carotenoid content in staple foods.

Molecular and physiological responses during thermal acclimation of leaf photosynthesis and respiration in rice.

To further our understanding of how sustained changes in temperature affect the carbon economy of rice (Oryza sativa), hydroponically grown plants of the IR64 cultivar were developed at 30°C/25°C (day/night) before being shifted to 25/20°C or 40/35°C. Leaf messenger RNA and protein abundance, sugar and starch concentrations, and gas-exchange and elongation rates were measured on preexisting leaves (PE) already developed at 30/25°C or leaves newly developed (ND) subsequent to temperature transfer. Following a shift in growth temperature, there was a transient adjustment in metabolic gene transcript abundance of PE leaves before homoeostasis was reached within 24 hr, aligning with Rdark (leaf dark respiratory CO2 release) and An (net CO2 assimilation) changes. With longer exposure, the central respiratory protein cytochrome c oxidase (COX) declined in abundance at 40/35°C. In contrast to Rdark , An was maintained across the three growth temperatures in ND leaves. Soluble sugars did not differ significantly with growth temperature, and growth was fastest with extended exposure at 40/35°C. The results highlight that acclimation of photosynthesis and respiration is asynchronous in rice, with heat-acclimated plants exhibiting a striking ability to maintain net carbon gain and growth when exposed to heat-wave temperatures, even while reducing investment in energy-conserving respiratory pathways.

A GDSL Esterase/Lipase Catalyzes the Esterification of Lutein in Bread Wheat.

Xanthophylls are a class of carotenoids that are important micronutrients for humans. They are often found esterified with fatty acids in fruits, vegetables, and certain grains, including bread wheat (Triticum aestivum). Esterification promotes the sequestration and accumulation of carotenoids, thereby enhancing stability, particularly in tissues such as in harvested wheat grain. Here, we report on a plant xanthophyll acyltransferase (XAT) that is both necessary and sufficient for xanthophyll esterification in bread wheat grain. XAT contains a canonical Gly-Asp-Ser-Leu (GDSL) motif and is encoded by a member of the GDSL esterase/lipase gene family. Genetic evidence from allelic variants of wheat and transgenic rice (Oryza sativa) calli demonstrated that XAT catalyzes the formation of xanthophyll esters. XAT has broad substrate specificity and can esterify lutein, ß-cryptoxanthin, and zeaxanthin using multiple acyl donors, yet it has a preference for triacylglycerides, indicating that the enzyme acts via transesterification. A conserved amino acid, Ser-37, is required for activity. Despite xanthophylls being synthesized in plastids, XAT accumulated in the apoplast. Based on analysis of substrate preferences and xanthophyll ester formation in vitro and in vivo using xanthophyll-accumulating rice callus, we propose that disintegration of the cellular structure during wheat grain desiccation facilitates access to lutein-promoting transesterification.plantcell;31/12/3092/FX1F1fx1.

Excess Light Priming in Arabidopsis thaliana Genotypes with Altered DNA Methylomes.

Plants must continuously react to the ever-fluctuating nature of their environment. Repeated exposure to stressful conditions can lead to priming, whereby prior encounters heighten a plant's ability to respond to future events. A clear example of priming is provided by the model plant Arabidopsis thaliana (Arabidopsis), in which photosynthetic and photoprotective responses are enhanced following recurring light stress. While there are various post-translational mechanisms underpinning photoprotection, an unresolved question is the relative importance of transcriptional changes toward stress priming and, consequently, the potential contribution from DNA methylation - a heritable chemical modification of DNA capable of influencing gene expression. Here, we systematically investigate the potential molecular underpinnings of physiological priming against recurring excess-light (EL), specifically DNA methylation and transcriptional regulation: the latter having not been examined with respect to EL priming. The capacity for physiological priming of photosynthetic and photoprotective parameters following a recurring EL treatment was not impaired in Arabidopsis mutants with perturbed establishment, maintenance, or removal of DNA methylation. Importantly, no differences in development or basal photoprotective capacity were identified in the mutants that may confound the above result. Little evidence for a causal transcriptional component of physiological priming was identified; in fact, most alterations in primed plants presented as a transcriptional 'dampening' in response to an additional EL exposure, likely a consequence of physiological priming. However, a set of transcripts uniquely regulated in primed plants provide preliminary evidence for a novel transcriptional component of recurring EL priming, independent of physiological changes. Thus, we propose that physiological priming of recurring EL in Arabidopsis occurs independently of DNA methylation; and that the majority of the associated transcriptional alterations are a consequence, not cause, of this physiological priming.

A Genome-Wide Association Study of Non-Photochemical Quenching in response to local seasonal climates in Arabidopsis thaliana.

Field-grown plants have variable exposure to sunlight as a result of shifting cloud-cover, seasonal changes, canopy shading, and other environmental factors. As a result, they need to have developed a method for dissipating excess energy obtained from periodic excessive sunlight exposure. Non-photochemical quenching (NPQ) dissipates excess energy as heat, however, the physical and molecular genetic mechanics of NPQ variation are not understood. In this study, we investigated the genetic loci involved in NPQ by first growing different Arabidopsis thaliana accessions in local and seasonal climate conditions, then measured their NPQ kinetics through development by chlorophyll fluorescence. We used genome-wide association studies (GWAS) to identify 15 significant quantitative trait loci (QTL) for a range of photosynthetic traits, including a QTL co-located with known NPQ gene PSBS (AT1G44575). We found there were large alternative regulatory segments between the PSBS promoter regions of the functional haplotypes and a significant difference in PsbS protein concentration. These findings parallel studies in rice showing recurrent regulatory evolution of this gene. The variation in the PSBS promoter and the changes underlying other QTLs could give insight to allow manipulations of NPQ in crops to improve their photosynthetic efficiency and yield.

Wheat drought tolerance in the field is predicted by amino acid responses to glasshouse-imposed drought.

Water limits crop productivity, so selecting for a minimal yield gap in drier environments is critical to mitigate against climate change and land-use pressure. We investigated the responses of relative water content (RWC), stomatal conductance, chlorophyll content, and metabolites in flag leaves of commercial wheat (Triticum aestivum L.) cultivars to three drought treatments in the glasshouse and in field environments. We observed strong genetic associations between glasshouse-based RWC, metabolites, and yield gap-based drought tolerance (YDT; the ratio of yield in water-limited versus well-watered conditions) across 18 field environments spanning sites and seasons. Critically, RWC response to glasshouse drought was strongly associated with both YDT (r2=0.85, P<8E-6) and RWC under field drought (r2=0.77, P<0.05). Moreover, multiple regression analyses revealed that 98% of genetic YDT variance was explained by drought responses of four metabolites: serine, asparagine, methionine, and lysine (R2=0.98; P<0.01). Fitted coefficients suggested that, for given levels of serine and asparagine, stronger methionine and lysine accumulation was associated with higher YDT. Collectively, our results demonstrate that high-throughput, targeted metabolic phenotyping of glasshouse-grown plants may be an effective tool for selection of wheat cultivars with high field-derived YDT.

Volatile apocarotenoid discovery and quantification in Arabidopsis thaliana: optimized sensitive analysis via HS-SPME-GC/MS.

INTRODUCTION: In the field of carotenoid metabolism researchers' focus has been directed recently toward the discovery and quantification of carotenoid cleavage products (i.e. apocarotenoids, excluding the well-studied carotenoid-derived hormones abscisic acid and strigolactones), due to their emerging roles as putative signaling molecules. Gas chromatography mass spectrometry (GC/MS) and sample preparation via headspace solid phase micro-extraction (HS-SPME) are widely used analytical techniques for broad untargeted metabolomics studies and until now, no optimized quantitative targeted HS-SPME-GC/MS method has been developed specifically for volatile apocarotenoids (VAs) in planta. OBJECTIVES: Optimization and subsequent validation of the HS-SPME technique for extracting and quantifying volatile apocarotenoids in planta. METHODS: Factors considered during method optimization were HS-SPME parameters; vial storage conditions; different adsorbent SPME fibre coating chemistries; plant tissue matrix effects; and fresh tissues to be analyzed. RESULTS: Mean linear regression in planta calibration correlation coefficients (R2) for VAs was 0.974. The resultant method mean limits of detection (LOD) and lower limits of quantification (LLOQ) for VAs using in planta standard additions were 0.384 ± 0.139 and 0.640 ± 0.231 µg/L, respectively. VAs remained stable at elevated SPME incubation temperatures, with no observable effects of thermal and photo-stereoisomerisation and oxidation. The bipolar 50/30 µm divinylbenzene/carboxen on polydimethylsiloxane (PDMS/DVB/CAR) was identified as the optimal fibre for broad molecular weight range VA analysis. CONCLUSIONS: An optimized HS-SPME-GC/MS method for VA detection and quantification was validated in vitro and in planta: based on biological replicates and stringent QA/QC approaches, thereby providing robust detection and quantification of VAs across a broad range of Arabidopsis tissues, fifteen of which were identified for the first time in Arabidopsis.

Predicting dark respiration rates of wheat leaves from hyperspectral reflectance.

Greater availability of leaf dark respiration (Rdark ) data could facilitate breeding efforts to raise crop yield and improve global carbon cycle modelling. However, the availability of Rdark data is limited because it is cumbersome, time consuming, or destructive to measure. We report a non-destructive and high-throughput method of estimating Rdark from leaf hyperspectral reflectance data that was derived from leaf Rdark measured by a destructive high-throughput oxygen consumption technique. We generated a large dataset of leaf Rdark for wheat (1380 samples) from 90 genotypes, multiple growth stages, and growth conditions to generate models for Rdark . Leaf Rdark (per unit leaf area, fresh mass, dry mass or nitrogen, N) varied 7- to 15-fold among individual plants, whereas traits known to scale with Rdark , leaf N, and leaf mass per area (LMA) only varied twofold to fivefold. Our models predicted leaf Rdark , N, and LMA with r2 values of 0.50-0.63, 0.91, and 0.75, respectively, and relative bias of 17-18% for Rdark and 7-12% for N and LMA. Our results suggest that hyperspectral model prediction of wheat leaf Rdark is largely independent of leaf N and LMA. Potential drivers of hyperspectral signatures of Rdark are discussed.