Operating at the extreme: estimating the upper yield boundary of winter wheat production in commercial practice.

Wheat farming provides 28.5% of global cereal production. After steady growth in average crop yield from 1950 to 1990, wheat yields have generally stagnated, which prompts the question of whether further improvements are possible. Statistical studies of agronomic parameters such as crop yield have so far exclusively focused on estimating parameters describing the whole of the data, rather than the highest yields specifically. These indicators include the mean or median yield of a crop, or finding the combinations of agronomic traits that are correlated with increasing average yields. In this paper, we take an alternative approach and consider high yields only. We carry out an extreme value analysis of winter wheat yield data collected in England and Wales between 2006 and 2015. This analysis suggests that, under current climate and growing conditions, there is indeed a finite upper bound for winter wheat yield, whose value we estimate to be 17.60 tonnes per hectare. We then refine the analysis for strata defined by either location or level of use of agricultural inputs. We find that there is no statistical evidence for variation of maximal yield depending on location, and neither is there statistical evidence that maximum yield levels are improved by high levels of crop protection and fertilizer use.

Hypoxia during incubation does not affect aerobic performance or haematology of Atlantic salmon (Salmo salar) when re-exposed in later life.

Hypoxia in aquatic ecosystems is becoming increasingly prevalent, potentially reducing fish performance and survival by limiting the oxygen available for aerobic activities. Hypoxia is a challenge for conserving and managing fish populations and demands a better understanding of the short- and long-term impacts of hypoxic environments on fish performance. Fish acclimate to hypoxia via a variety of short- and long-term physiological modifications in an attempt to maintain aerobic performance. In particular, hypoxia exposure during early development may result in enduring cardio-respiratory modifications that affect future hypoxia acclimation capacity, yet this possibility remains poorly investigated. We incubated Atlantic salmon (Salmo salar) in normoxia (~100% dissolved oxygen [DO, as percent air saturation]), moderate hypoxia (~63% DO) or cyclical hypoxia (100-25% DO daily) from fertilization until 113 days post-fertilization prior to rearing all groups in normoxia for a further 8 months. At ~11 months of age, subsets of each group were acclimated to hypoxia (50% DO) for up to 44 days prior to haematology, aerobic metabolic rate and hypoxia tolerance measurements. Hypoxia exposure during incubation (fertilization to 113 days post-fertilization) did not affect the haematology, aerobic performance or hypoxia tolerance of juvenile salmon in later life. Juveniles acclimated to hypoxia increased maximum aerobic metabolic rate and aerobic scope by ~23 and ~52%, respectively, when measured at 50% DO but not at 100% DO. Hypoxia-incubated juveniles also increased haematocrit and haemoglobin concentration but did not affect acute hypoxia tolerance (critical oxygen level and DO at LOE). Thus, while Atlantic salmon possess a considerable capacity to physiologically acclimate to hypoxia by improving aerobic performance in low oxygen conditions, we found no evidence that this capacity is influenced by early-life hypoxia exposure.

Physiological effects of dissolved oxygen are stage-specific in incubating Atlantic salmon (Salmo salar).

Oxygen availability is highly variable during salmonid incubation in natural redds and also in aquaculture incubation systems. Hypoxia generally decreases growth and aerobic metabolism prior to hatching, in parallel with eliciting physiological modifications that enhance oxygen delivery. However, it is less-well known whether developmental hyperoxia can drive the opposite effect. Moreover, there is insufficient understanding of stage-specific developmental windows during which ambient oxygen availability may be of greater or lesser impact to incubating embryos. Here, we tested the effects of hypoxia (50% dissolved oxygen: DO, % air saturation) and hyperoxia (150% DO) on the growth, routine aerobic metabolism ([Formula: see text]) and hypoxia tolerance (O2crit) of Atlantic salmon (Salmo salar) during seven developmental windows throughout incubation. Embryos exposed to hyperoxia (150% DO) did not differ from the normoxic group in growth, [Formula: see text] or O2crit at any developmental window. In contrast, embryos exposed to hypoxia grew slower and had a lower [Formula: see text], but had higher hypoxia tolerance (lower O2crit) than normoxic and hyperoxic counterparts. Interestingly, these differences were only apparent when the embryos were measured prior to hatching. Larvae (alevins) incubated in hypoxia following hatching grew similarly to normoxia-incubated alevins. Our results provide evidence that Atlantic salmon embryos are most sensitive to hypoxia prior to hatching, probably due to increasing (absolute) oxygen requirements concurrent with restricted oxygen diffusion through the egg. Moreover, the similarities between normoxia- and hyperoxia-incubated salmon demonstrate that embryos are not oxygen-limited under normoxic conditions.

Fiber length and concentration: Synergistic effect on mechanical and cellular response in wet-laid poly(lactic acid) fibrous scaffolds.

In the area of biomaterials, fibers not only offer increased mechanical response, but also serve as an extracellular matrix mimicking morphology to direct cellular attachment and proliferation. While biologically similar in morphology, soft, and flexible hydrogel materials have low mechanical properties. For applications in tissue engineering, the lack of directional cues and attachment regions within the biogels is undesired as cells require a guide for adequate attachment and organized proliferation. In this work, we have investigated the role of poly(lactic acid) (PLA) fiber length and concentration as a reinforcement phase in a gelatin hydrogel matrix and the resultant mechanical and cellular responses. With increasing fiber length and concentration, the ultimate tensile strength, modulus, and toughness increased for the samples. Similarly, for shorter fiber lengths, the loss and storage modulus increased with fiber concentration. After seeding human mesenchymal stem cells (hMSCs) onto the neat fibrous scaffolds it was found that the fabrication process imparted no cytotoxicity. Furthermore, it was the concentrations and lengths of fiber both caused discernable differences in cell viability at the extreme values. Fibers of all lengths, when in a 4.0 wt % concentration, had a decrease in cell viability after 10 days while the 12.7 mm fibers showed a similar response at 2.0 wt %, but all stayed about 90% viability. With increased incubation time, hMSCs became elongated with increased proliferation. These results indicate that the wet-lay process is a rapid and scalable method by which fibrous 3D-scaffolds can be produced to reinforce hydrogel matrices. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 332-341, 2019.

Acid-base regulation in the air-breathing swamp eel (Monopterus albus) at different temperatures.

Vertebrates reduce arterial blood pH (pHa) when body temperature increases. In water breathers, this response occurs primarily by reducing plasma HCO3- levels with small changes in the partial pressure of CO2 (PCO2 ). In contrast, air breathers mediate the decrease in pHa by increasing arterial PCO2  (PaCO2 ) at constant plasma HCO3- by reducing lung ventilation relative to metabolic CO2 production. Much less is known about bimodal breathers, which utilize both water and air. Here, we characterized the influence of temperature on arterial acid-base balance and intracellular pH (pHi) in the bimodal-breathing swamp eel, Monopterus albus This teleost uses the buccopharyngeal cavity for gas exchange and has very reduced gills. When exposed to ecologically relevant temperatures (20, 25, 30 and 35°C) for 24 and 48 h, pHa decreased by -0.025 pH units (U) °C-1 in association with an increase in PaCO2 , but without changes in plasma [HCO3-]. pHi was also reduced with increased temperature. The slope of pHi of liver and muscle was -0.014 and -0.019 U °C-1, while the heart muscle showed a smaller reduction (-0.008 U °C-1). When exposed to hypercapnia (7 or 14 mmHg) at either 25 or 35°C, M. albus elevated plasma [HCO3-] and therefore seemed to defend the new pHa set-point, demonstrating an adjusted control of acid-base balance with temperature. Overall, the effects of temperature on acid-base balance in M. albus resemble those in air-breathing amniotes, and we discuss the possibility that this pattern of acid-base balance results from a progressive transition in CO2 excretion from water to air as temperature rises.

Linear discriminant analysis reveals differences in root architecture in wheat seedlings related to nitrogen uptake efficiency.

Root architecture impacts water and nutrient uptake efficiency. Identifying exactly which root architectural properties influence these agronomic traits can prove challenging. In this paper, approximately 300 wheat (Triticum aestivum) plants were divided into four groups using two binary classifications, high versus low nitrogen uptake efficiency (NUpE), and high versus low nitrate in the growth medium. The root system architecture for each wheat plant was captured using 16 quantitative variables. The multivariate analysis tool, linear discriminant analysis, was used to construct composite variables, each a linear combination of the original variables, such that the score of the plants on the new variables showed the maximum between-group variability. The results show that the distribution of root-system architecture traits differs between low- and high-NUpE plants and, less strongly, between low-NUpE plants grown on low versus high nitrate media.

Developmental Hypoxia Has Negligible Effects on Long-Term Hypoxia Tolerance and Aerobic Metabolism of Atlantic Salmon (Salmo salar).

Exposure to developmental hypoxia can have long-term impacts on the physiological performance of fish because of irreversible plasticity. Wild and captive-reared Atlantic salmon (Salmo salar) can be exposed to hypoxic conditions during development and continue to experience fluctuating oxygen levels as juveniles and adults. Here, we examine whether developmental hypoxia impacts subsequent hypoxia tolerance and aerobic performance of Atlantic salmon. Individuals at 8°C were exposed to 50% (hypoxia) or 100% (normoxia) dissolved oxygen (DO) saturation (as percent of air saturation) from fertilization for ∼100 d (800 degree days) and then raised in normoxic conditions for a further 15 mo. At 18 mo after fertilization, aerobic scope was calculated in normoxia (100% DO) and acute (18 h) hypoxia (50% DO) from the difference between the minimum and maximum oxygen consumption rates ([Formula: see text] and [Formula: see text], respectively) at 10°C. Hypoxia tolerance was determined as the DO at which loss of equilibrium (LOE) occurred in a constantly decreasing DO environment. There was no difference in [Formula: see text], [Formula: see text], or aerobic scope between fish raised in hypoxia or normoxia. There was some evidence that hypoxia tolerance was lower (higher DO at LOE) in hypoxia-raised fish compared with those raised in normoxia, but the magnitude of the effect was small (12.52% DO vs. 11.73% DO at LOE). Acute hypoxia significantly reduced aerobic scope by reducing [Formula: see text], while [Formula: see text] remained unchanged. Interestingly, acute hypoxia uncovered individual-level relationships between DO at LOE and [Formula: see text], [Formula: see text], and aerobic scope. We discuss our findings in the context of developmental trajectories and the role of aerobic performance in hypoxia tolerance.

Brain translocator protein occupancy by ONO-2952 in healthy adults: A Phase 1 PET study using [11 C]PBR28.

ONO-2952, a novel antagonist of translocator protein 18 kDa (TSPO), binds with high affinity to TSPO in rat brain and human tumor cell line membrane preparations. This study used the TSPO-specific PET radioligand [11 C]PBR28 to confirm binding of ONO-2952 to brain TSPO in human subjects, and evaluate brain TSPO occupancy and its relationship with ONO-2952 plasma concentration. Sixteen healthy subjects received a single oral dose of 200, 60, 20, or 6 mg ONO-2952 (n = 4 per dose). Two PET scans with [11 C]PBR28 were conducted ≤7 days apart: at baseline and 24 h after ONO-2952 administration. [11 C]PBR28 regional distribution volume (VT ) was derived with kinetic modeling using the arterial input function and a two tissue compartment model. Nonspecific binding (VND ) was obtained on an individual basis for each subject using linear regression as the x-intercept of the Lassen plot. The binding potential relative to VND (BPND ) was derived as the difference between VT in the ROI (VT ROI) and VND , normalized to VND ; BPND = (VT ROI - VND )/VND . TSPO occupancy was calculated as the change in BPND (ΔBPND ) from individual's baseline scan to the on-medication scan to the baseline BPND value. TSPO occupancy by ONO-2952 was dose dependent between 20-200 mg, approaching saturation at 200 mg both in the whole brain and in 15 anatomic regions of interest (ROI). Estimated Ki values ranged from 24.1 to 72.2 nM. This open-label, single-center, single-dose study demonstrated engagement of ONO-2952 to brain TSPO. The relationship between pharmacokinetics and TSPO occupancy observed in this study support the hypothesis that ONO-2952 could potentially modulate neurosteroid production by binding to brain TSPO.

Wet-laid soy fiber reinforced hydrogel scaffold: Fabrication, mechano-morphological and cell studies.

Among materials used in biomedical applications, hydrogels have received consistent linear growth in interest over the past decade due to their large water volume and saliency to the natural extracellular matrix. These materials are often limited due to their sub-optimal mechanical properties which are typically improved via chemical or physical crosslinking. Chemical crosslinking forms strong inter-polymer bonds but typically uses reagents that are cytotoxic while physical crosslinking is more temperamental to environmental changes but can be formed without these toxic reagents. In this study, we added a fiber-reinforcement phase to a poly(vinyl alcohol) (PVA) hydrogel formed through successive freezing-thawing cycles by incorporating a non-woven microfiber mat formed by the wet-lay process. By reinforcing the hydrogel with a wet-laid fibrous mat, the ultimate tensile strength and modulus increased from 0.11 ± 0.01 MPa and 0.17 ± 0.02 kPa to 0.24 ± 0.02 MPa and 5.76 ± 1.12 kPa, respectively. An increase in toughness and elongation was also found increasing from 2.52 ± 0.37 MPa to 25.6 ± 3.84 and 51.89 ± 5.16% to 111.16 ± 9.68%, respectively. The soy fibers were also found to induce minimal cytotoxicity with endothelial cell viability showing 96.51% ± 1.91 living cells after a 48 h incubation. This approach to hydrogel-reinforcement presents a rapid, tunable method by which hydrogels can attain increased mechanical properties without sacrificing their inherent biologically favorable properties.

Assessing differences in toxicity and teratogenicity of three phthalates, Diethyl phthalate, Di-n-propyl phthalate, and Di-n-butyl phthalate, using Xenopus laevis embryos.

Phthalates, compounds used to add flexibility to plastics, are ubiquitous in the environment. In particular, the diethyl (DEP), di-n-propyl (DnPP), and di-n-butyl (DBP) phthalates were found to exert detrimental effects in both mammalian and non-mammalian studies, with toxic effects varying according to alkyl chain length. Embryos of Xenopus laevis, the African clawed frog, have been used to assess toxicity and teratogenicity of several compounds and serves as a model for assessing adverse and teratogenic effects of ortho-phthalate esters. The purpose of this study was to develop a model for comparison of developmentally toxic effects of ortho-phthalate esters using Xenopus embryos. In this study developing Xenopus laevis embryos were exposed to increasing concentrations of DEP, DnPP, and DBP using the 96-h Frog Embryo Teratogenesis Assay-Xenopus (FETAX), with 96-h lethal concentrations, effective concentrations to induce malformations, teratogenic indices, and concentrations to inhibit growth determined. DEP, DnPP, and DBP showed enhanced toxicity with increasing ester length. Developing Xenopus laevis exposed to DEP, DnPP, and DBP showed similar malformations that also occurred at lower concentrations with increasing alkyl chain length. Teratogenic risk did not change markedly with alkyl chain length, with data showing only DBP to be teratogenic.

Safety, Tolerability, and Pharmacokinetic Profile of the Novel Translocator Protein 18 kDa Antagonist ONO-2952 in Healthy Volunteers.

PURPOSE: To investigate safety, tolerability, and pharmacokinetic properties of single and multiple doses of novel translocator protein 18 kDa antagonist ONO-2952 in healthy subjects. METHODS: Double-blind, placebo-controlled single (SAD) and multiple (MAD) dose escalation studies were conducted. Healthy men and women aged 18 to 55 years inclusive and without history of psychiatric disorders were eligible. Forty-eight volunteers received single doses of ONO-2952 (3, 10, 30, 100, 200, or 400 mg) or placebo under fasted conditions (SAD study), and 36 received ONO-2952 (30, 60, or 100 mg/d) or placebo for 21 consecutive days under fed conditions (MAD study). ONO-2952 10 and 200 mg were administered under fasted and fed conditions in the SAD study to investigate the effect of food on the absorption of ONO-2952. Safety assessments included adverse events, vital signs, 12-lead ECGs, and clinical laboratory evaluations. Plasma and urine pharmacokinetic profiles of ONO-2952 were determined. FINDINGS: Across both studies, mean age ranged from 29.8 to 39.8 years, most participants were white, and the proportion of female volunteers was 52%. No treatment or dose-related trends in adverse events were observed. The most frequent adverse events were headache and presyncope (n = 2 each [SAD study]) and constipation and headache (n = 3 each [MAD study]). All headache and constipation episodes were possibly related to the study drug. Plasma ONO-2952 concentrations peaked 2.5 to 3.5 hours (SAD study) and 3.0 to 4.0 hours (MAD study) postdose. ONO-2952 systemic exposure increased less than dose proportionally under fasted conditions. Fed conditions significantly increased exposure compared with fasted conditions: geometric mean ratios of Cmax (90% CIs) were 229% (176-299 [10 mg]) and 778% (623-971 [200 mg]), and AUClast were 159% (131-192 [10 mg]) and 382% (288-506 [200 mg]). In the MAD study, the systemic exposure of ONO-2952 increased in a slightly greater than dose-proportional manner. Geometric mean accumulation ratios (95% CI) of AUC24 were 2.50 (2.09-2.98 [30 mg]), 2.23 (1.85-2.68 [60 mg]), and 2.73 (2.10-3.55 [100 mg]); and Cmax were 1.65 (1.43-1.90 [30 mg]), 1.56 (1.31-1.85 [60 mg]), and 1.85 (1.38-2.49 [100 mg]). IMPLICATIONS: ONO-2952 was safe and well tolerated in these early clinical studies investigating safety, tolerability, and pharmacokinetic properties of single and multiple doses. ONO-2952 systemic exposure increased in a less than dose-proportional manner under fasted conditions and in a slightly greater than dose-proportional manner under fed conditions. These results support the progression of ONO-2952 to further studies in humans. SAD study: ClinicalTials.gov identifier: NCT01364441. MAD study: ClinicalTrials.gov identifier: NCT01489345.

Digital Single-Cell Analysis of Plant Organ Development Using 3DCellAtlas.

Diverse molecular networks underlying plant growth and development are rapidly being uncovered. Integrating these data into the spatial and temporal context of dynamic organ growth remains a technical challenge. We developed 3DCellAtlas, an integrative computational pipeline that semiautomatically identifies cell types and quantifies both 3D cellular anisotropy and reporter abundance at single-cell resolution across whole plant organs. Cell identification is no less than 97.8% accurate and does not require transgenic lineage markers or reference atlases. Cell positions within organs are defined using an internal indexing system generating cellular level organ atlases where data from multiple samples can be integrated. Using this approach, we quantified the organ-wide cell-type-specific 3D cellular anisotropy driving Arabidopsis thaliana hypocotyl elongation. The impact ethylene has on hypocotyl 3D cell anisotropy identified the preferential growth of endodermis in response to this hormone. The spatiotemporal dynamics of the endogenous DELLA protein RGA, expansin gene EXPA3, and cell expansion was quantified within distinct cell types of Arabidopsis roots. A significant regulatory relationship between RGA, EXPA3, and growth was present in the epidermis and endodermis. The use of single-cell analyses of plant development enables the dynamics of diverse regulatory networks to be integrated with 3D organ growth.

Promotion of testa rupture during garden cress germination involves seed compartment-specific expression and activity of pectin methylesterases.

Pectin methylesterase (PME) controls the methylesterification status of pectins and thereby determines the biophysical properties of plant cell walls, which are important for tissue growth and weakening processes. We demonstrate here that tissue-specific and spatiotemporal alterations in cell wall pectin methylesterification occur during the germination of garden cress (Lepidium sativum). These cell wall changes are associated with characteristic expression patterns of PME genes and resultant enzyme activities in the key seed compartments CAP (micropylar endosperm) and RAD (radicle plus lower hypocotyl). Transcriptome and quantitative real-time reverse transcription-polymerase chain reaction analysis as well as PME enzyme activity measurements of separated seed compartments, including CAP and RAD, revealed distinct phases during germination. These were associated with hormonal and compartment-specific regulation of PME group 1, PME group 2, and PME inhibitor transcript expression and total PME activity. The regulatory patterns indicated a role for PME activity in testa rupture (TR). Consistent with a role for cell wall pectin methylesterification in TR, treatment of seeds with PME resulted in enhanced testa permeability and promoted TR. Mathematical modeling of transcript expression changes in germinating garden cress and Arabidopsis (Arabidopsis thaliana) seeds suggested that group 2 PMEs make a major contribution to the overall PME activity rather than acting as PME inhibitors. It is concluded that regulated changes in the degree of pectin methylesterification through CAP- and RAD-specific PME and PME inhibitor expression play a crucial role during Brassicaceae seed germination.

Transcriptional dynamics of two seed compartments with opposing roles in Arabidopsis seed germination.

Seed germination is a critical stage in the plant life cycle and the first step toward successful plant establishment. Therefore, understanding germination is of important ecological and agronomical relevance. Previous research revealed that different seed compartments (testa, endosperm, and embryo) control germination, but little is known about the underlying spatial and temporal transcriptome changes that lead to seed germination. We analyzed genome-wide expression in germinating Arabidopsis (Arabidopsis thaliana) seeds with both temporal and spatial detail and provide Web-accessible visualizations of the data reported (vseed.nottingham.ac.uk). We show the potential of this high-resolution data set for the construction of meaningful coexpression networks, which provide insight into the genetic control of germination. The data set reveals two transcriptional phases during germination that are separated by testa rupture. The first phase is marked by large transcriptome changes as the seed switches from a dry, quiescent state to a hydrated and active state. At the end of this first transcriptional phase, the number of differentially expressed genes between consecutive time points drops. This increases again at testa rupture, the start of the second transcriptional phase. Transcriptome data indicate a role for mechano-induced signaling at this stage and subsequently highlight the fates of the endosperm and radicle: senescence and growth, respectively. Finally, using a phylotranscriptomic approach, we show that expression levels of evolutionarily young genes drop during the first transcriptional phase and increase during the second phase. Evolutionarily old genes show an opposite pattern, suggesting a more conserved transcriptome prior to the completion of germination.

Transcriptional bursting diversifies the behaviour of a toggle switch: hybrid simulation of stochastic gene expression.

Hybrid models for gene expression combine stochastic and deterministic representations of the underlying biophysical mechanisms. According to one of the simplest hybrid formalisms, protein molecules are produced in randomly occurring bursts of a randomly distributed size while they are degraded deterministically. Here, we use this particular formalism to study two key regulatory motifs-the autoregulation loop and the toggle switch. The distribution of burst times is determined and used as a basis for the development of exact simulation algorithms for gene expression dynamics. For the autoregulation loop, the simulations are compared to an analytic solution of a master equation. Simulations of the toggle switch reveal a number of qualitatively distinct scenarios with implications for the modelling of cell-fate selection.

Exact and approximate distributions of protein and mRNA levels in the low-copy regime of gene expression.

Gene expression at the single-cell level incorporates reaction mechanisms which are intrinsically stochastic as they involve molecular species present at low copy numbers. The dynamics of these mechanisms can be described quantitatively using stochastic master-equation modelling; in this paper we study a generic gene-expression model of this kind which explicitly includes the representations of the processes of transcription and translation. For this model we determine the generating function of the steady-state distribution of mRNA and protein counts and characterise the underlying probability law using a combination of analytic, asymptotic and numerical approaches, finding that the distribution may assume a number of qualitatively distinct forms. The results of the analysis are suitable for comparison with single-molecule resolution gene-expression data emerging from recent experimental studies.

Multiscale stochastic modelling of gene expression.

Stochastic phenomena in gene regulatory networks can be modelled by the chemical master equation for gene products such as mRNA and proteins. If some of these elements are present in significantly higher amounts than the rest, or if some of the reactions between these elements are substantially faster than others, it is often possible to reduce the master equation to a simpler problem using asymptotic methods. We present examples of such a procedure and analyse the relationship between the reduced models and the original.

A guideline to family-wide comparative state-of-the-art quantitative RT-PCR analysis exemplified with a Brassicaceae cross-species seed germination case study.

Comparative biology includes the comparison of transcriptome and quantitative real-time RT-PCR (qRT-PCR) data sets in a range of species to detect evolutionarily conserved and divergent processes. Transcript abundance analysis of target genes by qRT-PCR requires a highly accurate and robust workflow. This includes reference genes with high expression stability (i.e., low intersample transcript abundance variation) for correct target gene normalization. Cross-species qRT-PCR for proper comparative transcript quantification requires reference genes suitable for different species. We addressed this issue using tissue-specific transcriptome data sets of germinating Lepidium sativum seeds to identify new candidate reference genes. We investigated their expression stability in germinating seeds of L. sativum and Arabidopsis thaliana by qRT-PCR, combined with in silico analysis of Arabidopsis and Brassica napus microarray data sets. This revealed that reference gene expression stability is higher for a given developmental process between distinct species than for distinct developmental processes within a given single species. The identified superior cross-species reference genes may be used for family-wide comparative qRT-PCR analysis of Brassicaceae seed germination. Furthermore, using germinating seeds, we exemplify optimization of the qRT-PCR workflow for challenging tissues regarding RNA quality, transcript stability, and tissue abundance. Our work therefore can serve as a guideline for moving beyond Arabidopsis by establishing high-quality cross-species qRT-PCR.