Genetic dissection of root architecture in Ethiopian sorghum landraces.

KEY MESSAGE: This study quantified genetic variation in root system architecture (root number, angle, length and dry mass) within a diversity panel of 1771 Ethiopian sorghum landraces and identified 22 genomic regions associated with the root variations. The root system architecture (RSA) of crop plants influences adaptation to water-limited conditions and determines the capacity of a plant to access soil water and nutrients. Four key root traits (number, angle, length and dry mass) were evaluated in a diversity panel of 1771 Ethiopian sorghum landraces using purpose-built root chambers. Significant genetic variation was observed in all studied root traits, with nodal root angle ranging from 16.4° to 26.6°, with a high repeatability of 78.9%. Genome wide association studies identified a total of 22 genomic regions associated with root traits which were distributed on all chromosomes except chromosome SBI-10. Among the 22 root genomic regions, 15 co-located with RSA trait QTL previously identified in sorghum, with the remaining seven representing novel RSA QTL. The majority (85.7%) of identified root angle QTL also co-localized with QTL previously identified for stay-green in sorghum. This suggests that the stay-green phenotype might be associated with root architecture that enhances water extraction during water stress conditions. The results open avenues for manipulating root phenotypes to improve productivity in abiotic stress environments via marker-assisted selection.

Coordination of stomata and vein patterns with leaf width underpins water-use efficiency in a C4 crop.

Despite its importance for crop water use and productivity, especially in drought-affected environments, the underlying mechanisms of variation in intrinsic water-use efficiency (iWUE = net photosynthesis/stomatal conductance for water vapour, gsw ) are not well understood, especially in C4 plants. Recently, we discovered that leaf width (LW) correlated negatively with iWUE and positively with gsw across several C4 grasses. Here, we confirmed these relationships within 48 field-grown genotypes differing in LW in Sorghum bicolor, a C4 crop adapted to dry and hot conditions. We measured leaf gas exchange and modelled leaf energy balance three times a day, alongside anatomical traits as potential predictors of iWUE. LW correlated negatively with iWUE and stomatal density, but positively with gsw , interveinal distance of longitudinal veins, and the percentage of stomatal aperture relative to maximum. Energy balance modelling showed that wider leaves needed to open their stomata more to generate a more negative leaf-to-air temperature difference, especially at midday when air temperatures exceeded 40°C. These results highlight the important role that LW plays in shaping iWUE through coordination of vein and stomatal traits and by affecting stomatal aperture. Therefore, LW could be used as a predictor of higher iWUE among sorghum genotypes.

Genetic control of leaf angle in sorghum and its effect on light interception.

Developing sorghum genotypes adapted to different light environments requires understanding of a plant's ability to capture light, determined through leaf angle specifically. This study dissected the genetic basis of leaf angle in 3 year field trials at two sites, using a sorghum diversity panel (729 accessions). A wide range of variation in leaf angle with medium heritability was observed. Leaf angle explained 36% variation in canopy light extinction coefficient, highlighting the extent to which variation in leaf angle influences light interception at the whole-canopy level. This study also found that the sorghum races of Guinea and Durra consistently having the largest and smallest leaf angle, respectively, highlighting the potential role of leaf angle in adaptation to distinct environments. The genome-wide association study detected 33 quantitative trait loci (QTLs) associated with leaf angle. Strong synteny was observed with previously detected leaf angle QTLs in maize (70%) and rice (40%) within 10 cM, among which the overlap was significantly enriched according to χ2 tests, suggesting a highly consistent genetic control in grasses. A priori leaf angle candidate genes identified in maize and rice were found to be enriched within a 1-cM window around the sorghum leaf angle QTLs. Additionally, protein domain analysis identified the WD40 protein domain as being enriched within a 1-cM window around the QTLs. These outcomes show that there is sufficient heritability and natural variation in the angle of upper leaves in sorghum which may be exploited to change light interception and optimize crop canopies for different contexts.

Genetic modification of PIN genes induces causal mechanisms of stay-green drought adaptation phenotype.

The stay-green trait is recognized as a key drought adaptation mechanism in cereals worldwide. Stay-green sorghum plants exhibit delayed senescence of leaves and stems, leading to prolonged growth, a reduced risk of lodging, and higher grain yield under end-of-season drought stress. More than 45 quantitative trait loci (QTL) associated with stay-green have been identified, including two major QTL (Stg1 and Stg2). However, the contributing genes that regulate functional stay-green are not known. Here we show that the PIN FORMED family of auxin efflux carrier genes induce some of the causal mechanisms driving the stay-green phenotype in sorghum, with SbPIN4 and SbPIN2 located in Stg1 and Stg2, respectively. We found that nine of 11 sorghum PIN genes aligned with known stay-green QTL. In transgenic studies, we demonstrated that PIN genes located within the Stg1 (SbPIN4), Stg2 (SbPIN2), and Stg3b (SbPIN1) QTL regions acted pleiotropically to modulate canopy development, root architecture, and panicle growth in sorghum, with SbPIN1, SbPIN2, and SbPIN4 differentially expressed in various organs relative to the non-stay-green control. The emergent consequence of such modifications in canopy and root architecture is a stay-green phenotype. Crop simulation modelling shows that the SbPIN2 phenotype can increase grain yield under drought.

Genetic basis of sorghum leaf width and its potential as a surrogate for transpiration efficiency.

KEY MESSAGE: Leaf width was correlated with plant-level transpiration efficiency and associated with 19 QTL in sorghum, suggesting it could be a surrogate for transpiration efficiency in large breeding program. Enhancing plant transpiration efficiency (TE) by reducing transpiration without compromising photosynthesis and yield is a desirable selection target in crop improvement programs. While narrow individual leaf width has been correlated with greater intrinsic water use efficiency in C4 species, the extent to which this translates to greater plant TE has not been investigated. The aims of this study were to evaluate the correlation of leaf width with TE at the whole-plant scale and investigate the genetic control of leaf width in sorghum. Two lysimetry experiments using 16 genotypes varying for stomatal conductance and three field trials using a large sorghum diversity panel (n = 701 lines) were conducted. Negative associations of leaf width with plant TE were found in the lysimetry experiments, suggesting narrow leaves may result in reduced plant transpiration without trade-offs in biomass accumulation. A wide range in width of the largest leaf was found in the sorghum diversity panel with consistent ranking among sorghum races, suggesting that environmental adaptation may have a role in modifying leaf width. Nineteen QTL were identified by genome-wide association studies on leaf width adjusted for flowering time. The QTL identified showed high levels of correspondence with those in maize and rice, suggesting similarities in the genetic control of leaf width across cereals. Three a priori candidate genes for leaf width, previously found to regulate dorsoventrality, were identified based on a 1-cM threshold. This study provides useful physiological and genetic insights for potential manipulation of leaf width to improve plant adaptation to diverse environments.

Hotter, drier, CRISPR: the latest edit on climate change.

KEY MESSAGE: Integrating CRISPR/Cas9 genome editing into modern breeding programs for crop improvement in cereals. Global climate trends in many agricultural regions have been rapidly changing over the past decades, and major advances in global food systems are required to ensure food security in the face of these emerging challenges. With increasing climate instability due to warmer temperatures and rising CO2 levels, the productivity of global agriculture will continue to be negatively impacted. To combat these growing concerns, creative approaches will be required, utilising all the tools available to produce more robust and tolerant crops with increased quality and yields under more extreme conditions. The integration of genome editing and transgenics into current breeding strategies is one promising solution to accelerate genetic gains through targeted genetic modifications, producing crops that can overcome the shifting climate realities. This review focuses on how revolutionary genome editing tools can be directly implemented into breeding programs for cereal crop improvement to rapidly counteract many of the issues affecting agriculture production in the years to come.

Neuregulin-Dependent Regulation of Fast-Spiking Interneuron Excitability Controls the Timing of the Critical Period.

Maturation of excitatory drive onto fast-spiking interneurons (FS INs) in the visual cortex has been implicated in the control of the timing of the critical period for ocular dominance plasticity. However, the mechanisms that regulate the strength of these synapses over cortical development are not understood. Here we use a mouse model to show that neuregulin (NRG) and the receptor tyrosine kinase erbB4 regulate the timing of the critical period. NRG1 enhanced the strength of excitatory synapses onto FS INs, which inhibited ocular dominance plasticity during the critical period but rescued plasticity in transgenics with hypoexcitable FS INs. Blocking the effects of endogenous neuregulin via inhibition of erbBs rescued ocular dominance plasticity in postcritical period adults, allowing recovery from amblyopia induced by chronic monocular deprivation. Thus, the strength of excitation onto FS INs is a key determinant of critical period plasticity and is maintained at high levels by NRG-erbB4 signaling to constrain plasticity in adulthood. SIGNIFICANCE STATEMENT: Despite decades of experimentation, the mechanisms by which critical periods of enhanced synaptic plasticity are initiated and terminated are not completely understood. Here we show that neuregulin (NRG) and the receptor tyrosine kinase erbB4 determine critical period timing by controlling the strength of excitatory synapses onto FS INs. NRG1 enhanced excitatory drive onto fast spiking interneurons, which inhibited ocular dominance plasticity in juveniles but rescued plasticity in transgenics with hypoexcitable FS INs. Blocking the effects of endogenous neuregulin via inhibition of erbBs rescued ocular dominance plasticity in adults, allowing recovery from amblyopia induced by chronic monocular deprivation. Thus, in contrast to prevailing views of the termination of the critical period, active maintenance of strong excitation onto FS INs constrains plasticity in adults.

Widespread sequence variations in VAMP1 across vertebrates suggest a potential selective pressure from botulinum neurotoxins.

Botulinum neurotoxins (BoNT/A-G), the most potent toxins known, act by cleaving three SNARE proteins required for synaptic vesicle exocytosis. Previous studies on BoNTs have generally utilized the major SNARE homologues expressed in brain (VAMP2, syntaxin 1, and SNAP-25). However, BoNTs target peripheral motor neurons and cause death by paralyzing respiratory muscles such as the diaphragm. Here we report that VAMP1, but not VAMP2, is the SNARE homologue predominantly expressed in adult rodent diaphragm motor nerve terminals and in differentiated human motor neurons. In contrast to the highly conserved VAMP2, BoNT-resistant variations in VAMP1 are widespread across vertebrates. In particular, we identified a polymorphism at position 48 of VAMP1 in rats, which renders VAMP1 either resistant (I48) or sensitive (M48) to BoNT/D. Taking advantage of this finding, we showed that rat diaphragms with I48 in VAMP1 are insensitive to BoNT/D compared to rat diaphragms with M48 in VAMP1. This unique intra-species comparison establishes VAMP1 as a physiological toxin target in diaphragm motor nerve terminals, and demonstrates that the resistance of VAMP1 to BoNTs can underlie the insensitivity of a species to members of BoNTs. Consistently, human VAMP1 contains I48, which may explain why humans are insensitive to BoNT/D. Finally, we report that residue 48 of VAMP1 varies frequently between M and I across seventeen closely related primate species, suggesting a potential selective pressure from members of BoNTs for resistance in vertebrates.

Stay-green traits to improve wheat adaptation in well-watered and water-limited environments.

A stay-green phenotype enables crops to retain green leaves longer after anthesis compared with senescent types, potentially improving yield. Measuring the normalized difference vegetative index (NDVI) during the whole senescence period allows quantification of component stay-green traits contributing to a stay-green phenotype. These objective and standardized traits can be compared across genotypes and environments. Traits examined include maximum NDVI near anthesis (Nmax), senescence rate (SR), a trait integrating senescence (SGint), plus time from anthesis to onset (OnS), mid-point (MidS), and near completion (EndS) of senescence. The correlation between stay-green traits and yield was studied in eight contrasting environments ranging from well watered to severely water limited. Environments were each classified into one of the four major drought environment types (ETs) previously identified for the Australian wheat cropping system. SGint, OnS, and MidS tended to have higher values in higher yielding environments for a given genotype, as well as for higher yielding genotypes within a given environment. Correlation between specific stay-green traits and yield varied with ET. In the studied population, SGint, OnS, and MidS strongly correlated with yield in three of the four ETs which included well-watered environments (0.43-0.86), but less so in environments with only moderate water-stress after anthesis (-0.03 to 0.31). In contrast, Nmax was most highly correlated with yield under moderate post-anthesis water stress (0.31-0.43). Selection for particular stay-green traits, combinations of traits, and/or molecular markers associated with the traits could enhance genetic progress toward stay-green wheats with higher, more stable yield in both well-watered and water-limited conditions.

Stay-green alleles individually enhance grain yield in sorghum under drought by modifying canopy development and water uptake patterns.

Stay-green is an integrated drought adaptation trait characterized by a distinct green leaf phenotype during grain filling under terminal drought. We used sorghum (Sorghum bicolor), a repository of drought adaptation mechanisms, to elucidate the physiological and genetic mechanisms underpinning stay-green. Near-isogenic sorghum lines (cv RTx7000) were characterized in a series of field and managed-environment trials (seven experiments and 14 environments) to determine the influence of four individual stay-green (Stg1-4) quantitative trait loci (QTLs) on canopy development, water use and grain yield under post-anthesis drought. The Stg QTL decreased tillering and the size of upper leaves, which reduced canopy size at anthesis. This reduction in transpirational leaf area conserved soil water before anthesis for use during grain filling. Increased water uptake during grain filling of Stg near-isogenic lines (NILs) relative to RTx7000 resulted in higher post-anthesis biomass production, grain number and yield. Importantly, there was no consistent yield penalty associated with the Stg QTL in the irrigated control. These results establish a link between the role of the Stg QTL in modifying canopy development and the subsequent impact on crop water use patterns and grain yield under terminal drought.

Drought adaptation of stay-green sorghum is associated with canopy development, leaf anatomy, root growth, and water uptake.

Stay-green sorghum plants exhibit greener leaves and stems during the grain-filling period under water-limited conditions compared with their senescent counterparts, resulting in increased grain yield, grain mass, and lodging resistance. Stay-green has been mapped to a number of key chromosomal regions, including Stg1, Stg2, Stg3, and Stg4, but the functions of these individual quantitative trait loci (QTLs) remain unclear. The objective of this study was to show how positive effects of Stg QTLs on grain yield under drought can be explained as emergent consequences of their effects on temporal and spatial water-use patterns that result from changes in leaf-area dynamics. A set of four Stg near-isogenic lines (NILs) and their recurrent parent were grown in a range of field and semicontrolled experiments in southeast Queensland, Australia. These studies showed that the four Stg QTLs regulate canopy size by: (1) reducing tillering via increased size of lower leaves, (2) constraining the size of the upper leaves; and (3) in some cases, decreasing the number of leaves per culm. In addition, they variously affect leaf anatomy and root growth. The multiple pathways by which Stg QTLs modulate canopy development can result in considerable developmental plasticity. The reduction in canopy size associated with Stg QTLs reduced pre-flowering water demand, thereby increasing water availability during grain filling and, ultimately, grain yield. The generic physiological mechanisms underlying the stay-green trait suggest that similar Stg QTLs could enhance post-anthesis drought adaptation in other major cereals such as maize, wheat, and rice.

QTL for root angle and number in a population developed from bread wheats (Triticum aestivum) with contrasting adaptation to water-limited environments.

Root architecture traits in wheat are important in deep soil moisture acquisition and may be used to improve adaptation to water-limited environments. The genetic architecture of two root traits, seminal root angle and seminal root number, were investigated using a doubled haploid population derived from SeriM82 and Hartog. Multiple novel quantitative trait loci (QTL) were identified, each one having a modest effect. For seminal root angle, four QTL (-log10(P) >3) were identified on 2A, 3D, 6A and 6B, and two suggestive QTL (-log10(P) >2) on 5D and 6B. For root number, two QTL were identified on 4A and 6A with four suggestive QTL on 1B, 3A, 3B and 4A. QTL for root angle and root number did not co-locate. Transgressive segregation was found for both traits. Known major height and phenology loci appear to have little effect on root angle and number. Presence or absence of the T1BL.1RS translocation did not significantly influence root angle. Broad sense heritability (h (2)) was estimated as 50 % for root angle and 31 % for root number. Root angle QTL were found to be segregating between wheat cultivars adapted to the target production region indicating potential to select for root angle in breeding programs.

Scorpion toxins for the reversal of BoNT-induced paralysis.

The botulinum neurotoxins, characterized by their neuromuscular paralytic effects, are the most toxic proteins known to man. Due to their extreme potency, ease of production, and duration of activity, the BoNT proteins have been classified by the Centers for Disease Control as high threat agents for bioterrorism. In an attempt to discover effective BoNT therapeutics, we have pursued a strategy in which we leverage the blockade of K(+) channels that ultimately results in the reversal of neuromuscular paralysis. Towards this end, we utilized peptides derived from scorpion venom that are highly potent K(+) channel blockers. Herein, we report the synthesis of charybdotoxin, a 37 amino acid peptide, and detail its activity, along with iberiotoxin and margatoxin, in a mouse phrenic nerve hemidiaphragm assay in the absence and the presence of BoNT/A.

Integrating stay-green and PIN-FORMED genes: PIN-FORMED genes as potential targets for designing climate-resilient cereal ideotypes.

Plant architecture modification (e.g. short-stature crops) is one of the key outcomes of modern crop breeding for high-yielding crop varieties. In cereals, delayed senescence, or stay-green, is an important trait that enables post-anthesis drought stress adaptation. Stay-green crops can prolong photosynthetic capacity during grain-filling period under post-anthesis drought stress, which is essential to ensure grain yield is not impacted under drought stress conditions. Although various stay-green quantitative trait loci have been identified in cereals, the underlying molecular mechanisms regulating stay-green remain elusive. Recent advances in various gene-editing technologies have provided avenues to fast-track crop improvement, such as the breeding of climate-resilient crops in the face of climate change. We present in this viewpoint the focus on using sorghum as the model cereal crop, to study PIN-FORMED (PIN) auxin efflux carriers as means to modulate plant architecture, and the potential to employ it as an adaptive strategy to address the environmental challenges posed by climate uncertainties.

Estimating Photosynthetic Attributes from High-Throughput Canopy Hyperspectral Sensing in Sorghum.

Sorghum, a genetically diverse C4 cereal, is an ideal model to study natural variation in photosynthetic capacity. Specific leaf nitrogen (SLN) and leaf mass per leaf area (LMA), as well as, maximal rates of Rubisco carboxylation (V cmax), phosphoenolpyruvate (PEP) carboxylation (V pmax), and electron transport (J max), quantified using a C4 photosynthesis model, were evaluated in two field-grown training sets (n = 169 plots including 124 genotypes) in 2019 and 2020. Partial least square regression (PLSR) was used to predict V cmax (R 2 = 0.83), V pmax (R 2 = 0.93), J max (R 2 = 0.76), SLN (R 2 = 0.82), and LMA (R 2 = 0.68) from tractor-based hyperspectral sensing. Further assessments of the capability of the PLSR models for V cmax, V pmax, J max, SLN, and LMA were conducted by extrapolating these models to two trials of genome-wide association studies adjacent to the training sets in 2019 (n = 875 plots including 650 genotypes) and 2020 (n = 912 plots with 634 genotypes). The predicted traits showed medium to high heritability and genome-wide association studies using the predicted values identified four QTL for V cmax and two QTL for J max. Candidate genes within 200 kb of the V cmax QTL were involved in nitrogen storage, which is closely associated with Rubisco, while not directly associated with Rubisco activity per se. J max QTL was enriched for candidate genes involved in electron transport. These outcomes suggest the methods here are of great promise to effectively screen large germplasm collections for enhanced photosynthetic capacity.

Selection in Early Generations to Shift Allele Frequency for Seminal Root Angle in Wheat.

A current challenge for plant breeders is the limited ability to phenotype and select for root characteristics to enhance crop productivity. The development of a high-throughput phenotyping method has recently offered new opportunities for the selection of root characteristics in breeding programs. Here, we investigated prospects for phenotypic and molecular selection for seminal root angle (SRA), a key trait associated with mature root system architecture in wheat ( L.). We first investigated genetic diversity for this trait in a panel of 22 wheat lines adapted to Australian environments. The angle between the first pair of seminal roots ranged from 72 to 106°. We then evaluated selection gain via direct phenotypic selection in early generations by comparing the resulting shift in population distribution in tail populations selected for "narrow" and "wide" root angle. Overall, two rounds of selection significantly shifted the mean root angle as much as 10°. Furthermore, comparison of allele frequencies in the tail populations revealed genomic regions under selection, for which marker-assisted selection appeared to be successful. By combining efficient phenotyping and rapid generation advance, lines enriched with alleles for either narrow or wide SRA were developed within only 18 mo. These results suggest that there is a valuable source of allelic variation for SRA that can be harnessed and rapidly introgressed into elite wheat lines.

Two distinct mechanisms for experience-dependent homeostasis.

Models of firing rate homeostasis such as synaptic scaling and the sliding synaptic plasticity modification threshold predict that decreasing neuronal activity (for example, by sensory deprivation) will enhance synaptic function. Manipulations of cortical activity during two forms of visual deprivation, dark exposure (DE) and binocular lid suture, revealed that, contrary to expectations, spontaneous firing in conjunction with loss of visual input is necessary to lower the threshold for Hebbian plasticity and increase miniature excitatory postsynaptic current (mEPSC) amplitude. Blocking activation of GluN2B receptors, which are upregulated by DE, also prevented the increase in mEPSC amplitude, suggesting that DE potentiates mEPSCs primarily through a Hebbian mechanism, not through synaptic scaling. Nevertheless, NMDA-receptor-independent changes in mEPSC amplitude consistent with synaptic scaling could be induced by extreme reductions of activity. Therefore, two distinct mechanisms operate within different ranges of neuronal activity to homeostatically regulate synaptic strength.

Genomic Regions Influencing Seminal Root Traits in Barley.

Water availability is a major limiting factor for crop production, making drought adaptation and its many component traits a desirable attribute of plant cultivars. Previous studies in cereal crops indicate that root traits expressed at early plant developmental stages, such as seminal root angle and root number, are associated with water extraction at different depths. Here, we conducted the first study to map seminal root traits in barley ( L.). Using a recently developed high-throughput phenotyping method, a panel of 30 barley genotypes and a doubled-haploid (DH) population (ND24260 × 'Flagship') comprising 330 lines genotyped with diversity array technology (DArT) markers were evaluated for seminal root angle (deviation from vertical) and root number under controlled environmental conditions. A high degree of phenotypic variation was observed in the panel of 30 genotypes: 13.5 to 82.2 and 3.6 to 6.9° for root angle and root number, respectively. A similar range was observed in the DH population: 16.4 to 70.5 and 3.6 to 6.5° for root angle and number, respectively. Seven quantitative trait loci (QTL) for seminal root traits (root angle, two QTL; root number, five QTL) were detected in the DH population. A major QTL influencing both root angle and root number (/) was positioned on chromosome 5HL. Across-species analysis identified 10 common genes underlying root trait QTL in barley, wheat ( L.), and sorghum [ (L.) Moench]. Here, we provide insight into seminal root phenotypes and provide a first look at the genetics controlling these traits in barley.

Phenotyping novel stay-green traits to capture genetic variation in senescence dynamics.

Stay-green plants retain green leaves longer after anthesis and can have improved yield, particularly under water limitation. As senescence is a dynamic process, genotypes with different senescence patterns may exhibit similar final normalised difference vegetative index (NDVI). By monitoring NDVI from as early as awn emergence to maturity, we demonstrate that analysing senescence dynamics improves insight into genotypic stay-green variation. A senescence evaluation tool was developed to fit a logistic function to NDVI data and used to analyse data from three environments for a wheat (Triticum aestivum L.) population whose lines contrast for stay-green. Key stay-green traits were estimated including, maximum NDVI, senescence rate and a trait integrating NDVI variation after anthesis, as well as the timing from anthesis to onset, midpoint and conclusion of senescence. The integrative trait and the timing to onset and mid-senescence exhibited high positive correlations with yield and a high heritability in the three studied environments. Senescence rate was correlated with yield in some environments, whereas maximum NDVI was associated with yield in a drought-stressed environment. Where resources preclude frequent measurements, we found that NDVI measurements may be restricted to the period of rapid senescence, but caution is required when dealing with lines of different phenology. In contrast, regular monitoring during the whole period after flowering allows the estimation of senescence dynamics traits that may be reliably compared across genotypes and environments. We anticipate that selection for stay-green traits will enhance genetic progress towards high-yielding, stay-green germplasm.