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.

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.

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.

Detecting Sorghum Plant and Head Features from Multispectral UAV Imagery.

In plant breeding, unmanned aerial vehicles (UAVs) carrying multispectral cameras have demonstrated increasing utility for high-throughput phenotyping (HTP) to aid the interpretation of genotype and environment effects on morphological, biochemical, and physiological traits. A key constraint remains the reduced resolution and quality extracted from "stitched" mosaics generated from UAV missions across large areas. This can be addressed by generating high-quality reflectance data from a single nadir image per plot. In this study, a pipeline was developed to derive reflectance data from raw multispectral UAV images that preserve the original high spatial and spectral resolutions and to use these for phenotyping applications. Sequential steps involved (i) imagery calibration, (ii) spectral band alignment, (iii) backward calculation, (iv) plot segmentation, and (v) application. Each step was designed and optimised to estimate the number of plants and count sorghum heads within each breeding plot. Using a derived nadir image of each plot, the coefficients of determination were 0.90 and 0.86 for estimates of the number of sorghum plants and heads, respectively. Furthermore, the reflectance information acquired from the different spectral bands showed appreciably high discriminative ability for sorghum head colours (i.e., red and white). Deployment of this pipeline allowed accurate segmentation of crop organs at the canopy level across many diverse field plots with minimal training needed from machine learning approaches.

Tall 3-dwarfs: oxymoron or opportunity to increase grain yield in sorghum?

MAIN CONCLUSION: Plant height was positively correlated with grain yield across a large set of 3-dwarf sorghum hybrids and production environments in north-eastern Australia. In industrialised countries, plant breeders tend to select for short plant stature in cereals like wheat, barley and rice, but also grain sorghum. This is mainly to prevent stalk lodging and to allow for machine harvesting. However, this counteracts an intrinsic positive relationship between plant height and yield potential often observed in cereals. We used data from multi-environment breeding trials comprising large sets of female sorghum lines from a range of pedigrees in hybrid combination with five different male testers. The hybrids were grown in 22 different rainfed environments in north-eastern Australia, which allowed us to thoroughly examine the relationship between plant height and yield across a range of productivity levels. Covariate analysis showed that in 38 out of the 90 tested relationships, grain yield was significantly (p < 0.05) positively and in only one case significantly negatively associated with plant height. This strong positive association between the traits was supported by the observation that 87% of the effects were either positive or zero. The effects of height on yield ranged from a decrease of 0.015 t ha-1 to an increase of 0.057 t ha-1 cm-1. The majority of the negative effects were observed in low-yielding trials and the positive effect of height tended to increase with increasing mean trial yield. Opportunities to increase yield potential by selecting for slightly taller sorghum hybrids therefore need to be explored in context with the target environments and in combination with other means to control the risks of lodging.

Genetic Diversity of C4 Photosynthesis Pathway Genes in Sorghum bicolor (L.).

C4 photosynthesis has evolved in over 60 different plant taxa and is an excellent example of convergent evolution. Plants using the C4 photosynthetic pathway have an efficiency advantage, particularly in hot and dry environments. They account for 23% of global primary production and include some of our most productive cereals. While previous genetic studies comparing phylogenetically related C3 and C4 species have elucidated the genetic diversity underpinning the C4 photosynthetic pathway, no previous studies have described the genetic diversity of the genes involved in this pathway within a C4 crop species. Enhanced understanding of the allelic diversity and selection signatures of genes in this pathway may present opportunities to improve photosynthetic efficiency, and ultimately yield, by exploiting natural variation. Here, we present the first genetic diversity survey of 8 known C4 gene families in an important C4 crop, Sorghum bicolor (L.) Moench, using sequence data of 48 genotypes covering wild and domesticated sorghum accessions. Average nucleotide diversity of C4 gene families varied more than 20-fold from the NADP-malate dehydrogenase (MDH) gene family (θπ = 0.2 × 10-3) to the pyruvate orthophosphate dikinase (PPDK) gene family (θπ = 5.21 × 10-3). Genetic diversity of C4 genes was reduced by 22.43% in cultivated sorghum compared to wild and weedy sorghum, indicating that the group of wild and weedy sorghum may constitute an untapped reservoir for alleles related to the C4 photosynthetic pathway. A SNP-level analysis identified purifying selection signals on C4 PPDK and carbonic anhydrase (CA) genes, and balancing selection signals on C4 PPDK-regulatory protein (RP) and phosphoenolpyruvate carboxylase (PEPC) genes. Allelic distribution of these C4 genes was consistent with selection signals detected. A better understanding of the genetic diversity of C4 pathway in sorghum paves the way for mining the natural allelic variation for the improvement of photosynthesis.

Genotypic variation in whole-plant transpiration efficiency in sorghum only partly aligns with variation in stomatal conductance.

Water scarcity can limit sorghum (Sorghum bicolor (L.) Moench) production in dryland agriculture, but increased whole-plant transpiration efficiency (TEwp, biomass production per unit of water transpired) can enhance grain yield in such conditions. The objectives of this study were to quantify variation in TEwp for 27 sorghum genotypes and explore the linkages of this variation to responses of the underpinning leaf-level processes to environmental conditions. Individual plants were grown in large lysimeters in two well-watered experiments. Whole-plant transpiration per unit of green leaf area (TGLA) was monitored continuously and stomatal conductance and maximum photosynthetic capacity were measured during sunny conditions on recently expanded leaves. Leaf chlorophyll measurements of the upper five leaves of the main shoot were conducted during early grain filling. TEwp was determined at harvest. The results showed that diurnal patterns in TGLA were determined by vapour pressure deficit (VPD) and by the response of whole-plant conductance to radiation and VPD. Significant genotypic variation in the response of TGLA to VPD occurred and was related to genotypic differences in stomatal conductance. However, variation in TGLA explained only part of the variation in TEwp, with some of the residual variation explained by leaf chlorophyll readings, which were a reflection of photosynthetic capacity. Genotypes with different genetic background often differed in TEwp, TGLA and leaf chlorophyll, indicating potential differences in photosynthetic capacity among these groups. Observed differences in TEwp and its component traits can affect adaptation to drought stress.

Field crop phenomics: enabling breeding for radiation use efficiency and biomass in cereal crops.

Plant phenotyping forms the core of crop breeding, allowing breeders to build on physiological traits and mechanistic science to inform their selection of material for crossing and genetic gain. Recent rapid progress in high-throughput techniques based on machine vision, robotics, and computing (plant phenomics) enables crop physiologists and breeders to quantitatively measure complex and previously intractable traits. By combining these techniques with affordable genomic sequencing and genotyping, machine learning, and genome selection approaches, breeders have an opportunity to make rapid genetic progress. This review focuses on how field-based plant phenomics can enable next-generation physiological breeding in cereal crops for traits related to radiation use efficiency, photosynthesis, and crop biomass. These traits have previously been regarded as difficult and laborious to measure but have recently become a focus as cereal breeders find genetic progress from 'Green Revolution' traits such as harvest index become exhausted. Application of LiDAR, thermal imaging, leaf and canopy spectral reflectance, Chl fluorescence, and machine learning are discussed using wheat and sorghum phenotyping as case studies. A vision of how crop genomics and high-throughput phenotyping could enable the next generation of crop research and breeding is presented.

Effect of Soil Moisture Regimes on Growth and Seed Production of Two Australian Biotypes of Sisymbrium thellungii O. E. Schulz.

Sisymbrium thellungii O. E. Schulz is an emerging problematic weed in the northern grain region of Australia. Several different biotypes exist in this region but not all biotypes exhibit the same growth and reproduction behavior. This might be due to local adaptation to the different agro-ecosystems, however, information on this aspect is limited. To determine whether adaptation to water stress was a factor in biotype demographic growth and reproduction behavior, we evaluated the physiological and biochemical responses of two Australian S. thellungii biotypes, selected from high (Dalby) and medium (St. George) rainfall areas, to different pot soil moisture levels corresponding to 100, 75, 50, and 25% of soil water holding capacity (WHC). Averaged across moisture levels, the St. George biotype (medium rainfall area) had 89% greater biomass and produced 321% more seeds than the Dalby biotype. The St. George biotype was less affected by increased levels of water stress than the Dalby biotype. The Dalby biotype produced 4,787 seeds plant-1 at 100% WHC and only 28 seeds plant-1 at 25% WHC. On the other hand, the St. George biotype produced 4,061 seeds plant-1 at 25% WHC and its seed production at 100% WHC was 9,834 seeds plant-1. On a per leaf area basis and averaged across all moisture levels, the St. George had significantly lower net carbon assimilation compared with the Dalby biotype, accompanied by a trend for lower stomatal conductance, which might indicate an adaptation to water stress. Across the moisture levels, the St. George biotype had higher phenolics and total soluble sugar, but free proline content was higher in the Dalby biotype compared with the St. George biotype. Like total soluble sugar, proline content increased with water stress in both biotypes, but it increased to a greater extent in the Dalby biotype, particularly at the 25% of WHC. Branching, flowering and maturity occurred earlier in the St. George biotype compared with the Dalby biotype, indicating relatively faster growth of the St. George biotype, which again seems to be an adaptation to water-limited environments. In conclusion, the St. George biotype of S. thellungii had higher reproductive capacity than the Dalby biotype across all the moisture regimes, which suggests greater invasiveness. Overall, the large size and rapid growth of the S. thellungii population from the medium rainfall area, together with its physiological response to water stress and its ability to maintain seed production in dry conditions, may enable this biotype to become widespread in Australia.

Novel Grain Weight Loci Revealed in a Cross between Cultivated and Wild Sorghum.

Grain weight has increased during domestication of cereals. Together with grain number it determines yield, but the two are often negatively correlated. Understanding the genetic architecture of grain weight and its relationship with grain number is critical to enhance crop yield. Sorghum is an important food, feed, and biofuel crop well-known for its adaptation to drought and heat. This study aimed to dissect the genetic basis of thousand grain weight (TGW) in a BCF population between a domesticated sorghum accession and its wild progenitor, subsp. and investigate its relationship with grain number. Thousand grain weight, grain number, and yield were measured in field trials in two successive years. A strong negative correlation between TGW and grain number was observed in both trials. In total, 17 TGW quantitative trait loci (QTL) were identified, with 11 of them exhibiting an opposing effect on grain number, implying the correlation between TGW and grain number is due to pleiotropy. Nine grain size candidate genes were identified within 6 TGW QTL, and of these 5 showed signatures of selection during sorghum domestication. Large-effect QTL in this study that have not been identified previously in cultivated sorghum were found to contain candidate genes with domestication signal, indicating that these QTL were affected during sorghum domestication. This study sheds new light on the genetic basis of TGW, its relationship with grain number, and sorghum domestication.

Multi-Spectral Imaging from an Unmanned Aerial Vehicle Enables the Assessment of Seasonal Leaf Area Dynamics of Sorghum Breeding Lines.

Genetic improvement in sorghum breeding programs requires the assessment of adaptation traits in small-plot breeding trials across multiple environments. Many of these phenotypic assessments are made by manual measurement or visual scoring, both of which are time consuming and expensive. This limits trial size and the potential for genetic gain. In addition, these methods are typically restricted to point estimates of particular traits, such as leaf senescence or flowering and do not capture the dynamic nature of crop growth. In water-limited environments in particular, information on leaf area development over time would provide valuable insight into water use and adaptation to water scarcity during specific phenological stages of crop development. Current methods to estimate plant leaf area index (LAI) involve destructive sampling and are not practical in breeding. Unmanned aerial vehicles (UAV) and proximal-sensing technologies open new opportunities to assess these traits multiple times in large small-plot trials. We analyzed vegetation-specific crop indices obtained from a narrowband multi-spectral camera on board a UAV platform flown over a small pilot trial with 30 plots (10 genotypes randomized within 3 blocks). Due to variable emergence we were able to assess the utility of these vegetation indices to estimate canopy cover and LAI over a large range of plant densities. We found good correlations between the Normalized Difference Vegetation Index (NDVI) and the Enhanced Vegetation Index (EVI) with plant number per plot, canopy cover and LAI both during the vegetative growth phase (pre-anthesis) and at maximum canopy cover shortly after anthesis. We also analyzed the utility of time-sequence data to assess the senescence pattern of sorghum genotypes known as fast (senescent) or slow senescing (stay-green) types. The Normalized Difference Red Edge (NDRE) index which estimates leaf chlorophyll content was most useful in characterizing the leaf area dynamics/senescence patterns of contrasting genotypes. These methods to monitor dynamics of green and senesced leaf area are suitable for out-scaling to enhance phenotyping of additional crop canopy characteristics and likely crop yield responses among genotypes across large fields and multiple dates.

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.

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.