ENHANCED GRAVITROPISM 2 encodes a STERILE ALPHA MOTIF–containing protein that controls root growth angle in barley and wheat

The root growth angle defines how roots grow toward the gravity vector and is among the most important determinants of root system architecture. It controls water uptake capacity, nutrient use efficiency, stress resilience, and, as a consequence, yield of crop plants. We demonstrated that the egt2 (enhanced gravitropism 2) mutant of barley exhibits steeper root growth of seminal and lateral roots and an auxin-independent higher responsiveness to gravity compared to wild-type plants. We cloned the EGT2 gene by a combination of bulked-segregant analysis and whole genome sequencing. Subsequent validation experiments by an independent CRISPR/Cas9 mutant allele demonstrated that egt2 encodes a STERILE ALPHA MOTIF domain–containing protein. In situ hybridization experiments illustrated that EGT2 is expressed from the root cap to the elongation zone. We demonstrated the evolutionary conserved role of EGT2 in root growth angle control between barley and wheat by knocking out the EGT2 orthologs in the A and B genomes of tetraploid durum wheat. By combining laser capture microdissection with RNA sequencing, we observed that seven expansin genes were transcriptionally down-regulated in the elongation zone. This is consistent with a role of EGT2 in this region of the root where the effect of gravity sensing is executed by differential cell elongation. Our findings suggest that EGT2 is an evolutionary conserved regulator of root growth angle in barley and wheat that could be a valuable target for root-based crop improvement strategies in cereals.

A Comparative Analysis of Quantitative Metrics of Root Architecture

High throughput phenotyping is important to bridge the gap between genotype and phenotype. The methods used to describe the phenotype therefore should be robust to measurement errors, relatively stable over time, and most importantly, provide a reliable estimate of elementary phenotypic components. In this study, we use functional-structural modeling to evaluate quantitative phenotypic metrics used to describe root architecture to determine how they fit these criteria. Our results show that phenes such as root number, root diameter, and lateral root branching density are stable, reliable measures and are not affected by imaging method or plane. Metrics aggregating multiple phenes such as total length, total volume, convex hull volume, and bushiness index estimate different subsets of the constituent phenes; they however do not provide any information regarding the underlying phene states. Estimates of phene aggregates are not unique representations of underlying constituent phenes: multiple phenotypes having phenes in different states could have similar aggregate metrics. Root growth angle is an important phene which is susceptible to measurement errors when 2D projection methods are used. Metrics that aggregate phenes which are complex functions of root growth angle and other phenes are also subject to measurement errors when 2D projection methods are used. These results support the hypothesis that estimates of phenes are more useful than metrics aggregating multiple phenes for phenotyping root architecture. We propose that these concepts are broadly applicable in phenotyping and phenomics.

ENHANCED GRAVITROPISM 2 encodes a STERILE ALPHA MOTIVE containing protein that controls root growth angle in barley and wheat

The root growth angle defines how roots grow towards the gravity vector and is among the most important determinants of root system architecture. It controls water uptake capacity, nutrient use efficiency, stress resilience and as a consequence yield of crop plants. We demonstrated that the egt2 (enhanced gravitropism 2) mutant of barley exhibits steeper root growth of seminal and lateral roots and an auxin independent higher responsiveness to gravity compared to wild type plants. We cloned the EGT2 gene by a combination of bulked segregant analysis and whole genome sequencing. Subsequent validation experiments by an independent CRISPR/Cas9 mutant allele demonstrated that egt2 encodes a STERILE ALPHA MOTIF domain containing protein. In situ hybridization experiments illustrated that EGT2 is expressed from the root cap to the elongation zone. Subcellular localization experiments revealed that EGT2 localizes to the nucleus and cytoplasm. We demonstrated the evolutionary conserved role of EGT2 in root growth angle control between barley and wheat by knocking out the EGT2 orthologs in the A and B genomes of tetraploid durum wheat. By combining laser capture microdissection with RNA-seq, we observed that seven expansin genes were transcriptionally downregulated in the elongation zone. This is consistent with a role of EGT2 in this region of the root where the effect of gravity sensing is executed by differential cell elongation. Our findings suggest that EGT2 is an evolutionary conserved regulator of root growth angle in barley and wheat that could be a valuable target for root-based crop improvement strategies in cereals.Significance StatementTo date the potential of utilizing root traits in plant breeding remains largely untapped. In this study we cloned and characterized the ENHANCED GRAVITROPISM2 (EGT2) gene of barley that encodes a STERILE ALPHA MOTIF domain containing protein. We demonstrated that EGT2 is a key gene of root growth angle regulation in response to gravity which is conserved in barley and wheat and could be a promising target for crop improvement in cereals.

A comparative analysis of quantitative metrics of root architectural phenotypes

High throughput phenotyping is important to bridge the gap between genotype and phenotype. The methods used to describe the phenotype therefore should be robust to measurement errors, relatively stable over time, and most importantly, provide a reliable estimate of elementary phenotypic components. In this study, we use functional-structural modeling to evaluate quantitative phenotypic metrics used to describe root architecture to determine how they fit these criteria. Our results show that phenes such as root number, root diameter, lateral root branching density are stable, reliable measures and are not affected by imaging method or plane. Metrics aggregating multiple phenes such as total length, total volume, convexhull volume, bushiness index etc. estimate different subsets of the constituent phenes, they however do not provide any information regarding the underlying phene states. Estimates of phene aggregates are not unique representations of underlying constituent phenes: multiple phenotypes having phenes in different states could have similar aggregate metrics. Root growth angle is an important phene which is susceptible to measurement errors when 2D projection methods are used. Metrics that aggregate phenes which are complex functions of root growth angle and other phenes are also subject to measurement errors when 2D projection methods are used. These results support the hypothesis that estimates of phenes are more useful than metrics aggregating multiple phenes for phenotyping root architecture. We propose that these concepts are broadly applicable in phenotyping and phenomics.

Intricate genetic variation networks control the adventitious root growth angle in apple

Background The root growth angle (RGA) typically determines plant rooting depth, which is significant for plant anchorage and abiotic stress tolerance. Several quantitative trait loci (QTLs) for RGA have been identified in crops. However, the underlying mechanisms of the RGA remain poorly understood, especially in apple rootstocks. The objective of this study was to identify QTLs, validate genetic variation networks, and develop molecular markers for the RGA in apple rootstock. Results Bulked segregant analysis by sequencing (BSA-seq) identified 25 QTLs for RGA using 1955 hybrids of the apple rootstock cultivars ‘Baleng Crab’ (Malus robusta Rehd., large RGA) and ‘M9’ (M. pumila Mill., small RGA). With RNA sequencing (RNA-seq) and parental resequencing, six major functional genes were identified and constituted two genetic variation networks for the RGA. Two single nucleotide polymorphisms (SNPs) of the MdLAZY1 promoter damaged the binding sites of MdDREB2A and MdHSFB3, while one SNP of MdDREB2A and MdIAA1 affected the interactions of MdDREB2A/MdHSFB3 and MdIAA1/MdLAZY1, respectively. A SNP within the MdNPR5 promoter damaged the interaction between MdNPR5 and MdLBD41, while one SNP of MdLBD41 interrupted the MdLBD41/MdbHLH48 interaction that affected the binding ability of MdLBD41 on the MdNPR5 promoter. Twenty six SNP markers were designed on candidate genes in each QTL interval, and the marker effects varied from 0.22°-26.11°. Conclusions Six diagnostic markers, SNP592, G122, b13, Z312, S1272, and S1288, were used to identify two intricate genetic variation networks that control the RGA and may provide new insights into the accuracy of the molecular markers. The QTLs and SNP markers can potentially be used to select deep-rooted apple rootstocks.

Genetic Relationship and Structural Variation of Root Growth Angle for Deep Rooting in Rice (Oryza sativa L.)

Breeding for drought tolerance revolves around selection of genotypes with desirable root characters. Root pattern studies in rice have shown significant relationship with drought tolerance. In order to understand the effects of deep rooting pattern and root growth angle in relation to water stress was assessed following basket method. The Backcross inbred lines (BILs) of ADT (R) 45*1/Apo and ADT (R) 45*1/Wayrarem with drought QTLs viz., qDTY1.1, qDTY3.1, qDTY4.1 and qDTY12.1 were evaluated for various root traits. Out of 20 BILs and three parental lines studied four BILs showed high values for root growth at deep angle 65-90 °and yield under drought stress. Based on association studies among the root traits positive and significant correlation was observed between yield and root traits viz., root dry weight, root growth at deep angle 65-90° (RA4), ratio of deep rooting and root length. Clustering of BILs and parents have grouped deep rooting BILs and drought tolerant donors into one cluster and drought susceptible ADT (R) 45 into a separate cluster II which clearly indicates, the importance of deep rooting and yield under drought stress. Strong association of root traits and drought tolerance clearly shows the importance in utilization of these traits as selection criteria for drought tolerance in rice.

Gravity-Sensing Tissues for Gravitropism Are Required for “Anti-Gravitropic” Phenotypes of lzy Multiple Mutants in Arabidopsis

Plant posture is controlled by various environmental cues, such as light, temperature, and gravity. The overall architecture is determined by the growth angles of lateral organs, such as roots and branches. The branch growth angle affected by gravity is known as the gravitropic setpoint angle (GSA), and it has been proposed that the GSA is determined by balancing two opposing growth components: gravitropism and anti-gravitropic offset (AGO). The molecular mechanisms underlying gravitropism have been studied extensively, but little is known about the nature of the AGO. Recent studies reported the importance of LAZY1-LIKE (LZY) family genes in the signaling process for gravitropism, such that loss-of-function mutants of LZY family genes resulted in reversed gravitropism, which we term it here as the “anti-gravitropic” phenotype. We assume that this peculiar phenotype manifests as the AGO due to the loss of gravitropism, we characterized the “anti-gravitropic” phenotype of Arabidopsis lzy multiple mutant genetically and physiologically. Our genetic interaction analyses strongly suggested that gravity-sensing cells are required for the “anti-gravitropic” phenotype in roots and lateral branches. We also show that starch-filled amyloplasts play a significant role in the “anti-gravitropic” phenotype, especially in the root of the lzy multiple mutant.