Root system architecture variation among barley (Hordeum vulgare L.) accessions at seedling stage under soil acidity condition.

MAIN CONCLUSION: Soil acidity in Ethiopian highlands impacts barley production, affecting root system architecture. Study on 300 accessions showed significant trait variability, with potential for breeding enhancement. Soil acidity poses a significant challenge to crop production in the highland regions of Ethiopia, particularly impacting barley, a crucial staple crop. This acidity serves as a key stressor affecting the root system architecture (RSA) of this crop. Hence, the objective of this study was to assess the RSA traits variability under acidic soil conditions using 300 barley accessions in a greenhouse experiment. The analysis of variance indicated substantial variations among the accessions across all traits studied. The phenotypic coefficient of variation ranged from 24.4% for shoot dry weight to 11.1% for root length, while the genotypic coefficient variation varied between 18.83 and 9.2% for shoot dry weight and root length, respectively. The broad-sense heritability ranged from 36.7% for leaf area to 69.9% for root length, highlighting considerable heritability among multiple traits. The genetic advances as a percent of the mean ranged from 13.63 to 29.9%, suggesting potential for enhancement of these traits through breeding efforts. Principal component analysis and cluster analysis grouped the genotypes into two major clusters, each containing varying numbers of genotypes with contrasting traits. This diverse group presents an opportunity to access a wide range of potential parent candidates to enhance genetic variablity in breeding programs. The Pearson correlation analysis revealed significant negative associations between root angle (RA) and other RSA traits. This helps indirect selection of accessions for further improvement in soil acidity. In conclusion, this study offers valuable insights into the RSA characteristics of barley in acidic soil conditions, aiding in the development of breeding strategies to enhance crop productivity in acidic soil environments.

Early gene expression response of barley root tip to toxic concentrations of cadmium.

KEY MESSAGE: Already a short-term Cd treatment induces changes in gene expression in barley root tips via IAA and ROS signaling during mild and severe Cd stress, respectively. Even a short, 30 min, Cd treatment of roots induced a considerable alteration in gene expression in the barley root tips within an hour after the treatments. The very early activation of MYB1 transcription factor expression is partially regulated by auxin signaling in mildly stressed seedlings. An increase in allene oxide cyclase and NADPH oxidase expression was a distinguishing feature of root tips response to mild Cd stress and their expression is activated via IAA signaling. Meanwhile, early changes in the level of dehydrin transcripts were detected in moderately and severely stressed root tips, and their induction is related to altered ROS homeostasis in cells. The early activation of glutathione peroxidase expression by mild Cd stress indicates the involvement of IAA in the signaling process. In contrast, early ascorbate peroxidase expression was induced only with Cd treatment causing severe stress and ROS play central roles in its induction. The expression of cysteine protease was activated similarly in both mildly and severely Cd-stressed roots; consequently, both increased IAA and ROS levels take part in the regulation of cysteine protease expression. The Cd-evoked accumulation of BAX Inhibitor-1 mRNA was characteristic for moderately and severely stressed roots. Whereas decreased IAA level did not affect its expression, rotenone-mediated ROS depletion markedly reduced the Cd-induced expression of BAX Inhibitor-1. An early increase of alternative oxidase levels in the root tip cells indicated that the reduction of mitochondrial superoxide generation is an important component of barley root response to severe Cd stress.

Genetic Loci Underlying Awn Morphology in Barley.

Barley awns are highly active in photosynthesis and account for 30-50% of grain weight in barley. They are diverse in length, ranging from long to awnless, and in shape from straight to hooded or crooked. Their diversity and importance have intrigued geneticists for several decades. A large collection of awnness mutants are available-over a dozen of them have been mapped on chromosomes and a few recently cloned. Different awnness genes interact with each other to produce diverse awn phenotypes. With the availability of the sequenced barley genome and application of new mapping and gene cloning strategies, it will now be possible to identify and clone more awnness genes. A better understanding of the genetic basis of awn diversity will greatly facilitate development of new barley cultivars with improved yield, adaptability and sustainability.

MADS1 maintains barley spike morphology at high ambient temperatures.

Temperature stresses affect plant phenotypic diversity. The developmental stability of the inflorescence, required for reproductive success, is tightly regulated by the interplay of genetic and environmental factors. However, the mechanisms underpinning how plant inflorescence architecture responds to temperature are largely unknown. We demonstrate that the barley SEPALLATA MADS-box protein HvMADS1 is responsible for maintaining an unbranched spike architecture at high temperatures, while the loss-of-function mutant forms a branched inflorescence-like structure. HvMADS1 exhibits increased binding to target promoters via A-tract CArG-box motifs, which change conformation with temperature. Target genes for high-temperature-dependent HvMADS1 activation are predominantly associated with inflorescence differentiation and phytohormone signalling. HvMADS1 directly regulates the cytokinin-degrading enzyme HvCKX3 to integrate temperature response and cytokinin homeostasis, which is required to repress meristem cell cycle/division. Our findings reveal a mechanism by which genetic factors direct plant thermomorphogenesis, extending the recognized role of plant MADS-box proteins in floral development.

Modulating tiller formation in cereal crops by the signalling function of fertilizer nitrogen forms.

Cereal crop yield comprises interrelated components, among which the number of tillers is highly responsive to nitrogen fertilization. We addressed the hypothesis of whether the supply of different nitrogen forms can be employed to manipulate the tiller number in cereal crops. Relative to urea or ammonium, exclusive supply of nitrate increased tiller number in hydroponically-grown barley plants. Thereby, tiller number correlated positively with the root-to-shoot translocation rate of endogenous cytokinins. External supply of a synthetic cytokinin analog further stimulated tillering in nitrate-containing but not in urea-containing nutrient solution. When the cytokinin analog 6-benzylaminopurine riboside was externally supplied to roots, its translocation to shoots was 2.5 times higher in presence of nitrate than in presence of urea or ammonium, suggesting that cytokinin loading into the xylem is affected by different nitrogen forms. We then translated this finding to field scale, cultivated winter wheat in four environments, and confirmed that nitrate fertilization significantly increased tiller number in a dose-dependent manner. As assessed in 22 winter wheat cultivars, nitrogen form-dependent tiller formation was subject to substantial genotypic variation. We conclude that cytokinin-mediated signaling effects of fertilizer nitrogen forms can be employed as a management tool to regulate the tiller number in cereal crops.

Conversion of hulled into naked barley by Cas endonuclease-mediated knockout of the NUD gene.

BACKGROUND: The naked caryopsis character in barley is a domestication-associated trait defined by loss-of-function of the NUD gene. The functional NUD gene encodes an Apetala 2/Ethylene-Response Factor (AP2/ERF) controlling the formation of a cementing layer between pericarp and both lemma and palea. The downstream genes regulated by the NUD transcription factor and molecular mechanism of a cementing layer formation are still not sufficiently described. A naturally occurring 17-kb deletion in the nud locus is associated with the emergence of naked barley. Naked barley has been traditionally used for food and nowadays is considered as a dietary component for functional nutrition. RESULTS: In the present study, we demonstrate that targeted knockout of the NUD gene using RNA-guided Cas9 endonuclease leads to the phenotype conversion from hulled to naked barley. Using in vivo pre-testing systems, highly effective guide RNAs targeting the first exon of the NUD gene were selected. Expression cassettes harboring the cas9 and guide RNA genes were used to transform barley cv. Golden Promise via Agrobacterium-mediated DNA transfer. The recessive naked grain phenotype was observed in 57% of primary transformants, which indicates a frequent occurrence of homozygous or biallelic mutations. T-DNA-free homozygous lines with independently generated mutations in the NUD gene were obtained in the T1 generation. At homozygous state, all obtained mutations including one- and two-amino acid losses with the translational reading frame being retained invariably caused the naked grain phenotype. CONCLUSIONS: The hulled and naked barley isogenic lines generated are a perfect experimental model for further studies on pleiotropic consequences of nud mutations on overall plant performance under particular consideration of yield-determining traits. Due to the high ß-glucan content of its grains, naked barley is considered as being of particular dietary value. The possibility to convert hulled into naked barley cultivars by targeted mutagenesis allows breeders to extend the potential utilization of barley by the provision of functional food.

Physiological and morphological responses of different spring barley genotypes to water deficit and associated QTLs.

Water deficit is one of the major limitations to food production worldwide and most climate change scenarios predict an aggravation of the situation. To face the expected increase in drought stress in the coming years, breeders are working to elucidate the genetic control of barley growth and productivity traits under water deficit. Barley is known as a relatively drought tolerant crop and genetic variability was observed for drought tolerance traits. The objectives of the present study were the quantification of morphological and physiological responses in a collection of 209 spring barley genotypes to drought stress, and the genetic analysis by genome-wide association study to find quantitative trait loci (QTL) and the allele contributions for each of the investigated traits. In six pot experiments, 209 spring barley genotypes were grown under a well-watered and water-limited regime. Stress phases were initiated individually for each genotype at the beginning of tillering and spiking for the vegetative- and the generative stage experiments, respectively, and terminated when the transpiration rates of stress treatments reached 10% of the well-watered control. After the stress phase, a total of 42 productivity related traits such as the dry matter of plant organs, tiller number, leaf length, leaf area, amount of water soluble carbohydrates in the stems, proline content in leaves and osmotic adjustment of corresponding well-watered and stressed plants were analysed, and QTL analyses were performed to find marker-trait associations. Significant water deficit effects were observed for almost all traits and significant genotype x treatment interactions (GxT) were observed for 37 phenotypic traits. Genome-wide association studies (GWAS) revealed 77 significant loci associated with 16 phenotypic traits during the vegetative stage experiment and a total of 85 significant loci associated with 13 phenotypic traits during the generative stage experiment for traits such as leaf area, number of green leaves, grain yield, harvest index and stem length. For traits with significant GxT interactions, genotypic differences for relative values were analysed using one way ANOVA. More than 110 loci for GxT interaction were found for 17 phenotypic traits explaining in many cases more than 50% of the genetic variance.

Genetic analysis reveals four interacting loci underlying awn trait diversity in barley (Hordeum vulgare).

Barley (Hordeum vulgare) awns contribute to grain yield, but the genetic basis of awn development remains largely unclear. Five barley lines differing in awn traits and row types were used to create four F2 populations. Genetic analyses revealed that four pairs of genes were involved in awn development: A/a (awnless/awned), B/b (awnless/awned), H/h (hooded/straight), and L/l (long/short). Of these four loci, A, H and L functioned on both central rows (CR) and lateral rows (LR) of the barley spikes, while B exhibited effect only on LR. A and B had duplicate effects on LR, and both showed dominant epistasis to loci H and L, whereas H was epistatic to L. Meanwhile, A and B were found to be genetically linked, with a row-type locus V located between them. The genetic distances of A-V and B-V were estimated to be 9.6 and 7.7 cM, respectively. Literature search suggested that A, H and V may correspond to the reported Lks1, Kap1 and Vrs1, respectively, whereas B is a novel gene specifically controlling awn development on LR, designated as Lsa1 for lateral spikelet awnless 1. The only barley homolog of wheat awn inhibitor gene B1, HORVU2Hr1G077570, is a potential candidate of Lsa1.

Affordable and robust phenotyping framework to analyse root system architecture of soil-grown plants.

The phenotypic analysis of root system growth is important to inform efforts to enhance plant resource acquisition from soils; however, root phenotyping remains challenging because of the opacity of soil, requiring systems that facilitate root system visibility and image acquisition. Previously reported systems require costly or bespoke materials not available in most countries, where breeders need tools to select varieties best adapted to local soils and field conditions. Here, we report an affordable soil-based growth (rhizobox) and imaging system to phenotype root development in glasshouses or shelters. All components of the system are made from locally available commodity components, facilitating the adoption of this affordable technology in low-income countries. The rhizobox is large enough (approximately 6000 cm2 of visible soil) to avoid restricting vertical root system growth for most if not all of the life cycle, yet light enough (approximately 21 kg when filled with soil) for routine handling. Support structures and an imaging station, with five cameras covering the whole soil surface, complement the rhizoboxes. Images are acquired via the Phenotiki sensor interface, collected, stitched and analysed. Root system architecture (RSA) parameters are quantified without intervention. The RSAs of a dicot species (Cicer arietinum, chickpea) and a monocot species (Hordeum vulgare, barley), exhibiting contrasting root systems, were analysed. Insights into root system dynamics during vegetative and reproductive stages of the chickpea life cycle were obtained. This affordable system is relevant for efforts in Ethiopia and other low- and middle-income countries to enhance crop yields and climate resilience sustainably.

An Acyl-CoA N-Acyltransferase Regulates Meristem Phase Change and Plant Architecture in Barley.

The modification of shoot architecture and increased investment into reproductive structures is key for crop improvement and is achieved through coordinated changes in the development and determinacy of different shoot meristems. A fundamental question is how the development of different shoot meristems is genetically coordinated to optimize the balance between vegetative and reproductive organs. Here we identify the MANY NODED DWARF1 (HvMND1) gene as a major regulator of plant architecture in barley (Hordeum vulgare). The mnd1.a mutant displayed an extended vegetative program with increased phytomer, leaf, and tiller production but a reduction in the number and size of grains. The induction of vegetative structures continued even after the transition to reproductive growth, resulting in a marked increase in longevity. Using mapping by RNA sequencing, we found that the HvMND1 gene encodes an acyl-CoA N-acyltransferase that is predominately expressed in developing axillary meristems and young inflorescences. Exploration of the expression network modulated by HvMND1 revealed differential expression of the developmental microRNAs miR156 and miR172 and several key cell cycle and developmental genes. Our data suggest that HvMND1 plays a significant role in the coordinated regulation of reproductive phase transitions, thereby promoting reproductive growth and whole plant senescence in barley.

Seminal and Nodal Roots of Barley Differ in Anatomy, Proteome and Nitrate Uptake Capacity.

The root system of barley plants is composed of embryogenic, seminal roots as well as lateral and nodal roots that are formed postembryonically from seminal roots and from the basal part of shoots, respectively. Due to their distinct developmental origin, seminal and nodal roots may differ in function during plant development; however, a clear comparison between these two root types has not yet been undertaken. In this study, anatomical, proteomic and physiological traits were compared between seminal and nodal roots of similar developmental stages. Nodal roots have larger diameter, larger metaxylem area and a larger number of metaxylem vessels than seminal roots. Proteome profiling uncovered a set of root-type-specific proteins, including proteins related to the cell wall and cytoskeleton organization, which could potentially be implicated with differential metaxylem development. We also found that nodal roots have higher levels of auxin, which is known to trigger metaxylem development. At millimolar nitrate supply, nodal roots had approximately 2-fold higher nitrate uptake and root-to-shoot translocation capacities than seminal roots, whereas no differences were found at micromolar nitrate supply. Since these marked differences were not reflected by the transcript levels of low-affinity nitrate transporter genes, we hypothesize that the larger metaxylem volume of nodal roots enhances predominantly the low-affinity uptake and translocation capacities of nutrients that are transported with the bulk flow of water, like nitrate.

Sheathing the blade: Significant contribution of sheaths to daytime and nighttime gas exchange in a grass crop.

Despite representing a sizeable fraction of the canopy, very little is known about leaf sheath gas exchange in grasses. Specifically, estimates of sheath stomatal conductance, transpiration and photosynthesis along with their responses to light, CO2 and vapour pressure deficit (VPD) are unknown. Furthermore, the anatomical basis of these responses is poorly documented. Here, using barley as a model system, and combining leaf-level gas exchange, whole-plant gravimetric measurements, transpiration inhibitors, anatomical observations, and biophysical modelling, we found that sheath and blade stomatal conductance and transpiration were similar, especially at low light, in addition to being genotypically variable. Thanks to high abaxial stomata densities and surface areas nearly half those of the blades, sheaths accounted for up to 17% of the daily whole-plant water use, which -surprisingly- increased to 45% during the nighttime. Sheath photosynthesis was on average 17-25% that of the blade and was associated with lower water use efficiency. Finally, sheaths responded differently to the environment, exhibiting a lack of response to CO2 but a strong sensitivity to VPD. Overall, these results suggest a key involvement of sheaths in feedback loops between canopy architecture and gas exchange with potentially significant implications on adaptation to current and future climates in grasses.

Construction of a high-density genetic map and fine mapping of a candidate gene locus for a novel branched-spike mutant in barley.

A Yunnan branched-spike (Ynbs) barley mutant is useful for study of the genetic mechanisms underlying variation in barley spike architecture. In the current study, a mutant (Ynbs-1), a recombinant inbred line (RIL-1), and a cultivar (BDM-8) were used as parents to develop populations. Ynbs-1 exhibits typical branched spike, whereas the others exhibit six-row spike. Genetic analysis on their F1, F2 and F3 populations showed that one recessive gene is responsible for the branched spike trait. SLAF marker generated from specific locus amplified fragment sequencing (SLAF-seq) was used to genotype the populations. A high-density genetic map of barley was constructed using 14,348 SLAF markers, which covered all 7 chromosomes at 1,347.44 cM in length with an average marker density of 0.09 cM between adjacent markers. Linkage analysis of the branched-spike trait using the genetic map indicated that branched spike trait in the Ynbs-1 is controlled by single locus on chromosome 2H at the interval between 65.00 and 65.47 cM that is flanked by Marker310119 and Marker2679451. Several candidate genes that may be responsible for barley multiple-spikelet degeneration, single-floret spikelet increase and seed set rate decrease were identified in the region. The high-density genetic map and the gene locus revealed in this study provide valuable information for elucidating the genetic mechanism of spike branching in barley.

Anatomical and ultrastructural responses of Hordeum sativum to the soil spiked by copper.

Effects of Cu toxicity from contaminated soil were analysed in spring barley (Hordeum sativum distichum), a widely cultivated species in South Russia. In this study, H. sativum was planted outdoors in one of the most fertile soils-Haplic Chernozem spiked with high concentration of Cu and examined between the boot and head emergence phase of growth. Copper toxicity was observed to cause slow ontogenetic development of plants, changing their morphometric parameters (shape, size, colour). To the best of our knowledge, the ultrastructural changes in roots, stems and leaves of H. sativum induced by excess Cu were fully characterized for the first time using transmission electron microscopy. The plant roots were the most effected, showing degradation of the epidermis, reduced number of parenchyma cells, as well as a significant decrease in the diameter of the stele and a disruption and modification to its cell structure. The comparative analysis of the ultrastructure of control plants and plants exposed to the toxic effects of Cu has made it possible to reveal significant disruption of the integrity of the cell wall and cytoplasmic membranes in the root with deposition of electron-dense material. The changes in the ultrastructure of the main cytoplasmic organelles-endoplasmic reticulum, mitochondria, chloroplasts and peroxisomes-in the stem and leaves were found. The cellular Cu deposition, anatomical and ultrastructural modifications could mainly account for the primary impact points of metal toxicity. Therefore, this work extends the available knowledge of the mechanisms of the Cu effect tolerance of barley.

Genetic components of root architecture and anatomy adjustments to water-deficit stress in spring barley.

Roots perform vital roles for adaptation and productivity under water-deficit stress, even though their specific functions are poorly understood. In this study, the genetic control of the nodal-root architectural and anatomical response to water deficit were investigated among diverse spring barley accessions. Water deficit induced substantial variations in the nodal root traits. The cortical, stele, and total root cross-sectional areas of the main-shoot nodal roots decreased under water deficit, but increased in the tiller nodal roots. Root xylem density and arrested nodal roots increased under water deficit, with the formation of root suberization/lignification and large cortical aerenchyma. Genome-wide association study implicated 11 QTL intervals in the architectural and anatomical nodal root response to water deficit. Among them, three and four QTL intervals had strong effects across seasons and on both root architectural and anatomical traits, respectively. Genome-wide epistasis analysis revealed 44 epistatically interacting SNP loci. Further analyses showed that these QTL intervals contain important candidate genes, including ZIFL2, MATE, and PPIB, whose functions are shown to be related to the root adaptive response to water deprivation in plants. These results give novel insight into the genetic architectures of barley nodal root response to soil water deficit stress in the fields, and thus offer useful resources for root-targeted marker-assisted selection.

Alternative splicing of a barley gene results in an excess-tillering and semi-dwarf mutant.

KEY MESSAGE: An excess-tillering semi-dwarf gene Hvhtd was identified from an EMS-induced mutant in barley and alternative splicing results in excess-tillering semi-dwarf traits. Tillering and plant height are important traits determining plant architecture and grain production in cereal crops. This study identified an excess-tillering semi-dwarf mutant (htd) from an EMS-treated barley population. Genetic analysis of the F1, F2, and F2:3 populations showed that a single recessive gene controlled the excess-tillering semi-dwarf in htd. Using BSR-Seq and gene mapping, the Hvhtd gene was delimited within a 1.8 Mb interval on chromosome 2HL. Alignment of the RNA-Seq data with the functional genes in the interval identified a gene HORVU2Hr1G098820 with alternative splicing between exon2 and exon3 in the mutant, due to a G to A single-nucleotide substitution at the exon and intron junction. An independent mutant with a similar phenotype confirmed the result, with alternative splicing between exon3 and exon4. In both cases, the alternative splicing resulted in a non-functional protein. And the gene HORVU2Hr1G098820 encodes a trypsin family protein and may be involved in the IAA signaling pathway and differs from the mechanism of Green Revolution genes in the gibberellic acid metabolic pathway.

Mapping dynamic QTL dissects the genetic architecture of grain size and grain filling rate at different grain-filling stages in barley.

Grain filling is an important growth process in formation of yield and quality for barley final yield determination. To explore the grain development behavior during grain filling period in barley, a high-density genetic map with 1962 markers deriving from a doubled haploid (DH) population of 122 lines was used to identify dynamic quantitative trait locus (QTL) for grain filling rate (GFR) and five grain size traits: grain area (GA), grain perimeter (GP), grain length (GL), grain width (GW) and grain diameter (GD). Unconditional QTL mapping is to detect the cumulative effect of genetic factors on a phenotype from development to a certain stage. Conditional QTL mapping is to detect a net effect of genetic factors on the phenotype at adjacent time intervals. Using unconditional, conditional and covariate QTL mapping methods, we successfully detected 34 major consensus QTLs. Moreover, certain candidate genes related to grain size, plant height, yield, and starch synthesis were identified in six QTL clusters, and individual gene was specifically expressed in different grain filling stages. These findings provide useful information for understanding the genetic basis of the grain filling dynamic process and will be useful for molecular marker-assisted selection in barley breeding.

Wheat and barley can increase grain yield in shade through acclimation of physiological and morphological traits in Mediterranean conditions.

Major cereal yields are expected to decline significantly in coming years due to the effects of climate change temperature rise. Agroforestry systems have been recognized as a useful land management strategy that could mitigate these effects through the shelter provided by trees, but it is unclear how shade affects cereal production. Most cereal species and cultivars have been selected for full light conditions, making it necessary to determine those able to acclimate to low irradiance environments and the traits that drive this acclimation. A greenhouse experiment was conducted in central Spain to assess the photosynthetic response, leaf morphology and grain yield of nine cultivars of winter wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) at three levels of photosynthetic active radiation (100%, 90% and 50%). Cultivars were selected according to three different precocity categories and were widely used in the studied area. The main objective was to assess whether the species and cultivars could acclimate to partial shade through physiological and morphological acclimations and thus increase their grain yield for cultivation in agroforestry systems. Both species increased grain yield by 19% in shade conditions. However, they used different acclimation strategies. Barley mostly performed a physiological acclimation, while wheat had a major morphological adjustment under shaded environment. Barley had lower dark respiration (42%), lower light compensation point (73%) and higher maximum quantum yield (48%) than wheat in full light conditions, revealing that it was a more shade-tolerant species than wheat. In addition, to acclimate to low irradiance conditions, barley showed a 21% reduction of the carotenoids/chlorophyll ratio in the lowest irradiance level compared to 100% light availability and adjusted the chlorophyll a/b ratio, photosystem II quantum efficiency, electron transport rate and non-photochemical quenching to shade conditions. On the other hand, wheat showed a 48% increase in single leaf area in the 50% irradiance level than in full light to maximize light capture. Our results showed that current commercialized wheat and barley cultivars had sufficient plasticity for adaptation to shade, supporting tree presence as a tool to reduce the negative effects of climate change.

CENTRORADIALIS Interacts with FLOWERING LOCUS T-Like Genes to Control Floret Development and Grain Number.

CENTRORADIALIS (CEN) is a key regulator of flowering time and inflorescence architecture in plants. Natural variation in the barley (Hordeum vulgare) homolog HvCEN is important for agricultural range expansion of barley cultivation, but its effects on shoot and spike architecture and consequently yield have not yet been characterized. Here, we evaluated 23 independent hvcen, also termed mat-c, mutants to determine the pleiotropic effects of HvCEN on developmental timing and shoot and spike morphologies of barley under outdoor and controlled conditions. All hvcen mutants flowered early and showed a reduction in spikelet number per spike, tiller number, and yield in the outdoor experiments. Mutations in hvcen accelerated spikelet initiation and reduced axillary bud number in a photoperiod-independent manner but promoted floret development only under long days (LDs). The analysis of a flowering locus t3 (hvft3) hvcen double mutant showed that HvCEN interacts with HvFT3 to control spikelet initiation. Furthermore, early flowering3 (hvelf3) hvcen double mutants with high HvFT1 expression levels under short days suggested that HvCEN interacts with HvFT1 to repress floral development. Global transcriptome profiling in developing shoot apices and inflorescences of mutant and wild-type plants revealed that HvCEN controlled transcripts involved in chromatin remodeling activities, cytokinin and cell cycle regulation and cellular respiration under LDs and short days, whereas HvCEN affected floral homeotic genes only under LDs. Understanding the stage and organ-specific functions of HvCEN and downstream molecular networks will allow the manipulation of different shoot and spike traits and thereby yield.

Evolution of Deeper Rooting 1-like homoeologs in wheat entails the C-terminus mutations as well as gain and loss of auxin response elements.

Root growth angle (RGA) in response to gravity controlled by auxin is a pertinent target trait for obtainment of higher yield in cereals. But molecular basis of this root architecture trait remain obscure in wheat and barley. We selected four cultivars two each for wheat and barley to unveil the molecular genetic mechanism of Deeper Rooting 1-like gene which controls RGA in rice leading to higher yield under drought imposition. Morphological analyses revealed a deeper and vertically oriented root growth in "NARC 2009" variety of wheat than "Galaxy" and two other barley cultivars "Scarlet" and "ISR42-8". Three new homoeologs designated as TaANDRO1-like, TaBNDRO1-like and TaDNDRO1-like corresponding to A, B and D genomes of wheat could be isolated from "NARC 2009". Due to frameshift and intronization/exonization events the gene structures of these paralogs exhibit variations in size. DRO1-like genes with five distinct domains prevail in diverse plant phyla from mosses to angiosperms but in lower plants their differentiation from LAZY, NGR and TAC1 (root and shoot angle genes) is enigmatic. Instead of IGT as denominator motif of this family, a new C-terminus motif WxxTD in the V-domain is proposed as family specific motif. The EAR-like motif IVLEM at the C-terminus of the TaADRO1-like and TaDDRO1-like that diverged to KLHTLIPNK in TaBDRO1-like and HvDRO1-like is the hallmark of these proteins. Split-YFP and yeast two hybrid assays complemented the interaction of TaDRO1-like with TOPLESS-a repressor of auxin regulated root promoting genes in plants-through IVLEM/KLHTLIPNK motif. Quantitative RT-PCR revealed abundance of DRO1-like RNA in root tips and spikelets while transcript signals were barely detectable in shoot and leaf tissues. Interestingly, wheat exhibited stronger expression of TaBDRO1-like than barley (HvDRO1-like), but TaBDRO1-like was the least expressing among three paralogs. The underlying cause of this expression divergence seems to be the presence of AuxRE motif TGTCTC and core TGTC with a coupling AuxRE-like motif ATTTTCTT proximal to the transcriptional start site in TaBDRO1-like and HvDRO1-like promoters. This is evident from binding of ARF1 to TGTCTC and TGTC motifs of TaBDRO1-like as revealed by yeast one-hybrid assay. Thus, evolution of DRO1-like wheat homoeologs might incorporate the C-terminus mutations as well as gain and loss of AuxREs and other cis-regulatory elements during expression divergence. Since root architecture is an important target trait for wheat crop improvement, therefore DRO1-like genes have potential applications in plant breeding for enhancement of plant productivity by the use of modern genome editing approaches.