Root-knot nematodes (RKNs) are important constraints to rice production worldwide. While Meloidogyne graminicola predominates in lowland rice, Meloidogyne incognita is a major pathogen of upland rice, where it can cause severe yield losses. These obligate endoparasites establish specialized feeding sites by secreting effector proteins that reprogram host cell development and suppress plant immunity. Here, we characterize Mi-SP5 (Minc14137), a M. incognita effector bearing a predicted i-type lysozyme/destabilase-like domain. In situ hybridization and quantitative RT-PCR demonstrate that Mi-SP5 is transcribed specifically in the subventral oesophageal glands of pre-parasitic second-stage juveniles (J2s) and is most highly expressed during early parasitism (6 days after inoculation). Transient expression of a Mi-SP5–GFP fusion in onion epidermal cells revealed a predominantly cell wall-associated distribution of the mature protein. In vitro siRNA soaking of J2s reduced Mi-SP5 transcript levels by ∼60% and significantly reduced the number of eggs produced per inoculated J2 by approximately 38%. Transgenic Nipponbare rice lines expressing artificial microRNA (amiRNA) targeting Mi-SP5 showed ∼80% reduction in Mi-SP5 transcripts in feeding nematodes and produced significantly fewer eggs per female (∼20% reduction; p ≤ 0.001), consistent across two independent single-copy transgenic events. Together, these findings support a role for Mi-SP5 as a virulence-associated effector contributing to nematode fecundity. These findings provide proof-of-concept that Mi-SP5 may represent one component of multi-target HIGS strategies aimed at improving resistance to root-knot nematodes in rice.

Cereal plants possess a fibrous root system in which crown roots form the major component. Crown roots develop post-embryonically from the lower, mostly underground nodes of the stem base. A strict spatiotemporal regulation of gene expression governs this process. Much of the knowledge about signaling pathways controlling crown root initiation (CRI) and development comes from rice. However, distinct regulatory mechanisms may have evolved in other cereals to adapt to different habitats. In this study, using a Crown Root Inducible System (CRIS), we aimed to investigate the early molecular regulation of barley CRI. We revealed dynamic transcriptomic changes within the first 24 hours following auxin stimulation. Among the differentially expressed genes, we identified orthologs of important CRI regulators from other cereals, demonstrating that CRIS is suitable for uncovering genes involved in CRI. Further, ATAC-seq revealed that CRI relies on changes in chromatin accessibility near root development-related genes and within distal intergenic regions. Finally, we focused on two transcription factors, HvNAC013 and CBF12C, which likely play roles in both CRI and abiotic stresses. By performing DAP-seq, we determined their genome-wide binding sites and identified their potential downstream targets. Data suggest that CBF12C is a putative target of HvNAC013, along with other auxin-responsive genes implicated in CRI. We propose that HvNAC013 and CBF12C function as part of a transcription factor network involved in CRI and potentially modulate root architecture in response to environmental conditions. This study enhances our understanding of the CRI mechanism in barley.

Plant-specific LOB-domain (LBD) transcription factors are crucial in post-embryonic root initiation. In cereals, the fibrous root system comprises primary and seminal roots that develop during embryogenesis and lateral- and crown roots that develop post-embryonically from root or stem, respectively. In rice, the CROWNROOTLESS1 (CRL1) LBD transcription factor is the core regulator of crown root initiation and a direct target of the auxin response factor (ARF)-mediated auxin signaling pathway. Orthologs of CRL1 have been identified and characterized in several species, where their role in crown root initiation has been validated. In barley, we identified two genes phylogenetically closely related to the rice CRL1 genes that we named HvCRL1 and HvCRL1L1. Using a crown root inducible system (CRIS), we identified that both HvCRL1 and HvCRL1L1 are expressed in response to auxin during the early steps of crown root differentiation in stem base, with HvCRL1 transcripts being accumulated quickly during the first hour of treatment. Transient activation assays in rice protoplast showed that HvCRL1 could bind the LBD-box, a consensus DNA sequence recognized by LBD transcription factors, whereas HvCRL1L1 did not. Both genes can partially complement the crl1 rice mutant. Loss-of-function mutation in each gene drastically impairs crown root formation in barley. These data show that HvCRL1 and HvCRL1L1 are both involved in the regulation of crown root formation in barley but that these two transcription factors likely act through distinct and complementary pathways in this developmental process.

Pearl millet (Pennisetum glaucum) thrives in arid and nutrient-poor environments, establishing its role as a crucial cereal crop for food security in sub-Saharan Africa. Despite its remarkable adaptability, its yields remain below genetic potential, primarily due to limited water and nutrient availability. In this study, we conducted ionomic profiling and genome-wide association studies (GWAS) in field conditions across two growing seasons to unravel the genetic basis of nutrient acquisition in pearl millet. Soil ion content analyses revealed significant differences in nutrient distribution between field sites, while certain ions, such as phosphorus (P) and zinc (Zn), consistently displayed stratified accumulation patterns across years, suggesting stable depth-dependent trends. Evaluation of a genetically diverse panel of inbred lines revealed substantial variation in leaf ion concentrations, with high heritability estimates. Correlations between leaf ion content and root anatomical or agromorphological traits highlighted the intricate interplay between genetic and environmental factors shaping leaf ion accumulation. These analyses also uncovered potential trade-offs in nutrient acquisition strategies. GWAS identified genomic regions associated with leaf ion concentrations, and the integration of genetic and gene expression data facilitated the identification of candidate genes implicated in ion transport and homeostasis. Our findings provide valuable insights into the genetic regulation of nutrient acquisition in pearl millet, offering potential targets for breeding nutrient-efficient and climate-resilient varieties. This study underscores the importance of integrating genetic, physiological, and root architectural traits to enhance agricultural productivity and sustainability in resource-constrained environments.

Pearl millet is a key food security grain crop in the world's drylands due to its tolerance to abiotic stresses. However, its yield remains low and is negatively impacted by climate change. Root phenes are potential targets to improve crop productivity and resilience to environmental stress. However, the sheer number of combinations resulting from interactions of multiple phenes is a challenge for empirical research. In silico approaches are a plausible alternative to assess the utility of different phene combinations in varying states over diverse environmental contexts. Here, we developed an implementation of the functional-structural plant/soil model – OpenSimRoot, for pearl millet in typical sub-Sahelian soil and environmental conditions. Root architectural, anatomical, and physiological parameters were measured using a popular pearl millet variety (Souna 3) and implemented in the model. The above-ground biomass and root length density predicted by the model were similar to data from field trials. The utility of different root phenes was then evaluated for improved phosphorus uptake and plant growth in P deficient soils. Doubled root hair length and density, shallower root angle (-15°) and doubled long lateral root density were found to improve plant growth by 76%, 33% and 33% respectively under low P conditions. Moreover, these phenes showed synergism when combined in silico and led to optimal biomass production in low P supply conditions that resulted in a 75% loss of biomass in the reference variety. Our study suggests that these phenotypes could be targeted to improve biomass production in pearl millet and consequently its yield in low-P availability conditions.

Seedling root traits impact plant establishment under challenging environments. Pearl millet is one of the most heat and drought tolerant cereal crops that provides a vital food source across the sub-Saharan Sahel region. Pearl millet’s early root system features a single fast-growing primary root which we hypothesize is an adaptation to the Sahelian climate. Using crop modeling, we demonstrate that early drought stress is an important constraint in agrosystems in the Sahel where pearl millet was domesticated. Furthermore, we show that increased pearl millet primary root growth is correlated with increased early water stress tolerance in field conditions. Genetics including genome-wide association study and quantitative trait loci (QTL) approaches identify genomic regions controlling this key root trait. Combining gene expression data, re-sequencing and re-annotation of one of these genomic regions identified a glutaredoxin-encoding gene PgGRXC9 as the candidate stress resilience root growth regulator. Functional characterization of its closest Arabidopsis homolog AtROXY19 revealed a novel role for this glutaredoxin (GRX) gene clade in regulating cell elongation. In summary, our study suggests a conserved function for GRX genes in conferring root cell elongation and enhancing resilience of pearl millet to its Sahelian environment.

Nitrogen is an essential nutrient for plants and a major determinant of plant growth and crop yield. Plants acquire nitrogen mainly in the form of nitrate and ammonium. Both nitrogen sources affect plant responses and signaling pathways in a different way, but these signaling pathways interact, complicating the study of nitrogen responses. Extensive transcriptome analyses and the construction of gene regulatory networks, mainly in response to nitrate, have significantly advanced our understanding of nitrogen signaling and responses in model plants and crops. In this study, we aimed to generate a more comprehensive gene regulatory network for the major crop, rice, by incorporating the interactions between ammonium and nitrate. To achieve this, we assessed transcriptome changes in rice roots and shoots over an extensive time course under single or combined applications of the two nitrogen sources. This dataset enabled us to construct a holistic co-expression network and identify potential key regulators of nitrogen responses. Next to known transcription factors, we identified multiple new candidates, including the transcription factors OsRLI and OsEIL1, which we demonstrated to induce the primary nitrate-responsive genes OsNRT1.1b and OsNIR1. Our network thus serves as a valuable resource to obtain novel insights in nitrogen signaling.

Rice (Oryza sativa L.) is a staple food for half of the world’s population, and its biofortification is a key factor in fighting micronutrient malnutrition. However, harmful heavy metals tend to accumulate in rice grains due to soil and water contamination. Therefore, it is important to improve beneficial micronutrient contents and reduce the accumulation of undesirable metals in rice grain. To better characterize the genetic control of mineral accumulation in rice, we conducted association genetics on the ion contents of white and brown grains using a collection of 184 Vietnamese rice landraces. In total, 27 significant associations were identified and delimited into quantitative trait loci associated with macronutrients such as phosphorus, potassium or calcium; micronutrients such as iron or zinc; or toxic heavy metals such as arsenic and cadmium. Several genes related to ion homeostasis or ion transport were identified in the different quantitative trait loci. LOC_Os10g30610, present in qRAs10-1 associated with arsenic content in brown rice, encodes an ABC transporter (OsABCG25), which is involved in the silicon-induced formation of the Casparian strip in the rice exodermis and could act as a barrier restricting As diffusion within the root cortex. LOC_Os05g04330, present in qRP5-1 and associated with phosphorus content in brown rice, encodes a CHH methylation maintenance protein, and its expression is downregulated in roots in the presence of the phosphorus uptake 1 (Pup1), suggesting a role for epigenetics in the regulation of phosphorus uptake and accumulation in grain. These findings reveal novel quantitative trait loci associated with grain ion content and candidate genes that are potentially valuable for breeding programs aimed at rice grain biofortification and reducing toxic metal accumulation.

Pearl millet is a key cereal for food security in drylands but its yield is strongly impacted by drought. We investigated how root anatomical traits contribute to mitigating the effects of vegetative drought stress in pearl millet. We examined associations between root anatomical traits and agronomical performance in a pearl millet diversity panel under irrigated and vegetative drought stress treatments in field trials. The impact of associated anatomical traits on transpiration was assessed using subpanels grown in different soil within a greenhouse. In the field, total metaxylem area was positively correlated with grain weight and its maintenance under drought. In the greenhouse, genotypes with larger metaxylem area grown in sandy soil exhibited a consumerist water use strategy under irrigation, which shifted to a conservative strategy under drought. Water savings was mediated by transpiration restriction under high evaporative demand. This mechanism was dependent on soil hydraulics as it was not observed in peat soil with higher hydraulic conductivity upon soil drying. We propose that water savings under drought, mediated by large metaxylem area and its interaction with soil hydraulics, help mitigate vegetative drought stress. Our findings highlight the role of soil hydraulic properties in shaping plant hydraulics and drought tolerance. ### Competing Interest Statement The authors have declared no competing interest.

Mini zinc fingers constitute a class of microproteins that appeared early in evolution and expanded in seeds plants. In this review, the phylogenetic history, the functions and the mode of action of Mini zinc fingers in plants are reported and discussed. It appears that mini zinc fingers play an important role in the control of plant development. They are involved in the control of cell division and expansion, in the switch between the determinate/indeterminate state of the meristems and in the regulation of vegetative growth and floral organ development. Their biochemical mode of action seems to be diverse. In some studies, it has been reported that mini zinc fingers can directly bind to DNA and activate target gene expression, whereas other studies have shown that they can interact with and inhibit the activity of specific zinc finger homeodomain transcription factors or act as adaptor proteins necessary to aggregate polymeric protein complexes corresponding to chromatin remodelling factors negatively regulating the expression of specific genes. The diversity of mode of action for mini zinc finger microproteins suggests a wider range of biological functions than what has been that described in the literature thus far, and their involvement in the response to biotic and abiotic stresses should be further investigated in future studies.

Lateral root (LR) formation is a postembryonic organogenesis process that is crucial for plant root system development and adaptation to heterogenous soil environments. Since the early 1990s, a wealth of experimental data on arabidopsis (Arabidopsis thaliana) has helped reveal the LR formation regulatory network, in which dynamic auxin distribution and transcriptional cascades direct root cells through their organogenesis pathway. Some parts of this network appear conserved across diverse plant species or distinct developmental contexts. Recently, our knowledge of this process dramatically expanded thanks to technical advances, from single cell profiling to whole-root system phenotyping. Interestingly, new players are now emerging in this network, such as fatty acids and reactive oxygen species (ROS), transforming our knowledge of this hidden half of plant biology.

Background Roots play an important role during plant growth and development, ensuring water and nutrient uptake. Understanding the mechanisms regulating their initiation and development opens doors towards root system architecture engineering. Results Here, we investigated by RNA-seq analysis the changes in gene expression in the barley stem base of 1 day-after-germination (DAG) and 10DAG seedlings when crown roots are formed. We identified 2,333 genes whose expression was lower in the stem base of 10DAG seedlings compared to 1DAG seedlings. Those genes were mostly related to basal cellular activity such as cell cycle organization, protein biosynthesis, chromatin organization, cytoskeleton organization or nucleotide metabolism. In opposite, 2,932 genes showed up-regulation in the stem base of 10DAG seedlings compared to 1DAG seedlings, and their function was related to phytohormone action, solute transport, redox homeostasis, protein modification, secondary metabolism. Our results highlighted genes that are likely involved in the different steps of crown root formation from initiation to primordia differentiation and emergence, and revealed the activation of different hormonal pathways during this process. Conclusions This whole transcriptomic study is the first study aiming at understanding the molecular mechanisms controlling crown root development in barley. The results shed light on crown root emergence that is likely associated with a strong cell wall modification, death of the cells covering the crown root primordium, and the production of defense molecules that might prevent pathogen infection at the site of root emergence.

COI1-mediated perception of jasmonate is critical for plant development and responses to environmental stresses. Monocots such as rice have two groups of COI genes due to gene duplication: OsCOI1a and OsCOI1b that are functionally equivalent to the dicotyledons COI1 and OsCOI2 whose function remains unclear. In order to assess the function of OsCOI2 and its functional redundancy with COI1 genes, we developed a series of rice mutants in the 3 genes OsCOI1a, OsCOI1b and OsCOI2 by CRISPR Cas9-mediated editing and characterized their phenotype and responses to jasmonate. Characterization of OsCOI2 uncovered its important roles in root, leaf and flower development. In particular, we show that crown root growth inhibition by jasmonate relies on OsCOI2 and not on OsCOI1a nor on OsCOI1b, revealing a major function for the non-canonical OsCOI2 in jasmonate-dependent control of rice root growth. Collectively, these results point to a specialized function of OsCOI2 in the regulation of plant development in rice and indicate that sub-functionalisation of jasmonate receptors has occurred in the monocot phylum.

Water availability is undoubtedly one of the most important environmental factors affecting crop production. Drought causes a gradual deprivation of water in the soil from top to deep layers and can occur at diverse stages of plant development. Roots are the first organs that perceive water deficit in soil and their adaptive development contributes to drought adaptation. Domestication has contributed to a bottleneck in genetic diversity. Wild species or landraces represent a pool of genetic diversity that has not been exploited yet in breeding program. In this study, we used a collection of 230 two-row spring barley landraces to detect phenotypic variation in root system plasticity in response to drought and to identify new quantitative trait loci (QTL) involved in root system architecture under diverse growth conditions. For this purpose, young seedlings grown for 21 days in pouches under control and osmotic-stress conditions were phenotyped and genotyped using the barley 50k iSelect SNP array, and genome-wide association studies (GWAS) were conducted using three different GWAS methods (MLM GAPIT, FarmCPU, and BLINK) to detect genotype/phenotype associations. In total, 276 significant marker-trait associations (MTAs; p-value (FDR)< 0.05) were identified for root (14 and 12 traits under osmotic-stress and control conditions, respectively) and for three shoot traits under both conditions. In total, 52 QTL (multi-trait or identified by at least two different GWAS approaches) were investigated to identify genes representing promising candidates with a role in root development and adaptation to drought stress.

The rhizosheath, the layer of soil that adheres strongly to roots, influences water and nutrients acquisition. Pearl millet is a cereal crop that plays a major role for food security in arid regions of sub-Saharan Africa and India. We previously showed that root-adhering soil mass is a heritable trait in pearl millet and that it correlates with changes in rhizosphere microbiota structure and functions. Here, we studied the correlation between root-adhering soil mass and root hair development, root architecture, and symbiosis with arbuscular mycorrhizal fungi and we analysed the genetic control of this trait using genome wide association (GWAS) combined with bulk segregant analysis and gene expression studies. Root-adhering soil mass was weakly correlated only to root hairs traits in pearl millet. Twelve QTLs for rhizosheath formation were identified by GWAS. Bulk segregant analysis on a biparental population validated five of these QTLs. Combining genetics with a comparison of global gene expression in the root tip of contrasted inbred lines revealed candidate genes that might control rhizosheath formation in pearl millet. Our study indicates that rhizosheath formation is under complex genetic control in pearl millet and suggests that it is mainly regulated by root exudation.

Abstract Lateral root organogenesis is a key process in the development of a plant’s root system and its adaptation to the environment. During lateral root formation, an early phase of cell proliferation first produces a four-cell-layered primordium, and only from this stage onwards is a root meristem-like structure, expressing root stem cell niche marker genes, being established in the developing organ. Previous studies reported that the gene regulatory network controlling lateral root formation is organized into two subnetworks whose mutual inhibition may contribute to organ patterning. PUCHI encodes an AP2/ERF transcription factor expressed early during lateral root primordium development and required for correct lateral root formation. To dissect the molecular events occurring during this early phase, we generated time-series transcriptomic datasets profiling lateral root development in puchi-1 mutants and wild types. Transcriptomic and reporter analyses revealed that meristem-related genes were expressed ectopically at early stages of lateral root formation in puchi-1 mutants. We conclude that, consistent with the inhibition of genetic modules contributing to lateral root development, PUCHI represses ectopic establishment of meristematic cell identities at early stages of organ development. These findings shed light on gene network properties that orchestrate correct timing and patterning during lateral root formation.