CROWN ROOTLESS1 binds DNA with a relaxed specificity and activates OsROP and OsbHLH044 genes involved in crown root formation in rice.

In cereals, the root system is mainly composed of post-embryonic shoot-borne roots, named crown roots. The CROWN ROOTLESS1 (CRL1) transcription factor, belonging to the ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES DOMAIN (ASL/LBD) family, is a key regulator of crown root initiation in rice (Oryza sativa). Here, we show that CRL1 can bind, both in vitro and in vivo, not only the LBD-box, a DNA sequence recognized by several ASL/LBD transcription factors, but also another not previously identified DNA motif that was named CRL1-box. Using rice protoplast transient transactivation assays and a set of previously identified CRL1-regulated genes, we confirm that CRL1 transactivates these genes if they possess at least a CRL1-box or an LBD-box in their promoters. In planta, ChIP-qPCR experiments targeting two of these genes that include both a CRL1- and an LBD-box in their promoter show that CRL1 binds preferentially to the LBD-box in these promoter contexts. CRISPR/Cas9-targeted mutation of these two CRL1-regulated genes, which encode a plant Rho GTPase (OsROP) and a basic helix-loop-helix transcription factor (OsbHLH044), show that both promote crown root development. Finally, we show that OsbHLH044 represses a regulatory module, uncovering how CRL1 regulates specific processes during crown root formation.

Physiological and genetic control of transpiration efficiency in African rice, Oryza glaberrima Steud.

Improving crop water use efficiency, the amount of carbon assimilated as biomass per unit of water used by a plant, is of major importance as water for agriculture becomes scarcer. In rice, the genetic bases of transpiration efficiency, the derivation of water use efficiency at the whole-plant scale, and its putative component trait transpiration restriction under high evaporative demand remain unknown. These traits were measured in 2019 in a panel of 147 African rice (Oryza glaberrima) genotypes known to be potential sources of tolerance genes to biotic and abiotic stresses. Our results reveal that higher transpiration efficiency is associated with transpiration restriction in African rice. Detailed measurements in a subset of highly contrasted genotypes in terms of biomass accumulation and transpiration confirmed these associations and suggested that root to shoot ratio played an important role in transpiration restriction. Genome wide association studies identified marker-trait associations for transpiration response to evaporative demand, transpiration efficiency, and its residuals, with links to genes involved in water transport and cell wall patterning. Our data suggest that root-shoot partitioning is an important component of transpiration restriction that has a positive effect on transpiration efficiency in African rice. Both traits are heritable and define targets for breeding rice with improved water use strategies.

A New Phenotyping Pipeline Reveals Three Types of Lateral Roots and a Random Branching Pattern in Two Cereals.

Recent progress in root phenotyping has focused mainly on increasing throughput for genetic studies, while identifying root developmental patterns has been comparatively underexplored. We introduce a new phenotyping pipeline for producing high-quality spatiotemporal root system development data and identifying developmental patterns within these data. The SmartRoot image-analysis system and temporal and spatial statistical models were applied to two cereals, pearl millet (Pennisetum glaucum) and maize (Zea mays). Semi-Markov switching linear models were used to cluster lateral roots based on their growth rate profiles. These models revealed three types of lateral roots with similar characteristics in both species. The first type corresponds to fast and accelerating roots, the second to rapidly arrested roots, and the third to an intermediate type where roots cease elongation after a few days. These types of lateral roots were retrieved in different proportions in a maize mutant affected in auxin signaling, while the first most vigorous type was absent in maize plants exposed to severe shading. Moreover, the classification of growth rate profiles was mirrored by a ranking of anatomical traits in pearl millet. Potential dependencies in the succession of lateral root types along the primary root were then analyzed using variable-order Markov chains. The lateral root type was not influenced by the shootward neighbor root type or by the distance from this root. This random branching pattern of primary roots was remarkably conserved, despite the high variability of root systems in both species. Our phenotyping pipeline opens the door to exploring the genetic variability of lateral root developmental patterns.

Chitinase-resistant hydrophilic symbiotic factors secreted by Frankia activate both Ca(2+) spiking and NIN gene expression in the actinorhizal plant Casuarina glauca.

Although it is now well-established that decorated lipo-chitooligosaccharide Nod factors are the key rhizobial signals which initiate infection/nodulation in host legume species, the identity of the equivalent microbial signaling molecules in the Frankia/actinorhizal association remains elusive. With the objective of identifying Frankia symbiotic factors we present a novel approach based on both molecular and cellular pre-infection reporters expressed in the model actinorhizal species Casuarina glauca. By introducing the nuclear-localized cameleon Nup-YC2.1 into Casuarina glauca we show that cell-free culture supernatants of the compatible Frankia CcI3 strain are able to elicit sustained high frequency Ca(2+) spiking in host root hairs. Furthermore, an excellent correlation exists between the triggering of nuclear Ca(2+) spiking and the transcriptional activation of the ProCgNIN:GFP reporter as a function of the Frankia strain tested. These two pre-infection symbiotic responses have been used in combination to show that the signal molecules present in the Frankia CcI3 supernatant are hydrophilic, of low molecular weight and resistant to chitinase degradation. In conclusion, the biologically active symbiotic signals secreted by Frankia appear to be chemically distinct from the currently known chitin-based rhizobial/arbuscular mycorrhizal signaling molecules. Convenient bioassays in Casuarina glauca are now available for their full characterization.

Inhibition of auxin signaling in Frankia species-infected cells in Casuarina glauca nodules leads to increased nodulation.

Actinorhizal symbioses are mutualistic interactions between plants and the soil bacteria Frankia spp. that lead to the formation of nitrogen-fixing root nodules. The plant hormone auxin has been suggested to play a role in the mechanisms that control the establishment of this symbiosis in the actinorhizal tree Casuarina glauca. Here, we analyzed the role of auxin signaling in Frankia spp.-infected cells. Using a dominant-negative version of an endogenous auxin-signaling regulator, INDOLE-3-ACETIC ACID7, we established that inhibition of auxin signaling in these cells led to increased nodulation and, as a consequence, to higher nitrogen fixation per plant even if nitrogen fixation per nodule mass was similar to that in the wild type. Our results suggest that auxin signaling in Frankia spp.-infected cells is involved in the long-distance regulation of nodulation in actinorhizal symbioses.

The Casuarina NIN gene is transcriptionally activated throughout Frankia root infection as well as in response to bacterial diffusible signals.

Root nodule symbioses (RNS) allow plants to acquire atmospheric nitrogen by establishing an intimate relationship with either rhizobia, the symbionts of legumes or Frankia in the case of actinorhizal plants. In legumes, NIN (Nodule INception) genes encode key transcription factors involved in nodulation. Here we report the characterization of CgNIN, a NIN gene from the actinorhizal tree Casuarina glauca using both phylogenetic analysis and transgenic plants expressing either ProCgNIN::reporter gene fusions or CgNIN RNAi constructs. We have found that CgNIN belongs to the same phylogenetic group as other symbiotic NIN genes and CgNIN is able to complement a legume nin mutant for the early steps of nodule development. CgNIN expression is correlated with infection by Frankia, including preinfection stages in developing root hairs, and is induced by culture supernatants. Knockdown mutants were impaired for nodulation and early root hair deformation responses were severely affected. However, no mycorrhizal phenotype was observed and no induction of CgNIN expression was detected in mycorrhizas. Our results indicate that elements specifically required for nodulation include NIN and possibly related gene networks derived from the nitrate signalling pathways.

CRISPR/Cas9-Mediated Gene Editing of the Jasmonate Biosynthesis OsAOC Gene in Rice.

The function of Jasmonate (JA) is well documented in different plant physiological processes as well as in the interactions with their environment. Mutants impaired in JA production and/or signaling are useful materials to study the function of this phytohormone. Genes involved in the JA biosynthesis pathway in rice have been described, but few mutants deficient in JA production and signaling have been identified. Moreover, these mutants are mostly generated through random mutagenesis approaches, such as irradiation, EMS treatment, or T-DNA insertion, and potentially harbor undesired mutations that could affect other biological processes. The CRISPR/Cas9 system is a precise and efficient genome editing tool that creates DNA modification at specific loci and limit undesired mutations.In this chapter, we describe a procedure to generate new JA-deficient mutant using CRISPR/Cas9 system in rice. The Allene Oxide Cyclase (OsAOC) gene is targeted since it is a single copy gene in the JA biosynthesis pathway in rice. The widely used variety Oryza sativa japonica Kitaake has been chosen due to its short life cycle and its ease of genetic transformation. This protocol describes the selection of the 20-nt target sequence, construction of the binary vector, and strategy for selecting the T-DNA-free mutant.

Characterization of Pearl Millet Root Architecture and Anatomy Reveals Three Types of Lateral Roots.

Pearl millet plays an important role for food security in arid regions of Africa and India. Nevertheless, it is considered an orphan crop as it lags far behind other cereals in terms of genetic improvement efforts. Breeding pearl millet varieties with improved root traits promises to deliver benefits in water and nutrient acquisition. Here, we characterize early pearl millet root system development using several different root phenotyping approaches that include rhizotrons and microCT. We report that early stage pearl millet root system development is characterized by a fast growing primary root that quickly colonizes deeper soil horizons. We also describe root anatomical studies that revealed three distinct types of lateral roots that form on both primary roots and crown roots. Finally, we detected significant variation for two root architectural traits, primary root lenght and lateral root density, in pearl millet inbred lines. This study provides the basis for subsequent genetic experiments to identify loci associated with interesting early root development traits in this important cereal.