Far-red light-enhanced apical dominance stimulates flower and fruit abortion in sweet pepper.

Far-red radiation affects many plant processes, including reproductive organ abortion. Our research aimed to determine the role of apical dominance in far-red light-induced flower and fruit abortion in sweet pepper (Capsicum annuum L.). We conducted several climate room experiments where plants were grown under white- or red-rich LED light, with or without additional far-red light. Additional far-red light enhanced apical dominance: it increased auxin levels in the apices of dominant shoots, and caused a greater difference in internode length and apical auxin levels between dominant and subordinate shoots. Additional far-red light stimulated fruit abortion in intact plants but not in decapitated plants, suggesting a crucial role of shoot apices in this effect. However, reducing basipetal auxin transport in the stems with N-1-naphthylphthalamic acid did not influence far-red light-stimulated fruit abortion, although auxin levels in the stem were largely reduced. Applying the synthetic auxin 1-naphthaleneacetic acid on decapitated apices did not influence fruit abortion. However, applying the auxin biosynthesis inhibitor yucasin to shoot apices reduced fruit abortion regardless of the light conditions, accompanied by slight shoot growth retardation. These findings suggest that the basipetal auxin stream does not mediate far-red light-stimulated fruit abortion. Far-red light-stimulated fruit abortion was associated with reduced sucrose accumulation and lower invertase activities in flowers. We suggest that under additional far-red light conditions, increased auxin levels in shoot apices promote fruit abortion probably through enhanced competition for assimilates between apices and flowers, which limits assimilate import into flowers.

The symbiosome-a transient organelle in evolution.

This article comments on: Casaes PA, Ferreira dos Santos JM, Silva VC, Rhem MFK, Teixeira Cota MM, de Faria SM, Rando JG, James EK, Gross E. 2024. The radiation of nodulated Chamaecrista species from the rainforest into more diverse habitats has been accompanied by a reduction in growth form and a shift from fixation threads to symbiosomes. Journal of Experimental Botany 75, 3643-3662.

The Compact Root Architecture 2 systemic pathway is required for the induction of cytokinins and of the miR399 in Medicago truncatula N-satisfied plants.

Plants use a combination of sophisticated local and systemic pathways to optimize growth depending on heterogeneous nutrient availability in the soil. Legume plants can acquire mineral nitrogen (N) either through their roots or via a symbiotic interaction with N-fixing rhizobia bacteria housed in so-called root nodules. To identify shoot-to-root systemic signals acting in Medicago truncatula plants at N-deficit or N-satiety, plants were grown in a split-root experimental design, in which either high or low N was provided to a half of the root system, allowing the analysis of systemic pathways independently of any local N response. Among the plant hormone families analyzed, the cytokinin trans-Zeatin accumulated in plants at N-satiety. Cytokinin application by petiole feeding led to an inhibition of both root growth and nodulation. In addition, an exhaustive analysis of miRNAs revealed that miR2111 accumulates systemically under N-deficit in both shoots and non-treated distant roots, whereas a miRNA related to inorganic Phosphate (Pi)-acquisition, the miR399, does so in plants grown at N-satiety. These two accumulation patterns are dependent on CRA2 (Compact Root Architecture 2), a receptor required for CEP (C-terminally Encoded Peptide) signaling. Constitutive ectopic expression of the miR399 reduced nodule numbers and root biomass depending on Pi availability, suggesting that the miR399-dependent Pi-acquisition regulatory module controlled by N-availability affects the development of the whole legume plant root system.

Medicago SPX1 and SPX3 regulate phosphate homeostasis, mycorrhizal colonization, and arbuscule degradation.

To acquire sufficient mineral nutrients such as phosphate (Pi) from the soil, most plants engage in symbiosis with arbuscular mycorrhizal (AM) fungi. Attracted by plant-secreted strigolactones (SLs), the fungi colonize the roots and form highly branched hyphal structures called arbuscules inside inner cortex cells. The host plant must control the different steps of this interaction to maintain its symbiotic nature. However, how plants sense the amount of Pi obtained from the fungus, and how this determines the arbuscule lifespan, are far from understood. Here, we show that Medicago truncatula SPX-domain containing proteins SPX1 and SPX3 regulate root Pi starvation responses, in part by interacting with PHOSPHATE RESPONSE REGULATOR2, as well as fungal colonization and arbuscule degradation. SPX1 and SPX3 are induced upon Pi starvation but become more restricted to arbuscule-containing cells upon the establishment of symbiosis. This induction in arbuscule-containing cells is associated with the presence of cis-regulatory AW-boxes and transcriptional regulation by the WRINKLED1-like transcription factor WRI5a. Under Pi-limiting conditions, SPX1 and SPX3 facilitate the expression of the SL biosynthesis gene DWARF27, which could help explain the increased fungal branching in response to root exudates. Later, in arbuscule-containing cells, SPX1 and SPX3 redundantly control arbuscule degradation. Thus, SPX proteins play important roles as phosphate sensors to maintain a beneficial AM symbiosis.

A rare non-canonical splice site in Trema orientalis SYMRK does not affect its dual symbiotic functioning in endomycorrhiza and rhizobium nodulation.

BACKGROUND: Nitrogen-fixing nodules occur in ten related taxonomic lineages interspersed with lineages of non-nodulating plant species. Nodules result from an endosymbiosis between plants and diazotrophic bacteria; rhizobia in the case of legumes and Parasponia and Frankia in the case of actinorhizal species. Nodulating plants share a conserved set of symbiosis genes, whereas related non-nodulating sister species show pseudogenization of several key nodulation-specific genes. Signalling and cellular mechanisms critical for nodulation have been co-opted from the more ancient plant-fungal arbuscular endomycorrhizal symbiosis. Studies in legumes and actinorhizal plants uncovered a key component in symbiotic signalling, the LRR-type SYMBIOSIS RECEPTOR KINASE (SYMRK). SYMRK is essential for nodulation and arbuscular endomycorrhizal symbiosis. To our surprise, however, despite its arbuscular endomycorrhizal symbiosis capacities, we observed a seemingly critical mutation in a donor splice site in the SYMRK gene of Trema orientalis, the non-nodulating sister species of Parasponia. This led us to investigate the symbiotic functioning of SYMRK in the Trema-Parasponia lineage and to address the question of to what extent a single nucleotide polymorphism in a donor splice site affects the symbiotic functioning of SYMRK. RESULTS: We show that SYMRK is essential for nodulation and endomycorrhization in Parasponia andersonii. Subsequently, it is revealed that the 5'-intron donor splice site of SYMRK intron 12 is variable and, in most dicotyledon species, doesn't contain the canonical dinucleotide 'GT' signature but the much less common motif 'GC'. Strikingly, in T. orientalis, this motif is converted into a rare non-canonical 5'-intron donor splice site 'GA'. This SYMRK allele, however, is fully functional and spreads in the T. orientalis population of Malaysian Borneo. A further investigation into the occurrence of the non-canonical GA-AG splice sites confirmed that these are extremely rare. CONCLUSION: SYMRK functioning is highly conserved in legumes, actinorhizal plants, and Parasponia. The gene possesses a non-common 5'-intron GC donor splice site in intron 12, which is converted into a GA in T. orientalis accessions of Malaysian Borneo. The discovery of this functional GA-AG splice site in SYMRK highlights a gap in our understanding of splice donor sites.

Long-lasting impact of chitooligosaccharide application on strigolactone biosynthesis and fungal accommodation promotes arbuscular mycorrhiza in Medicago truncatula.

The establishment of arbuscular mycorrhiza (AM) between plants and Glomeromycotina fungi is preceded by the exchange of chemical signals: fungal released Myc-factors, including chitooligosaccharides (CO) and lipo-chitooligosaccharides (LCO), activate plant symbiotic responses, while root-exuded strigolactones stimulate hyphal branching and boost CO release. Furthermore, fungal signaling reinforcement through CO application was shown to promote AM development in Medicago truncatula, but the cellular and molecular bases of this effect remained unclear. Here, we focused on long-term M. truncatula responses to CO treatment, demonstrating its impact on the transcriptome of both mycorrhizal and nonmycorrhizal roots over several weeks and providing an insight into the mechanistic bases of the CO-dependent promotion of AM colonization. CO treatment caused the long-lasting regulation of strigolactone biosynthesis and fungal accommodation-related genes. This was mirrored by an increase in root didehydro-orobanchol content, and the promotion of accommodation responses to AM fungi in root epidermal cells. Lastly, an advanced downregulation of AM symbiosis marker genes was observed at the latest time point in CO-treated plants, in line with an increased number of senescent arbuscules. Overall, CO treatment triggered molecular, metabolic, and cellular responses underpinning a protracted acceleration of AM development.

A Remote cis-Regulatory Region Is Required for NIN Expression in the Pericycle to Initiate Nodule Primordium Formation in Medicago truncatula.

The legume-rhizobium symbiosis results in nitrogen-fixing root nodules, and their formation involves both intracellular infection initiated in the epidermis and nodule organogenesis initiated in inner root cell layers. NODULE INCEPTION (NIN) is a nodule-specific transcription factor essential for both processes. These NIN-regulated processes occur at different times and locations in the root, demonstrating a complex pattern of spatiotemporal regulation. We show that regulatory sequences sufficient for the epidermal infection process are located within a 5 kb region directly upstream of the NIN start codon in Medicago truncatula Furthermore, we identify a remote upstream cis-regulatory region required for the expression of NIN in the pericycle, and we show that this region is essential for nodule organogenesis. This region contains putative cytokinin response elements and is conserved in eight more legume species. Both the cytokinin receptor 1, which is essential for nodule primordium formation, and the B-type response regulator RR1 are expressed in the pericycle in the susceptible zone of the uninoculated root. This, together with the identification of the cytokinin-responsive elements in the NIN promoter, strongly suggests that NIN expression is initially triggered by cytokinin signaling in the pericycle to initiate nodule primordium formation.

HISTONE DEACETYLASE 9 stimulates auxin-dependent thermomorphogenesis in Arabidopsis thaliana by mediating H2A.Z depletion.

Many plant species respond to unfavorable high ambient temperatures by adjusting their vegetative body plan to facilitate cooling. This process is known as thermomorphogenesis and is induced by the phytohormone auxin. Here, we demonstrate that the chromatin-modifying enzyme HISTONE DEACETYLASE 9 (HDA9) mediates thermomorphogenesis but does not interfere with hypocotyl elongation during shade avoidance. HDA9 is stabilized in response to high temperature and mediates histone deacetylation at the YUCCA8 locus, a rate-limiting enzyme in auxin biosynthesis, at warm temperatures. We show that HDA9 permits net eviction of the H2A.Z histone variant from nucleosomes associated with YUCCA8, allowing binding and transcriptional activation by PHYTOCHROME INTERACTING FACTOR 4, followed by auxin accumulation and thermomorphogenesis.

Duplication of Symbiotic Lysin Motif Receptors Predates the Evolution of Nitrogen-Fixing Nodule Symbiosis.

Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2 These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.

Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses.

Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete Frankia as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using Parasponia species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between Parasponia andersonii and its nonnodulating relative Trema tomentosa demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.

The Medicago truncatula nodule identity gene MtNOOT1 is required for coordinated apical-basal development of the root.

BACKGROUND: Legumes can utilize atmospheric nitrogen by hosting nitrogen-fixing bacteria in special lateral root organs, called nodules. Legume nodules have a unique ontology, despite similarities in the gene networks controlling nodule and lateral root development. It has been shown that Medicago truncatula NODULE ROOT1 (MtNOOT1) is required for the maintenance of nodule identity, preventing the conversion to lateral root development. MtNOOT1 and its orthologs in other plant species -collectively called the NOOT-BOP-COCH-LIKE (NBCL) family- specify boundary formation in various aerial organs. However, MtNOOT1 is not only expressed in nodules and aerial organs, but also in developing roots, where its function remains elusive. RESULTS: We show that Mtnoot1 mutant seedlings display accelerated root elongation due to an enlarged root apical meristem. Also, Mtnoot1 mutant roots are thinner than wild-type and are delayed in xylem cell differentiation. We provide molecular evidence that the affected spatial development of Mtnoot1 mutant roots correlates with delayed induction of genes involved in xylem cell differentiation. This coincides with a basipetal shift of the root zone that is susceptible to rhizobium-secreted symbiotic signal molecules. CONCLUSIONS: Our data show that MtNOOT1 regulates the size of the root apical meristem and vascular differentiation. Our data demonstrate that MtNOOT1 not only functions as a homeotic gene in nodule development but also coordinates the spatial development of the root.

Auxin transport, metabolism, and signalling during nodule initiation: indeterminate and determinate nodules.

Most legumes can form a unique type of lateral organ on their roots: root nodules. These structures host symbiotic nitrogen-fixing bacteria called rhizobia. Several different types of nodules can be found in nature, but the two best-studied types are called indeterminate and determinate nodules. These two types differ with respect to the presence or absence of a persistent nodule meristem, which consistently correlates with the cortical cell layers giving rise to the nodule primordia. Similar to other plant developmental processes, auxin signalling overlaps with the site of organ initiation and meristem activity. Here, we review how auxin contributes to early nodule development. We focus on changes in auxin transport, signalling, and metabolism during nodule initiation, describing both experimental evidence and computer modelling. We discuss how indeterminate and determinate nodules may differ in their mechanisms for generating localized auxin response maxima and highlight outstanding questions for future research.

A petunia ABC protein controls strigolactone-dependent symbiotic signalling and branching.

Strigolactones were originally identified as stimulators of the germination of root-parasitic weeds that pose a serious threat to resource-limited agriculture. They are mostly exuded from roots and function as signalling compounds in the initiation of arbuscular mycorrhizae, which are plant-fungus symbionts with a global effect on carbon and phosphate cycling. Recently, strigolactones were established to be phytohormones that regulate plant shoot architecture by inhibiting the outgrowth of axillary buds. Despite their importance, it is not known how strigolactones are transported. ATP-binding cassette (ABC) transporters, however, are known to have functions in phytohormone translocation. Here we show that the Petunia hybrida ABC transporter PDR1 has a key role in regulating the development of arbuscular mycorrhizae and axillary branches, by functioning as a cellular strigolactone exporter. P. hybrida pdr1 mutants are defective in strigolactone exudation from their roots, resulting in reduced symbiotic interactions. Above ground, pdr1 mutants have an enhanced branching phenotype, which is indicative of impaired strigolactone allocation. Overexpression of Petunia axillaris PDR1 in Arabidopsis thaliana results in increased tolerance to high concentrations of a synthetic strigolactone, consistent with increased export of strigolactones from the roots. PDR1 is the first known component in strigolactone transport, providing new opportunities for investigating and manipulating strigolactone-dependent processes.

Auxin Depletion from the Leaf Axil Conditions Competence for Axillary Meristem Formation in Arabidopsis and Tomato.

The enormous variation in architecture of flowering plants is based to a large extent on their ability to form new axes of growth throughout their life span. Secondary growth is initiated from groups of pluripotent cells, called meristems, which are established in the axils of leaves. Such meristems form lateral organs and develop into a side shoot or a flower, depending on the developmental status of the plant and environmental conditions. The phytohormone auxin is well known to play an important role in inhibiting the outgrowth of axillary buds, a phenomenon known as apical dominance. However, the role of auxin in the process of axillary meristem formation is largely unknown. In this study, we show in the model species Arabidopsis thaliana and tomato (Solanum lycopersicum) that auxin is depleted from leaf axils during vegetative development. Disruption of polar auxin transport compromises auxin depletion from the leaf axil and axillary meristem initiation. Ectopic auxin biosynthesis in leaf axils interferes with axillary meristem formation, whereas repression of auxin signaling in polar auxin transport mutants can largely rescue their branching defects. These results strongly suggest that depletion of auxin from leaf axils is a prerequisite for axillary meristem formation during vegetative development.

Lateral suppressor and Goblet act in hierarchical order to regulate ectopic meristem formation at the base of tomato leaflets.

In seed plants, new axes of growth are established by the formation of meristems, groups of pluripotent cells that maintain themselves and initiate the formation of lateral organs. After embryonic development, secondary shoot meristems form in the boundary zones between the shoot apical meristem and leaf primordia, the leaf axils. In addition, many plant species develop ectopic meristems at different positions of the plant body. In the compound tomato leaf, ectopic meristems can initiate at the base of leaflets, which are delimited by two distinct boundary zones, referred to as the proximal (PLB) and distal (DLB) leaflet boundaries. We demonstrate that the two leaflet boundaries differ from each other and that ectopic meristem formation is strictly limited to the DLB. Our data suggest that the DLB harbours a group of pluripotent cells that seems to be the launching pad for meristem formation. Initiation of these meristems is dependent on the activities of the transcriptional regulators Goblet (Gob) and Lateral suppressor (Ls), specifically expressed in the DLB. Gob and Ls act in hierarchical order, because Ls transcript accumulation is dependent on Gob activity, but not vice versa. Ectopic meristem formation at the DLB is also observed in other seed plants, like Cardamine pratensis, indicating that it is part of a widespread developmental program. Ectopic meristem formation leads to an increase in the number of buds, enhances the capacity for survival and opens the route to vegetative propagation.

Quantitative modelling of legume root nodule primordium induction by a diffusive signal of epidermal origin that inhibits auxin efflux.

BACKGROUND: Rhizobium nitrogen fixation in legumes takes place in specialized organs called root nodules. The initiation of these symbiotic organs has two important components. First, symbiotic rhizobium bacteria are recognized at the epidermis through specific bacterially secreted lipo-chitooligosaccharides (LCOs). Second, signaling processes culminate in the formation of a local auxin maximum marking the site of cell divisions. Both processes are spatially separated. This separation is most pronounced in legumes forming indeterminate nodules, such as model organism Medicago truncatula, in which the nodule primordium is formed from pericycle to most inner cortical cell layers. RESULTS: We used computer simulations of a simplified root of a legume that can form indeterminate nodules. A diffusive signal that inhibits auxin transport is produced in the epidermis, the site of rhizobium contact. In our model, all cells have the same response characteristics to the diffusive signal. Nevertheless, we observed the fastest and strongest auxin accumulation in the pericycle and inner cortex. The location of these auxin maxima correlates with the first dividing cells of future nodule primordia in M. truncatula. The model also predicts a transient reduction of the vascular auxin concentration rootward of the induction site as is experimentally observed. We use our model to investigate how competition for the vascular auxin source could contribute to the regulation of nodule number and spacing. CONCLUSION: Our simulations show that the diffusive signal may invoke the strongest auxin accumulation response in the inner root layers, although the signal itself is strongest close to its production site.

Thermoperiodic control of hypocotyl elongation depends on auxin-induced ethylene signaling that controls downstream PHYTOCHROME INTERACTING FACTOR3 activity.

We show that antiphase light-temperature cycles (negative day-night temperature difference [-DIF]) inhibit hypocotyl growth in Arabidopsis (Arabidopsis thaliana). This is caused by reduced cell elongation during the cold photoperiod. Cell elongation in the basal part of the hypocotyl under -DIF was restored by both 1-aminocyclopropane-1-carboxylic acid (ACC; ethylene precursor) and auxin, indicating limited auxin and ethylene signaling under -DIF. Both auxin biosynthesis and auxin signaling were reduced during -DIF. In addition, expression of several ACC Synthase was reduced under -DIF but could be restored by auxin application. In contrast, the reduced hypocotyl elongation of ethylene biosynthesis and signaling mutants could not be complemented by auxin, indicating that auxin functions upstream of ethylene. The PHYTOCHROME INTERACTING FACTORS (PIFs) PIF3, PIF4, and PIF5 were previously shown to be important regulators of hypocotyl elongation. We now show that, in contrast to pif4 and pif5 mutants, the reduced hypocotyl length in pif3 cannot be rescued by either ACC or auxin. In line with this, treatment with ethylene or auxin inhibitors reduced hypocotyl elongation in PIF4 overexpressor (PIF4ox) and PIF5ox but not PIF3ox plants. PIF3 promoter activity was strongly reduced under -DIF but could be restored by auxin application in an ACC Synthase-dependent manner. Combined, these results show that PIF3 regulates hypocotyl length downstream, whereas PIF4 and PIF5 regulate hypocotyl length upstream of an auxin and ethylene cascade. We show that, under -DIF, lower auxin biosynthesis activity limits the signaling in this pathway, resulting in low activity of PIF3 and short hypocotyls.

The strigolactone biosynthesis gene DWARF27 is co-opted in rhizobium symbiosis.

BACKGROUND: Strigolactones are a class of plant hormones whose biosynthesis is activated in response to phosphate starvation. This involves several enzymes, including the carotenoid cleavage dioxygenases 7 (CCD7) and CCD8 and the carotenoid isomerase DWARF27 (D27). D27 expression is known to be responsive to phosphate starvation. In Medicago truncatula and rice (Oryza sativa) this transcriptional response requires the GRAS-type proteins NSP1 and NSP2; both proteins are essential for rhizobium induced root nodule formation in legumes. In line with this, we questioned whether MtNSP1-MtNSP2 dependent MtD27 regulation is co-opted in rhizobium symbiosis. RESULTS: We provide evidence that MtD27 is involved in strigolactone biosynthesis in M. truncatula roots upon phosphate stress. Spatiotemporal expression studies revealed that this gene is also highly expressed in nodule primordia and subsequently becomes restricted to the meristem and distal infection zone of a mature nodules. A similar expression pattern was found for MtCCD7 and MtCCD8. Rhizobium lipo-chitooligosaccharide (LCO) application experiments revealed that of these genes MtD27 is most responsive in an MtNSP1 and MtNSP2 dependent manner. Symbiotic expression of MtD27 requires components of the symbiosis signaling pathway; including MtDMI1, MtDMI2, MtDMI3/MtCCaMK and in part MtERN1. This in contrast to MtD27 expression upon phosphate starvation, which only requires MtNSP1 and MtNSP2. CONCLUSION: Our data show that the phosphate-starvation responsive strigolactone biosynthesis gene MtD27 is also rapidly induced by rhizobium LCO signals in an MtNSP1 and MtNSP2-dependent manner. Additionally, we show that MtD27 is co-expressed with MtCCD7 and MtCCD8 in nodule primordia and in the infection zone of mature nodules.

Strigolactone biosynthesis in Medicago truncatula and rice requires the symbiotic GRAS-type transcription factors NSP1 and NSP2.

Legume GRAS (GAI, RGA, SCR)-type transcription factors NODULATION SIGNALING PATHWAY1 (NSP1) and NSP2 are essential for rhizobium Nod factor-induced nodulation. Both proteins are considered to be Nod factor response factors regulating gene expression after symbiotic signaling. However, legume NSP1 and NSP2 can be functionally replaced by nonlegume orthologs, including rice (Oryza sativa) NSP1 and NSP2, indicating that both proteins are functionally conserved in higher plants. Here, we show that NSP1 and NSP2 are indispensable for strigolactone (SL) biosynthesis in the legume Medicago truncatula and in rice. Mutant nsp1 plants do not produce SLs, whereas in M. truncatula, NSP2 is essential for conversion of orobanchol into didehydro-orobanchol, which is the main SL produced by this species. The disturbed SL biosynthesis in nsp1 nsp2 mutant backgrounds correlates with reduced expression of DWARF27, a gene essential for SL biosynthesis. Rice and M. truncatula represent distinct phylogenetic lineages that split approximately 150 million years ago. Therefore, we conclude that regulation of SL biosynthesis by NSP1 and NSP2 is an ancestral function conserved in higher plants. NSP1 and NSP2 are single-copy genes in legumes, which implies that both proteins fulfill dual regulatory functions to control downstream targets after rhizobium-induced signaling as well as SL biosynthesis in nonsymbiotic conditions.

Lost in space: what single-cell RNA sequencing cannot tell you.

Plant scientists are rapidly integrating single-cell RNA sequencing (scRNA-seq) into their workflows. Maximizing the potential of scRNA-seq requires a proper understanding of the spatiotemporal context of cells. However, positional information is inherently lost during scRNA-seq, limiting its potential to characterize complex biological systems. In this review we highlight how current single-cell analysis pipelines cannot completely recover spatial information, which confounds biological interpretation. Various strategies exist to identify the location of RNA, from classical RNA in situ hybridization to spatial transcriptomics. Herein we discuss the possibility of utilizing this spatial information to supervise single-cell analyses. An integrative approach will maximize the potential of each technology, and lead to insights which go beyond the capability of each individual technology.