John Innes Centre

Nitrogen (N) is a major plant nutrient, and its supply is very often limiting growth.The main forms of inorganic N in soil supplying plants are ammonium and nitrate ions.Although the soil availability of N can vary greatly, the cytoplasmic nutrient ion activities in a typical plant cell are maintained at set points that are independent of changes in supply.By contrast, the storage of N as protein and vacuolar nitrate, depends on the external supply.Measurements of cellular homeostasis of ammonium and nitrate are limited by methodology.The upper limits for cytoplasmic set points are likely to depend on toxicity, and for ammonium this is well known, but less clear for nitrate.An intracellular set point for N must be maintained by membrane transport systems and assimilation processes.Crop N use efficiency has uptake and assimilation components, and understanding homeostasis is fundamentally important for improving this important trait.

Abstract Plant viruses interfere with host signaling pathways, but it remains unclear how calcium (Ca 2+ ) signaling, reactive oxygen species (ROS), and changes in the plasma membrane interact during viral infection. Here, we investigated how plantago asiatica mosaic virus (PlAMV) modulates host Ca 2+ and ROS-associated signaling in Arabidopsis thaliana . Using live-cell imaging and the R-GECO1.2 Ca 2+ sensor, we observed a rapid increase in cytoplasmic Ca 2+ before the virus was detected, indicating that Ca 2+ release occurs early in infection. Genetic analysis showed that GLR, CPK3, and CNGC, core components of Ca 2+ signaling, limit PlAMV spread between cells, while the usual pattern-triggered immunity (PTI) co-receptors were not needed. This means that Ca 2+ -based antiviral restriction operates independently of PTI. With the plasma membrane-tethered and cytosolic HyPer7 biosensor, we found that ROS levels were lower inside infection foci in the inoculated leaves, but higher in nearby cells, respectively. The NADPH oxidases RBOHD and RBOHF, which produce ROS, slowed down the local viral propagation. The PM sphingolipid biosynthetic enzyme MOCA1 altered ROS patterns and reduced the virus’s spread. Epistasis analysis revealed a functional interaction between RBOHD and MOCA1, suggesting that ROS signaling and plasma membrane sphingolipid homeostasis are interconnected in antiviral defense. Overall, our findings suggest that PlAMV triggers Ca 2+ influx and ROS signaling at the plasma membrane, which induces sphingolipid reorganization and helps restrict the propagation of the virus. This study shows how Ca 2+ , ROS, and membrane sphingolipid signaling work together in plant antiviral immunity and points to possible ways to improve resistance to viruses.

Starch Synthase 1 (SS1) participates in the synthesis of amylopectin. To determine its role in hexaploid bread wheat, we selected and combined TILLING mutations for each homoeologue to create two independent SS1-deficient lines. The lines, which have different combinations of ss1 mutations, both lacked SS1 protein. Both lines exhibited mild but significant changes to starch phenotype, including fewer short amylopectin chains, a slight increase in B-type starch granules and a modest increase in amylose content. Lack of SS1 also led to changes in the thermal properties of starch measured by differential scanning calorimetry including reduced enthalpy, and increased gelatinization temperature. Despite the changes to starch properties, the starch contents of the mutant lines compared to wild types were within the normal range, as were grain weight and protein content. However, the concentration of total- and water-extractable arabinoxylan, and MLG, were increased in white flour compared to wild-type controls.

Cell-to-cell communication underpins pattern formation and organ function in multicellular organisms. Plant cells can communicate directly through cytoplasmic channels called plasmodesmata. The distribution, abundance, and density of plasmodesmata on plant cell interfaces impact the flow of molecules between plant cells; yet the extent to which these properties are genetically and dynamically regulated remains poorly understood at an organ scale. We developed a quantitative approach to map plasmodesmata pit fields across roots in 3D at cell type and cell interface-specific resolution. Multiple parameters are captured simultaneously, including plasmodesmata pit field abundance, density, and spatial distribution, enabling parallel multiscale analyses at cellular resolution across this organ. During root maturation, plasmodesmata abundance increases, with the greatest biogenesis occurring within the inner cell layers. This is coupled with changes in the degree of clustering of the pit fields on these inner cell layers: becoming more dispersed on specific cell interface types and more clustered on others. Significant differences in plasmodesmata pit field spatial patterning were detected at cell type-specific resolution in the BRASSINOSTEROID INSENSITIVE1 mutant, demonstrating a role for this hormone pathway in channel patterning. The ability to quantify pit field abundance and patterning at cell type-specific resolution provides novel insight into the developmental and hormonal regulation of potential symplastic connectivity across plant organs, while providing a powerful tool toward the investigation of quantitative systems-level plasmodesmata distribution and macro-communication between cells in a complex multicellular system.

Abstract Background: Nucleotide-binding leucine-rich-repeat receptors (NLRs) are key components of plant innate immunity. These intracellular proteins specifically recognize pathogen effectors and subsequently activate a defense response. The genes encoding these proteins comprise one of the largest and most diverse gene families in plants. Understanding the various forms and functions of these proteins is imperative to the goal of generating disease resistant crops. This is particularly important for cultivated strawberry, which is heavily impacted by disease. Results: Using conserved motifs and functional domains, we identified 823 NLR-encoding genes (NLRs) in the ( Fragaria \((\times)\) ananassa ) `FaRR1' genome and categorized them according to their association with the anciently diverged RNL, CNL, and TNL classes. Among these, 45 were newly annotated and 149 were predicted to be full-length. 94 genes exhibited a previously unreported architecture, containing both CNL-specific and TNL-specific sequences. This architecture was supported by full-length transcript data and was found to be present in additional Rosaceous species but not in any non-Rosaceous species examined. Expression analyses revealed that 583 NLRs, including nearly all those with the novel mixed architecture, were differentially expressed following inoculation with at least one of five pathogens. Conclusions: Our genome-wide analysis expands the catalog of NLR genes in cultivated strawberry and uncovers a previously undescribed form of domain organization that appears to be unique to the Rosaceae family. The results suggest that recombination or domain swapping among N-terminal regions has contributed to NLR diversification in this lineage, providing insight into the evolutionary and functional basis of pathogen recognition in strawberry.

The present study reports the metabolic profiling and antimicrobial evaluation of Vitex negundo seed extract. UHPLC-QTOF-MS/MS analysis identified seventeen bioactive phytoconstituents, correlated with the observed antimicrobial and antifungal activities. Among them, isoorientin (-7.3 kcal mol ⁻ ¹), quercetin (-7.8 kcal mol ⁻ ¹), and orientin (-7.4 kcal mol ⁻ ¹) exhibited strong binding affinities towards Staph Gyrase B (24 kDa). Similarly, isoorientin (-8.1 kcal mol ⁻ ¹), quercetin (-8.4 kcal mol ⁻ ¹), and orientin (-8.3 kcal mol ⁻ ¹) displayed significant interactions with secreted aspartic proteinase (SAP2) enzyme, confirming their antimicrobial potential. The aqueous-methanolic seed extract demonstrated notable inhibitory activity against Staphylococcus aureus (26.4 ± 0.3 mm; 44.08% inhibition) and Candida albicans (25.7 ± 0.4 mm; 29.73% inhibition). Density functional theory (DFT) calculations at B3LYP/6-31G level were used to optimize the ground state geometries of the identified phytochemicals and analyze their frontier molecular orbitals (FMOs) and global reactivity descriptors. Time-dependent DFT (TDDFT) calculations at the B3LYP/6-311G level (solvent: DMSO) further explored their biological relevance and nonlinear optical (NLO) properties, including ionization potential (IP), molecular electrostatic potential (MEP), and HOMO-LUMO energy gaps. These quantum chemical parameters provided mechanistic insights into the antimicrobial potential of the identified constituents. Molecular docking simulations further confirmed strong geometric complementarity and favorable binding affinities, highlighting the Vitex negundo seed extract as a promising source of a novel medicinal agent with previously unreported antifungal and antibacterial activities.

Plant non-specific lipid transfer proteins (nsLTPs) are small secretory proteins that bind and transport hydrophobic molecules. nsLTPs are crucial players in various plant physiological processes, including lipid metabolism and defense. In <italic>Artemisia annua</italic>, glandular trichomes serve as the primary sites for artemisinin biosynthesis and storage. Although several nsLTPs have been reported in <italic>Artemisia annua</italic>, a comprehensive genome-wide analysis was previously lacking. In this study, 55 <italic>nsLTP</italic> genes were identified in the <italic>A</italic>. <italic>annua</italic> genome and classified into eight types (I, II, III, IV, V, VI, VII, and IX) through phylogenetic analysis. Notably, expression profiling revealed that certain <italic>AaLTP</italic> genes, especially <italic>AaLTP1</italic> and <italic>AaLTP2</italic>, exhibit glandular trichome-specific expression, as confirmed by promoter::GUS assays. Overexpression of <italic>AaLTP1</italic> significantly increased artemisinin content by 1.5-fold (<italic>p</italic> &lt; 0.05), suggesting its direct role in facilitating artemisinin accumulation. Furthermore, an HD-ZIP transcription factor, AaHD8, was confirmed as a positive regulator of <italic>AaLTP1</italic> and <italic>AaLTP2</italic> through direct promoter binding. Overall, this study provides the first comprehensive characterization of the <italic>nsLTP</italic> gene family in <italic>A</italic>. <italic>annua</italic> and establishes a functional link between nsLTPs, glandular trichome biology, and artemisinin biosynthesis, offering new insights for metabolic engineering to enhance artemisinin production.

Abstract Flowering plants respond to multiple environmental and endogenous cues to determine the timing of their transition from the vegetative to floral state. Most of our knowledge of the gene regulatory network (GRN) controlling flowering time has been derived from the model plant Arabidopsis thaliana . This knowledge needs to be translated to crop plants to support the development of varieties that can be grown in different and rapidly changing climatic conditions. However, due to increased genome complexity and limited prior knowledge, translation into crops is not always straightforward. Here, we present a study of the GRN controlling flowering time in Brassica napus (oilseed rape), an allotetraploid crop that is a close relative of Arabidopsis. Using a comparative transcriptomics approach, we show that the majority of the orthologous gene pairs have similar expression dynamics over development between Arabidopsis and Brassica napus . Some genes, however, have experienced regulatory changes, with flowering time genes in Brassica napus having higher than average differences in their expression profiles from their Arabidopsis orthologues. Despite these differences, the inferred GRN for flowering in Brassica napus exhibits a similar network topology to the network known in Arabidopsis. This is likely due to preferential retention of these genes in higher paralogue numbers, which allows subtle changes in the regulation of individual paralogues, while still conserving the overall regulatory structure through evolution. We discover and present a detailed analysis of one such example where orthologues of SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 ( SOC1 ) have similar expression patterns under normal conditions, but different dynamics under cold temperature conditions, suggesting possible subfunctionalisation among paralogues in response to temperature change.

Abstract Multiple studies have identified genes affecting grain morphology, yet their capacity to deliver yield gains under field conditions remains unclear. We performed a multiyear, multilocation factorial evaluation of GRAIN WIDTH2 ( TaGW2 ) mutants in hexaploid wheat using BC 4 near-isogenic lines, sowing-density treatments and semi-dwarfing RHT1 backgrounds. Loss-of-function mutations in TaGW2 increased grain size and thousand grain weight (TGW) additively; with the aaBBDD single mutant showing the most stable singlelocus effect, while the aabbdd triple mutant achieved ~20% higher TGW across twelve field trials. However, overall grain yield remained unchanged or slightly reduced, reflecting a compensatory trade-off with grain number. Spike phenotyping of both main and secondary tillers showed comparable increases in TGW and spike yield despite fewer grains per spike, indicating that limited yield gain primarily reflects reduced spike number per unit area rather than decreased spike-level productivity. Effects were stable across sowing densities, whereas interactions with semi-dwarfing alleles were allele-specific: RHT-B1b partially suppressed TGW gains and accentuated yield penalties, whereas RHT-D1b maintained the large-grain phenotype and productivity. Across experiments, the TaGW2-A1D1 double mutant increased TGW (~14%) while maintaining yield stability, identifying it as a promising genotype for breeding. We conclude that TaGW2 is a reliable modifier of grain size but not yield in isolation.

Obligate host-associated bacteria with reduced genomes, such as phytoplasmas, face strong evolutionary constraints, including metabolic dependence on hosts, limited opportunities for horizontal gene transfer (HGT), and frequent population bottlenecks. Despite these limitations, phytoplasmas, which are parasitic, insect-transmitted plant pathogens, maintain a diverse arsenal of secreted effectors that manipulate both plant and insect hosts to promote infection and transmission. These effectors can suppress immunity and reprogram plant development, inducing alterations such as witch's broom and leaf-like flowers, through ubiquitin-independent degradation of key transcription factors. However, how phytoplasmas diversify and maintain these effectors in the absence of frequent genetic exchange remains unclear. To address this, we analysed the effectoromes of 239 phytoplasma genomes and identified a diverse set of secreted proteins, which we designated as putative Phytoplasma Effectors (PhAMEs). We found that PhAMEs targeting evolutionarily conserved and structurally constrained surfaces of host proteins are widespread across phytoplasmas. These effectors adopt compact, efficient folds. They often function as molecular scaffolds with dual interaction surfaces capable of linking host proteins or integrating signalling pathways. Such scaffolding PhAMEs have evolved multiple times independently, providing clear evidence of convergent evolution. Despite severe genomic constrains imposed by genome reduction and limited HGT, gene duplications, interface variations, domain fusions, and repeat expansions have helped the shaping effector fold and diversity. While the overall effector repertoire of phytoplasmas appeared largely unique, some PhAME domains share similarities with proteins from other mollicutes and pathogens. Collectively, our findings shed light on how genome-reduced bacteria innovate molecular functions and offer insights into phytoplasma biology, effector evolution, and host-pathogen dynamics. They also lay the groundwork for protein engineering approaches aimed at discovering or designing novel biomolecules with biotechnological potential.