Crop productivity relies on synthetic nitrogen fertilizers, which entail significant economic and environmental costs. Engineering crop plants to produce nitrogenase allowing them to acquire nitrogen from the atmosphere offers a transformative approach to sustainable agriculture. However, engineering the nitrogenase pathway into eukaryotic cells poses significant challenges due to the enzyme metal clusters, high energy demands, and extreme sensitivity to oxygen. This review summarizes recent advances in nitrogenase engineering in yeast and plants. Progress has been made using a stepwise modular approach and a functional validation pipeline for core and ancillary nitrogenase proteins. Key milestones include recovery of NifH with in vitro activity from mitochondria and chloroplasts, production of NifB that is active upon isolation or after in vitro cluster reconstitution, production of NifB-co in yeast mitochondria, in vitro FeMo-co biosynthesis using proteins purified from yeast mitochondria, and recovery of nitrogenase components from transgenic rice with activity demonstrated in vitro. Complete nitrogenase activity in engineered yeast or plants has not yet been demonstrated. We discuss the importance of organelle targeting, the exploitation of natural diversity and engineered proteins, and synthetic biology tools for the assembly of multigene pathways. Finally, we present the main remaining obstacles, such as the assembly of functional NifDK in plants, integration with host metabolism and coordinated regulation, and we outline the prospects for the development of nitrogen-fixing crops.

Systemic immunity induced by beneficial bacteria enhances plant resilience to subsequent pathogen attacks. However, the mechanisms by which plants perceive these bacteria and translate this perception into a systemic response remain poorly understood. The Brassicaceae LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) receptor kinase is the direct sensor of bacterial medium-chain-length 3-hydroxy fatty acids (mc-3-OH-FAs) and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), both of which activate local Brassicaceae immunity. Here, we show that the HAA-producing bacterium Pantoea ananatis BRT175 can colonize Arabidopsis thaliana roots independently of the production of HAAs, but these compounds induce a lore-dependent local immune response in roots. P. ananatis triggers induced systemic resistance (ISR) against the necrotrophic fungus Botrytis cinerea, but not against the hemibiotrophic bacterium Pseudomonas syringae pv. tomato DC3000. This ISR can be mimicked by HAAs or 3-hydroxy-decanoic acid (3-OH-C10), the strongest agonist of LORE. Both bacterium- and 3-hydroxylated fatty acid MAMP-induced ISR against B. cinerea rely on LORE-mediated sensing, involve salicylic acid, jasmonic acid/ethylene signaling pathways, and activate similar defense gene expression profiles in infected leaves. Our results demonstrate that lipidic microbe-associated molecular patterns (MAMPs) produced by beneficial microorganisms are necessary and sufficient to trigger rhizobacteria-mediated ISR and could differentially activate systemic resistance against biotrophic and necrotrophic pathogens.

Root rot caused by Fusarium oxysporum (F. oxysporum) severely constrains Codonopsis pilosula (C. pilosula) production. Indigenous culturable bacteria associated with C. pilosula were screened for antifungal activity, plant growth-promoting traits, and interstrain compatibility, and two defined three-member synthetic microbial communities, SCI and SCII, were constructed. Both SynComs alleviated root rot, with SCI showing stronger overall performance and a biocontrol efficacy of 57.8%, together with improved root growth and photosynthetic performance. Rhizosphere bacterial profiling was integrated with rhizosphere metabolite profiling and root tissue metabolomics. SynCom treatment was associated with limited changes in overall bacterial diversity but variation in specific rhizosphere taxa. In roots, pathway-enrichment patterns shifted from sulfur metabolism and oxidative stress-related processes under pathogen stress toward amino acid-related metabolism after SynCom treatment, accompanied by treatment-associated variation in medicinally relevant metabolite features. Associations between metabolic changes and potentially beneficial bacteria indicated coordinated variation between host metabolism and rhizosphere bacterial composition. In vitro assays further showed that L-valine elicited chemotactic and growth responses in a representative Sphingomonas isolate. Collectively, SynCom-mediated root rot suppression was associated with constituent-strain antagonism, plant recovery, rhizosphere bacterial shifts, and amino acid-related metabolic variation.

Growth and death of vegetation control both the amount of carbon held in living biomass and much of the flow between the atmosphere and soils, the largest terrestrial carbon reservoir. However, long-term field datasets on belowground growth dynamics are rare. Knowledge is lacking on how root growth patterns relate to the environment, fluctuating carbon and water availability, and internal plant physiological control, at both seasonal and diel scales. We imaged roots in a temperate grassland using automated minirhizotrons and interpreting root length density (RLD) automatically twice a day for nine months. From winter to autumn, we always observed some root growth in individual images, but with decreasing net RLD (indicative of net turnover) becoming predominant later in the season. Roots grew day-round except during a period of about one month in spring when root growth was only in daytime. This was a period without rain, during which soil moisture depleted rapidly, but not at its driest point. We discuss potential causes for both overall and short-term growth patterns, particularly the change in drivers of growth from internal storage to new photosynthates, or limits imposed by water availability - both soil moisture and rainfall - under shifting environmental conditions.

Bitter pit of apple is commonly ascribed to calcium dilution because symptoms are positively associated with fruit mass. A dilution effect implies that calcium transport or supply cannot keep pace with fruit growth but does not identify the mechanism. Disparate sink:source ratios or transient photosynthetic inhibition were used to alter 'Honeycrisp' fruit growth rate (FGR) to study fruit xylem transport kinetics and peel calcium concentration. FGR and xylem dysfunction increased with decreasing sink:source ratio. Xylem transport declined to 50% of maximum 1.7 weeks earlier under Low than High sink:source ratios and peel calcium decreased concomitantly with xylem transport. Treatment effects on bitter pit incidence were explained by integrating time-dependent xylem transport curves into an empirical 'cumulative xylem' function. Regressing this function on FGR conflated treatment differences. When photosynthetic light reactions were inhibited by episodic limb shading, FGR was reduced and xylem transport increased; shade removal caused transient surges in FGR and concomitant reduction in xylem transport. Changes in calcium concentration were less responsive to shade due to stomatal limits to water transport. Combined, the results support that FGR compromises xylem transport, effectively limiting calcium supply and exacerbating bitter pit, the magnitude of which depends on the onset of xylem dysfunction.

Understanding the root-soil interface is essential for determining when plants become hydraulically isolated from their soil environment during drought. Emerging evidence suggests roots shrink substantially during dehydration, but the drivers and water-flow implications remain elusive, with few data linking shrinkage across soil and plant water status. Using Pinus radiata, a widespread isohydric conifer, we use synchrotron-based Micro-CT to scan intact roots < 2.5 mm in soil during mild plant drying (prior to embolism) to isolate relationships between shrinkage, soil (Ψsoil) and xylem (Ψplant) water potential. Fine roots in situ began shrinking during the earliest stages of Ψplant drying (-0.2 MPa to -0.88 MPa), losing 14.8 ± 10.3% initial transverse-sectional area (62% of observed shrinkage) prior to incipient soil drying (0-72 kPa Ψsoil). Ψplant was a significantly better predictor of shrinkage than Ψsoil, showing a linear shrinkage-Ψplant relationship across this early drying gradient when normalised to shrinkage at -1 MPa, accounting for variable initial Ψplant. Ψplant decline was linearly coordinated with cortical lacunae formation. This Ψplant-driven pattern identifies root shrinkage as an essential component of plant hydraulic function during commonly experienced water potential gradients, occurring long prior to stomatal closure and xylem embolism. This reveals water use from cortical tissues as a key physiological process defining plant-soil disconnection during water deficit.

The rapid rise in atmospheric CO2 concentration presents a critical paradox for global agriculture: while it stimulates photosynthesis and plant productivity, it simultaneously compromises crop nutritional quality. This decline in quality is linked to a lower concentration of essential macro- and micronutrients in crop edible parts, involving complex regulatory shifts that go beyond the simple growth dilution effect caused by the decoupling between growth and nutrient uptake. In this review, we summarize the current knowledge regarding the impact of elevated CO2 (eCO2) on nitrogen (N) nutrition, among other aspects, discussing how the availability of nitrate (NO3-) or ammonium (NH4+) affects plant growth and N status under eCO2, a long-standing debate in the field. We outline recent advances in the signalling mechanisms regulating NO3- uptake under eCO2 and discuss the interaction between photorespiration and the assimilation of both N sources. Likewise, we address the main effects and regulatory mechanisms associated with eCO2 and the homeostasis of sulphur (S), phosphorus (P) and the micronutrients iron (Fe) and zinc (Zn). Collectively, we intend to provide a comprehensive framework to guide future crop breeding and climate-resilient agronomic management.

Short dormancy period of sweetpotato (Ipomoea batatas) storage roots causes significant economic losses. To unravel the molecular mechanism of sweetpotato sprouting, a comparative transcriptome was performed using storage roots incubated under non-sprout (15 °C) and sprout favouring conditions (25 °C). Our analysis revealed that 7404 genes were differentially expressed between non-sprouting and sprouted storage roots incubated at 25 °C. Further, we observed that gibberellin (GA) biosynthesis gene IbGA3OXIDASE and abscisic acid (ABA) catabolism gene ABA 8'-HYDROXYLASE were upregulated by > 10-fold and > 40-fold, respectively, at the onset of sprouting. Metabolite profiling showed increased GA and cytokinin levels, but reduced ABA content in the sprout initials. Based on conserved expression patterns of phytohormone-related genes during sprouting in potato tubers and sweetpotato storage roots, two genes that were downregulated during sweetpotato sprouting - IbPYL4 (ABA receptor) and IbGA2OXIDASE (GA catabolic enzyme) - were independently overexpressed in potato. Transgenic potato plants overexpressing IbPYL4 or IbGA2OXIDASE exhibited low percentages of tuber sprouting and reduced sprout length. Furthermore, knockdown of the ABA 8'-HYDROXYLASE gene (ABA catabolism) significantly reduced sprout length in sweetpotato storage roots compared to wild-type plants. Overall, our study suggests that selective candidate genes (ABA 8'-HYDROXYLASE, IbPYL4, IbGA3OXIDASE, and IbGA2OXIDASE) could be targeted for developing sweetpotato genotypes with enhanced shelf-life.

Gibberellins (GAs) are widely used in grape cultivation to promote berry enlargement and induce seedlessness, but their effects on seed development and the regulatory mechanism remain poorly understood. In this study, exogenous GA3 significantly reduced seed weight in grapevine, indicating impaired seed development. To investigate the underlying mechanism, we characterized the grape DELLA protein SLENDER RICE 1 (VvSLR1). Overexpression of VvSLR1 in tomato caused a dwarf and compact phenotype but had limited effects on fruit and seed development under normal conditions. However, GA3 treatment caused a stronger reduction in seed formation in VvSLR1-overexpressing plants, whereas paclobutrazol (PAC), an inhibitor of GA biosynthesis, allowed relatively normal seed formation, indicating that the effect of VvSLR1 on reproductive development is GA-dependent. In grape calli, VvSLR1 overexpression altered GA metabolism and the expression of several VvGA20ox and VvGA3ox genes, suggesting a role for VvSLR1 in GA homeostasis. We further identified GAMETOPHYTE DEFECTIVE 1 (VvGAD1), whose Arabidopsis homolog is involved in gametophyte development, as a nuclear-localized VvSLR1-interacting protein. VvGAD1 associated with the promoter of GA 20-oxidase 4 (VvGA20ox4), and co-expression of VvGAD1 and VvSLR1 significantly enhanced VvGA20ox4 promoter activity. Moreover, heterologous expression of VvGA20ox4 in Arabidopsis increased silique length and seed number, while exogenous GA3 suppressed VvGA20ox4 promoter activity. Together, these findings suggest that VvSLR1 acts as a regulatory node linking GA signaling and homeostasis, with the VvSLR1-VvGAD1-VvGA20ox4 relationship representing a potential regulatory branch involved in GA-dependent seed development.

Two isozymes of granule-bound starch synthase (GBSS), GBSSI and GBSSII, are present in rice. While GBSSI is solely responsible for amylose synthesis in endosperm and gbss1 mutants accumulate glutinous starch in grains, the role of GBSSII remains unclear. In this study, rice gbss2 mutants were isolated for the first time in cereals and gbss1 gbss2 double mutants were generated to clarify the respective roles of GBSS isozymes. Iodine staining of starch confirmed that amylose in pollen is synthesized by GBSSI and those in leaf and root tip are by GBSSII. Gel filtration chromatography of debranched starch from endosperm and leaf sheath revealed that GBSSI and GBSSII appear to be the primary amylose synthases in endosperm and in leaf sheath, respectively. However, the absence of the primary GBSS in the respective organs did not result in the complete elimination of amylose. A small peak detected in the fractions corresponding to amylose was shown to be α-glucans by glucoamylase digestion, and the presence of branches was revealed by detailed structure analyses. The amylose-like, branched α-glucans found in the double mutants, which probably also existed in single mutants and wild-type starches, were suggested to be synthesized by other enzymes than GBSSs.

Betaine lipids (BLs) are a class of non-phosphorus glycerolipids widely found in microalgae, fungi, bacteria, mosses, and ferns. For decades, the central paradigm regarding the biological function of BLs has been their role in substituting for phospholipids under phosphate starvation-a crucial adaptive mechanism for survival in oligotrophic environments. However, accumulating evidence indicates that the functional diversity of BLs is far more extensive. Recent studies have demonstrated their distinct roles in fatty acid metabolism, adaptation to various environmental stresses, and ecological interactions such as symbiosis and predation. This review summarizes current insights into the pleiotropic roles of BLs in microalgal physiology and ecology, moving beyond their role as mere phospholipid surrogates. Furthermore, we highlight the recently discovered bioactive function of BL derivative in mammals.

The MucR/Ros family are DNA-interacting proteins and pleiotropic regulators of key aspects of the lifecycle in α-proteobacteria, particularly in plant-associated bacteria. The early finding that MucR/Ros proteins contain a C-terminal domain resembling the Cys2His2 eukaryotic Zn-finger domain, indicated MucR/Ros were classical transcription factors binding specific DNA sequences. However, the pleiotropic phenotypes displayed by ros/mucR mutants and the involvement in regulating diverse functions, including cell cycle progression, symbiosis, virulence, motility, production of cell surface components, pointed MucR/Ros as global regulators. Structural and functional studies have positioned MucR/Ros as novel H-NS (Histone-like Nucleoid Structuring)-like proteins in α-proteobacteria, that bind AT-rich DNA sequences and are able of bridging DNA through formation of higher order oligomers. MucR/Ros have also been positioned as xenogeneic silencers (XS), with key roles in horizontal gene acquisition and genome evolution. At least one MucR member is reported to bind the bacterial universal second messenger c-di-GMP, what could thus represent an anti-silencing mechanism as proposed for other bacterial XS. The co-existence of functionally and structurally heterogeneous MucR homologs in the same genome adds further complexity but also warrants renovated interest to decipher the multiple roles of MucR in this important group of bacteria.

Far-red (FR) light can enhance photosynthesis over short timescales, even at irradiances above the light-limited range. However, it remains unclear whether increasing FR beyond the fraction naturally present in direct sunlight further stimulates photosynthesis, and whether changes in mesophyll conductance to CO2 (gm) contribute to this response. Tomato leaves developed under artificial sunlight-mimicking PAR (16 h; 300 μmol PAR m-2 s-1) with a far-red fraction representative of direct sunlight (R:FR ratio 1.1) were exposed to two actinic light regimes (constant and sinusoidal intensity patterns) combined with three R:FR ratios (10.5, 1.1 and 0.48), representing FR fractions below, similar to, and above those occurring in direct sunlight. High-temporal-resolution gas exchange and carbon isotope (δ13C) measurements (0.2 Hz) were used to quantify gm, stomatal conductance (gs), net CO2 assimilation (Anet) and chloroplastic CO2 concentration. Increasing FR from a low level to the fraction occurring in direct sunlight enhanced both gm and Anet throughout the photoperiod in both actinic light regimes. However, a further increase in FR to levels characteristic of foliar shade did not result in additional increases in either gm or Anet. Estimates of chloroplastic CO2 concentration suggest that the FR-induced increase in apparent gm may help sustain chloroplastic CO2 supply as assimilation increases, but is not sufficient to explain the stimulation of Anet. These results indicate that the short-term photosynthetic response to FR is already established at the FR fraction occurring in direct sunlight and does not increase further with additional FR.

Nitrogen availability is one of the main factors limiting crop productivity, and the low nitrogen use efficiency (NUE) of crops imposes significant economic and environmental costs. Research on NUE has traditionally focused on improving nitrogen uptake and assimilation; however, the efficient redistribution of assimilated nitrogen within the plant is equally important for growth, yield, and crop quality. Amino acids are the main form in which reduced nitrogen is transported over long distances. Their movement between source and sink tissues depends on a diverse group of membrane transporters belonging to the Amino Acid/Auxin Permeases (AAAP), Amino Acid/Polyamine/Choline transporters (APC), and Usually Multiple Acids Move In and Out Transporters (UMAMIT) families. These transporters mediate amino acid uptake, export, vascular loading and unloading, intracellular transport, and redistribution to developing organs, thereby influencing plant growth, reproductive development and the composition of harvested products. Recent work in cereals, legumes and horticultural crops has shown that modifying amino acid transport can alter biomass production, grain filling, seed protein content, fruit quality, and NUE. In this review, we summarize current knowledge of plant amino acid transporters, from their molecular and physiological functions to their roles in crop yield, nutritional quality and NUE. We also discuss how advances in genome editing, exploitation of natural genetic variation, high-throughput phenotyping and systems biology may facilitate the development of crops with more efficient nitrogen use and improved agricultural sustainability.

Strigolactones (SLs) are known to regulate shoot architecture and to be involved in plant responses to environmental stress, whereas their specific contributions to drought adaptation in barley remain incompletely defined. In this study, we analysed transcriptional, hormonal, and physiological responses to water deficit in barley SL mutants affected in early biosynthesis (Hvd10 and Hvd17), late biosynthesis (Hvmax1a), or signalling (Hvd14). The Hvd10, Hvd17, and Hvd14 mutants exhibited the typical high-tillering phenotype of SL deficiency, whereas Hvmax1a displayed characteristics similar to the wild type (WT), indicating functional differences within the SL biosynthetic pathway. Transcriptome analysis showed a clear overlap in gene expression among the high-tillering SL mutants under both control and drought conditions. We also used computational methods to identify potential transcription factors that might regulate SL-dependent gene expression. A drought experiment showed that SL mutants exhibited reduced biomass, relative water content, and photosynthetic efficiency, with the most pronounced effects observed in the high-tillering lines. Drought also activated the abscisic acid (ABA) pathway in all genotypes, with particularly high accumulation of ABA metabolites in the high-tillering SL mutants. Notably, Hvmax1a resembled the mutant-like metabolic profile, despite maintaining a wild-type-like architecture. Taken together, these results provide new insights into the roles of SL pathway components in drought responses and highlight functional differences among individual genes influencing both plant architecture and stress-related transcriptional programmes. Furthermore, the mutants generated in this study using Cas9-mediated genome editing represent a valuable genetic collection for future research into SL-mediated development and stress responses in barley.