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Aluminium (Al) toxicity in acid soils severely limits forage productivity, and dissecting Al-tolerance mechanisms is crucial for securing forage supply in acid-soil regions. White clover (Trifolium repens L.), an excellent acid-soil-adapted forage with pronounced Al tolerance, serves as an ideal model for studying legume Al-tolerance mechanisms. We exposed white clover seedlings to gradient Al ³⁺ concentrations (0, 2, 4, 6, 10 mmol·L ⁻¹ ) to characterize the core physiological and molecular responses underlying its Al adaptation. Al ³⁺ stress inhibited growth in a concentration-dependent manner, suppressing both root and shoot development. Roots adopted a prioritised defence strategy, with enhanced antioxidant enzyme activity and soluble sugar accumulation mitigating oxidative damage. Transcriptome analysis revealed coordinated regulation of key pathways: flavonoid biosynthesis showed a core inhibition–branch-specific activation pattern, photosystem-related genes were upregulated to reinforce photosynthetic function, and hormone signalling networks were extensively rewired with divergent responses among auxin, gibberellin, cytokinin, ABA and JA pathways. White clover copes with Al ³⁺ stress via an integrated mechanism featuring root-prioritised defence, photosynthetic maintenance and hormone network remodelling. These findings provide new insights into legume Al tolerance and a framework for breeding Al-tolerant forages. Future studies will quantify Al content in shoots and roots, and perform functional validation of upregulated hormone-related genes to clarify their roles in the Al-tolerance regulatory network.

The research objective of this study was to determine the physiological condition of selected legume species growing under natural conditions, based on analysis in them: photosynthetic efficiency, chlorophyll fluorescence lifetime and photosynthetic pigment content. Evaluation of these parameters allows early detection of stress factors affecting the physiology of the plants and the determination of their adaptive capacity. The plants selected for the study included: white clover, red clover, alfalfa, and common sainfoin. The presented results revealed that the photosynthetic activity was lower in red clover but higher in common sainfoin. Longer τ 1 and τ 2 fluorescence lifetimes of Chl a were measured in red clover and common sainfoin, while shorter lifetimes were recorded for white clover and alfalfa. Shorter lifetimes suggest greater activity of other chlorophyll excitation pathways, including in particular ones related to energy dissipation in the photosynthetic antennae. The content of chlorophyll a , a + b was noticeably higher in the leaves of red clover, and lower in the leaves of common sainfoin. The varied values of photosynthetic parameters measured in the analyzed perennial plants from the same Fabaceae family indicate the ease with which they adapt to environmental conditions. The studied plants are excellent sources of nitrogen and fodder protein, rendering them interesting in the context of sustainable agriculture. Graphical abstract

Soil nutrients exhibit heterogeneity in their spatial distribution, presenting challenges to plant acquisition. Notably, phosphorus (P) heterogeneity is a characteristic feature of soil, necessitating the development of adaptive strategies by plants to cope with this phenomenon. To address this, fully crossed three-factor experiments were conducted using red clover within rhizoboxes. Positions of P in three conditions, included P even distribution (even P), P close distribution (close P), and P far distribution (far P). Concurrently, N addition was two amounts(0 and 20 mg kg− 1), both with and without AMF inoculation. The findings indicated a decrease in aboveground biomass attributable to uneven P distribution, whereas N and AMF demonstrated the potential to affect aboveground biomass. In a structural equation model, AMF primarily increased aboveground biomass by enhancing nodule number and specific leaf area (SLA). In contrast, N addition improved aboveground biomass through increased nodule number or direct effects. Subsequently, a random forest model indicated that under the far P treatment, fine root length emerged as the primary factor affecting aboveground biomass, followed by thickest root length. Conversely, in the even P treatment, the thickest root length was of paramount importance. In summary, when confronted with uneven P distribution, clover plants adopted various root foraging strategies. AMF played a pivotal role in elevating nodule number, and SLA.

Key messageAccurate genomic prediction of yield within and across generations was achieved by estimating the genetic merit of individual white clover genotypes based on extensive genetic replication using cloned material.White clover is an agriculturally important forage legume grown throughout temperate regions as a mixed clover–grass crop. It is typically cultivated with low nitrogen input, making yield dependent on nitrogen fixation by rhizobia in root nodules. Here, we investigate the effects of clover and rhizobium genetic variation by monitoring plant growth and quantifying dry matter yield of 704 combinations of 145 clover genotypes and 170 rhizobium inocula. We find no significant effect of rhizobium variation. In contrast, we can predict yield based on a few white clover markers strongly associated with plant size prior to nitrogen fixation, and the prediction accuracy for polycross offspring yield is remarkably high. Several of the markers are located near a homolog of Arabidopsis thaliana GIGANTUS 1, which regulates growth rate and biomass accumulation. Our work provides fundamental insight into the genetics of white clover yield and identifies specific candidate genes as breeding targets.

The nutrient and carbon (C) allocation dynamics in mycorrhizal hyphal networks cause variation in costs and benefits for individual plants and fungi and influence the productivity, diversity and C cycling in ecosystems. We manipulated light and phosphorus (P) availability in a pot experiment with Trifolium subterraneum colonised by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. Stable ¹³C-labelling was used to trace assimilated CO₂ to the mycorrhizal fungus in roots and soil using compound-specific isotope ratio mass spectrometry. We used the neutral lipid fatty acid 16:1ω5 as a signature for AM fungal storage lipids. Both P and shading reduced the AM fungal lipid accumulation in the intraradical mycelium, while only P reduced the amount of lipids in the extraradical mycelium. Recently assimilated plant C was only allocated to the mycorrhizal fungus to a small extent when plant mycorrhizal benefit was reduced by P fertilization, while increasing the plant C cost by shading did not reduce the C flow to the fungus. These results are of importance for our conception of mycorrhizal dynamics during periods of shade in nature.

• Inorganic arsenic (iAs) is a ubiquitous human carcinogen, and rice (Oryza sativa) is the main contributor to iAs in the diet. Methylated pentavalent As species are less toxic and are routinely found in plants; however, it is currently unknown whether plants are able to methylate As. • Rice, tomato (Solanum lycopersicum) and red clover (Trifolium pratense) were exposed to iAs, monomethylarsonic acid (MMA(V)), or dimethylarsinic acid (DMA(V)), under axenic conditions. Rice seedlings were also grown in two soils under nonsterile flooded conditions, and rice plants exposed to arsenite or DMA(V) were grown to maturity in nonsterile hydroponic culture. Arsenic speciation in samples was determined by HPLC‐ICP‐MS. • Methylated arsenicals were not found in the three plant species exposed to iAs under axenic conditions. Axenically grown rice was able to take up MMA(V) or DMA(V), and reduce MMA(V) to MMA(III) but not convert it to DMA(V). Methylated As was detected in the shoots of soil‐grown rice, and in rice grain from nonsterile hydroponic culture. GeoChip analysis of microbial genes in a Bangladeshi paddy soil showed the presence of the microbial As methyltransferase gene arsM. • Our results suggest that plants are unable to methylate iAs, and instead take up methylated As produced by microorganisms.

Aims Root colonization by arbuscular mycorrhizal fungi (AMF) enhances plant growth and the symbiosis can play a major role in enhancing drought tolerance of host plants. Our aim was to determine whether AMF can mitigate negative effects of drought stress on productivity in annual legume species used in seed mixtures for permanent pastures in agroecosystems in the Iberian Peninsula. Methods Five species – Trifolium subterraneum , Trifolium incarnatum , Trifolium resupinatum , Trifolium vesiculosum and Ornithopus sativus – were grown in pots, non-inoculated or inoculated with AMF, at different watering regimes (no-stress, moderate drought stress, severe drought stress). We measured colonization levels, mycorrhizal dependency, biomass, and plant and substrate phosphorus (P) status. Results In all species, AMF led to a significantly higher biomass and tissue P, and water deficit significantly decreased biomass, with the responses to mycorrhization and watering being species-specific. In all Trifolium spp. watering did not affect mycorrhizal growth dependency (MGD), but mycorrhizal P-dependency (MPD) was significantly higher with drought. However, in Ornithopus , MGD was lower with severe drought, while MPD in this species was not affected by watering. Conclusions Biomass accumulation in the studied species benefitted from AMF symbiosis, although mycorrhizae did not mitigate drought effects on growth. Drought stress severity determined the carbon allocation to the different fungal structures. Under moderate drought stress carbon allocation to fungal structures increased, outweighing plant growth benefits by P or water gain of the symbiosis, while severe drought stress led to carbon limitation for both plant and fungi.

Companion planting of white clover ( Trifolium repens L.) with orchard grass ( Dactylis glomerata L.), a famous hay grass, improves the forage quality of orchard grass. Microbiome profiling techniques can reveal the specific role of white clover companion planting with orchard grass. This study aimed to explore the microbiome distribution and gene functions of rhizosphere and non-rhizosphere soil via companion planting systems of white clover and orchard grass. From metagenomics sequencing analysis, we confirmed the significant role of white clover on soil environment modeling during companion planting with orchard grass. Twenty-eight biomarkers of rhizosphere soil organisms were identified during companion planting, including Proteobacteria , Betaproteobacteria , Flavobacteriia , and Caulobacterales . The number of gene functions of nitrogen and carbon fixation in companion planting was higher than that in single plants, indicating new functional flora for companion planting. We characterized specific rhizosphere effects, typical biomarker flora, and potential regulatory mechanisms for white clover-related companion planting by metagenomics analyses.

Background Auxin may have a positive effect on plants under drought stress. White clover is widely cultivated and often prone to water shortages. In the present study, we investigated the effects of exogenous indole − 3-acetic acid (IAA) on growth and physiological changes of white clover under drought stress condition. The contents of endogenous IAA and other hormones including ABA, CTK, JA, GA, IAA, and SA were assayed. Moreover, expressions of auxin-responsive genes, drought-responsive genes and leaf senescence-associated genes were detected in response to exogenous IAA. Results Compared to control, drought stress alone significantly diminished stem dry weigh, relative water content (RWC) and total chlorophyll content (Chl). Exogenous IAA treatment significantly increased RWC and Chl, whereas L-AOPP treatment drastically decreased stem dry weight, RWC and Chl under drought stress condition. Additionally, exogenous IAA treatment significantly increased ABA content and JA content, up-regulated expression of auxin responsive genes ( GH3.1 , GH3.9 , IAA8 ), drought stress responsive genes ( bZIP11 , DREB2 , MYB14 , MYB48 , WRKY2 , WRKY56 , WRKY108715 and RD22 ), and down-regulated expressions of auxin-responding genes ( GH3.3 , GH3.6 , IAA27 ) and leaf senescence genes ( SAG101 and SAG102 ) in the presence of PEG. Contrarily, L-AOPP treatment significantly reduced contents of ABA, GA3 and JA, down-regulated expressions of GH3.1 , GH3.9 , IAA8 , bZIP11 , DREB2 , MYB14 , MYB48 , WRKY2 , WRKY56 , WRKY108715 , ERD and RD22 , and up-regulated SAG101 and SAG102 . Conclusions Exogenous IAA improved drought tolerance of white clover possibly due to endogenous plant hormone concentration changes and modulation of genes involving in drought stress response and leaf senescence. These results provided useful information to understand mechanisms of IAA improved drought tolerance in white clover.

The objective of this study was to determine the effect of soaking with γ-aminobutyric acid (GABA) on white clover (Trifolium repens cv. Haifa) seed germination under salt stress induced by 100 mM NaCl. Seeds soaking with GABA (1 μM) significantly alleviated salt-induced decreases in endogenous GABA content, germination percentage, germination vigor, germination index, shoot and root length, fresh and dry weight, and root activity of seedling during seven days of germination. Exogenous application of GABA accelerated starch catabolism via the activation of amylase and also significantly reduced water-soluble carbohydrate, free amino acid, and free proline content in seedlings under salt stress. In addition, improved antioxidant enzyme activities (SOD, GPOX, CAT, APX, DHAR, GR and MDHR) and gene transcript levels (Cu/ZnSOD, FeSOD, MnSOD, CAT, GPOX, APX, MDHR, GPX and GST) was induced by seeds soaking with GABA, followed by decreases in O2∙−, H2O2, and MDA accumulation during germination under salt stress. Seeds soaking with GABA could also significantly improve Na+/K+ content and transcript levels of genes encoding Na+/K+ transportation (HKT1, HKT8, HAL2, H+-ATPase and SOS1) in seedlings of white clover. Moreover, exogenous GABA significantly induced the accumulation of dehydrins and expression of genes encoding dehydrins (SK2, Y2K, Y2SK, and dehydrin b) in seedlings under salt stress. These results indicate that GABA mitigates the salt damage during seeds germination through enhancing starch catabolism and the utilization of sugar and amino acids for the maintenance of growth, improving the antioxidant defense for the alleviation of oxidative damage, increasing Na+/K+ transportation for the osmotic adjustment, and promoting dehydrins accumulation for antioxidant and osmotic adjustment under salt stress.