Abstract
Premise
β‐Cyanoalanine synthase (β‐CAS) and alternative oxidase (AOX) play important roles in the ability of plants to detoxify and tolerate hydrogen cyanide (HCN). These functions are ...critical for all plants because HCN is produced at low levels during basic metabolic processes, and especially for cyanogenic species, which release high levels of HCN following tissue damage. However, expression of β
‐CAS
and
Aox
genes has not been examined in cyanogenic species, nor compared between cyanogenic and acyanogenic genotypes within a species.
Methods
We used a natural polymorphism for cyanogenesis in white clover to examine β
‐CAS
and
Aox
gene expression in relation to cyanogenesis‐associated HCN exposure. We identified all β
‐CAS
and
Aox
gene copies present in the genome, including members of the
Aox1, Aox2a
, and
Aox2d
subfamilies previously reported in legumes. Expression levels were compared between cyanogenic and acyanogenic genotypes and between damaged and undamaged leaf tissue.
Results
β‐
CAS
and
Aox2a
expression was differentially elevated in cyanogenic genotypes, and tissue damage was not required to induce this increased expression.
Aox2d
, in contrast, appeared to be upregulated as a generalized wounding response.
Conclusions
These findings suggest a heightened constitutive role for HCN detoxification (via elevated β‐
CAS
expression) and HCN‐toxicity mitigation (via elevated
Aox2a
expression) in plants that are capable of cyanogenesis. As such, freezing‐induced cyanide autotoxicity is unlikely to be the primary selective factor in the evolution of climate‐associated cyanogenesis clines.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Urbanization is a global threat to biodiversity due to its large impact on environmental changes. Recently, urban environmental change has been shown to impact the evolution of many species. However, ...much remains unknown about how urban environments influence evolutionary processes and outcomes due to the non‐linearity and discontinuity of environmental variables along urban–rural gradients. Here, we focused on the evolution of hydrogen cyanide (HCN) production and its components (presence/absence of cyanogenic glycosides and the hydrolytic enzyme linamarase) in the herbaceous plant white clover Trifolium repens, which thrive in both urban and rural areas. To comprehensively elucidate how plants evolve and adapt to heterogenous urban environments, we collected 3299 white clover plants from 122 populations throughout Sapporo, Japan. We examined the spatial variation in environmental factors, such as herbivory, sky openness, impervious surface cover, snow depth, and temperature, and how variation in these factors was related to the production of HCN, cyanogenic glycosides, and linamarase. Environmental factors showed complex spatial variation due to the heterogeneity of the urban landscape. Among these factors, herbivory, sky openness, and impervious surface cover were highly related to the frequency of plants producing HCN in populations. We also found that impervious surface cover was related to the frequency of plants producing cyanogenic glycosides, while herbivory pressure was not. As a result, the cyanogenic glycoside frequency showed a clearer trend along urban–rural gradient rather than HCN frequency, and thus, the predicted spatial distributions of HCN and cyanogenic glycosides were inconsistent. These results suggest that urban landscape heterogeneity and trait multifunctionality determines mosaic‐like spatial distribution of evolutionary traits.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The extent to which species can adapt to spatiotemporal climatic variation in their native and introduced ranges remains unresolved. To address this, we examined how clines in cyanogenesis (hydrogen ...cyanide HCN production—an antiherbivore defense associated with decreased tolerance to freezing) have shifted in response to climatic variation in space and time over a 60‐year period in both the native and introduced ranges of Trifolium repens. HCN production is a polymorphic trait controlled by variation at two Mendelian loci (Ac and Li). Using phenotypic assays, we estimated within‐population frequencies of HCN production and dominant alleles at both loci (i.e., Ac and Li) from 10,575 plants sampled from 131 populations on five continents, and then compared these frequencies to those from historical data collected in the 1950s. There were no clear relationships between changes in the frequency of HCN production, Ac, or Li and changes in temperature between contemporary and historical samples. We did detect evidence of continued evolution to temperature gradients in the introduced range, whereby the slope of contemporary clines for HCN and Ac in relation to winter temperature became steeper than historical clines and more similar to native clines. These results suggest that cyanogenesis clines show no clear changes through time in response to global warming, but introduced populations continue to adapt to their contemporary environments.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
White clover (
L) is a high-quality forage grass with a high protein content, but it is vulnerable to cold stress, which can negatively affect its growth and development. WRKY transcription factor is ...a family of plant transcription factors found mainly in higher plants and plays an important role in plant growth, development, and stress response. Although WRKY transcription factors have been studied extensively in other plants, it has been less studied in white clover.
In the present research, we have performed a genome-wide analysis of the WRKY gene family of white clover, in total, there were 145 members of WRKY transcription factors identified in white clover. The characterization of the TrWRKY genes was detailed, including conserved motif analysis, phylogenetic analysis, and gene duplication analysis, which have provided a better understanding of the structure and evolution of the TrWRKY genes in white clover. Meanwhile, the genetic regulation network (GRN) containing TrWRKY genes was reconstructed, and Gene Ontology (GO) annotation analysis of these function genes showed they contributed to regulation of transcription process, response to wounding, and phosphorylay signal transduction system, all of which were important processes in response to abiotic stress. To determine the TrWRKY genes function under cold stress, the RNA-seq dataset was analyzed; most of TrWRKY genes were highly upregulated in response to cold stress, particularly in the early stages of cold stress. These results were validated by qRT-PCR experiment, implying they are involved in various gene regulation pathways in response to cold stress.
The results of this study provide insights that will be useful for further functional analyses of TrWRKY genes in response to biotic or abiotic stresses in white clover. These findings are likely to be useful for further research on the functions of TrWRKY genes and their role in response to cold stress, which is important to understand the molecular mechanism of cold tolerance in white clover and improve its cold tolerance.
•Yara-N oblique sensor successfully estimated N uptake in all developmental stages.•Yara-N oblique sensor can satisfactorily estimate forage yield and crude protein concentration.•SVM outperformed ...PLS for the estimation of forage dry matter yield and nutitional quality.
The project aim was to estimate N uptake (Nup), dry matter yield (DMY) and crude protein concentration (CP) of forage crops both during typical harvest times and at a very early developmental stage. Canopy spectral reflectance of legume and grass mixtures was measured in Sweden using a commercialized radiometer (400–1000 nm range). In total, 377 plant samples were tested in-situ in different grass and legume mixtures (6 grass species and 2 clover species) across two years, two locations and five N rates. Two mathematical methods, namely partial least squares (PLS) and support vector machine (SVM) were used to build prediction models between Nup, DMY and CP, and canopy spectral reflectance. Of the total 377 samples, 251 were randomly selected and used for calibration, and the remaining 126 samples were used as an independent dataset for validation. Results showed that the performance of SVM was better than PLS (based on mean absolute error (MAE) for both calibration and validation datasets) for the estimation of all investigated variables. Results for the validation set showed that the MAEs of PLS and SVM for Nup estimation were 17 and 9.2 kg/ha, respectively. The MAEs of PLS and SVM for DMY estimation were 587 and 283 kg/ha, respectively. The MAEs of PLS and SVM for CP estimation were 2.8 and 1.8%, respectively. In addition, a subsample, which corresponded to an early developmental stage, was analysed separately with PLS and SVM as for the whole dataset. Results showed that SVM was better than PLS for the estimation of all investigated variables. The high performance of SVM to estimate legume and grass mixture N uptake and dry matter yield could provide support for varying management decisions including fertilization and timing of harvest.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Symbiotic microorganisms such as arbuscular mycorrhizal fungi (AMF) and rhizobia (Rh) both promote host plant N acquisition, but whether and how the combination affects host plant nitrogen (N) ...assimilation remains unclear. To clarify the role of AMF and rhizobia on N assimilation in white clover (Trifolium repens), the effect of single or combined inoculation with an arbuscular mycorrhizal fungus Paraglomus occultum and a rhizobium Rhizobium trifolii on plant growth, photosynthesis, leaf N and amino acid contents, and the activities of N assimilation-related enzymes were analyzed in white clover under potted conditions. The results obtained were as follows: (1) The plant height, stolon length, leaf number, shoot biomass, total biomass, chlorophyll b and total chlorophyll contents, steady-state light quantum efficiency, and leaf N contents of white clover were significantly increased by single inoculation with Paraglomus occultum, single Rhizobium trifolii, and combined inoculations of Paraglomus occultum and
This study was designed to determine the effect of exogenous spermidine (Spd) (30 μM) on white clover seed germination under water stress induced by polyethylene glycol 6000. Use of seed priming with ...Spd improved seed germination percentage, germination vigor, germination index, root viability and length, and shortened mean germination time under different water stress conditions. Seedling fresh weight and dry weight also increased significantly in Spd-treated seeds compared with control (seeds primed with distilled water). Improved starch metabolism was considered a possible reason for this seed invigoration, since seeds primed with Spd had significantly increased α-amylase/β-amylase activities, reducing sugar, fructose and glucose content and transcript level of β-amylase gene but not transcript level of α-amylase gene. In addition, the physiological effects of exogenous Spd on improving seeds' tolerance to water deficit during germination were reflected by lower lipid peroxidation levels, better cell membrane stability and significant higher seed vigour index in seedlings. Enhanced antioxidant enzyme activities (superoxide dismutase, peroxidase, catalase and ascorbate peroxidase), ascorbate-glutathione cycle (ASC-GSH cycle) and transcript level of genes encoding antioxidant enzymes induced by exogenous Spd may be one of the critical reasons behind acquired drought tolerance through scavenging of reactive oxygen species (ROS) in water-stressed white clover seeds. The results indicate that Spd plays an important function as a stress-protective compound or physiological activator.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Published analyses of enteric methane (CH4) emissions from sheep and cattle show an inverse relationship between feed intake and CH4 yield (gCH4/kg dry matter (DM) intake), which suggests ...opportunities for reducing CH4 emissions from feed eaten and per unit of animal production. Most relationships between feed intake and CH4 yield have been based on animals fed conserved feeds, especially silages and grains. Our research is a series of experiments with fresh white clover (Trifolium repens) and perennial ryegrass (Lolium perenne; ryegrass) forages fed to sheep at a range of feed intake levels. This study was comprised of four experiments where good quality freshly harvested white clover or ryegrass were fed to sheep over a three-fold range in DM intake, and CH4 emissions were measured in respiration chambers for two consecutive days in each experiment. Measurements were made from 16 sheep in Experiment 1 (fed at 1.6×metabolizable energy requirements for maintenance; MEm), 28 sheep in Experiment 2 (at 0.8 and 2.0×MEm), eight sheep and two measurement periods in Experiment 3 (at 1.6×MEm), and 30 sheep in Experiment 4 (fed at 0.8, 1.2, 1.6, 2.0 and 2.5×MEm). Prior to each experiment, sheep had a 10d acclimatization period to diets. Apparent digestibility was measured over 7d from sheep in Experiments 1, 3 and 4, along with collection of rumen digesta for volatile fatty acid (VFA) determination. Although CH4 yields differed when sheep were fed white clover or ryegrass at similar intakes, the differences were inconsistent and mean values similar across all experiments. This, and a similar structure of all experiments, enabled combined analysis of data from all four experiments using the restricted maximum likelihood (REML) procedure to estimate effects of feed intake level on digestibility, digestible nutrient intake, gas emissions, and VFA concentrations in the rumen. The REML analysis showed that when DM intake increased from 0.40 to 1.60kg/d, the predicted responses were an increase in CH4 production (g/d) of 187% (12.4–35.6g/d; P<0.001), and a decline in CH4 yield of 21% (25.6–20.2g/kg DM intake; P<0.001). High feed intake levels were associated with increased molar proportions (mM of total VFA) of propionate from 0.17 to 0.21 (P=0.038). Single and multiple regressions were completed on the data from all experiments, with organic matter (OM) intake predicting 0.87 of the variation in CH4 production, and molar proportion of propionate predicting 0.60 of the variation in CH4 yield. Increasing feed intakes by 1kg/d of DM reduced CH4 yield by 4.5g/kg DM intake. Plant chemical composition was weakly related to CH4 yield. High intakes of fresh forages will lower CH4 yield from fermentation, but effects of feed composition on CH4 emissions were minor. The interaction between effects of feed intake and rumen function requires further investigation to understand relationships with CH4 emissions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Nitrogen (N) is an essential nutrient for plant growth. In grazed pasture systems in New Zealand, N fertiliser is regularly applied to maintain high productivity of ryegrass (Lolium perenne L.)/white ...clover (Trifolium repens L.) pastures. Several decades of trials/research have established the scientific basis for N management and N fertiliser application in grass/clover pasture systems. However, despite, or perhaps because of the rich diversity of this work, it isn't always easy to see the full picture of this research; the seminal research and sometimes more recent advances are not always easily discoverable. This paper therefore seeks to amalgamate and summarise some of the vast amount of research on N fertiliser use in grazed pasture systems undertaken in New Zealand over the last 50 years. It provides an overview of research, raising the awareness of key findings, and importantly providing a pathway for decision makers to find and access the research on N fertiliser use and management. It also assists in the identification of knowledge gaps for new avenues of enquiry that could improve the efficiency of N fertiliser use and reduce environmental impacts of N loss from grazed pasture systems.
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BFBNIB, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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