A genome sequence for the threatened whitebark pine.

Whitebark pine (WBP, Pinus albicaulis) is a white pine of subalpine regions in the Western contiguous United States and Canada. WBP has become critically threatened throughout a significant part of its natural range due to mortality from the introduced fungal pathogen white pine blister rust (WPBR, Cronartium ribicola) and additional threats from mountain pine beetle (Dendroctonus ponderosae), wildfire, and maladaptation due to changing climate. Vast acreages of WBP have suffered nearly complete mortality. Genomic technologies can contribute to a faster, more cost-effective approach to the traditional practices of identifying disease-resistant, climate-adapted seed sources for restoration. With deep-coverage Illumina short reads of haploid megagametophyte tissue and Oxford Nanopore long reads of diploid needle tissue, followed by a hybrid, multistep assembly approach, we produced a final assembly containing 27.6 Gb of sequence in 92,740 contigs (N50 537,007 bp) and 34,716 scaffolds (N50 2.0 Gb). Approximately 87.2% (24.0 Gb) of total sequence was placed on the 12 WBP chromosomes. Annotation yielded 25,362 protein-coding genes, and over 77% of the genome was characterized as repeats. WBP has demonstrated the greatest variation in resistance to WPBR among the North American white pines. Candidate genes for quantitative resistance include disease resistance genes known as nucleotide-binding leucine-rich repeat receptors (NLRs). A combination of protein domain alignments and direct genome scanning was employed to fully describe the 3 subclasses of NLRs. Our high-quality reference sequence and annotation provide a marked improvement in NLR identification compared to previous assessments that leveraged de novo-assembled transcriptomes.

Conserving a threatened North American walnut: a chromosome-scale reference genome for butternut (Juglans cinerea).

With the advent of affordable and more accurate third-generation sequencing technologies, and the associated bioinformatic tools, it is now possible to sequence, assemble, and annotate more species of conservation concern than ever before. Juglans cinerea, commonly known as butternut or white walnut, is a member of the walnut family, native to the Eastern United States and Southeastern Canada. The species is currently listed as Endangered on the IUCN Red List due to decline from an invasive fungus known as Ophiognomonia clavigignenti-juglandacearum (Oc-j) that causes butternut canker. Oc-j creates visible sores on the trunks of the tree which essentially starves and slowly kills the tree. Natural resistance to this pathogen is rare. Conserving butternut is of utmost priority due to its critical ecosystem role and cultural significance. As part of an integrated undergraduate and graduate student training program in biodiversity and conservation genomics, the first reference genome for Juglans cinerea is described here. This chromosome-scale 539 Mb assembly was generated from over 100 × coverage of Oxford Nanopore long reads and scaffolded with the Juglans mandshurica genome. Scaffolding with a closely related species oriented and ordered the sequences in a manner more representative of the structure of the genome without altering the sequence. Comparisons with sequenced Juglandaceae revealed high levels of synteny and further supported J. cinerea's recent phylogenetic placement. Comparative assessment of gene family evolution revealed a significant number of contracting families, including several associated with biotic stress response.

A Genome Sequence for the Threatened Whitebark Pine.

Whitebark pine (WBP, Pinus albicaulis ) is a white pine of subalpine regions in western contiguous US and Canada. WBP has become critically threatened throughout a significant part of its natural range due to mortality from the introduced fungal pathogen white pine blister rust (WPBR, Cronartium ribicola ) and additional threats from mountain pine beetle ( Dendroctonus ponderosae ), wildfire, and maladaptation due to changing climate. Vast acreages of WBP have suffered nearly complete mortality. Genomic technologies can contribute to a faster, more cost-effective approach to the traditional practices of identifying disease-resistant, climate-adapted seed sources for restoration. With deep-coverage Illumina short-reads of haploid megametophyte tissue and Oxford Nanopore long-reads of diploid needle tissue, followed by a hybrid, multistep assembly approach, we produced a final assembly containing 27.6 Gbp of sequence in 92,740 contigs (N50 537,007 bp) and 34,716 scaffolds (N50 2.0 Gbp). Approximately 87.2% (24.0 Gbp) of total sequence was placed on the twelve WBP chromosomes. Annotation yielded 25,362 protein-coding genes, and over 77% of the genome was characterized as repeats. WBP has demonstrated the greatest variation in resistance to WPBR among the North American white pines. Candidate genes for quantitative resistance include disease resistance genes known as nucleotide-binding leucine-rich-repeat receptors (NLRs). A combination of protein domain alignments and direct genome scanning was employed to fully describe the three subclasses of NLRs (TNL, CNL, RNL). Our high-quality reference sequence and annotation provide a marked improvement in NLR identification compared to previous assessments that leveraged de novo assembled transcriptomes.

Identification of Cd-resistant microorganisms from heavy metal-contaminated soil and its potential in promoting the growth and Cd accumulation of bermudagrass.

Phytoremediation has been increasingly used as a green technology for the remediation of heavy metal contaminated soils. Microorganisms could enhance phytoremediation efficiency by solubilizing heavy metal and improve plant growth by producing phytohormones in the heavy metal contaminated soils. In this study, we investigated the abundance and composition of soil microbial communities in heavy metal contaminated soils. Furthermore, we identified a Cd-resistant fungal strain Penicillium janthinellum ZZ-2 and assessed its potential in improving plant growth, Cd accumulation and Cd tolerance in bermudagrass. The results indicated that long-term heavy metal pollution decreased microbial biomass and activity by inhibiting microbial community diversity, but did not significantly affect community composition. Mainly, the relative abundance of some specific bacterial and fungal taxa, such as Actinobacteria, Chloroflexi, Bacteroidetes, Ascomycota and Basidiomycota, changes under metal pollution. Furthermore, at genus level, certain microbial taxa, such as Pseudonocardiaceae, AD3, Latescibacteria, Apiotrichum and Paraboeremia, only exist in polluted soil. One Cd-resistant fungus ZZ-2 was isolated and identified as Penicillium janthinellum. Further characterization revealed that ZZ-2 had a greater capacity for Cd2+ absorption, produced indole-3-acid (IAA), and facilitated plant growth in the presence of Cd. Interestingly, ZZ-2 inoculation significantly increased Cd uptake in the stem and root of bermudagrass. Thus, ZZ-2 could improve plant growth under Cd stress by reducing Cd-toxicity, increasing Cd uptake and producing IAA. This study suggests a novel fungus-assisted phytoremediation approach to alleviate Cd toxicity in heavy metals contaminated soils.

FaHSP17.8-CII orchestrates lead tolerance and accumulation in shoots via enhancing antioxidant enzymatic response and PSII activity in tall fescue.

Tall fescue (Festuca arundinacea Schreb.) shows huge potential for lead (Pb) phytoremediation, while little is known on the molecular mechanisms involved in Pb tolerance and accumulation. Here, genetic engineering strategy was firstly used to investigate Pb tolerance and accumulation in tall fescue. The transgenic tall fescue overexpressing a class II (CII) sHSP gene FaHSP17.8-CII was generated. After exposure to 1000 mg/L Pb(NO3)2, two FaHSP17.8-CII overexpressing lines, OE#3 and OE#7, showed higher tolerance to Pb as illustrated by the reduced levels of electrolyte leakage (EL) and malondialdehyde (MDA) as compared to the wild-type (WT) plants under Pb stress. Moreover, the FaHSP17.8-CII overexpression lines, OE#3 and OE#7, exhibited 36.3% and 46.6% higher shoot Pb accumulation relative to the WT grasses. When the grasses were exposed to Pb stress, the two OE lines had higher CAT, POD and SOD activities as compared to WT. Additionally, overexpression of FaHSP17.8-CII improved the synthesis of chlorophyll and transcript abundance of FapsbC, FapsbD and FapsbE, and alleviated the photoinhibition of PSII in tall fescue under Pb stress. This study provides an initial genetic engineering strategy to improve Pb phytoremediation efficiency in tall fescue by FaHSP17.8-CII overexpression.

Stress memory gene FaHSP17.8-CII controls thermotolerance via remodeling PSII and ROS signaling in tall fescue.

High temperature is the most limiting factor in the growth of cool-season turfgrass. To cope with high-temperature stress, grass often adopt a memory response by remembering one past recurring stress and preparing a quicker and more robust reaction to the next stress exposure. However, little is known about how stress memory genes regulate the thermomemory response in cool-season turfgrass. Here, we characterized a transcriptional memory gene, Fa-heat shock protein 17.8 Class II (FaHSP17.8-CII) in a cool-season turfgrass species, tall fescue (Festuca arundinacea Schreb.). The thermomemory of FaHSP17.8-CII continued for more than 4 d and was associated with a high H3K4me3 level in tall fescue under heat stress (HS). Furthermore, heat acclimation or priming (ACC)-induced reactive oxygen species (ROS) accumulation and photosystem II (PSII) electron transport were memorable, and this memory response was controlled by FaHSP17.8-CII. In the fahsp17.8-CII mutant generated using CRISPR/Cas9, ACC+HS did not substantially block the ROS accumulation, the degeneration of chloroplast ultra-structure, and the inhibition of PSII activity compared with HS alone. However, overexpression of FaHSP17.8-CII in tall fescue reduced ROS accumulation and chloroplast ultra-structure damage, and improved chlorophyll content and PSII activity under ACC+HS compared with that HS alone. These findings unveil a FaHSP17.8-CII-PSII-ROS module regulating transcriptional memory to enhance thermotolerance in cool-season turfgrass.

Comparative physiological and metabolic analyzes of two Italian ryegrass (Lolium multiflorum) cultivars with contrasting salinity tolerance.

Italian ryegrass (Lolium multiflorum) is a widely cultivated forage with high nutritional value and good palatability. Salinity, however, is a negative factor to lessen output and quality in Italian ryegrass. The aim of this study was to elucidate the salt tolerance mechanism of two Italian ryegrass cultivars, 'Abundant' and 'Angus'. Under hydroponic conditions, two cultivars of Italian ryegrass with different salt tolerance were exposed to 0 and 300 mM NaCl solution for 1 week, respectively. The results showed that salt stress decreased relative growth rate and relative water content, especially in salt-sensitive 'Angus'. The salt-tolerant 'Abundant' cultivar alleviated reactive oxygen species (ROS) induced burst and cell damage. However, 'Angus' exhibited a greater activity of superoxide dismutase (SOD) and peroxidase (POD) than 'Abundant'. Additionally, 'Abundant' exhibited higher photosynthetic efficiency than 'Angus' under salt stress condition. Salt treatment significantly increased the Na/K, Na/Mg, and Na/Ca ratios in the leaves and roots of both cultivars, with a pronounced effect in salt-sensitive 'Angus'. The metabolite analysis of leaf polar extracts revealed 41 salt responsive metabolites in both cultivars, mainly consisting of amino acids, organic acids, fatty acids, and sugars. Following exposure to salt conditions, salt-sensitive 'Angus' had a higher level of metabolites and more uniquely upregulated metabolites were detected. Based on these findings, we conclude that the 'Abundant' cultivar emerged as a favorite in saline-alkali soil, while the 'Angus' cultivar is suitable for planting in normal soil. It appears that the high salt tolerance of 'Abundant' is partly to prevent the plant from ionic homeostasis disruption.

SMRT and Illumina RNA sequencing reveal novel insights into the heat stress response and crosstalk with leaf senescence in tall fescue.

BACKGROUND: As a cool-season grass species, tall fescue (Festuca arundinacea) is challenged by increasing temperatures. Heat acclimation or activation of leaf senescence, are two main strategies when tall fescue is exposed to heat stress (HS). However, lacking a genome sequence, the complexity of hexaploidy nature, and the short read of second-generation sequencing hinder a comprehensive understanding of the mechanism. This study aims to characterize the molecular mechanism of heat adaptation and heat-induced senescence at transcriptional and post-transcriptional levels. RESULTS: Transcriptome of heat-treated (1 h and 72 h) and senescent leaves of tall fescue were generated by combining single-molecular real-time and Illumina sequencing. In total, 4076; 6917, and 11,918 differentially expressed genes (DEGs) were induced by short- and long-term heat stress (HS), and senescence, respectively. Venn and bioinformatics analyses of DEGs showed that short-term HS strongly activated heat shock proteins (Hsps) and heat shock factors (Hsfs), as well as specifically activated FK506-binding proteins (FKBPs), calcium signaling genes, glutathione S-transferase genes, photosynthesis-related genes, and phytohormone signaling genes. By contrast, long-term HS shared most of DEGs with senescence, including the up-regulated chlorophyll catabolic genes, phytohormone synthesis/degradation genes, stress-related genes, and NACs, and the down-regulated photosynthesis-related genes, FKBPs, and catalases. Subsequently, transient overexpression in tobacco showed that FaHsfA2a (up-regulated specifically by short-term HS) reduced cell membrane damages caused by HS, but FaNAC029 and FaNAM-B1 (up-regulated by long-term HS and senescence) increased the damages. Besides, alternative splicing was widely observed in HS and senescence responsive genes, including Hsps, Hsfs, and phytohormone signaling/synthesis genes. CONCLUSIONS: The short-term HS can stimulate gene responses and improve thermotolerance, but long-term HS is a damage and may accelerate leaf senescence. These results contribute to our understanding of the molecular mechanism underlying heat adaptation and heat-induced senescence.

Phytohormones-induced senescence efficiently promotes the transport of cadmium from roots into shoots of plants: A novel strategy for strengthening of phytoremediation.

Due to the long growth period of plants, phytoremediation is time costly. Improving the accumulation of cadmium (Cd) in shoots of plants will promote the efficiency of phytoremediation. In this study, two senescence-relative phytohormones, abscisic acid (ABA) and salicylic acid (SA), were applied to strengthening phytoremediation of Cd by tall fescue (Festuca arundinacea S.). Under hydroponic culture, phytohormones treatment increased the Cd content of shoots 11.4-fold over the control, reaching 316.3 mg/kg (dry weight). Phytohormones-induced senescence contributes to the transport of heavy metals, and HMA3 was found to play a key role in this process. Additionally, this strategy could strengthen the accumulation of Cu and Zn in tall fescue shoots. Moreover, in soil pot culture, the strategy increased shoot Cd contents 2.56-fold over the control in tall fescue, and 2.55-fold over the control in Indian mustard (Brassica juncea L.), indicating its comprehensive adaptability and potential use in the field. In summary, senescence-induced heavy metal transport is developed as a novel strategy to strengthen phytoremediation. The strategy could be applied at the end of phytoremediation with an additional short duration (7 days) with comprehensive adaptability, and markedly strengthen the phytoremediation in the field.

Toxicity of soil labile aluminum fractions and aluminum species in soil water extracts on the rhizosphere bacterial community of tall fescue.

Different forms of aluminum (Al) in soil can be toxic to plants and the bacterial community. In our previous study, the distribution and toxicity to plants of soil Al species and soil labile Al fractions were examined. However, the toxicity of different forms of Al on the bacterial community has not been completely studied. In this study, five soil samples (pH: 4.92, 6.17, 6.62, 6.70, 8.51) were collected from Lichuan, China. Tall fescue was planted in rhizosphere boxes with those soils for 120 days. The toxicity of soil Al species and soil labile Al fractions on the bacterial community of near-rhizosphere (NR) soils and far-rhizosphere (FR) soils were analyzed. The effect of different forms of Al on bacterial community between NR and FR soils was small, but the difference was obvious according to the different spatial distribution of samples. An individual bacterial community has eosinophilia, and most bacterial communities are tolerant of heavy metals (e.g., Cu, Zn, Cd). The toxicity of exchangeable Al has a strong effect on the bacterial community. Meanwhile, the toxicity of Al3+ to the bacterial community is strong. In this study, the key finding was that the toxicity of the Al-F- complex toward the bacterial community and plants was different. AlF2+, AlF2+, AlF3, and AlF4- are toxic for the bacterial community, and the correlation decreases with the addition of F-. This finding is of considerable significance to the treatment of acid-contaminated soil and the study of the tolerance mechanism of plants toward Al.

Lead-induced oxidative stress triggers root cell wall remodeling and increases lead absorption through esterification of cell wall polysaccharide.

Tall fescue (Festuca arundinacea Schreb) shows remarkable tolerance to lead (Pb), but the mechanisms involved in metal tolerance are not yet well understood. Here, tall fescue were firstly cultivated hydroponically with Pb2+ (0, 50, 200 and 1000 mg/L) for 14 days. The results showed that remodeling of root architecture plays important roles in tolerance of tall fescue to Pb2+ stress. Increased cell wall (CW) components contribute to restrict high amount of Pb2+ in roots. Additionally, the uronic acid contents of pectin, hemicellulose 1 (HC1) and hemicellulose 2 (HC2) increased under Pb2+ stress. We further observed that tall fescue cultivated with H2O2 showed similar remodeling of root architecture as Pb2+ treatment. Furthermore, pectin, HC1 and HC2 fractions were sequentially extracted from 0 and 10 mM H2O2 treated roots, and Pb2+ adsorption capacity and contents of carboxyl groups of pectin and HC2 fractions were steadily increased under H2O2 treatment in vitro. Our results suggest that degrees of esterification of pectin and HC2 are regulated by H2O2. High amount of low-esterified pectin and HC2 offer more carboxyl groups, provide more Pb2+ binding sites, and restrict more Pb2+ in the CW, which may enhance tolerance of tall fescue to Pb2+ stress.

Salt-Induced Damage is Alleviated by Short-Term Pre-Cold Treatment in Bermudagrass (Cynodon dactylon).

Excess salinity is a major environmental stress that limits growth and development of plants. Improving salt stress tolerance of plants is important in order to enhance land utilization and crop yield. Cold priming has been reported to trigger the protective processes in plants that increase their stress tolerance. Bermudagrass (Cynodon dactylon) is one of the most widely used turfgrass species around the world. However, the effect of cold priming on salt tolerance of bermudagrass is largely unknown. In the present study, wild bermudagrass was pre-treated with 4 °C for 6 h before 150 mM NaCl treatment for one week. The results showed that the cell membrane stability, ion homeostasis and photosynthesis process which are usually negatively affected by salt stress in bermudagrass were alleviated by short-term pre-cold treatment. Additionally, the gene expression profile also corresponded to the change of physiological indexes in bermudagrass. The results suggest that cold priming plays a positive role in improving salt stress tolerance of bermudagrass.

Comparative physiological and metabolomic analyses reveal mechanisms of Aspergillus aculeatus-mediated abiotic stress tolerance in tall fescue.

Aspergillus aculeatus has been shown to stimulate plant growth, but its role in plants abiotic stress tolerance and the underlying mechanisms are not fully documented. In this study, we investigated the mechanisms of A.aculeatus-mediated drought, heat and salt stress tolerance in tall fescue. The results showed that A.aculeatus inoculation improved drought and heat stress tolerance in tall fescue as observed from its effect on turf quality (TQ) and leaf relative water content (LWC). In the same stress conditions, A.aculeatus alleviated reactive oxygen species (ROS) induced burst and cell damage, as indicated by lower H2O2, electrolyte leakage (EL) and malondialdehyde (MDA) levels. Additionally, the A.aculeatus inoculated plants exhibited higher photosynthetic efficiency than uninoculated plants under drought, heat and salt stress conditions. The fungus reduced the uptake of Na+, and inoculated plants showed lower Na+/K+, Na+/Ca2+and Na+/Mg2+ ratios compared to uninoculated ones under salt stress. Furthermore, comparative metabolomic analysis showed that A.aculeatus exclusively increased amino acid (such as proline and glycine) and sugar (such as glucose, fructose, sorbose, and talose) accumulation under drought and heat stress. However, there were no differences between inoculated and uninoculated plants except for changes in H2O2 level, Na+ in the root and photosynthetic efficiency under salt stress. Taken together, this study provides the first evidence of the protective roles of A.aculeatus in the tall fescue response to abiotic stresses, partially via protection of photosynthesis and modulation of metabolic homeostasis.