Integrated transcriptomic and metabolomic data reveal the cold stress responses molecular mechanisms of two coconut varieties.

Among tropical fruit trees, coconut holds significant edible and economic importance. The natural growth of coconuts faces a challenge in the form of low temperatures, which is a crucial factor among adverse environmental stresses impacting their geographical distribution. Hence, it is essential to enhance our comprehension of the molecular mechanisms through which cold stress influences various coconut varieties. We employed analyses of leaf growth morphology and physiological traits to examine how coconuts respond to low temperatures over 2-hour, 8-hour, 2-day, and 7-day intervals. Additionally, we performed transcriptome and metabolome analyses to identify the molecular and physiological shifts in two coconut varieties displaying distinct sensitivities to the cold stress. As the length of cold stress extended, there was a prominent escalation within the soluble protein (SP), proline (Pro) concentrations, the activity of peroxidase (POD) and superoxide dismutase (SOD) in the leaves. Contrariwise, the activity of glutathione peroxidase (GSH) underwent a substantial reduction during this period. The widespread analysis of metabolome and transcriptome disclosed a nexus of genes and metabolites intricately cold stress were chiefly involved in pathways centered around amino acid, flavonoid, carbohydrate and lipid metabolism. We perceived several stress-responsive metabolites, such as flavonoids, carbohydrates, lipids, and amino acids, which unveiled considerably, lower in the genotype subtle to cold stress. Furthermore, we uncovered pivotal genes in the amino acid biosynthesis, antioxidant system and flavonoid biosynthesis pathway that presented down-regulation in coconut varieties sensitive to cold stress. This study broadly enriches our contemporary perception of the molecular machinery that contributes to altering levels of cold stress tolerance amid coconut genotypes. It also unlocks several unique prospects for exploration in the areas of breeding or engineering, aiming to identifying tolerant and/or sensitive coconut varieties encompassing multi-omics layers in response to cold stress conditions.

Genome-wide identification, classification and expression analysis of MYB gene family in coconut (Cocos nucifera L.).

MYB transcription factors regulate the growth, development, and secondary metabolism of plant species. To investigate the origin of color variations in coconut pericarp, we identified and analyzed the MYB gene family present in coconut. According to the sequence of MYB genes in Arabidopsis thaliana, homologous MYB gene sequences were found in the whole genome database of coconut, the conserved sequence motifs within MYB proteins were analyzed by Motif Elicitation (MEME) tool, and the sequences without conservative structure were eliminated. Additionally, we employed RNA-seq technology to generate gene expression signatures of the R2R3-MYB genes across distinctive coconut parts exhibiting diverse colors. To validate these profiles, we conducted quantitative PCR (qPCR). Through comprehensive genome-wide screening, we successfully identified a collection of 179 MYB genes in coconut. Subsequent phylogenetic analysis categorized these 179 coconut MYB genes into 4-subfamilies: 124 R2R3-MYB, 4 3R-MYB types, 4 4R-MYB type, and 47 unknown types. Furthermore, these genes were further divided into 34 subgroups, with 28 of these subgroups successfully classified into known subfamilies found in Arabidopsis thaliana. By mapping the CnMYB genes onto the 16 chromosomes of the coconut genome, we unveiled a collinearity association between them. Moreover, a preservation of gene structure and motif distribution was observed across the CnMYB genes. Our research encompassed a thorough investigation of the R2R3-MYB genes present in the coconut genome, including the chromosomal localization, gene assembly, conserved regions, phylogenetic associations, and promoter cis-acting elements of the studied genes. Our findings revealed a collection of 12 R2R3-MYB candidate genes, namely CnMYB8, CnMYB15, CnMYB27, CnMYB28, CnMYB61, CnMYB63, CnMYB68, CnMYB94, CnMYB101, CnMYB150, CnMYB153, and CnMYB164. These genes showed differential expressions in diverse tissues and developmental stages of four coconut species, such as CnMYB68, CnMYB101, and CnMYB28 exhibited high expression in majority of tissues and coconut species, while CnMYB94 and CnMYB164 showed lower expression. These findings shed light on the crucial functional divergence of CnMYB genes across various coconut tissues, suggesting these genes as promising candidate genes for facilitating color development in this important crop.

Shoot organogenesis and somatic embryogenesis from leaf and petiole explants of endangered Euryodendron excelsum.

Euryodendron excelsum H.T. Chang is a rare and endangered woody plant endemic to China. It is very important to conserve and propagate this species from extinction. In this study, leaves and petioles from the axillary shoots in vitro were used as explants to culture on the different plant growth regulator (PGR) woody plant medium (WPM) and establish an efficient shoot proliferation and plant regeneration system. WPM supplemented with 1.0 mg/L 2,4-D induced callus dedifferentiated into buds and somatic embryos on various media,including PGR-free WPM. However, only adventitious shoots formed on WPM with 1.0 mg/L of cytokinins such as 6-benzyladenine (BA), kinetin (KIN) or thidiazuron (TDZ). When another cytokinin, zeatin, was used, somatic embryos were induced directly from From cut surface of these explants. Adventitious roots could be induced from both explants on WPM with 1.0 mg/L α-naphthaleneacetic acid (NAA). Somatic embryos cultured in PGR-free WPM or WPM with 0.2 mg/L NAA developed roots. Plantlets derived from somatic embryos were transferred to a peat: sand (1:1, v/v) substrate, and showed survival rates of 64.3% at 30 days and 54.6% at 90 days. Callus clumps with adventitious shoot buds that were transferred to WPM containing 1.0 mg/L BA and 0.2 mg/L NAA generated a mean 3.3 multiple shoots. Callus-derived shoots regenerated and rooted successfully (100%) on agar-free vermiculite-based WPM with 0.5 µM NAA after 30 d. Plantlets transplanted to peat soil: vermiculite (1:1, v/v) displayed the highest survival (96.7%) after three months.

Ectopic Expression of a Salt-Inducible Gene, LcSAIN3, from Sheepgrass Improves Seed Germination and Seedling Growth under Salt Stress in Arabidopsis.

Sheepgrass is a perennial native grass species in China, and it can tolerate high levels of salt stress with an aggressive and vigorous rhizome system. Many salt-stress-responsive genes have been identified in sheepgrass. In this study, we report the cloning and characterization of a novel salt-induced gene, LcSAIN3 (Leymus chinensis salt-induced 3), from sheepgrass. Expression analysis confirmed that LcSAIN3 was induced by PEG, ABA, and salt treatments, and the expression of LcSAIN3 was significantly increased in salt-tolerant germplasms under salt treatment. Subcellular localization analysis indicated that the GFP-LcSAIN3 protein was mainly localized in the chloroplasts. The heterologous expression of LcSAIN3 in Arabidopsis increased the seed germination rate of transgenic plants under salt, ABA, and mannitol treatments. The seedling survival rate, plant height, and fresh weight of the transgenic plants were higher than those of WT plants under salt stress. The overexpression of LcSAIN3 caused a relatively high accumulation of free proline, enhanced SOD activity, and led to the upregulation of several stress-responsive genes such as AtRD26, AtRD29B, AtSOS1, and AtP5CS1. These results suggest that LcSAIN3 could be a potential target for molecular breeding to improve plants' salt tolerance.

Axillary shoot proliferation and plant regeneration in Euryodendron excelsum H. T. Chang, a critically endangered species endemic to China.

Euryodendron excelsum H. T. Chang is a single-type, rare and endangered woody plant unique to China. In this study, young stems were used as explants and cultured on Woody Plant Medium (WPM) supplemented with 5.0 µM 6-benzyladenine (BA), were subcultured for more than 15 times over a total of more than 3 years and finally an efficient axillary shoot proliferation and plantlet regeneration system was established in which one shoot could proliferate an average of 5.1 axillary shoots every 2 months on the medium supplemented with 5.0 µM BA and 0.5 µM α-naphthaleneacetic acid (NAA). Shoots rooted at a moderate frequencies (50.1%) on agarized WPM supplemented with 0.5 µM NAA but 100% of shoots rooted in agar-free vermiculite-based WPM after culture for 2 months. Plantlets, when transplanted to peat soil: vermiculite (1:1), showed the highest 95.1% survival within 1 month.

LcMYB4, an unknown function transcription factor gene from sheepgrass, as a positive regulator of chilling and freezing tolerance in transgenic Arabidopsis.

BACKGROUND: Sheepgrass (Leymus chinensis (Trin.) Tzvel) is a perennial forage grass that can survive extreme freezing winters (- 47.5 °C) in China. In this study, we isolated an unknown function MYB transcription factor gene, LcMYB4, from sheepgrass. However, the function of LcMYB4 and its homologous genes has not been studied in other plants. RESULTS: The expression of the LcMYB4 gene was upregulated in response to cold induction, and the LcMYB4 fusion protein was localized in the nucleus, with transcriptional activation activity. Biological function analysis showed that compared with WT plants, LcMYB4-overexpressing Arabidopsis presented significantly increased chilling and freezing tolerance as evidenced by increased germination rate, survival rate, and seed setting rate under conditions of low temperature stress. Furthermore, LcMYB4-overexpressing plants showed increased soluble sugar content, leaf chlorophyll content and superoxide dismutase activity but decreased malondialdehyde (MDA) under chilling stress. Moreover, the expression of the CBF1, KIN1, KIN2 and RCI2A genes were significantly upregulated in transgenic plants with chilling treatment. These results suggest that LcMYB4 overexpression increased the soluble sugar content and cold-inducible gene expression and alleviated oxidative damage and membrane damage, resulting in enhanced cold resistance in transgenic plants. Interestingly, our results showed that the LcMYB4 protein interacts with fructose-1,6-bisphosphate aldolase protein1 (LcFBA1) and that the expression of the LcFBA1 gene was also upregulated during cold induction in sheepgrass, similar to LcMYB4. CONCLUSION: Our findings suggest that LcMYB4 encodes MYB transcription factor that plays a positive regulatory role in cold stress.

Adventitious shoot organogenesis from leaf explants of Portulaca pilosa L.

This study established, for the first time, shoot proliferation and plant regeneration protocols via shoot organogenesis from leaf explants of a medical and ornamental plant, Portulaca pilosa L. The optimal proliferation of axillary shoots was 6.2-fold within 30 days on Murashige and Skoog (MS) medium supplemented with 3.0 µM 6-benzyladenine (BA). Shoots could be induced directly from leaf explants, forming an average of 3.8 adventitious shoots per explant, on optimal MS medium supplemented with 1.0 µM thidiazuron (TDZ) and 0.1 µM α-naphthaleneacetic acid (NAA). A higher concentration of TDZ (3.0 µM), alone or in combination with 0.1 µM NAA, induced somatic embryo-like shoot buds and then developed into real shoots. Rooting was easier since roots were induced on all rooting media within one month. Half-strength MS medium free of plant growth regulators was best for rooting. Rooted plantlets were transferred to a sand: perlite (1:1, v/v) substrate, resulting in highest survival (90%). Plantlets showed more robust growth, however, on substrates of yellow mud: perlite (1:1, v/v) or peat soil: vermiculite: perlite (1:1:1, v/v).

A MYB-related transcription factor from sheepgrass, LcMYB2, promotes seed germination and root growth under drought stress.

BACKGROUND: Drought is one of the most serious factors limiting plant growth and production. Sheepgrass can adapt well to various adverse conditions, including drought. However, during germination, sheepgrass young seedlings are sensitive to these adverse conditions. Therefore, the adaptability of seedlings is very important for plant survival, especially in plants that inhabit grasslands or the construction of artificial grassland. RESULTS: In this study, we found a sheepgrass MYB-related transcription factor, LcMYB2 that is up-regulated by drought stress and returns to a basal level after rewatering. The expression of LcMYB2 was mainly induced by osmotic stress and was localized to the nucleus. Furthermore, we demonstrate that LcMYB2 promoted seed germination and root growth under drought and ABA treatments. Additionally, we confirmed that LcMYB2 can regulate LcDREB2 expression in sheepgrass by binding to its promoter, and it activates the expression of the osmotic stress marker genes AtDREB2A, AtLEA14 and AtP5CS1 by directly binding to their promoters in transgenic Arabidopsis. CONCLUSIONS: Based on these results, we propose that LcMYB2 improves plant drought stress tolerance by increasing the accumulation of osmoprotectants and promoting root growth. Therefore, LcMYB2 plays pivotal roles in plant responses to drought stress and is an important candidate for genetic manipulation to create drought-resistant crops, especially during seed germination.

Transcriptomic Analysis Reveals a Comprehensive Calcium- and Phytohormone-Dominated Signaling Response in Leymus chinensis Self-Incompatibility.

Sheepgrass (Leymus chinensis (Trin.) Tzvel.) is an economically and ecologically important forage in the grass family. Self-incompatibility (SI) limits its seed production due to the low seed-setting rate after self-pollination. However, investigations into the molecular mechanisms of sheepgrass SI are lacking. Therefore, microscopic observation of pollen germination and pollen tube growth, as well as transcriptomic analyses of pistils after self- and cross-pollination, were performed. The results indicated that pollen tube growth was rapidly inhibited from 10 to 30 min after self-pollination and subsequently stopped but preceded normally after cross-pollination. Time course comparative transcriptomics revealed different transcriptome dynamics between self- and cross-pollination. A pool of SI-related signaling genes and pathways was generated, including genes related to calcium (Ca2+) signaling, protein phosphorylation, plant hormone, reactive oxygen species (ROS), nitric oxide (NO), cytoskeleton, and programmed cell death (PCD). A putative SI response molecular model in sheepgrass was presented. The model shows that SI may trigger a comprehensive calcium- and phytohormone-dominated signaling cascade and activate PCD, which may explain the rapid inhibition of self-pollen tube growth as observed by cytological analyses. These results provided new insight into the molecular mechanisms of sheepgrass (grass family) SI.

Comparative transcriptome analysis provides insights into the distinct germination in sheepgrass (Leymus chinensis) during seed development.

Sheepgrass (Leymus chinensis ((Trin.) Tzvel)) is an important perennial forage grass that is widely distributed in the Eurasia steppe. The seed germination percentage show significant variation among the different germplasm in sheepgrass. However, the underlying molecular mechanisms of distinct germination during seed development are still mostly unknown. Here, we performed comparative transcriptomic analyses of high seed germination percentage (H) and low seed germination percentage (L) at 14, 28, and 42 days after pollination. After comparing 3 consecutive development stages, 9255, 5366, and 4306 genes were found to be significantly differently expressed between H and L. Pathway analysis indicated that transcripts related to starch and sucrose metabolism, phenylpropanoid biosynthesis, plant hormone signal transduction, amino sugar and nucleotide sugar metabolism, and photosynthesis were significantly changed between the two germplasm at three stages. ABA and GA metabolism- and signaling transduction-related genes were differentially expressed between two germplasm at development stages, suggesting that the reduced signaling of GA and ABA is likely to be related to seed germination and dormancy in sheepgrass. We also identified 81 transcription factor (TF) families, and some TFs genes such as NAC48, NAC78, WRKY80, ZnFP, C3H14 and ILR3 were significantly differential expressed in two germplasm. Our results provide insights into seed development, germination and dormancy in sheepgrass at the transcriptional level.

LcFIN2, a novel chloroplast protein gene from sheepgrass, enhances tolerance to low temperature in Arabidopsis and rice.

Adverse environmental stresses affect plant growth and crop yields. Sheepgrass (Leymus chinensis (Trin.) Tzvel), an important forage grass that is widely distributed in the east of Eurasia steppe, has high tolerance to extreme low temperature. Many genes that respond to cold stress were identified in sheepgrass by RNA-sequencing, but more detailed studies are needed to dissect the function of those genes. Here, we found that LcFIN2, a sheepgrass freezing-induced protein 2, encoded a chloroplast-targeted protein. Expression of LcFIN2 was upregulated by freezing, chilling, NaCl and abscisic acid (ABA) treatments. Overexpression of LcFIN2 enhanced the survival rate of transgenic Arabidopsis after freezing stress. Importantly, heterologous expression of LcFIN2 in rice exhibited not only higher survival rate but also accumulated various soluble substances and reduced membrane damage in rice under chilling stress. Furthermore, the chlorophyll content, the quantum photochemistry efficiency of photosystem II (ΦPSII), the non-photochemical quenching (NPQ), the net photosynthesis rate (Pn) and the expression of some chloroplast ribosomal-related and photosynthesis-related genes were higher in the transgenic rice under chilling stress. These findings suggested that the LcFIN2 gene could potentially be used to improve low-temperature tolerance in crops.

bHLH92 from sheepgrass acts as a negative regulator of anthocyanin/proanthocyandin accumulation and influences seed dormancy.

LcbHLH92, a pleiotropic gene from sheepgrass, negatively regulates anthocyanins/proanthocyandins and reduces seed dormancy in transgenic Arabidopsis.

MADS-box family genes in sheepgrass and their involvement in abiotic stress responses.

BACKGROUND: MADS-box genes are categorized into A, B, C, D and E classes and are involved in floral organ identity and flowering. Sheepgrass (Leymus chinensis (Trin.) Tzvel) is an important perennial forage grass and adapts well to many adverse environments. However, there are few studies on the molecular mechanisms of flower development in sheepgrass, especially studies on MADS-domain proteins. RESULTS: In this study, we cloned 11 MADS-box genes from sheepgrass (Leymus chinensis (Trin.) Tzvel), and phylogenetic analysis of the 11 genes with their homologs revealed that they are divided into nine subclades. Tissue-specific expression profile analysis showed that most of these MADS-box genes were highly expressed in floral organs. LcMADS1 and LcMADS3 showed higher expression in the stamen than in the other tissues, and LcMADS7 showed high expression in the stamen, glume, lemma and palea, while expression of LcMADS2, LcMADS9 and LcMADS11 was higher in vegetative organs than floral organs. Furthermore, yeast two-hybrid analyses showed that LcMADS2 interacted with LcMADS7 and LcMADS9. LcMADS3 interacted with LcMADS4, LcMADS7 and LcMADS10, while LcMADS1 could interact with only LcMADS7. Interestingly, the expression of LcMADS1 and LcMADS2 were significantly induced by cold, and LcMADS9 was significantly up-regulated by NaCl. CONCLUSION: Hence, we proposed that LcMADS1, LcMADS2, LcMADS3, LcMADS7 and LcMADS9 play a pivotal role in sheepgrass sexual reproduction and may be involved in abiotic stress responses, and our findings provide useful information for further exploration of the functions of this gene family in rice, wheat and other graminaceous cereals.

Jasmonate-Elicited Stress Induces Metabolic Change in the Leaves of Leucaena leucocephala.

The plant Leucaena leucocephala was exposed to four jasmonate elicitors, i.e., jasmonic acid (JA), methyl jasmonic acid (MeJA), jasmonoyl-l-isoleucine (JA-Ile) and 6-ethyl indanoyl glycine conjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acid methyl ester) (CGM). The treatment was to mimic the herbivores and wounding stresses. By using NMR spectroscopy along with chemometric analysis, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), the changes of metabolites in the leaves of L. leucocephala were determined under the stress as induced by the four elicitors. The challenge of JA-Ile caused an accumulation of lactic acid (6), ß-glucose (10), alanine (12), threonine (13), steroids (18), 3,4-dihydroxypyridine (19) and an unidentified compound 20. The chemometric analysis of the PCA and PLS-DA models indicated that the alternation of metabolites triggered by JA, MeJA, and CGM treatments were very minimum. In contrast, the treatment by JA-Ile could induce the most significant metabolic changes in the leaves. Moreover, there was very minimal new metabolite being detected in responding to the jasmonate-induced stresses. The results showed some metabolite concentrations changed after application of the elicitors, which may be related to a high level of tolerance to stress conditions as well as the strong ecological suitability of L. leucocephala.

New Insights on Drought Stress Response by Global Investigation of Gene Expression Changes in Sheepgrass (Leymus chinensis).

Water is a critical environmental factor that restricts the geographic distribution of plants. Sheepgrass [Leymus chinensis, (Trin.) Tzvel] is an important forage grass in the Eurasia Steppe and a close germplasm for wheat and barley. This native grass adapts well to adverse environments such as cold, salinity, alkalinity and drought, and it can survive when the soil moisture may be less than 6% in dry seasons. However, little is known about how sheepgrass tolerates water stress at the molecular level. Here, drought stress experiment and RNA-sequencing (RNA-seq) was performed in three pools of RNA samples (control, drought stress, and rewatering). We found that sheepgrass seedlings could still survive when the soil water content (SWC) was reduced to 14.09%. Differentially expressed genes (DEGs) analysis showed that 7320 genes exhibited significant responses to drought stress. Of these DEGs, 2671 presented opposite expression trends before and after rewatering. Furthermore, ~680 putative sheepgrass-specific water responsive genes were revealed that can be studied deeply. Gene ontology (GO) annotation revealed that stress-associated genes were activated extensively by drought treatment. Interestingly, cold stress-related genes were up-regulated greatly after drought stress. The DEGs of MAPK and calcium signal pathways, plant hormone ABA, jasmonate, ethylene, brassinosteroid signal pathways, cold response CBF pathway participated coordinatively in sheepgrass drought stress response. In addition, we identified 288 putative transcription factors (TFs) involved in drought response, among them, the WRKY, NAC, AP2/ERF, bHLH, bZIP, and MYB families were enriched, and might play crucial and significant roles in drought stress response of sheepgrass. Our research provided new and valuable information for understanding the mechanism of drought tolerance in sheepgrass. Moreover, the identification of genes involved in drought response can facilitate the genetic improvement of crops by molecular breeding.

Gibberellin stimulates regrowth after defoliation of sheepgrass (Leymus chinensis) by regulating expression of fructan-related genes.

Gibberellins (GAs) affect forage growth and development; however, it is largely unknown how GAs regulate the metabolism of fructan (an important polysaccharide reserve in many cereals) and the regrowth of forage plants after defoliation. To explore the mechanism of the responses of defoliated sheepgrass [Leymus chinensis (Trin.) Tzvel] to GA, we sprayed defoliated sheepgrass with GA3 and/or paclobutrazol (PAC; an inhibitor of GA biosynthesis) and analyzed the growth characteristics, carbohydrate contents, and transcript levels of genes related to GA metabolism, GA signal transduction, and fructan metabolism. The results showed that spraying exogenous GA3 onto defoliated sheepgrass promoted leaf and internode elongation, while spraying with PAC inhibited leaf and internode elongation, compared with the control. Spraying GA3 onto defoliated sheepgrass also altered the fructan content by extending the period of fructan utilization. At the transcriptional level, exogenous GA3 increased the transcript levels of genes related to GA metabolism in the sheath. Taken together, our results suggest that exogenous GA3 stimulates the regrowth of defoliated sheepgrass regrowth by regulating GA and fructan-related genes, and by promoting endogenous GA synthesis, fructan metabolism, and signaling.

Overexpression of a novel cold-responsive transcript factor LcFIN1 from sheepgrass enhances tolerance to low temperature stress in transgenic plants.

As a perennial forage crop broadly distributed in eastern Eurasia, sheepgrass (Leymus chinensis (Trin.) Tzvel) is highly tolerant to low-temperature stress. Previous report indicates that sheepgrass is able to endure as low as -47.5 °C,allowing it to survive through the cold winter season. However, due to the lack of sufficient studies, the underlying mechanism towards the extraordinary low-temperature tolerance is unclear. Although the transcription profiling has provided insight into the transcriptome response to cold stress, more detailed studies are required to dissect the molecular mechanism regarding the excellent abiotic stress tolerance. In this work, we report a novel transcript factor LcFIN1 (L. chinensis freezing-induced 1) from sheepgrass. LcFIN1 showed no homology with other known genes and was rapidly and highly induced by cold stress, suggesting that LcFIN1 participates in the early response to cold stress. Consistently, ectopic expression of LcFIN1 significantly increased cold stress tolerance in the transgenic plants, as indicated by the higher survival rate, fresh weight and other stress-related indexes after a freezing treatment. Transcriptome analysis showed that numerous stress-related genes were differentially expressed in LcFIN1-overexpressing plants, suggesting that LcFIN1 may enhance plant abiotic stress tolerance by transcriptional regulation. Electrophoretic mobility shift assays and CHIP-qPCR showed that LcCBF1 can bind to the CRT/DRE cis-element located in the promoter region of LcFIN1, suggesting that LcFIN1 is directly regulated by LcCBF1. Taken together, our results suggest that LcFIN1 positively regulates plant adaptation response to cold stress and is a promising candidate gene to improve crop cold tolerance.

Global transcriptome profiling analysis reveals insight into saliva-responsive genes in alfalfa.

KEY MESSAGE: We studied the genome-wide multiple time-course transcriptome dynamics after saliva deposition in alfalfa and demonstrate that saliva deposition functions as a stress that negatively affects the regrowth of alfalfa. Saliva deposition is one of the key factors influencing plant-herbivore interactions during grazing. Although many studies have focused on the effects of saliva deposition on plant regrowth, no consistent conclusions have been reached. Alfalfa is the most extensively cultivated forage legume, yet most alfalfa cultivars, thus far, are not grazing-tolerant. To better understand the underlying mechanism, we undertook a study to evaluate the global changes in the transcriptome of alfalfa after cow saliva deposition treatment. In this study, cDNA libraries from alfalfa seedlings at 0, 4, 8, and 24 h after cow saliva deposition were constructed and sequenced, resulting in the identification of 53,195 annotated unigenes, from which 4,814 unigenes were significantly differentially expressed. A metabolic pathway enrichment analysis demonstrated that saliva deposition functions as a stress that negatively affects the regrowth of alfalfa by modifying jasmonic acid synthesis, enhancing the susceptibility to pathogens and reducing the expression levels of ribosomal protein genes. In the present study, we demonstrate the potential effects of saliva deposition on alfalfa regrowth at the transcriptome level. These fundamental and important findings could facilitate further investigations into the molecular mechanisms underlying the responses of alfalfa and other related species to herbivore grazing.

Bovine serum albumin in saliva mediates grazing response in Leymus chinensis revealed by RNA sequencing.

BACKGROUND: Sheepgrass (Leymus chinensis) is an important perennial forage grass across the Eurasian Steppe and is adaptable to various environmental conditions, but little is known about its molecular mechanism responding to grazing and BSA deposition. Because it has a large genome, RNA sequencing is expensive and impractical except for the next-generation sequencing (NGS) technology. RESULTS: In this study, NGS technology was employed to characterize de novo the transcriptome of sheepgrass after defoliation and grazing treatments and to identify differentially expressed genes (DEGs) responding to grazing and BSA deposition. We assembled more than 47 M high-quality reads into 120,426 contigs from seven sequenced libraries. Based on the assembled transcriptome, we detected 2,002 DEGs responding to BSA deposition during grazing. Enrichment analysis of Gene ontology (GO), EuKaryotic Orthologous Groups (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed that the effects of grazing and BSA deposition involved more apoptosis and cell oxidative changes compared to defoliation. Analysis of DNA fragments, cell oxidative factors and the lengths of leaf scars after grazing provided physiological and morphological evidence that BSA deposition during grazing alters the oxidative and apoptotic status of cells. CONCLUSIONS: This research greatly enriches sheepgrass transcriptome resources and grazing-stress-related genes, helping us to better understand the molecular mechanism of grazing in sheepgrass. The grazing-stress-related genes and pathways will be a valuable resource for further gene-phenotype studies.