Priming seeds with salicylic acid modulates membrane integrity, antioxidant defense, and gene expression in Medicago sativa grown under iron deficiency and salinity.

Exposure of plants to adverse environmental conditions reduces their growth and productivity. Currently, seed priming with phytohormones is considered one of the most reliable and cost-effective approaches that can help alleviate the toxic effects of environmental stress. In this context, the present study aims to investigate the effect of priming alfalfa seeds with salicylic acid (SA) on oxidative stress markers, including malonyldialdehyde, protein content, activities of antioxidant enzymes, and expression of genes encoding these enzymes in leaves and roots of alfalfa (Gabes ecotype) grown under saline stress, iron deficiency, or both. Our results showed that the application of salt stress and iron deficiency separately or simultaneously induces changes in the activities of antioxidant enzymes, but these are organ- and stress-dependent. The Gabes ecotype was able to increase the activities of these enzymes under salt stress to alleviate oxidative damage. Indeed, priming seeds with 100 µM SA significantly increases the enzymatic activities of APX, GPX, CAT, and SOD. Therefore, this concentration can be considered optimal for the induction of iron deficiency tolerance. Our results showed not only that Gabes ecotype was able to tolerate salt stress by maintaining high expression of the Fe-SOD isoform, but also that the pretreatment of seeds with 100 µM SA improved the tolerance of this ecotype to iron deficiency by stimulating Fe-SOD expression and inhibiting CAT and APXc.

Elucidation of Physiological, Transcriptomic and Metabolomic Salinity Response Mechanisms in Medicago sativa.

Alfalfa (Medicago sativa L.) is a widely grown perennial leguminous forage crop with a number of positive attributes. However, despite its moderate ability to tolerate saline soils, which are increasing in prevalence worldwide, it suffers considerable yield declines under these growth conditions. While a general framework of the cascade of events involved in plant salinity response has been unraveled in recent years, many gaps remain in our understanding of the precise molecular mechanisms involved in this process, particularly in non-model yet economically important species such as alfalfa. Therefore, as a means of further elucidating salinity response mechanisms in this species, we carried out in-depth physiological assessments of M. sativa cv. Beaver, as well as transcriptomic and untargeted metabolomic evaluations of leaf tissues, following extended exposure to salinity (grown for 3-4 weeks under saline treatment) and control conditions. In addition to the substantial growth and photosynthetic reductions observed under salinity treatment, we identified 1233 significant differentially expressed genes between growth conditions, as well as 60 annotated differentially accumulated metabolites. Taken together, our results suggest that changes to cell membranes and walls, cuticular and/or epicuticular waxes, osmoprotectant levels, antioxidant-related metabolic pathways, and the expression of genes encoding ion transporters, protective proteins, and transcription factors are likely involved in alfalfa's salinity response process. Although some of these alterations may contribute to alfalfa's modest salinity resilience, it is feasible that several may be disadvantageous in this context and could therefore provide valuable targets for the further improvement of tolerance to this stress in the future.

MsSPL9 Modulates Nodulation under Nitrate Sufficiency Condition in Medicago sativa.

Nodulation in Leguminous spp. is induced by common environmental cues, such as low nitrogen availability conditions, in the presence of the specific Rhizobium spp. in the rhizosphere. Medicago sativa (alfalfa) is an important nitrogen-fixing forage crop that is widely cultivated around the world and relied upon as a staple source of forage in livestock feed. Although alfalfa's relationship with these bacteria is one of the most efficient between rhizobia and legume plants, breeding for nitrogen-related traits in this crop has received little attention. In this report, we investigate the role of Squamosa-Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in nodulation in alfalfa. Transgenic alfalfa plants with SPL9-silenced (SPL9-RNAi) and overexpressed (35S::SPL9) were compared to wild-type (WT) alfalfa for phenotypic changes in nodulation in the presence and absence of nitrogen. Phenotypic analyses showed that silencing of MsSPL9 in alfalfa caused an increase in the number of nodules. Moreover, the characterization of phenotypic and molecular parameters revealed that MsSPL9 regulates nodulation under a high concentration of nitrate (10 mM KNO3) by regulating the transcription levels of the nitrate-responsive genes Nitrate Reductase1 (NR1), NR2, Nitrate transporter 2.5 (NRT2.5), and a shoot-controlled autoregulation of nodulation (AON) gene, Super numeric nodules (SUNN). While MsSPL9-overexpressing transgenic plants have dramatically increased transcript levels of SUNN, NR1, NR2, and NRT2.5, reducing MsSPL9 caused downregulation of these genes and displayed a nitrogen-starved phenotype, as downregulation of the MsSPL9 transcript levels caused a nitrate-tolerant nodulation phenotype. Taken together, our results suggest that MsSPL9 regulates nodulation in alfalfa in response to nitrate.

Effects of Transparent Testa8 (TT8) gene and Homeobox12 (HB12) gene silencing in alfalfa (Medicago sativa L.) on molecular structure spectral profile in relation to energy, degradation, and fermentation characteristics in ruminant systems.

Alfalfa (Medicago sativa L.) is a legume forage that is widely cultivated owing to its high biomass yield and favorable nutrient values. However, alfalfa contains relatively high lignin, which limits its utilization. Downregulation of two transcriptional factors, Transparent Testa8 (TT8) and Homeobox12 (HB12), has been proposed to reduce lignin content in alfalfa. Therefore, silencing of TT8 (TT8i) and HB12(HB12i) in alfalfa was achieved by RNAi technology. The objective of this project was to determine effect of gene modification through silencing of TT8 and HB12 genes in alfalfa plants on lignin and phenolic content, bioenergic value, nutrient supply from rumen degradable and undegradable fractions, and in vitro ammonia production in response to the silencing of TT8 and HB12 genes in alfalfa. All gene silenced alfalfa plants (5 TT8i and 11 HB12i) were grown under greenhouse conditions with wild type as a control. Samples were analyzed for bioactive compounds, degradation fractions, truly digestible nutrients, energetic values and in vitro ammonia productions in ruminant systems. Furthermore, relationships between physiochemical, metabolic and fermentation characteristics and molecular spectral parameters were determined using vibrational molecular spectroscopy. Results showed that the HB12i had higher lignin, while TT8i had higher phenolics. Both silenced genotypes had higher rumen slowly degraded carbohydrate fractions and truly digestible neutral detergent fiber, but lower rumen degradable protein fractions. Moreover, the HB12i had lower truly digestible crude protein, energetic values and ammonia production compared with other silenced genotypes. In addition, in relation to the nutritive values of alfalfa, structural carbohydrate parameters were negatively correlated, whereas alpha/beta ratio in protein structure was positively correlated. Furthermore, good predictions were obtained for degradation of protein and carbohydrate fractions and energy values from molecular spectral parameters. In conclusion, silencing of the TT8 and HB12 genes decreased protein availability and increased fiber availability. Silencing of the HB12 gene also increased lignin and decreased energy and rumen ammonia production. Moreover, nutritional alterations were closely correlated with molecular spectral parameters. Therefore, gene modification through silencing the TT8 and HB12 genes in alfalfa influenced physiochemical, metabolic and fermentation characteristics.

Eliciting Targeted Mutations in Medicago sativa Using CRISPR/Cas9-Mediated Genome Editing: A Potential Tool for the Improvement of Disease Resistance.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has become a breeding tool of choice for eliciting targeted genetic alterations in crop species as a means of improving a wide range of agronomic traits, including disease resistance, in recent years. With the recent development of CRISPR/Cas9 technology in Medicago sativa (alfalfa), which is an important perennial forage legume grown worldwide, its use for the enhancement of pathogen resistance is almost certainly on the horizon. In this chapter, we present detailed procedures for the generation of a single nonhomologous end-joining-derived indel at a precise genomic locus of alfalfa via CRISPR/Cas9. This method encompasses crucial steps in this process, including guide RNA design, binary CRISPR vector construction, Agrobacterium-mediated transformation of alfalfa explants, and molecular assessments of transformed genotypes for transgene and edit identification.

SPL12 Regulates AGL6 and AGL21 to Modulate Nodulation and Root Regeneration under Osmotic Stress and Nitrate Sufficiency Conditions in Medicago sativa.

The highly conserved plant microRNA, miR156, affects root architecture, nodulation, symbiotic nitrogen fixation, and stress response. In Medicago sativa, transcripts of eleven SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE, SPLs, including SPL12, are targeted for cleavage by miR156. Our previous research revealed the role of SPL12 and its target gene, AGL6, in nodulation in alfalfa. Here, we investigated the involvement of SPL12, AGL6 and AGL21 in nodulation under osmotic stress and different nitrate availability conditions. Characterization of phenotypic and molecular parameters revealed that the SPL12/AGL6 module plays a negative role in maintaining nodulation under osmotic stress. While there was a decrease in the nodule numbers in WT plants under osmotic stress, the SPL12-RNAi and AGL6-RNAi genotypes maintained nodulation under osmotic stress. Moreover, the results showed that SPL12 regulates nodulation under a high concentration of nitrate by silencing AGL21. AGL21 transcript levels were increased under nitrate treatment in WT plants, but SPL12 was not affected throughout the treatment period. Given that AGL21 was significantly upregulated in SPL12-RNAi plants, we conclude that SPL12 may be involved in regulating nitrate inhibition of nodulation in alfalfa by targeting AGL21. Taken together, our results suggest that SPL12, AGL6, and AGL21 form a genetic module that regulates nodulation in alfalfa under osmotic stress and in response to nitrate.

Genetic variation and structural diversity in major seed proteins among and within Camelina species.

MAIN CONCLUSION: Genetic variation in seed protein composition, seed protein gene expression and predictions of seed protein physiochemical properties were documented in C. sativa and other Camelina species. Seed protein diversity was examined in six Camelina species (C. hispida, C. laxa, C. microcarpa, C. neglecta, C. rumelica and C. sativa). Differences were observed in seed protein electrophoretic profiles, total seed protein content and amino acid composition between the species. Genes encoding major seed proteins (cruciferins, napins, oleosins and vicilins) were catalogued for C. sativa and RNA-Seq analysis established the expression patterns of these and other genes in developing seed from anthesis through to maturation. Examination of 187 C. sativa accessions revealed limited variation in seed protein electrophoretic profiles, though sufficient to group the majority into classes based on high MW protein profiles corresponding to the cruciferin region. C. sativa possessed four distinct types of cruciferins, named CsCRA, CsCRB, CsCRC and CsCRD, which corresponded to orthologues in Arabidopsis thaliana with members of each type encoded by homeologous genes on the three C. sativa sub-genomes. Total protein content and amino acid composition varied only slightly; however, RNA-Seq analysis revealed that CsCRA and CsCRB genes contributed > 95% of the cruciferin transcripts in most lines, whereas CsCRC genes were the most highly expressed cruciferin genes in others, including the type cultivar DH55. This was confirmed by proteomics analyses. Cruciferin is the most abundant seed protein and contributes the most to functionality. Modelling of the C. sativa cruciferins indicated that each type possesses different physiochemical attributes that were predicted to impart unique functional properties. As such, opportunities exist to create C. sativa cultivars with seed protein profiles tailored to specific technical applications.

Deciphering the role of SPL12 and AGL6 from a genetic module that functions in nodulation and root regeneration in Medicago sativa.

KEY MESSAGE: Our results show that SPL12 plays a crucial role in regulating nodule development in Medicago sativa L. (alfalfa), and that AGL6 is targeted and downregulated by SPL12. Root architecture in plants is critical because of its role in controlling nutrient cycling, water use efficiency and response to biotic and abiotic stress factors. The small RNA, microRNA156 (miR156), is highly conserved in plants, where it functions by silencing a group of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. We previously showed that transgenic Medicago sativa (alfalfa) plants overexpressing miR156 display increased nodulation, improved nitrogen fixation and enhanced root regenerative capacity during vegetative propagation. In alfalfa, transcripts of eleven SPLs, including SPL12, are targeted for cleavage by miR156. In this study, we characterized the role of SPL12 in root architecture and nodulation by investigating the transcriptomic and phenotypic changes associated with altered transcript levels of SPL12, and by determining SPL12 regulatory targets using SPL12-silencing and -overexpressing alfalfa plants. Phenotypic analyses showed that silencing of SPL12 in alfalfa caused an increase in root regeneration, nodulation, and nitrogen fixation. In addition, AGL6 which encodes AGAMOUS-like MADS box transcription factor, was identified as being directly targeted for silencing by SPL12, based on Next Generation Sequencing-mediated transcriptome analysis and chromatin immunoprecipitation assays. Taken together, our results suggest that SPL12 and AGL6 form a genetic module that regulates root development and nodulation in alfalfa.

Alfalfa transcriptome profiling provides insight into miR156-mediated molecular mechanisms of heat stress tolerance.

Heat is one of the major environmental stressors that negatively affects alfalfa production. Previously, we reported the role of microRNA156 (miR156) in heat tolerance; however, the mechanism and downstream genes involved in this process were not fully studied. To provide further insight, we compared an empty vector control and miR156-overexpressing alfalfa plants (miR156+) after exposing them to heat stress (40 °C) for 24 h. We collected leaf samples for transcriptome analysis to illustrate the miR156-regulated molecular mechanisms underlying the heat stress response. A total of 3579 differentially expressed genes (DEGs) were detected exclusively in miR156+ plants under heat stress using the Medicago sativa genome as a reference. GO and KEGG analysis indicated that these DEGs were mainly involved in "polysaccharide metabolism", "response to chemical", "secondary metabolism", "carbon metabolism", and "cell cycle". Transcription factors predicted in miR156+ plants belonged to the TCP family, MYB, ABA response element-binding factor, WRKY, and heat shock transcription factor. We also identified two new SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family gene members (SPL8a and SPL12a), putatively regulated by miR156. The present study provided a comprehensive transcriptome profile of alfalfa, identified a number of genes and pathways, and revealed an miR156-regulated network of mechanisms at the gene expression level to modulate heat responses in alfalfa.

Identification of Differential Drought Response Mechanisms in Medicago sativa subsp. sativa and falcata through Comparative Assessments at the Physiological, Biochemical, and Transcriptional Levels.

Alfalfa (Medicago sativa L.) is an extensively grown perennial forage legume, and although it is relatively drought tolerant, it consumes high amounts of water and depends upon irrigation in many regions. Given the progressive decline in water available for irrigation, as well as an escalation in climate change-related droughts, there is a critical need to develop alfalfa cultivars with improved drought resilience. M. sativa subsp. falcata is a close relative of the predominantly cultivated M. sativa subsp. sativa, and certain accessions have been demonstrated to exhibit superior performance under drought. As such, we endeavoured to carry out comparative physiological, biochemical, and transcriptomic evaluations of an as of yet unstudied drought-tolerant M. sativa subsp. falcata accession (PI 641381) and a relatively drought-susceptible M. sativa subsp. sativa cultivar (Beaver) to increase our understanding of the molecular mechanisms behind the enhanced ability of falcata to withstand water deficiency. Our findings indicate that unlike the small number of falcata genotypes assessed previously, falcata PI 641381 may exploit smaller, thicker leaves, as well as an increase in the baseline transcriptional levels of genes encoding particular transcription factors, protective proteins, and enzymes involved in the biosynthesis of stress-related compounds. These findings imply that different falcata accessions/genotypes may employ distinct drought response mechanisms, and the study provides a suite of candidate genes to facilitate the breeding of alfalfa with enhanced drought resilience in the future.

Involvement of the miR156/SPL module in flooding response in Medicago sativa.

The highly conserved plant microRNA, miR156, affects plant development, metabolite composition, and stress response. Our previous research revealed the role of miR156 in abiotic stress response in Medicago sativa exerted by downregulating SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE transcription factors. Here we investigated the involvement and possible mechanism of action of the miR156/SPL module in flooding tolerance in alfalfa. For that, we used miR156 overexpressing, SPL13RNAi, flood-tolerant (AAC-Trueman) and -sensitive (AC-Caribou) alfalfa cultivars exposed to flooding. We also used Arabidopsis ABA insensitive (abi1-2, abi5-8) mutants and transgenic lines with either overexpressed (KIN10-OX1, KIN10-OX2) or silenced (KIN10RNAi-1, KIN10RNAi-2) catalytic subunit of SnRK1 to investigate a possible role of ABA and SnRK1 in regulating miR156 expression under flooding. Physiological analysis, hormone profiling and global transcriptome changes revealed a role for miR156/SPL module in flooding tolerance. We also identified nine novel alfalfa SPLs (SPL1, SPL1a, SPL2a, SPL7, SPL7a, SPL8, SPL13a, SPL14, SPL16) responsive to flooding. Our results also showed a possible ABA-dependent SnRK1 upregulation to enhance miR156 expression, resulting in downregulation of SPL4, SPL7a, SPL8, SPL9, SPL13, and SPL13a. We conclude that these effects induce flooding adaptive responses in alfalfa and modulate stress physiology by affecting the transcriptome, ABA metabolites and secondary metabolism.

The CRISPR/Cas9-Mediated Modulation of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 in Alfalfa Leads to Distinct Phenotypic Outcomes.

Alfalfa (Medicago sativa L.) is the most widely grown perennial leguminous forage and is an essential component of the livestock industry. Previously, the RNAi-mediated down-regulation of alfalfa SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 (MsSPL8) was found to lead to increased branching, regrowth and biomass, as well as enhanced drought tolerance. In this study, we aimed to further characterize the function of MsSPL8 in alfalfa using CRISPR/Cas9-induced mutations in this gene. We successfully generated alfalfa genotypes with small insertions/deletions (indels) at the target site in up to three of four MsSPL8 alleles in the first generation. The efficiency of editing appeared to be tightly linked to the particular gRNA used. The resulting genotypes displayed consistent morphological alterations, even with the presence of up to two wild-type MsSPL8 alleles, including reduced leaf size and early flowering. Other phenotypic effects appeared to be dependent upon mutational dosage, with those plants with the highest number of mutated MsSPL8 alleles also exhibiting significant decreases in internode length, plant height, shoot and root biomass, and root length. Furthermore, MsSPL8 mutants displayed improvements in their ability to withstand water-deficit compared to empty vector control genotypes. Taken together, our findings suggest that allelic mutational dosage can elicit phenotypic gradients in alfalfa, and discrepancies may exist in terms of MsSPL8 function between alfalfa genotypes, growth conditions, or specific alleles. In addition, our results provide the foundation for further research exploring drought tolerance mechanisms in a forage crop.

Overexpression of miR156 and Silencing SPL6RNAi and SPL13RNAi Genes in Medicago sativa on the Changes of Carbohydrate Physiochemical, Fermentation, and Nutritional Profiles.

This study aimed to explore the comparative effects of overexpressing miR156 with individually silencing SPL6RNAi and SPL13RNAi genes on carbohydrate physiochemical, fermentation, and nutritional profiles of alfalfa (Medicago sativa). Three sub-genotypes of miR156 overexpressed (miR156 OE), SPL6RNAi, and SPL13RNAi grown with the wild type (WT) in a greenhouse were harvested 3 times at an early vegetative stage. Samples were freeze-dried, ground, and analyzed for carbohydrate nutritional profiles in terms of chemical composition, CNCPS fractions, energetic values, in vitro degradation, and fermentation characteristics. Results showed that miR156 OE had lower fiber and higher energy compared to all other genotypes. Moreover, miR156 OE had higher starch compared to SPL13RNAi and higher DM degradation compared to WT and SPL13RNAi. In conclusion, overexpression of miR156 decreased the fiber content of alfalfa but increased energy and DM degradation. SPL6RNAi was more similar to miR156 OE alfalfa in chemical composition and degradation, indicating that the SPL6RNAi gene plays an important role in the miR156 overexpression event.

Label-free quantitative proteomic analysis of alfalfa in response to microRNA156 under high temperature.

BACKGROUND: Abiotic stress, including heat, is one of the major factors that affect alfalfa growth and forage yield. The small RNA, microRNA156 (miR156), regulates multiple traits in alfalfa during abiotic stress. The aim of this study was to explore the role of miR156 in regulating heat response in alfalfa at the protein level. RESULTS: In this study, we compared an empty vector control and miR156 overexpressing (miR156OE) alfalfa plants after exposing them to heat stress (40 °C) for 24 h. We measured physiological parameters of control and miR156OE plants under heat stress, and collected leaf samples for protein analysis. A higher proline and antioxidant contents were detected in miR156OE plants than in controls under heat stress. Protein samples were analyzed by label-free quantification proteomics. Across all samples, a total of 1878 protein groups were detected. Under heat stress, 45 protein groups in the empty vector plants were significantly altered (P < 0.05; |log2FC| > 2). Conversely, 105 protein groups were significantly altered when miR156OE alfalfa was subjected to heat stress, of which 91 were unique to miR156OE plants. The identified protein groups unique to miR156OE plants were related to diverse functions including metabolism, photosynthesis, stress-response and plant defenses. Furthermore, we identified transcription factors in miR156OE plants, which belonged to squamosa promoter binding-like protein, MYB, ethylene responsive factors, AP2 domain, ABA response element binding factor and bZIP families of transcription factors. CONCLUSIONS: These results suggest a positive role for miR156 in heat stress response in alfalfa. They reveal a miR156-regulated network of mechanisms at the protein level to modulate heat responses in alfalfa.

Transcriptome-IPMS analysis reveals a tissue-dependent miR156/SPL13 regulatory mechanism in alfalfa drought tolerance.

BACKGROUND: We previously reported on the interplay between miR156/SPL13 and WD40-1/DFR to improve response to drought stress in alfalfa (Medicago sativa L.). Here we aimed to investigate whether the role of miR156/SPL13 module in drought response is tissue-specific, and to identify SPL13-interacting proteins. We analyzed the global transcript profiles of leaf, stem, and root tissues of one-month old RNAi-silenced SPL13 (SPL13RNAi) alfalfa plants exposed to drought stress and conducted protein-protein interaction analysis to identify SPL13 interacting partners. RESULT: Transcript analysis combined with weighted gene co-expression network analysis showed tissue and genotype-specific gene expression patterns. Moreover, pathway analysis of stem-derived differentially expressed genes (DEG) revealed upregulation of genes associated with stress mitigating primary and specialized metabolites, whereas genes associated with photosynthesis light reactions were silenced in SPL13RNAi plants. Leaf-derived DEG were attributed to enhanced light reactions, largely photosystem I, II, and electron transport chains, while roots of SPL13RNAi plants upregulated transcripts associated with metal ion transport, carbohydrate, and primary metabolism. Using immunoprecipitation combined with mass spectrometry (IPMS) we showed that SPL13 interacts with proteins involved in photosynthesis, specialized metabolite biosynthesis, and stress tolerance. CONCLUSIONS: We conclude that the miR156/SPL13 module mitigates drought stress in alfalfa by regulating molecular and physiological processes in a tissue-dependent manner.

Synchrotron-radiation sourced SR-IMS molecular spectroscopy to explore impact of silencing TT8 and HB12 genes in alfalfa leaves on the molecular structure and chemical mapping.

Advanced synchrotron-based vibrational molecular spectroscopy (SR-IMS) has been developed to image molecular chemistry in biological tissues within cellular and subcellular dimension. However, it is seldomly used in gene-transformation and gene-silencing study. The objectives of this study were to apply synchrotron-based vibrational molecular spectroscopy (SR-IMS) to determine the molecular structural changes and chemical mapping of alfalfa leaves induced by silencing of TT8 and HB12 genes in alfalfa in comparison with wild type of alfalfa. Five alfalfa leaves from each alfalfa genotype were selected for FTIR spectra collection and chemical mapping with synchrotron-based FTIR microspectroscopy (SR-IMS). Peak heights and areas of empirical regions were analyzed, and peak areas of previous regions were mapped for each sample using OMNIC 7.3. Results showed that transformed alfalfa had higher peak height and area of carbonyl CO (CCO), compared with wild type (WT). Chemical groups maps for carbohydrate, amide and lipid-related regions were successfully obtained. HB12-silenced (HB12i) had higher carbohydrate intensity both in the mesophyll and epidermises, whereas TT8-silenced (TT8i) and WT only had higher carbohydrate spectral peak intensity in epidermises. In addition, HB12i had higher CCO intensity and lower lignin intensity compared with TT8i and WT. All alfalfa genotypes had higher intensity of amide and asymmetric and symmetric CH2 and CH3 (ASCC) area in mesophylls. In conclusion, silencing of HB12 and TT8 genes in alfalfa both increased CCO profiles of alfalfa leaves, while silencing of HB12 had more impacts on chemical localization in alfalfa leaves.

Characterization of the Role of SPL9 in Drought Stress Tolerance in Medicago sativa.

Extreme environmental conditions, such as drought, are expected to increase in frequency and severity due to climate change, leading to substantial deficiencies in crop yield and quality. Medicago sativa (alfalfa) is an important crop that is relied upon as a staple source of forage in ruminant feed. Despite its economic importance, alfalfa production is constrained by abiotic stress, including drought. In this report, we investigate the role of Squamosa Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in drought tolerance. Transgenic alfalfa plants with RNAi-silenced MsSPL9 (SPL9-RNAi) were compared to wild-type (WT) alfalfa for phenotypic changes and drought tolerance indicators. In SPL9-RNAi plants, both stem thickness and plant height were reduced in two- and six-month-old alfalfa, respectively; however, yield was unaffected. SPL9-RNAi plants showed less leaf senescence and had augmented relative water content under drought conditions, indicating that SPL9-RNAi plants had greater drought tolerance potential than WT plants. Interestingly, SPL9-RNAi plants accumulated more stress-alleviating anthocyanin compared to WT under both drought and well-watered control conditions, suggesting that MsSPL9 may contribute to drought tolerance in alfalfa, at least in part, by regulating anthocyanin biosynthesis. The results suggest that targeting MsSPL9 is a suitable means for improving alfalfa resilience towards drought conditions.

The interplay between miR156/SPL13 and DFR/WD40-1 regulate drought tolerance in alfalfa.

BACKGROUND: Developing Medicago sativa L. (alfalfa) cultivars tolerant to drought is critical for the crop's sustainable production. miR156 regulates various plant biological functions by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. RESULTS: To understand the mechanism of miR156-modulated drought stress tolerance in alfalfa we used genotypes with altered expression levels of miR156, miR156-regulated SPL13, and DIHYDROFLAVONOL-4-REDUCTASE (DFR) regulating WD40-1. Previously we reported the involvement of miR156 in drought tolerance, but the mechanism and downstream genes involved in this process were not fully studied. Here we illustrate the interplay between miR156/SPL13 and WD40-1/DFR to regulate drought stress by coordinating gene expression with metabolite and physiological strategies. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40-1 to fine-tune DFR expression for enhanced anthocyanin biosynthesis. This, in combination with other accumulated stress mitigating metabolites and physiological responses, improved drought tolerance. We also demonstrated that SPL13 binds in vivo to the DFR promoter to regulate its expression. CONCLUSIONS: Taken together, our results reveal that moderate relative miR156 transcript levels are sufficient to enhance drought resilience in alfalfa by silencing SPL13 and increasing WD40-1 expression, whereas higher miR156 overexpression results in drought susceptibility.

Effects of silencing TT8 and HB12 on in vitro nutrients degradation and VFA production in relation to molecular structures of alfalfa (Medicago sativa).

BACKGROUND: Transparent Testa8 (TT8) and Homeobox12 (HB12) are two transcriptional factors in plant phenylpropanoid pathways and were reported to be positively related to lignin content. Alfalfa with silenced TT8 (TT8i) and HB12 (HB12i) was therefore generated using the RNA interference (RNAi) technique. Although lignin was found to be high in HB12i, such gene-silencing of alfalfa resulted in nutrient profiles that might be suitable for grazing. To extend the nutritional evaluation of transformed alfalfa, ground samples of 11 HB12i, 5 TT8i and 4 wild type (WT) were incubated in rumen fluid : buffer solution for 0, 2, 4, 8, 12, 24 and 48 h at 39 °C. Dry matter (DM) and neutral detergent fiber (NDF) degradations at each time point, and production of volatile fatty acids (VFA) at 4, 12, 24 and 48 h were analyzed, as well as degradation and production kinetics. The correlations and regressions between nutritive profiles and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectral parameters were determined. RESULTS: Both transformed genotypes had lower DM degradation and HB12i had lower VFA production compared with WT. Structural carbohydrate (STC) parameters were found to be negatively correlated with DM degradation and VFA production. The kinetics of DM degradation and VFA production were predicted from spectral parameters with good estimation power. CONCLUSION: Silencing of HB12 and TT8 affected fermentation characteristics of alfalfa and some fermentation characteristics were predictable from spectral parameters using ATR-FTIR spectroscopy. © 2019 Society of Chemical Industry.

Silencing TT8 and HB12 Decreased Protein Degradation and Digestion, Microbial Synthesis, and Metabolic Protein in Relation to Molecular Structures of Alfalfa (Medicago sativa).

This study aimed to explore the effects of silencing HB12 and TT8 genes on protein utilization characteristics of alfalfa. Ground samples of 11 HB12-silenced (HB12i), 5 TT8-silenced (TT8i) and 4 wild type (WT) were incubated in a Daisy II incubator with N15 labeled ammonium sulfate for 0, 4, 8, 12, and 24 h. CP degradation and degradational kinetics, microbial nitrogen fractions, and protein metabolic profiles were determined. Moreover, relationships between protein profiles and FTIR spectral parameters were estimated. Results showed that transgenic alfalfa had lower CP degradation, microbial protein, and total available protein compared with WT, especially for HB12i. In addition, CP degradation and protein metabolic profiles were closely correlated with FTIR spectral parameters and thereby could be predicted from spectral parameters. In conclusion, silencing of HB12 and TT8 genes in alfalfa decreased protein degradational and metabolic profiles, which were predictable with FTIR spectral parameters.