Comparative Analysis of Alfalfa (Medicago Sativa L.) Seedling Transcriptomes Reveals Genotype-specific Drought Tolerance Mechanisms

doi:10.21203/rs.3.rs-75482/v1

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Research article

Comparative Analysis of Alfalfa (Medicago Sativa L.) Seedling Transcriptomes Reveals Genotype-specific Drought Tolerance Mechanisms

https://doi.org/10.21203/rs.3.rs-75482/v1

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posted 06 Oct, 2020

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Background

Drought is one of the main abiotic factors that affect alfalfa yield. The identification of genes that control this complex trait can provide important insights for alfalfa breeding. However, little is known about how alfalfa responds and adapts to drought stress, particularly in cultivars of differing drought tolerance.

Results

In this study, the drought-tolerant cultivar Dryland ‘DT’ and the drought-sensitive cultivar WL343HQ ‘DS’ were used to characterize leaf and root physiological responses and transcriptional changes in response to water deficit. Under drought stress, Dryland roots (DTR) showed more differentially expressed genes than WL343HQ roots (DSR), whereas WL343HQ leaves (DSL) showed more differentially expressed genes than Dryland leaves (DTL). Many of these genes were involved in stress-related pathways, carbohydrate metabolism, and lignin and wax biosynthesis, which may have improved the drought tolerance of alfalfa. We also observed that several genes related to ABA metabolism, root elongation, peroxidase activity, cell membrane stability, ubiquitination, and genetic processing responded to drought stress in alfalfa. We highlighted several candidate genes, including sucrose synthase, xylan 1,4-beta-xylosidase, primary-amine oxidase, and alcohol-forming fatty acyl-CoA reductase, for future studies on drought stress resistance in alfalfa and other plant species.

Conclusions

In summary, our results reveal the unique drought adaptation and resistance characteristics of two alfalfa genotypes. These findings, which may be valuable for drought resistance breeding, warrant further gene functional analysis to augment currently available information and to clarify the drought stress regulatory mechanisms of alfalfa and other plants.

Plant Molecular Biology and Genetics

Plant Physiology and Morphology

Alfalfa

Genotype

Drought stress

Transcriptome

Leaves

Roots

We assembled leaf and root transcriptomes of drought-tolerant and drought-sensitive alfalfa cultivars under control and drought conditions.
Under drought, the drought-tolerant cultivar exhibited more differentially expressed metabolites than the drought-sensitive cultivar.
Genes associated with carbon metabolism, amino acid metabolism, hormones, and secondary metabolites were differentially expressed under drought stress.
The drought-tolerant cultivar exhibited higher expression of genes encoding sucrose synthase, xylan 1,4-beta-xylosidase, primary-amine oxidase, and alcohol-forming fatty acyl-CoA.

Drought is one of the most important environmental stresses in today’s agricultural landscape, and it exacerbates the damage caused by other abiotic stresses such as heat, salt, and cold[1]. Widespread water pollution and global climate change have increased the frequency of water deficits[2]. Drought stress alters various plant biochemical and physiological processes, including those related to photosynthesis[3], glucose metabolism[4], and secondary metabolism [5], thereby inhibiting plant growth and yield. Plants use multiple mechanisms to respond to drought stress, and there are therefore many candidate pathways through which stress tolerance can be engineered and enhanced. For example, early flowering[6] and changes in the root-to-shoot dry mass ratio reduced root and shoot growth[7] and decreased the leaf expansion rate[8]. Adding to the complexity, crops respond quite differently to drought stress depending on their developmental stage, and drought can occur at different times during the growing season.

Alfalfa (Medicago sativa L.) is an important leguminous forage crop grown worldwide. Its deep root system not only prevents soil erosion but also improves soil fertility [9]. Alfalfa is also a promising crop for use as a bioenergy feedstock[10]. Compared with other crops, alfalfa is relatively tolerant to drought[11]. Although alfalfa varieties can adapt to water-limited regions, water deficits still have a negative impact on productivity[12]. Therefore, improving the drought resistance of alfalfa and increasing its potential as a forage remain the focus of many research groups[13, 14]. The benefits of biotechnology in improving crop yields in stressful environments have recently become evident with the first report of improved drought tolerance in field crops[15]. Recently, many transgenic alfalfa lines with enhanced tolerance to drought stress have been produced[16-18] through the manipulation of transcription factors (TF, Alfin1, GsWRKY20, MsWRKY33, WXP1), oxidases (CodA, MnSOD, MsALR, AtNDPK2, AVP1, EsMcsu1MsZEP), and other genes (MicroRNA156, SPL6, SPL12 and SPL13, IbOr GsZFP1, ZxNHX/ZxVP1-1). Transcriptional profiling in plants with drought-tolerant and drought-susceptible genotypes provides greater insights into the complexity of plant responses to drought stress at the molecular level[19-21]. Transcriptome profiling data from alfalfa have been reported in several microarray studies and, more recently, RNA-seq studies[22-25]. However, alfalfa is a heterogeneous synthetic population[26]. This trait makes it a genetically complex species, recalcitrant to direct genomics studies. Although some specific genes have been identified, the heterogeneous genetic backgrounds of tolerant and susceptible germplasms often obscure the relationship between genetic variation and drought tolerant phenotypes; thus, using the same alfalfa genetic background is very important in the identification of genes involved in drought stress. Furthermore, the transcriptional regulatory network of the water stress response in alfalfa remains unstudied.

Here, we used cloned populations with the same genetic background to screen out drought-tolerant and drought-susceptible genotypes through many long-term indoor and outdoor experiments. Our objectives were to identify drought-responsive differentially expressed genes and to investigate the molecular and physiological mechanisms of adaptation to drought stress in alfalfa using physiological experiments and RNA-seq data. The resulting drought tolerance candidate genes can be subjected to further functional characterization and used for genetic engineering or breeding new drought-tolerant cultivars for use in arid environments.

Preliminary drought stress experiment

To investigate the transcriptional response of alfalfa to drought stress, we chose two varieties with different drought tolerances and performed RNA-seq transcriptome profiling. When alfalfa seedlings were grown with restricted water availability, the two varieties showed different phenotypes. The drought-tolerant variety DT showed relatively normal growth, whereas the drought-sensitive variety DS appeared to wilt (Fig. 1A). To choose a suitable sample collection time, we first selected six samples for use in a preliminary 6-d drought experiment. We observed that stem growth rate decreased significantly under drought stress; it declined more in DS than in DT, even shrinking on the fourth day (Fig. 1C). We therefore chose 4 d as the stress duration for the full experiment. DT had a greater root length than DS (Fig. 1B), and there were no significant differences between the varieties after drought stress. We also monitored ion changes under drought conditions. Levels of Ca²⁺increased in roots but decreased in leaves after drought stress, and there was greater Ca²⁺ accumulation in DSR. DT had higher K⁺ content than DS, and K⁺ content decreased in DTR and DTL under drought stress. In DS, drought caused no significant changes in roots but a small increase in leaves. (Fig.1D, E).

Overview of transcriptome changes in two alfalfa genotypes under drought stress

To investigate the molecular responses of alfalfa to drought stress, we compared gene transcription of seedlings under drought and control conditions. A total of 73,059 unique genes were identified and quantified by RNA-seq. We used thresholds of P < 0.05 and ≥2-fold expression difference to identify genes whose expression differed significantly between control and drought treatments. Drought stress resulted in more transcriptional changes in DS than DT in comparison to normal growth conditions. Interestingly, there were more DEGs in DT roots than DS roots but more DEGs in DS leaves than DT leaves (Fig. 2A).

The differing transcriptome profiles of DT and DS under drought stress suggested that DEGs between the two cultivars were also associated with plant characteristics other than drought stress responses. Therefore, we first compared the DEGs across genotypes and organs and found that 395 DEGs were genotype- and organ-independent (Fig. 2B). Based on KEGG analysis, these DEGs were enriched in members of the carbohydrate metabolism, amino acid metabolism and signal transduction pathways (Supplementary Data Fig 1). In addition, most genes that were differentially expressed in both organs belonged to the category of plant hormone signal transduction (Supplementary Table 2). Another highly represented process was biosynthesis of amino acids. Within this pathway, Arabidopsis histidine kinase 2/3/4 (a cytokinin receptor) was upregulated only in DTR. GO term analysis indicated that two terms were significantly enriched: ABA-activated signaling pathway and response to water deprivation (Supplementary Data Fig. 2). Expression trends were similar for most of those genes, and all of them belonged to the biological process GO category. By contrast, a gene encoding the transcription factor KUA1 OS, which participates in plant hormone signal transduction, was upregulated only in DTR (Fig. 4).

Second, we compared organ-specific DEGs. In roots of both genotypes, the most significant difference in gene enrichment was in plant hormone signal transduction (Supplementary Data Table 3). Among these genes, 5'-AMP-activated protein kinase (AMPK) was slightly upregulated in DTR but significantly downregulated in DSR, and the expression of genes associated with histidine and ethylene increased more in DTR than in DSR. Expression of genes encoding SAUR family protein, mitotic-specific cyclin-B1, and NADPH decreased more in DTR than in DSR. Among lipid metabolism genes, those encoding 3-ketoacyl-CoA synthase and lysophosphatidic acid acyltransferase were both upregulated in DTR but showed little change in DSR. Based on GO term analysis, numerous DEGs in both DTR and DSR were associated with response to water deprivation and response to abscisic acid (Supplementary Data Fig. 2). Among the genes associated with response to abscisic acid, those encoding protein phosphatase 2C, U-box domain, and low-temperature-induced 65 kDa protein OS were expressed at a higher level in DTR. Some genes encoding homeobox-leucine zipper proteins and protein phosphatase 2C were also involved in the response to water deprivation pathway. Nine genes were annotated as protein phosphatase 2C, and three of them were upregulated more in DTR than DSR. Moreover, genes encoding aquaporin PIP, Arabidopsis histidine kinase 2/3/4 (cytokinin receptor), and another unannotated gene were upregulated in DTR but downregulated in DSR, and these genes may have potential for the improvement of plant drought resistance.

KEGG pathway and GO term analysis results were similar for leaves, and many DEGs were also associated with photosynthesis and the cell wall. More DEGs in DSL were associated with the term chloroplast, whereas more DEGs in DTL were associated with the term extracellular region (Supplementary Data Table 4, Fig. 3).

Genes differentially expressed between the two varieties

To search for important drought-response genes, we identified DEGs that were unique to the roots or leaves of individual varieties. DT roots had a larger number of DEGs than DS roots. Among these, the number of DEGs in all KEGG pathways was greater in DTR than in DSR, with the exception of the signal sensing and transduction pathway. In contrast to DSR, DEGs in signal sensing and transduction were all downregulated in DTR (Supplementary Data Fig. 3). It is also interesting to note that the GO term “structural constituent of ribosome” showed the most significant change in DTR under drought. By comparison, the term signal transduction appeared more frequently in DSR (Supplementary Data Fig. 2).

When comparing leaves of the two varieties, two secondary metabolite pathways were significantly changed in DTL only: stilbenoid, diarylheptanoid and gingerol biosynthesis, and flavonoid biosynthesis (Supplementary Data Table 5). Genes in the stilbenoid, diarylheptanoid and gingerol biosynthesis pathway were downregulated. Only one gene in the flavonoid biosynthesis pathway was upregulated. There were more DEGs in DSL than in DTL, and the expression of genes involved in fatty acid degradation changed markedly (Supplementary Data Table 6). Consistent with the KEGG analysis, GO term analysis showed that DEGs were enriched in the term photosynthesis in both genotypes. DTL showed the greatest change in the term extracellular region, but DSL showed the greatest change in the term chloroplast (Fig. 3). In DTL, the most significantly enriched pathway was S-methyl-5-thioribose kinase activity: four DEGs were involved in this pathway, and all were upregulated.

Genes that shape drought tolerance in alfalfa

To better explore drought tolerance genes, we assumed that the DT genotype, which demonstrated greater drought tolerance based on physiological analyses, exhibited a higher level of expression of certain genes than DS under drought conditions. We therefore considered genes upregulated in DT but downregulated in DS to be drought tolerance genes; such genes were most prevalent in roots. The log₂(fold-change) values of genes with similar expression patterns were averaged, and genes were divided into six groups based on gene function: carbon metabolism, amino acid metabolite, lipid metabolite, genetic processing, reactive oxygen species (ROS) and secondary metabolism (Fig. 4). These results suggested that carbohydrate metabolism, amino acid metabolites, and secondary metabolism may play important roles in alfalfa drought stress resistance. In addition, to better understand the coordinated regulation of genes in all tissues, we mapped the drought tolerance gene network regulation (Fig. 5). The results showed that the DT genotype significantly accumulated genes involved in lignin biosynthesis and wax layer metabolism. To further confirm these findings, we measured the contents of amino acids, soluble sugars, peroxidase, and lignin in different tissues and genotypes. The soluble sugar contents increased after drought stress, and there was greater soluble sugar accumulation in DT than in DS (Fig. 6A), a trend consistent with the RNA-seq results. After drought, amino acid contents increased in DTR, DSR and DSL but decreased non-significantly in DTL, a result that did not correlate with the RNA-seq data (Fig. 6B). POD content was significantly lower in DTR, DSR and DSL after drought, consistent with the RNA-seq data, which showed that POD gene expression was higher in DTL than in other groups (Fig. 6C). DTR had a higher SOD content than the other groups (Fig. 6D). The content of acid-soluble lignin was decreased under drought in all groups but was higher in DT than in DS (Fig. 6E), consistent with the RNA-seq and qPCR data (Fig. 7). Changes in cuticular wax on the lower leaf epidermis were observed by scanning electron microscopy. The wax distribution in DS was relatively sparse compared with DT (Fig. 6F), a result that was also in accord with the RNA-seq and qPCR data. Taken together, these results suggest that the main causes of drought resistance in DT may be higher levels of soluble sugar, peroxidase, lignin, and waxes.

qRT–PCR validation of differentially expressed transcripts from RNA-seq

To verify the reliability of the RNA-seq data, the expression of 17 DEGs involved in carbon metabolism, amino acid metabolism, SOD, response to water, lignin, and wax synthesis were analyzed by qRT–PCR (Supplementary Table 7). A correlation coefficient analysis showed that their expression changes were highly correlated between the two analyses (Fig. 7), confirming the reliability of the RNA-seq data.

Plants react to drought stress at multiple levels, including biochemical, physiological, and developmental. Drought induces the transcription of metabolic genes, which contribute to the production and accumulation of osmotic substances that help to retain water and cellular antioxidants that protect cells from stress-related reactive oxygen species[34, 35]. In the present study, transcriptome analysis showed marked changes in gene expression of roots and leaves of alfalfa seedlings after 4 d of severe drought stress. Leaves exhibited more transcriptional changes than roots in drought-sensitive alfalfa, whereas roots exhibited more transcriptional changes than leaves in drought-tolerant alfalfa.

Genes involved in photosynthesis and carbon metabolism

Drought stress reduces the photosynthetic rate of plants, changes the distribution and metabolism of plant carbon, and causes decreased energy consumption and yield[36]. Here, downregulated expression of photosynthesis-related genes in drought-stressed leaves may have been associated with decreased photosynthetic capacity under drought stress[37]. The high number of downregulated genes associated with photosynthesis may also indicate that oxidative stress was higher in DSL than in DTL. Fructose-1,6-bisphosphatase (FBA) is an important metabolic enzyme involved in glycolysis, gluconeogenesis, and the Calvin cycle[38], and 6-phosphofructokinase 1 is a novel regulator indispensable for early chloroplast development[39]. Overexpression of FBA1 can alter growth, photosynthesis, and stress responses in higher plants[40, 41]. These two genes were upregulated in DTL, suggesting that they may have the potential to improve drought tolerance.

Drought stress is known to alter the activities of carbon-metabolizing enzymes[42]. Increased levels of soluble sugar and starch under drought stress indicates they are a preferred mechanism for maintaining root growth and metabolic processes[43]. Raffinose synthase is one of the key enzymes for the biosynthesis of sucrose into oligosaccharides[44]. It has been reported that raffinose synthase plays an important role in the osmotic regulation of nut seedling roots under drought stress[45]. ABA can increase raffinose biosynthesis and promote desiccation tolerance[46]. The expression of a raffinose synthase gene was markedly increased in DSL but decreased in DTR. From these results, we hypothesize that leaves of drought-sensitive alfalfa may produce more raffinose to resist severe drought stress. Moreover, 1,4-α-glucan, a branching enzyme, plays an important role in the biosynthesis of branched polysaccharides, glycogen, and amylopectin in carbohydrate metabolism[47]. A gene encoding this enzyme was up-regulated in DTL, suggesting that 1,4-α-glucan may contribute to its improved drought tolerance. Aldehyde dehydrogenase (NAD⁺) is an important enzyme whose overexpression can enhance drought and salt tolerance[48]. The upregulation of the corresponding genes in DT was consistent with its higher level of drought tolerance. Two genes encode β-D-fructofuranosidase, which promotes cell growth[49], and we speculate that it is also very important for alfalfa drought tolerance.

In addition, carbohydrates regulate drought stress. In our study, genes encoding fructokinase and trehalose 6-phosphate were upregulated in DT. Fructokinase plays an important role in plant tolerance to abiotic stresses, and it can promote the accumulation of osmoprotectants under drought[50]. Trehalose 6-phosphate phosphatase controls the biosynthesis of trehalose[51], which functions as an osmolyte, carbon reserve, transport sugar, stress protectant, and signaling and homeostatic regulator of sucrose in plants[52].

Lignin is the main structural component of thickened secondary cell walls in almost all vascular plants[53], and it is very important for drought tolerance[54]. Ferulate-5-hydroxylase is responsible for the final hydroxylation of syringyl-type lignin precursors. Laccase catalyzes the oxidation of various substrates while reducing molecular oxygen to water[55]and participates in lignin biosynthesis[56]. All of these genes were increased in DT, suggesting that increased lignin metabolism improves drought-tolerance.

Multiple cellulose-related genes were also upregulated. Endoglucanase is the main enzyme involved in the hydrolysis of biomass; it initiates cellulose hydrolysis and increases the cellulose hydrolysis rate. Ten genes encoded β-glucosidase, which hydrolyzes cellobiose into glucose[57, 58] and is the rate-limiting component of the cellulase-hydrolyzing reaction[59]. The biosynthesis of cellulose can be promoted by sucrose synthase[60], and three genes encoded sucrose synthase. Xylan is the main component of hemicellulose[61], and application of β-xylosidase together with xylanase improves xylan hydrolysis[62]. Together, we suggest that the cellulose-related genes may positively regulated the drought stress response in DT genotype.

Genes involved in hormone metabolism and root elongation

Many drought-responsive genes from both genotypes were involved in the signal transduction of plant hormones, especially ABA. ABA plays an important role in plant adaptation to environmental stress such as drought, cold, and high salinity[63]. β-Glucosidase functions with inactive ABA to produce active ABA, thereby increasing ABA levels in plants[64] and improving drought tolerance[64]. Six β-glucosidase genes were up-regulated in DTR only, indicating that these related genes may serve as candidate gene for alfalfa drought tolerance.

The root system is the first site of drought perception, and this environmental perception is reflected in changes in gene expression[65]. Ethylene can inhibit root elongation and promote the formation and elongation of root hairs[66]. There is increasing evidence that light and ethylene signaling pathways interact with auxin and other plant hormone signaling pathways[67, 68]. This system is usually composed of two proteins, a histidine kinase and a response regulator[69]. Interestingly, the expression of genes encoding Arabidopsis histidine kinase (HKs) and the transcription factor KUA1 OS were increased in DTR only, indicating that HKs may improve drought stress. HKs forms part of the multistep His–Asp phospho-relay that controls CK signaling in Arabidopsis[70]. HKs are important receptors widely involved in cellular reactions of bacteria, fungi, and plants[71]. In addition, HK can regulate root elongation by an ETR1-dependent abscisic acid signaling pathway. Arabidopsis HK 2/3/4 is a type of cytokinin receptor. Thus, we speculate that HK may have improved drought stress tolerance by promoting elongation of alfalfa roots. However, further studies are needed to determine the role of KUA1 OS in drought stress. In previous studies, drought stress inhibited the synthesis and transport of CK, decreasing the CK concentration and accelerating leaf senescence[72]. The exogenous application or endogenous increase of CK content can significantly delay leaf senescence[73]. Therefore, upregulation of CK-related genes in roots may improve drought survival. Another ethylene-responsive element binding protein is EREBP, which belongs to a large transcription factor family that is unique and widely distributed in plants[74]. EREBP plays important roles in abiotic stress responses[75]. Increased EREBP expression in DTR may indicate increased drought tolerance. It is interesting that there was greater upregulation of histidine- and ethylene-related genes in DTR than in DSR, perhaps indicating that such genes promote root elongation in drought-tolerant alfalfa. Aquaporins (AQPs) are intrinsic plant proteins[76]that form channels in the plasma membrane and intracellular membranes, facilitating the passive movement of water among compartments[77]. In maize, an aquaporin gene has been shown to be involved in root elongation and stomatal movement[78]. Our results suggest that the aquaporin PIP may improve alfalfa drought tolerance. In summary, DT showed greater expression of root extension-related genes, which may have contributed to its greater drought tolerance.

Genes involved in ROS and the stability of the plant cell membrane

It is well known that drought stress increases the production of reactive oxygen species (ROS) in intercellular compartments such as chloroplasts, peroxisomes, and mitochondria[79]. SOS2 is the central signaling element of the SOS pathway and represents a large family of protein kinases with catalytic domains similar to that of yeast sucrose nonfermenting 1 (SNF1) and mammalian amp activated kinase (AMPK)[80]. Therefore, upregulation of genes responsible for AMPK synthesis in roots may result in better drought survival. Peroxidase plays an important role in plant stress responses by efficiently scavenging H₂O₂ in the cytosol and chloroplasts[81]. Polyamine-related genes were upregulated only in DTR and have been shown to inhibit lipoxygenase activity in lentils[82]. In addition, 4-hydroxyphenylacetaldehyde can be produced by the activity of primary-amine oxidase[83], an enzyme involved in the biosynthesis of various secondary metabolites and compounds including plastoquinone[84, 85]. Primary-amine oxidase is also widely regarded as a drug target for the treatment of inflammatory, vascular, and neurodegenerative diseases[86]. Therefore, an increased abundance of these genes may improve drought tolerance.

The stability of plant cell membranes and ionic stability are also very important under drought conditions. Lysophosphatidic acid acyltransferases are known to function in the new glycerolipid biosynthesis pathway (Kennedy pathway), using lysophosphatidic acid (LPA) and acyl coenzyme a to form phosphatidic acid (PA)[87]. Hence, greater amounts of lysophosphatidic acid acyltransferase promote greater plant cell membrane stability. On this basis, alfalfa with more 3-ketoacyl-CoA synthase (KTCS) and lysophosphatidic acid acyltransferase should exhibit greater drought tolerance.

Drought stress can lead to insufficient supply of essential elements, thereby impairing plant metabolism and function[88]. H⁺-ATPase is an important transporter for the maintenance of membrane potential and the regulation of K⁺ transmembrane gradients in mesophyll cells. Early activation of root hair cell PM H⁺-ATPase triggers may increase the biosynthesis of major osmolytes, leading to upregulation of the water maintenance system[89]. It has been reported that maintenance of mesophyll K⁺ in tea plants under drought and rehydration is associated with regulation of plasma membrane H⁺-ATPase activity. In tea mesophyll cells, drought stress inhibited plasma membrane H⁺-ATPase activity, induced net H⁺ inflow, and led to membrane potential depolarization, resulting in a large K⁺outflow[90]. Under drought conditions, the diffusion of potassium ions from soil to plant roots is impaired[91]. Therefore, maintaining adequate cellular K⁺ has a positive effect on drought resistance of plants under water limitation[92]. The increased expression of genes encoding H⁺-ATPases observed here may have therefore been helpful in reducing K⁺ outflow under drought conditions and may have played a role in the improved drought tolerance of DTR. Ca²⁺ content normally increases in plants under abiotic stress[93]. In our results, Ca²⁺ accumulated in DT, especially in the roots, but decreased in the drought-sensitive cultivar. This result suggests that concentrations of Ca²⁺may improve drought stress tolerance, but severe drought stress may cause decreased Ca²⁺ levels, and this hypothesis requires further study.

Genes involved insecondarymetabolism

Secondary metabolites play an important role in plant drought tolerance[94], and ubiquitination-mediated protein degradation can improve drought tolerance[95]. Speckle-type POZ protein is a key adaptor molecule of ubiquitination[96]and can promote ubiquitination[97]. Our results therefore suggest that speckle-type POZ protein may improve drought tolerance by promoting ubiquitination. RING-box protein 1 (RBX1) regulates physiological cellular functions in animal cells[98]and plays a key role in organismal development[99]. RING-box proteins contain a RING finger domain; as the catalytic components of cullin E3 ligases, they participate in the ubiquitination of target proteins for subsequent proteasome-dependent degradation[12]. E3 plays a unique role in the recognition of ubiquitinated target proteins[100]. Some E3 ubiquitin-protein ligase-like F-box proteins have important roles in drought stress[101]. RING-box protein 1 was upregulated in DT under drought stress, which may indicate that it has a role in the response of alfalfa to drought stress.

Cuticular wax helps limit non-stomatal transpiration and has been used as a marker in breeding and selection of drought-resistant wheat cultivars[102]. Alcohol-forming fatty acyl reductases produce fatty alcohols that have a single hydroxyl group at the terminal position; these are often components of plant extracellular lipid barriers like cell walls or cuticular waxes[103]. Four fatty acid omega-hydroxylase genes were upregulated in DT; their products are known as signaling molecules that act as mediators of plant defense reactions[104]. In plants, fatty acid omega-hydroxylase is essential for the synthesis of the cuticle[105], and fatty acids have also been found to improve drought tolerance[106]. 3-ketoacyl-CoA synthase encodes the first component of the fatty acid elongation complex[107] that is responsible for the synthesis of wax precursors and is therefore involved in limiting non-stomatal water loss and responding to drought stress[108]. Significantly upregulated wax metabolism genes may constitute additional candidate genes for drought tolerance.

In addition to the genes above, we also identified additional drought-related genes in DT. Branched chain amino acid aminotransferases (BCATs) specialize in the degradation of the l-branched chain amino acids (BCAAs) leucine, isoleucine and valine using 2-oxoglutarate as an amino acceptor[109]. They have been reported to increase growth[110]. Chalcone synthase (CHS) catalyzes the first committed step in the biosynthesis of flavonoids[111], important secondary metabolites that play a key role in many aspects of plant growth and development[112]. Homogentisate phytyltransferase (HPT) is an important enzyme in the biosynthesis of vitamin E[113]. Vitamin E appears to be essential for plant development and participates in the response to a number of environmental stresses[114]. Three genes encoding tyrosine aminotransferase were upregulated in DTL. L-tyrosine is required for protein synthesis and also serves as a precursor for several plant metabolites, including alkaloids, phenylquinones, and cyanoglycosides[115]. Tyrosine aminotransferase (TAT) is the first key enzyme for the synthesis of important secondary metabolites [116] and has a proposed role in abiotic stress response[117]. It catalyzes the reversible conversion of tyrosine and 4-hydroxyphenylpyruvate in the tyrosine-derived pathway. Our results suggest that TAT may help to improve drought tolerance. In addition, two tropinone reductase (TR) genes were differentially expressedsmall proteins that belong to the short chain dehydrogenase/reductase family[118]. Their role in drought stress has received little attention and requires further study.

Genes involved in genetic processing

It is interesting that some genes involved in cell division were differentially expressed only in DT. NF-kappa B is a nuclear factor-κB (NF-κB) that participates in the regulation of inflammatory enzymes and cytokines[119]. According to our results, expression of the NF-kappa B gene may also improve drought stress tolerance. Three DEGs encoded origin recognition complex subunit 1 (ORC1), which is reported to be closely associated with the cell cycle[120] and is essential for the initiation of DNA replication[121]. Mitotic-specific cyclin-B1 and NADPH can activate CDK1 throughout early mitosis[122], and CDK1 can ensure repression of macroautophagy during mitosis[123]. Decreased expression of these genes in DT may have been associated with reduced macroautophagy in DTR. Carbon degradation repression (Crc) can directly inhibit the translation of mRNAs that encode enzymes. It can also indirectly inhibit the entry into cells of enzymes and transporters required for the translation of mRNAs encoding transcriptional regulators that drive the expression of genes whose products decompose specific substrates[124]. Dmc1 acts synergistically with the recombinase Rdh54 to mediate inter-homologue recombination during meiosis[125], and Dmc1 gene sequences are useful for resolution of the molecular phylogenetic relationships of tetraploid wheat[126]. We found few reports about the role of these cell cycle genes in drought tolerance, and additional research is needed to clarify their roles in alfalfa’s drought response.

Alfalfa has great economic and ecological importance, and drought stress is one of the major limitations on its growth and geographic distribution. Here, phenotypic, physiological, and transcriptomic analyses suggested that high contents of soluble sugars, lignin and wax were associated with greater drought stress tolerance in alfalfa. A number of upregulated genes appeared to have potential roles in drought tolerance, including those associated with ABA metabolism, root elongation, peroxidase activity, cell membrane stability, ubiquitination, and genetic processing. However, the drought-tolerance effects of these candidate genes require further empirical confirmation. The results obtained here provide new insight into regulatory pathways of drought resistance, and functional analysis of these drought tolerance-related genes is an important avenue for future research.

Plant materials and drought treatments

Two genotype alfalfa cloned populations, the drought-tolerant DT cultivar and the drought-sensitive DS cultivar, were selected for use in this study based on results from preliminary experiments. All plant materials were selected and cloned by cutting to ensure genotype consistency. The plants were transferred to plastic culture pots (10 cm height, 8 cm diameter, one plant per pot) that contained perlite and vermiculite (v/v, 1:1), with 18 pots per cultivar. The propagated plants grew in a greenhouse at 22°C and 40% relative humidity, and all plants were cut twice to ensure that their sizes were uniform. After 4 weeks, the plants were randomly assigned to control or drought stress groups. The control pots were continuously watered to maintain 100% field soil water capacity, whereas the drought pots were not continuously watered. The soil moisture level of each pot was measured in the morning and afternoon using a ProCheck with GS3 sensor. When the soil moisture level was lower than average and the variation was greater than 10%, we adjusted the soil moisture level to the average level by watering. Therefore, the soil moisture level of all pots was uniform, and all plants were exposed to similar drought stress conditions. Stress conditions were maintained until plants showed visible signs of wilting.

Phenotypic and biochemical analysis

Measurements of stem elongation rate and chlorophyll content were taken each morning at 9:00 AM for 6 d from the initiation to the cessation of drought stress. The stem length was measured each morning using a ruler, and the stem elongation rate was calculated as previously described[27]. The chlorophyll content was measured using a SPAD-502 meter (Konica Minolta, Osaka, Japan). Root length and all other physiological and biochemical parameters were measured after 4 d of drought stress. The root length was measured using a ruler. Leaf and root specimens were collected for biochemical measurements and transcriptome sequencing, and each replicate contained all leaves from an individual plant. The roots were dug out and washed free of soil under a stream of cold water. Six individual plants from each replicate of each treatment and cultivar were used, and the experiment was repeated three times. All specimens were collected between 10:00 AM and 12:00 PM and immediately frozen in liquid nitrogen. The concentrations of superoxide dismutase (SOD) and peroxidase (POD) were determined as previously described[28]. The content of free amino acids was determined by the ninhydrin method[29]. The sugar analysis was performed as previously described[30]with minor modifications. In brief, leaves and roots (0.1 g) were homogenized and transferred into test tubes to which 1 mL of 23% phenol solution was added. After incubation in a boiling water bath for 30 min, the supernatants were collected, and the absorbance was measured by UV spectrophotometry at 540 nm (Shimadzu, Japan). The concentrations of K⁺ and Ca²⁺ in leaves were determined by flame spectrophotometry (ColeParmer Instrument Co, Vernon Hills, IL, USA) as previously described[31].

The lignin concentration was measured as previously described [17] with minor modifications. In brief, dry specimens (100 mg) were ground, the cell wall materials were isolated using 0.01% sodium azide, and cell wall starch was digested with amylase and pullulans. The lignin level was quantified using the acetyl bromide method with an absorbance of 280 nm. An extinction coefficient of 15.693 was used to calculate the lignin concentration. The leaf wax content was determined by ESEM imaging. The middle sections of the second true leaves of alfalfa seedlings were randomly selected from control and treatment groups. The leaves were minced into fragments of 5 mm² and immediately fixed in 3% glutaric dialdehyde. The dehydrated samples were coated with gold particles using a sputtering machine in a vacuum dryer (Bal-Tec GmbH, Germany), and the epidermis was observed by environmental scanning electron microscopy (ESEM, Quanta-200, Fei Co., USA).

RNA sequencing and data analysis

Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion) according to the manufacturer’s instructions. RNA quality and quantity were evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Transcriptome sequencing and analysis were performed by OE Biotech Co., Ltd. (Shanghai, China). Raw reads were processed using Trimmomatic[32]. Both a P-value <0.05 and a fold-change >2 were used as the threshold to identify genes with significantly different levels of expression. Hierarchical clustering analysis of DEGs was performed to explore gene expression patterns. Gene Ontology (GO) and KEGG pathway enrichment analyses of all DEGs detected during different periods of drought were performed in R based on the hypergeometric distribution. The reads were reassembled using StringTie [33].

Validation of DEGs by qRT–PCR

To validate the RNA-seq data, 16 significant DEGs were selected for validation by qRT–PCR. The treatments and RNA isolation steps were performed as previously described. The primers for these genes were designed using Primer-BLAST at NCBI (Supplementary Table 1). We selected the actin gene as the endogenous control. The reverse-transcription reactions were performed using the PrimeScript Reagent Kit with gDNA Eraser (Takara, USA). Quantitative real time PCR was performed using TB Green Premix Ex Taq II (Takara). All analyses were performed using three biological replicates.

Conflicts of interest

The authors declare no conflicts of interest.

Authors’ contributions

QM Conceptualization, Validation, Resources, Writing: original draft, Writing: review & editing, Visualization. XX Validation, Visualization, Supervision. YX Visualization, Supervision. LZ performed the experiments, Software. WW analyzed the data, Formal analysis. DM: Validation, Visualization, Supervision, Project administration, Funding acquisition. All authors read

and approved the final manuscript.

Funding

This research was supported by the Project of First-Class Discipline Construction (Ecology) for Ningxia Higher Education (NXYLXK2017B06), National Natural Science Foundation of China (31760698) and Ningxia Agricultural Breeding Project 2019 (BBF02022-04), the Project of First-Class University of Western China for Key Laboratory of Ningxia University (GZXM2017001).

Acknowledgements

We also thank Prof. Xiqing Ma (China Agricultural University, College of Grass Science) for editorial suggestions.

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Comparative Analysis of Alfalfa (Medicago Sativa L.) Seedling Transcriptomes Reveals Genotype-specific Drought Tolerance Mechanisms

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