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. Here, downregulated expression of photosynthesis-related genes in drought-stressed leaves may have been associated with decreased photosynthetic capacity under drought stress. 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, and 6-phosphofructokinase 1 is a novel regulator indispensable for early chloroplast development. 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. Increased levels of soluble sugar and starch under drought stress indicates they are a preferred mechanism for maintaining root growth and metabolic processes. Raffinose synthase is one of the key enzymes for the biosynthesis of sucrose into oligosaccharides. It has been reported that raffinose synthase plays an important role in the osmotic regulation of nut seedling roots under drought stress. ABA can increase raffinose biosynthesis and promote desiccation tolerance. 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. 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. 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, 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. Trehalose 6-phosphate phosphatase controls the biosynthesis of trehalose, which functions as an osmolyte, carbon reserve, transport sugar, stress protectant, and signaling and homeostatic regulator of sucrose in plants.
Lignin is the main structural component of thickened secondary cell walls in almost all vascular plants, and it is very important for drought tolerance. 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 waterand participates in lignin biosynthesis. 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. The biosynthesis of cellulose can be promoted by sucrose synthase, and three genes encoded sucrose synthase. Xylan is the main component of hemicellulose, and application of β-xylosidase together with xylanase improves xylan hydrolysis. 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. β-Glucosidase functions with inactive ABA to produce active ABA, thereby increasing ABA levels in plants and improving drought tolerance. 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. Ethylene can inhibit root elongation and promote the formation and elongation of root hairs. 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. 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. HKs are important receptors widely involved in cellular reactions of bacteria, fungi, and plants. 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. The exogenous application or endogenous increase of CK content can significantly delay leaf senescence. 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. EREBP plays important roles in abiotic stress responses. 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 proteinsthat form channels in the plasma membrane and intracellular membranes, facilitating the passive movement of water among compartments. In maize, an aquaporin gene has been shown to be involved in root elongation and stomatal movement. 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. 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). 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. Polyamine-related genes were upregulated only in DTR and have been shown to inhibit lipoxygenase activity in lentils. In addition, 4-hydroxyphenylacetaldehyde can be produced by the activity of primary-amine oxidase, 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. 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). 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. 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. 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. Under drought conditions, the diffusion of potassium ions from soil to plant roots is impaired. Therefore, maintaining adequate cellular K+ has a positive effect on drought resistance of plants under water limitation. 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. 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, and ubiquitination-mediated protein degradation can improve drought tolerance. Speckle-type POZ protein is a key adaptor molecule of ubiquitinationand can promote ubiquitination. 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 cellsand plays a key role in organismal development. 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. E3 plays a unique role in the recognition of ubiquitinated target proteins. Some E3 ubiquitin-protein ligase-like F-box proteins have important roles in drought stress. 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. 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. 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. In plants, fatty acid omega-hydroxylase is essential for the synthesis of the cuticle, and fatty acids have also been found to improve drought tolerance. 3-ketoacyl-CoA synthase encodes the first component of the fatty acid elongation complex that is responsible for the synthesis of wax precursors and is therefore involved in limiting non-stomatal water loss and responding to drought stress. 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. They have been reported to increase growth. Chalcone synthase (CHS) catalyzes the first committed step in the biosynthesis of flavonoids, important secondary metabolites that play a key role in many aspects of plant growth and development. Homogentisate phytyltransferase (HPT) is an important enzyme in the biosynthesis of vitamin E. Vitamin E appears to be essential for plant development and participates in the response to a number of environmental stresses. 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. Tyrosine aminotransferase (TAT) is the first key enzyme for the synthesis of important secondary metabolites  and has a proposed role in abiotic stress response. 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. 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. 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 and is essential for the initiation of DNA replication. Mitotic-specific cyclin-B1 and NADPH can activate CDK1 throughout early mitosis, and CDK1 can ensure repression of macroautophagy during mitosis. 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. Dmc1 acts synergistically with the recombinase Rdh54 to mediate inter-homologue recombination during meiosis, and Dmc1 gene sequences are useful for resolution of the molecular phylogenetic relationships of tetraploid wheat. 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.