Comprehensive Transcriptome and Metabolome Analysis of Hemp (Cannabis Sativa L.) in Soil Under NaCl Stress

ABSTRACT Planting economic crops in soil with salt imbalances can improve land use efficiency. Hemp, which can be planted in low-salinity soil, is a crop with a very high economic value. To reveal the salt tolerance mechanism of hemp, the leaves of Longma #3 and #9 were collected at 0, 2, 4, and 6 days after salt stress in this study, and transcriptome and metabolome joint analysis were performed. The concentration of glutamic acid, succinic acid and gamma-aminobutyric acid (GABA) was 12 times that of the control group. The large increase in succinic acid led to the maintenance of peroxidase and superoxide dismutase levels at normal values and a decrease in H2O2 and catalase. The enriched KEGG pathway citrate cycle (TCA cycle) contains the metabolic regulation of succinic acid. Succinic acid was positively correlated with the MAPK gene. The MAPK pathway is a key pathway of plant tolerance in a high-salt environment. This study provides a reference for research on NaCl tolerance in plants and can also provide an important reference for the cultivation of NaCl-tolerant hemp varieties.


Introduction
Salt stress is an abiotic factor that endangers the sustainable development of global agriculture. Twenty percent of cultivated lands are affected, causing great harm to the growth and yield of plants (Sevilla et al. 2022). NaCl stress induces physiological and metabolic changes in most plants (Khan et al. 2023). Hemp generates at least 120 different cannabinoids, which change under abiotic stress (Park et al. 2022). In addition to salt stress, other abiotic stress can also lead to changes in hemp metabolism. Drought (Park et al. 2022), fertilizer (Anderson et al. 2021), light (Srajer Gajdosik et al. 2022), heavy metals Yin et al. 2022) can all cause changes in the physiology of the hemp.
Hemp (Cannabis sativa Linn.) is an annual herb that has become one of the crops popularized in saline alkali areas because of its strong growth adaptability and excellent stress resistance (Dixit 2022). Marijuana has been fully legalized in some countries, e.g., Canada (Fell et al. 2022), and cannabidiol (CBD) is listed as a legal drug for children in the United States (Failing, Boehnke, and Riebschleger 2021). The planting of hemp should thus be further promoted. The hemp we studied is mainly used for CBD extraction and plant fiber use. Guerriero et al. (2017) showed that 200 mM NaCl can lead to changes in gene expression in hemp leaf tissue. Some studies have also measured the physiological and biochemical indexes of hemp under salt stress (Dixit 2022;Yep, Gale, and Zheng 2020). Transcriptome and metabolome analysis revealed the mechanisms of the response of Nitraria sibirica Pall. to NaCl stress . Therefore, this research adopted the method of transcriptome and metabolome joint analysis. The purpose is to reveal the molecular response mechanism of hemp under NaCl stress.
Soil salinity leads to the accumulation of salt in the rhizosphere soil of plants, and adversely affects plants through osmotic and ionic stress. These stressors also lead to the production of reactive oxygen species (ROS) and hydrogen peroxide (H 2 O 2 ) in organelles (Alharbi, Al-Osaimi, and Alghamdi 2022). Therefore, the physiological and biochemical indexes measured in this study include peroxidase (POD), superoxide dismutase (SOD), H 2 O 2 , and catalase (CAT) (Kostyn et al. 2020). The salt tolerance of hemp varieties differ (Berni et al. 2021;Dixit 2022). In this study, the pot culture method was used to screen hemp varieties, and the effect of NaCl on hemp growth was further studied using the joint analysis method to provide basic research data for the study of hemp salt and alkaline tolerance.

Hemp salt stress experiment
The experimental site was located in the greenhouse of the Harbin Academy of Agricultural Sciences. The temperature in the greenhouse was 25°C; the humidity was 50-70%, and natural light was used. The salt stress experiment on industrial hemp was performed using pot culture. Longma #3 and #9 were used as experimental varieties. Seeds of uniform size, health, and plumpness for sowing were selected. The composition ratio of soil was organic soil: vermiculite = 5:1 (premixed). Each pot was filled with 150 g of potting soil, sown with 10 seeds, and watered every 7 days. The control group (CK) and treatment group comprised 6 pots with 3 replicates each. When the seedlings reached 8 cm, they were intercropped, and 4 seedlings were left in each pot. When the seedlings had 3-4 pairs of true leaves, the middle leaves of the plants were collected to determine biochemical indicators for transcriptome and metabolome analysis. CK was marked as 0 d. For the irrigation treatment, 400 mM NaCl solution was used, with 200 ml for each pot applied once, and no irrigation was applied for the next 6 days. CK was irrigated with 200 ml of deionized water and was not irrigated for the next 6 days to observe changes in its growth and appearance. The middle leaves of hemp plants irrigated with NaCl solution were collected at 2, 4, and 6 days under salt stress. The collected leaves were quickly frozen in liquid nitrogen and placed in a − 80°C refrigerator for subsequent experiments.

Measurement of physiological and biochemical indexes of hemp under salt stress
The leaves of Longma #3 and #9 were collected at 0, 2, 4, and 6 days of salt stress and sent to Suzhou Keming Biotechnology Co., Ltd. (www.cominbio.com, Suzhou, Zhejiang, China). The total protein content (CPR), POD, SOD, malondialdehyde (MDA), H 2 O 2 , CAT, proline (Pro), plant total potassium, and plant total sodium were measured. The test samples were random repeated 3 times. Used SPASS 21.0 software to eliminate outliers. GraphPad Prism 9.2.0 to display the average value as a concentration change curve and marked standard error (SE).

Hemp metabolome and transcriptome analysis under salt stress
According to the results of physiological and biochemical indexes, the leaves collected at 0, 2, 4, and 6 days of salt stress of Longma #9 were selected. The leaves were sent to Suzhou Panomic Biomedical Technology Co., Ltd. (http://www.biodeep.cn/, Suzhou, Zhejiang, China). Metabolome analysis comparing the Human Metabolome Database (HMDB), massbank, LipidMaps, mzclound, and Kyoto Encyclopedia of Genes and Genomes (KEGG) public databases was used to identify substances. The R v3.5.1 software package "Ropls" was used to compare and analyze the differences between samples. The Benjamini -Hochberg procedure false discovery rate (FDR) was used; when FDR≤0.05 and log2 (fold-changes) ≥ 1, metabolites increased, but when FDR≤0.05 and log2 (fold-changes) ≤ −1, they decreased. The MetaboAnalyst software package was used to enrich the functional pathways of the screened differential metabolic molecules. The 19 channels with the highest p values were used to create a bubble chart.
Transcriptome analysis was performed using HISAT2 software (http://ccb.jhu.edu/software/hisat2/ index.shtml) to align the reads obtained from the experiment to the reference genome. The R v3.5.1 software package "DESeq" was used to compare and analyze mRNA differences among various samples. When P ≤ .05 and log2 (fold-changes) ≥ 1, gene expression increased, and when FDR≤0.05 and log2 (fold-changes) ≤ −1, it decreased.
Metabolites and gene expression mRNA correlation analyses were performed. Based on the difference in metabolite content, representative metabolites were selected. The regulatory genes of this representative metabolite were determined according to the published literature. The regulatory genes related to metabolites were compared with transcriptome data obtained from the experiments (Silska 2020). The metabolites were correlated with the mRNA of the regulatory genes.

Changes in morphology and physiological and biochemical indexes of hemp under salt stress
After 4 days of salt stress, there was no obvious abnormality in the control group, but the plant height in the NaCl solution treatment group was relatively dwarfed, the overall leaf color was dim, the middle leaf was yellow and wilted, and some lower leaves became brown or even dry ( Figure 1). Under salt stress, the Na+ content in plants increased significantly and reached its peak on the 4th to 6th day ( Figure 2A). Figure 2B shows that in Longma #3 and #9, POD content did not change more than 1.5 times, and SOD in Longma #9 did not show a significant difference. Figure 2c shows that the K+ concentration first decreased, then gradually increased in Longma #9. For Longma #3 and #9, the H 2 O 2 and CPR content decreased, and CPR reached a stable value on days 4-6. Figure 2D shows that Pro improved. The CAT content decreased. Figure 2E shows that MDA increased and peaked on day 6. No outliers were found for the above-mentioned measurements. Through comprehensive comparison, Longma # 9 showed stronger salt tolerance and was used for further experiments.

Hemp metabolome analysis under salt stress
Figure 3A-C shows that the metabolites that increase in hemp under salt stress mainly include glutathione, myristic acid, 16-hydroxy hexadecanoic acid, retinol, formiminoglutamic acid, succinic acid, geranylgeraniol, gamma-aminobutyric acid (GABA), and glutamic acid. Among these, the succinic acid concentration of the NaCl treatment group reached 12 times that of CK at 2, 4, and 6 days. The concentrations of both glutamic acid and GABA in the NaCl-treated group were 12 times higher than those in the CK group. The main metabolites with reduced content were 6-methoxymellein, HC-toxin, saccharopine, lupan-3beta, 20-diol, (S)-abscisic acid, penciclovir, N-acetylbialaphos, apiole, hexadecanedioate, and 3-hydroxy-5-methyl-L-tyrosine. The KEGG pathway enrichment results, as shown in Figure 3D-F, were enriched in the following pathways: alanine, aspartate, and glutamate metabolism; lysine degradation; linoleic acid metabolism; cysteine and methionine metabolism; ABC transporters; citrate cycle (TCA cycle); glycine, serine, and threonine metabolism; carbon fixation in photosynthetic organisms; and arginine biosynthesis.

Hemp transcriptome analysis under salt stress
A 2-day comparison of CK showed that 3605 genes were upregulated and 3173 were downregulated ( Figure 4A). Figure 4B shows a 4-day comparison of upregulated and downregulated genes in CK (5245 and 5030, respectively). Figure 4C shows that after 6 days, in CK, there were 5399 upregulated and 5281 downregulated genes. Figure 4D shows the differential expression of genes. Figure 4E indicates the differentially expressed genes.
Two-day comparative CK association analysis showed that the succinic acid gene was positively related to LOC115721231 (mitogen-activated protein kinase, MAPK) and LOC115705779 (transcription factor DIVARICATA).

Discussion
Nearly 33% of irrigated lands in the world are affected by salt stress, and countermeasures have always been the focus of research (Sevilla et al. 2022). Specific methods include the cultivation of salt alkaliresistant rice varieties (Das et al. 2019;Gupta and Shaw 2020), the planting of salt-tolerant Tamarix ) and hemp (Cabral et al. 2022), and soil remediation (Mann, Rutter, and Zeeb 2020). Most of the Na+ in the soil is dissociated by NaCl, as well as Na 2 SO 4 , Na 2 CO 3 , and NaHCO 3 (Cabral et al. 2022). Salt stress is more harmful to plants in hot climates or dry and water-deficient conditions than in cold and cool conditions . Under strong light irradiation, salt stress has a greater inhibitory effect on plant growth and development than under weak light conditions. In this study, considering that rainfall would interfere with the results of the field experiments, pot experiments were adopted.
Halophytes can complete the life cycle from germination to seed production at a 200 mM NaCl concentration (Flowers and Colmer 2008). Plants have two methods to resist salt stress: reduce the accumulation of salt in the plant and enhance physiological activities or metabolic pathways to adapt to high-salt environments. Some halophytes, such as Limonium bicolor (Gao et al. 2021), Recretohalophytes (Lu et al. 2021), Tamarix chinensis , and Cynodon dactylon L (Parthasarathy et al. 2015), have salt glands that can expel salt from the body. Thus far, no salt glands have been found in hemp.  One other study showed that 200 mM NaCl was used to treat hemp for 15 days (Guerriero et al. 2017). In that report, Hemp watered 200 mM NaCl twice a week. The concentration of NaCl is continuous superposition. In other words, on 14 days, the lamp treats with 800 mM NaCl (Guerriero et al. 2017). In the greenhouse used in this study, dewdrops were condensed on the plant leaves every morning, so the designed experimental lamp was only watered once, with a total of 400 mM NaCl. The first few hours of watering NaCl were the time of stress reaction. No obvious changes in plant appearance were observed at this stage. Then hemp entered the NaCl adaptation stage. According to the observation results, on the the fourth day, the leaves of the treatment group were obviously dry. Therefore, the sampling time was set at 2 days and 4 days. The results of this study also reflected that transcriptome and metabolome analysis can explain the observed phenomenon of dry leaves.
Longma # 9 was a seed using hemp variety, and the content of tetrahydrocannabinol (THC) was less than 0.3%. The seed was called Huomaren (Maren), which was a traditional Chinese herbal medicine (Lu et al. 2022). Salt stress/adaptation could lead to changes in cannabinoid accumulation (Islam et al. 2022). The research team has been engaged in the planting of industrial hemp. We believed that when Hemp was planted in saline alkali land, the seeds were already under salt stress during germination, and the accumulation of cannabinoid occurred in the middle and late stages of development, so this stage was salt adaptation. For careful consideration, further research should focus on the content change of CBD and THC caused by NaCl stress.

Changes in the metabolome and transcriptome in hemp leaves under NaCl stress
This study showed that GABA in hemp leaves increased under NaCl stress. The enriched KEGG pathway Alanine, aspartate, and glutamate metabolism also includes the metabolic regulation of GABA. Other researchers have obtained similar results to those of this research. Under NaCl stress, GABA levels in Barclay seedings, soybean sprouts, sugar beet, and poplar also increased (Behr et al. 2021;Ji et al. 2020;Wang et al. 2021;. In plants, GABA can act as a signal molecule to promote plant growth and alleviate abiotic stress. Exogenous GABA can be applied to alleviate the stimulation of Sesbania rostrata resistance to salt stress . Succinic acid in poplar and rice also increased under NaCl stress (Das et al. 2019;Ji et al. 2020). In this study, the enriched citrate cycle (TCA cycle) of the KEGG pathway involved the metabolic regulation of succinic acid. The NaCl stress group also had increased succinic acid levels. This study also found that succinic acid was positively correlated with the MAPK gene. The MAPK pathway is a key pathway of plant tolerance in a high-salt environment (Xiong et al. 2020). Ginkgo can also activate genes related to the MAPK signal transduction pathway under the stress of 100 mM NaCl (Xu et al. 2020). Arabidopsis pumila transcriptome sequences during continuous salt stress showed that the MAPK gene was continuously upregulated (Yang et al. 2018).
1-Aminocyclopropanecarboxylic acid in tobacco decreases under 0.2 M NaCl (Pan et al. 2019). The experimental results of this study were similar, and 1-Aminocyclopropanecarboxylic acid in the NaCl stress group also decreased.

Changes in oxidative stress and physiological indexes of hemp under NaCl stress
The salt tolerance of hemp varieties differs (Berni et al. 2021;Dixit 2022). These results also show that the physiological and biochemical indexes of Longma #3 and #9 differed. In addition to NaCl for 48 and 168 hours, proline in Tamarix ramosissima also increases ). Our analysis of physiological and biochemical indicators showed that proline was upregulated, and KEGG analysis showed enriched arginine and proline metabolism. The concentration of glutamic acid, the precursor to proline synthesis, increased 12-fold. Proline is a protective amino acid. In the process of resisting osmotic stress caused by salt stress, proline can act as an osmotic protector .
NaCl induced H 2 O 2 production decline in muskmelon (Oryza sativa L.) (Welbaum, Tissaoui, and Bradford 1990). The H 2 O 2 concentration of different varieties of hemp increased or decreased under low salt concentrations and decreased significantly with a high concentration (Dixit 2022). When NaCl is applied to soybean seeds, SOD increases in soybean sprouts ). This experiment lasted for 6 days, during which the SOD change in Longma #3 was significantly different because after the salt stress phase, the SOD content returned to the previous level after entering the salt adaptation phase. SOD was not found in the 549 differentially expressed proteins, with the most significant difference. The large increase in succinic acid in this study led to the maintenance of POD and SOD levels at normal values, and decreased H 2 O 2 and CAT.
Similar to the results of this study, in an NaCl resistance experiment for Arabidopsis pumila, SOD increased to the highest value at 0-12 hours and then decreased, while K+ remained stable (Yang et al. 2018). Measurements in this study began on day 2, when the hemp had changed from NaCl stress to NaCl adaptation. Similar results can be seen from the fact that Na+ concentration remained stable from day 4 to day 6.

Ion transport changes under NaCl stress
Ions entering the cytoplasm are transported to vacuoles by reverse transport of Na+/H+. There are two types of channels on the vacuole membrane: FT-ATP (V-ATPase) (Siddiqui et al. 2021) and pyrogenase (V-PPase) (Theerawitaya et al. 2020). When treated with 200 mM NaCl, V-ATPase decreased in tomato seeding roots compared to the control (Siddiqui et al. 2021). In salt-tolerant rice breeding, V-ATPase is also recorded as an important reference index (Gupta and Shaw 2020). Sugarcane V-ATPase and V-PPase increase simultaneously within 3-7 days of 150 mM NaCl treatment (Theerawitaya et al. 2020). Under salt stress, due to the gradual increase of Na+ concentration in the soil, Na+ ions compete with K+ for transport carriers because they have the same transport mechanism, which reduces the absorption of K+ and leads to the loss and lack of K+ in plants Siddiqui et al. 2021). However, this study found that while NaCl concentration increased, K+ remained unchanged, indicating that hemp can resist salt stress.

Proposed hemp response to NaCl stress
NaCl stress activates the MAPK gene (Li et al. 2016;Xiong et al. 2020), and this study showed that MAPK mRNA content increased significantly. MAPK signals destroy the balance of chloroplast synthesis/decomposition (Liu and He 2017); chloroplasts are reduced, and the appearance of the hemp shows that the leaves lose their green pigment and wither. The decomposed chloroplast content was dispersed to other parts of the hemp. The main components of chlorophyll are alanine, arginine, glutamic acid, glycine, and lysine (Wei et al. 2021). In this study, the contents of succinic acid, GABA, and glutamic acid significantly increased. These amino acids can be transformed into each other. This can be interpreted as the proposed mode in which hemp responds to NaCl stress.

Conclusion
This study preliminarily elucidated the molecular mechanism of hemp salt resistance. While Na+ significantly increased, K+, POD, and SOD were relatively stable, suggesting that the NaCl tolerance of hemp may exceed expectations. In addition, joint analysis has found some candidate genes, and this can provide a reference for future research on plant tolerance to NaCl and can also provide important references for the cultivation of NaCl-tolerant varieties. Furthermore, new problems have emerged, such as whether K+ remains relatively stable while Na+ increases significantly. Solving these problems will be the goal of future research.