Transcriptome Profiling Reveals the Response of Seed Germination of Peganum harmala to Drought Stress

Peganum harmala L. is a perennial herbaceous plant that plays critical roles in protecting the ecological environment in arid, semi-arid, and desert areas. Although the seed germination characteristics of P. harmala in response to environmental factors (i.e., drought, temperature, and salt) have been investigated, the response mechanism of seed germination to drought conditions has not yet been revealed. In this study, the changes in the physiological characteristics and transcriptional profiles in seed germination were examined under different polyethylene glycol (PEG) concentrations (0–25%). The results show that the seed germination rate was significantly inhibited with an increase in the PEG concentration. Totals of 3726 and 10,481 differentially expressed genes (DEGs) were, respectively, generated at 5% and 25% PEG vs. the control (C), with 1642 co-expressed DEGs, such as drought stress (15), stress response (175), and primary metabolism (261). The relative expression levels (RELs) of the key genes regulating seed germination in response to drought stress were in accordance with the physiological changes. These findings will pave the way to increase the seed germination rate of P. harmala in drought conditions.


Introduction
Peganum harmala L. (family Zygophyllaceae) is a perennial herbaceous plant mainly distributed in arid, semi-arid, and desert areas in China (e.g., Xinjiang, Ningxia, and the Inner Mongolia province), and it plays critical roles in preventing land degradation [1].Furthermore, it has been widely used to treat diabetes, hypertension, nervous system disorders, etc.; these uses largely rely on its bioactive compounds, including alkaloids, flavonoids, and anthraquinones [2].
As is known, drought significantly limits plant biomass, crop yield, and agricultural cultivation [3].Plants can respond to drought stress through a series of physiological, biochemical, and nutritional networks [4].Extensive studies have demonstrated that drought stress inhibits plant growth while improving the activity of antioxidant enzymes (e.g., SOD, CAT, and APX) by inducing the expression levels of related genes and stressinducible genes encoding the enzymes involved in ABA biosynthesis [5,6].Previous studies on Peganum multisectum Bobr (the same genus with P. harmala) have found that drought stress significantly increased the SOD and CAT activities as well as the soluble sugar, MDA, and Pro contents [7].
To date, although the seed germination characteristics of P. harmala in response to different stress factors (i.e., drought, temperature, and salt) have been investigated [12][13][14], the regulatory mechanisms in the seed germination stage have not yet been revealed.In this study, the physiological characteristics and transcriptional profiles of P. harmala at the seed germination stage were examined under PEG 6000-induced drought stress.

Germination Characteristics and Physiological Indices under PEG Treatments
The seed germination was significantly affected by the PEG-induced drought stress (Figure 1).The germination characteristics (i.e., seed germination rate, length of hypocotyl and radicle, and fresh weight) significantly decreased with an increase in the PEG concentration (Figure 1A-D).Meanwhile, the PEG treatments significantly adjusted the activities of antioxidant enzymes (i.e., SOD, POD, CAT, and APX) and the contents of osmolytes (i.e., soluble sugar, protein, MDA, and Pro) (Figures S1 and S2).show the germination rate, hypocotyl length, radicle length, and fresh weight, respectively.Different letters indicate a significant difference at the p < 0.05 level.

Other Functional DEGs
Moreover, 490 DEGs were identified from the P. harmala in response to drought stress.The specific data are shown in Figure 3 and Tables S12-S16.

Discussion
Plants are frequently exposed to unfavorable environments, restricting their growth Plants 2024, 13, 1649 8 of 14 2.3.7.Other Functional DEGs Moreover, 490 DEGs were identified from the P. harmala in response to drought stress.The specific data are shown in Figure 3 and Tables S12-S16.

Discussion
Plants are frequently exposed to unfavorable environments, restricting their growth and development [15].Drought seriously inhibits plant growth and development by interrupting various biological processes [16].In this study, the physiological characteristics and transcriptional profiles of P. harmala were significantly affected during seed germination in response to PEG-induced drought stress.
Previous studies on drought have found that plants undergo changes in their phenotypes, antioxidant enzyme activities, and osmolyte contents [17].For example, the germination rate of P. harmala significantly decreased under water limitations, PGE treatments, and dry cold storage [12,18].The antioxidant enzymes and osmolytes in Peganum multisectum Bobr were affected under soil drought stress [7].The contents of soluble sugar, free amino acids, and Pro significantly increased, while MDA gradually increased in Seabuckthorn under drought stress [19].Furthermore, significant fluctuations of genes involved in protein kinases and TFs have been identified in response to drought stress [20]; for example, the expression levels of DHN1, RCD1, and LIPC were found to be related to the response to water-deficient up-regulation under PEG treatments [9].
In this study, the seed germination was significantly inhibited with an increase in the PEG concentrations, and the activities of antioxidant enzymes and contents of osmolytes were significantly altered under different PEG treatments.Meanwhile, 1642 DEGs were observed to be co-expressed in P. harmala, with 11 categories classified (e.g., drought stress, stress response, and primary metabolism) (see Figure 3).Specifically, 15 genes directly participate in drought stress.For example, ADH1 and ADHIII are involved in the response to water deprivation [21]; ANN1 is differentially regulated under drought conditions [22]; CRY1 and CRY2 participate in the response to water deprivation [23]; ERD7 and ERD14 participate in the rapid response to dehydration [24]; DRPD participates in the response to desiccation and is abundantly expressed in dried leaves [25]; EDL3 participates in the response to drought stress via the plant hormones [26]; HVA22E plays a role in stress tolerance and is differentially regulated by dehydration stress [27]; PLAT1 acts as a positive regulator in response to abiotic stress [28]; ARP1 is involved in the response to water deprivation, affecting ABA-regulated seed germination [29]; ASPG1 is involved in drought avoidance through ABA signaling [30]; REM4.2 plays a role in various abiotic stresses and participates in the SnRK1-mediated signaling pathway [31]; and CDSP32 participates in the plastid defense against oxidative damage in response to water deprivation [32].Based on the results of this study, we believe that these DEGs largely contribute to the seed germination under drought stress.
Generally, plants have evolved an antioxidant enzyme system to protect them from damage [33].Here, 20 genes were found to be directly associated with antioxidant enzyme activities.For example, BAS1, PER31, and PRXIIE-1 play critical roles in cell protection against oxidative stress [34]; PEX11C is involved in peroxisomal proliferation [35]; Gpx3 participates in catalyzing the reduction of peroxides [36]; Gpx4 participates in preventing membrane lipid peroxidation [37]; APX1 and APX3 play key roles in hydrogen peroxide removal [38]; AFRR and MDAR4 are involved in the detoxification of H 2 O 2 [39,40]; CATA and CAT2 protect cells from peroxide toxicity [41]; and PNC1 and PNC2 are involved in the response to the removal of H 2 O 2 [42].Based on the results of this study, these DEGs participate in regulating the antioxidant enzyme activities under drought stress.
Regarding the 12 genes directly associated with seed germination, briefly, ACT7 and AC97 are involved in seed germination and play an important role in cell division, organelle movement, and extension growth [52]; AP2 is required during seed development [53]; SBP65 plays roles in determining the seed germination capacity [54]; pec2a1a and At2g18540 act as seed storage proteins and play critical roles in seed development [55].In this study, these DEGs must play dominant roles in seed germination under drought stress.
TFs play significant roles in regulating plant growth and development as well as signal networks [56].Based on the results of this study, 42 TFs participate in seed germination and stress response.For example, MYB TFs participate in the drought, salt, and cold stress responses [57]; BZIP TFs (BZIP34 and BZIP53) act as transcriptional activators of seed development [58]; WRKY TFs (WRKY6 and WRKY71) are involved in the control of processes related to senescence and pathogen defense [59]; NAC TFs (NAC019, NAC92, and NAC056) are involved in the response to water deprivation and gene regulation during seed germination [60,61]; and BHLH TFs (BHLH94 and BHLH143) are involved in the response to abiotic stresses [62].The results of this study indicate that these IFs may play critical roles in regulating the seed germination of P. harmala.
As is known, plant hormones play critical roles in the adaptation to various stresses [9].Based on the results of this study, 52 genes participate in the hormone response; for example, GID1B acts as a GA receptor and is required for GA signaling, which controls seed germination [63]; AHK2 regulates several developmental processes, including seed germination, cell division, and shoot promotion [64]; and PYL4 is involved in ABA-mediated responses, such as germination inhibition [65].These DEGs must regulate the fluctuation of hormones to control the seed germination of P. harmala.
Based on the physiological characteristics and transcriptome profiles, a model of the seed germination of P. harmala in response to drought stress is outlined in Figure 10.Briefly, when seeds are exposed to drought stress, stress genes are induced to show differential expressions to switch on bio-signaling.Subsequently, low expression levels of genes participating in drought stress responses, soluble sugar metabolism, protein metabolism, and TFs will result in lower antioxidant enzyme activities and osmolyte contents, leading to membrane injury under drought stress; finally, these changes lead to cell morphogenesis, inhibiting the seed germination of P. harmala.seed germination [60,61]; and BHLH TFs (BHLH94 and BHLH143) are involved in the response to abiotic stresses [62].The results of this study indicate that these IFs may play critical roles in regulating the seed germination of P. harmala.
As is known, plant hormones play critical roles in the adaptation to various stresses [9].Based on the results of this study, 52 genes participate in the hormone response; for example, GID1B acts as a GA receptor and is required for GA signaling, which controls seed germination [63]; AHK2 regulates several developmental processes, including seed germination, cell division, and shoot promotion [64]; and PYL4 is involved in ABA-mediated responses, such as germination inhibition [65].These DEGs must regulate the fluctuation of hormones to control the seed germination of P. harmala.
Based on the physiological characteristics and transcriptome profiles, a model of the seed germination of P. harmala in response to drought stress is outlined in Figure 10.Briefly, when seeds are exposed to drought stress, stress genes are induced to show differential expressions to switch on bio-signaling.Subsequently, low expression levels of genes participating in drought stress responses, soluble sugar metabolism, protein metabolism, and TFs will result in lower antioxidant enzyme activities and osmolyte contents, leading to membrane injury under drought stress; finally, these changes lead to cell morphogenesis, inhibiting the seed germination of P. harmala.pgdC, 6-phosphogluconate dehydrogenase, decarboxylating 1; SUSs, sucrose synthase; FBAs, fructosebisphosphate aldolases; APA1, aspartic proteinase A1; FTSHs, ATP-dependent zinc metalloprotease FTSHs; RPT1, 26S proteasome regulatory subunit 7; MYBs, transcription factor MYBs; BZIPs, bZIP transcription factors; WRKYs, WRKY transcription factors; GMPM1, 18 kDa seed maturation protein; ASP, 21 kDa seed protein; At4g25140, oleosin 18.5 kDa.

Measurement of Germination Characteristics
After 3 d, the seed germination rate was measured.After 6 d, the lengths of the hypocotyls and radicles as well as the fresh weights of germinated seedlings were measured.Forty independent biological replicates were used for each measurement.

Transcriptomic Analysis
High-quality RNA samples of the C, 5, and 25% PEG-6000 seedlings were used to construct the cDNA library, and the biosynthesis of second-strand cDNA and purification of cDNA fragments were performed according to previous protocols [75].Their transcriptome profiles were analyzed using the Illumina HiSeqTM 4000 platform, with the filtration of raw reads, the assembly of clean reads, the normalization of transcripts to RPKM values, the expression analysis of different treatments, and the annotation of DEGs [76][77][78][79].

qRT-PCR Validation of Selected Genes
Primer-BLAST in the NCBI was used to design the primer sequence (Table S17).The cDNA synthesis, PCR amplification, and melting curve analysis were performed according to the manufacturers of the kits.The RELs of genes were calculated using the 2 −∆∆Ct method, with actin used as a reference control [80].

Statistical Analysis
Significant differences at the p < 0.05 level were analyzed using ANOVA and Duncan's post hoc test in SPSS 22.0.

Conclusions
The above observations reveal that the seed germination of P. harmala was inhibited under drought stress, with significant physiological changes in its antioxidant enzyme activities and osmolyte contents.The differential expressions of related genes play critical roles in regulating seed germination under drought stress.To identify the specific roles of genes related to drought resistance, additional studies are required.

Figure 1 .
Figure 1.Seed germination characteristics of Peganum harmala under PEG treatments.Images (A-D) show the germination rate, hypocotyl length, radicle length, and fresh weight, respectively.Different letters indicate a significant difference at the p < 0.05 level.

Figure 1 .
Figure 1.Seed germination characteristics of Peganum harmala under PEG treatments.Images (A-D) show the germination rate, hypocotyl length, radicle length, and fresh weight, respectively.Different letters indicate a significant difference at the p < 0.05 level.

Figure 2 .
Figure 2. Volcano plot of unigenes and number of DEGs under 5% and 25% PEG vs. C. Images (A,B) show the volcano plots; image (C) shows the number of DEGs.

Figure 2 .
Figure 2. Volcano plot of unigenes and number of DEGs under 5% and 25% PEG vs. C. Images (A,B) show the volcano plots; image (C) shows the number of DEGs.

Figure 3 .
Figure 3. Classification of DEGs under PEG treatments.Images (A,B) show the identified and unidentified DEGs; image (C) shows their Venn diagram; and image (D) shows the classification of the co-expressed DEGs.

Figure 3 .
Figure 3. Classification of DEGs under PEG treatments.Images (A,B) show the identified and unidentified DEGs; image (C) shows their Venn diagram; and image (D) shows the classification of the co-expressed DEGs.

Figure 3 .
Figure 3. Classification of DEGs under PEG treatments.Images (A,B) show the identified and unidentified DEGs; image (C) shows their Venn diagram; and image (D) shows the classification of the co-expressed DEGs.