Seung Yong Shin,Chae-Min Lee,Hyun-Soon Kim,Changsoo Kim,Jae-Heung Jeon,Hyo-Jun Lee
Ethylene signals modulate the survival of Arabidopsis leaf explants
Ethylene,Survival of explants,Defense gene expression,Anthocyanin,Arabidopsis
Background Leaf explants are major materials in plant tissue cultures. Incubation of detached leaves on phytohormone-containing media, which is an important process for producing calli and regenerating plants, change their cell fate. Although hormone signaling pathways related to cell fate transition have been widely studied, other molecular and physiological events occurring in leaf explants during this process remain largely unexplored. Results Here, we identified that ethylene signals modulate expression of pathogen resistance genes and anthocyanin accumulation in leaf explants, affecting their survival during culture. Anthocyanins accumulated in leaf explants, but were not observed near the wound site. Ethylene signaling mutant analysis revealed that ethylene signals are active and block anthocyanin accumulation in the wound site. Moreover, expression of defense-related genes increased, particularly near the wound site, implying that ethylene induces defense responses possibly by blocking pathogenesis via wounding. We also found that anthocyanin accumulation in non-wounded regions is required for drought resistance in leaf explants. Conclusions Our study revealed the key roles of ethylene in the regulation of defense gene expression and anthocyanin biosynthesis in leaf explants. Our results suggest a survival strategy of detached leaves, which can be applied to improve the longevity of explants during tissue culture. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-023-04299-4.
Ethylene signals modulate the survival of Arabidopsis leaf explants

Leaf explants are major materials in plant tissue cultures. Incubation of detached leaves on phytohormone-containing media, which is an important process for producing calli and regenerating plants, change their cell fate. Although hormone signaling pathways related to cell fate transition have been widely studied, other molecular and physiological events occurring in leaf explants during this process remain largely unexplored.

Here, we identified that ethylene signals modulate expression of pathogen resistance genes and anthocyanin accumulation in leaf explants, affecting their survival during culture. Anthocyanins accumulated in leaf explants, but were not observed near the wound site. Ethylene signaling mutant analysis revealed that ethylene signals are active and block anthocyanin accumulation in the wound site. Moreover, expression of defense-related genes increased, particularly near the wound site, implying that ethylene induces defense responses possibly by blocking pathogenesis via wounding. We also found that anthocyanin accumulation in non-wounded regions is required for drought resistance in leaf explants.

Our study revealed the key roles of ethylene in the regulation of defense gene expression and anthocyanin biosynthesis in leaf explants. Our results suggest a survival strategy of detached leaves, which can be applied to improve the longevity of explants during tissue culture.

The online version contains supplementary material available at 10.1186/s12870-023-04299-4.

Plant tissue culture is an important technique to produce calli and regenerated plants, which are used as materials in various industries. For example, regenerated Sophora flavescens Aiton and Rhodiola imbricata contain higher levels of medicinal phytochemicals, including maackiain and cinnamyl alcohol, than the normally grown plants [1, 2], suggesting the importance of plant regeneration in the food and pharmaceutical industries. In addition, Bougainvillea glabra and Artemisia annua L. calli produce flavonoids and phenolic acids as antioxidants [3, 4]. To induce callus formation or shoot/root organogenesis, plant tissues are excised and incubated on a medium containing various phytohormones. Low levels of auxins induce adventitious root organogenesis, whereas high levels of auxins promote cell dedifferentiation to induce callus formation [5–8]. In contrast, a high cytokinin:auxin ratio changes the cell fate and promotes the formation of shoot meristems [9]. In these processes, the explant conditions are important for efficient tissue culture. In Arabidopsis thaliana (Arabidopsis), the efficiency of root organogenesis in leaf explants is highly dependent on the leaf age [5]. In addition, salinity stress in leaf explants largely decreases the rates of callus formation and shoot regeneration in tomatoes [10]. However, molecular and physiological events other than those affecting callus formation and organogenesis in leaf explants remain largely elusive. Ethylene is a gaseous hormone that regulates various physiological responses in plants. Ethylene biosynthesis is mediated by 1-aminocyclopropane-1-carboxylic acid synthases (ACSs) [11]. In Arabidopsis, the expression of ACS2, ACS6, ACS7, and ACS8 is up-regulated by mitogen-activated and calcium-dependent protein kinases after wounding or infection by pathogens, such as Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 [12–14]. Ethylene activates downstream signaling pathways via endoplasmic reticulum-localized ethylene receptors and nearby signaling proteins [15]. ETHYLENE INSENSITIVE 2 (EIN2) acts as a signaling hub [16, 17]. Mutations in EIN2 cause defects in ethylene responses, including resistance to B. cinerea [18, 19]. EIN2 up-regulates the ETHYLENE RESPONSE FACTOR 1 (ERF1) expression in B. cinerea-infected plants [20, 21]. ERF1 induces the expression of defense-related genes, including PLANT DEFENSIN 1.2 (PDF1.2) and PATHOGENESIS-RELATED 3 (PR3), to confer resistance to B. cinerea. EIN2 is also involved in resistance to Fusarium [19, 22]. The ein2 mutant plants exhibit defects to induce the expression of ERF1 and PDF1.2 after infection with Fusarium graminearum and F. oxysporum [19], indicating that EIN2 plays an important role in pathogen resistance responses. Anthocyanins are phytochemicals that accumulate in various tissues of plants under abiotic stress conditions [23]. Accumulation of anthocyanins protects the plants from stress-induced damage, possibly via their roles as antioxidants [24]. In addition to pathogen resistance response, ethylene also plays a key role in abiotic stress-induced anthocyanin biosynthesis [25–27]. Inhibition of ethylene signaling by ethylene signaling inhibitors increases sugar-mediated anthocyanin accumulation in plants [25]. In contrast, the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1)-deficient mutant that exhibits constitutive ethylene responses shows reduced anthocyanin accumulation [25]. In addition, mutations in ethylene signaling transcription factors, ETHYLENE-INSENSITIVE 3 (EIN3) and EIN3-LIKE 1 (EIL1), increase anthocyanin levels under high sucrose conditions [27]. Moreover, ethylene-responsive transcription factors, PpERF9 and PpERF105, inhibit anthocyanin biosynthesis by regulating MYB transcription factors in pear (Pyrus spp.) [28, 29]. These results suggest that ethylene negatively regulates anthocyanin accumulation. However, there are opposite results that ethylene promotes anthocyanin biosynthesis in several studies. In Arabidopsis, ERF4 and ERF8-deficient mutants show reduced anthocyanin content and decreased expression of genes involved in anthocyanin biosynthesis under high light stress conditions [30]. In apple (Malus domestica), MdEIL1 directly activates MdMYB1 to induce anthocyanin accumulation [31]. These studies show the importance of ethylene signals in controlling anthocyanin content, yet the regulatory mechanisms can differ depending on plant species and environmental conditions. In this study, we identified that ethylene signaling controls the pathogen defense responses and anthocyanin biosynthesis in leaf explants, which would be important for survival of explants during tissue culture. We observed that anthocyanin biosynthesis and pathogen resistance responses are regulated differently depending on the distance from the wound in leaf explants. In addition, accumulated anthocyanins were necessary for drought stress resistance in leaf explants, but they were not involved in de novo root organogenesis. Our study shows the role of ethylene in affecting the survival of leaf explants, which would be important to improve longevity of leaf explants during tissue culture.

We previously reported that wound-induced reactive oxygen species (ROS) and Ca2+ are essential for de novo root organogenesis in leaf explants [32]. Here, incubation of leaf explants on phytohormone-free medium for de novo root organogenesis resulted in the accumulation of anthocyanins (Fig. 1A). Anthocyanin accumulation started one day after culture (DAC) and increased throughout the incubation period. Notably, anthocyanins were not observed at the site near the wound (SNW) until 7 DAC regardless of the excision site (Fig. 1A). To confirm the accumulation patterns of anthocyanins in leaf explants, we separately measured anthocyanin levels at SNW and at a site distant from the wound (SDW). Anthocyanin levels gradually increased at both SNW and SDW, but SDW exhibited significantly higher anthocyanin levels than SNW until 5 DAC (Fig. 1B). These results suggest that anthocyanins accumulated in leaf explants, but wound-induced signals somehow suppress anthocyanin accumulation near the wound site. As wounding induces jasmonate and ethylene production within 2 h [14, 33], we hypothesized that early phytohormone responses to wounding are related to anthocyanin accumulation patterns in leaf explants. Therefore, we analyzed the anthocyanin levels in mutants defective in ethylene (ein2-1) and jasmonate (coi1-21) signaling with the ctr1-1 mutant that exhibits constitutively active ethylene signaling [34–36]. We also used the tt4-11 mutant that exhibits defective anthocyanin biosynthesis as a control [37]. We found that anthocyanin levels at SDW were significantly reduced by coi1-21 mutation 5 DAC (Fig. 2A, B). However, the anthocyanin levels at SNW in the coi1-21 mutant were similar to those in Col-0 wild-type plant, suggesting that only jasmonate signals are required for anthocyanin accumulation at SDW. Moreover, the ein2-1 mutant showed significantly higher anthocyanin levels than Col-0 at both SDW and SNW. However, the ctr1-1 mutant showed the opposite trend as its anthocyanin levels were significantly lower than those in Col-0 at both SDW and SNW, similar to tt4-11. These results suggest that ethylene signals negatively regulate anthocyanin accumulation in leaf explants, which is consistent with a previous report that ethylene suppresses abiotic stress-induced anthocyanin biosynthesis [25].

As anthocyanin levels at the SNW were elevated by ein2-1 mutation (Fig. 2A, B), we hypothesized that ethylene signals are specifically activated near the wound site. To verify whether ethylene production is activated by wounding, we analyzed the expression of representative ethylene biosynthesis genes including ACS2, ACS6, ACS7, and ACS8, which are involved in wound-induced ethylene production [14]. Expression of ACS2 was elevated after leaf excision only at SNW (Fig. 3A). Expression of ACS7 increased at both SDW and SNW, but expression levels at SNW were significantly higher than those at SDW. Expression of ACS8 was largely suppressed after leaf excision at SDW, but expression levels became similar at SDW and SNW at 5 DAC. Expression of ACS6 did not show any significant differences during incubation. These results suggest that ACS2- and ACS7-mediated ethylene biosynthesis might be activated particularly near the wound site during the incubation of leaf explants. Ethylene has been reported to inhibit anthocyanin biosynthesis by suppressing the transcription of anthocyanin biosynthesis genes [25]. To further analyze the role of ethylene in the spatial regulation of anthocyanin accumulation, we determined the expression of anthocyanin biosynthesis genes. Genes encoding phenylalanine ammonia-lyases (PALs) belong to the phenylpropanoid metabolic pathway, which produces secondary metabolites, including anthocyanins [38]. The PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) and TRANSPARENT TESTA 8 (TT8) are transcription factors that up-regulate the expression of anthocyanin biosynthesis genes such as DIHYDROFLAVONOL 4-REDUCTASE (DFR), ANTHOCYANIDIN SYNTHASE (ANS) and UDP-GLUCOSE:FLAVONOID 3-O-GLUCOSYLTRANSFERASE (UF3GT) [39]. Among PAL genes, only PAL2 showed reduced expression at the SNW 0 and 3 DAC, whereas other PALs showed similar and complex expression patterns (Fig. 3B). Notably, expression levels of TT8 and PAP1, along with their downstream target genes DFR, ANS, and UF3GT, were largely increased following leaf excision at SDW. However, the expression levels at SNW were significantly lower than those at SDW. (Fig. 3B). These expression patterns were consistent with low anthocyanin levels and high ACSs expression at SNW, suggesting that wound-induced local ethylene production could potentially suppress anthocyanin biosynthesis in the leaf explants. Next, we examined the specific roles of ethylene in leaf explants. Ethylene plays a key role in defense responses to pathogens [18, 19]. Therefore, we hypothesized that ethylene-mediated pathogen resistance is differentially activated at SDW and SNW in leaf explants. To verify our hypothesis, we analyzed the spatial expression patterns of marker genes related to plant defense responses. Expression of all analyzed ethylene-responsive defense genes, PR3, PR4, and PDF1.2 [18, 19, 22], was elevated after leaf excision at both SDW and SNW, but the expression levels were significantly higher at SNW than at SDW at least once during incubation (Fig. 3C). In contrast, the expression of salicylic acid marker genes, PR1, PR2, and PR5 [40], were significantly higher at SDW than at SNW. To confirm the role of ethylene in the local expression of pathogen resistance genes, we analyzed gene expression in ein2-1 mutant. Expression of PR3, PR4, and PDF1.2 was significantly decreased at SNW, whereas that at SDW was unaltered by ein2-1 mutation (Fig. 4). In addition, expression of salicylic acid-responsive genes, PR1 and PR5, was not affected by ein2-1 mutation. Together with our observation that the expression of ethylene biosynthesis genes was elevated at SNW, these results suggest that wound-induced ethylene signals stimulate the defense responses locally near the wound site.

Anthocyanin accumulation is induced by abiotic stresses, such as high light and drought [23, 41, 42]. Accumulated anthocyanins act as a ROS scavengers to protect cells from oxidative damage [23, 41]. As leaf explants do not develop roots until they regenerate, we hypothesized that reduced water intake causes drought stress-induced anthocyanin accumulation. To verify our hypothesis, we incubated aerial parts of the seedlings without entire roots, with half of the roots, and intact seedlings on B5-agar and liquid media. Anthocyanin accumulation was observed only in the seedlings without roots incubated on agar medium (Fig. 5A, B). However, anthocyanin levels were significantly reduced in the seedlings incubated in liquid media, suggesting that decreased water intake through the roots is the major cause for anthocyanin accumulation in the aerial parts of the seedlings. To investigate whether anthocyanin accumulation in leaf explants is also due to drought stress, we incubated leaf explants on B5-agar and liquid media. As observed in the seedlings, anthocyanin levels were significantly reduced in leaf explants incubated in liquid media (Fig. 5C, D), suggesting that drought stress-induced anthocyanin accumulation also occurs in leaf explants during tissue culture. Next, we examined whether anthocyanin levels affect root organogenesis in leaf explants. Rooting rates of leaf explants on agar and liquid media did not show any significant differences until 14 DAC (Fig. 5E), suggesting that anthocyanin levels do not affect root organogenesis. Flavonoids, including anthocyanins, confer drought tolerance to plants [43, 44]. As our results showed that anthocyanin accumulation in leaf explants is not related to root organogenesis, we hypothesized that accumulated anthocyanins affect drought resistance. We thus examined the survival of leaf explants under drought stress using anthocyanin-deficient tt4-11 and ctr1-1 mutants. Excised leaves were incubated on B5-agar medium for 5 days and subjected to drought stress on dry filter paper. Intensity of green color was measured as an indicator of chlorophyll content and resistance to drought-induced cell death after 2 days of recovery. Although anthocyanin-deficient tt4-11 and ctr1-1 lost their green color, Col-0 explants showed a relatively higher intensity of green color (Fig. 6A), suggesting that anthocyanins might be required for drought resistance in leaf explants. However, we cannot entirely rule out the possibility that reduced drought tolerance in tt4-11 and ctr1-1 mutants is caused by other functions of TT4 and CTR1, which are not related to anthocyanin biosynthesis. Next, we compared the rooting rates of leaf explants between anthocyanin-deficient tt4-11 and ctr1-1 mutants and anthocyanin over-accumulating ein2-1 mutant. Similar to our observation that anthocyanin content did not affect root regeneration (Fig. 5C to E), tt4-11 and Col-0 were found to exhibit similar rooting rates until 14 DAC (Fig. 6B). However, the ein2-1 exhibited slow rooting rate compared to Col-0, but the final rooting rates at 14 DAC were similar in ein2-1 and Col-0. Meanwhile, ctr1-1 exhibited significantly reduced rooting rate compared to Col-0 at all time points in our experiments. Because altered anthocyanin levels did not affect root regeneration, these results might be due to the altered ethylene signaling in these mutants [45, 46].

Anthocyanins and lignins share upstream biosynthetic pathways starting with phenylalanine [47]. As lignins act as barriers to block pathogen infections [48], we examined whether lignins accumulate near the wound site in leaf explants using phloroglucinol-HCl, which stains the 4-O-linked hydroxycinnamyl aldehyde structures of lignins [49, 50]. Phloroglucinol staining revealed primary lignin deposition at the wound site, where anthocyanins did not accumulate, 1 DAC (Fig. 1A and S1A). Lignin signals increased during incubation until 5 DAC. However, similar pink-red colors were also observed at SDW where anthocyanins accumulated, possibly because the low pH of phloroglucinol-HCl turns anthocyanin color to red [51]. To distinguish between anthocyanins and lignins, we used anthocyanin-deficient tt4-11 mutant as a control. Although the pink-red color at SDW after phloroglucinol staining was diminished by tt4-11 mutation, staining signals at the wound site were still observed in both Col-0 and tt4-11 explants 5 DAC (Fig. S1B). These results indicate that, unlike anthocyanins, lignins are deposited at the wound site of leaf explants. Next, we performed phloroglucinol staining using ein2-1 mutants to determine whether ethylene signals affect lignin deposition. We found that lignin signals at the wound site were not affected by ein2-1 mutation (Fig. S1C), suggesting that ethylene is not mainly involved in lignin deposition in leaf explants. To further identify the upstream regulators of lignin deposition at the wound site, we analyzed NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG (RBOH)-deficient mutants as RBOHD- and RBOHF-produced ROS control lignin deposition in flowers [52]. Although the rbohD mutation did not affect lignin deposition, rbohDF double mutations diminished the lignin signals at the wound site 5 DAC (Fig. S1D, E). These results indicate that RBOHD- and RRBOHF-mediated ROS production after wounding induces lignin deposition at the wound site independent of anthocyanin and ethylene signals.

In this study, we found that ethylene signals regulate anthocyanin accumulation and expression of pathogen resistance genes in leaf explants. In our signaling scheme, wounding may induce ethylene biosynthesis mainly at SNW through upregulation of ACS expressions (Fig. S2). Ethylene then inhibits anthocyanin accumulation and induces expression of PDF1.2, PR3, and PR4 at this region. In contrast, relatively weak ethylene signals allow anthocyanin accumulation at SDW. As induction of PR and PDF expressions confers pathogen resistance [53–55] and anthocyanin accumulation improves drought resistance (Fig. 5) [43], our observations indicate that wounding triggers these responses via ethylene signals for the survival of leaf explants under both abiotic and biotic stresses. Wounding is unavoidable to generate explants for plant tissue culture. However, pathogens can easily invade the plant tissues via the wounded parts [56]. Therefore, in many plant species, defense responses are activated by wounding. In Arabidopsis, wounding promotes camalexin production for defense against B. cinerea [57]. In tomato, accumulation of feruloyltyramine and p-coumaroyltyramine, which confer resistance against Xanthomonas campestris, is increased in leaves after wounding [58–60]. In this study, we found that wound-induced defense responses are also activated in leaf explants during tissue culture. Expression of PR3, PR4, and PDF1.2 genes, which are involved in ethylene-mediated defense responses [18, 19, 22], increased at SNW in an EIN2-dependent manner (Figs. 3 and 4). Therefore, our observations suggest that ethylene locally induces expression of these genes near the wound site, where pathogen invasion is suspected. During tissue culture, leaf explants face drought stress despite being cultured on agar medium because of the absence of roots. Therefore, leaf explants activate auxin signals for root organogenesis [61]. However, the explants may need to induce drought stress resistance responses prior to root development. As ROS production is critical for drought-induced cell death [62], ROS-scavenging processes would be essential in leaf explants. We found that leaf explants accumulate anthocyanins particularly at SDW (Fig. 1). Anthocyanins act as antioxidants that scavenge ROS and enhance tolerance to drought stress [39, 43]. Indeed, anthocyanin over-accumulating plants exhibit high survival rates, whereas anthocyanin-deficient plants exhibit low survival rates under drought and ROS stress conditions [39, 43]. Consistent with previous reports, anthocyanin-deficient tt4-11 and ctr1-1 explants exhibited significantly low survival rates under drought conditions in our data (Fig. 6A). These results suggest that antioxidants accumulate at non-wounded sites, where ethylene signals are relatively weak, to confer drought tolerance to leaf explants. Notably, lignin signals were not observed in rbohDF double mutants (Fig. S1E), indicating the important roles of RBOHD and RBOHF in lignin accumulation. As RBOHs are NADPH oxidases that produce ROS in plant cells [63, 64], ROS would be required for lignin biosynthesis in leaf explants. Indeed, ROS induce lignin biosynthesis in roots and flowers and accumulate at the wound site in leaf explants [32, 50, 52]. Here, anthocyanins accumulated very slowly near the wound site (Fig. 1A, B), indicating that ROS-mediated lignin deposition may be supported by ethylene-mediated inhibition of anthocyanin biosynthesis at the wound site. Lignins are physical barriers that restrict pathogen invasion in plant cells [48]; therefore, lignin deposition at the wound site may be another defense mechanism to minimize pathogen infection via disrupted tissues in leaf explants.

Arabidopsis thaliana ecotype Columbia (Col-0) was used in this study. The coi1-21 (N68754), rbohD (N9555), rbohDF (N9558), and tt4-11 (N2105573) seeds were obtained from the Nottingham Arabidopsis Stock Centre (NASC, Nottingham, UK). The ctr1-1 [34], and ein2-1 [65] seeds were a gift from Dr. Young-Joon Park. Seeds were surface-sterilized in 75% (v/v) ethanol with 0.03% (v/v) Triton X-100, and then washed with 70% (v/v) ethanol two times. After 3 days of stratification at 4 °C, seeds were transferred to growth room set at 24 °C with 40–50% humidity under long-day conditions. The seedlings were grown on 1/2 Murashige and Skoog (MS)-agar plates containing 0.05% (w/v) of MES and 0.7% (w/v) of plant agar with pH 5.7. The plates were exposed to white light with an intensity of 100 µmol m− 2 s− 1 using fluorescent FL40EX-D tubes (Focus, Bucheon, Korea).

Seedlings were grown on MS-agar plates for 9 days. Leaves or seedlings excised at the indicated site in figures. Mainly, blade-petiole junctions were excised for leaf explants. For leaf explants, first rosette leaves were used. The explants were incubated on Gamborg B5-agar plates containing 0.05% (w/v) of MES and 0.7% (w/v) of plant agar with pH 5.7 for the indicated time periods in figures. The explants were exposed to same light intensity of growth conditions. The plant materials were placed on MS-agar plates and photographed using a Nikon D5600 digital camera and STEMI 2000-C stereo microscope (Carl Zeiss). About 1.2 mm from the wound site was defined as SNW and the remaining part was defined as SDW. For measuring anthocyanin content, SNW and SDW were separately harvested and incubated in 300 µl of methanol containing 1% (v/v) HCl for 16 h at 4 °C. After the extraction, 200 µl of distilled water and 200 µl of chloroform were added for separating chlorophylls from anthocyanins. The mixture was centrifuged at 16,000 X g for 10 min at 4 °C. The supernatant was transferred to a new microcentrifuge tube and 500 µl of distilled water was added. Absorbance was measured at 535 nm (A535) using a spectrophotometer (Beckman Coulter, Brea, CA, United States). Anthocyanin contents were calculated as follows: Anthocyanin contents = A535 / fresh weight (mg)

Seedlings were grown on MS-agar plates for 9 days and first rosette leaves were detached. The leaf explants were incubated on Gamborg B5-agar plates for the indicated time periods in figures. The explants were immersed in phloroglucinol staining solution containing 1% (w/v) of phloroglucinol in 50% (v/v) of ethanol and 50% (v/v) HCl. After inverting several times, the plant materials were mounted on MS-agar-plates and photographed using a STEMI 2000-C stereo microscope (Carl Zeiss).

Seedlings were grown on MS-agar plates for 9 days and first rosette leaves were detached. The explants were incubated on Gamborg B5-agar plates or floated on B5 liquid for up to 14 days to induce de novo root organogenesis. The rooting rate of explants was measured at intervals of two days starting at 6th day after culture.

Seedlings were grown on MS-agar plates for 9 days and first rosette leaves were detached. The leaf explants were incubated on Gamborg B5-agar plates for 5 days. To induce drought stress, the explants were placed on filter paper at room temperature for 2 h. For recovery, explants were incubated in growth room for 2 days after supplying distilled water. The plant materials were photographed using a Nikon D5600 digital camera at the indicated time periods in figures. The ImageJ software was used to measure the leaf color intensity.

The leaf explants from the seedling grown for 9 days on MS-agar plates were incubated on Gamborg B5-agar plates for the indicated time periods in figures. The SNW and SDW were harvested separately. Total RNA was extracted from the plant materials using Trizol (Thermo Fisher Scientific) according to the manufacturer’s recommendations. First-strand complementary DNA synthesis was performed using AccuPower CycleScript RT PreMix (Bioneer) according to the manufacturer’s protocol. Quantitative PCR (qPCR) was performed using TOPreal qPCR 2X PreMIX (SYBR Green with low 5-carboxy-x-rhodamine, Enzynomics) in CFX Connect PCR device (Bio-Rad). The comparative ΔΔCT method was used to calculate relative quantities of each amplified PCR product. The threshold cycle (CT) was determined using the Bio-Rad CFX Manager software with default parameters. Primers used for qPCR were listed in Table S1. The gene expression was normalized using UBQ10 as a reference gene.

To determine statistically significant differences, one-way analysis of variance (ANOVA) with post hoc Tukey’s test and Student’s t-test were performed using Rstudio and Excel software, respectively.

Below is the link to the electronic supplementary material. Supplementary Material 1