Previous studies showed that RcMYB8 was significantly induced in both roots and leaves of rose when challenged by drought (Li et al. 2021). We firstly isolated RcMYB8 using specific primers (Supplemental Table. S1). The full length of RcMYB8 is 837 bp, which encodes 278 amino acids (aa). Phylogenetic analysis revealed that RcMYB8 has high homology with Arabidopsis AtMYB96 (Seo et al. 2011), Gossypium hirsutum GhMYB36 (Liu et al. 2022a), and Zea mays ZmMYB48 (Wang et al. 2017), which all have similar roles in the response to abiotic stresses (Fig. 1A). Sequence alignment with RcMYB8 and seven other MYB proteins (LhMYB6, OsMYB4, SbMYB8, AtPAP1, AtPAP2, PtrMYB134, and VvMYBPA2) revealed that these proteins belong to R2R3-MYBs, which have conserved MYB domains in N-terminus and nonconserved variable domains in C-terminus. A conserved [D/E]LX2[R/K]X3LX6LX3R motif was encompassed in the sequence’s N-terminal zone, which acted as the site for bHLH protein interplays (Fig. S1).

As a next step, the expression patterns of RcMYB8 upon drought and salinity challenges were assessed at various points of time (0, 3, 6, 12, 24, and 48 h). Compared with the expression in the control, RcMYB8 was significantly induced to different degrees under drought conditions for 3 h (3.66-fold), 6 h (1.66-fold), 12 h (2.78-fold), 24 h (9.76-fold), and 48 h (5.94-fold) (Fig. 1B). Under salinity stress, RcMYB8 did not increase intensively from 0 h to 3 h, while higher expressions were noted at 6 h (8.4-fold) and 12 h (14.53-fold) and the highest level was reached at 24 h (27-fold) (Fig. 1C). In addition, we measured the transactivation of RcMYB8. As is clear from Fig. 1D, the entire transformants exhibited preferable growth on tryptophan (Trp)-free synthetic dropout (SD) medium. Moreover, yeast colonies of the positive control and BD-RcMYB8 grew normally under SD conditions lacking histidine (His) and Trp (SD/−Trp/−His), implying that RcMYB8 is a transactivator. We next conducted the subcellular localization analysis RcMYB8 (Fig. 1E). The RcMYB8-green fluorescent protein (GFP) signal distribution was noted in tobacco leave nuclear cells, agreeing with its role as a TF. As suggested by the foregoing findings, RcMYB8 is a nucleus localized transcriptional activator.

To assess RcMYB8 biofunction, the VIGS approach was adopted to silence it in rose plants. We firstly silenced RcMYB8 in rose leaves that were treated normally and with NaCl (200 mM) (Fig. S2A). Compared to the tobacco rattle virus (TRV) control, TRV-RcMYB8 exhibited 54% lower expression level of RcMYB8 (Fig. S2B), as well as prominently higher electrolyte leakage (Fig. S2C), implying that cell membrane leakage occurred quickly when RcMYB8 was silenced. Moreover, the chlorophyll (Chl) content in TRV-RcMYB8 was significantly lower (1073 μg·g⁻¹) than that in the TRV control (1593 μg·g⁻¹) (Fig. S2D). The results of NBT and DAB staining showed that TRV-RcMYB8 accumulated more brown and blue colors than TRV (Fig. S2E), suggesting that RcMYB8 influenced the ROS levels when treated with salinity stress. We further silenced RcMYB8 in rose seedlings treated for 3 d normally and with NaCl (200 mM). The RcMYB8 level in TRV-RcMYB8 decreased to 50% of the control group (Fig. 2A). Compared to TRV, the leaves of TRV-RcMYB8 showed more curled and shrinkage, implying TRV-RcMYB8 suffered more damage than TRV (Fig. 2B).

Next, the Na⁺, K⁺, and Ca²⁺ contents in TRV-RcMYB8 and TRV were assessed as well. The inter-group difference in Ca²⁺ content was found to be insignificant (Fig. 2C). The K⁺ content was slightly higher in TRV-RcMYB8 than in TRV. With a value of 8.31 mg·g⁻¹, TRV contained pronouncedly lower Na⁺ compared to the TRV-RcMYB8 (13.83 mg·g⁻¹) (Fig. 2D, E). The balance of Na⁺/K⁺ was identified as the pivotal factor upon salinity challenge (Li et al. 2023). When stressed by salinity, the Na⁺/K⁺ ratio in TRV was 1.34, showing a markedly lower value in comparison to TRV-RcMYB8 (2.1) (Fig. 2F). Overall, silencing RcMYB8 in rose causes broader peroxidation of membrane lipids and weakened stability of cellular membranes upon salinity challenge, and disrupts the Na⁺/K⁺ balance by increasing the Na⁺ content, resulting in decreased salinity tolerance.

Next, upon salinity challenge, RcMYB8 was transiently overexpressed in rose seedlings under a constitutive Superpromoter (VC), thereby deriving RcMYB8-overexpressing (pSuper:RcMYB8) plants. Compared to the VC control, the pSuper:RcMYB8 plants exhibited 2.8-fold higher RcMYB8 level (Fig. 3B). In the 0 mM NaCl scenario, phenotypic differences were absent between VC and pSuper:RcMYB8 plants. Contrastively, following 3 days of 200 mM NaCl treatment, pSuper:RcMYB8 plants exhibited fewer wilting phenotypes and a smaller degree of root blackening (Fig. 3A). In addition, VC accumulated higher ion leakage (Fig. 3C) and lower Chl content compared to pSuper:RcMYB8 plants (Fig. 3D). More callose accumulation in pSuper:RcMYB8 was also noted compared to the VC control (Fig. 3E, F), indicating that RcMYB8 may influence the deposition of callose.

We next overexpressed RcMYB8 in rose calli under normal and 200 mM NaCl, respectively (Fig. 3G). We obtained the pSuper:RcMYB8 through genomic PCR analysis using the GFP and RcMYB8 specific primers (Fig. 3H, Supplemental Table S1). Further Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed RcMYB8 was upregulated 2.9-fold in pSuper:RcMYB8 plants (Fig. S3). Under 200 mM NaCl for 15 d, the VC control callus exhibited severe blackening compared to pSuper:RcMYB8 callus (Fig. 3G), and the pSuper:RcMYB8 calli exhibited significantly higher fresh weight (Fig. 3I). The results of NBT and DAB staining showed less concentration of blue and brown colors in pSuper:RcMYB8 than in VC (Fig. 3J). As implied by these findings, the RcMYB8 overexpression leads to improved tolerance against salinity by regulating ROS homeostasis.

As RcMYB8 exhibited drought-induced expression, we next clarified its role in response to the drought challenge. Firstly, RcMYB8 in rose leaves was silenced using the VIGS approach under dehydration (0, 6, 12, and 24 h) and rehydration (3 h) conditions (Fig. S4B). After dehydration for 24 h and rehydration for 3 h, TRV-RcMYB8 exhibited a greater degree of curling than the control group (Fig. S4A). TRV-RcMYB8 contained significantly less Chl (1319 μg·g⁻¹) compared to the TRV control (1551 μg·g⁻¹) (Fig. S4C). It is clear from Fig. S4D that compared with TRV controls, the relative fresh weight in TRV-RcMYB8 was significantly lower at 6, 24 h of dehydration and 6 h of rehydration. Moreover, TRV-RcMYB8 displayed prominently higher electrolyte leakage in contrast to TRV (Fig. S4F), implying that cell membrane leakage occurred quickly when RcMYB8 was silenced. Staining with NBT and DAB revealed that TRV-RcMYB8 accumulated more blue and brown color than TRV (Fig. S4E), respectively, suggesting that RcMYB8 influenced ROS levels when challenged by drought.

To better examine the role of RcMYB8 upon drought challenge, we silenced RcMYB8 in rose seedlings and subjected them to 0-d and 3-d drought stress (20% polyethylene glycol-6000 (PEG6000)), followed by a 1-d rewatering (Fig. 4A). The expression of RcMYB8 in TRV-RcMYB8 was only 0.43-fold of that in the TRV control (Fig. 4B). In the normal growth setting, the differences between TRV and TRV-RcMYB8 were insignificant, while TRV-RcMYB8 showed more wilting and curling leaves after drought for 3 d (Fig. 4A). Compared to the TRV control, TRV-RcMYB8 displayed significantly lower enzymatic activities of CAT, POD and SOD (29.8, 17.95 and 26.7%, respectively) (Fig. 4C, D and E), whereas a pronouncedly higher content of MDA, with a value of 19.89 nmol·g⁻¹FW (Fig. 4F). Moreover, compared to that in the control, the proline content in TRV-RcMYB8 was only 360.6 μg·g⁻¹FW, showing a markedly lower value compared to the TRV control (487 μg·g⁻¹FW) (Fig. 4G). Staining with NBT and DAB revealed that TRV-RcMYB8 accumulated more blue and brown color than TRV (Fig. 4H).

We also examined the changes in photosynthesis capacity between TRV and TRV-RcMYB8 after rewatering for 1 d. The photochemical indices, including Fv/Fm, NPQ, qP, and YII, were all significantly higher in the TRV control than in TRV-RcMYB8, suggesting that silencing RcMYB8 resulted in a weaker photosynthesis capacity (Fig. 4I to M). These results clearly suggest that silencing RcMYB8 decreased tolerance to drought by reducing the proline content, increasing the MDA content, and decreasing the CAT, SOD and POD enzyme activities to balance the ROS level, resulting in weaker photosynthesis capacity.

C to G CAT activity (C), POD activity (D), SOD activity (E), MDA content (F) and proline content (G) of leaves in TRV and TRV-RcMYB8 under drought stress. H NBT and DAB staining of TRV and TRV-RcMYB8 under drought stress. Scale bar: 1 cm. I to M Chlorophyll imaging analysis of TRV and TRV-RcMYB8 rose seedlings under drought stress conditions. Fv/Fm (J), NPQ (K), qP (L), and YII (M) indicated in H. Scale bar: 1 cm. Data are the mean ± SD (n = 3).

To probe deeper into the function of RcMYB8 upon drought challenge, we utilized RcMYB8 overexpression (pSuper:RcMYB8) to study the role of drought challenged rose seedlings. In contrast to the VC control, the pSuper:RcMYB8 plants exhibited a 2.5-fold higher RcMYB8 level (Fig. 5B). In normal growth settings, insignificant differences were observed between VC and pSuper:RcMYB8, while VC showed more wilting and curling phenotype after drought for 3 d. After rewatering for 1 d, pSuper:RcMYB8 recovered slightly better than VC (Fig. 5A). We also found that the VC controls showed higher ion leakage (Fig. 5C) and lower Chl content compared with pSuper:RcMYB8 (Fig. 5D). We also measured the proline content and found that pSuper:RcMYB8 had a significantly higher proline content than the TRV control (Fig. 5E). In addition, we also overexpressed RcMYB8 in rose calli in the absence or presence of 10% PEG6000, respectively. The pSuper:RcMYB8 calli grew much faster than the VC control calli (Fig. 5F). In addition, the pSuper:RcMYB8 calli exhibited significantly higher fresh weight than that of controls (Fig. 5G). DAB and NBT staining results showed that pSuper:RcMYB8 accumulated less brown and blue colors than VC (Fig. 5H). These findings illustrate that overexpression of RcMYB8 enhanced tolerance against drought stress through the ROS scavenging facilitation.

To identify the downstream genes of RcMYB8, we firstly identified the genes related to the physiological indicators we tested in RcMYB8-silenced and RcMYB8-overexpressing plants. We performed a BLAST search on Rosaceae genome database (https://www.rosaceae.org/), where the amino acid sequences of related genes in Arabidopsis and rice served as a query. RcMYB8 improved drought and salt tolerance by influencing ROS dynamic changes and proline accumulation. In addition, previous studies have shown a connection between RcMYB8 and RcPR5/1 (Su et al. 2021), and RcPR5/1 was proved to enhance salinity resistance in rose (Su et al. 2023). We next examined the relevant gene expression corresponding to ROS scavenging, proline synthase and pathogenesis-related-5 (PR5) in both RcMYB8-silenced and RcMYB8-overexpressing plants. We firstly analyzed POD, SOD, CAT, and proline synthesis and PR5 gene expression using public drought (accession number: PRJNA663119) and salt (accession number: PRJNA42884) transcriptomic datasets (Li et al. 2021; Tian et al. 2018). These genes included 83 RcPODs, 15 RcSODs, one RcCAT, two RcP5CSs, and 27 RcPR5s (Fig. S5A and B).

We then selected the significant drought- or salt-inducible genes to accomplish additional qRT-PCR assessment in TRV-RcMYB8 and pSuper:RcMYB8 after drought or salt treatment, respectively (Table. S3). As shown in Fig. 6, four genes (RcPOD1, RcSOD2, RcSOD3, and RcP5CS1) exhibited significantly depressed gene expression levels, and only RcP5CS1 was downregulated by more than 50% in TRV-RcMYB8 and was upregulated by 4.3-fold in pSuper:RcMYB8 compared with controls (Fig. 6A). For the expression of six PR5 genes, only RcPR5/1 was discovered to display a nearly 50% downregulation in TRV-RcMYB8, as well as a prominent 5-fold upregulation in pSuper:RcMYB8 (Fig. 6B). In addition, under salt stress, we found that the K⁺ and Ca²⁺ contents in TRV and TRV-RcMYB8 did not change greatly, although TRV-RcMYB8 contained significantly more Na⁺ compared to the TRV. We also identified 32 ion-related genes, including ten RcAKTs, nine RcCAXs, and 13 RcNHXs in the transcriptome database (Fig. S5C). We selected significantly drought- or salt-induced genes, and further qRT-PCR analysis was carried out on TRV-RcMYB8 and pSuper:RcMYB8 after salt treatment. Four Na⁺ channel protein genes were significantly downregulated in TRV-RcMYB8 and upregulated to varying degrees in pSuper:RcMYB8. However, the K⁺ channel protein gene did not change significantly in either TRV-RcMYB8 or pSuper:RcMYB8, respectively. Of the four Ca²⁺ channel protein genes (RcCAX1, RcCAX2, RcCAX3, RcCAX4), only RcCAX4 was significantly downregulated in TRV-RcMYB8, and the other three genes showed no significant change (Fig. 6C). Based on these results, we infer that RcMYB8 can modulate RcPR5/1 and RcP5CS1 positively in rose plants.

RcP5CS1 and RcPR5/1 exhibited significantly downregulated expression patterns in TRV-RcMYB8 and upregulated expression patterns in pSuper:RcMYB8. We hypothesize that these two genes might be the downstream genes of RcMYB8. Couples of MYB binding sites were evenly distributed in the 2 kb RcPR5/1 promoter regions (Fig. S6). We then selected four fragments (P1, P2, P3, and P4) that contained different numbers of MYB CREs for Y1H analysis. In yeast cells, 300 ng/mL Aureobasidin A (AbA) suppressed the RcPR5/1 self-activation (Fig. S7). All yeast cotransformed strains with RcMYB8 + RcPR5/1-P1, RcMYB8 + RcPR5/1-P2, RcMYB8 + RcPR5/1-P3, RcMYB8 + RcPR5/1-P4 fragments displayed prefereable growth in SD medium deficient in leucine (Leu) and uracil (Ura), but after 300 ng/mL AbA was added to the culture medium. Only the cotransformed RcMYB8 + RcPR5/1-P2 and RcMYB8 + RcPR5/1-P4 showed normal growth (Fig. 7B), implying that RcMYB8 can bind to the promoter of RcPR5/1.

We then used firefly luciferase reporter gene (LUC) analysis to test the binding ability with RcMYB8. The RcPR5/1-P2 and RcPR5/1-P4 promoter fragments were fused to LUC, and subsequently transformed into Agrobacterium GV3101 (Fig. S8), followed by cotransformation of pSuper:RcMYB8 with RcPR5/1-P2-LUC or RcPR5/1-P4-LUC in tobacco, respectively (Fig. 7C, E). When pSuper:RcMYB8 was cotransformed with RcPR5/1-P2-LUC or RcPR5/1-P4-LUC, RcMYB8 distinctly accumulated relative LUC/REN activity (Fig. 7D, F). To verify the interplay, the full-length RcMYB8 protein was purified and an electrophoretic mobility shift assay (EMSA) was undertaken, where the labeled probe adopted was biotin-labeled RcPR5/1 promoter encompassing the sequence of TAACCA. RcMYB8 bound to the RcPR5/1 promoter (Lane 3, Fig. 7G) and, after addition of competition, the binding ability exhibited a decrease. Additionally, when the binding cis-element TAACCA mutated to AAAAAA, no competition was detected with the addition of mutation probes, confirming the specific binding of the sequence TAACCA. The foregoing results suggest binding of RcMYB8 to the promoter of RcPR5/1.

There are two proline synthesis genes (RcP5CS1 and RcP5CS2) in rose (Fig. S9), and they exhibited differential expression patterns under drought stress (Fig. S5A). qRT-PCR analysis revealed that RcP5CS1 exhibited an induced expression pattern under drought for 24 h and 48 h (Fig. S10). We then selected RcP5CS1 for further binding analysis. Two putative MYB binding sites (RcP5CS1-P5 and RcP5CS1-P6) were found in its 2 kb promoter regions (Fig. S11). Then, we carried out a self-activation experiment on RcP5CS1, and 3-Amino-1,2,4-triazole (3-AT; 60 mM) suppressed the RcP5CS1 self-activation in yeast cells (Fig. S12). Two yeast strains with RcMYB8 + RcP5CS1-P5 and RcMYB8 + RcP5CS1-P6 fragments exhibited preferable growth in SD medium deficient in His, Trp and Leu. However, after 60 mM 3-AT was added to the culture medium, only the cotransformed RcMYB8 + RcP5CS1-P5 showed normal growth (Fig. 7H). We then performed an EMSA to determine whether RcMYB8 binds directly to the RcP5CS1 promoter. A 46 bp biotin-labeled DNA fragment in the RcP5CS1-P5 regions, which contains the MYB CREs (TAACCA), was used as a probe. When labeled probes were preincubated with GST-RcMYB8 protein, a shifted band was noticed. Contrastively, the GST-RcMYB8 protein binding to the promoter was abolished by adding excess unlabeled probe. Moreover, mutation of the core sequences abolished the binding activity (Fig. 7I). Next we used firefly luciferase reporter gene (LUC) analysis to test the binding ability with RcMYB8. The RcP5CS1-P5 promoter fragments were fused to LUC, and subsequently transformed into Agrobacterium strain GV3101 (Fig. S13), followed by cotransformation of pSuper:RcMYB8 with RcP5CS1-P5:LUC in tobacco (Fig. 7J). When pSuper:RcMYB8 was cotransformed with RcP5CS1-P5:LUC, RcMYB8 distinctly accumulated relative LUC/REN activity (Fig. 7K). These results suggest that RcMYB8 physically interacts with RcPR5/1 and RcP5CS1.

To determine the role of RcP5CS1 under drought stress, the VIGS method was used to silence RcP5CS1 in rose seedlings (Fig. 8). After 3 days of drought, TRV-RcP5CS1 showed more obvious damage in terms of leaf curl and water loss, especially in the tender leaves, which were withered and brittle (Fig. 8A). qRT-PCR analysis showed RcP5CS1 in TRV-RcP5CS1 was only 0.36-fold of that in the TRV control (Fig. 8B). We also measured the contents of proline, MDA, CAT, POD, and SOD in both TRV and TRV-RcP5CS1 (Fig. 8C, D, E, F and G). Compared to the TRV control, the CAT, POD and SOD enzyme activities in TRV-RcP5CS1 were markedly lower (suppressed by 25.3, 30.7, and 31.6%, respectively). However, the MDA content in TRV-RcP5CS1 was 20.75 nmol·g⁻¹, which prominently surpassed the TRV control value. Compared to the control, the proline content in TRV-RcP5CS1 was only 305.3 μg·g⁻¹ FW, showing a markedly lower value compared to the TRV control (Fig. 8C). Staining with NBT and DAB revealed that TRV-RcP5CS1 accumulated more blue and brown color than TRV (Fig. 8H).

Furthermore, we examined the changes in photosynthesis capacity between TRV and TRV-RcP5CS1 after rewatering for 1 d. The photochemical indices, including Fv/Fm, NPQ, qP, and YII, were all distinctly lower in TRV-RcP5CS1 compared to the TRV control, suggesting that silenced RcP5CS1 resulted in a weaker photosynthesis capacity (Fig. 8I to M). These results revealed that silencing RcP5CS1 in rose decreased tolerance to drought.

The in vitro propagation buds of rose (Rosa chinensis ‘Old Blush’) from Qingdao Agricultural University were used as materials, which were grown for 7 weeks on Murashige and Skoog (MS) alkali salts. The cultivation settings were 25 °C, 16-h light/8-h dark photocycle, as well as 50-60% RH. Next step was a 2-week cultivation of rose plants using 1/4 Hoagland solution, followed by treatment of the plants for 0, 3, 6, 12, 24 and 48 h using NaCl (200 mM) or PEG6000 (20%). Utilizing the sterile young rose leaves, callus induction was accomplished on callus induction medium (CIM), while subsequent somatic embryo induction was undertaken on embryos induction medium (EIM) according to Liu et al. (2021). The callus was cultured without light at 26 °C and renewed every 4 weeks. Each experiment consisted of three independent plants.

For sequence analysis, Genedoc was used to perform a visual alignment of the homologous RcMYB8 protein. The phylogenetic tree of 29 MYB proteins with known functions from other plant species (Supplementary Table. S2) were constructed in MEGA X using the neighbor connection (NJ) method (Kumar et al. 2018).

A FastPure Isolation Kit (Vazyme Biotech, Nanjing, China) was utilized for the total RNA sampling from rose leaves and, subsequently, a HiScript III All-in-One RT SuperMix Perfect for qPCR from the same manufacture was used to reverse transcribe the total RNA, thereby deriving cDNA. qRT-PCR was accomplished following a prior procedure (Su et al. 2023). Gene expression was normalized against the internal control RcUBI2, while the level of each gene expressed was computed by 2^-∆∆CT approach. Supplementary Table S1 details the adopted qRT-PCR primers.

With the assistance of specific primers (Supplemental Table. S1), RcMYB8-GFP was formed by inserting RcMYB8 coding region between the Sal I and Hind III sites in pCAMBIA 1300 vector. After transforming both RcMYB8-GFP and the vector into Agrobacterium tumefaciens CV3101, infection proceeded into tobacco (Nicotiana benthamiana) leaves that were aged 4 weeks. Following 2 days of placement away from light, the lower epidermal samples were gathered from tobacco leaves, which were subjected to 4′, 6-diamidine-2′-phenylindole dihydrochloride (DAPI, 10 μg/mL) staining, and finally photographed under a TCS SP8 laser scanning confocal microscope (Leica, Germany).

With the utilization of specific primers (Supplementary Table. S1), RcMYB8 coding sequence was subjected to PCR amplification, and subsequently connected to pGBKT7 vector encompassing the DNA binding domain of GAL4. After transforming fusion vector pGBKT7-RcMYB8 (BD-RcMYB8), empty body (BD), as well as positive and negative controls into yeast strain yeast two-hybrid (Y2H) GOLD (Weidi, Shanghai, China), coating proceeded with SD/−Trp and SD/−Trp-His growth medium, respectively. After growing in a constant temperature oven at 28 °C for 3-5 days, the samples were photographed.

The VIGS approach was conducted as previously reported (Su et al. 2023). The 200- and 483-bp fragments in the non-conserved zone of the 3′ region of RcMYB8 and the 5′ region of RcP5CS1 were selected as silent fragments. Through their insertion into pTRV2 vector, plasmids pTRV2-RcMYB8 and pTRV2-RcP5CS1 were derived, which were transformed subsequently into Agrobacterium tumefaciens strain GV3101. After supplementing with AS (40 μM), MES (10 mM), kanamycin (100 mg/L) and rifampicin (100 mg/L), every transformant was cultured in YEB medium. Folling overnight culture (200 rpm) at 28 °C, we collected the cellular strain and centrifuged the cell body (5000×g, 8 min) using suspended bacteria (200 μM AS, each 10 mM MgCl₂ and MES), followed by rectification of the OD600 to 0.8-1.0. Cells containing pTRV2-RcMYB8, pTRV2-RcP5CS1 or pTRV2 were blended with pTRV1 cells in a 1:1 volume and incubated in darkness for 4 h. Then, the nutrient solution-cultured rose plants were infiltrated with strains containing TRV-RcMYB8 or TRV-RcP5CS1 as the TRV plasmid (negative control) under 0.7 atm for 10 min and then circulated twice. After soaking, deionized water was used to wash the plants, in order to eliminate the osmotic buffer. Finally, the plants were cultured in darkness at 8 °C for 3 days and then treated under an aqueous scenario containing NaCl (0 or 200 mM) or PEG6000 (20%) in identical cultivation conditions.

Transient overexpression of RcMYB8 was accomplished following a former procedure (Luo et al. 2021). First step was cloning of open reading frame (ORF) of RcMYB8 into the pCAMBIA1300 vector, thereby yielding RcMYB8-overexpressing plants (pSuper:RcMYB8). Through centrifugation, A. tumefaciens GV3101 carrying pCAMBIA 1300 (VC) or pSuper:RcMYB8 was gathered, which was resuspended using buffer solutions (200 μM AS, each 10 mM MgCl₂ and MES), followed by rectification of the OD600 to 0.8 for 3-4 h. Then, hydroponic plantlets with similar growth conditions were selected for infiltration in a vacuum of 0.7 atm pressure. Next, the infiltrated plants were incubated in darkness at 8 °C for 3 days and treated with PEG6000 (20%) or NaCl (0 or 200 mM) in identical growth settings.

Genetic transformation of rose calli was conducted following previous studies procedure with little modification (Liu et al. 2021). The bacterial solution was first cultured overnight on a shaker, gathered through centrifugation and resuspended using buffer solutions (MS medium with 100 μM AS and 60 g/L glucose), and OD600 was adjusted to 0.5. The culture was then incubated at 200 r/min and 28 °C for 2 h. Then, the same growth state of callus was soaked for 15 minutes in the bacterial suspension using a vacuum pump of 0.7 Mpa, and then subjected to a 4-d cocultivation at 22 °C devoid of light using a co-culture medium (CM). For cultivation of somatic embryos, a screening medium involving antibiotics (300 mg/L Cefotaxim and 20 mg/L Hygromycin) was utilized. Through both qRT-PCR and genomic PCR, identification of positive pSuper:RcMYB8 plants was accomplished with the utilization of specific primers (Supplementary Table S1). The screened pSuper:RcMYB8 and VC calli were treated on a medium containing 200 mM NaCl or 10% PEG6000, respectively. At least 16 calli were used for each experiment.

Chlorophyll quantification was accomplished as per a prior procedure (An et al. 2021). A chlorophyll fluorescence imager (IMAG-MAX/L; WALZ, Germany) was used to measure the image and parameters of qP, NPQ, Fv/Fm, and Y(II), with each 3 technical and biological replicates.

To assess the rate of ion leakage, plant sample was weighed (0.2 g) and placed for 8-12 h in deionized water (10 mL) to measure their conductivity. The mixture was boiled for 30 minutes and then cooled to room temperature, and the conductivity was measured again. The relative electrical conductivity was calculated as the conductivity prior to high boiling as a percentage of post-boiling conductivity.

Diaminobenzidine (DAB) along with nitro blue tetrazolium (NBT) were used to determine the H₂O₂ and O₂⁻ depositions in leaves by histochemical staining. The relative fresh weight is referred to as the indicated timepoint (drought 6, 12, 24, rewatering 3 h) weight versus the weight at 0 h. The enzymatic activity assessment for Peroxidase (POD), Superoxide Dismutase (SOD), Catalase (CAT) and proline was undertaken separately via the corresponding assay kits (Solarbio, China). Thiobarbituric acid method (TBA) was employed for quantifying MDA levels. Three biological replicates were used for each physiological index.

For the contents of Na⁺, K⁺ and Ca²⁺, we firstly dried the salt-stressed sample in an 80 °C dryer, ground and weighed 0.1 g, put it into a digestive tube, added 12 mL of HNO₃:HCLO₄ (V:V = 5:1), mixed it with acid, covered it, and let it stand overnight. Acid digestion proceeded on the next day at 160-170 °C on a temperature-controlled digestion instrument, and finally, the sample solution was transferred to a volumetric flask without loss, and deionized water was used to bring the volume to 30 mL. Finally, an optical emission spectrometer (Perkin Elmer, USA) was utilized to assess the Na⁺, K⁺ and Ca²⁺ levels in the liquid, respectively. Each test was conducted using three biological replicates.

Callose staining was determined as described previously (Su et al. 2023). After treating leaves infected with TRV and TRV-RcMYB8 using NaCl solution (0 and 200 mM), they were subjected to a 1-h aniline blue solution (Solarbio, Beijing, China) staining devoid of light. Green fluorescent samples were deemed as callose (Luna et al. 2011), which were surveilled using an Axio Scope A1 microscope (Carl Zeiss). With the aid of ImageJ (https://imagej.nih·gov/ij/), the callose quantity was identified.

To produce an effector construct, cloning of RcMYB8 coding sequence into pGADT7 vector was accomplished. After cloning RcP5CS1 promoter fragments into pHIS2 vector, they were transferred into Y1H yeast cells with pGADT7-RcMYB8. For spot analysis, we chose and grew the transformants on SD/−Trp-His-Leu and SD/−Trp-His-Leu + 3-AT plates. The RcPR5/1 promoter fragment was cloned into the BstB I-digested pABAi vector and cotransformed into Y1H yeast using pGADT7-RcMYB8. We chose and grew the transformed positive yeast colonies on SD/−Ura plates, and subsequently transferred them severally to SD/−Ura/−Leu + AbA and SD/−Ura/−Leu plates for spot analysis.

Cloning of RcMYB8 CDS as an effector into the pCAMBIA 1300 vector was accomplished. The pGreenII 0800-LUC vector was inserted with RcPR5/1 promoter sequence (P2, P4) and RcP5CS1 promoter sequence (P5) as a reporter. After transformation of the plasmid into Agrobacterium strain GV3101, cultivation of vector-transformed A. tumefaciens proceeded under 200 rpm at 28 °C until OD600 = 1.0. Then, the Agrobacterium was suspended in liquid medium (200 μM AS, each 10 mM MgCl₂ and MES), incubated for 3 h and filtered into the back of the tobacco leaf. After 3 days of growth in the dark, the leaves were sprayed with sodium d-fluorescein (Sangon Biotech, Shanghai, China). A live plant fluorescence detector (SH-Advance 523, Hangzhou) was employed for the fluorescence surveillance, while a dual luciferase reporting reagent (Vazyme Biotech, Nanjing, China) was utilized to evaluate the activity levels of luciferase (LUC) and renal luciferase (REN) in fireflies. Each test was conducted using three biological replicates.

The ORF of RcMYB8 was inserted into the vector expressing pGEX4T-1, followed by transfer into E. coli BL21(DE3) for expression, added 100 mM Isopropyl-β-D-thiogalactopyranoside (IPTG), thereby producing recombinant GST-RcMYB8 protein. A chemiluminescent EMSA kit (Beyotime, Shanghai, China) was used for the binding reaction. The probe and mutant fragments of the RcPR5/1 and RcP5CS1 promoters were selected and labeled with biotin, and the same unlabeled fragments were used as competitors. Single-chain 5′ end biotin-labeled probes were synthesized by Shanghai Sangon Biotech (China).

SPSS ver. 25.0 (SPSS Inc., Chicago, IL, USA) was used to undertake the entire statistical analyses. Data were processed by Student’s t test or univariate analysis of variance (ANOVA), and subsequently through least significant difference (LSD) approach. Individual experimental statistics are presented as means ± standard deviations (SDs).

Not applicable.

All authors approve the manuscript and consent to the publication of the work.

The authors declare that they have no competing interests.