Effects of GR24 on growth performance under low light stress

Low light stress significantly inhibited the growth performance and biomass of cucumber seedlings (Fig. 1). As shown in Table 2, compared with the control, the plant height, stem diameter, shoot dry weight and shoot fresh weight decreased by 25.1, 36.3, 64.2 and 49.1% under low light, respectively. Low light stress also significantly decreased leaf length, leaf width, leaf area, root dry weight and root fresh weight by 12.9, 24.6, 34.51, 57.1 and 58.7%, respectively. However, exogenous application of GR24 significantly improved the adverse effects of low light stress on the inhibition of plant growth. Under the control conditions, GR24 alone significantly increased leaf size but had no significant effects on other growth parameters.

Fig. 1
figure1

Visual assessment of cucumber seedlings under low light and GR24 treatment conditions. Note: Photographs of the cucumber seedlings were taken at the end of different treatments. The growth of cucumber seedlings was significantly inhibited under different treatments for 7 d. Cont, 0 μM GR24 + 500 μmolm− 2 s− 1 PPFD; Cont+GR24, 10 μM GR24 + 500 μmolm− 2 s− 1 PPFD; LL, 0 μM GR24 + 60 μmolm− 2 s− 1 PPFD; LL + GR24, 0 μM GR24 + 60 μmolm− 2 s− 1 PPFD

Table 2 Effects of exogenous GR24 treatments on the growth parameters of cucumber seedlings under low light stress

Effects of GR24 on the photosynthetic pigment content and gas exchange parameters under low light stress

As shown in Table 3, compared with the control, low light stress increased chlorophyll b (Chl b) by 22.3% but decreased the chlorophyll a (Chl a), total chlorophyll content (Chl a + b) and Chl a/b of cucumber seedling leaves by 30.00, 9.8 and 42.8%, respectively. Exogenous GR24 induced a significant increase in the levels of Chl a, Chl a + b and Chl a/b. There was no significant difference in Chl content between the control leaves and GR24-treated leaves. With increasing treatment time, the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) of cucumber leaves significantly decreased under low light stress. On the seventh day, these values decreased to 33.03, 30.51, and 37.03% of the control levels, but the intercellular carbon dioxide concentration (Ci) increased by 10.49%. However, exogenous GR24 alleviated the negative effects caused by low light stress, thus increasing the values of Pn, Gs and Tr by 63.35, 133.33 and 121.86%, respectively. No significant differences were observed in photosynthetic parameters between the control and GR24-treated seedlings (Fig. 2).

Table 3 Effects of exogenous GR24 treatments on chlorophyll contents in leaves of cucumber seedlings under low light stress
Fig. 2
figure2

Effects of exogenous GR24 on the gas exchange parameters Pn (A), Gs (B), Ci (C) and Tr (D) in cucumber seedlings under low light stress. Note: The respective parameters were measured at 0, 1, 4, and 7 days after the start of low light stress and/or 10 μM GR24 treatments. Each histogram represents the mean value of three independent experiments, and the vertical bars indicate SEs (n = 3). Different letters indicate significant differences at P < 0.05, according to Duncan’s multiple range tests. Cont, 0 μM GR24 + 500 μmolm− 2 s− 1 PPFD; Cont+GR24, 10 μM GR24 + 500 μmolm− 2 s− 1 PPFD; LL, 0 μM GR24 + 60 μmolm− 2 s− 1 PPFD; LL + GR24, 10 μM GR24 + 60 μmolm− 2 s− 1 PPFD. Here, Pn, net photosynthetic rate; Gs, stomatal conductance; Ci, intercellular CO2 concentration; Tr, transpiration rate

Effects of GR24 on the chlorophyll fluorescence parameters of cucumber leaves under low light stress

As shown in Fig. 3, under normal growth conditions, GR24 had no significant effects on the maximum quantum yield of PSII (Fv/Fm), actual photochemical efficiency of PSII (ФPSII), modulated heat dissipation of PSII, (ФNPQ), nonmodulated heat dissipation of PSII (ФNO), photochemical quenching coefficient (qP), or nonphotochemical quenching coefficient (NPQ). However, compared with the control, low light stress reduced the values of Fv/Fm, ФPSII, ФNPQ, qP and NPQ by 13.65, 41.29, 27.23, 46.19 and 48.88%, respectively, but significantly increased ФNO. However, the application of GR24 under low light increased the values of Fv/Fm, ФPSII, ФNPQ, qP and NPQ by 6.47, 46.19, 14.11, 39.45 and 46.55%, respectively, but reduced the value of ФNO by 21.96% under low light stress.

Fig. 3
figure3

Effects of exogenous GR24 on chlorophyll fluorescence in cucumber seedlings under low light stress. Note: Here, Fv/Fm, the maximum quantum yield of PSII; ФPSII, actual photochemical efficiency of PSII; ФNPQ, modulated heat dissipation of PSII; ФNO, nonmodulated heat dissipation of PSII; qP, photochemical quenching coefficient; NPQ, nonphotochemical quenching coefficient. Each image in the same column represents the same leaf. The bars represent the standard errors. The colour scale at the bottom indicates values from 0 (black) to 1 (pink)

Effects of GR24 on the carbohydrate level and sucrose metabolism of cucumber seedlings under low light stress

As shown in Fig. 4A and B, compared with the control, low light stress caused a significant decrease in the soluble sugar and sucrose contents of cucumber leaves. However, compared with low light stress alone, GR24 supplementation under low light stress significantly increased the levels of soluble sugar and sucrose by 100.83 and 31.67%, respectively. To gain a better understanding of how GR24 enhances sucrose metabolism, we quantified sucrose synthase enzyme and sucrose phosphate synthase enzyme activity in cucumber leaves (Fig. 4C and D) and SS (sucrose synthase) and SPS (sucrose phosphate synthase) gene expression (Fig. 5). Compared with the control, low light stress reduced the activities of sucrose synthase and sucrose phosphate synthase by 57.50 and 71.54%, respectively, and the corresponding SS and SPS gene expression levels were 0.42- and 0.28-fold higher than those of the control, respectively. Interestingly, supplementing GR24 under low light stress increased the activities of sucrose synthase and sucrose phosphate synthase by 59.28 and 109.12%, respectively, compared with those under only low light stress, while the expression levels of the corresponding coding genes SS and SPS were 1.59- and 2.09-fold higher than those under low light stress.

Fig. 4
figure4

Effects of exogenous GR24 on the content of soluble total sugar (A) and sucrose (B) and enzyme activity related to sucrose metabolism in cucumber seedlings under low light stress. Note: The respective parameters were measured at 7 days after the start of low light stress and/or 10 μM GR24 treatments. Each histogram represents the mean value of three independent experiments, and the vertical bars indicate SEs (n = 3). Different letters indicate significant differences at P < 0.05, according to Duncan’s multiple range tests. Here, SS, sucrose synthase; SPS, sucrose phosphate synthase. Cont, 0 μM GR24 + 500 μmolm− 2 s− 1 PPFD; Cont+GR24, 10 μM GR24 + 500 μmolm− 2 s− 1 PPFD; LL, 0 μM GR24 + 60 μmolm− 2 s− 1 PPFD; LL + GR24, 10 μM GR24 + 60 μmolm− 2 s− 1 PPFD

Fig. 5
figure5

Heatmap representing the relative transcript abundance of differentially expressed antioxidant enzyme-encoding genes, strigolactone Signaling genes, and sucrose metabolism enzyme genes in the leaves and roots of cucumber seedlings under low light stress with or without GR24 treatment. Note: The gene expression intensity is represented with a colour gradient from blue (low) to red (high). The respective parameters were measured at 7 days after the start of low light stress and/or 10 μM GR24 treatments. Cont, 0 μM GR24 + 500 μmolm− 2 s− 1 PPFD; Cont+GR24, 10 μM GR24 + 500 μmolm− 2 s− 1 PPFD; LL, 0 μM GR24 + 60 μmolm− 2 s− 1 PPFD; LL + GR24, 10 μM GR24 + 60 μmolm− 2 s− 1 PPFD. Here, MDHAR R, monnodehydroascorbate in roots; MDHAR L, monnodehydroascorbate in leaves; DHAR R, dehydroascorbate reductase in roots; DHAR L, dehydroascorbate reductase in leaves; GR R, glutathione reductase in roots; GR L, glutathione reductase in leaves; APX R, ascorbate peroxidase in roots; APX L, ascorbate peroxidase in leaves; MAX2 R, more axillary growth 2 in roots; MAX2 L, more axillary growth 2 in leaves; SPS L, sucrose phosphate synthase in leaves; SS L, sucrose synthase in leaves

Effects of GR24 on the biosynthesis and signal transduction of strigolactone under low light stress

As shown in Fig. 6A, the strigolactone content in the roots and leaves of the cucumber treated with GR24 alone was 1.28- and 1.36-fold that of the untreated cucumber, respectively. Compared with the control, the strigolactone content in the roots and leaves of cucumber seedlings increased by 8.13 and 15.48% under low light stress, respectively. However, under low light stress, the content of strigolactone in cucumber roots treated with GR24 increased significantly, by 31.46% compared with the low light-only treatments. (Fig. 6A).

Fig. 6
figure6

Effects of exogenous GR24 on the expression of strigolactone (A) and the strigolactone synthesis genes CsMAX1 (B), CsMAX3 (C) and CsMAX4 (D) in cucumber seedlings under low light stress. Note: The respective parameters were measured at 7 days after the start of low light stress and/or 10 μM GR24 treatments. Each histogram represents the mean value of three independent experiments, and the vertical bars indicate SEs (n = 3). Different letters indicate significant differences at P < 0.05, according to Duncan’s multiple range tests. Here, SLs, strigolactone; MAX1, more axillary growth 1; MAX3, more axillary growth 3; MAX4, more axillary growth 4. Cont, 0 μM GR24 + 500 μmolm− 2 s− 1 PPFD; Cont+GR24, 10 μM GR24 + 500 μmolm− 2 s− 1 PPFD; LL, 0 μM GR24 + 60 μmolm− 2 s− 1 PPFD; LL + GR24, 10 μM GR24 + 60 μmolm− 2 s− 1 PPFD

In the roots and leaves of cucumber seedlings under GR24 under low light stress, strigolactone synthesis and the transcriptional abundance of signal transduction genes, namely, MAX1, MAX3, MAX4 and MAX2, were significantly upregulated (Figs. 5 and 6B-D). In cucumber seedlings that were not treated with GR24, the expression of strigolactone synthesis genes in roots was slightly higher than that in leaves, but the differences were not significant. However, in cucumber seedlings under the combined treatment of low light stress and GR24, the expression of the strigolactone synthesis genes MAX1, MAX3 and MAX4 in roots was increased by 37.51, 88.89 and 52.34%, respectively, compared with the expression in leaves. Compared with low light stress alone, the expression levels of the MAX1, MAX2, MAX3 and MAX4 genes in leaves under the combined treatment of low light stress and GR24 were significantly increased by 37.91, 125.88, 108.04 and 112.75% on the 7th day. At the same time, the expression levels of the MAX1, MAX2, MAX3 and MAX4 genes in cucumber roots were significantly increased by 89.07, 159.23, 271.85 and 156.63%, respectively, which implied that exogenous GR24 application might participate in endogenous strigolactone induction and regulation of strigolactone biosynthesis and signal transduction to mitigate low light-induced damage to cucumber seedlings.

Effects of GR24 on oxidative damage and RBOH gene expression under low light stress

As shown in Fig. 7A and B, after GR24 was used alone, the H2O2 and MDA contents in cucumber leaves and roots did not change significantly. However, compared with the control, the contents of H2O2 and MDA in cucumber leaves/roots significantly increased by 69.12/183.60% and 189.04/232.34% under low light stress, respectively. Strikingly, GR24 supplementation under low light stress significantly reduced the H2O2 and MDA contents in leaves/roots by 25.00/21.39% and 44.00/56.50%, respectively, compared with those under low light stress. It has been widely demonstrated that the RBOH gene encoding an ROS-forming enzyme is induced under stress conditions, and the relative expression of RBOH in the leaves and roots was markedly elevated throughout the low light stress duration (Fig. 7C and D), reaching approximately 6.11- and 4.69-fold from the initial time to 7 d of stress treatment, respectively. In contrast, GR24-treated seedlings showed downregulation of the expression of the same genes in the leaves and roots compared to that in low light-stressed seedlings, and the expression was reduced by 47.43 and 34.90% at 7 days of stress, respectively.

Fig. 7
figure7

Effects of exogenous GR24 on H2O2 (A), MDA (B) and RBOH gene expression in cucumber seedling roots (C) and leaves (D) under low light stress. Note: The contents of H2O2 and MDA were measured at 7 days after the start of low light stress and/or 10 μM GR24 treatments. The samples used to determine gene expression were taken at 0 h, 12 h, 1 d, 4 d and 7 d after the start of low light stress and/or 10 μM GR24 treatments. Each histogram represents the mean value of three independent experiments, and the vertical bars indicate SEs (n = 3). Different letters indicate significant differences at P < 0.05, according to Duncan’s multiple range tests. Cont, 0 μM GR24 + 500 μmolm− 2 s− 1 PPFD; Cont+GR24, 10 μM GR24 + 500 μmolm− 2 s− 1 PPFD; LL, 0 μM GR24 + 60 μmolm− 2 s− 1 PPFD; LL + GR24, 10 μM GR24 + 60 μmolm− 2 s− 1 PPFD. Here, RBOH, respiratory burst oxidase homologue

Effects of GR24 on the antioxidant enzyme activity of cucumber seedlings under low light stress

To study the role of strigolactone in alleviating low light stress through antioxidant activity, we analysed the transcriptional abundance of APX, GR, DHAR and MDHAR in the leaves and roots of cucumber seedlings (Fig. 5) and the antioxidant enzyme activity (Fig. 8). Low light stress significantly stimulated the transcriptional abundance of the APX, GR, DHAR and MDHAR genes compared with the control, while the expression levels of these genes were further positively modulated in GR24-treated seedlings under low light stress (Fig. 5). Compared with the control conditions, the activities of APX, GR, DHAR and MDHAR in leaves/roots under low light stress increased by 34.34/61.37%, 74.49/79.49%, 58.73/65.48% and 59.20/48.34%, respectively. Importantly, treatment with GR24 under low light stress markedly increased the activities of APX, GR, DHAR and MDHAR compared with those under low light stress alone by 88.98/45.97%, 108.70/65.29%, 60.37/31.35% and 98.15/61.66%, respectively. In contrast, the GR24 treatment under low light stress increased the activities of APX, GR, DHAR and MDHAR in leaves/roots by 57.84/41.04%, 45.03/45.46, 10.70/10.35% and 31.73/39.84%, respectively, compared with those under low light stress. In addition, under only GR24 treatment, the activities of APX and GR were increased by 12.20/55.92% and 21.26/57.95% in leaves/roots compared to the control levels, respectively.

Fig. 8
figure8

Effects of exogenous GR24 on the enzyme activities of APX (A), GR (B), DHAR (C) and MDHAR (D) in cucumber seedlings under low light stress. Note: The respective parameters were measured at 7 days after the start of low light stress and/or 10 μM GR24 treatments. Each histogram represents the mean value of three independent experiments, and the vertical bars indicate SEs (n = 3). Different letters indicate significant differences at P < 0.05, according to Duncan’s multiple range tests. Cont, 0 μM GR24 + 500 μmolm− 2 s− 1 PPFD; Cont+GR24, 10 μM GR24 + 500 μmolm− 2 s− 1 PPFD; LL, 0 μM GR24 + 60 μmolm− 2 s− 1 PPFD; LL + GR24, 10 μM GR24 + 60 μmolm− 2 s− 1 PPFD. Here, APX, ascorbate peroxidase; GR, glutathione reductase; DHAR, dehydroascorbate reductase; MDHAR, monnodehydroascorbate

Effects of GR24 on the antioxidant contents of cucumber seedlings under low light stress

Compared with those in control seedlings, treatment with GR24 only reduced the contents of ASA, AsA + DAsA, GSH and GSH + GSSG in leaves/roots by 22.58/22.96%, 8.44/8.73%, 7.72/13.58% and 1.15/3.27% (Fig. 9) but increased the contents of DAsA and GSSG in leaves/roots by 35.46/34.84% and 7.68/11.35%, thereby reducing the values of AsA/DAsA and GSH/GSSG in leaves/roots by 42.85/43.43% and 14.66/22.86%, respectively. Low light stress significantly reduced the AsA and GSH contents in leaves by 52.70 and 29.32%, respectively, and in roots by 53.01 and 40.73%, respectively. In contrast, compared with those in the control seedlings, the DAsA contents increased by 111.89 and 111.07% in leaves and roots, respectively, while the GSSG contents increased by 27.81 and 44.45%, respectively. Compared with low light stress alone, the application of GR24 under low light stress increased the levels of ASA, AsA + DAsA, GSH and GSH + GSSG in leaves/roots by 51.07/54.97%, 2.90/0.52%, 8.74/38.18% and 1.82/2.20% but reduced the contents of DAsA and GSSG in leaves/roots by 30.46/36.60% and 3.33/18.76%, thereby increasing the values of AsA/DAsA and GSH/GSSG in leaves/roots by 117.24/144.58% and 12.45/70.08%, respectively.

Fig. 9
figure9

Effects of GR24 treatment on AsA(A), DAsA(B), and AsA + DAsA contents (C), the AsA/DAsA ratio (D), GSH(E), GSSG(F), and GSH + GSSG(G) contents and the GSH/GSSG ratio (H) in cucumber seedlings under low light stress. Note: The respective parameters were measured at 7 days after the start of low light stress and/or 10 μM GR24 treatments. Each histogram represents the mean value of three independent experiments, and the vertical bars indicate SEs (n = 3). Different letters indicate significant differences at P < 0.05, according to Duncan’s multiple range tests. Cont, 0 μM GR24 + 500 μmolm− 2 s− 1 PPFD; Cont+GR24, 10 μM GR24 + 500 μmolm− 2 s− 1 PPFD; LL, 0 μM GR24 + 60 μmolm− 2 s− 1 PPFD; LL + GR24, 10 μM GR24 + 60 μmolm− 2 s− 1 PPFD. Here, AsA, reduced ascorbate; DAsA, oxidized ascorbate; GSH, reduced glutathione; GSSG, oxidized glutathione

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