Ethylene-mediated metabolic priming increases photosynthesis and metabolism to enhance plant growth and stress tolerance

Abstract

Enhancing crop yields is a major challenge because of an increasing human population, climate change, and reduction in arable land. Here, we demonstrate that long-lasting growth enhancement and increased stress tolerance occur by pretreatment of dark grown Arabidopsis seedlings with ethylene before transitioning into light. Plants treated this way had longer primary roots, more and longer lateral roots, and larger aerial tissue and were more tolerant to high temperature, salt, and recovery from hypoxia stress. We attributed the increase in plant growth and stress tolerance to ethylene-induced photosynthetic-derived sugars because ethylene pretreatment caused a 23% increase in carbon assimilation and increased the levels of glucose (266%), sucrose/trehalose (446%), and starch (87%). Metabolomic and transcriptomic analyses several days posttreatment showed a significant increase in metabolic processes and gene transcripts implicated in cell division, photosynthesis, and carbohydrate metabolism. Because of this large effect on metabolism, we term this "ethylene-mediated metabolic priming." Reducing photosynthesis with inhibitors or mutants prevented the growth enhancement, but this was partially rescued by exogenous sucrose, implicating sugars in this growth phenomenon. Additionally, ethylene pretreatment increased the levels of CINV1 and CINV2 encoding invertases that hydrolyze sucrose, and cinv1;cinv2 mutants did not respond to ethylene pretreatment with increased growth indicating increased sucrose breakdown is critical for this trait. A model is proposed where ethylene-mediated metabolic priming causes long-term increases in photosynthesis and carbohydrate utilization to increase growth. These responses may be part of the natural development of seedlings as they navigate through the soil to emerge into light.

Keywords: ethylene; growth; metabolism; photosynthesis; stress tolerance.

Fig. 1.

Phenotypes of plants after exposure to ethylene in the dark in the presence and absence of added sugar. Germinating Arabidopsis seeds were exposed to 0.7 ppm ethylene or ethylene-free air in the dark. At this time (day 0), they were transferred to ethylene-free conditions and grown under a 16-h photoperiod. In some cases, exogenous sugar was included as indicated. A) Images of wild-type (Col) seedlings were acquired 10 days after transfer to light. Scale bar = 5 mm. B, C) The effects of ethylene pretreatment at the indicated concentrations of sucrose on wild-type B) new primary root growth and C) lateral root density at the indicated times after transfer to light and ethylene-free conditions. D) The effects of ethylene pretreatment on leaf fresh weight of wild-type and ein2-5 seedlings in the presence or absence of 0.8% (w/v) sucrose at 12 days after transfer to light and ethylene-free conditions. E, F) Images of plants grown in soil for D) 22 days or E) 33 days after transfer to light and ethylene-free conditions. Scale bars = 1 cm. G) Images of growing roots were captured every 15 min for the first 22 h after transfer to light. The average growth rate ± SEM for each time interval is plotted (n ≥ 10). H) The effects of ethylene pretreatment on new root growth of wild-type, ein2-5, and ein3-1;eil1-1 seeds in the presence or absence of 0.8% (w/v) sucrose at 9 days after transfer to light and ethylene-free conditions. I) The effects of ethylene pretreatment on new primary root growth in wild-type seedlings in the presence or absence of 0.8% (w/v) sucrose, glucose, or fructose at 9 days after transfer to light and ethylene-free conditions. J) The effects of ethylene pretreatment on new primary root growth in wild-type seedlings in the presence or absence of 0.02% (w/v) glucose or mannose at 9 days after transfer to light and ethylene-free conditions. Data in B–D) and H–J) represent the mean ± SEM (n ≥ 15), the data were analyzed by ANOVA, and the different letters indicate significant difference (P < 0.05).

Fig. 2.

Ethylene enhances the growth of several plant species. Germinating tomato (S. lycopersicum, cultivar Floridade) A–D) and cucumber (C. sativus, cultivar Beit Alpha Burpless) E) seeds sown in soil were treated with ethylene or ethylene-free air in the dark for 4 days and wheat (T. aestivum) seeds grown on agar F, G) for 3.5 days. At this time, the seedlings were transferred to light and ethylene-free conditions. Photos show representative plants A) 7 days, B) 9 days, E) 11 days, and F) 1.5 days after transfer to light. E) Arrows point to first true leaves. Seedlings on the left in each panel are ethylene-free controls and on the right pretreated with 0.7 ppm ethylene. Scale bars = 1 cm. C, D) Quantification of tomato height and leaf area of tomato seedlings 9 days after transfer to light. G) Quantification of wheat primary root length 1.5 days after transfer to light. Data in C), D), and G) are the mean ± SEM (n ≥ 6) and statistical differences from the untreated controls determined with Student's t test (*P < 0.05; ***P < 0.001).

Fig. 3.

Ethylene pretreatment increases carbon assimilation. Germinating Arabidopsis seeds were treated with 0.7 ppm ethylene or ethylene-free air for 3 days in the dark and then transferred to ethylene-free air and light. Unless otherwise indicated, no exogenous sugar was added. A) Wild-type seeds were sown in the presence or absence of 0.2% sucrose in the presence of 2.5 µm DCMU to block electron transport. Solvent-treated samples are included as controls. B, C) Wild-type and cop1-4 B) or rbcs C) mutants were sown in the absence or presence of 0.2% (w/v) sucrose. A–C) New root primary root growth was measured 9 days after transfer to light. Data are the average ± SEM of at least 15 seedlings. Different letters denote statistical difference (P < 0.05) using ANOVA. D) The F_v/F_m was determined in tissue of wild-type seedlings at the indicated times after transfer to white light. Data represent the mean ± SEM (n ≥ 9). E) Chlorophyll was extracted from excised cotyledons of wild-type seedlings and quantified at different times after transfer to white light as indicated. Data were normalized to tissue fresh weight and represent the mean ± SEM (n ≥ 6). D, E) Data were analyzed by Student's t test and found to be statistically different from seedlings not treated with ethylene with a *P < 0.05 and **P < 0.005. F) Transcript levels of selected genes that encode proteins involved in photosynthesis and chlorophyll metabolism were evaluated by qPCR as described in the Materials and methods at 5 days after transfer to light. Each gene was normalized to its levels in the control condition and to housekeeping genes. G) Measurements of carbon assimilation and stomatal conductance in individual leaves 3 weeks after transfer to light were made in three separate experiments and normalized to the amount in control samples. Different symbols represent individual data points from the different experiments. The mean ± SEM is plotted (n ≥ 29). In D), F), and G), data were analyzed by Student's t test and found to be statistically different from seedlings not treated with ethylene with a *P < 0.05 or ***P < 0.001.

Fig. 4.

Ethylene pretreatment increases starch and glucose levels. Germinating Arabidopsis seeds in the absence of added sugar were treated with 0.7 ppm ethylene or ethylene-free air for 3 days in the dark and then transferred to ethylene-free air and white light with a 16-h photoperiod. A) Control (left) and ethylene-treated (right) seedlings stained for starch as described in the Materials and methods 9 days after transfer to light. Scale bar is 3 mm. B) Quantification of starch levels normalized to fresh weight of the tissue at 9 days after transfer to light (n = 6). C) Transcript levels of genes that encode enzymes involved in starch and sucrose biosynthesis were evaluated 5 days after transfer to ethylene-free air and light by qPCR as described in the Materials and methods. Each gene was normalized to its levels in the control condition and to housekeeping genes. D) Quantification of glucose levels normalized to fresh weight of the tissue at the indicated days after transfer to light (n = 5). E) Transcript levels of CINV1 and CINV2 were evaluated 5 days after transfer to ethylene-free air and light by qPCR as described in the Materials and methods. Each gene was normalized to its levels in the control condition and to housekeeping genes. In B–E), Student's t test was used to determine statistically significant change from the control samples (*P < 0.05; **P < 0.005; ***P < 0.001). F) The amount of new primary root growth was compared in Col (wild-type) and cinv1;cinv2 double mutants 9 days after transfer to light in the presence or absence of 0.8% (w/v) sucrose. Different letters denote statistically significant differences (P < 0.05) as determined by ANOVA. Data in B–F) are the mean ± SEM (n ≥ 15).

Fig. 5.

Ethylene pretreatment increases tolerance to abiotic stresses. Germinating Arabidopsis seeds in the absence of added sugar were treated with 0.7 ppm ethylene or ethylene-free air for 3 days in the dark and then transferred to ethylene-free air and white light with a 16-h photoperiod. A) Five days after transfer to light, Col (wild-type) and cinv1;cinv2 seedlings were exposed to high temperature (43°C) for 22 min. Survival rates 2 days after exposure to high temperature are shown compared with seedlings not stressed with high temperature (22°C). B) Five days after transfer to light, seedlings were exposed to 150 mm NaCl stress and survival assessed 1 week later compared with no stress controls. **P < 0.01 indicates statistical difference from the no ethylene condition using Student's t test. C) Five days after transfer to light, the seedlings were exposed to hypoxia stress for 12 h in darkness and then transferred back to white light and normoxia and allowed to recover. Normoxia controls are shown for comparison. Survival was assessed each day for 3 days. Different letters denote statistically significant differences (P < 0.05) on each day as determined by ANOVA. D) Wild-type seedlings were treated as in A) in the presence or absence 0.2% (w/v) sucrose. In A) and D), different letters denote statistically significant differences (P < 0.05) as determined by ANOVA.

Fig. 6.

Model for enhanced plant vigor from ethylene pretreatment in darkness. In this model, exposure to ethylene while seeds are germinating in darkness activates ethylene signaling resulting in higher EIN3/EIL1 activity. In the dark, this results in the “triple response.” Also, an unknown factor or factors downstream of EIN3 and EIL1 lead to long-lasting changes that, upon illumination and removal of ethylene, result in increased photosynthesis and sugar metabolism resulting in more stress tolerance and growth. It is currently unclear if ethylene is directly affecting all of these pathways or if some effects are secondary to a primary response. In either case, increased photosynthesis leads to higher glucose levels in the leaves to increase starch accumulation. The synthesis of sucrose is also increased which is transported from source to sink tissues where it is broken down to glucose (and fructose). Metabolomic data indicate other metabolic pathways are also affected. Although COP1 is known to affect EIN3 levels in the dark, our data suggest that COP1 affects priming via its role in photomorphogenesis. The changes in photosynthesis and carbohydrate metabolism might occur naturally when seeds germinate underground in darkness and ethylene levels are high due to mechanostimulation from the soil. Upon emergence into light aboveground, the seedlings are exposed to less ethylene because of less mechanostimulation and diffusion away from the aboveground parts of the plant leading to long-lasting developmental changes.