Ethylene-Orchestrated Circuitry Coordinates a Seedlingts Response

Ethylene-orchestrated circuitry coordinates INAUGURAL ARTICLE a seedling’s response to soil cover and etiolated growth

Shangwei Zhonga,b, Hui Shia,b, Chang Xuea, Ning Weib,1, Hongwei Guoa,1, and Xing Wang Denga,b,1

aPeking–Yale Joint Center for Plant Molecular Genetics and Agro-biotechnology, National Key Laboratory of Protein and Plant Gene Research, and Peking–Tsinghua Center for Life Sciences, College of Life Sciences, Peking University, Beijing 100871, China; and bDepartment of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2013.

Contributed by Xing Wang Deng, February 10, 2014 (sent for review January 2, 2014)

The early life of terrestrial seed plants often starts under the soil in In the absence of light signals, the growth modulation of eti- subterranean darkness. Over time and through adaptation, plants olated seedlings is determined by the coaction of a variety of have evolved an elaborate etiolation process that enables seedlings plant hormones (12–16). For example, Gibberellin (GA) is to emerge from soil and acquire autotrophic ability. This process, known to promote hypocotyl elongation and repress photomor- however, requires seedlings to be able to sense the soil condition phogenic gene expression in darkness within the context of the and relay this information accordingly to modulate both the seed- etiolation program (17–19). The mechanism by which seedlings lings’ growth and the formation of photosynthetic apparatus. The monitor soil depth and texture, and modulate their growth pat- mechanism by which soil overlay drives morphogenetic changes in tern accordingly, however, remains poorly understood. Previous plants, however, remains poorly understood, particularly with research has suggested that ethylene gas is likely to play an im- regard to the means by which the cellular processes of different portant role in modulating plant growth and development, par- organs are coordinated in response to disparate soil conditions. ticularly in light of the fact that ethylene, as the smallest plant hormone, can rapidly spread throughout the plant (10, 20–26). Here, we illustrate that the soil overlay quantitatively activates PLANT BIOLOGY ’ – Ethylene is perceived by five receptors that are related to bac- seedlings ethylene production, and an EIN3/EIN3-like 1 dependent – ethylene-response cascade is required for seedlings to successfully terial two-component regulators (27 29), whereas the biological responses to ethylene are known to be mediated by ethylene emerge from the soil. Under soil, an ERF1 pathway is activated in – the hypocotyl to slow down cell elongation, whereas a PIF3 path- insensitive 3 and ethylene insensitive 3-like 1 (EIN3/EIL1) (30 33), two plant-specific transcription factors that are rapidly induced way is activated in the cotyledon to control the preassembly of by ethylene at the protein level (34–36). Loss of EIN3/EIL1 photosynthetic machinery. Moreover, this latter PIF3 pathway function has been known to lead to complete insensitivity to appears to be coupled to the ERF1-regulated upward-growth rate. ethylene (37, 38). In early physiological experiments, pea seed- The coupling of these two pathways facilitates the synchronized lings and bean roots have been shown to produce an increased progression of etioplast maturation and hypocotyl growth, which, amount of ethylene in response to mechanical stress (39, 40), in turn, ultimately enables seedlings to maintain the amount of whereas ethylene pathway mutants are found to exhibit defects in protochlorophyllide required for rapid acquisition of photoauto- their soil emergence capabilities (41). Moreover, ethylene has trophic capacity without suffering from photooxidative damage also been shown to induce dramatic morphological changes during the dark-to-light transition. Our findings illustrate the ex- known as “the triple response” on typical dark-grown seedlings istence of a genetic signaling pathway driving soil-induced plant (20, 38, 42). These changes include inhibition of hypocotyl and morphogenesis and define the specific role of ethylene in orches- root elongation, radial swelling (horizontal growth) of hypocotyl trating organ-specific soil responses in Arabidopsis seedlings. Significance plant hormone ethylene | chlorophyll biosynthesis | ROS | cell death

Seedlings’ ability to both adapt to their soil environment and errestrial flowering plants often drop their seeds under soil or acquire photoautotrophic capacity under various buried con- Tlitter, which serves to protect the propagation process from ditions is a life-or-death issue for terrestrial flowering plants. hostile conditions such as cold temperatures and/or predators By designing and utilizing a standardized real-soil assay, we – (1 3). As a result of this soil cover and/or dense canopy, seeds identify the key features of germinating seedlings’ soil re- often germinate in subterranean darkness. Unlike seedlings that sponse and deduce that the gaseous phytohormone ethylene undergo germination in the light, which immediately undergo acts as the primary regulator of soil-induced plant morphoge- photomorphogenesis, seedlings that germinate under soil follow netic changes. Moreover, our study illustrates that an EIN3/ an adaptive growth program known as skotomorphogenesis or EIL1-conducted PIF3–ERF1 molecular circuitry enables seedlings etiolation (4–6). When seedlings finally emerge from soil, light to synchronize the rate of protochlorophyllide biosynthesis terminates the etiolation program and activates the conversion with upward growth, a mechanism critical to the prevention of of etioplasts to chloroplasts, which, in turn, enable the plants to photooxidative damage during seedlings’ initial transition gain photoautotrophic ability (7, 8). This transition from dark- from dark to light in natural conditions. ness to light is a point of particular vulnerability in the life cycle of a higher land plants, however, due to the fact that light energy Author contributions: S.Z., N.W., H.G., and X.W.D. designed research; S.Z., H.S., and C.X. performed research; S.Z., H.S., N.W., H.G., and X.W.D. analyzed data; and S.Z., H.S., absorbed by protochlorophyllide (Pchlide) (a precursor of chlo- N.W., H.G., and X.W.D. wrote the paper. rophyll) is extremely phototoxic and can potentially cause seedling The authors declare no conflict of interest. death by light-induced photooxidative damage (6, 9–11). To deal 1To whom correspondence may be addressed. E-mail: [email protected], ning. with these obstacles, higher land plants have adapted an elaborate [email protected], or [email protected]. etiolation program that allows for precise control of the progression This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of etioplast development before their conversion into chloroplasts. 1073/pnas.1402491111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1402491111 PNAS Early Edition | 1of8 Downloaded by guest on September 26, 2021 cells, and exaggeration of the apical hook (20, 43). This “triple response” seedling morphology is thought to optimize the seedling’s ability to push through the soil without damaging its shoot meristem. Nonetheless, little is known about the specific role of ethylene plays over the course of etiolation program under soil. Here, we demonstrate that the documented “triple response” is actually an adaptive response to soil overlay, and that ethylene is the primary mediator of plants’ soil response during growing out of soil. In soil-induced etiolation program, EIN3/EIL1 acts a point of convergence from which cellular activities of the cotyledon and hypocotyl are coordinated in accordance to the depth and texture of the soil in which a seedling finds itself. Once activated by the soil, EIN3/EIL1 stimulates ethylene response factor (ERF1) in the hypocotyl, which, in turn, inhibits cell elongation and slows down the upward-growth rate. Similarly, EIN3/EIL1 concurrently activates phytochrome-interacting factor 3 (PIF3) in the cotyledon to repress Pchlide biosynthesis, and thus cope with the extended time in subterranean darkness. As a result of this synchronized mechanism, the progression of the Pchlide accumulation is optimized so as to ensure seedlings rapidly acquire photosynthetic capacity without experiencing photooxidative damage once they emerge from the soil. Fig. 1. Soil overlay quantitatively activates ethylene production and the endogenous level of EIN3 protein, which is essential for soil-induced mor- Results phogenetic changes in seedlings. (A) Ethylene production from 4-d-old dark Soil Overlay Quantitatively Activates Ethylene Production and EIN3 grown Col-0 seedlings on MS medium without soil covering (0 mm) or buried ± Protein Accumulation. To examine how Arabidopsis seedlings re- under soft soil (silicon powder) or firm soil (sand) of indicated depth. Mean spond to various buried conditions, we designed a set of conve- SD; n = 3. (B) Image analysis of 4-d-old dark grown EIN3p:EIN3-Luciferase/ nient and highly reproducible soil assays with which we ein3eil1 seedlings under white light (Left) or bioluminescence (Right). The monitored the emergence of seedlings from soil of divergent depth bioluminescence of EIN3p:EIN3-Luciferase/ein3eil1 indicates the EIN3 protein level. The seedlings were grown on MS medium without soil (No soil) or and texture (Fig. S1). We found that soil induced ethylene pro- Arabidopsis buried in 1 or 2 mm of soft soil. The color-coded bars display the intensity of duction in seedlings in a manner that correlated with luciferase activity. Two independent T2 transgenic lines #7 and #20 were the depth and firmness of the soil (Fig. 1A). To monitor the in planta used. Hypocotyl phenotypes (C) and hypocotyl length (D) of 5-d-old etiolated activity of ethylene signaling activity in real time, we gen- Col-0 and ein3eil1 seedlings on MS medium without soil (0 mm), buried in 2 erated EIN3p:EIN3-Luciferase/ein3eil1 transgenic plants, in which mm (2 mm) or 3 mm (3 mm) of firm soil. Mean ± SD; n ≥ 20. (E) Hypocotyl the EIN3-Luciferase fusion protein driven by the EIN3 native length of 5-d-old etiolated Col-0 and ein3eil1 seedlings on MS medium promoter was expressed in ein3eil1 (Fig. 1B). As indicated by the without ACC (0 μM), with gradient of ACC (0.1 or 1 μM). Mean ± SD; n ≥ 20. heat map images shown in Fig. 1B, EIN3 protein levels were observed to dramatically increase over the course of etiolated seedlings’ growth through the soil overlay. Similarly, this increase in the seedlings failed to emerge from 3 mm of firm soil (Fig. 2 A–D). EIN3proteinlevelwasalsoobservedtocorrelatewiththedepthof Taken together, these results demonstrate that the EIN3- the soil (Fig. 1B). Thus, it appears that ethylene signaling activity dependent ethylene pathway is essential to seedlings’ suc- can be sensitively and effectively induced by the presence of soil. cessful emergence from soil. Interestingly, when ein3eil1 mutant seeds were germinated in the light without soil covering, the The Soil Responses of Etiolated Seedling Are Dependent on EIN3/EIL1. seedlings’ survival rate were comparable to that of the WT (Fig. We next observed how soil overlay influenced the growth pattern 2 A–D). This result indicates that ethylene signaling is imperative of seedlings. In comparison with soil-free etiolated seedlings, to the seedling growth process only when the germinating seedlings growing through soil overlay exhibited a morphology seedlings are buried in soil. characterized by a shorter, thicker hypocotyl with a distinct apical The survival of soil-covered seedlings hinges on their ability to hook (Fig. 1C). Furthermore, the severity of these morphological both break through the soil and turn green upon exposure to changes was observed to increase with soil depth (Fig. 1 C and D). light. Although almost 100% of the WT seedlings effectively No changes were observed, however, in the ethylene pathway of penetrated their soil cover, less than 50% of ein3eil1 seedlings the ein3eil1 mutant (Fig. 1 C and D), which suggests that the in- grown under the same soil overlay in darkness were observed to ducement of soil-elicited morphological change is dependent on successfully emerge (Fig. 2E), indicating that the mutants were the function of EIN3/EIL1. Moreover, the soil-induced phenotype less capable of penetrating the soil. In addition, after the plates was observed to be phenocopied in soil-free seedlings treated were transferred into light for an additional 2 d, those ein3eil1 with 1-aminocyclopropane-1-carboxylic acid (ACC), a bio- seedlings that had made it to the soil surface died on account of synthetic precursor of ethylene (Fig. 1E) (also see Fig. 4D). photobleaching (Fig. 2F). Thus, ethylene signaling appears to be These observations suggest not only that ethylene is required for important for two different processes of etiolation growth: soil-induced growth modification but also that it can, on its own, strengthening of the hypocotyl to better penetrate the soil, and induce comparable growth responses in etiolated seedlings. preventing photooxidation of the cotyledons.

Ethylene Signaling Is Critical for Seedlings Germinating Under Soil Soil Overlay Activates Gene Expression of ERF1 in the Hypocotyl and Cover. To address the functional significance of the ethylene– PIF3 in the Cotyledon. Given that previous research has shown that EIN3 pathway under soil conditions, WT and ein3eil1 seeds were ERF1 represses hypocotyl elongation in etiolated seedlings (26, buried at different depths (Fig. 2 A and B) or within different 44) and that PIF3 is known to play a role in regulating the textures (Fig. 2 C and D) of soil. The seeds were then grown for 7 greening process (45–47), we examined the soil-dependent acti- or 9 d under long-day conditions (16 h light/8 h dark). In contrast vation of ERF1 and PIF3 in different seedling organs. Using to WT seedlings, which successfully emerged from 3 mm of firm reporter transgenic plants ERF1p:GUS and PIF3p:GUS in our soil, the survival rate of ein3eil1 seedlings was observed to soil assays, we found that soil overlay dramatically stimulated drastically decrease with increasing soil depth (Fig. 2 A and B) ERF1 gene expression in the hypocotyl and increased PIF3 and firmness (Fig. 2 C and D). Similarly, almost all of the ein3eil1 expression most strongly in the cotyledon (Fig. 3 A and B).

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1402491111 Zhong et al. Downloaded by guest on September 26, 2021 ERF1 acts downstream of EIN3/EIL1 and mediates the re-

pressive effect of ethylene on hypocotyl cell elongation. INAUGURAL ARTICLE

PIF3 Acts Downstream of EIN3/EIL1 to Prevent Light-Induced Photooxidation. One of the central components of the deetiolation process is the regulation of cotyledon greening, which determines whether seedlings turn green or are killed by photobleaching after their initial exposure to light (6, 9, 10). After being transferred to light, ERF1ox exhibited a normal cotyledon greening response (Fig. S3), which suggests that the initiation of chlorophyll biosynthesis is not regulated by the ERF1-mediated pathway. Previous research has demonstrated that PIF3 plays a critical role in regulating the greening process (45–47). In light of the findings that EIN3/EIL1 is required for cotyledon greening (10, 48) and that PIF3 is activated by soil and ethylene in the cotyledon (Fig. 3 B and D), we set out to determine whether the function of PIF3 is driven by ethylene during chlorophyll biosynthesis. Over the course of our greening experiment, which was conducted with 5-d-old seedlings transferred from dark to light, both ein3eil1 and pif3ein3eil1 mutants underwent photobleached cell death with a high level of reactive oxygen species (ROS) accumulation (Fig. 5 A–C and Fig. S4). The mutants of pif3 also perished on account of photooxidative damage, regardless of their ACC treatment status (Fig. 5 A–C and Fig. S4). Over- expression of PIF3, however, was able to rescue the greening defect of ein3eil1 (Fig. 5 A–C and Fig. S4). Thus, it appears that EIN3/ EIL1’s regulation of the greening process is mediated through PIF3. Interestingly, although the pif3 mutants suffered photooxidative Fig. 2. EIN3/EIL1 is critical to seedlings’ emergence from soil and survival. damage in the cotyledons, they displayed normal hypocotyl growth PLANT BIOLOGY Phenotypes (A) and survival percentage (B) of Col-0 and ein3eil1 seedlings in response to ACC treatment (Fig. 5D and Fig. S5). These results on MS medium without soil (0 mm), buried in 2 or 3 mm of firm soil and ± = thus indicate that the processes of cotyledon greening and hypocotyl grown under white light for 7 d. Mean SD; n 3. Phenotypes (C) and elongation are regulated via separate pathways. survival percentage (D) of Col-0 and ein3eil1 seedlings grown on MS medium without soil (0 mm), buried in 3 mm of silicon powder (soft soil) or 3 mm of sand (firm soil), and grown under white light for 9 d. Mean ± SD; n = 3. (E) EIN3/EIL1 Represses Pchlide Biosynthesis Through PIF3. Overaccu- Percentages of etiolated seedlings emerging from the soil for Col-0 and mulation of Pchlide is known to cause photooxidative damage ein3eil1 seedlings on MS medium without soil (0 mm) or buried in 3 mm of to etiolated seedlings during the greening process (9, 49, 50). To firm soil (3 mm). The seedlings were grown in the dark for 5 d, and the investigate whether the photobleaching death of pif3 and ein3eil1 percentage of seedlings that successfully emerged from the soil was calcu- was caused by Pchlide overaccumulation, we examined the ex- lated. Mean ± SD; n = 3. (F) Greening percentage of Col-0 and ein3eil1 pression of specific genes in these mutants known to be in- seedlings on MS medium without soil (0 mm) or buried in 3 mm of firm soil (3 volved in Pchlide and chlorophyll biosynthesis. HEMA1 is known mm). The seedlings were grown in the dark for 5 d and then irradiated with to encode the first committed enzyme of the chlorophyll bio- white light for an additional 2 d. Mean ± SD; n = 3. synthesis pathway of higher land plants (Fig. 6A)(51–53), whereas genomes uncoupled 4/5 (GUN4/GUN5) are known to determine Ethylene was also observed to have similar effects on the same reporters (Fig. 3 C and D) and was found to activate the expression of both of these genes via an EIN3/EIL1-dependent pathway in etiolated seedlings (Fig. S2). Taken together, these results suggest that ERF1 and PIF3 may play a role in modulating hypocotyl growth and cotyledon development, respectively, during seedlings’ adaptation to their soil environment.

ERF1 Acts Downstream of EIN3/EIL1 to Mediate Ethylene-Inhibited Hypocotyl Cell Elongation. Given that ethylene (ACC) treatment is able to elicit soil-dependent growth responses in etiolated seedlings, we used ACC treatment to mimic soil-induced phe- notypic changes during our additional mechanistic studies. To elucidate the role of ERF1 in hypocotyl growth, we generated inducible ERF1 transgenic plants (pER8-ERF1) that expressed ERF1-MYC in a β-estradiol concentration-dependent manner (Fig. 4A). As the expression of ERF1-MYC was increased in etiolated seedlings, the hypocotyls appeared to be progressively stunted (Fig. 4B). The observed effect of induced ERF1 in- Fig. 3. Soil overlay dramatically stimulates ERF1 and PIF3 expression, whereas the comparable expression can be attained using exogenous hibition on hypocotyl elongation resembled the effect of soil or ethylene treatment and no soil. GUS staining images of 4-d-old dark grown ethylene (ACC) on the hypocotyl cell elongation of etiolated ERF1p:GUS (A) and PIF3p:GUS (B) seedlings on MS medium without soil seedlings (Figs. 4 C and D and 1 C–E). Moreover, constitutive ERF1 ein3eil1 ein3eil1 (0 mm) or buried in 3 mm of firm soil (3 mm). (C) GUS staining of 3-d-old dark- overexpression of in mutants (ERF1ox/ ) grown ERF1-promoter:GUS (ERF1p:GUS) seedlings on MS with or without 10 was found to override the ein3eil1 phenotype, and thus converted μM ACC. The #1 and #11 are two independent T2 transgenic lines of ERF1p: the long hypocotyl characteristic of a deficient ethylene response GUS. (D) GUS staining of 3-d-old dark grown PIF3-promoter:GUS (PIF3p:GUS) to the shortened hypocotyl characteristic of a constitutive eth- seedlings. The seedlings were grown on MS (Air) or on MS treated with ylene response (Fig. 4E). These results thus demonstrate that ∼20 ppm ethylene (Ethylene).

Zhong et al. PNAS Early Edition | 3of8 Downloaded by guest on September 26, 2021 development and hypocotyl growth in response to soil condition is a particularly advantageous adaptive response for plants. Although the pif3 mutant was observed to suffer from photooxidative damage to its cotyledons, this mutant displayed a normal ethylene response with regard to hypocotyl growth (Fig. 5 and Fig. S5). Similarly, although ERF1ox displayed repressed hypocotyl elongation, it developed a normal cotyledon during the greening process (Fig. S3). Consequently, these results seem to indicate that PIF3-regulated cotyledon greening and ERF1- mediated hypocotyl elongation are two independent pathways that are orchestrated downstream of EIN3/EIL1. Nonetheless, given that the ERF1 and PIF3 pathways are activated by the same stimulus, it seems likely that these two pathways are regulated in a synchronized manner. We next examined whether the coregulation of the PIF3 and ERF1pathways is necessary for seedlings’ adaptation to their soil environment. Prior research has shown that firm, deep soil induces more ethylene production than soft soil (Fig. 1A). Given that both soil and exogenous ACC treatment have both been shown to comparably activate ERF1 and PIF3 expression (Fig. 3 and Fig. S2), we used ACC treatment to simulate the firmness of different soil conditions. In the absence of ACC treatment (simulation of soft soil), dark-grown seedlings that had reached a stipulated length (to simulate depth of soil) within 4 d of Fig. 4. ERF1 acts downstream of EIN3/EIL1 to mediate ethylene-inhibited hy- β – growth were transferred into light. Upon exposure to light, ap- pocotyl cell elongation. (A) -Estradiol induced expression of ERF1-MYC in pER8- proximately one-half of the pif3 seedlings died from photo- ERF1#7 as shown by the anti-MYC immunoblot in 4-d-old etiolated seedlings. (B) bleaching due to their lack of PIF3 activity (Fig. 7A). Overactivation β-Estradiol–dependent inhibition of hypocotyl growth in 4-d-old etiolated seedlings. Mean ± SD; n ≥ 20. (C) Propidium iodide (PI) staining hypocotyl cell of ERF1 resulted in ERF1ox exhibiting slower hypocotyl growth wall of 4-d-old etiolated seedlings shows inhibition of cell elongation treated than the WT. Specifically, ERF1ox required 7 d to reach a hypo- with 10 μM ACC or 10 μM β-estradiol. DMSO was added in the same amount in cotyl length comparable to that which the WT achieved in 4 d. uninduced control. (D) Hypocotyl phenotype of 4-d-old etiolated seedlings Furthermore, due to the lack of corresponding compensation of the treated with 10 μM ACC or 10 μM β-estradiol. The pER8-ERF1#7 and #27 are two PIF3 pathway in repressing etioplast maturation, one-half of the independent T2 transgenic lines. (E) Hypocotyl length of 4-d-old dark-grown seedlings were photobleached upon irradiation with light (Fig. 7A). seedlings on MS with or without 10 μM ACC. The #22 and #31 are two in- In the presence of ACC, which was used to mimic firm soil, seed- dependent T2 transgenic lines of ERF1ox/ein3eil1.Mean± SD; n ≥ 20. lings required an extended time period (6 d) to attain hypocotyl lengths that were comparable to the 4-d-old WT. Although most WT seedlings were observed to still green normally, all of the pif3 the branch pathways leading to Pchlide/chlorophyll synthesis (54– seedlings died due to photobleaching (Fig. 7A). 56). As a result of chlorophyll synthesis occurring in the dark, We further examined the effect of simulated soil depths on a pool of Pchlide accumulates in the etioplasts. Upon exposure to seedling growth. To accomplish this task, seedling hypocotyls light, this accumulated Pchlide is then rapidly converted to were allowed to grow to different lengths in the dark (from 5.5 to chlorophyll by Pchlide oxidoreductases (PORs) (encoded by 15 mm to simulate soil depth) before being transferred to the PORA,B,C genes). This chlorophyll, in turn, enables the plant to light (Fig. 7B). Under soft soil conditions [in this case, Murashige instantly achieve photoautotrophic capacity (9, 10, 57). PIF3 has and Skoog (MS) medium], less ethylene was produced and WT previously been reported to inhibit the expression of HEMA1, seedlings were able to turn green at hypocotyl lengths up to 15 mm GUN4, and GUN5 (45, 46). Here, we found that the expression (Fig. 7B). In firm soil (in this case, MS with ACC supplement), of these genes was stimulated in both the pif3 and ein3eil1 only a slight reduction in the greening percentage was observed mutants in darkness (Fig. 6B). Unlike pif3, however, ein3eil1 also when seeds were buried deeper than 10 mm (Fig. 7B). In contrast, exhibited a markedly reduced expression of PORA and PORB abolishing the PIF3 pathway (pif3) and constitutively activating (Fig. S6). These results thus suggest that EIN3/EIL1 suppresses ERF1 (ERF1ox) were observed to render the seedlings hyper- Pchlide biosynthesis via PIF3, but activates PORA and PORB sensitive to simulated soil depths of greater that 5.5 mm (Fig. 7B). independently of PIF3. Therefore, it appears that seedlings, with either a constitutively We next measured the degree of Pchlide accumulation in activated ERF1 pathway or a defective PIF3 pathway, face a clear ein3eil1 and pif3 etiolated seedlings. Our results indicated that disadvantage in terms of greening when they are grown in either far more Pchlide was accumulated in ein3eil1 than the WT. soft or firm soil at a simulated depth greater than 5.5 mm. Furthermore, an even higher level of Pchlide was accumulated in pif3, which displayed Pchilide levels comparable to the pif3ei- Genetically Reconstructing PIF3–ERF1 Circuitry in an Ethylene- n3eil1 triple mutant (Fig. 6 C and D). Consistent with the ob- Insensitive Mutant Restores a Normal Ethylene Response. Finally, served greening phenotypes of ein3eil1 and PIF3ox/ein3eil1 (Fig. to further determine the role of the PIF3–ERF1 circuitry in me- 5A), overexpression of PIF3 (PIF3ox) was found to dramatically diating seedlings’ survival as they emerge from soil, we artificially decrease the Pchlide levels in ein3eil1, and fully rescue ein3eil1 in reconstructed the PIF3 and ERF1 pathways in an ethylene- terms of Pchlide accumulation (Fig. 6 C and D). These data thus insensitive ein3eil1 mutant background. Our results demon- suggest that PIF3 plays a key role in the ethylene-mediated strated that overactivation of ERF1 in ein3eil1 (ERF1ox/ein3eil1) suppression of Pchlide biosynthesis. constitutively suppressed hypocotyl elongation to such an extent that 6 d of growing time were required for ERF1ox/ein3eil1 Ethylene-Orchestrated PIF3–ERF1 Circuitry Optimizes Seedling seedlings to attain the same hypocotyl length that ein3eil1 seed- Survival in a Variety of Buried Environments. Although it is logical lings reached within 4 d. Upon exposure to light, almost all of the that ethylene responds to soil pressure by modulating the hypocotyl ERF1ox/ein3eil1 seedlings died due to photobleaching (Fig. 7 C growth pattern (via ERF1), it is intriguing that ethylene also and D). Importantly, however, although seedlings in which both appears to actively repress Pchlide biosynthesis (via PIF3). Conse- PIF3 and ERF1 had been artificially coactivated (ein3eil1/PIF3ox/ quently, we next investigated whether the coregulation of cotyledon ERF1ox) still required a longer growth period (6 d) to attain

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Fig. 5. PIF3 acts downstream of EIN3/EIL1 to prevent lethal photooxidative damage upon light irradiation, and this pathway is independent of hypocotyl elongation. (A) Greening percentage of 5-d-old dark grown seedlings on MS with or without 1 μMACCor 10 μM ACC, followed by 2 d of continuous white-light irradiation. Mean ± SD; n = 3. (B) Fluorescence microscopy images of ROS accumulation (indicated by

H2DCFDA fluorescence) in the cotyledons of 5-d-old dark-grown seedlings on MS with or without 1 or 10 μM ACC, followed by 12 h of white-light irradiation. (C) Trypan blue staining of the cotyledons of 5-d-old dark-grown seedlings on MS with or without 10 μM ACC followed by 1 d of white-light irradiation. (D) Hypocotyl and cotyledon phenotypes of 5-d-old dark-grown seedlings on MS with or without 1 μM ACC or 10 μM ACC, followed by 2 d of continuous white-light irradiation.

a hypocotyl length comparable to the WT, these seedlings still crucial to seedlings’ ability to survive in different buried environ- PLANT BIOLOGY successfully turned green at a percentage comparable to WT ments. Therefore, breaking the coregulation of PIF3–ERF1 cir- (Fig. 7 C and D). In fact, the ein3eil1/PIF3ox/ERF1ox seedlings cuitry by constitutive activation of the ERF1 pathway only, which exhibited a phenotype that resembled that of ethylene-treated causes prolonged period of dark growth but without repressing C D WT (Fig. 7 and ). This result thus demonstrates that when Pchlide biosynthesis accordingly, or mutation of PIF3 pathway that the ERF1 pathway is activated by either soil or ethylene, coac- ’ abolishing the repression of Pchlide accumulation, could result in tivation of PIF3 is absolutely crucial to seedlings ability to lethal photooxidative damage upon light irradiation. survive the greening process. The etiolation process in plants is terminated when seedlings – – are exposed to light, which induces the degradation of PIF3 (58 PIF3 ERF1 Circuitry Provides a Genetic Foundation for Germinating F Seedlings to Adapt to Both Etiolation and Deetiolation Processes. 60) (Fig. 7 ), and thus leads to the derepression of Pchlide bio- The importance of PIF3 and ERF1 coactivation is due to the fact synthesis. In addition, light also stabilizes ERF1 protein (26) (Fig. F that the ERF1-controlled hypocotyl elongation rate ultimately 7 ) to suppress hypocotyl elongation. Together, the repression determines the duration of etiolation. Given that Pchlide continu- of hypocotyl elongation and activation of chlorophyll bio- ously accumulates in the cotyledons throughout the entire etiolation synthesis make the seedlings be able to rapidly develop cotyle- period (Fig. 7E), ethylene’s suppression of the rate of Pchlide ac- dons, and thus achieve the capacity of photoautotrophic growth, cumulation and hypocotyl elongation must be coactivated, which is once seedlings emerge from the soil.

Fig. 6. PIF3 mediates EIN3/EIL1-repressed Pchlide biosynthesis in etiolated seedlings. (A) A simplified schematic of the tetrapyrrole biosynthesis process in higher land plants. PɸB, phytochromobilin, shares a common biosynthetic pathway from glutamate to protoporthyrin IX (Proto IX) with chlorophyll. The glutamyl-tRNA reductase, encoded by HEMA1 gene, is the first committed enzyme and rate-limiting step in tetrapyrrole biosynthesis. The branching point from protoporthyrin IX to chlorophyll is catalyzed by the Mg-chelatase, which is regulated by genomes uncoupled 4 (GUN4) and GUN5. The photoreduc- tion of Pchlide to chlorophyll is catalyzed by NADPH: Pchlide oxidoreductases (PORs) (encoded by PORA,B,C genes). (B) Quantitative RT-PCR showing the expression of key tetrapyrrole biosynthesis genes in 3-d-old dark-grown seedlings. Mean ± SD; n = 3. (C)Fluores- cence of Pchlide in 4-d-old etiolated seedlings. (D) The Pchlide fluorescence at 634 nm of three biological replicates in 4-d-old dark-grown seedlings. Mean ± SD; n = 3.

Zhong et al. PNAS Early Edition | 5of8 Downloaded by guest on September 26, 2021 Taken together, our results provide compelling genetic evi- and thus limit Pchlide accumulation. Both of these regulatory dence that plants adapt to various buried conditions by using functions are crucial to plants’ ability to successfully emerge from ethylene signaling to coactivate a PIF3–ERF1 circuitry that the soil and survive the transition from dark to light. Overall, the couples the rate of Pchlide accumulation to the hypocotyl ethylene-directed PIF3–ERF1 circuitry coordinates the tran- elongation process. scription networks that govern a seedlings’ heterotrophic growth in preparation for its transition to the photoautotrophic phase. Discussion It has been hypothesized that photomorphogenesis is the de- Although a great deal is known about how plant seedlings grow fault developmental pathway, whereas etiolation (skotomor- in the dark, little is known about how seedlings respond and phogenesis) is an adaptive developmental program that evolved adapt to the various soil conditions they may face. In addition, later in plants (61). The majority of terrestrial seed plants ger- even less is known about the mechanisms by which these soil minate under the soil and thus initially grow in a subterranean, responses are integrated into the full etiolation program. By dark environment. To adapt to this reality, plants have de- studying the effect of soil cover on the growth of Arabidopsis veloped the etiolation program. The process of etiolation both seedlings, we have identified ethylene as the primary mediator of allows germinating seedlings to grow in darkness against the soil responses during plant etiolation. Plants proportionally mechanical pressure of the soil and prepares the photosynthetic produce ethylene in response to soil pressure, and then use the apparatus for the critical dark-to-light transition, so as to enable ethylene signaling pathway to relay the information necessary for the plant to immediately acquire photoautotrophic capacity growth modulations in both the hypocotyl and cotyledons. Thus, upon exposure to light. Our study indicated that ethylene plays we suggest that the ethylene-induced “triple response” (20, 43) is an essential role in mediating the various soil responses actually etiolated seedlings’ typical response to soil. throughout the etiolation process. Mutation of EIN3/EIN3-like 1 Under the soil, the duration of dark growth time is determined was observed to abolish seedlings’ soil response. In addition, we by the rate of hypocotyl elongation and the depth of soil, which demonstrated that the morphological responses and tissue- varies according the burial conditions a given seedling faces. The specific gene expression of seedlings grown in soil are compa- biosynthesis of Pchlide must also be precisely controlled so as to rable to that exhibited by the WT under exogenous ethylene enable the rapid conversion of accumulated Pchilide into func- treatment without soil. Moreover, the ethylene pathway was also tional chlorophyll by light at the exact moment the shoots pro- found to be not essential to plants’ ability to establish vegetative trude from the soil surface. Underaccumulation of Pchilde can growth when the plants are germinated under light and without potentially delay a seedlings’ transition to photoautotrophic soil. Consequently, we suggest that plants developed the ethylene growth (6, 45), whereas overaccumulated Pchlide poses an even pathway as a means of specifically coping with the soil environ- greater danger due to its lethal phototoxicity (6, 9). Our studies ment, at least at seedling stage. Although ethylene plays a variety revealed that the EIN3/EIL1-directed PIF3–ERF1 circuitry of key regulatory roles over the course of plant development, synchronizes the mechanisms of cotyledon development and hy- including promoting both fruit ripening and the senescence of pocotyl growth in response to different soil environments (Fig. 8). leaves and flowers (12, 20), based on our results, the ethylene For example, seedlings were observed to produce a greater amount pathway is crucial to the growth of etiolated seedlings only when of ethylene under firmly packed soil than they did under soft soil. they must push their way out from under the soil. In turn, this ethylene, most likely via the ERF1 pathway, serves to strengthen the hypocotyl cell wall and slow down the hypo- Materials and Methods cotyl elongation rate. At the same time, this increased ethylene Plant Materials and Growth Conditions. All of the plants used in this study were also acts via the PIF3 pathway to repress Pchlide biosynthesis Columbia-0 ecotype. The ein3eil1, pif3, pif3ein3eil1, PIF3ox, EIN3ox, ERF1ox,

Fig. 7. The coactivation of PIF3 and ERF1 is essential for etiolated seedlings’ ability to survive the dark- to-light transition. (A) Hypocotyl lengths (mean ± SD; n ≥ 20) of etiolated seedlings grown on MS with or without 1 μM ACC for the indicated number of days (D4, D6, and D7 mean 4, 6, and 7 d grown in the dark) and their greening percentages (mean ± SD; n = 3) after being transferring to white light for an additional 2 d. (B) Greening percentage of etiolated seedlings on MS (/M) or M with 1 μM ACC (/A) as a function of the hypocotyl length at the time of being transferred from darkness to white light. Mean ± SD; n = 3. Phenotypes (C) and the hypocotyl lengths (mean ± SD; n ≥ 20) with greening percentages (mean ± SD; n = 3) (D) of seedlings that were grown in the dark for the indicated number of days (D4 and D6 mean 4 and 6 d grown in the dark) and transferred to white light for an additional 2 d. The #3 and #4 are two independent T2 transgenic lines of ein3eil1/PIF3ox/ERF1ox. (E) Fluorescence of Pchlide (Pchlide) in WT seedlings grown in darkness for the indicated number of days. (F) Light has opposing regulatory effects on the accumulation of PIF3 and ERF1 proteins. Immunoblots of PIF3-MYC and ERF1-MYC proteins (by anti-MYC antibody) in 5-d-old dark-grown ein3eil1/PIF3ox/ERF1ox#4 seedlings. Dark to light means that the etiolated seedlings were transferred into white light for 0–4h.WL4toD means after 4 h of white light exposure, the seedlings were transferred back to darkness for the indicated number of hours (hr). Ponceau S staining is used as a loading control.

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1402491111 Zhong et al. Downloaded by guest on September 26, 2021 PIF3p:GUS (#7, 3 kb upstream of ATG), and PIF3ox/ein3eil1 lines have all been previously reported in the literature (10, 26, 62). To generate ERF1ox/ INAUGURAL ARTICLE ein3eil1 and ERF1ox/PIF3ox/ein3eil1 lines, the full length of ERF1 coding DNA sequence (CDS) was amplified from cDNA of Col-0 and cloned into a pENTR/D-TOPO vector (Invitrogen). The entry vectors were then subcloned into gateway compatible binary vector pGWB17 (65) and transformed into ein3eil1 or PIF3ox/ein3eil1 plants. To generate ERF1p:GUS, the promoter of ERF1 was amplified from genomic DNA of Col-0 and cloned into a pENTR/D- TOPO vector (Invitrogen). The entry vectors were then subcloned into gateway compatible binary vector pGWB3 (63) and transformed into Col- 0 plants. To generate EIN3p:EIN3-Luciferase/ein3eil1, the full length of EIN3 CDS without a stop codon was amplified from cDNA of Col-0, and connected to the promoter of EIN3 that had been amplified from genomic DNA of Col- 0. The EIN3p-EIN3 fragment was then further subcloned into a compatible Fig. 8. A working model for the integrated regulation of Pchlide accumula- binary vector and transformed to ein3eil1 plants. The ERF1 CDS, which was tion and hypocotyl elongation in etiolated seedlings under soil. (Left) A dia- × fused with a 4 MYC tag, was then cloned into a pER8-derived plasmid (64) gram of the seedlings grown in different soil environments. (Right) The model β – to generate -estradiol induced ERF1 transgenic plants. showing the signaling pathway that enables seedlings to grow out of soil and Seeds were surface sterilized, and plated on a MS medium with a half survive the critical dark-to-light transition. After germination under soil, the dosage of MS salts and sucrose (2.2 g/L MS salt, 5 g/L sucrose, pH 5.7, and 8 g/L etiolated seedlings produce ethylene in response to the mechanical stress of agar). The plates were then stored in the dark at 4 °C for 3 d before ger- the soil. Through the EIN3/EIL1 proteins, ethylene activates a PIF3–ERF1 tran- mination was induced by irradiating the plates with 6 h of white light at 22 °C. scriptional circuitry to synchronize tissue-specific developments in etiolated seedlings. The ERF1 pathway most likely regulates hypocotyl cell elongation so Phenotype, Histochemistry, and Microscopy. Hypocotyl lengths were measured that the etiolated seedlings can better penetrate the soil cover, whereas the from the point at which the cotyledons join to the hypocotyl to the hypo- PIF3 pathway simultaneously modulates the preassembly of photosynthetic cotyl–root junction using ImageJ software. More than 20 seedlings were machinery in the cotyledon. The coupling of these two processes enables measured for each set of experiments. To determine the greening per- seedlings to rapidly acquire photosynthetic capacity without being damaged centage, the seedlings were grown in darkness for the indicated number of by photooxidation during their initial transition from dark to light. − − days, and then exposed to continuous white light (120–150 μmol·m 2·s 1)for an additional 2 d. The greening percentage was then calculated by dividing

the number of successfully greened seedlings by the total number of seed- buffer [90% (vol/vol) acetone containing 0.1% NH3]. The pigments were

lings (10). More than 100 seedlings were examined each time with at least then extracted in the dark at room temperature for 24 h. After the sample PLANT BIOLOGY four biological repeats. had been centrifuged, the fluorescence emission spectra of the supernatant After the etiolated seedlings had been irradiated with continuous white light were analyzed at room temperature. The excitation wavelength was 440 for 24 h, the seedlings were collected to perform trypan blue staining. Samples nm, and the emission spectra from 610 to 750 nm with a bandwidth of 1 nm were submerged in lactic acid phenol trypan blue (LPTB) solution (the LPTB solution were recorded. All Pchlide measurements were independently performed in was made by mixing 9.3 mL of not–water-saturated phenol, 10 mL of lactic acid, 10 biological triplicate, and representative results were shown. mL of glycerol, and 10 mL of 10 mg/mL water-dissolved trypan blue stock; the solution was freshly prepared within a week of its use, and prewarmed to 65 °C RNA Extraction and qRT-PCR. Total RNA was extracted from 3-d-old dark- before use). The samples were placed under vacuum for 5 min, and then allowed grown etiolated seedlings using a Spectrum plant total RNA kit (Sigma) (3). to return to normal atmospheric pressure for 5 min. The vacuum infiltration was The gene expression results were normalized according to a PP2A control repeated three times to remove air bubbles from the leaves. The samples were gene using the primers shown in Table S1. EBF2, a known EIN3-regulated then placed into boiling water for 10 min. After the samples had been cooled to gene, was used as an ethylene activation control (65). All quantitative PCR room temperature, the LPTB solution was removed, and 1 mL of chloral hydrate experiments were independently performed in triplicate, and representative solution (25 g dissolved in 10 mL of ddH2O) was added. The samples were then results with three technical replicas are shown in the figures of this paper. gently shaken for 4–8 h to remove any unspecific staining. Multiple washes of chloral hydrate solution were used if necessary. Finally, the samples were Soil Experiment. Seeds were surface sterilized and plated on a MS medium. mounted and photographed on a dissecting microscope with a CCD camera. The silicon dioxide powder (Sigma) and sand (50–70 mesh particle size; For the histochemical β-glucuronidase (GUS) staining assays, seedlings Sigma) were autoclaved, and the amounts were measured using a 50-mL were pretreated with 90% (vol/vol) acetone at −20 °C for 30 min, and then Falcon tube. The silicon powder or sand was then evenly spread onto the rinsed three times with a 1× PBS buffer. The pretreated seedlings were then agar medium to cover the plated seeds. Given that the area of the plate was × 2 incubated in the substrate buffer [1 PBS, 1 mM K3Fe(III)(CN)6, 0.5 mM K4Fe ∼50 cm , we measured out 5-, 10-, and 15-mL portions of powder or sand, (II)(CN)6, 1 mM EDTA, 1% Triton X-100, and 1 mg/mL X-gluc] at 37 °C. After and used them to form a cover over the seeds that was 1, 2, and 3 mm in incubation, the seedlings were submerged in a graded series of ethanol depth, respectively. The plates were then placed in the dark for 3 d at 4 °C treatments (30%, 70%, 95%) and stored in 95% (vol/vol) ethanol at 4 °C. before being illuminated under white light for 6 h to induce germination. Biological triplicates were performed for the two independent transgenic For the light-grown condition, the bottom and rim of the plate were cov- lines, and representative images were selected for each of these lines. ered with aluminum foil to prevent the roots from being exposed to light. Fluorescence microscopy images of ROS accumulation [indicated by 2′,7′- The top of the plate was then illuminated with white light for the indicated dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence] in the coty- number of days. For the dark-grown condition, the plates were incubated in ledons were captured after the 5-d-old dark-grown seedlings had been ex- darkness for the indicated number of days. posed to white light for 12 h. About 20 seedlings from each sample For soil experiments, 150 seeds of each sample were plated with at population were incubated in 0.8 mL of ROS staining solution (100 μM least three biological repeats. The survival percentage of light-grown · H2DCFDA in 10 mM Tris HCl, pH 7.22) (Sigma) at room temperature. After seedlings was calculated by dividing the number of green seedlings that being shaken on an incubator for 5 min in the dark, the staining solution successfully emerged from soil by the total number of plated seeds. was removed, and the seedlings were washed with 10 mM Tris·HCl (pH 7.22). Similarly, the percentage of dark-grown seedlings emerging from the soil The seedlings were then washed three times over the course of being was calculated by dividing the number of etiolated seedlings that grew shaken for 10 min at room temperature to eliminate any unspecific staining. out of soil after incubation in darkness for the indicated number of days Fluorescence microscopic images were captured using a DMI6000 B fluo- by the total number of plated seeds. For the greening experiments, the rescence microscope with a CCD camera (Leica). plates were incubated in darkness for the indicated number of days, and To examine the hypocotyl cell morphology, dark-grown seedlings were then transferred into white light for an additional 2 d. Then greening stained with 10 μg/mL propidium iodide (PI) (Sigma) for 5–10 min, washed percentage was calculated by dividing of number of successfully greened with ddH2O, and then observed using a LSM 510 Meta confocal laser scan- seedlings by the total number seedlings that emerged from soil. To ex- ning microscope (Carl Zeiss). The wavelength for excitation was 535 nm, and amine the EIN3-Luciferase protein levels, EIN3p:EIN3-Luciferase/ein3eil1 a bandpass filter of 580–617 nm was used for emission. transgenic plants were incubated under dark-grown conditions for 4 d.

To measure the levels of Pchlide, 20 dark-grown etiolated seedlings were The plates were then washed three times with ddH2O to remove the soil. collected under a dim-green safe light, and soaked in 1 mL of extraction Thirty milliliters of 0.5 M D-Luciferin potassium salt solution (BIOTIUM)

Zhong et al. PNAS Early Edition | 7of8 Downloaded by guest on September 26, 2021 were then added to the plate to submerge the seedlings, which were ACKNOWLEDGMENTS. We thank Abigail Coplin for critically reading the then incubated for 10 min in the dark. The luciferase bioluminescence manuscript; Yanpeng Xi and Lei Wang for help in experiments; Dr. Dongqin images were obtained using a Xenogen IVIS Spectrum imaging system Chen for help in trypan blue staining assay; Dr. T. Nakagawa for providing us (Caliper). To measure the production of ethylene, seeds of ∼5mgwere the pGWBs vector; and Ying Zhu and Xiangzhong Sun for help in the ethylene amount determination. This work was supported by grants from evenly plated in 10-mL vials and covered with soil of varying depth and National Natural Science Foundation (31330048), National Basic Research texture. After the seeds had been incubated under dark-grown con- Program of China (973 Program) (2012CB910900), National Institutes of Health ditions for 4 d, gas chromatography (6890N Network GC System; Agilent (GM-47850), and National Science Foundation (MCB-0929100) (to X.W.D.); and Technologies) was used to determine the amount of ethylene present in National Natural Science Foundation (91017010) and Ministry of Agriculture the headspace of the vials. (2010ZX08010-002) of China (to H.G.). H.S. is a Monsanto fellow.

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