A dual mode of ethylene actions contributes to the optimization of hypocotyl growth under fluctuating temperature environments

ABSTRACT

The gaseous phytohormone ethylene plays versatile roles in sustaining plant growth and fitness in response to environmental changes, such as light illumination, flooding, and mechanical pressure. Interestingly, it is well known that the effects of ethylene on plant growth vary profoundly, depending on external conditions. For example, light/dark conditions alter the directionality of ethylene action on hypocotyl growth. Similarly, a recent study has shown that the effects of ethylene on hypocotyl growth are reversed during temperature increases: ethylene attenuates hypocotyl elongation in the light at warm temperatures (28°C), while promoting it at normal temperatures (22°C). The ethylene-activated ETHYLENE-INSENSITIVE 3 (EIN3) transcription factor directly promotes the transcription of both PHYTOCHROM INTERACTING FACTOR 3 (PIF3) and ARABIDOPSIS PP2C CLADE D7 (APD7) genes. At 22°C, the auxin activity is tuned down, and thus ethylene promotes hypocotyl growth via the PIF3-mediated microtubule reorganization. On the other hand, when auxin highly accumulates at 28°C, the ethylene-directed growth repression is potentiated through the APD7-mediated repression of auxin responses. APD7 plays a role in integrating ethylene cues into auxin signaling. We propose that the dual mode of EIN3-mediated ethylene actions enables plants to optimize growth under constantly changing environments.

Accumulating evidence in recent years indicate that ethylene plays diverse roles in facilitating plants to adapt to a broad spectrum of environmental changes, ranging from non-threatening cues, such as light/dark cycles,¹ to harsh conditions, including mechanical stress and flooding.^2,3 Among these, molecular mechanisms governing ethylene actions are best understood in plant architectural adaptive behaviors.^1–6

In particular, it is notable that ethylene often imposes contrasting effects on plant morphogenesis, depending on the physical nature of environmental cues. It has been reported that ethylene promotes hypocotyl growth in the light, while repressing it in darkness.¹ It is also known that ethylene suppresses plant growth during flash flooding, thus leading to energy saving that is essential for plant survival, whereas under deep submergence, ethylene activates stem elongation, which helps plants to deviate from the underwater harmfulness.^5,6 Furthermore, it has been recently reported that ethylene-mediated hypocotyl growth is contingent on temperature contexts in that ethylene promotes hypocotyl growth at 22°C but represses it at 28°C (Table 1).⁷ However, it is largely unknown how ethylene differentially controls hypocotyl growth, depending on temperature conditions.

Table 1.

Opposing effects of ethylene actions on hypocotyl growth under varying environments. The promotive and repressive effects of ethylene actions were indicated by + and –, respectively. Note that plant stems respond to flooding in rice

	Light		Temperature		Flooding
	Light	Dark	Normal (22°C)	Warm (28°C)	Flash	Deep
Effects of ethylene on hypocotyl growth	+	–	+	–	–	+

It is known that ethylene promotes hypocotyl growth via the transcriptional activation of a bHLH transcription factor PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) in the light.^1,8 Meanwhile, it has been observed that ethylene suppresses hypocotyl growth during skotomorphogenesis and the ethylene action is mediated by several ethylene-responsive regulators, such as ETHYLENE RESPONSE FACTOR 1, WAVE-DAMPENED 5, and OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF 59.^1,9,10 Notably, none of these regulators are not involved in the ethylene-mediated repression of hypocotyl growth in the light under warm environments. These observations raise a possibility that the thermo-induced reversal of ethylene action is regulated by molecular mechanisms that are distinct from those involved in light signaling and responses.

Light and temperature are two major environmental cues that broadly affect hypocotyl growth.^11,12 In particular, temperature effects on plant growth and architecture, termed thermomorphogenesis, have been actively studied in recent decades.¹³ Elevated, but nonstressful, temperatures induce distinct morphological alterations in plants, which include hypocotyl overgrowth, increased leaf hyponasty, and formation of small, thin leaves.^14–17 These thermomorphogenic traits are regulated by a variety of phytohormones, such as auxin, gibberellic acid, and brassinosteroid, and extensive signaling crosstalks also occur in fine-tuning plant thermomorphogenic adaptation behaviors.¹³ An interesting question is whether and how the ethylene functioning in repressing hypocotyl thermomorphogenesis is inter-connected with other hormone signaling events.

Auxin is a primary growth hormone functioning during plant thermomorphogenesis.^12,15 It is known that auxin biosynthetic pathways are stimulated by PIF4 and the thermo-responsive PIF4-mediated pathways reinforce the induction of thermomorphogenic growth.^15,16 A challenging question is whether ethylene signaling affects auxin signaling and responses by regulating the PIF4 activity during hypocotyl thermomorphogenesis. Molecular genetic studies have shown that the ethylene-mediated repression of hypocotyl thermomorphogenesis is comprised in PIF4-deficient mutants, suggesting that PIF4 is critical for the reversed action of ethylene under warm temperature conditions.

A following question is how ethylene controls the PIF4 function under warm environments. It has been found that a master transcription factor of ethylene responses, ETHYLENE-INSENSITIVE 3 (EIN3), mediates the ethylene-directed suppression of hypocotyl thermomorphogenesis.⁷ In addition, gene expression studies demonstrate that ethylene represses the transcription of PIF4 and its downstream genes at warm temperatures. However, the reduction of PIF4 transcription does not significantly affect PIF4 protein abundance and its action during hypocotyl thermomorphogenesis. Collectively, it is likely that other auxin signaling steps, other than the PIF4-mediated auxin pathways, are repressed by the ethylene-activated EIN3 transcription factor.

It is known that auxin effects on hypocotyl growth is exerted by cell elongation. Accumulated auxin activates SMALL AUXIN UP-RNA 19 (SAUR19) and thus SAUR19 induces plasma membrane (PM) H⁺-ATPase activation.¹⁸ The activated PM H⁺-ATPase promotes proton efflux to acidify cell wall apoplast region, resulting in cell expansion by water-swelling, which is proposed in auxin acid-growth theory.¹⁸ Interestingly, media acidification assay showed that apoplast acidification is reduced by treatment of an ethylene precursor 1-aminocyclopropane-1-carboxylic acid at 28°C and this reduction is diminished by EIN3 mutation. Thus, these results suggest that EIN3-mediated ethylene signaling attenuates auxin action by repressing auxin-induced apoplast acidification.

A next question is how ethylene attenuates auxin action in cell expansion by inhibiting auxin-induced apoplast acidification. Chromatin immunoprecipitation (ChIP) assays revealed that EIN3 directly binds to promoter of ARABIDOPSIS PP2C CLADE D7 (APD7), encoding a phosphatase that inhibits auxin-induced apoplast acidification, and induces its gene transcription at both 22°C and 28°C.^7,19 Thus, these results suggest that the ethylene-activated EIN3 attenuates hypocotyl thermomorphogenesis by inducing APD7 inhibition of cell expansion.

It is notable that the ethylene-activated EIN3 promotes both the transcription of growth-promoting PIF3 and growth-repressing APD7 genes.⁷ At 22°C, auxin level is relatively low, and thus ethylene promotes hypocotyl growth through the PIF3 pathway. In contrast, under warm temperature conditions, when auxin is highly accumulated via the PIF4-mediated pathway, the repressive action of the EIN3-APD7 module on the auxin-induced hypocotyl cell expansion is evident (Figure 1).

Figure 1.

Optimization of plant adaptive fitness under challenging environments via a web of signaling crosstalks between ethylene and auxin or environmental stimuli

Various environmental cues, such as light-dark cycles, ambient temperatures, and flooding, profoundly affect plant growth and adaptive behaviors, among which their effects on hypocotyl elongation have been most extensively studied (left diagram). It is interesting that the effects of ethylene are reversed, depending on external temperatures: while hypocotyl growth is accelerated at normal temperatures, it is repressed at warm temperatures. The ethylene-mediated reversible control of hypocotyl growth is intimately linked with auxin signaling (right diagram). In addition, the effects of light-dark cycles are associated with plant thermal responses. It is currently unclear whether signaling events governing plant responses to light-dark cycles and ambient temperatures are functionally interrelated with those mediating flooding responses. We propose that the complicated network of signaling crosstalks during hypocotyl growth orchestrate optimal hypocotyl growth and adaptive behaviors under environmental fluctuations. It has not been explored yet whether signaling networks governing plant adaptation to flooding are directly linked with those mediating ethylene-auxin signaling crosstalks during hypocotyl growth.

Growth optimization is a representative adaptive strategy, by which plants maintain fitness in response to environmental changes. However, it might also be a threat to plant survival in that plant overgrowth is prone to fracture hypocotyls or stems by imposing mechanical stress.²⁰ Thus, plants have evolved various regulatory devices to tightly control plant overgrowth. As expected, hypocotyl thermomorphogenesis is also controlled precisely through multiple promotive and repressible pathways.^20,21 Meanwhile, it is known that ethylene biosynthesis is promoted by mechanical stresses, such as mechanical impedance imposed by obstructs and water wave during flooding.^4,22 In addition, ethylene responses are reinforced when temperatures are elevated: the EIN3-binding F-box proteins 1/2, repressors of EIN3, are degraded under warm temperature conditions.²³ Together, it is envisioned that the ethylene-mediated attenuation of hypocotyl thermomorphogenesis is a powerful mechanism that enables plants to successfully adapt to unforeseen external stimuli under warm temperature conditions.

It is particularly interesting that the ethylene activation of PIF3 and APD7 is largely temperature-independent. Why the ethylene-APD7-mediated growth inhibition is turned on regardless of temperature conditions? It is possible that when plants encounter both mechanical stresses and other growth-promoting conditions, rather than temperature cues, the ethylene-APD7 module functions as a growth attenuator and thus contributes to saving metabolic energy, which helps plants to overcome external harsh events. We propose a universal growth attenuation process, which is facilitated via the auxin-ethylene signaling crosstalks, that optimizes plant architectural adaptation to various external cues. We believe that a web of signaling crosstalks between ethylene and other growth hormones or adaptive signaling events contributes to reshaping environmental plasticity in plants under fluctuating environments (Figure 1).

Funding Statement

This work was supported by the National Research Foundation of Korea [NRF-2021R1A2B5B03001476].