The regulation of photosynthesis by ethylene in tomato

Ethylene is a volatile plant hormone that plays a regulatory role in many aspects of vegetative plant growth and development, including the regulation of photosynthesis. It is also regarded as an 'aging' hormone, as it is required for processes such as climacteric fruit ripening, leaf and flower senescence, and abscission. In addition, ethylene is also involved in regulating plant responses to various biotic and abiotic stresses.

It is established that enhanced ethylene production (e.g. by stress) and signaling can result in decreased growth and ultimately reduced crop yield. In part, this is due to the downregulation of photosynthesis through direct and/or indirect responses to ethylene. There is still no definitive mode of action proposed for the regulatory mechanism through which ethylene exerts control over photosynthesis. Furthermore, most of the molecular work related to the effect of ethylene on photosynthesis has been done in the model species such as Arabidopsis thaliana using ethylene perception and signaling mutants, which cannot reveal the actual temporal effect of ethylene.

In this study, we have undertaken a profound characterization of the temporal effect of ethylene on the photosynthesis dynamics of young tomato plants. To achieve this, we have performed real-time photosynthesis measurements under controlled environmental conditions, also taking into account the temporal aspect of the ethylene effect. We have focused on the 4th leaf of 5-6 leaves stage tomato plants which represents a young, non-senescing, photosynthetically active leaf.

We developed an automated ethylene gassing system to monitor plant responses throughout the ethylene treatment and utilized a combination of physiological, biochemical, and molecular (RNA- and ChIP-seq) experiments to untangle the temporal dynamics of the ethylene effect on plant photosynthesis. In addition, we assessed the impact of an ethylene pretreatment (also called priming) on the tolerance of tomato plants to external environmental stressors, such as salinity, drought, and waterlogging, that are known to influence photosynthesis and lead to a reduction of tomato crop yield.

Our study reveals that ethylene exerts a dose-dependent effect on photosynthesis and confirms that prolonged exposure to saturating concentrations of ethylene suppresses plant growth by inhibiting photosynthesis. The reduction of photosynthesis triggered by ethylene ultimately leads to the depletion of the hexose pool, which is essential to fuel metabolic pathways responsible for energy production, leading to inhibited growth and development. Furthermore, the integration of physiological and biochemical data with time-course transcriptomics revealed that ethylene inhibits photosynthesis in a time-dependent manner, through a complex interplay of early and direct ethylene reactions and indirect secondary responses, which we can group into three distinct phases.

In the first phase (0 – 8 hours), ethylene evokes an immediate response leading to epinasty and stomatal closure, limiting light perception and gas exchange, respectively. Stomatal closure occurred fast, suggesting it is one of the earliest physiological responses towards ethylene, and that ethylene can directly act on mechanisms that control stomatal closure, probably involving a series of secondary messengers such as reactive oxygen species (ROS), nitric oxide (NO), and protein kinases. Besides the reduction of gas exchange, ethylene also evokes epinasty which is one of the most recognizable physiological responses. Epinasty causes a reduced light perception, which can lead to a lower expression of genes involved in the photosynthetic light reactions, including antenna protein complexes, PSI, PSII, cytochrome b6/f, electron transport proteins, and F-type ATPase, also confirmed by our RNA-seq data. A combination of the direct ethylene effect and these secondary, indirect responses (decreased CO2 and light capturing), or an overall inhibition of light reactions (and chloroplastic ATP/NADPH production) further reduces carboxylation rates leading to reduced contents of soluble sugars (mainly glucose and fructose).

In the second phase (8 – 32 hours), plants switch from growth to survival mode in response to sugar deficit and an ethylene-induced energy crisis. Our data shows that ethylene caused a coordinated upregulation of several TCA cycle genes. We believe that, despite the limited availability of sugars, ethylene can stimulate the upregulation of respiration enzymes which may be crucial in sustaining the production of substrates necessary to fuel energy production during periods of low carbon availability. Our data further show that genes involved in lipolysis and fatty acid degradation, as well as protein degradation, are upregulated by ethylene, allowing plants to use alternative respiratory substrates for energy production. Surprisingly, our data reveal enhanced sucrose levels, especially after 8 and 32 h of the ethylene treatment. This might suggest that sucrose is imported into the 4th leaf when carboxylation levels drop.

The third phase, (56+ hours), is initiated as the leaf ages, and premature senescence is triggered by ethylene in combination with the prolonged energy crisis. We want to highlight that other factors such as low light conditions and a carbon crisis together with ethylene exert a cumulative effect on triggering and accelerating senescence, constrained by age-related factors. This leads to the induction of genes associated with senescence (SAG-genes), that promote starch breakdown and chlorophyll degradation.

Our transcriptomics data revealed more than 3000 differentially expressed genes during the early ethylene treatment. To reveal which genes are directly regulated by ethylene, we performed a ChIP-seq experiment using antibodies against one of four ethylene master transcription factors - EIL1 (Ethylene Insensitive Like 1). Our findings indicate that EIL1 can directly bind to the promotor of several genes, but we only pulled down a relatively small number of genes (10). This implies that the majority of differentially expressed genes regulated by ethylene may be due to secondary transcriptional waves, or controlled by other EIL transcription factors. Moreover, two ethylene-enriched EIL1-controlled genes are negative regulators of the ethylene signaling pathway (ETR3 and EBF1), which indicate that the ethylene treatment evokes a feedback mechanism on its signaling pathway to lower its sensitivity. Furthermore, the ChIP-seq experiment also revealed putative candidate regulatory transcription factors (e.g. bHLH87-like, TBG5, RAX3-like) that are ethylene-regulated and can evoke secondary transcriptional waves to influence gene expression related to photosynthesis.

To evaluate the tissue-specific action of ethylene, we generated an EBS:GFP-GUS (EBS - EIN3 binding site) reporter in the Ailsa Craig background. Surprisingly, there was no strong difference in the GUS signal between control-untreated and ethylene-treated (1 ppm for 4 hours) plants after staining (for 6-7 h). We observed a weak increase of the overall GUS staining in the treated plants, especially in some leaflet tips and older, senescing leaves/cotyledons. This, along with ChIP-seq results, indicates that most of the responses to ethylene are a result of secondary, rather than direct ethylene effects. But this also makes us wonder if there is a single or a few central hubs (transcription factors) that are ethylene-regulated and amplify downstream responses.

During normal development, plants only produce a low basal level of ethylene, but ethylene synthesis is strongly enhanced during biotic and abiotic stress conditions. Ethylene plays a crucial role in activating stress survival mechanisms, and therefore we wanted to study the effect of an ethylene pretreatment (priming) on plant performance during salinity, drought, and waterlogging. We assessed physiological responses (e.g. epinastic bending, transpiration, CO2 assimilation rates) and the effect on plant development (fresh and dry weight). Our results show that these stresses all negatively affect all measured parameters which lead to the inhibition of photosynthesis and lower biomass production and crop yield. A short pretreatment with ethylene (1 ppm for 4 h) did not significantly alleviate the negative effects during waterlogging stress, while ethylene made the plants more sensitive to salt stress (by reducing stomatal closure and increasing water losses). When exposed to drought, a pretreatment with ethylene resulted in slightly reduced transpiration rates, enabling plants to retain water more effectively in conditions of limited water availability.

While much research has been conducted on the effects of ethylene on photosynthesis, there is still a lack of information on what are the direct effects of ethylene and which molecular players are involved downstream of the EILs. Our ChIP-seq and time-course RNA-seq results provide a first glimpse into this matter, but future mechanistic follow-up studies are needed to understand how ethylene activates a cascade of responses that lead to the inhibition of photosynthesis and ultimately senescence. Furthermore, our findings indicate that pretreating tomato plants with ethylene can potentially regulate their response to upcoming abiotic stress. However, it remains to be determined whether this modulation has a net positive or negative effect or no effect at all on photosynthesis and overall plant metabolism. Further investigations are needed to fully elucidate the benefits and drawbacks of ethylene pretreatment in this context.