Actually, several thousand years ago when people knew nothing about the chemical nature of ethylene, they began to produce ethylene using various methods to accelerate maturation of plants. The ancient Egyptians, after the figs yielded fruits, would cut some openings on the fruits so as to accelerate the maturation; while our ancestors put green pears in houses with burning incenses, and they would become mature more rapidly. These experiences and methods were effective because the plants were placed into an environment full of ethylene. The relation between ethylene and plant growth was discovered by the ancients without understanding it, but it is applied in modern agriculture purposefully. Those “green” fruits in preservation require a “maturation acceleration” operation before they are distributed. In the meantime, ethylene plays an important role in coping with biotic or abiotic stresses such as diseases and pests, low temperature and salt stress. Therefore, an in-depth study of the signal pathway of the plant hormone ethylene has significant theoretical meaning and practical value.

The Research Group of Professor Hongwei Guo in the Department of Biology of Southern University of Science and Technology (SUSTech) has long been dedicated to the study of regulatory mechanisms of signal transduction molecules and biochemical mechanisms of the plant hormone ethylene and senescence of plant organs, focusing on analyzing the working mechanism of core components in ethylene conduction, and artificial regulation of ethylene synthesis and senescence of plant organ.

Secret Molecular “Domino” Reveals the Action of Ethylene

How does the plant relate to the ethylene? In the plant cells there are protein receptors binding directly with ethylene gas. The two proteins ETR1 and CTR1 involved in this process were identified early in 1993. When there is no ethylene, the proteins are in active state and repress expression of the downstream genes responsible for the maturation of the fruits. Once ethylene is generated or applied, such repression would be relieved, and the signal will be transmitted downstream through a transmembrane protein called EIN2 on the endoplasmic reticulum, sequentially leading to the transcriptional cascade controlled by the transcription factor EIN3 in nucleus, and finally resulting in the expression of the enzymes, such as pectinase, which could promote maturation, as well as the conversion of sugar to make the fruits sweet.

Such a series of molecules seem just like Dominoes (ETR1-CTR1-EIN2-EIN3- downstream gene) transmitting the biological signals from the outside to the inside, This linear ethylene signal transduction from the receptor on endoplasmic reticulum of cells to transcription factor in the cell nucleus has been established by the scientists in the plant Arabidopsis thaliana more than 30 years ago. Among the numerous dominoes, the most mysterious one is the EIN2 protein, a transmembrane protein in the endoplasmic reticulum and the central component in the ethylene signal pathway, because once it loses its function, the plant will be completely insensitive to ethylene. Since EIN2 gene was cloned in 1999, scientists have attempted to know how the EIN2 protein on the endoplasmic reticulum membrane regulates and controls the ethylene signal, and how the EIN2 itself participates in the transmission of the ethylene signal. (Hao D et al., Hormone Metabolism and Signaling in Plants, 2017)

In 2012, three independent research groups including the research group of Hongwei Guo reported the “cutting and shuttling” model of EIN2 in activating the downstream ethylene signal nearly at the same time. When the concentration of ethylene raises higher in plant cell, EIN2 will be activated and its C terminal (CEND) will be cut by some protease and then break away from the endoplasmic reticulum and enter the cell nucleus, activating EIN3/EIL1 and the ethylene reaction in a certain way (Wen X et al., Cell Research, 2012). When the research group of Hongwei Guo used fluorescent proteins to trace the distribution of EIN2 proteins in cells, they also discovered that ethylene could not only induce CEND of EIN2 to enter the cell nucleus, but also promote it to form bright and randomly-distributed dots structure, which implied that EIN2 may have an important function in cytoplasm. In 2015, the research group of Hongwei Guo discovered that in the cytoplasm, EIN2 interacted with 3’UTR of mRNA (messenger RNA) of the protein responsible for degrading EIN3, repressed its translation process, promoted accumulation of EIN3, and activated the ethylene reaction at last (Li W et al., Cell, 2015). This achievement expounded a new function of EIN2 protein in cytoplasm, and discovered a kind of new PolyU sequence of RNA elements responding to the ethylene signal. In the meantime, their research achievement has indicated in the plant signal transduction field for the first time that the 3’UTR of mRNA could perceive the upstream signal and transmit downward like a “receptor”, having high significance to plant studies. In addition, this research achievement also has important application prospects, and the regulatory effect of CEND of EIN2 on 3’UTR of 1/2 mRNA in proteins responsible for degrading EIN3 would be used to make an artificial “ON” or “OFF” regulqation of the ethylene signal, so as to resist various kinds of threats or to delay maturation of fruits and senescence of crops, thus serving the agricultural practice.

Know the Law and Use the Best

Plant hormone ethylene participates in regulating maturation of fruits and senescence of organs, and has an important influence on physiological activity and the quality of the post-harvested plants during the storage period including fruits, vegetables and flowers. The ethylene generated in large volumes could shorten the shelf life of these plants, causing their premature deterioration and resulting in food safety problems and giant post-harvest loss (the annual post-harvest loss of fruits and vegetables in China only reaches hundreds of billions of yuan). Therefore, there would be significant economic and social benefits in developing an effective and safe ethylene response repressor. Based on this, by means of chemical genetics and taking over-production mutant eto1-2 and ethylene signal activated mutant ctr1-1 as the screening materials, Guo’s group screened out three small molecular compounds repressing ethylene synthesis or reaction from a library of 2,000 kinds of chemicals: they are kynurenine (KYN), ponalrestat (PRT) and pyrazinamide (PZA).

Their early studies revealed that KYN could directly repress a key enzyme in the auxin biosynthetic route induced by the ethylene (He W et al., Plant Cell, 2011), and recently they discovered that PRT also acted on downstream of ethylene signaling, repressing another key enzyme in the auxin biosynthetic route (related work is under organization for publication). Different from KYN and PRT, the third molecule PZA could specially repress an enzyme involved in ethylene biosynthesis——ACC oxidase (ACO)——thus repressing ethylene production. It is discovered through in vitro biochemical analysis that, PZA cannot repress the catalytic activity of ACO directly and it needs to be transformed to pyrazinoic acid (POA) in planta first, and then weakens the catalytic activity of ACO. Up to now, PZA/POA is the most efficient small molecule discovered in repressing synthesis of ethylene.

In the meantime, Guo’s group cooperated with the research Group of Dr. Junyu Xiao from Peking University, analyzing the high-resolution crystal structure (2.1Å) of ACO family members in Arabidopsis thaliana: ACO2 and POA compounds, and revealed the repression mechanism of POA at the atomic level. The crystal structure indicates that POA combines with ACO2 through forming a coordinate bond with one zinc ion or iron ion in the active center. In addition, the hydrogen bond, hydrophobic interaction and van der waals force (VDW) between POA and its surrounding amino acid have also consolidated its combination with protein. Through mutation of key amino acid of ACO2 protein, it has been proved that POA or similar analogue 2-PA could simulate endogenous substrate ACC of ACO, thus having made a competitive repression of activity of ACO. These results have not only expounded the repression mechanism of POA at atomic level, but also provided a theoretical basis for further optimizing the small molecular structure and improving its repression activity (Sun X et al., Nature Communication, 2017).

Deciphering the Secret of Plant Senescence

The maturing or senescence progress of plants may influence the efficiency of agricultural production dramatically. Take the grain yield and quality for example,  the yield may increase 2-10% for each day of delayed senescence of the functional leaves in later period according to estimation of main crops (such as corn, soybean, cotton, rice and wheat). Ethylene has been regarded as a plant senescence hormone for a long time as it could promote senescence of leaves together with internal and environmental signals, but its molecular mechanism of the action in detail is still not known.

The research group of Hongwei Guo has carried out studies on senescence of plant organs, established the first LSD (Leaf Senescence Database) (Liu X et al., Nucleic Acids Research, 2011) in the academic community, and discovered the role of the transcription factor EIN3 of ethylene signal pathway in the process of leaf senescence. During the senescence process, expression and transcriptional activity of EIN3 increased gradually, and its overexpression plant and loss-of-function mutant were manifested by early leaf senescence and late leaf senescence, respectively. It has been reported that microRNA (miR164) plays an important role in delaying leaf senescence. They found that EIN3 regulates transcription of miR164 directly, accordingly repressing its level in the process of leaf senescence, promoting the expression level of the target gene NAC2 of miR164 and accelerating the leaf senescence at last (Zhong H et al., Plant Cell, 2013). In the meantime, it was discovered by the group in their recent research that during the progress of ethylene-regulated leaf senescence, there is also another important transcription factor, WRKY75, which could form a synergistic regulatory ring in pairs with salicylic acid and hydrogen peroxide (Guo P et al., Plant Cell, 2017). The ethylene interacts with multiple hormones to control this senescence regulatory ring, and promotes the leaf senescence forward irreversibly.

The above figures are model diagrams of ethylene signal transduction pathway: The left figure is a description of modular Arabidopsis thaliana used in the lab, while the right figure is the “cutting and shuttling” model (a) and translational repression model (b) of EIN2 protein discovered by the Research Group of Hongwei Guo. (Wen X et al., Cell Research, 2012; Li W et al., Cell, 2015)