Ethylene (C2H4) takes part in the hormonal cross-talk that regulate growth, development, senescence and more throughout a plant’s life in response to environmental or internal conditions (Ivanchenko, et al., 2008). Ethylene itself is a dominant hormone in conditions requiring senescence and abscission (Růžička et al., 2007) but its interaction with the hormone auxin in root length elongation, is the main component of interest in this experiment. Notably, root width will not be the primary subject of research since ethylene strongly influences elongation rather than radial expansion unlike auxin, although they act through a common hormonal pathway mechanism that regulates general growth (Smulders, et al., 1991). Ethylene does so by reducing cell division. Its signalling pathway involves the activation of ethylene through binding of the kinase-receptors and the ethylene transcription factors activating to initiate the transcriptional cascade (Schaller, 2012).
Different concentrations of ethephon will serve as treatments as well as the ethylene inhibitor, Amino-oxyacetic acid (AoA). These treatments will be used on lettuce seeds (Lactuca sativa L. var. Grand Rapids) after germination in water to observe for root cell and total root length growth in various ethylene concentrations. Hence, the hypothesis is that with increasing amounts of ethylene, root cell length and root length growth should decrease in germinated seedlings because of ethylene’s inhibitory effect on growth. The purpose is to observe any trends directly caused by adjustments in ethylene concentrations and synthesis in root length growth.
Treatment choices include Eth as a plant growth
In addition, Petruzzelli et al. (1995) conducted research using an ethylene inhibitor which resulted in its root length being longer than the control treatment itself and reducing radial growth. Here, a similar trend is expected in lettuce seedlings when AoA inhibits ethylene biosynthesis. While experiments do prove that Eth inhibits lateral root growth, a few studies show that certain low concentrations of Eth promotes radial root growth instead (Ivanchenko et al., 2008), possibly due to the higher ratio of auxin to ethylene. This subsequent experiment’s purpose is to increase reliability from past research on the effects of ethylene and its inhibitor in lateral root growth compared to the control.
Materials and methods
Refer to Lab Manual: Physiology of Seed Germination and Hypocotyl Cell Growth in Lettuce (Singh, 2020). Some changes have been made to the original procedure. Sample size is changed to 36 petri dishes instead of 6 petri dishes. Only root cell growth and total root growth is measured and not germination. Treatments include 4 concentrations of ethephon (500 μm, 100 μm, 10 μm, 1 μm) and an ethylene inhibitor (0.1 mM). Germinated seedlings are put into the fridge after 36-72 hours, depending on root growth progress and root growth was measured instead of hypocotyl growth.
The root cell growth varied between treatment types and concentrations. The general trend for cell length (Figure 1A) shows a 6.8% decrease in growth from the control growth to Eth (500 μm) and a 7.7% increase in cell width from the control to Eth (500 μm) (Figure 1A). Most growth is seen in Eth (10 μm) both laterally and radially with a 4.6% length increase and 14% width increase respectively, despite the decreasing trend in length elongation with the addition of ethephon (A). The least lateral growth is seen in Eth (500 μm) with a decrease in 6.8% with the least width growth seen in water at 23 μm (A). A clear trend is seen (B) in lettuce root length where the greatest elongation is found in Eth (1 μm) and the control with the most reduced elongation in Eth (500 μm) by 24%. Total width is greatest in water and least successful in Eth at 1 μm and 10 μm equally by 46% (B). The results are statistically significant as determined by the independent t-test and the null hypothesis may be rejected (P 500 μm) treatment.
Microscopic images in Figure 2 are provided to demonstrate the greatest cellular morphological changes. Water root cells (C) are thin and elongated, whereas Eth (500 μm ) treatment is thicker and stunted in lateral growth (D). Although the primary purpose of this experiment is to observe the effects of ethylene in lateral elongation, width is noted as well because of the crosstalk between ethylene and auxin could explain some of the phenotype seen.
The most consistent trend seen and supported by the experimental hypothesis is that elongation generally decreases overall with increasing amounts of ethylene. Many existing experiments have proven these findings. For instance, De Cnodder et al., 2005 treated Arabidopsis thaliana roots with ACC which resulted in roots with poor cell length and elongation. ACC is oxidized into ethylene by ACC oxidase and it is crucial in the last step of ethylene biosynthesis (Vanderstraeten, Depaepe, Bertrand, 2019). High concentrations of this ethylene precursor reduces the initiation of new lateral root primordia and subsequently reduces pericycle cell length by inhibiting pericycle cells (Ivanchenko, 2008). Existing lateral root primordia created by the division of the pericycle, can be promoted by ACC, resulting in increased cell width (Figure 2D) (Ivanchenko, 2008). Hence, high concentrations of ACC as seen in ethephon (500 μm), represses root growth.
A mechanism that results in inhibition of lateral root growth is through auxin accumulation in the zone of elongation rather than in the meristem (Růžička, 2007). The significance in these results help distinguish the optimal levels of ethephon in plants which is between 1-10μm. At lower concentrations, ethylene stimulates auxin biosynthesis in the hormonal crosstalk regulation of growth and promotes lateral root initiation by utilizing the auxin response mechanisms (e.g. ARF proteins) (Ivanchenko, et al.,2008). ARFs act as transcription factors and is responsible for assigning specificity to the auxin signalling pathway using target genes (Li, Xie, Hu Zhang, 2016). Larger doses than this results in a supra-optimal concentration of ethylene and stress induced mechanisms are initiated that inhibit lateral root growth and reduces the division of pericycle cells required to create lateral root primordia. Hence, the control and higher doses of ethylene (100, 500 μm) roots have significant difference seen in Figure 1 with a P 500μm) causes a reduction in elongation caused by an accumulation of ACC intracellularly due to the inability of ACO to convert large amounts of ACC into ethylene (Vanderstraeten et al., 2019).
Ethylene regulates many processes in root elongation in addition to controlling microtubule orientation and expansion of the cell wall (Ivanchenko, 2008). Microtubules can only be reoriented into one direction after expansins loosen the cell wall. Normally, cells are oriented longitudinally but in elongating cells, its orientation must be across the cell (Pierik, 2007). This process explains why lettuce roots treated with ethephon have greater cell root width than in water or with AoA (Figure 1A). Ethylene and ACC inhibit elongation but can activate these mechanisms subsequently which results in width expansion. However, a plausible correlation between the data observed in Figure 1A and 1B in root width is difficult to explain since the trends are not similar between treatments and further experimentation and changes should be executed.
Furthermore, ethylene inhibits root growth synergistically at the cellular level with the addition of auxin to stimulate the hormonal growth crosstalk (Růžička et al., 2007). Auxin must be accumulated in the elongation zone of the root to demonstrate the ethylene triple response through auxin polar transport (Růžička et al., 2007). Specifically, PIN1, PIN2, PIN4, AUX1 found in the auxin transport mechanism is upregulated by ethylene in the vasculature (Růžička et al., 2007). In their study, auxin efflux carrier PIN2 and influx AUX1 carrier accumulation has been crucial in basipetal auxin transport and redirection in the elongation zone, causing elongation inhibition because of the change in auxin distribution.
As for the inhibitor, it was hypothesized that based on previous research, root cells treated with an ethylene inhibitor should have greater length than the control itself (De Cnodder et al., 2005). Ethylene inhibitors block the conversion of ACC to ethylene by ACC oxidase. Xu et al., 2008 studied the effects of ACC in cell expansion in a specific FEI/SOS5 pathway. The mutant fei1fei2 on roots suggests that the effects of ethylene in cell expansion is reversible using AoA to inhibit the synthesis of ethylene. Although AoA treated roots had greater growth than ethephon generally, it contradicts this hypothesis, or perhaps a different approach must be used.
Overall, the stated hypothesis is supported by this experiment, despite some discrepancies that may be due to limitations or the method of experimentation chosen. For instance, aminoethoxyvinylglycine (AVG) could be used instead of AoA as an ethylene inhibitor. In hindsight, many experiments have used AVG instead and have observed greater elongation in the inhibitor than with water, a trend that was not supported in this experiment. Ethephon was the core treatment used but experimenting with a gaseous form of ethylene with the correct materials instead would enhance the effects through the accumulation of the gaseous particles in the lettuce seedling.
The methods were modified from the original experiment on shoot elongation in order to be specialized for root elongation in Eth treated seedlings. Lettuce seedlings were germinated solely in water and then treated with solutions to ensure that all seeds germinate evenly. Three replicates of each treatment were conducted simultaneously to account for variation when conducting a statistical analysis test. In detail, ethylene’s impact on lettuce root elongation were well observed at the cellular level consistently with past studies; however, observing its effect on total root width and cell width would give rise to exciting experimentation ideas.