Chapter 3 Transcriptomic changes in Arabidopsis leaves suggest possible causes for loss
3.3 Discussion
3.3.2 Senescence-inducing ARCs decrease tolerance to stress with increased age of Arabidopsis leaves
Arabidopsis leaves
To study the effects of senescence-inducing ARCs in EEL, MEL and FEL, Arabidopsis WT plants were exposed to drought stress at 10, 15 and 20 DAG, and the RWC was measured after a 6-day drought period. The percentage of RWC in drought-stressed plants decreased more with age, when compared to the well-watered plants (Figure 3.9D). Also, the first and second rosette leaves were detached from the control and stressed plants to observe the influence of drought stress in leaves of distinct ages. This was chosen because the first rosette leaf that emerges is older than the second rosette leaf, so it was likely that age-related symptoms would be observed primarily on the first rosette leaf pair and later in the second rosette leaf pair. The water deficit in 10+6 DAG first rosette leaf pairs did not show any wilting, while the drought-stressed 15+6 DAG first rosette leaves displayed moderate level of wilting, and the 20+6 DAG stressed samples showed the greatest display of wilting. The same results were found on the second rosette drought-stressed leaves, where wilting was only visible on the 20+6 DAG leaves (Figure 3.9). Altogether, these results clearly show that as Arabidopsis leaves age, the occurrence of senescence-inducing ARCs cause reduced desiccation tolerance.
Salt shock was another experiment used to examine the consequence of stress on different aged
Arabidopsis leaves.Plants can be exposed to salt stress in two ways, either by exposure to mild salt levels causing osmotic stress, or by sudden exposure to concentrated salt that leads to osmotic shock (Shavrukov, 2013). In this study plants were exposed to osmotic shock by watering with 300 mM NaCl solution for 6 days at 10, 15 and 20 DAG, and the progression of senescence was recorded by measuring the chlorophyll content from the first rosette leaf pair (Figure 3.11D). The obvious effect of salt was viewed in all three stages (10, 15 and 20 DAG), where plant samples showed darker colour and diminished leaf growth (small rosette), likely due to reduced photosynthetic capacity, affected cell division and cell enlargement (Osakabe et al., 2014). However, the results showed a significant difference between the salt shocked samples of different ages, where leaf yellowing was more pronounced in 20+6 DAG first rosette leaves than 15+6 DAG samples (Figure 3.11). This result
correlates with the observation that the chlorophyll level declined progressively with age. Hence, this experiment also suggests that tolerance to stress decreases with leaf age because of gradual occurrence of senescence-inducing ARCs. Additionally, the effect of darkness was examined in different aged plants by exposing them to dark conditions (Figure 3.12D). It was also determined whether plant recovery was affected by increased leaf age (Figure 3.12E). The results of this experiment were consistent with the previous observation where chlorophyll degraded progressively with age, and leaf recovery also decreased with age.
It has previously been found that young plants are more tolerant to stress than mature plants. For example, detached Xanthium young leaves did not show wilting after drought stress, but mature leaves showed signs of leaf dryness and stress-induced ABA accumulation (Cornish and Zeevaart, 1984). Also, young cucumber leaves were shown to display remarkable resistance to sulphur dioxide (SO2) stress, whereas mature leaves were more susceptible (Sekiya et al., 1982). The research conducted by Wang et al., (2012) also revealed that young rice leaves adapt well to alkaline stress, but the cell membranes of adult rice leaves were severely injured, and showed reduction in chlorophyll pigment. Moreover, recent research has shown that paraquat sprayed on young Arabidopsis leaves results in reduced ROS accumulation due to higher antioxidant activity, while adult leaves exhibited damage from high ROS abundance and reduced ROS scavenging activity (Moustaka et al., 2015). The results of these studies confirm that decreased stress tolerance with age is a consequence of developmental senescence- inducing ARCs. Thus, young seedlings were resistant to stress because of reduced senescence-inducing ARCs, whereas adult leaves displayed sensitivity because of elevated senescence-inducing ARCs. In plants, ageing itself does not lead to death under stress but causes the decrease in stress tolerance in plants and therefore later increases a susceptibility to death (Mueller-Roeber and Balazadeh, 2014). Altogether, these findings justify the hypothesis that EEL are more tolerant to stress because of highly active DNA repair mechanisms and reduced endogenous stress. Low expression of oxidative stress and senescence-related genes signify that EEL have negligible levels of endogenous stress at this age. On the contrary, the expression of genes linked to DNA repair are reduced, and genes associated to oxidative stress and senescence are increased in MEL and FEL. Here, I propose that, this is one of the reasons the first rosette MEL and FEL samples display poor stress tolerance. The downregulation of genes linked to stress is well supported by the elevated stress hormones and ROS accumulation in first rosette MEL and FEL samples. Whereas, the constant increase in endogenous oxidative stress, senescence, stress hormones and ethylene-related genes is a finding that strengthens our understanding that reduced resistance to stress increases in leaves with age.
I propose that, in a stressed environment the occurrence of senescence-inducing ARCs causes death of the oldest leaves with remobilisation of nutrients to the young growing tissues for the survival.
Therefore, together the transcriptomic study presented here suggests that occurrence of senescence- inducing ARCs is an intrinsic process that causes timely and certain death in plants.