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It was confirmed that old13 mutants are sensitive to stressed conditions. The results from the previous section illustrate that old13 FEL are also susceptible to sucrose treatment. Next, I hypothesise that old13

mutants are more susceptible to sugar because the old13 cellular sugar levels are higher than those of the WT. Therefore, to determine if sugars are high in old13, primary metabolite profiling of old13 EEL, MEL and FEL using Gas Chromatography-Mass Spectrometry (GC-MS) was performed. If old13

mutants contain high concentrations of sugars, then it is expected to see increased sugar metabolites in

old13 leaf samples compared to the WT.

*

*

For primary metabolic profiling, samples were extracted from WT and old13 first rosette EEL, MEL and FEL, as described in methods section 2.12. To observe the difference between metabolites, 10, 15, and 20 DAG old13 samples were compared with WT samples. Among the 60 metabolites analysed, only sugar metabolites (23 in number) are shown in the heat map (Figure 5.10). The heat map shows that most of the sugar metabolites (15/23) are low in old13 10 DAG samples compared to the old13 15 and 20 DAG samples. Interestingly, raffinose and galactinol which were undetectable in old13 10 DAG samples, significantly increased in 15 and 20 DAG old13 leafsamples. Also, the heat map and data bars clearly illustrate that most of the sugar-related metabolites are significantly higher while some sugars like maltose and threitol are considerably lower in 20 DAG old13 leaf samples, compared to old13 10 and 15 DAG samples. The log2 values of significantly different carbohydrates in old13 10, 15 and 20 DAG leaf samples are listed in Appendix 4. These results confirm that sugar levels are high in old13

FEL leaf samples and are generally low in old13 EEL.

Figure 5.10. Primary sugar metabolite profiling of old13 first rosette EEL, MEL and FEL

(A) Heat map representing changes in primary metabolite content quantified by GC-MS. Metabolite levels were determined in old13 first rosette leaf pairs 10, 15 and 20 DAG. The samples are expressed as log2. Scale bar

represents red maximum (between 0.0 and 3.0) and green minimum (between 0.0 and -3.0). (B) The data bars represent log2 transformed fold changes. * indicates the values are significantly different between 10 DAG WT

and old13, 15 DAG WT and old13, 20 DAG WT and old13 samples at P < 0.05 using Student t-test. Values were obtained from five biological replicates.

Carbohydrate Glucose

Sugar 8 (Galactose or Manitol) Fucose Sugar 1 Sugar(Glucoso_6_phosph) Sugar 3 Sugar 2 Fructose Galactinol Sugar 5 Sugar 6

Sugar 9 (Galactose or Manitol) Mannose

Glycerol Raffinose Maltose Threitol

Glucose, 1,6-anhydro, beta- Sugar 7 Inositol, myo- Sucrose Sugar 4 Trehalose, alpha,alpha o ld 1 3 -10 DAG o ld 1 3 -1 5 DAG o ld 1 3 -2 0 DAG * old13 -1 0 DA G old13 -1 5 DA G old13 -2 0 DA G

5.3 Discussion

Research performed on various plant species such as Arabidopsis thaliana, Oriza sativa, Solanum lycopersicum, Zea mays and Nicotiana tabacum provide information about the number of genes involved in regulating senescence processes (Lim et al., 2007). It has been shown that mutant plants with delayed developmental growth, flowering and extended longevity, often exhibit greater stress resistance. Whereas, phenotypes showing early growth, reproductive phase and premature ageing, often show sensitivity to stressed environments. This suggests that when plants are young, they show enhanced resistance to stress, but as they age, they become more susceptible. For instance, transgenic plants overexpressing the JUB1 NAC transcription factor, exhibited extended leaf longevity, strongly delayed reproductive growth, and greater tolerance to stress inducing treatments (Wu et al., 2012). As a result, young JUB1-overexpressers might display increased tolerance to stress factors and a delayed senescence phenotype. Similarly, recent research on Arabidopsis lines overexpressing AtERF019,

showed enhanced drought tolerance compared to wild type (WT) plants (Scarpeci et al., 2017). Overexpression of AtERF019 resulted in delayed vegetative and reproductive growth, along with delayed natural senescence. These studies also support the concept that improved tolerance to stress in transgenic plants is because of extended leaf longevity. The function of very few genes have been identified that display leaf longevity and increased stress resistance, without altering the growth and development of a plant. In Arabidopsis, mutants like drd1, ddm1 and oresara1 are well characterised, and show an association with plant ageing. These mutants show greater stress tolerance and longevity without alterations to developmental growth (Cho et al., 2016; Hye et al., 2004b). Results shown in chapter 3 also confirm that young leaves are more tolerant to stress compared to mature and old leaves. Also, it is confirmed that stress tolerance decreases with age in Arabidopsis because of gradual occurrence of senescence-inducing ARCs. In chapter 4, it was confirmed that old13 shows no early ageing when grown in standard laboratory conditions, but displays early initiation of senescence when exposed to stress. In this chapter, the old13 mutant was selected to better understand the ageing and stress responses because it is highly similar to the WT when grown in standard laboratory conditions, and is hypersensitive to stressed environments. To test whether the hypersensitive response to stress in

old13 is observed in all developmental stages, or whether the susceptibility increases with age, stress- induced experiments, followed by transcriptomic and metabolomic studies were carried out.