Chilling hypersensitivity of the IZS 288

Considering the fact that at optimum growing condition IZS 288 already showed transcriptional misregulation of 74 genes that were chilling responsive in WT roots, the chilling hypersensitivity phenotype of IZS 288 could be a secondary effect of the mutation.

Among the 74 misregulated genes 65 of them showed similar pattern of expression in both IZS 288 roots at optimum growing condition and in the chilling exposed WT roots.

Particularly, two transcription factors: WRKY transcription factor 75/ WRKY7 (AT5G13080) and MYB-like transcription factor /RVE8 (AT3G09600) that showed suppressed expression were part of this group. In Arabidopsis WRKY75 transcription factor is involved in regulating the phosphate starvation response. It has also been shown that in RNAi lines with suppressed expression of WRKY75 lateral root length and number, as well as root hair number, were significantly increased (Devaiah et al., 2007) which is also true in case of IZS 288. The second transcription factor, REV8, is a Myb-like transcription factor that shows high sequence similarity to CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and ELONGATED HYPOCOTYL (LHY), which are essential regulators of the Arabidopsis circadian clock (Rawat et al., 2011). Loss of RVE8 causes a delay and reduction in levels of evening-phased clock gene transcripts and significant lengthening of circadian clock pace (Hsu et al., 2013).

Similarly, among the remaining 9 misregulated genes that showed opposing pattern of expression was found REV2/CIR1 (AT5G37260) another MYB family transcription factor

WT roots after chilling stress however it was down-regulated in IZS 288 roots under optimal growing condition.

Interestingly, four sulfur nutrition related genes ( i.e. sulfate transporter 1.1 (AT4G08620), 5'-adenylylsulfate reductase 3/APR3 (AT4G21990), response to low sulfur 2 (AT5G24660) and glutathione S-transferase U17(AT1G10370)) that showed up-regulation in WT roots after chilling stress were repressed in IZS 288 roots at optimum growing condition. In literature there are reports where inhibition of glutatione (GSH) synthesis and reduced glutatione reductase activity has led to reduced chilling tolerance in maize (Zea mays) (Kocsy et al., 2000). In Chorispora bungeana (an alpine plant) low-temperature induced GSH conferred chilling tolerance by maintaining high enzymatic activity and the fluid state of plasma membrane via increasing unsaturated fatty acids composition of the membrane (Wu et al., 2008). In contrast, increasing glutathione content through application of safeners (herbicide) in a chilling-sensitive maize inbred line increased protection against chilling-induced injury (Kocsy et al., 2001). Hence, in IZS 288 misregulation of the expression of sulfur uptake and assimilation genes could possibly lead to a reduction in GSH pool, which compromises their ability to degrade the high level of H2O2 created during chilling stress making them more susceptible to chilling stress. As a follow up one can verify this possibility by applying exogenous cysteine or γ-glutamylcysteine and increase the amount of GSH and investigate its impact in improving the chilling hypersensitivity of IZS 288.

Chilling stress induced profound transcriptional changes in IZS 288 influencing 11% of the genome while affecting 8% in the WT. In literature there are reports stating that 4%-20% of the Arabidopsis transcriptome are cold responsive (Chinnusamy et al., 2007). The regulation of 70% of the cold responsive genes was similar between IZS 288 and the WT, therefore the key to IZS 288 chilling hypersensitivity is hypothesized to lie in few of the differences observed between the two genotypes. One of such differences is the transcript level of cold response genes (like CBF3/DREB1A, LIT30, LIT78 and KIN1) not showing the same level of transcriptional induction after chilling stress in IZS 288. These genes showed two three fold up-regulation at optimum temperature in IZS 288 but after chilling stress they showed up to 20 fold lower transcript levels than in WT. One possible explanation for this variation could be the low transcript abundance of ICE1 (inducer of CBF expression1) in IZS 288 after chilling stress. ICE1 is a constitutive transcription factor that can bind to MYC recognition

cis-elements (CANNTG) in the promoter of CBF3/DREB1A and induce the expression of CBF3/DREB1A and its regulons during cold acclimation (Lee et al., 2005). In our case after chilling stress ICE1 showed a two fold up-regulation in WT facilitating the higher induction of CBF3 but in case of IZS 288 such effect was not observed. In further support for this notion, MYB15, an upstream transcription factor that negatively regulates the transcription of CBF genes was five fold up-regulated in IZS 288 after chilling stress. Here again, ICE1 appears to negatively regulate the transcription of MYB15 (Agarwal et al., 2006). Therefore, the low level ICE1 transcript abundance in IZS 288 after chilling stress might have led to higher transcript level of MYB15 and consequently to the repression of CBF3 (Fig. 4.2). The second possible explanation would be, since cbf2 null mutant showed increased expression level of CBF1/DREB1B and CBF3/DREB1A and demonstrated freezing tolerance, CBF2/ DREB1C is suggested to be a negative regulator of both CBF1/DREB1B and CBF3/DREB1A (Novillo et al., 2004). Thus the increased expression level of CBF2/ DREB1C in IZS 288 could also have caused the lack of strong induction in CBF3/DREB1A. It is worth mentioning here that two zinc finger stress-response proteins, ZAT12 (At5g59820) and ZAT10/STZ (At1g27730), also showed higher level of induction in IZS 288 after chilling stress. These two stress-response proteins have been implicated to have a central role in reactive oxygen and abiotic stress signaling in Arabidopsis (Davletova et al., 2005; Mittler et al., 2006).

The other variation observed in chilling response of IZS 288 was that early and transient cold response genes were still up- or down-regulated after the 24 hours chilling stress. Based on a comparison made between the chilling responsive gene set of IZS 288 (i.e. 2560 genes up or down regulated as a result of chilling stress) and the cold stress response gene sets of the AtGenExpress global stress expression data set (Kilian et al., 2007) 31 genes that showed differential expression particularly at earlier time points of chilling stress (i.e. at 30 minutes, 1, 3, 6 and 12 hours) were part of the IZS 288 chilling responsive gene set (which was identified after exposing the plants to chilling stress (4°C) for 24 hours); which could be indicative of the lack of proper regulation of the expression of chilling responsive genes.

Among these 31 genes three ethylene responsive element binding factor (ERF13 (AT2G44840), ERF6 (AT4G17490) and ERF2 (AT5G47220)), two MYB transcription factors (MYB4 (AT4G38620) and MYB44 (AT5G67300)) and a potential calcium sensor (TCH2/CLM24 (AT5G37770)) were included (Appendix. list 6). A previous study reported that CLM24 shows 15 fold increase after 4 hours exposure to 4°C, whereas

CML24-underexpressing transgenic lines are resistant to ABA inhibition of germination and seedling growth, show late flowering and have enhanced tolerance to MgCl2, ZnSO4, CoCl2. Consequently, it has been postulated CML24 may have a role in redox signaling, whereby altering the sensitivity to and/or production of reactive oxygen species (Delk et al., 2005).

Therefore, the constitutive up-regulation of C ML24 together with other factors may have negative impact on the chilling tolerance of IZS 288.

Figure 4.2. Schematic representation of the cold response pathway. Low temperature activates ICE1 and ICE1 like proteins that consequently induce the transcription of CBFs and at the same time block the transcription of MYB15. MYB15 when expressed negatively regulates the transcription of CBFs. This picture is adapted from Gong et al., 2009.