Differential expression of transcripts mapping to key genes with

with known involvement in abiotic stress responses

Expression levels of selected GD33DH transcripts, whose Arabidopsis orthologs have been shown to have a key role in the response to abiotic stress conditions, particularly dehydration stress are shown in Figure 4.13. This group includes some important genes which have been proposed to be involved in the abiotic stress response through the ABA, auxin or ethylene signalling pathways in Arabidopsis and other plant species. Transcript expression was compared to expression of Arabidopsis orthologs from the AtGenExpress dataset in salt stress and osmotic stress conditions using the Arabidopsis eFP Browser through bar.utoronoto.ca (Winter et al., 2007).

GD33DH orthologs of several ABA inducible genes were found to be differentially expressed in this study, reaffirming the important role of this hormone in the response to

salt shock. Of these MYC2 (encoded by Bo5g086990.1; Fig. 4.13a) is an important bHLH

TF involved in the cross talk between multiple stress response pathways. The MYC2 protein functions by binding to MYC recognition sites in the promoters of genes under its control. It has a well established role in the regulation of ABA inducible genes such as

RD22 (encoded by Bo9g011300.1; Fig. 4.13b) (Abe et al., 2003), of which the protein is a

positive regulator of JA signalling (Pauwels et al., 2010). RD22 expression has been used

as a marker of ABA induced expression in drought conditions (Yamaguchi-Shinozaki and

Shinozaki, 1993). In theB. oleracea GD33DH time series experiment, both MYC2 and

RD22 gene expression levels were similar in both the treated sample and the control until

16 hpt, after which expression levels of both genes increased in the salt treated plants (Fig. 4.13a and b). The transcripts had a BF score of BF=29.2 and BF=15.8, respectively.

ATAF2 (encoded by Bo2g009250.1; Fig. 4.13c) is a member of the NAC transcription factor family that has been widely implicated in the biotic and abiotic stress responses (Ooka et al., 2003). The ATAF2 protein has been found to repress the expression of PR genes in biotic stress responses and is induced by dehydration independently of ABA

(a) Bo5g086990.1 (MYC2) (b) Bo9g011300.1 (RD22)

(c) Bo2g009250.1 (ATAF2) (d) Bo9g014980.1 (MYB96)

(e) Bo1g007700.1 (ABF3) (f ) Bo3g142840.1 (STZ)

(g) Bo3g032500.1 (WRKY33) (h) Bo9g098940.1 (ERD1)

(i) Bo5g030290.1 (ERD10)

Figure 4.13: Expression profiles of differentially expressedB. oleracea GD33DH transcripts

whose orthologs have previously reported functions in the abiotic stress response in other plant species

Plots of a selection ofGD33DH differentially expressed salt shock genes, with the closest Ara- bidopsis ortholog. (a) Bo5g086990.1 (MYC2); (b) Bo9g011300.1 (RD22); (c) Bo2g009250.1

(ATAF2); (d) Bo9g014980.1 (MYB96); (e) Bo1g007700.1 (ABF3); (f) Bo3g142840.1 (STZ);

(g) Bo3g032500.1 (WRKY33); (h) Bo9g098940.1 (ERD1) and (i) Bo5g030290.1 (ERD10). Log2 expression on the y-axis and time on the x-axis. Red corresponds to the salt-treated

score of BF=14.0, which is at the bottom threshold level for differential expression. The up-regulation of this repressor protein could be an example of the plant lowering its basal

immunity to redirect energies to rebalance following an increase in Na+ ions in the leaves.

MYB96 (encoded by Bo9g014980.1; Fig. 4.13d) has a role in stomatal movement, as well as being an important regulator in the cross talk between the ABA and auxin response pathway during lateral root development under water stress conditions (Seo et al., 2009). When the Arabidopsis ortholog was examined using the Arabidopsis eFP browser,

it was shown that MYB96 was differentially expressed mildly in leaf and strongly in the

root at 3 hpt and levels were maintained up to 24 hpt, in both salt and osmotic stress treatments. In this experiment, accumulation of the MYB96 transcript occurred at 16 hpt in GD33DH following salt shock conditions. The protein likely plays an important role in stomatal movement and root development in response to salt shock in GD33DH.

Genes such asABF3 (encoded by Bo1g007700.1; Fig. 4.13e), along withAREB1 and

AREB2 (plots not shown) encode proteins that have been found to be master regulators

in ABRE-dependent ABA signalling during water stress conditions. They are bZIP transcription factors which work either as homodimers or heterodimers and require ABA for full activation of downstream gene expression (Yoshida et al., 2010). In this experiment,

ABF3 was instantly up-regulated and expression remained high for the duration of the

experiment. It is likely the proteins that these transcripts encode play important roles in ABA-dependent gene expression, suggesting cross talk between stress response pathways in response to salt shock. Expression of the Arabidopsis ortholog in the eFP browser shows that the gene was differentially expressed in the leaf, and mildly in the root for both salt and osmotic stress between 0.5 and 6 hpt suggesting an early role for this gene in Arabidopsis.

SALT TOLERANCE ZINC FINGER (STZ, encoded by Bo3g142840.1; Fig. 4.13f) is

an abiotic marker gene whose protein has been implicated in salt and cold stress tolerance and is rapidly up-regulated under these conditions (Sakamoto et al., 2004; Seki et al., 2002; Teige et al., 2004). It is thought to have a role in repressing photosynthesis and carbohydrate metabolism and transgenic over-expressers show reduced growth (Maruyama et al., 2004). When the expression patterns of the Arabidopsis ortholog were examined in the Arabidopsis eFP browser, it was shown that the gene was differentially expressed in the roots in response to salt shock between 3 and 6 hpt. In the GD33DH experiment, the transcript steadily accumulated and became differentially expressed at around 16 hpt, suggesting that it plays a role in the repression of photosynthesis in response to salt shock. WRKY33 (encoded by Bo3g142840.1;Fig. 4.13g) has been reported to play key roles in multiple stress responses including salt stress (along with WRKY25) (Jiang and

glutathione-S-transferases (Jiang and Deyholos, 2008) making it a key regulator in osmotic stress conditions. Viewing the expression patterns in the Arabidopsis eFP browser shows that the gene is differentially expressed in the roots at 3 hpt in response to salt treatment

but not in the leaves. In this experiment, WRKY33 became differentially expressed at

around 18 hpt, where it accumulated above the control, suggesting additional roles in GD33DH, causing differential expression in the leaves and not in the leaves of Arabidopsis under similar experimental conditions.

Bo9g098940.1 and Bo5g030290.1 encode the EARLY RESPONSE TO DESICCATION

proteins, ERD1 and ERD10, respectively; Fig. 4.13h, i). The ERD genes were rapidly

activated upon drought stress in Arabidopsis (Kiyosue et al., 1994; Taji et al., 1999) and were highly up-regulated in the time series experiment, suggesting an important role in the response to salt shock in GD33DH. ERD1 encodes a chloroplast ATP-dependent protease (Soitamo et al., 2008), and is seen here to be up-regulated at 18 hpt after treatment. The Arabidopsis ortholog of this gene was differentially expressed in the leaves of osmotic stress

conditions from 12 hpt. ERD10 is a member of the late embryogenesis abundant protein

(LEA) family that is up-regulated immediately, within 2 hpt. The expression pattern of this gene in Arabidopsis using the eFP browser shows that this gene is rapidly differentially expressed under both salt and osmotic stress conditions in both the leaf and root, though the effect is stronger in the leaf. The function of this gene family remains unclear, but roles have been proposed in the sequestration of ions (Bray, 1993) and a chaperone role protecting and refolding of proteins following water stress (Kovacs et al., 2008).