Costos de Recuperación de residuos

TFs are proteins that bind to DNA upstream of the coding region of a gene and regulate its expression by either recruiting or blocking the assembly of basal transcriptional machinery and of RNA polymerase II, which catalyses translation of DNA into mRNA. TFs bind to DNA binding domains, which are usually found upstream of genes. Multiple genes with related function can have the same DNA binding motifs and hence a single TF can regulate a multitude of genes. The TFs ability to regulate gene expression is dynamic, allowing rapid changes in the expression levels of the target genes depending on external stimuli. In order to assess the complex network of signalling pathways in GD33DH in response to salt shock, further analysis of TF families in particular was carried out.

A total of 737 differentially expressed transcripts encoding putative TFs were identified. Of particular abundance and interest were the bHLH, MYB and MYB-related, AP2-EREBP, bZIP, WRKY and NAC TF families which have been widely implicated in the response to abiotic stress conditions in genome-wide analyses of abiotic stress condition in various plant species (Hu et al., 2015; Liu et al., 2015a; Peng et al., 2014), and have been extensively reviewed (Baldoni et al., 2015; Chen et al., 2012; Llorca et al., 2014; Mizoi et al., 2012;

20 24 25 12 13 14 11 18 4 8 9 12 16 6 85

(a) Up-regulated TF families

23! 16! 39! 26! 22! 31! 12! 29! 25! 10! 18! 8! 12! 7! 7! 170!

bZIP! NAC! bHLH! AP2-EREBP!

MYB-related! MYB! WRKY! HB!

Orphans! C2C2-GATA! C2H2! MADS!

G2-like! TCP! ARF! Other!

(b) Down-regulated TF families

Figure 4.14: Differentially expressed TF families

(a) Up-regulated and (b) down-regulated differentially expressed TF families. The numbers in each segment represent the number of transcripts in the category, as per the key.

Nakashima et al., 2014; Puranik et al., 2012).

Transcription factors were identified by a querying the list of differentially expressed transcripts against the Plant Transcription Factor DataBase (Plant TFDB3.0) (Jin et al., 2013) and against the Arabidopsis TAIR10 assembly to obtain the closest ortholog, and was broken into up- and down-regulated, as summarised in Figure 4.14. A selection of transcripts with a named Arabidopsis ortholog will be discussed in the literature analysis of the results below, as shown in Appendix E.

AP2/EREBP transcription factor family

APETALA 2/ ethylene response element binding protein (AP2/EREBP) transcription factors are involved in stress acclimation by modulating cross talk between hormone signalling pathways (Dietz et al., 2010; Yang et al., 2005). They signal through the ethylene signalling pathway, which is often referred to as the ABA-independent stress

responsive pathway. InB. rapa spsp. pekinensis 291 putative AP2/EREBP TF proteins

were identified which could be further resolved into 15 groups - ARP2, ERF, RAV and Soloist (Song et al., 2013). Of the differentially expressed transcript list, 36 transcripts mapped to the AP2/EREBP TF family. The most well known genes in this TF family are

theDREB1 and DREB2 TFs, belonging to the ERF group. Despite these genes being

key in the salt response signalling through the ABA-independent pathway in Arabidopsis

(Lata and Prasad, 2011; Oh et al., 2005), only DREB2B was differentially expressed in

GD33DH during the first 36h of salt shock. Other AP2/EREBP genes were differentially

expressed including theETHYLENE RESPONSE FACTOR 4 and 5 (ERF4 and ERF5)

genes. ERF4 is a repressor of expression and is capable of modulating both ethylene and ABA signalling (Yang et al., 2005) and ERF5 has been highly implicated in JA/Ethlene

signalling in defence against pathogens such as B. cinera (Moffat et al., 2012) and in

response to chitin (Son et al., 2011). bHLH transcription factor family

The most abundant TF family in both up- and down-regulated differentially expressed transcripts was the bHLH TF family. In Arabidopsis, this group consists of a total of 162 bHLH genes which can be further split into 21 subfamilies (Toledo-Ortiz et al., 2003). In addition 167 bHLH genes have been identified in rice (Li et al., 2006). The functions of the bHLH TF cover a broad range of growth, developmental and maintenance processes that occur at all stages of the plant life cycle. Here, 62 transcripts mapping to bHLH TFs were identified as differentially expressed in the experiment. Down-regulated transcripts have roles in phytochrome signalling (PIF3 and PIF4), dark-induced senescence (PIF4 and PIF5) (Sakuraba et al., 2014a), and the response to far red light (controlled by

PIF3, PIF4 and PIF7) (Leivar et al., 2008). Photoreceptors such as phytochromes have been shown to modulate responses to both biotic and abiotic stress (Carvalho et al.,

2011; Indorf et al., 2007). Up-regulated transcripts mapping to ABA-INDUCIBLE bHLH-

TYPE (AIB) and MYC2 encode proteins that involved in regulating ABA-induced gene

expression in Arabidopsis (Abe et al., 2003; Li et al., 2007), again indicating the influence

of ABA signalling following salt shock in GD33DH. Transcripts mapping to theLOTUS

JAPONICUS ROOTHAIRLESS1-LIKE (LRL1 and LRL2) TFs have been shown to

positively regulate development of the tips of root hair cells and are controlled by auxin signalling (Tam et al., 2015). These genes were both up-regulated following salt shock treatment in GD33DH suggesting that the presence of increased salt in the soil affects the root architecture.

bZIP transcription factor family

The bZIP TF family is one of the largest TF families in plants and takes part in multiple processes, particularly abiotic stress responses, mediated through the ABA signalling

pathway. The cis-acting ABRE element (ABA Responsive Element) and ABF TFs that

bind the ABRE element (ABRE-binding protein/ABRE-binding factors) are bZIP which are key in ABA-dependent gene expression (Yoshida et al., 2014b). In this study there were 42 differentially expressed transcripts mapping to bZIP TFs, of which the ABA

responsive genesABF3, ABF4, AREB3 and ABI5 were up-regulated. ABF3 (Fig. 4.13e)

and ABF4 proteins are part of trio of master regulators of ABA-induced gene expression in response to abiotic stress conditions (Yoshida et al., 2010). ABI5 is active during seed maturation and germination, regulating late embryogenesis-abundant genes during both developmental stages (Bensmihen et al., 2002). The observation that a selection of master regulators of the ABA dependent gene expression pathway were up-regulated under salt shock again highlights the importance of this signalling hormone in stress tolerance. MYB and MYB-related transcription factor family

The functionally diverse MYB and MYB-related TF families constitute a large proportion of the differentially expressed TFs with 77 transcripts mapped to this family found to be differentially expressed. These TF families have been well characterised to have a role in developing tolerance to abiotic stresses (Li et al., 2015a), particularly drought stress (Baldoni et al., 2015). As described in Section 4.4.8, MYB96 (Fig. 4.13d) is ABA responsive and is involved in lateral root growth and in decreasing stomatal aperture in

to drought stress inB. napus (Liu et al., 2015a). MYB108 has a key role in the response to

infection by B. cinera as well as roles in abiotic stress response and is induced in response

to ABA, JA and ethylene (Mengiste et al., 2003). Here, the transcript mapping to this gene is down-regulated following salt shock, suggesting that MYB108 could be acting in its role of negative regulator of ABA induced death, as has been seen in response to wounding and in defence against necrotrophic pathogens (Cui et al., 2013). MYB60 is a regulator of stomatal movement and root growth and is down-regulated under drought stress (Oh et al., 2011). It is also down-regulated in response to salt shock in GD33DH, possibly resulting in an effect on root architecture and stomatal closure to protect the plant from further desiccation.

NAC transcription factor family

Plant specific NAC (NAM, ATAF1/2, and CUC2) TFs contain a highly conserved NAC DNA binding domain with variable C-terminal domains and are plant specific. They play key roles in plant development, senescence and abiotic and biotic stress responses (Breeze et al., 2011; Hickman et al., 2013; Windram et al., 2012; Xu et al., 2013b). In this experiment, 39 transcripts mapping to NAC TFs were identified as differentially expressed, indicating the importance of the TF family in the response to salt shock in

GD33DH. Up-regulated in this experiment was the NAC102 gene, which has a role in

senescence (Breeze et al., 2011) and is activated by EIN2 (ETHYLENE INSENSITIVE 2) (Kim et al., 2014) and ATAF2, whose role was described in Section 4.4.8 (Fig. 4.13c).

A transcript mapping to NAC096 was down-regulated in response to salt shock. This

is a surprising result given that a major proportion of abscisic acid (ABA) responsive genes are under the transcriptional regulation of NAC096, in response to dehydration and osmotic stresses in Arabidopsis (Xu et al., 2013b). The NAC096 protein has been shown to work together with ABF2 (a bZIP TF, which up-regulated in this study), activates

important dehydration response genes such as RD29 (Xu et al., 2013b). This could either

be as a result of cross talk between the stress response pathways or this gene is present

in triplicate in B. oleracea and the orthologs were not annotated, or not included on the

array due to high levels of sequence similarity. WRKY transcription factor family

Members of the WRKY protein family contain a highly conserved amino acid sequence motif WRKYGQK, responsible for the WRKY name. WRKY proteins act as transcriptional activators of key ABA-responsive genes e.g. ABI4, ABI5, ABF4, MYB2, DREB1A, DREB2A and RD29A. Members of the WRKY family also play an important role in defence and cross talk between signalling pathways (Deng-Hui et al., 2008; Li et al., 2010;

Wu et al., 2011). In this experiment, 19 transcripts mapping to WRKY genes were found to be differentially expressed in GD33DH in response to salt shock. Of these, the most well characterised is WRKY33, discussed in Section 4.4.8 (Fig. 4.13g). Other salt induced

WRKYs include WRKY15, which is induced by oxidative and salt stress in Arabidopsis

and the protein negatively regulates salt and osmotic stress tolerance (Vanderauwera et al.,

2012). Also WRKY28, which is rapidly induced by ROS and the protein protects against

fungal pathogens such as B. cinera that are known ROS producers (Wu et al., 2011).