Herbivore feeding influences the expression of transcription factors

Transcription factors act as switches of regulatory cascades (Scott 2000) and a combination of cis-acting elements are needed for the appropriate responses to stimuli (El-Shehawi et al.

2011). In Arabidopsis, up-regulation of transcriptions factors was demonstrated to cause a general shift in transcriptional regulation in response to insect damage (Reymond et al.

2004). In maize, we found a total number of 90 differentially regulated transcription factors in the leaves of herbivore-induced maize within 0.5 h and 4 h after damage. Out of that number, 12 genes had multiple hits. The majority of herbivore-regulated transcription factors were found at 0.5 h and 1 h while the number of differentially regulated metabolic enzymes started to increase after 1 h.

After an 18 h treatment with S. littoralis, only 7 transcription factors were identified in the leaves. The discrepancies in these observations are due to the sustained time of damage by the herbivore. Some of the early induced transcription factors were already down-regulated after 18 h. A distinct picture was observed in roots of maize. Many transcription factors

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displayed a differential regulation pattern independently of the induction type (D. virgifera herbivory, mechanical damage, and systemic induction). A difference in the total number of transcription factors could be observed between D. virgifera feeding and mechanical damage in roots, indicating that some transcription factors function in an herbivore-specific pathway.

Transcription factors induced by herbivory and mechanical damage might mediate a general wound-response of the roots. Roots of maize react strongly in response to above-ground herbivory. Still, the function and relevance of this type of response remains to be resolved.

The results from these data implicate that there are similarities as well as differences in the signal transduction pathways between a systemic and a local damage reaction to herbivore attack (Erb et al. 2009).

The transcription factors identified in this study belong to several classes that are linked to plant stress responses. These include AP2/EREBP transcription factors, WRKY transcription factors, MYB transcription factors, and bZIP factors (Stracke et al. 2001; Singh et al. 2002).

AP2/EREBPs, for example, were found in insect-induced defense in poplar (Ralph et al.

2006). In Catharanthus roseus, members of the AP2/EREBP family responded to jasmonic acid (Menke et al. 1999). A homolog of the maize transcription factor, tf1, was first identified in Arabidopsis (ATAF1/2). It shares a 74 % homology (amino acid level) to the ARAF1/2 in Arabidopsis and 94 % homology to rice. Both proteins were found to be expressed after herbivore attack (Delessert et al. 2005; Yuan et al. 2008). A similar expression pattern for tf1 and its homologs was found in herbivore-challenged species like Arabidopsis (Reymond et al. 2004) and poplar (Ralph et al. 2006). Binding to a drought-responsive element, the DRE transcription factors were first related to the response to cold stress and salt stress (Zhao et al.

2006). Newer studies placed the DRE transcription factors into the group of AP2/EREB proteins, connecting them to the ethylene-dependent signaling pathway (Sun et al. 2008).

One DRE transcription factor found in this study is tf40 which showed a strong early induction after continuous herbivore feeding. A 53 % homolog from Aloe vera was found to be induced 12 h after cold stress (Wang and He 2007). It is therefore unlikely that the Aloe DREB1 and maize tf40 have similar functions.

Also, maize CAF1-like protein tf2, is involved in defense responses in Arabidopsis and rice (Walley et al. 2007; Yuan et al. 2008). The AtCAF1a/b factor responds to wounding and biotic stresses within 5 min after induction stimuli and is under a circadian control (Walley et

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al. 2007; Walley et al. 2010). The homology to tf2 is 51 % based on amino acid level. The family of CCR4 associated factors (CAF) are factors involved in the post transcriptional regulation of genes. In yeast, they are part of the major cytoplasmic deadenylase, initiating mRNA turnover by removing the poly(A)-tail (Tucker et al. 2001). Recent studies indicate that CAF1 proteins might degrade repressors of the pathogen-related genes PR1 and PR2, assuming that this factor plays a role in the plant defense response (Liang et al. 2009). In this work, tf2 showed an early induction in response to herbivore feeding and mechanical damage, suggesting a general role in maize defense responses.

MYB-transcription factors are involved in several developmental steps of the plant. It has been demonstrated that the transcription factor ODORANT1, a R2R3 MYB-transcription factor, controls the synthesis of volatile benzenoids present in the floral scent of petunia (Verdonk et al. 2005). We also identified two MYB-(like) factors in maize, tf12 and tf28.

While tf12 was suppressed after herbivore feeding, tf28 showed an induced expression. A 48

% amino acid identity was found between tf12 and the R2R3 MYB-transcription factors that play a role in abiotic stress responses in Triticum aestivum (Zhang et al. 2011). But the low amino acid identity to bread wheat raises the question about the specific function of tf12 in maize. Only 17 % sequence similarity of the maize tf28 and TaMYB13 of Triticum aestivum was predicted by NCBI. TaMYB13 functions as an activator of fructosyltransferase genes (Xue et al. 2011), but due to the low similarity a functional consensus can not be assumed.

An I-box binding factor has first been described in tomato and was associated to the class of MYB-like proteins (Rose et al. 1999). The transcription factor tf35 from maize was annotated as putative I-box binding factor and can therefore be grouped to the class of MYB transcription factors. Tf35 was highly induced by herbivory and its homolog in bread wheat is implicated in abiotic stress responses (Zhang et al. 2011). Due to the low amino acid sequence similarity of 24 %, a prediction about the specific role of tf35 in maize cannot be made.

One zinc-finger transcription factor of maize, tf29, showed a negative regulation of gene expression after herbivore attack. This factor shares a 95% amino acid sequence identity to OsLDS1 from Oryza sativa. Here, it has been described as a negative regulator of programmed cell death in plants (Wang et al. 2005). Programmed cell death is often associated with a hypersensitive response of plants to pathogen attack (Greenberg et al.

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1994). The down-regulation of tf29 after herbivory could be a precautious step to increase resistance towards a subsequent pathogen attack.

The class of bHLH proteins is represented by tf8 in maize. This transcription factor is strongly induced after herbivore attack, but no homolog of tf8 has been described in other plant species yet.

An example of WRKY transcription factors induced by herbivory is the maize transcription factor tf34. WRKY transcription factors are unique to the plant kingdom (Eulgem et al. 1999) and have been identified to bind to W-boxes (Chen et al. 2002) which are found in the promoters of many plant defense genes (Kalde et al. 2003). A homolog of maize tf34, OsWRKY71 (90 % amino acid identity), encodes a transcriptional repressor of the gibberellin signaling pathway in rice (Zhang et al. 2004).

Further factors include an ETTIN-like auxin response factor (maize tf20) and putative phi-1-proteins (maize tf42) which respond both with an induced expression after herbivore attack.

Characterization of tf20 or putative homologs has not been conducted so far. As a homolog to the transcription factor tf42, phi-1 has only be implemented in the process of phosphorylation (Sano et al. 1999).

Although the maize transcription factors found in this study are regulated by herbivory on the level of expression, they might not necessarily be directly involved in terpene production or plant defenses. Generally, specific functions or putative targets cannot be predicted based on the low sequence similarities to other species. To unravel the functionality of these factors, further studies have to be conducted.