myb-like protein with DNA-binding domain (GRMZM5G86998) is downregulated after

The myb gene family is a family of transcriptional activators, the name being derived from the disease myeloblastosis. Members of this family share an N-terminal DNA-binding domain, a central transactivation domain, and a C-terminal regulation domain (Oh, et al., 1999). Myb proteins were discovered in animals, but plants have their own subfamily of myb factors (Stracke, et al., 2001). They are involved in the regulation of secondary metabolism processes, such as flavonoid production in maize with myb proteins both as activators and repressors (Goettel, et al.), and regulation of anthocyanin production in citrus fruits (Butelli, et al., 2012). An involvement of myb factors in terpene regulation has not been demonstrated before.

The myb-like gene GRMZM5G86998 is downregulated after herbivory. This suggests a role as a suppressor, whose absence allows for the upregulation of terpene production steps. This gene has sequence differences between B73, CML333 and Oh7b. An insertion of six base pairs in an exon in the CML333 sequence keeps the reading frame intact, and adds two amino acids. This can lead to an altered secondary structure, possibly changing the binding site and thus the activity of the protein.

There is a deletion of 30 base pairs in the CML333 sequence of the myb factor gene. A deletion of 30 base pairs results in ten missing amino acids in the CML333 protein. This difference is large enough to alter the 3D structure more significantly than the small insert mentioned before. This can change the shape and interaction sites of an active site to the extent that it cannot bind its target anymore, or binds a different target. A loss of binding activity in a repressing factor would result in a constant increased level of its target. This kind of change in the regulatory chain would be unfavorable to the plant, as a constant upregulation of a metabolic process that isn’t constantly needed is a waste of energy and resources. It can be used to identify the target of the myb factor – a panel of the expression level of all genes related to terpene synthesis would show differences between plants with and without the 30 bp insertion in the myb gene. The target of the myb factor, and all downstream factors that are affected by this step, would show an altered expression level.

90 5.2.7.1 Pentatricopeptide (Zm.25643 (GRMZM2G077420)) is strongly induced after herbivory, but does not have any sequence difference between maize lines

Pentatricopeptides are the biggest class of RNA-binding proteins in plants. They share a motif of 35 amino acids (the pentatricopeptide repeat, PPR) which is repeated up to 30 times. These repeats form a binding platform for targets – the precise binding mechanism and target is as yet unknown. Pentatricopeptides are involved in translation, recruitment of other catalytically active effector proteins, and binding and modifying target RNAs (Beick, 2010). These functions mean the pentatricopeptide could play a role in a signal transduction cascade on the RNA level or protein level. It could directly influence the expression of the next gene in the regulatory cascade by altering the RNA processing or translation. Alternatively, it can recruit another enzyme to start a reaction in the synthesis of terpenes and their precursor molecules.

The expression level of the gene Zm.25643 (GRMZM2G077420) is strongly induced upon herbivory. This hints at a role in the regulatory chain of insect-induced terpene production.

Upregulation of a pentatricopeptide can lead to increased translation or local recruitment of other proteins in a signal chain.

The HapMap sequences for this gene were identical between B73, CML333 and Oh7b. Both the translated region and a 1 kbp promotor region were compared. The sequences matching in these lines probably rules out that this gene is relevant for the QTL. In order to come up as a QTL, a genetic difference which is related to a different phenotype would be needed.

This does not mean that the gene is not part of the signal cascade. Rather, the increased expression after herbivory strongly suggests that it does play a role in the terpene production.

But the identical sequences mean it is not part of the differential regulation of terpene production between the maize lines.

5.2.7.2 UFMU lines for the pentatricopeptide gene have a different terpene emission than background lines without transposons

There are two UFMU maize lines containing a Mu transposon in the pentatricopeptide gene.

These transposons interrupt the genetic sequence, thereby rendering the resulting protein less active or inactive. Both lines carry the transposon at the beginning of the translated region of

91 the pentatricopeptide gene. A transposon insertion in the beginning of the translated region of a gene is expected to result in a change of functionality of the gene product.

The measurement of volatiles indicates a lowered amount of bergamotene and farnesene in both UFMU lines, compared with the background line without transposon insertion.

E-β-caryophyllene emission is increased in one line, and slightly lowered in the other. For all terpenes, the UFMU lines exhibit a strong biological variability. An UFMU line may contain more than one transposon, so additional transposons having an unforeseen influence on terpene emission cannot be ruled out. Moreover, additional transposons can lead to difference in plant growth and primary metabolism, which can indirectly influence the plants capability to produce secondary metabolites. To exclude this, the whole maize line needs to be sequenced, and the position and function of other transposon inserts needs to be identified.

The fact that inserts in this gene lead to a difference in terpene emission supports the theory that the gene GRMZM2G077420 is part of the signal chain. For bergamotene and farnesene - both products of TPS10 - its function has a positive influence on the level of production.

Inactivity of the pentatricopeptide leads to a lowered emission of these two volatiles. This suggests that it plays a role in the regulation of TPS10, or the production of its substrates.

The volatile mix induced after herbivory also contains E-β-caryophyllene. This sesquiterpene is the product of TPS23. The two UFMU lines produce and emit different amounts of E-β-caryophyllene. It is improbable that the few base pair difference in the position of the transposon between the two UFMU lines creates a strong difference in the functionality of the gene product. This probably means that at least one of the UFMU lines contains another transposon influencing the production of E-β-caryophyllene or one of its precursors. This makes a clear statement about the involvement of the pentatricopeptide in E-β-caryophyllene regulation impossible.

92 5.2.8 Kinase LOC100383522 (GRMZM2G032694) is downregulated after herbivory,