Secondary metabolites and metabolic enzymes

The avoidance of non-hosts by many insects is based on volatile metabolites, which can be detected at a distance or by initial attempts at colonization or feeding, which are terminated due to, lack of appropriate physiology, nutritional aspects or by the detection of potentially toxic secondary metabolites (Agelopoulos et al. 1999).

Secondary metabolites are those compounds, which tend not to be so essential for the basic growth of plants, and for which function is not always known (Bernays and Chapman 1994).

Cyanogenic glycosides

Various nitrogenous protective compounds other than alkaloids are found in plants (Taiz and Zeiger 2002). Approximately 2500 plant species produce hydrogen cyanide (HCN).

HCN is a general respiratory poison, which is stored in the plant in a nontoxic form, often combined with a sugar to form a cyanogenic glycoside. When the tissue is damaged the vacuolar glycoside is contacted by the cytoplasmic hydrolyzing enzymes and the damaged plant releases HCN (Bernays and Chapman 1994)

Cyanogenic glycosides cause serious problems to numerous herbivores since they are widely distributed and generate toxic HCN as the plant tissues are masticated or ingested.

Cyanogenesis is especially dangerous for generalist phytophagous insects because the cyanide, in a process similar to carbon monoxide, nitrogen oxide or azides replaces oxygen binding to heme units of the terminal cytochrome oxidase and blocks electron transport through the respiratory chain. Thus the generalists usually do not feed on plants rich in the cyanogenic glycosides and in this role cyanogenesis might play an important role in plant chemical defence. Monophagous insects that are specialized to feed on cyanogenic plants have developed specific enzymes: cyanoalanine synthase, sulphur transferase (rhodanase) and/or linamarase that allows them to detoxify the highly toxic cyanide.

A very high content of cyanogenic glycosides is found in the youngest leaves of bird cherry when the first fundatrices of R. padi appear. While the aphid population starts to build up, the cyanogenic glycoside level decreases rapidly until almost all the aphids have left the primary host (Leszczyński et al. 2003).

Polyphenoloxidase

Polyphenoloxidase (PPO) plays an important role in plant resistance to insects, and in the detoxification of phenolic compounds taken in the nutrient components.

Polyphenoloxidase is a widespread enzyme found in plant cells, located in the chloroplast thylacoid membranes. Oxidation of phenolic compounds in plant cells is responsible for initiating the browning reaction of the tissues and is characteristic of the pathogen factor or of pest feeding. Within the tissues liable to damage by feeding insects there is an increased concentration of phenolic compounds and moreover, PPO induces metabolization of these phenolic compounds into more toxic forms. Some aphid resistant wheat varieties, for example spring wheat Eta, have markedly decreased PPO activity which is caused by aphid feeding. That is maybe because too high degree of phenol oxidation may induce a rapid loss of toxicity by further transformation into harmless polymers or be induced by insertion of oxidases in the aphid saliva into tissues of the host plant (Chrzanowski et al.

2003). Some varieties have a different reaction to aphid feeding; for example in Tugela DN, which expresses the Dn1 gene for resistance to the RWA the PPO activity was more than double than in the susceptible variety (Mohase and Van der Westhuizen 2002).

Hydroxamic acid (HAs)

Hydroxamic acids are the main group of secondary metabolites involved in the resistance of certain cereals against bacteria, fungi and several insects including aphids (Thackray et al. 1991, Nicol et al. 1992, Rustamani et al. 1996). They were first discovered in 1955 in rye in relation to fungal diseases (Virtanen and Hietala 1955) and later found in maize where they were associated with resistance to the European corn borer, Ostrinia nubilalis (Niemeyer et al. 1992) and corn leaf aphid Rhopalosiphum maidis (Beck et al. 1983). HAs can be found in the cultivated monocotyledons, in maize, wheat (Nicol et al. 1992) and rye but are not present in barley, however DIBOA can be found in the wild Hordeum species (Barria et al. 1992). HAs are not present in the seed (Cambier et al. 2000).

Hydroxamic acids are concentrated in the mesophyll protoplasts, the vascular bundles (Givovich and Niemeyer 1995) and in the sieve elements. The compounds are present in the plant as glucosides, which are enzymatically hydrolysed by endo-β-glucosides to DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) when the tissue is injured (Hofman and Hofmanová 1969, Givovich et al. 1994) (Figure 5). DIBOA (2,4-Dihydroxy-1,4-benzoxazin-3-one) is the main hydroxamic acid in rye, whereas DIMBOA is the predominant form in wheat and maize (Frey et al. 1997, Figure 6.). The HA level is highest in the early seedling stage and decreases rapidly as the seedling matures (Klun and Robinson 1969). The accumulation of these compounds may be influenced by the growing environment. High light intensity (Manuwoto and Scriber 1985), long photoperiod, elevated growth temperature (Epstein et al. 1986) and soil moisture can reduce the HA level in the seedling (Richardson and Bacon 1993).

β-glucosidase

Figure 5. Aphid feeding triggers the conversion from glucoside to the main aglucone in the cell. Blue arrows show the punctured cells, 1-3 injected watery saliva during probing and feeding, 4 taken up phloem sap

which contains DIMBOA-glucoside through the stylet during feeding (Tjallingii 2006).

Biosynthesis of hydroxamic acids has several steps. Fray et al. (1997) showed that five genes are required for the biosynthesis of 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) from indole-3-glycerol phosphate. Leighton, Niemeyer and Jonsson’s (1994) work showed that the glucosylation of hydroxamic acids would be the last step of the pathway (Cambier et al. 2000) (Figure 6.).

DIMBOA-glucoside DIMBOA

DIMBOA-glucoside

Figure 6. Hypothetical pathway of hydroxamic acids in maize (Frey et al. 1997 modified by Gordon-Weeks et al. 2010.)

Studies by Niemeyer and co-workers suggest that genes on chromosome 4A and 4B are involved in the transformation of DIBOA into DIMBOA. Comparison of the HA levels in tetraploid and hexaploid wheat shows that the incorporation of the D genome into the latter leads to a considerable decrease in HA accumulation (Niemeyer and Jerez 1997).

bx1 mutant should be defective in the production of free indole and DIMBOA (Frey et al. 1997)

bx3 mutant (homozygous for the recessive mutant allele) no DIMBOA was detected in the maize seedlings (Frey et al. 1997)

DIMBOA-glucoside can be converted into HDMBOA-glucoside, which could be important in the defence against insect and disease (Cambier et al. 1999). HDMBOA-glucoside accumulation was noted after stem rust infection, jasmonic acid treatment (Oikawa et al. 2002) and armyworm feeding (Oikawa et al. 2004). Artificial diet studies showed HDMBOA-glucoside is toxic. A diet containing 2mM HDMBOA-glucoside significantly reduced the number of Metopolophium dirhodum after 2 days (Cambier et al.

2001). The aglucone of HDMBOA-glucoside is HDMBOA which is produced by hydrolysis (Oikawa et al. 2001) (Figure 7.), this molecule however proves to be very unstable and degrades rapidly into MBOA which is also the degradation product of DIMBOA (Cambier et al. 2000).

Figure 7. Hypothetical pathway leading to the release of MBOA (Oikawa et al. 2004)

DIMBOA is the main HA aglucone in wheat. Extracts of DIMBOA produce antibiosis, feeding deterrence, decreased performance and reduced reproduction in aphids. It has mutagenic effects and affects the level of genetic polymorphism in aphid populations (Figueroa et al. 2004). Hydroxamic acids have also been associated with detoxification of triazine herbicides (Hamilton 1964, Frey et al. 1997). The maximum recorded HA level in cultivated wheat is between 1.4 – 10.9 mmol/kg fresh weight (Copaja et al. 1991). Thus, in the ancestors of the hexaploid wheat the highest level of HAs was found in the B genome species Aegilops speltoides 16 mmol/kg fresh weight by Niemeyer (1988) and nearly 40mmol/kg fresh weight was found in wild rye (Nicol et al. 1992, Nicol et al. 1993).

Nicol and team confirmed the potential of HAs as aphid resistance factors in modern cultivars based on seedling antixenosis bioassays that tested the suitability of the host plants to the aphids (Nicol et al. 1992). Givovich and Niemeyer published similar results where they showed R. padi preferentially settled on wheat seedlings with lower DIMBOA levels in a choice test (Givovich and Niemeyer 1991). In artificial diet the increased level of DIBOA and DIMBOA concentration decreased the survival rate of cereal aphids (Barria et al. 1992, Niemeyer et al. 1992)

As the concentration of HA increases, aphids need a longer time to search for suitable phloem vessels (Givovich and Niemeyer 1995, Givovich and Niemeyer1994), because they feed from a single sieve tube and the HA concentration can be different between sieve tubes (Givovich et al. 1994). They also spend a longer period ingesting xylem fluid (Ramìrez and Niemeyer1999), which doesn’t contain HAs (Givovich and Niemeyer 1995).

HA levels affect virus transmission as well because aphids takes a longer time to reach the phloem and are therefore less able to transmit the BYDV (Givovich and Niemeyer 1991, Nicol et al. 1992).