STANTON, MINNESOTA
Novartis Seeds, Inc.
ST. PAUL, MINNESOTA
University of Minnesota

Differential expression of chitinase in maize kernels
--Hanten, J; Kendra, D; Mettler, I; Harlander, S; Fulcher, RG

Several different plant species (i.e., corn, peas, wheat, barley, beans, etc.) contain significant levels of chitinases in seed and vegetative tissues (Collinge et al., Plant J 3:31-40, 1993). The physiological role of plant chitinases is uncertain because plants do not possess an endogenous chitin substrate (b-1,4 linked N-acetylglucosamine). However, it has been shown that plant chitinases act as defensive agents responding to environmental stress factors such as chitin containing fungal pathogens and certain abiotic elicitors (Boller, Dev Plant Path 2:391-397, 1993). An assessment of the level of chitinase present in maize kernels at two different stages of maturity was performed in order to establish relative abundance in these tissues.

Maize kernel chitinase was partially purified by 60% ammonium sulfate precipitation from shrunken-2 (sh2) and field corn kernels at 21 days after pollination (dap)-(R3 stage) and at physiological maturity (R6 stage). Comparisons in hydrolytic activity towards chitin substrate were made between R3 and R6 kernels with a colorimetric microtiter plate assay. Significant increases in chitinase activity were apparent in mature sh2 and field corn kernels (Table 1).

These results indicate that chitinase expression increases during maturation of the kernel. Additional studies also provide evidence that chitinase expression levels increase during seed maturation and germination. For example, chitinase mRNA levels are higher in mature kernels as compared to immature embryos (Huynh et al., J Biol Chem 267:6635-6640, 1992). In addition, chitinase expression levels increase dramatically during early germination of soybean seeds (Teichgraber et al., Agro-Food Ind Hi-Tech 3:11-14, 1991). The physiological significance of such differential expression is not completely understood. It is believed that the regulation of chitinases and a whole host of additional enzymes and secondary metabolites is controlled by internal and external growth signals, contributing to the defense response. Therefore, it is plausible that chitinase expression would increase during kernel maturation and early embryonic development to ensure survival against the numerous pathogens encountered.

Table 1. Chitinase activity in sh2 and field corn kernels. The amount of enzyme yielding 1 mol s-1 of N-acetylglucosamine is defined as a Katal (kat). Each column of data represents the mean values of two replicates of 10 kernel samples. sh2 mature kernels were F1 hybrid seed and 21 dap kernels were progeny of the F1 seed (F2). Field corn kernels at 21 dap and maturity were inbreds.
 
Chitinase Specific Activity
(Kkat/mg sol. protein)
21 dap - (R3)  Mature - (R6) 
Fieldcorn  53.95 (±9.15) 1355.93 (±17.28)
Sweetcorn (sh2) 39.60 (±8.7) 525.91 (±4.13)
Chitinase Activity  21 dap - (R3)  Mature - (R6) 
(Kkat/gm dry weight)
Fieldcorn  783.30 (±126.16) 1493.81 (±284.44)
Sweetcorn (sh2) 536.70 (±90.63) 1211.73 (±254.38)


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