Enhanced Protease Inhibitor Expression in Plant Residues Slows Nitrogen Mineralization

Organic N mineralization by extracellular proteases affects inorganic N availability and loss. Soil Nmineralization is slowed by addition of purified protease inhibitors. We hypothesized that elevated concentrations of protease inhibitors in plant residues would reduce soil and plant residue N mineralization. Isogenic controls and transgenic plants of Brassica (Brassica napus L.), Japonicum rice (Oryza sativa L.), and tobacco (Nicotiana tabaccum L.) showing enhanced wound-inducible protease inhibitor production were grown in a greenhouse, and leaves were mechanically wounded 3 d before shoot removal. Transgenic plants and their isogenic controls did not differ in N concentration, C/N ratio, or lignin concentration in shoot residues, but protease inhibitor concentration was 1.5 to 2.3 times greater in the transgenic lines. In laboratory incubations in a loamy sand soil, inorganic N in leachate from transgenic plants was significantly lower than isogenic controls for the first 30 d when the residues remained on the soil surface and were higher at one or more dates thereafter.When residues weremixed with soil, differences were observed only for Brassica. Cumulative N mineralization in static incubations of residues mixedwith soil followed the order Brassica . tobacco . rice residues. In general, transgenic residues mineralized between 22 and 27% less N than control plant residues in the first 30 d, but no differences in soil N mineralization were detected. Thus, protease inhibitor concentration of plant residues should be included with measures of total N concentration, C/N ratio, and lignin concentration to improve prediction and potentially management of short-term N mineralization from plant residues. MOST N PRESENT in soil is in organic forms, primarily proteinaceous compounds. Soil acts as a sink for proteins from plants, animals, and microorganisms and also is an environment characterized by rapid protein hydrolysis under relatively wide ranges of soil temperature, water content, and pH (Hankin and Hill, 1978). Soil proteases originate from the same sources as proteins and are a mixture of heterogeneous, primarily extracellular, enzymes with different molecular weights, structures, cofactor requirements, and substrate specificities (Loll and Bollag, 1983). Proteolytic microorganisms may comprise 22 to 89% of the total soil microbial biomass (Hankin and Hill, 1978; Bach and Munch, 2000). Protein hydrolysis is a necessary first step in soil N mineralization and therefore regulates inorganic N availability to soil organisms and plants and to processes leading to N loss. Most of the research on controlling the rate of N mineralization from crop residues, manures, and other organic materials that are added to the soil has concentrated on their management (e.g., degree of incorporation in the soil or timing of application) and their chemical characteristics (e.g., C/N ratio, lignin concentration, or presence of polyphenols) (Kumar and Goh, 2000). These approaches have met with limited success, in part because of the difficulty in predicting N mineralization rates and extent. Furthermore, there have been no tactics to regulate soil organic matter N mineralization, which can be a significant source of N loss in annual grain and row crop systems (Keeney and DeLuca, 1993; David et al., 1997; Haynes, 1999). The ability to manage N mineralization would help reduce environmental contamination from N losses and improve N uptake efficiency by plants. This is where protease inhibitors may play a role. Inhibitors of proteases are naturally present in plants, and their role as a defensemechanism against insects and disease organisms has been recognized (Geoffroy et al., 1990; Green andRyan, 1992; Duan et al., 1996). In plants belonging to Gramineae, Leguminosae, Solanaceae, and other families, protease inhibitors are produced in response to pathogen attack, herbivory, or mechanical damage (Ryan, 1990; Green and Ryan, 1992). Protease inhibitors reduce the growth and survival of many insect herbivores when present in artificial diets and reduce both insect feeding rate and performance when expressed in transgenic plants (McManus et al., 1994; Cipollini and Bergelson, 2000). Transgenic modifications have enhanced protease inhibitor expression to develop insectresistant crop cultivars in several important crops. These plant protease inhibitors have specificities for animal and microbial proteases that are similar to the proteases in soils. Thus, these protease inhibitors may also affect the activity of soil proteases, which are responsible for early steps in soil N mineralization. Of protease inhibitors, Loll and Bollag (1983, p. 367) stated that, “Little is known about the survival of these compounds in soil, but it is possible that they could affect proteolysis.”; however, little has been published on this topic intheinterveningtwodecades.Doneganetal. (1997) found no difference in N mineralization from leaves of tobacco engineered to express the tomato (Lycopersicum esculentum L.) protease inhibitor I (pJN3) belonging to serine type inhibitors.More recently, Cowgill et al. (2002) concluded that expression of cysteine protease inhibitors in potato (Solanum tuberosum L.) residues did not alter residue decomposition in soil. In both studies with transgenic plants, dried tissues were used, which may have altered protease inhibitor activity. Neither study focused on N mineralization per se. We have found short-term reduction in soil Nmineralizationwhen purified protease inhibitors were added to soil and discovered that some K. Kumar and C.J. Rosen; Dep. of Soil, Water, & Climate, 1991 Upper Buford Circle, Room 439, Univ. of Minnesota, Saint Paul, MN 55108, USA; and M.P. Russelle, USDA-ARS Plant Sci. Res. Unit, 1991 Upper Buford Circle, Room 439, Univ. of Minnesota, Saint Paul, MN 55108, USA. K. Kumar, current address: Res. and Dev., Metropolitan Water Reclamation District of Greater Chicago, 6001 West Pershing Rd., Cicero, IL 60804-4112. Received 8 Sept. 2005. *Corresponding author ([email protected]). Published in Agron. J. 98:514–521 (2006). Nitrogen Management doi:10.2134/agronj2005.0261 a American Society of Agronomy 677 S. Segoe Rd., Madison, WI 53711 USA R e p ro d u c e d fr o m A g ro n o m y J o u rn a l. P u b lis h e d b y A m e ri c a n S o c ie ty o f A g ro n o m y . A ll c o p y ri g h ts re s e rv e d . 514 Published online April 11, 2006