THE CIRCADIAN ACTIVITY OF NITRATE REDUCTASE IN TOMATO IS DRIVEN BY CHANGES IN THE PROTEIN LEVEL

Authors: TUCKER, DAWN - PLANT BIOLOGY UOFI URBANA; Ort, Donald

Submitted to: Crop Development for the Cool and Wet Regions of Europe
Publication Type: Book / Chapter
Publication Acceptance Date: 10/20/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: Many of the commercially most significant crops in temperature North America (e.g., corn, soybean, cotton and others) are referred to as 'chilling sensitive'. Plants that fall into this category are botanical immigrants from tropical and subtropical origins where selection pressures to deal with low temperature do not exist. Photosynthetic metabolism is among the most chill sensitive process in these plants and the chilling sensitivity of photosynthesis plays a critical role both in limiting the geographical range where these crops are grown as well as accounting for the annual variation in the economic success of these crops grown at the northern border of their cultivation. An improvement of even one degree in the low temperature tolerance would have a far reaching beneficial impact on the agronomy of these important crop species. Our earlier work demonstrated that chilling interrupts the internal timing mechanism (i.e. circadian rhythm) of chilling sensitive plants. Since this internal clock is responsible for controlling when during the course of a day that certain genes are expressed and specific proteins made, the interruption caused by chilling would be expected to have adverse effects on photosynthetic metabolism. We found that the activity nitrate reductase, a key enzyme controlling nitrogen metabolism in leaves, is controlled at the level of the NR gene. Our current work also reveals that the natural rhythm in NR activity is severely delayed by chilling. This finding is a very important clue in understanding the molecular basis for the chilling sensitive of photosynthesis in crop plants and presents exciting avenues for research designed to improve chilling tolerance.

Technical Abstract: Nitrate reductase (NR) catalizes the first and rate limiting reaction in nitrogen assimilation converting nitrate to nitrite using NAD(P)H as the electron donor. NR activity exhibits a circadian oscillation in activity that involves a complex combination of protein level changes and covalent modification such as phosphorylation. The NR activity rhythm in chilling sensitive species such as tomato shifts after exposure to a dark chill. Even though the rhythm resumes upon rewarming it becomes uncoordinated with the ambient diurnal cycle potentially contributing to reductions in photosynthetic rates observed in chilling sensitive species following an evening chill. The presence of Mg2+ in NR reactions allows the binding of an inhibitor protein necessary to inactivate the phosphorylated enzyme. NR activity rates without Mg2+ are interpreted as reflective of total protein levels because of the inability of the NR present to be inactivated by protein phosphorylation under these conditions. The ratio of NR activity in the presence of Mg2+ to activity in the absence of Mg2+ reflects activity changes due to covalent modification by phosphorylation. These assays indicate that NR protein levels do oscillate in constant conditions and little, if any, of the activity rhythm is due to the enzyme's phosphorylation state. Northern blot analysis conduct over a constant condition time course illustrate a robust rhythm in NR mRNA levels that directly shadows the activity oscillation in tomato.