Ribulose Bisphosphate Carboxylase and Proteolytic Activity in Wheat Leaves from Anthesis through Senescence

Abstract

Changes in ribulose bisphosphate carboxylase (RuBPCase) and proteolytic activity were folowed in the flag leaf and second leaf of field-grown winter wheat (cv. Arthur). These changes were foUlowed in relation to changes in leaf chlorophyll, protein, and photosynthesis, and seed development. Levels of RuBPCase were determined by rocket immunoelectrophoresis as described previously (Wittenbach 1978 RuBPCase in the flag leaf and the leaf immediately below it have been followed from full leaf expansion through senescence. These changes have been followed with respect to changes in leaf Chl, protein, and photosynthesis. In addition, proteolytic activity in these leaves was followed, since these enzymes are responsible for the breakdown of RuBPCase. MATERIALS AND METHODSPlant Material. Samples of flag leaves, second leaves (leaves immediately below the flag leaves), and seed heads were collected from field-grown winter wheat (Triticum aestivum L. cv. Arthur) beginning 1 week prior to anthesis and ending at harvest during the 1977 and 1978 seasons. All collections and photosynthesis measurements were made between 2 and 4 PM. Samples were immediately frozen in liquid N2 and transported to the lab on dry ice. They were then stored in liquid N2 until assayed. Freezing in liquid N2 had no significant effect on RuBPCase activity, and no activity was lost following storage for at least 1 month.Photosynthesis and RuBPCase Assays. Photosynthetic measurements were made using a 14C02-pulsing apparatus similar to that of Naylor and Teare (1 1). All determinations were made on clear days in full sunlight (-1,800 ,uE/m2. s) with the temperature between 22 and 30 C. Leaves were pulsed for 15 s with air (364 ,ul/l CO2) containing 14CO2. Leaf discs were removed with a cork borer, placed in scintillation vials with I ml of tissue solubilizer, and left ovemight on a shaker to digest. Counting solution was then added and the dpm determined by liquid scintillation spectroscopy.RuBPCase activity was determined by following 04C02 incorporation into acid-stable products as described previously (16). Leaf samples of 300 mg were extracted in 5 ml of 25 mm Hepes (pH 7.5) containing 4 mm DTT, 1 mm Na2EDTA, and 1% (w/v) PVP using a Polytron homogenizer. The extracts were centrifuged at 30,000g for 20 min, and the supernatant fractions were used to assay for RuBPCase activity.The levels of RuBPCase were quantitated using the procedure of rocket immunoelectrophoresis (15

Change in Content of Ribulose 1,5-Bisphosphate Carboxylase during Development and Senescence of Mulberry (Moms albaL.) Leaves

“…Among plant species, inconsistent experimental results have been obtained for the changes in the specific enzyme activity during leaf senescence. It has been reported that the specific activity increased in barley primary leaves (3), declined in wheat leaves (16), and remained constant in rice leaves (7). The changes in the specific enzyme activity in mulberry leaves during their formation and degradation differed somewhat from the results obtained from other plant species.…”

“…STODDART and THOMAS (15) noted that nitrogen is removed from aging leaves and redistributed to young expanding leaves according to their requirements. Senescenceassociated degradation of RuBPCase and redistribution of nitrogen within a plant have generally been confirmed in annuals (5,(7)(8)(9)(10)16). In vitro RuBPCase-degrading activity examined in rice leaves, however, did not show a close relation to protein loss during leaf senescence (6).…”

“…The extract was assayed for RuBPCase activity as described elsewhere (3,17). The level of RuBPCase protein was determined by rocket immunoelectrophoresis (4,16,18). A specific antibody against RuBPCase was raised in white rabbits by injections of RuBPCase purified from mulberry leaves.…”

Nitrogen and Photosynthesis in the Flag Leaf of Wheat (Triticum aestivum L.)

“…Wittenbach (30) and Peoples et al (19) also found that the ratio ofRuP2 carboxylase to soluble protein remained constant until late senescence but then declined. With barley, Friedrich and Huffaker (4) found a relatively constant RuP2 carboxylase specific activity, but found that the ratio of RuP2 carboxylase to soluble protein declined throughout senescence.…”

Protein Degradation in Lemna with Particular Reference to Ribulose Bisphosphate Carboxylase

“…The same group has shown that, during the dark-induced senescence of detached leaves of barley, the initial loss of protein is almost entirely due to the degradation of RuBPCase (28). This behavior has also been observed in the dark-induced senescence of attached wheat leaves (37,38). The stability of RuBPCase prior to senescence, and its rapid degradation during senescence, has led to its classification as a leaf storage protein ( 19,21).…”