The Degradation Of Rubisco Large Subunit And The Associated Proteases In Wheat Leaves

Ribulose-l,5-bisphosphate carboxylase/oxgenase (Rubisco, EC 4.1.1.39), is a bifunctional enzyme that catalyzes two competing reactions, namely photosynthetic CO₂ assimilation and photorespiratory carbon oxidation, in the stroma of the chloroplasts. This enzyme is the most abundant protein, accounting for 12-35% of total leaf protein and more than 50% of the soluble protein in C3 plants (Makino et al., 1983 ). The degradation of Rubisco is closely related to the rate of photosynthesis as well as nitrogen economy in leaves. Rubisco in senescing leaves of plants is thought as the major nitrogen source providing amino acids for developing organs (Feller and Fischer 1994). So an understanding of the degradation mechanism of Rubisco in senescing leaves is an important subject to understand the physiological function of Rubisco. Rubisco reaches its maximum content soon after full expansion of the leaf. Thereafter, it declines gradually, and is degraded rapidly in the late period of leaf senescence. Although the synthesis, assembly, structure and regulation of Rubisco have been studied extensively, the mechanisms of Rubisco degradation in leaves are as yet not known clearly (Hortensteiner and Feller 2002; Houtz and Portis 2003). The degradation of Rubisco LSU and the associated proteases were studied in this paper.Firstly, Rubisco was separated and purified from wheat leaves by ammonium sulfate precipitation, gel filtration, ion-exchange chromatography on the basis of Rubisco characteristic, such as dissolvability, molecular size and shape, and electric charge. On the basis of PAGE, Rubisco was recovered and purified through modified electrophoresis in the protein recovery apparatus. This method is simple with the high purification. Two predominant bands were detected by SDS-PAGE. One was the large subunit whose molecular weight was about 53kDa and the other was the small subunit, whose molecular weight was about 14.8kDa. Immunized the rabbits by subcutaneous, multi-spot, interval injection and the antibody of Rubisco was obtained.Secondly, the degradation of the large subunit ( LSU) of Rubisco in mature wheat leaves was studied. A 51kDa fragment, the portion of the LSU of Rubisco, was detected by SDS-PAGE and immunoblotting with antibody against Rubisco in crude extracts of mature wheat leaves. The appearance of the 51kDa fragment was the most obvious at 30-35℃and pH 5.5. The LSU and its 51kDa fragment both existed when the crude extracts were incubated for 60min. The amount of LSU decreased with incubation time from 0 to 3h in the crude extracts. In addition, through treatment with various inhibitors, the reaction was inhibited by cysteine proteinase inhibitor E-64 and leupeptin. However, the 51kDa fragment could not be found at all pHs from pH4.5 to pH8.5 in chloroplasts of mature wheat leaves.Protein degradation is an important event in the senescence, thus the further research on the degradation of Rubisco LSU in senescing wheat leaves was investigated. The 51kDa fragment was detected in leaf crude extracts and in chloroplast lysates from leaves with dark-induced senescence. Further studies showed that the 51kDa fragment was found in the reaction solution with stroma fraction, but not found in that with the chloroplast membrane fraction and in the chloroplast lysates from mature wheat leaves. The reaction of producing the 51-kDa fragment was inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 1,10-phenanthroline and EDTA. N-terminal sequence analysis indicated that the LSU was cleaved at the peptide bond between Lys-14 and Ala-15. The results obtained in this study implied that the appearance of the 51kDa fragment could be due to the involvement of a new senescence-associated protease which is located in the stroma of chloroplasts in the senescing wheat leaves. In addition, a 50-kDa fragment of LSU formed obviously at pH 6.0-6.5 was detected in the crude extracts of leaves with dark-induced, but not found in lysates of chloroplasts. The degradation was prevented by AEBSF, leupeptin and transepoxysucciny1-L-leucyla-mido (4-guanidino) butane (E-64). The protease degrading the LSU into 50kDa frament could be located in vacuole.The effect of ATP and Zn²⁺ on the degradation of Rubisco LSU in wheat leaves was investigated. The 51kDa fragment was detected when the leaf crude extracts of mature leaves were incubated at pH 7.5 buffer with 1mM ATP or the chloroplast lysates of mature leaves were incubated at pH 7.5 buffer with 1mM ATP and 1μM Zn²⁺. The 51kDa fragment was detected at all the pH values in the range of 5.5-8.5 in the leaf crude extracts, but in the chloroplast lysates the 51kDa fragment was only found at bias alkaline pH values. The experiment about sensitivity of the degradation to different inhibitors implied that the protease in crude extracts and in the chloroplast lysates were not the same type protease. It suggested that the protease in the crude extracts should not be the same protease in the chloroplasts, and that the protease in the chloroplast lysates from senescing leaves should not be associated senescence. In addition, the Tricine-SDS-PAGE with urea was used to investigate the another 2-kDa fragment. It showed that no 1kDa or 2kDa polypeptides were detected. It was speculated that the target protease(s) might cleave the Rubisco LSU to a 51 kDa fragment and several small polypeptides below 1 kDa.To further study the characters of the protease, quantitive RT-PCR approach was applied to examine the change of ClpPmRNA in the natural senescence. The results showed that ClpPmRNA decreased in the natural senescence. It suggested that the Clp should not be the protease degrading Rubisco LSU into 51kDa fragment. It could be an unknown protease.All the results could contribute to understand the protein degradation and the proteases in the leaf, expecially in the chloroplast. It would expound the molecular mechanism of Rubisco rapid degradation during leaf senescence. And our work is of great fundamental importance for future agronomic improvement, such as delaying cereal crops senescence, improving its output and quality, and so on.