Analysis of nitrogen reallocation from senescing barley leaves : characterization of the influence of a high-grain protein content locus on chromosome six, and molecular cloning and heterologous expression of a serine carboxypeptidase

Abstract

In cereals, senescence is a highly regulated and organized process in which the nutrients located in the leaves are translocated to the developing seeds. During this process multiple signaling cascades are activated either up-regulating or down-regulating senescence related genes. The majority of the nitrogen to be remobilized is located in the leaves in plastidial proteins, especially Rubisco. These proteins must be degraded before their components can be translocated to be reutilized in the seeds. The mechanism for plastidial protein degradation is largely unknown at this time. Proteases located in lytic vacuolar compartments, including carboxypeptidases, have been found to be up-regulated during senescence, which may indicate their involvement in the degradation of plastidial proteins. In a recent genomic study a serine carboxypeptidase, cp-mIII, was found to be up-regulated in senescing leaves. Further characterization and localization of this protease could lead to an increased understanding of the mechanisms of protein degradation within the leaves during senescence. After the proteins are degraded they are transported to the developing seeds to be reincorporated into grain storage proteins.

The amount of grain protein is noted to vary widely within a species depending on genotype and environmental conditions. Both high and low grain protein content (GPC) are economically important. Previously, a locus on chromosome 6 in barley was discovered that affects GPC. This locus has also been found to be homologous to chromosome 6B in wheat. To determine the effects of this locus, near isogenic lines (NIL) were generated from two varieties, one commonly grown high GPC variety and another known to have relatively low GPC. Several NIL were created where the high GPC allele(s) is isolated in the low GPC background and vice versa. Detailed physiological characterization of this germplasm indicated that the high-GPC trait in barley is associated with (a) earlier/accelerated leaf senescence and N remobilization; (b) enhanced availablity of amino nitrogen in kernels; (c) earlier grain total N accumulation; and (d) differences in whole-plant allocation and reallocation of nitrate. This analysis serves as a basis for the further, molecular analysis of the processes controlling (re)allocation of vegetative nitrogen to seeds in barley.