Isolation of cDNA clones for genes showing enhanced expression in barley leaves during dark-induced senescence as well as during senescence under field conditions

Senescence of barley (Hordeum vulgare L. cv. Carina) primary foliage leaves was induced by transfer of the plants into darkness for 2 d. Under these conditions senescence was characterized by a light-reversible decline in the efficiency of photosystem II, and in chlorophyll and protein contents. To isolate senescence-associated genes a differential display of cDNA fragments amplified from reversely transcribed RNA was employed. By this method, gene expression in leaves of control plants collected at the onset of the dark period was compared with gene expression in senescing leaves collected at the end of the extended dark period. The expression of the genes represented by various differentially displayed cDNA fragments was examined by Northern blot hybridizations with RNA derived from primary foliage leaves before and after induction of senescence by darkness. In order to test whether these genes with enhanced expression during dark-induced senescence also show enhanced expression during natural senescence, Northern blot hybridizations were carried out with RNA samples prepared from flag leaves of barley plants during maturation and senescence under field conditions. Five of the cDNA fragments representing transcripts associated with dark-induced senescence, as well as with natural senescence, were selected as probes for screening a cDNA library from senescent flag leaves. With one probe a larger cDNA including a complete open reading frame with homology to the sequence of a known proteinase inhibitor was found. Another cDNA isolated by this means showed high sequence similarity with a gene coding for a 4-hydroxyphenylpyruvate dioxygenase. The other three larger cDNA clones isolated by this procedure so far do not show significant homologies with known sequences.