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

Abstract

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.

Senescence Is Induced in Individually Darkened Arabidopsis Leaves, but Inhibited in Whole Darkened Plants

“…All reports with which we are familiar agree that the transfer of whole plants to darkness induces chlorophyll and/or protein loss in true leaves McKeon, 1991a, 1991b;Oh et al, 1996;Kleber-Janke and Krupinska, 1997;Weaver et al, 1998), and all those in which reversibility was assayed agree that chlorophyll and protein losses could be reversed by returning plants to the light, at least within several days of the dark treatment McKeon, 1991a, 1991b;Kleber-Janke and Krupinska, 1997). When the expression of various senescence-and photosynthesis-associated genes was examined in response to both whole-plant darkness and natural senescence, however, the results were more varied, with some genes responding similarly to both treatments and others responding very differently (Blank and Mckeon, 1991b;Oh et al, 1996;Kleber-Janke and Krupinska, 1997;Weaver et al, 1998). Kleber-Janke and Krupinska (working in the primary leaves of barley seedlings) have interpreted this to mean that when whole plants are darkened a portion of the senescence program is induced, perhaps one corresponding to an early stage of senescence.…”

The Role of Homogentisate Phytyltransferase and Other Tocopherol Pathway Enzymes in the Regulation of Tocopherol Synthesis during Abiotic Stress

“…Regulation of tocopherol biosynthesis in senescing and stressed plants may occur at multiple steps of the pathway. HPPD activity limits tocopherol synthesis in non-stressed Arabidopsis plants (Tsegaye et al, 2002), and HPPD mRNA levels are up-regulated in senescing barley (Hordeum vulgare) leaves (KleblerJanke and Krupinska, 1997). Similarly, various biotic and abiotic stresses elevate Tyr aminotransferase (TAT) mRNA and protein levels and enzyme activity in Arabidopsis (Lopukhina et al, 2001;Sandorf and Hollander-Czytko, 2002).…”

Nitric Oxide Signaling During Senescence and Programmed Cell Death in Leaves

“…Besides leaf age, darkness is more commonly considered to be an inducer of leaf senescence and induces loss of chlorophyll Biswal, 1980, 1981), nucleic acid and protein (Misra and Biswal, 1982b), and alterations in the thylakoid membrane function such as photosystem I (PS I) and PS II activities (Biswal et al, 2001;Misra and Biswal, 1982a;Misra, 1993a). The expression of various photosynthesis associated genes was reduced (Misra, 1993b;Kleber-Janke and Krupinska, 1997;Weaver et al, 1998). The dark induced senescence is supposed to be be reversed by returning plants to the light, at least within a thresh hold dark treatment (Kleber-Janke and Krupinska, 1997).…”

“…The dark induced senescence is supposed to be be reversed by returning plants to the light, at least within a thresh hold dark treatment (Kleber-Janke and Krupinska, 1997). The SAG gene expression in response to both whole-plant darkness and natural senescence, showed varied responses, with some genes responding similarly to both treatments and others responding very differently (Kleber-Janke and Krupinska, 1997;Weaver et al, 1998). When whole plants were darkened, in contrast, by all criteria except loss of total protein and chlorophyll (which still occurred less quickly and less strongly than with individual leaf darkness), senescence was not induced (Weaver and Amasino,2001).…”