Isolation of cDNA clones for genes that are expressed during leaf senescence in Brassica napus. Identification of a gene encoding a senescence-specific metallothionein-like protein.

cDNA clones representing genes that are expressed during leaf senescence in Brassica napus were identified by differential screening of a cDNA library made from RNA isolated from leaves at different stages of senescence. The expression of these genes at different stages of leaf development was examined by northern blot analysis, and several different patterns of expression were observed. One of the clones, LSC54, represented a gene that is expressed at high levels during leaf senescence. Analysis of this gene indicated strong expression in flowers as well as in senescing leaves. DNA sequence analysis of the LSC54 cDNA indicated a similarity between the deduced amino acid sequence and several metallothionein-like proteins previously identified in plants.     

Expression of a nitric oxide degrading enzyme induces a senescence programme in Arabidopsis

Nitric oxide (NO) has been proposed to act as a factor delaying leaf senescence and fruit maturation in plants. Here we show that expression of a NO degrading dioxygenase (NOD) in Arabidopsis thaliana initiates a senescence-like phenotype, an effect that proved to be more pronounced in older than in younger leaves. This senescence phenotype was preceded by a massive switch in gene expression in which photosynthetic genes were down-regulated, whereas many senescence-associated genes (SAGs) and the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene ACS6 involved in ethylene synthesis were up-regulated. External fumigation of NOD plants with NO as well as environmental conditions known to stimulate endogenous NO production attenuated the induced senescence programme. For instance, both high light conditions and nitrate feeding reduced the senescence phenotype and attenuated the down-regulation of photosynthetic genes as well as the up-regulation of SAGs. Treatment of plants with the cytokinin 6-benzylaminopurin (BAP) reduced the down-regulation of photosynthesis, although it had no consistent effect on SAG expression. Metabolic changes during NOD-induced senescence comprehended increases in salicylic acid (SA) levels, accumulation of the phytoalexin camalexin and elevation of leaf gamma-tocopherol contents, all of which occurred during natural senescence in Arabidopsis leaves as well. Moreover, NO fumigation delayed the senescence process induced by darkening individual Arabidopsis Columbia-0 (Col-0) leaves. Our data thus support the notion that NO acts as a negative regulator of leaf senescence.     

Large-scale identification of leaf senescence-associated genes

Leaf senescence is a form of programmed cell death, and is believed to involve preferential expression of a specific set of "senescence-associated genes" (SAGs). To decipher the molecular mechanisms and the predicted complex network of regulatory pathways involved in the senescence program, we have carried out a large-scale gene identification study in a reference plant, Arabidopsis thaliana. Using suppression subtractive hybridization, we isolated approximately 800 cDNA clones representing SAGs expressed in senescing leaves. Differential expression was confirmed by Northern blot analysis for 130 non-redundant genes. Over 70 of the identified genes have not previously been shown to participate in the senescence process. SAG-encoded proteins are likely to participate in macromolecule degradation, detoxification of oxidative metabolites, induction of defense mechanisms, and signaling and regulatory events. Temporal expression profiles of selected genes displayed several distinct patterns, from expression at a very early stage, to the terminal phase of the senescence syndrome. Expression of some of the novel SAGs, in response to age, leaf detachment, darkness, and ethylene and cytokinin treatment was compared. The large repertoire of SAGs identified here provides global insights about regulatory, biochemical and cellular events occurring during leaf senescence.     

Leaf senescence is accompanied by an early disruption of the microtubule network in Arabidopsis

The dynamic assembly and disassembly of microtubules (MTs) is essential for cell function. Although leaf senescence is a well-documented process, the role of the MT cytoskeleton during senescence in plants remains unknown. Here, we show that both natural leaf senescence and senescence of individually darkened Arabidopsis (Arabidopsis thaliana) leaves are accompanied by early degradation of the MT network in epidermis and mesophyll cells, whereas guard cells, which do not senesce, retain their MT network. Similarly, entirely darkened plants, which do not senesce, retain their MT network. While genes encoding the tubulin subunits and the bundling/stabilizing MT-associated proteins (MAPs) MAP65 and MAP70-1 were repressed in both natural senescence and dark-induced senescence, we found strong induction of the gene encoding the MT-destabilizing protein MAP18. However, induction of MAP18 gene expression was also observed in leaves from entirely darkened plants, showing that its expression is not sufficient to induce MT disassembly and is more likely to be part of a Ca(2+)-dependent signaling mechanism. Similarly, genes encoding the MT-severing protein katanin p60 and two of the four putative regulatory katanin p80s were repressed in the dark, but their expression did not correlate with degradation of the MT network during leaf senescence. Taken together, these results highlight the earliness of the degradation of the cortical MT array during leaf senescence and lead us to propose a model in which suppression of tubulin and MAP genes together with induction of MAP18 play key roles in MT disassembly during senescence.