Chlorophyll b reduction during senescence of barley seedlings

During senescence of flowering plants, only breakdown products derived from chlorophyll a were detected although  b disappears, too (Matile et al., 1996, Plant Physiol 112: 1403–1409). We investigated the possibility of chlorophyll b reduction during dark-induced senescence of barley (Hordeum vulgare L.) leaves. Plastids isolated from senescing leaves were lysed and incubated with NADPH. We found 7¹-hydroxy-chlorophyll a, 7¹-hydroxy-chlorophyllide a, and, after incubation with Zn-pheophorbide b, also Zn-7¹-hydroxy-pheophorbide a, indicating activity of chlorophyll(ide) b reductase. The highest activity was found at day 2 of senescence when chlorophyll breakdown reached its highest rate. Chlorophyllase reached its highest activity under the same conditions only at days 4–6 of senescence. Based on the chlorophyll b reductase activity of plastids at day 2.5 of senescence (=100%), the bulk of activity (83%) was found in the thylakoids and only traces (5%) in the envelope fraction. Chlorophyll b reduction is considered to be an early and obligatory step of chlorophyll b breakdown. Received: 22 February 1999 / Accepted: 24 March 1999     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Identification and characterization of novel senescence-associated genes from barley (Hordeum vulgare) primary leaves

Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence-specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up-regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence-associated expression was confirmed by Northern analyses or quantitative RealTime-PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence-induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1-like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF-like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1-GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed.     

Leaf senescence in rice plants: cloning and characterization of senescence up-regulated genes.

To identify senescence-associated genes (SAGs) in rice leaves, senescence was induced by transferring rice seedlings into darkness. Senescence up-regulated cDNAs were obtained by PCR-based subtractive hybridization. Among 14 SAG clones characterized, 11 were found to be associated with both dark-induced and natural leaf senescence. Three clones were associated only with dark-induced leaf senescence. The possible physiological roles of these SAGs during rice leaf senescence are discussed.     

Expression of the beta-oxidation gene 3-ketoacyl-CoA thiolase 2 (KAT2) is required for the timely onset of natural and dark-induced leaf senescence in Arabidopsis

The onset of leaf senescence is regulated by a complex mechanism involving positive and negative regulators. Among positive regulators, jasmonic acid (JA) accumulates in senescing leaves and the JA-insensitive coi1-1 mutant displays delayed leaf senescence in Arabidopsis. A strong activated expression of the gene coding for the JA-biosynthetic beta-oxidation enzyme 3-ketoacyl-CoA thiolase 2 (KAT2) in natural and dark-induced senescing leaves of Arabidopsis thaliana is reported here. By using KAT2::GUS and KAT2::LUC transgenic plants, it was observed that dark-induced KAT2 activation occurred both in excised leaves as well as in whole darkened plants. The KAT2 activation associated with dark-induced senescence occurred soon after a move to darkness, and it preceded the detection of symptoms and the expression of senescence-associated gene (SAG) markers. Transgenic plants with reduced expression of the KAT2 gene showed a significant delayed senescence both in natural and dark-induced processes. The rapid induction of the KAT2 gene in senescence-promoting conditions as well as the delayed senescence phenotype and the reduced SAG expression in KAT2 antisense transgenic plants, point to KAT2 as an essential component for the timely onset of leaf senescence in Arabidopsis.     

A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence

We have characterized the structure and expression of a senescence-associated gene (sen1) of Arabidopsis thaliana. The protein-coding region of the gene consists of 5 exons encoding 182 amino acids. The encoded peptide shows noticeable similarity to the bacterial sulfide dehydrogenase and 81% identity to the peptide encoded by the radish din1 gene. The 5-upstream region contains sequence motifs resembling the heat-shock- and ABA-responsive elements and the TCA motif conserved among stress-inducible genes. Examination of the expression patterns of the sen1 gene under various senescing conditions along with measurements of photochemical efficiency and of chlorophyll content revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence. During the normal growth phase, the gene is strongly induced in leaves at 25 days after germination when inflorescence stems are 2–3 cm high, and then the mRNA level is maintained at a comparable level in naturally senescing leaves. In addition, dark-induced senescence of detached leaves or of leaves in planta resulted in a high-level induction of the gene. Expression of the sen1 gene was also strongly induced in leaves subjected to senescence by 0.1 mM abscisic acid or 1 mM ethephon treatment. The induced expression of the gene by dark treatment was not significantly repressed by treatment with 0.1 mM cytokinin or 50 mM CaCl₂ which delayed loss of chlorophyll but not that of photochemical efficiency.     

The impact of light intensity on shade-induced leaf senescence

Plants often have to cope with altered light conditions, which in leaves induce various physiological responses ranging from photosynthetic acclimation to leaf senescence. However, our knowledge of the regulatory pathways by which shade and darkness induce leaf senescence remains incomplete. To determine to what extent reduced light intensities regulate the induction of leaf senescence, we performed a functional comparison between Arabidopsis leaves subjected to a range of shading treatments. Individually covered leaves, which remained attached to the plant, were compared with respect to chlorophyll, protein, histology, expression of senescence-associated genes, capacity for photosynthesis and respiration, and light compensation point (LCP). Mild shading induced photosynthetic acclimation and resource partitioning, which, together with a decreased respiration, lowered the LCP. Leaf senescence was induced only under strong shade, coinciding with a negative carbon balance and independent of the red/far-red ratio. Interestingly, while senescence was significantly delayed at very low light compared with darkness, phytochrome A mutant plants showed enhanced chlorophyll degradation under all shading treatments except complete darkness. Taken together, our results suggest that the induction of leaf senescence during shading depends on the efficiency of carbon fixation, which in turn appears to be modulated via light receptors such as phytochrome A.     

Regulation of the expression of ferredoxin-glutamate synthase in barley

We have investigated the regulation of ferredoxin–glutamate synthase (Fd-GOGAT) in leaves of barley (Hordeum vulgare L. cv. Maris Mink) at the mRNA, protein and enzyme activity levels. Studies of the changes in Fd-GOGAT during plant development showed that the activity in shoots increases rapidly after germination to reach a maximum (on a fresh-weight basis) at day 10 and then declines markedly to less than 50% of the maximal activity by day 30, this decline being correlated with an equivalent loss of Fd-GOGAT protein. Growing the plants in darkness reduced the maximum activity attained in the shoots, but did not affect the overall pattern of the changes or their timing. The activity of Fd-GOGAT increased two- to three-fold within 48 h when etiolated leaves were exposed to light, and Northern blots indicated that the induction occurred at the mRNA level. However, whilst a carbon source could at least partially substitute for light in the induction of nitrate reductase activity, no induction of Fd-GOGAT activity was seen when etiolated leaves were treated with either sucrose or glucose. Interestingly, the levels of Fd-GOGAT mRNA and activity remained high up to a period of 16 h or 72 h darkness, respectively. Compared with plants grown in N-free medium, light-grown plants supplied with nitrate had almost two-fold higher Fd-GOGAT activities and increased Fd-GOGAT mRNA levels, but nitrate had no effect on the abundance of the enzyme or its mRNA in etiolated plants, indicating that light is required for nitrate induction of barley Fd-GOGAT. Received: 23 April 1997 / Accepted: 28 May 1997