Mechanisms of Ozoneinduced Accelerated Leaf Senescence

During normal senescence, photosynthesis-associated genes (PAGs) decline. Ozone caused a rapid loss in rbcS transcript levels in potato (Solanum tubersosum L.) leaves immediately after acute exposures to the pollutant (Reddy et al., 1993), and this response was also observed in Arabidopsis and tobacco (Bahl and Kahl, 1995; Conklin and Last, 1995). A prematuare decline in rbcS was observed after more prolonged exposures to lower doses of the gas (Glick et al., 1995; Miller et al., 1999). A drop in rbcL was also observed, but this occurred after the initial drop in rbcS. In addition, O3 has been associated with a reduction in the transcript level of chlorophyll a/b binding protein (cab) and two senescence-down regulated genes of unidentified function (Conklin and Last, 1995; Miller et al., 1999).

Several laboratories have been pursuing the identification of senescence-associated genes (SAGs) in a number of botanical systems; this subject is covered in detail elsewhere in this volume (Chapters 4 and 5). We have examined the ability of O3 to induce a subset of SAGs isolated from Arabidopsis. When Landsberg erecta plants were treated with 0.15 ^L L-1 O3 for 6 h day-1 for two weeks, lower leaves in the rosette showed signs of yellowing in the absence of any foliar injury while companion control plants remained green (Miller et al., 1999). When we probed comparable aged leaves from plants in O3-treated or control environments, we found that seven of 11 SAGs were induced (Fig. 20-1).

The gene products of two SAGs induced by O3 have in common metal binding functions, namely copper chaperone (CCH) and blue copper-binding protein (BCB). These proteins may be important in the recycling of essential metals once the leaf is targeted for senescence, or may act as protectants against metal-catalyzed oxidation that leads to generation of free radicals (Weaver etal., 1997; Himelblau etal., 1998). Himelblau etal. (1998) have reported that 0.80 ^LL-1 O3 increased mRNA levels of CCH by 30% in 30 min. We found that CCH was induced after 6 days of the chronic exposure described above (Miller et al., 1999). CCH is a functional homologue of the Anti-oxidant 1 (ATX1) yeast gene, a gene identified for its ability to protect against oxygen toxicity in yeast lacking superoxide dismutase (Himelblau etal., 1998).

BCB expression was induced within 2 days of the chronic O3 exposure (Miller et al., 1999). In another report, Richards etal. (1998) treated Arabidopsis with 0.30 ^LL-1 O3 for 3 and 6 h and were able to demonstrate a 2-3-fold increase in transcript level of BCB. BCB is membrane bound with extensive similarity to blue Cu2+-binding proteins plastocyanin and stellacyanin, and likely functions as an electron carrier (Van Gysel et al., 1993). Retention of electron transport capabilities may be important during senescence for completion of degradative processes and maximum remobilization of ions (Weaver et al., 1997).

ERD1, a protease regulator, encodes a ClpC-like protein that may interact with the ATP-dependent ClpP protease to facilitate proteolysis in the chloroplast (Weaver et al., 1997) and was induced within 2 days of O3 treatment (Miller et al., 1999). More rapid decline in Rubisco levels during O3-induced accelerated leaf senescence is attributed to proteolysis

Ethylene And Senescence

Figure 20-1. Ozone (O3) induction of leaf senescence in older tissue. Reactive oxygen species (ROS), either directly or through induction of secondary signals, e.g. ethylene, abscisic acid (ABA), calcium, salicylic acid (SA), initiate a senescence program characterized by repression of photosynthesis-associated genes (PAGs) and induction of senescence-associated genes (SAGs) (Weaver et al., 1997). Resultant physiological and biochemical effects are associated with impacts on younger foliage in the plant.

Figure 20-1. Ozone (O3) induction of leaf senescence in older tissue. Reactive oxygen species (ROS), either directly or through induction of secondary signals, e.g. ethylene, abscisic acid (ABA), calcium, salicylic acid (SA), initiate a senescence program characterized by repression of photosynthesis-associated genes (PAGs) and induction of senescence-associated genes (SAGs) (Weaver et al., 1997). Resultant physiological and biochemical effects are associated with impacts on younger foliage in the plant.

(Brendley and Pell, 1998; Eckardt and Pell, 1994). It is not known whether ERD1 is involved in this response since the protein targets of ClpP are not known (Desimone et al., 1998). SAG12, a gene that codes for a protease, was not induced by O3 (Miller et al., 1999). All proteases may not participate in O3-induced accelerated senescence or may be induced at different times during the process.

SAG21 is similar to members of the late embryogenesis abundant (Lea) gene family (Weaver et al., 1998), a family of proteins that protect developing seeds from dehydration. In our experiments, SAG21 was strongly induced within 2 days of O3 treatment. If SAG21 has a similar protective function as Lea proteins, it might protect cells from damage during senescence (Weaver et al., 1997), including O3-induced senescence.

Two other genes, SAGs 18 and 20, were induced by O3 after 6 days of exposure (Miller et al., 1999). SAGs 18 and 20 show no strong similarity to other sequences in the database, although SAG20 did show weak similarity to a potato wound-induced gene (Weaver et al., 1998).

In the studies of Miller etal. (1999), SAG13 was one of the first genes detected after 2 days of O3 exposure. There is currently no known function of SAG13 in senescence, although the gene does have sequence similarity to short-chain alcohol dehydrogenases (Weaver et al., 1997). Gan and Amasino (personal communication) have transformed Arabidopsis with a SAG13 promoter-GUS fusion construct. When these plants were treated with O3, GUS expression was detected within the same time period as was the detection of the SAG13 transcript (Miller et al., 1999). The O3-induced expression of SAG13-driven GUS activity was observed throughout the leaf. In contrast, SAG13-driven GUS activity in control tissue was first observed only along the leaf margins, in the same location where senescence-associated chlorosis initially was observed. The spatial difference between O3-induced and normal expression of SAG13 suggests that the O3 response, at least in part, differs from that of natural senescence.

While transcripts for many SAGs were expressed early in response to O3, this was not a universal response. As discussed above, SAG12 was not detected in either O3-treated or control samples during the 14 day exposure; a similar observation was made for SAG19 (Miller et al., 1999). Atgsr2, a cytosolic isoform of glutamine synthetase, is important for the assimilation of nitrogen and increases in senescing and dark-treated Arabidopsis leaves (Bernhard and Matile, 1994); however, Atgsr2 was not induced by O3 treatment (Miller et al., 1999). MT1 is a SAG able to restore copper resistance in metallothionein-deficient yeast (Weaver et al., 1997). Transcript levels for MT1 increased in abundance in both O3-treated and non-treated leaves. Metallothioneins are thought to protect cells from the toxic effects of metals by binding and sequestering or removing the metals from undesirable locations, such as in the nucleus where sensitive DNA is located (Chubatsu and Meneghini, 1993). There are several explanations for the apparent absence of O3-induced expression of some SAGs. It is possible that the genes in question, which are induced later during normal senescence, might have exhibited an O3-accelerated response had sampling occurred later. Alternatively, plants at the age studied may have produced sufficient gene product to cope with the added O3 stress or in cases where a SAG was part of a multi-gene family, other members of the gene family may have been induced by O3.

It is likely that O3 elicits a signal(s) that is necessary for the subsequent induction of SAGs. It is well known that O3 treatment is associated with the emission of ethylene from leaves (Tingey et al., 1976). More recently there have been reports that O3 can induce genes involved in ethylene biosynthesis including the rapid induction of ACC oxidase in tomato (Lycopersicon esculentum Mill.) (Tuomainen et al., 1997) and the induction of

ACC synthase in potato (ACS4 and ACS5), tomato (LE-ACS2) and Arabidopsis (ACS6) (Schlagnhaufer et al., 1995, 1997; Tuomainen et al., 1997; Vahala et al., 1998). In experiments described above, O3 did induce ACS6, but the transcript appeared later than did the transcript for some of the SAGs, namely, SAG13, ERD1, BCB and SAG21, and concurrent with SAG18, SAG20 and CCH (Miller etal., 1999). Ethylene may play a role as a secondary signal in the O3 response since this hormone can induce the appearance of SAG13, SAG20, SAG21, BCB and ERD1 transcripts (Weaver et al., 1998).

There are many mutants of Arabidopsis that lack ethylene perception, due to mutations in receptors or in down stream signaling components, e.g. etr1-3 (Guzman and Ecker, 1990). No difference in SAG expression was found between the Columbia ecotype of Arabidopsis and etr1-3, following O3 treatment (Miller, Arteca and Pell unpublished data). This work suggests that ethylene perception by the etr1-3 receptor is not required for induction of the O3-responsive SAGs. The role of ethylene perception cannot be completely ruled out, due to the redundancy of ethylene perception in Arabidopsis. Other ethylene insensitive mutants are being studied to better define the role of this hormone in O3-induced SAG expression (Miller, Arteca and Pell, unpublished data).

The potential for other hormones to act as signals in the induction of O3 -responsive SAGs remains to be examined. Abscisic acid (ABA), for example, is known to induce ERD1 and SAG13 (Weaver et al., 1998). Since O3 can induce stomatal closure (Pearson and Mansfield, 1993) and accelerate the rate of foliar abscission (Pell et al., 1995), involvement of ABA is possible. Salicylic acid is induced during exposures to high O3 concentrations and is involved in the induction of antioxidant gene expression and cell death (Rao and Davis, 1999; Sharma et al., 1996; Yalpani et al., 1994). However, the importance of salicylic acid in regulating the response to O3 at concentrations that induce accelerated senescence is not known.

Calcium serves as a secondary messenger in many signal transduction pathways and changes in cytosolic calcium have been associated with senescence (Huang et al., 1997). There are several reports that O3 can induce changes in cytosolic calcium levels (Castillo and Heath, 1990; Clayton et al., 1999). Clayton et al. (1999) report an increase in transcript accumulation of glutathione-S-transferase (GST), in conjunction with the second of two O3-induced calcium peaks, suggesting a potential role of this cation as a signal. Whether changes in cytosolic calcium are necessary for induction of SAGs remains to be determined.

Oxidative stress may serve a signaling function in the induction of SAGs through generation of ROS, as discussed earlier in this chapter. Aluminum, which can also induce oxidative stress, increased BCB transcript levels (Richards et al., 1998). The ROS generated during an O3 exposure could directly regulate gene expression. Schubert et al. (1997) demonstrated that a specific region in the promoter of stilbene synthase, an enzyme responsible for synthesis of the secondary grape metabolite resveratrol, was required for induction by O3 but not by a biological pathogen.

In recent years, sugars, including glucose and sucrose, have been identified as regulatory signals in a variety of metabolic processes including photosynthesis (Lalonde et al., 1999). Ono and Watanabe (1997) have suggested that when sugar levels increase in the leaf, photosynthetic proteins may decline more rapidly. This may be related to a sugar-induced repression of protein synthesis. There is evidence that when plants are treated with O3, foliar carbohydrates can accumulate (Lux etal., 1997). Fialho and B├╝cker (1996) found transient increases in the levels of several sugars including glucose, fructose and sucrose in leaves of black poplar (Populus nigra cv. Loenen) treated with O3 and SO2 when compared with control tissue. The mechanism by which carbohydrates accumulate in the leaf in response to

O3 is not understood, although it has been proposed that phloem loading may be inhibited (Darrall, 1989). However, a mechanistic linkage between O3-induced accumulation of sugars, down regulation of photosynthesis-associated genes, and accelerated senescence is worthy of further consideration.

The hallmarks of programmed cell death (PCD) including DNA laddering and TUNEL positive cells, were found in naturally senescing leaves of five different plant species (Yen and Yang, 1998). There is evidence that relatively high concentrations of O3 leading to hypersensitive-like lesions can induce PCD in hybrid poplar based upon the appearance of TUNEL positive cells (Koch and Davis, personal communication). Markers of PCD have not been studied during the response to low doses of O3 in the induction of accelerated leaf senescence.

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    How ethylene induces senescence?
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