Discussion Directory UMM :Data Elmu:jurnal:E:Environmental and Experimental Botany:Vol44.Issue2.Oct2000:

3 . 4 . 2 . Time course of soluble protein content Time-course of total soluble proteins in barley flag leaves Fig. 3 was very similar to that of chlorophyll. Concentrations were in general lower when the plants were grown under CO 2 enrich- ment. Under ambient CO 2 , a reduction of protein contents to 50 of maximum was achieved on JD 197.5, whereas at 650 mmol mol − 1 CO 2 protein content was reduced to 50 already on JD 196. Thus, protein breakdown appeared to be acceler- ated by CO 2 enrichment, but was somewhat less affected than chlorophyll disassembling. 3 . 4 . 3 . Degradation of RubisCO LSU, SSU, and cytochrom b 559 LSU breakdown was clearly accelerated under CO 2 enrichment Figs. 4 and 5. No LSU could be detected any longer on JD 195, and afterwards, in plants grown under 650 mmol mol − 1 CO 2 , whereas flag leaves from plants grown under am- bient CO 2 still had nearly maximum LSU con- tents at that time. Decline of the SSU level differed signficantly from that of LSU. At ele- vated CO 2 concentrations, degradation of SSU started only after JD 192. On JD 198, there was still some SSU 6 of maximum contents de- tectable. At ambient CO 2 , no clear degradation of SSU towards the end of the sampling period could be observed. Cyt b559 was longer detectable than LSU, but its level started to decline some- what earlier than the level of SSU. Opposite to LSU and SSU, levels of Cyt b559 appeared to decline somewhat more rapidly at ambient CO 2 than at elevated CO 2 .

4. Discussion

Biomass and yield of barley crops increased by 38 when grown under 650 instead of 366 mmol mol − 1 CO 2 which resulted solely from an en- hancement of tiller production and tiller survival. This CO 2 -fertilization is lower than reported in previous studies. Poorter et al. 1996 compiled data from various CO 2 enrichment experiments and cite one study for H. 6ulgare where an in- crease in biomass of 104 occurred. However, only plants that were not in the reproductive phase were included into that data compilation. Thus, limitations occurring at later stages of de- velopment nutrients, competition for light were not included in the evaluation. In a season-long study under CO 2 enrichment, Weigel et al. 1994 found increases in spring barley yield of up to 89. However, these authors used plants in small pots with a low plant density. This might cause an over-estimation of CO 2 effects due to a lack of LAI limitations Ko¨rner, 1995. The results pre- sented in this study were obtained under condi- tions closely resembling field conditions. Thus, the 38 increase in yield appears to be realistic. Plants from the high fertilizer treatment were not subjected to nitrogen limitation, as 1 there was no increase in tissue nitrogen concentrations when nitrogen supply was increased from 8 to 14 g N m − 2 compare Table 2, 2 the crops fertil- ized with 14 g N m − 2 did not take up all of the N applied total nitrogen amount in the crop at final harvest: 13.3 g m − 2 , whereas crops receiving only 8 g N m − 2 had a higher nitrogen content on an area basis 11.2 g m − 2 than was applied. In spite of sufficient nitrogen being available at least in the high N fertilizer treatment, CO 2 Fig. 3. Concentrations of total soluble protein during flag leaf senescence in spring barley crops exposed to ambient or elevated CO 2 and fertilized with 140 kg N ha − 1 . Data are from four flag leaves randomly selected from three replicate OTC at each harvest date, respectively. Fig. 4. Contents of different proteins relative units per cm 2 leaf area during flag leaf senescence in spring barley crops exposed to ambient or elevated CO 2 and fertilized with 140 kg N ha − 1 . Data are from four flag leaves randomly selected from three replicate OTC at each harvest date, respectively. LSU, large subunit of RubisCO; SSU, small subunit of RubisCO; Cyt b559, cytochrome b559. enrichment significantly depressed nitrogen con- centrations found in the tissues. In a recent data compilation, CO 2 enrichment has been found to decrease the nitrogen concentration in green leaves of non-woody C 3 plants by 17, and in litter by 9 Cotrufo et al., 1998. We found even greater reductions 40 in straw at high nitrogen supply, 27 in grains which compares well to earlier findings in cereals McKee and Woodward, 1994; Manderscheid et al., 1995; Fangmeier et al., 1997. Several hypotheses have been proposed to ex- plain the reduced tissue nitrogen concentrations under CO 2 enrichment Conroy and Hocking, 1993. The most likely candidates to explain these findings are: 1 an optimisation of the photosyn- thetic apparatus in plants grown under high CO 2 concentrations, by which less nitrogen is invested in RubisCO and more nitrogen is allocated to RuBP-regeneration and to P i -regeneration Sage et al., 1989; Webber et al., 1994; Moore et al., 1999 though this type of acclimation does not always take place and is less obvious in plants well supplied with nitrogen Theobald et al., 1998; Stitt and Krapp, 1999, and 2 a reduction of the photosynthetic carbon oxidation PCO pathway, and thereby a reduction of the requirement for PCO enzymes, under altered CO 2 O 2 partial pres- sure relations Sharkey, 1988; Webber et al., 1994. Consequently, both a reduction in Ru- bisCO contents which represents up to 60 of soluble leaf protein Jacob et al., 1995; Moore et al., 1999, and of PCO enzymes Fangmeier and Ja¨ger, 1998 has often been observed in plant leaves grown at elevated CO 2 . The phenological development of the barley crops before grain filling was not responsive to CO 2 enrichment. Dates of plant emergence, of terminal spikelet formation, of flag leaf appear- ance and of anthesis were identical at either treat- ment. Nevertheless, flag leaf senescence was significantly affected by CO 2 enrichment. En- hancement of flag leaf senescence was detectable from both estimations of chlorophyll degradation and of the contents of soluble proteins. The latter estimations suggest an earlier protein breakdown and re-allocation away from the leaves. Among the three plastid proteins assessed in our study, the amounts of the LSU of RubisCO declined first. Contents of Cyt b559 clearly declined later than LSU. SSU kept rather high amounts until the end of the harvest period. On JD 205, i.e. 27 days after anthesis, still 31 of maximum SSU contents were detected at ambient CO 2 . The lack of coordination in the decline of RubisCO sub- units has also been observed with flag leaves of field-grown barley crops Humbeck et al., 1996. CO 2 enrichment accelerated the decline of both LSU and SSU contents by approximately four days. In contrast, the decline of Cyt b559 ap- peared to be somewhat delayed under CO 2 enrich- ment. This might be related to a possible acclimation effect of CO 2 enrichment on the pho- tosynthetic apparatus. However, the data are to scarce to draw further conclusions. Earlier senescence under CO 2 enrichment has been shown in many other studies with annual C 3 crops. Miller et al. 1997 exposed tobacco to 350 or 950 mmol mol − 1 CO 2 and followed leaf CO 2 exchange over the course of leaf development. The authors observed an earlier achievement of maximum photosynthesis rates at elevated CO 2 concentrations, but also a faster progress of devel- opment and an earlier beginning of senescence. From their data, Miller et al. 1997 conclude that faster leaf development under CO 2 enrichment might explain the often observed photosynthetic acclimation to elevated CO 2 . This statement is in part supported by findings from wheat exposure to CO 2 enrichment Sicher and Bunce, 1998 where also an earlier flag leaf senescence was observed. The authors state that premature Fig. 5. Immunoblot analyses of plastid proteins during flag leaf senescence in spring barley crops exposed to ambient or elevated CO 2 and fertilized with 140 kg N ha − 1 . Abbreviations as shown in Fig. 4. senescence contributed to decreased photosyn- thetic rates at elevated CO 2 concentrations. There is further experimental evidence for a faster de- crease of photosynthetic properties and of en- zymes of the photosynthetic apparatus as a result of CO 2 exposure. Sicher and Bunce 1997 found decreased RubisCO contents and photosynthesis rates, and an acceleration of senescence, in wheat and barley leaves grown in elevated CO 2 , and Garcia et al. 1998 observed a decline in flag leaf photosynthesis and an earlier senescence at final stages of wheat crop development under free air CO 2 enrichment. We do not believe that a general enhancement of leaf development and, thus, an earlier onset of leaf senescence can be used as a simple explana- tion of photosynthetic down-regulation due to CO 2 enrichment. Functional and molecular changes, and the redistribution of nutrients, oc- curring during senescence are too complex for such a simple explanation. Rather, one must search for a signal triggering senescence which is affected due to growth at elevated CO 2 concentrations. In monocarpic annual species like barley or wheat, a rather rapid transition takes place from a vegetative ‘green‘ plant acquiring CO 2 from the atmosphere and other nutrients from the soil, to a ‘generative‘ plant which does not acquire soil nutrients at significant amounts any longer be- cause of the breakdown of the root system Fangmeier et al. 1996, but only redistributes these from vegetative tissues to the generative organs, i.e. to the grains, and which gradually reduces carbon acquisition from the atmosphere. As stated above, CO 2 enrichment reduces the nitrogen demand of green tissues by several mech- anisms, causing an increase in nitrogen use effi- ciency. In our study and in hardly any previous experiment with C 3 plants, growth at elevated CO 2 concentrations resulted in higher biomass Poorter et al., 1996 and, finally, in greater yield, irrespective of possible down-regulation of the photosynthetic apparatus at leaf level. For the whole plant, yield increase of the barley crops occurred due to increased tillering. Because of increased nitrogen use efficiency under CO 2 en- richment, nitrogen acquisition of the crops did not keep pace with carbon gain. Rather, crop nitrogen uptake was dependent on nitrogen sup- ply but did not respond to CO 2 treatment. In cereals, nearly all the nitrogen resources required for grain filling originate from vegetative tissues and there is hardly any further uptake of nitrogen from the soil after anthesis Van Kraalingen, 1990; Fangmeier et al., 1999. Thus, the grains in plants grown at elevated CO 2 concentrations, al- though they had 48 more biomass at 140 kg ha − 1 of N fertilization, had to cope with the same amount of nitrogen in vegetative pools as the grains from ambient CO 2 . There is no reason to assume that CO 2 enrichment reduces the nitro- gen demand of grains, since none of the mecha- nisms reducing nitrogen demand in green tissues is working in grains. Rather, for optimal grain vitality and ecological quality, a certain amount of grain proteins is required and monocarpic plants in particular have been selected to achieve this protein content in order to ensure survival of the population. Thus, we believe that the higher nitrogen sink capacity of the growing population of grains un- der CO 2 enrichment works as a trigger to induce nitrogen release from the leaves and can explain the earlier senescence of barley flag leaves ob- served in our study. Nitrogen deficiency has been proved to induce senescence in previous studies Smart, 1994; Noode´n et al., 1997. In this case, CO 2 enrichment works as a tool to induce nitro- gen deficiency as ’seen‘ by the grains in late developmental stages, though it does not cause a true deficiency in green tissues, in spite of lowered concentrations, at earlier developmental stages when the leaves act as carbon sources. Our study was not designed to clarify the molecular mechanisms initiating the senescence process. It is clear, however, that developing seeds represent the most important sink for nutrients in monocarpic plants and that the onset of fruit development must be regarded as the most impor- tant event initiating senescence Noode´n et al., 1997. For survival of the populations of mono- carpic species, the nutrient salvage for the off- spring involved by the senescence process appears to be most important, and the death of the leaves might be regarded as an inevitable by-product Bleecker, 1998.

5. Conclusions