Nitrogen resorption and protein degradation ... - CSIRO Publishing

CSIRO PUBLISHING

Functional Plant Biology, 2007, 34, 409–417

www.publish.csiro.au/journals/fpb

Nitrogen resorption and protein degradation during leaf senescence in Chenopodium album grown in different light and nitrogen conditions Yuko YasumuraA,B,D , Kouki HikosakaA and Tadaki HiroseA,C A

Graduate School of Life Sciences, Tohoku University, 6-3 Aoba, Aramaki, Sendai 980-8578, Japan. Current address: Department of Plant Ecology, Forestry and Forest Products Research Institute (FFPRI), Tsukuba, Ibaraki 305-8687, Japan. C Current address: Department of International Agriculture Development, Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156-8502, Japan. D Corresponding author. Email: [email protected] B

Abstract. The extent of nitrogen (N) resorption and the degradability of different protein pools were examined in senescing leaves of an annual herb, Chenopodium album L., grown in two light and N conditions. Both N resorption efficiency (REFF ; the proportion of green-leaf N resorbed) and proficiency (RPROF ; the level to which leaf N content is reduced by resorption) varied among different growth conditions. During leaf senescence, the majority of soluble and membrane proteins was degraded in all growth conditions. Structural proteins were also highly degradable, implying that no particular protein pool constitutes a non-retranslocatable N pool in the leaf. Leaf carbon/N ratio affected the timing and duration of senescing processes, but it did not regulate the extent of protein degradation or N resorption. Sink–source relationships for N in the plant exerted a more direct influence, depressing N resorption when N sink strength was weakened in the low-light and high-N condition. N resorption was, however, not enhanced in high-light and low-N plants with the strongest N sinks, possibly because it reached an upper limit at some point. We conclude that a combination of several physiological factors determines the extent of N resorption in different growth conditions. Additional keywords: C/N ratio, membrane protein, sink–source relationships, soluble protein, structural protein.

Introduction Nitrogen (N) resorption from senescing leaves, a series of events associated with mobilisation and translocation of leaf N, is one of the most important processes regulating the N economy of plants. It has been shown that N resorption provides a substantial amount of N to developing tissues (May and Killingbeck 1992; Eckstein et al. 1998) and enables growth even at times of low N uptake from the soil (Dong et al. 2001; Grassi et al. 2002). Efficient N resorption thus leads to higher N-use efficiency (Eckstein et al. 1999; Aerts and Chapin 2000). Plants, however, do not resorb all the N from a senescing leaf. For example, forbs resorb only approximately 40% of leaf N during leaf senescence (the average for 33 species; Aerts 1996). The extent of N resorption is often evaluated by two different but complementary parameters; the proportion of green-leaf N that is resorbed during senescence (N resorption efficiency, REFF ), and the amount of N remaining in the dead leaf after resorption has taken place (N resorption proficiency, RPROF ; Killingbeck 1996). While REFF focuses on the ability of the plant to retrieve N initially invested in the leaf, RPROF focuses on the ability to minimise N loss associated with leaf abscission. RPROF is regarded as high when N resorption reduces leaf N concentration © CSIRO 2007

to a low level (i.e. dead-leaf N concentration is low), irrespective of the initial leaf N concentration. Both REFF and RPROF can vary within the same species. Many studies examined variation across a gradient of environmental factors, but they did not find a consistent pattern. For example, REFF increased (Boerner 1986; Nambiar and Fife 1987), decreased (Boerner 1984; Shaver and Melillo 1984) or did not change (Helmisaari 1992; Aerts and De Caluwe 1994) with increasing soil N availability. Similarly, REFF and RPROF did not respond consistently to water status (Pugnaire and Chapin 1992), CO2 concentration (Norby et al. 2000) or growth irradiance (Yasumura et al. 2005, 2006). As no single environmental factor is likely to control N resorption, it is important to focus on physiological processes that take place during N resorption to gain a better understanding of intraspecific variation across environmental factors. In a leaf, the majority of N is associated with protein. Leaf proteins must be converted to amino acids before their N can be translocated from the leaf (Stoddart and Thomas 1982). Therefore, the extent of N resorption may be determined by the degradability of leaf proteins (Aerts 1996), which is related to the accessibility of substrate proteins as well as

10.1071/FP06307

1445-4408/07/050409

410

Functional Plant Biology

the levels of proteolytic activities (Hörtensteiner and Feller 2002). Leaf proteins can be divided into two functional pools; soluble and insoluble proteins (Makino et al. 2003). It has been suggested that soluble proteins are far more degradable and thus contribute more to N resorption than insoluble proteins (Pugnaire and Chapin 1993). Soluble proteins, however, do not always fully explain the amount of N resorbed from a leaf (Millard and Thomson 1989; Pugnaire and Chapin 1993), and therefore insoluble proteins should also contribute some N to resorption. Insoluble proteins can further be divided into two functional pools: membrane and structural proteins (Takashima et al. 2004). Membrane proteins are mostly associated with thylakoid membranes, and they are indeed degradable (Huffaker 1990). In contrast, structural proteins that are associated with cell walls are thought to be non-degradable and non-retranslocatable (Anten and Werger 1996; Hikosaka 2003; Niinemets and Tamm 2005). However, it remains unclear to what relative extent soluble, membrane and structural proteins are degraded in senescing leaves. In fact, these protein fractions have rarely been distinguished explicitly from one another in previous studies. Environmental factors such as light and soil N availability affect protein composition of the leaf (Terashima and Evans 1988; Evans 1989; Hikosaka 1996). For example, high light and high N increase the proportion of soluble proteins in Spinacia oleracea (Evans 1989). These environmental factors may affect the extent of N resorption by modifying the composition of leaf protein with different degradability. Light and soil N availability are also expected to affect other physiological factors related to N resorption processes; carbon-to-N (C/N) ratio of individual leaves and sink–source relationships for N at the whole-plant level. Leaf C/N ratio plays a role in the regulation of leaf senescence (Pourtau et al. 2006; Wingler et al. 2006), with high C/N triggering a decline in photosynthetic rates and degradation of photosynthetic proteins (Paul and Driscoll 1997). Sink–source relationships affect the rate of matter translocation in the phloem (Marschner 1986), and the rate of phloem loading and transport can limit removal of N from senescing leaves (Stoddart and Thomas 1982; Pugnaire and Chapin 1992). C/N ratio and the size of N sink relative to N source would increase in high light that stimulates photosynthesis and plant growth. In contrast, C/N ratio and the size of the N sink would decrease with abundant N supply, if photosynthesis and growth are not enhanced in proportion to plant N uptake. In the present study, we examined the extent of N resorption and the degradability of soluble, membrane and structural proteins in senescing leaves of an annual herb, Chenopodium album, grown in two light and soil N conditions. We also evaluated how C/N ratio of the leaf and sink–source relationships for N in the plant are related to the extent of protein degradation and N resorption during leaf senescence. Materials and methods Growth condition Chenopodium album L. is an erect annual herb that favours Nrich environments. Seeds were sown in 1.4-L pots filled with washed river sand and placed in a greenhouse on 2 May 2002. All plants were grown at the same high irradiance (90% of natural sun light at the top of the canopy) and fertilised with

Y. Yasumura et al.

50 mL of nutrient solution containing 12 mM NO− 3 and other necessary elements (for details of nutrient solution composition see Hikosaka et al. 1994) twice a week from 25 May until 18 June. On 22 June, plants were randomly allotted to one of the following growth conditions: high light and high N (HL-HN); high light and low N (HL-LN); low light and high N (LL-HN); and low light and low N (LL-LN). High-light plants were continuously grown at high irradiance, whereas low-light plants were shaded with shade cloth (30% of natural sun light). From 22 June, high-N plants were fertilised with a high-N solution − (HN; 12 mM NO− 3 until 29 July and 24 mM NO3 thereafter) and low-N plants were with a low-N solution (LN; 3 mM NO− 3 ) twice a week until death. Destructive harvesting of the whole plant Plants (n = 5) were harvested on 21 June, 23 July, 22 August and 22 September and additionally on 2–27 November when they died (Fig. 1). Samples were dried at 70◦ C for at least 72 h, then weighed and ground before their N concentration was determined with an NC analyser (Sumigraph NC-80, Sumika Chemical Analysis Service Ltd, Tokyo, Japan) connected to a gas chromatograph (GC-8A, Shimadzu Ltd, Kyoto, Japan). The whole-plant N concentration (PNC) incorporates dry mass of organs that require N (the size of N sink) and the amount of N taken up from the soil and available for remobilisation (the size of N source). Therefore we used PNC as an indicator of N sink strength relative to source. Leaf sampling During their lives, C. album plants produced approximately 35 leaves during growth. Older leaves senesced and died gradually as they were shaded by new leaves. N resorption and protein degradability were determined in the 10th, 18th and 22nd (low-N treatments) or 25th (high-N treatments) leaves that were produced and abscised at different times during the

22/25

High light 18

10

Death

Flowering 22/25

Low light 10

18

Death

Flowering M

J

J

A

S

O

N

Fig. 1. Phenology of Chenopodium album plants and their leaves grown in different light conditions. Seeds were sown on 2 May 2002 and grown in high light until 22 June when half of the plants were transferred to low light. Bold lines, plants; thin lines, the 10th, 18th and 22nd/25th leaves. ( ), The onset of flowering; arrowheads, dates of destructive harvesting of the whole plant; ( ), dates of leaf sampling.

◦

•

N resorption and protein degradation during leaf senescence in C. album

growing season (Fig. 1). Leaf C/N ratio and nitrate content were determined in green leaves adjacent to these target leaves, assuming that they were at a similar physiological state. Green leaves (n = 5) were sampled on the same dates as destructive harvesting of the whole plant. Dead leaves (n = 5) were collected as soon as they were abscised. Determination of leaf C/N ratio and nitrate content of leaves adjacent to target leaves Leaf C/N ratio was calculated from carbon and N concentration determined with the NC analyser. Nitrate was extracted from dry ground samples in purified water at 45◦ C for 3 h. The extracts were mixed first with 5% salicylic acid in H2 SO4 and then with 2 M NaOH, and the absorbance was read with a spectrophotometer (Cataldo et al. 1975). Determination of dry mass, chlorophyll, N and protein content of the target leaves Immediately after sampling, we punched out discs of 0.8 cm diameter from the target leaves using a leaf punch (FujiwaraSeisakujo, Tokyo, Japan). Leaf mass per area (LMA; n = 5) was calculated as a mass/area ratio of leaf discs dried at 70◦ C for at least 72 h. N content (n = 5) was determined on the same discs with the NC analyser. Chlorophyll (Chl; n = 3) was extracted from a fresh disc in 3 mL of dimethylformamide and determined spectrophotometrically (Porra et al. 1989) except for the dead 25th leaves of HL-LN plants, which were too small to punch out enough discs. Protein content (n = 3) was determined with leaf discs stored at −80◦ C, except for the dead 25th leaves of HL-LN plants. Leaf protein was divided into three different pools according to solubility in the extraction buffer and the detergent SDS (Takashima et al. 2004). Frozen leaf discs were powdered in liquid N in a mortar with a pestle and homogenised in 100 mM Na-phosphate buffer (pH 7.5) containing 0.4 M sorbitol, 2 mM MgCl2 , 10 mM NaCl, 5 mM iodo-acetate and 10 mM DTT. The homogenate was centrifuged at 18 000g for 30 min and its supernatant was used for determination of soluble proteins. The pellet was suspended in the buffer with 1% (w/v) SDS, heated at 90◦ C for 5 min and centrifuged at 8000g for 15 min. The resultant supernatant and pellet were used for determination of membrane and structural proteins, respectively. Soluble and membrane proteins were precipitated with 10% (w/v) and 17.5% (w/v) trichloroacetic acid (TCA), respectively. Structural proteins were washed with ethanol. After hydrolysis with Ba(OH)2 and purified water in an autoclave (120◦ C, 0.12 MPa) for 30 min, protein contents were determined by the ninhydrin method (McGrath 1972). BSA was used as the protein standard. Definition of N resorption efficiency and proficiency REFF is the proportion of leaf N resorbed during leaf senescence (Aerts 1996): REFF = (green-leaf Narea − dead-leaf Narea )/green-leaf Narea , where Narea is N content per leaf area. For calculation, we chose green-leaf data on 23 July for the 10th leaves, and those on 22 August for the 18th and 22nd/25th leaves. Based on their N and Chl content, we considered that the leaves were nearest


411

to their physiological maturity on these sampling dates. RPROF is represented by the amount of N remaining in dead leaves (Killingbeck 1996). We regard low values of dead-leaf N content per unit mass (Nmass ) as high RPROF . Statistical analyses Statistical tests were performed with StatView software version 5.0 (SAS Institute, Inc., Cary, NC, USA). Differences among treatments were first analysed with ANOVA, and then by the Tukey–Kramer test for multiple comparisons. The data for proportional protein degradation and REFF were arcsinetransformed before statistical analysis. Results Plant growth and N status as affected by growth conditions Light conditions affected the phenology of plant growth (Fig. 1). In low light, individual leaves survived longer on the plant, and the onset of reproductive growth (flowering) and the whole plant death were delayed. Soil N availability affected the phenology to a lesser extent, but plants in low N tended to abscise leaves and die earlier than those in high N (data not shown). Both light and N conditions affected plant mass (Fig. 2a). As high light and high N stimulated growth, HL-HN and LL-LN plants had the greatest and least mass, respectively, throughout the experimental period. In all growth conditions plant mass decreased at the end of the season when all of their leaves and axillary leaves were abscised. Plants took up most of the N applied to the soil during the phase of vegetative growth (Fig. 2b) and therefore their N content was primarily determined by soil N condition and only slightly affected by light. PNC varied considerably among growth conditions (Fig. 2c). LLHN plants had by far the highest PNC, whereas HL-LN plants had the lowest PNC. HL-HN and LL-LN plants had similar, moderate N concentrations. PNC values suggest that plants had different N sink–source relationships, with LL-HN and HL-LN plants having the lowest and highest N sink strength, respectively. At seed maturation, reproductive tissues had similar N concentrations (2.3–2.8% dry mass) in all growth conditions (data not shown). Characteristics of individual leaves before and after senescence C/N ratio of green leaves varied among different growth conditions (Fig. 3). HL-LN and LL-HN plants had the highest and lowest C/N ratio, respectively. LMA, Narea , Chl and nitrate content of target green leaves also varied among growth conditions (Table 1). LMA was very responsive to light availability and LMA of leaves in high light was more than twice as large as that of leaves in low light. A considerable amount of nitrate was detected only from LL-HN leaves. In dead leaves, LMA, Narea and Chl content were smaller than in green leaves (Table 2). With leaf senescence, LMA decreased by 25% (the average for all leaves) owing to resorption of dry matter and respiration, Narea decreased by 69% as a result of N resorption and Chl content decreased by 89% owing to degradation.

412


Y. Yasumura et al.

50

20

HL-HN HL-LN LL-HN LL-LN

b

40

15

a

b

a a

C/N ratio

Dry mass (g plant–1)

a

10

30

d

b

20

a a

b

5

c

c

10 0

N content (mgN plant–1)

b

0 10

200

22/25

Fig. 3. C/N ratio of green leaves adjacent to target leaves. Samples were taken on 23 July (leaves adjacent to the 10th leaves) and on 22 August 2002 (leaves adjacent to the 18th and 22nd/25th leaves). HL-HN, high light and high N; HL-LN, high light and low N; LL-NH; low light and high N; LL-LN, low light and low N. Error bars denote s.d. Different letters indicate a significant difference at P < 0.05 (ANOVA followed by Tukey–Kramer test).

150

100

50

0

c

HL-HN HL-LN

N concentration (%)

3

LL-HN LL-LN

2

1

0

J

J

A

S

O

N

Fig. 2. (a) Biomass, (b) N content, (c) N concentration of Chenopodium album plants grown in different light and N conditions. HL-HN, high light and high N ( ); HL-LN, high light and low N (); LL-NH; low light and high N ( ); LL-LN, low light and low N (N ). Error bars denote s.d.

•

18

Leaf position

◦

Leaf protein degradation during leaf senescence Under the assumption that leaf protein has average N concentration of 16% (Field and Mooney 1986), total protein content (see Table 1) would account for 58, 58, 65 and 66% of green-leaf Narea in HL-HN, HL-LN, LL-HN and LL-LN plants, respectively. The composition of leaf protein differed among growth conditions (Fig. 4). Plants in high light had a

higher proportion of soluble proteins and a lower proportion of membrane proteins than in plants in low light, and plants in high N had a lower proportion of structural proteins than plants in low N. During leaf senescence, the majority of leaf protein was degraded in all leaves examined (Fig. 5a). Membrane proteins were as highly degradable as soluble proteins (Fig. 5b, c). Structural proteins were degraded to a lesser extent, but they could also be degraded by up to 90% (Fig. 5d). Temporal changes in soluble, membrane and structural protein content were linearly correlated with those in Narea (P < 0.001, R2 > 0.75 in all cases; Fig. 6). In dead leaves, total protein content explained 40, 12, 18 and 11% of leaf N in HL-HN, HL-LN, LL-HN and LL-LN plants, respectively. Thus, the amount of protein did not increase in proportion to the amount of N remaining in dead leaves. The extent of N resorption from senescing leaves The extent of N resorption was highly variable; REFF ranged from 44 to 81% and RPROF (dead-leaf Nmass ) ranged from 0.46 to 2.60% dry mass (Fig. 7). Both REFF and RPROF were significantly affected by growth conditions, being lowest in LL-HN plants. RPROF was also lower in LL-LN plants than in HL-HN and HL-LN plants. In low light, REFF and RPROF were apparently reduced in the 22nd/25th leaves compared with the 10th or 18th leaves. Discussion The extent of N resorption did not show a consistent pattern with light or N availability (Fig. 7). For example, REFF and RPROF decreased with increasing N availability in low light (LL-HN v. LL-LN), but did not change in high light (HL-HN v. HL-LN).



413

Table 1. Characteristics of green leaves taken from different positions LMA, leaf mass per area; Narea , N content per leaf area. Data are means with ± s.d. HL-HN, high light and high N; HL-LN, high light and low N; LL-HN, low light and high N; LL-LN, low light and low N. The 10th leaves were sampled on 23 July, the 18th and 22nd/25th leaves on 22 August 2002. Different susperscript letters indicate significant differences at P < 0.05 (ANOVA followed by Tukey–Kramer test) Treatment

LMA (g m−2 )

Narea (g m−2 )

Chl (mmol m−2 )

Protein (g m−2 )

Nitrate-N (%)A

10


62.4 ± 9.5a 69.0 ± 6.0a 31.7 ± 2.4b 32.8 ± 2.1b

0.87 ± 0.08ab 0.78 ± 0.11a 0.85 ± 0.13a 1.04 ± 0.10b

0.27 ± 0.02a 0.21 ± 0.05a 0.57 ± 0.05b 0.50 ± 0.13b

3.32 ± 0.11a 3.12 ± 0.54a 4.39 ± 0.43a 4.55 ± 1.51a

0.001 ± 0.003a 0.001 ± 0.002a 0.015 ± 0.009b 0.001 ± 0.002a

18


75.2 ± 4.4a 77.6 ± 15.8a 28.4 ± 1.3b 35.5 ± 3.5b

1.40 ± 0.08a 0.91 ± 0.18b 1.14 ± 0.04c 0.95 ± 0.09bc

0.43 ± 0.04a 0.20 ± 0.01b 0.62 ± 0.03c 0.47 ± 0.05a

5.01 ± 0.36a 3.02 ± 0.40b 4.51 ± 0.06a 3.64 ± 0.49b

0.000 ± 0.000a 0.000 ± 0.000a 0.014 ± 0.016a 0.000 ± 0.000a

22/25


81.5 ± 3.8a 89.2 ± 3.7b 31.8 ± 4.0c 38.4 ± 5.2c

1.89 ± 0.14a 1.29 ± 0.10b 1.33 ± 0.11b 1.03 ± 0.04c

0.54 ± 0.06a 0.31 ± 0.05b 0.55 ± 0.04a 0.45 ± 0.02a

6.71 ± 0.74a 4.59 ± 0.76b 5.36 ± 0.58ab 4.29 ± 0.15b

0.000 ± 0.000a 0.000 ± 0.000a 0.103 ± 0.100b 0.000 ± 0.000a

Leaf position

A Nitrate

concentration was determined in leaves adjacent to the target leaves. Table 2. Characteristics of dead leaves taken from different positions Abbreviations are as in Table 1. Data means with ± s.d. Different superscript letters indicate significant differences at P < 0.05 (ANOVA followed by Tukey–Kramer test). na, data not available Treatment

LMA (g m−2 )

Narea (g m−2 )

Chl (mmol m−2 )

Protein (g m−2 )

10


43.2 ± 4.5a 46.9 ± 5.1a 26.8 ± 3.5b 23.9 ± 1.4b

0.26 ± 0.02a 0.23 ± 0.02ab 0.39 ± 0.08c 0.19 ± 0.01b

0.00 ± 0.00a 0.00 ± 0.00a 0.12 ± 0.05b 0.00 ± 0.00a

0.54 ± 0.10a 0.39 ± 0.12a 0.42 ± 0.06a 0.14 ± 0.02b

18


53.6 ± 6.7a 55.5 ± 5.3a 25.0 ± 3.0b 22.6 ± 2.8b

0.31 ± 0.03a 0.26 ± 0.01a 0.46 ± 0.11c 0.21 ± 0.03b

0.00 ± 0.00a 0.00 ± 0.00a 0.16 ± 0.04b 0.01 ± 0.00a

0.82 ± 0.17a 0.15 ± 0.01b 0.55 ± 0.10a 0.21 ± 0.09b

22/25


59.4 ± 6.9a 63.1 ± 4.8a 29.2 ± 2.6b 27.7 ± 1.2b

0.36 ± 0.04a 0.32 ± 0.01a 0.75 ± 0.10b 0.41 ± 0.14a

0.00 ± 0.00a na 0.30 ± 0.04b 0.05 ± 0.04a

0.97 ± 0.10a na 0.85 ± 0.08a 0.19 ± 0.03b

Leaf position

Protein composition

1.0

0.8

0.6

Soluble Membrane

0.4

Structural

0.2

0

HL-HN HL-LN LL-HN LL-LN Fig. 4. Protein composition in green leaves grown in different light and N conditions. HL-HN, high light and high N; HL-LN, high light and low N; LL-NH; low light and high N; LL-LN, low light and low N. Data of the 10th, 18th and 22nd/25th leaves are averaged for each growth condition.

The results again confirm that the environmental factors do not directly control the extent of N resorption (Aerts 1996). LL-LN plants had a significantly lower RPROF than HL-HN and HL-LN plants, even though their REFF was comparably high. Thus plants cannot always enhance REFF and RPROF at the same time. Low RPROF values seemed to be associated with low LMA in low light, where plants packed more N into a unit of leaf mass (i.e. high green-leaf Nmass ). REFF , however, seemed to be only minutely affected by such physiological changes in leaf traits. Total protein content accounted for 58–66% of N in green leaves. These values are smaller than generally reported (e.g. >75%, Evans and Seemann 1989). Protein content may have been underestimated by disregarding proteins that are soluble in TCA or that were broken down before precipitation with TCA. Alternatively, the assumption that all protein had N concentration of 16% may have not been appropriate. Nevertheless, we consider that the estimated value for proportional protein degradation is acceptable because protein contents would have been similarly underestimated in green and dead leaves.

414


Y. Yasumura et al.

a

Total protein

100 a

ab

b

c

b ab b

c a

Membrane protein c

b a

a ab

a

b

bc ac b

b

a

a

a

80 60

Proportion degraded (%)

40 20 0 10

18

22/25

Soluble protein

b 100

a a a

b

bc a

ac

10

a

a

80

22/25

Structural protein

d b

b

18

b b

ab

b b


b

a a a

a a

60 40 20 0 10

18

22/25

10

18

22/25

Leaf position Fig. 5. The proportions in which (a) total protein, (b) soluble protein, (c) membrane protein and (d ) structural protein were degraded in the 10th, 18th and 22nd/25th leaves in different light and N conditions. HL-HN, high light and high N; HL-LN, high light and low N; LL-NH; low light and high N; LL-LN, low light and low N. Error bars denote s.d. Different letters indicate a significant difference among growth conditions at P = 0.05 (Tukey–Kramer test).

Previous studies have presumed that soluble, membrane and structural proteins have completely different degradability (Aerts 1996; Killingbeck 2004; Kobe et al. 2005). In particular, structural proteins had been considered almost non-degradable (Hikosaka 2003; Niinemets and Tamm 2005). However, we found that soluble and membrane proteins were comparably degradable (Fig. 5b, c), and that even structural proteins could be degraded to a large extent (Fig. 5d) in C. album. Thus none of the protein pools limited the amount of N available for N resorption. The strong linear correlations between protein content and Narea (Fig. 6) suggest that soluble, membrane and structural proteins are all degraded concomitantly with the progress of N resorption. Interestingly, no such correlation between structural protein content and Narea was found in a deciduous woody species Lindera umbellata (Yasumura et al. 2006). In this species, structural proteins were much more resistant to proteolytic activities than in C. album. Degradability of structural proteins may be related to leaf structural properties that differ among leaf habits and life forms (Garnier and Laurent 1994; Castro-Diéz et al. 2000). We expect that structural proteins are less accessible and degradable

in long-lived leaves with more rigid structures than in short-lived leaves. Even though proteins comprise a major N pool in green leaves, they accounted for only a minor part of total N in dead leaves in C. album. RPROF was likely to be determined by the sum of various N compounds such as protein, amino acid, lipid, nucleic acid (Chapin and Kedrowski 1983) and chlorophyll catabolite (Matile 2000). In the case of LL-HN plants, part of the N may have been associated with nitrate accumulated in leaves (see Table 1). It has been suggested that the rise in leaf C/N ratio, caused by sugar accumulation and/or by low N supply, induces protein degradation and N resorption (Paul and Driscoll 1997; Pourtau et al. 2006; Wingler et al. 2006). Although C/N ratio differed markedly among different light and N conditions (Fig. 3), the extent of protein degradation was comparably high in all leaves (Fig. 5a). Neither did N resorption change with C/N ratio; e.g. REFF and RPROF were completely different between the 10th LL-HN and LL-LN leaves, which had similar C/N ratio. The results suggest that although leaf C/N ratio plays a role in initiating leaf senescence, it does not directly control the extent


4


a

100

a

80

a a

3

2

a

a

b

a

a

60

b

40

1 20

P < 0.0001 R 2 = 0.91–0.98

0

0

b

HL-HN

b

HL-LN

b

LL-HN

3

3

R PROF (% d.m.)

Membrane protein (g m–2)

a a

b

R EFF (%)

Soluble protein (g m–2)

a

415

2

1

LL-LN

b

2

c

b c

1

P < 0.001 R 2 = 0.87–0.97

a a

c a a

a

a

0 0

Structural protein (g m–2)

c

10

HL-HN HL-LN

22/25

Leaf position

LL-HN

3

Fig. 7. (a) Nitrogen resorption efficiency (REFF ) and (b) proficiency (RPROF ; dead-leaf N concentration) in the 10th, 18th and 22nd/25th leaves in different light and N conditions. HL-HN, high light and high N; HL-LN, high light and low N; LL-NH; low light and high N; LL-LN, low light and low N. Error bars denote s.d. Different letters indicate a significant difference among growth conditions at P = 0.05 (Tukey–Kramer test).

LL-LN

2 P < 0.001 R 2 = 0.76–0.91

1

0 0

0.5

1

1.5

2

–2

Leaf N content (g m ) Fig. 6. (a) Soluble protein, (b) membrane protein and (c) structural protein content as functions of leaf N content in leaves grown under different light and N conditions. Data of all green and dead leaves are combined for each growth condition. HL-HN, high light and high N ( ); HL-LN, high light and low N (); LL-LH; low light and low N ( ); LL-HN, low light and high N ( ).

•

18

•

◦

of protein degradation or N resorption. Leaves with higher C/N ratio tended to abscise earlier than leaves with lower C/N ratio (see Fig. 1), suggesting that leaf C/N ratio affects the rate of leaf senescence and timing of leaf abscission, which can indirectly affect the extent of N resorption (Killingbeck et al. 1990). As sink–source relationships affect the rate of phloem transport (Marschner 1986), the relative size of N sink is expected to determine the extent of N resorption (Aerts 1996).

Previous studies showed that removal of a strong N sink depressed N resorption (Crafts-Brandner and Egli 1987; Chapin and Moilanen 1991), while an increase in N sink size resulted in higher N resorption (Nambiar and Fife 1987; Miyazawa et al. 2004). In this study, REFF and RPROF were indeed depressed when N sink was weakened in LL-HN conditions, but they were not enhanced in HL-LN plants, which had a stronger N sink than HL-HN plants (Fig. 7). These results may imply that sink– source relationships influence the extent of N resorption within a certain range, but not beyond the upper limit reached by HLHN plants. In LL-HN and LL-LN plants, REFF and RPROF were depressed in the 22nd/25th leaves compared with the 10th and 18th leaves. This is also likely to be associated with the drop in N sink size with the onset of reproductive growth. Reproductive tissues, which had similar final N concentrations in different conditions, served as a weaker N sink than vegetative tissues that could take up and accumulate more N than they require for current growth (Rossato et al. 2001). In this study, we showed that the majority of leaf protein was degraded during leaf senescence in C. album. Soluble, membrane and structural proteins were all highly degradable,

416


and thus no specific protein pool constituted a recalcitrant N pool in the leaf. There was only a small amount of protein remaining in dead leaves. It remains unclear whether all degraded proteins were resorbed; some protein-N may have remained in the form of amino acids or non-translocatable peptides. The total amount of N resorption from senescing leaves is likely to be determined by a combination of physiological factors including enzymatic activities that convert proteins to mobile forms, leaf C/N ratio that triggers senescing processes, and sink–source relationships that affect the rate of phloem transport. Detailed study on deadleaf N composition and phloem transport would provide us with a deeper understanding of N resorption processes. Acknowledgements We thank Mr K. Sato and Mr T. Ozaki for their assistance and three anonymous referees for their valuable comments. This work was supported by JSPS Research Fellowships for Young Scientists (YY) and Grants-inAid from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

References Aerts R (1996) Nutrient resorption from senescing leaves of perennials: are there general patterns? Journal of Ecology 84, 597–608. doi: 10.2307/2261481 Aerts R, Chapin FS (2000) The mineral nutrition of wild plants revisited: a re-evaluation of processes and patterns. Advances in Ecological Research 30, 402–407. Aerts R, De Caluwe H (1994) Nitrogen use efficiency of Carex species in relation to site fertility. Journal of Applied Ecology 21, 1029–1040. Anten NPR, Werger MJA (1996) Canopy structure and nitrogen distribution in dominant and subordinate plants in a dense stand of Amaranthus dubius L. with a size hierarchy of individuals. Oecologia 105, 30–37. doi: 10.1007/BF00328788 Boerner REJ (1984) Foliar nutrient dynamics and nutrient use efficiency of four deciduous tree species in relation to site fertility. Journal of Applied Ecology 21, 1029–1040. doi: 10.2307/2405065 Boerner REJ (1986) Seasonal nutrient dynamics, nutrient resorption and mycorrhizal infection intensity of two perennial forest herbs. American Journal of Botany 73, 1249–1257. doi: 10.2307/2444058 Castro-Diéz P, Puyravaud JP, Cornelissen JHC (2000) Leaf structure and anatomy as related to leaf mass per area variation in seedlings of a wide range of woody plant species and types. Oecologia 124, 476–486. doi: 10.1007/PL00008873 Cataldo DA, Haroon M, Schrader LE, Youngs VL (1975) Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Communications in Soil Science and Plant Analysis 6, 71–80. Chapin FS, Kedrowski RA (1983) Seasonal changes in nitrogen and phosphorus fractions and autumn retranslocation in evergreen and deciduous taiga trees. Ecology 64, 376–391. doi: 10.2307/1937083 Chapin FS, Moilanen L (1991) Nutritional controls over nitrogen and phosphorus resorption from Alaskan birch leaves. Ecology 72, 709–715. doi: 10.2307/2937210 Crafts-Brandner SJ, Egli DB (1987) Modification of seed growth in soybean by physical restraint: effect on leaf senescence. Journal of Experimental Botany 38, 2043–2049. doi: 10.1093/jxb/38.12.2043 Dong SF, Scagel CF, Cheng LL, Fuchigami LH, Rygiewicz PT (2001) Soil temperature and plant growth stage influence nitrogen uptake and amino acid concentration of apple during early spring growth. Tree Physiology 21, 541–547. Eckstein RL, Karlsson PS, Weih M (1998) The significance of resorption of leaf resources for shoot growth in evergreen and deciduous woody plants from a subarctic environment. Oikos 81, 567–575. doi: 10.2307/3546777

Y. Yasumura et al.

Eckstein RL, Karlsson PS, Weih M (1999) Leaf life span and nutrient resorption as determinants of plant nutrient conservation in temperatearctic regions. New Phytologist 143, 177–189. doi: 10.1046/j.14698137.1999.00429.x Evans JR (1989) Partitioning of nitrogen between and within leaves grown under different irradiances. Australian Journal of Plant Physiology 16, 533–548. Evans JR, Seemann JR (1989) The allocation of protein nitrogen in the photosynthetic apparatus: costs, consequences, and control. In ‘Photosynthesis’. (Ed. WR Brigs) pp. 183–205. (Alan R. Liss: New York) Field C, Mooney HA (1986) The photosynthesis–nitrogen relationship in wild plants. In ‘On the economy of plant form and function’. (Ed. TJ Givnish) pp. 25–55. (Cambridge University Press: Cambridge) Garnier E, Laurent G (1994) Leaf anatomy, specific mass and water content in congeneric annual and perennial grass species. New Phytologist 128, 725–736. doi: 10.1111/j.1469-8137.1994.tb04036.x Grassi G, Millard P, Wendler R, Minotta G, Tagliavini M (2002) Measurement of xylem sap amino acid concentrations in conjunction with whole tree transpiration estimates spring N remobilization by cherry (Prunus avium L.) trees. Plant, Cell & Environment 25, 1689–1699. doi: 10.1046/j.1365-3040.2002.00949.x Helmisaari HS (1992) Nutrient retranslocation with the foliage of Pinus sylvestris. Tree Physiology 10, 45–58. Hikosaka K, Terashima I, Katoh S (1994) Effects of leaf age, nitrogen nutrition and photon flux density on the distribution of nitrogen among leaves of a vine (Ipomoea tricolor Cav.) grown horizontally to avoid mutual shading of leaves. Oecologia 97, 451–457. doi: 10.1007/BF00325881 Hikosaka K (1996) Effects of leaf age, nitrogen nutrition and photon flux density on the organization of the photosynthetic apparatus in leaves of a vine (Ipomoea tricolor Cav.) grown horizontally to avoid mutual shading of leaves. Planta 198, 144–150. doi: 10.1007/ BF00197597 Hikosaka K (2003) A model of dynamics of leaves and nitrogen in a plant canopy: an integration of canopy photosynthesis, leaf life span, and nitrogen use efficiency. American Naturalist 162, 149–164. doi: 10.1086/376576 Hörtensteiner S, Feller U (2002) Nitrogen metabolism and remobilization during senescence. Journal of Experimental Botany 53, 927–937. doi: 10.1093/jexbot/53.370.927 Huffaker RC (1990) Proteolytic activity during senescence of plants. New Phytologist 116, 199–231. doi: 10.1111/j.1469-8137.1990.tb04710.x Killingbeck KT, May JD, Nyman S (1990) Foliar senescence in an aspen (Populus tremuloides) clone – the response of element resorption to interramet variation and timing of abscission. Canadian Journal of Forest Research 20, 1156–1164. Killingbeck KT (1996) Nutrients in senesced leaves: keys to the search for potential resorption and resorption proficiency. Ecology 77, 1716–1727. doi: 10.2307/2265777 Killingbeck KT (2004) Nutrient resorption. In ‘Plant cell death processes’. (Ed. LD Noodén) pp. 215–226. (Elsevier Academic Press: San Diego) Kobe RK, Lepczyk CA, Iyer M (2005) Resorption efficiency decreases with increasing green leaf nutrients in a global data set. Ecology 86, 2780–2792. Makino A, Sakuma H, Sudo E, Mae T (2003) Differences between maize and rice in N-use efficiency for photosynthesis and protein allocation. Plant & Cell Physiology 44, 952–956. doi: 10.1093/pcp/pcg113 Marschner H (1986) Yield and the source-sink relationship. In ‘Mineral nutrition in higher plants’. pp. 115–154 (Academic Press Inc.: London) Matile P (2000) Biochemistry of Indian summer: physiology of autumnal leaf coloration. Experimental Gerontology 35, 145–158. doi: 10.1016/S0531-5565(00)00081-4


May JD, Killingbeck KT (1992) Effects of preventing nutrient resorption on plant fitness and foliar nutrient dynamics. Ecology 73, 1868–1878. doi: 10.2307/1940038 McGrath R (1972) Protein measurement by ninhydrin determination of amino acids released by alkaline hydrolysis. Analytical Biochemistry 49, 95–102. doi: 10.1016/0003-2697(72)90245-X Millard P, Thomson CM (1989) The effect of the autumn senescence of leaves on the internal cycling of nitrogen for the spring growth of apple trees. Journal of Experimental Botany 40, 1285–1289. doi: 10.1093/jxb/40.11.1285 Miyazawa SI, Suzuki AA, Sone K, Terashima I (2004) Relationships between light, leaf nitrogen and nitrogen remobilization in the crowns of mature evergreen Quercus glauca trees. Tree Physiology 24, 1157–1164. Nambiar EKS, Fife DN (1987) Growth and nutrient retranslocation in needles of radiata pine in relation to nitrogen supply. Annals of Botany 60, 147–156. ¨ Tamm U ¨ (2005) Species differences in timing of leaf fall and Niinemets U, foliage chemistry modify nutrient resorption efficiency in deciduous temperate forest stands. Tree Physiology 25, 1001–1014. Norby RJ, Long TM, Hartz-Rubin JS, O’Neill EG (2000) Nitrogen resorption in senescing tree leaves in a warmer, CO2 -enriched atmosphere. Plant and Soil 224, 15–29. doi: 10.1023/A:1004629231766 Paul MJ, Driscoll SP (1997) Sugar repression of photosynthesis: the role of carbohydrates in signalling nitrogen deficiency through source: sink imbalance. Plant, Cell & Environment 20, 110–116. doi: 10.1046/j.1365-3040.1997.d01-17.x Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophyll a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochimica et Biophysica Acta 975, 384–394. Pourtau N, Jennings R, Pelzer E, Pallas J, Wingler A (2006) Effect of sugar-induced senescence on gene expression and implications for the regulation of senescence in Arabidopsis. Planta 224, 556–568. doi: 10.1007/s00425-006-0243-y


417

Pugnaire FI, Chapin FS (1992) Environmental and physiological factors governing nutrient resorption efficiency in barley. Oecologia 90, 120–126. doi: 10.1007/BF00317817 Pugnaire FI, Chapin FS (1993) Controls over nutrient resorption from leaves of evergreen mediterranean species. Ecology 74, 124–129. doi: 10.2307/1939507 Rossato L, Laine P, Ourry A (2001) Nitrogen storage and remobilization in Brassica napus L. during the growth cycle: nitrogen fluxes within the plant and changes in soluble protein patterns. Journal of Experimental Botany 52, 1655–1663. doi: 10.1093/jexbot/52.361.1655 Shaver GR, Melillo JM (1984) Nutrient budgets of marsh plants: efficiency concepts and relation to availability. Ecology 65, 1491–1510. doi: 10.2307/1939129 Stoddart JL, Thomas H (1982) Leaf senescence. In ‘Encyclopedia of plant physiology (New series). Volume 14B’. (Eds B Parthier, D Boulter) pp. 592–636. (Springer-Verlag: Berlin) Takashima T, Hikosaka K, Hirose T (2004) Photosynthesis or persistence: nitrogen allocation in leaves of evergreen and deciduous Quercus species. Plant, Cell & Environment 27, 1047–1054. doi: 10.1111/j.13653040.2004.01209.x Terashima I, Evans JR (1988) Effect of light and nitrogen nutrition on the organization of the photosynthetic apparatus in spinach. Plant & Cell Physiology 29, 143–156. Wingler A, Purdy S, MacLean JA, Pourtau N (2006) The role of sugars integrating environmental signals during the regulation of leaf senescence. Journal of Experimental Botany 57, 391–399. doi: 10.1093/jxb/eri279 Yasumura Y, Onoda Y, Hikosaka K, Hirose T (2005) Nitrogen resorption from leaves under different growth irradiance in three deciduous woody species. Plant Ecology 178, 29–37. doi: 10.1007/s11258-004-2485-8 Yasumura Y, Hikosaka K, Hirose T (2006) Seasonal changes in photosynthesis, nitrogen content and nitrogen partitioning in Lindera umbellata leaves grown in high or low irradiance. Tree Physiology 26, 1315–1323.

Manuscript received 17 November 2006, accepted 14 March 2007

http://www.publish.csiro.au/journals/fpb