Plant, Cell and Environment (2007) 30, 1269–1283
`
`doi: 10.1111/j.1365-3040.2007.01701.x
`
`Increase in respiratory cost at high growth temperature is
`attributed to high protein turnover cost in
`Petunia ¥ hybrida petals
`
`TAKUSHI HACHIYA1,2, ICHIRO TERASHIMA2 & KO NOGUCHI2
`
`1Department of Biological Science, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka,
`Osaka, 560-0043 and 2Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo,
`Bunkyo-ku, Tokyo, 113-0033 Japan
`
`ABSTRACT
`
`It is widely believed that turnover of nitrogenous (N) com-
`pounds (especially proteins) incurs a high respiratory cost.
`Thus, if protein turnover costs change with temperature, this
`would influence the dependence of respiration rate on
`growth temperature. Here, we examined the extent to which
`protein turnover cost explained differences in N-utilization
`costs (nitrate uptake/reduction, ammonium assimilation,
`amino acid and protein syntheses, protein turnover and
`amino acid export) and in respiration rate with changes in
`growth temperature. By measurements and literature data,
`we evaluated each N-utilization cost in Petunia ¥ hybrida
`petals grown at 20, 25 or 35 °C throughout their whole
`lifespans. Protein turnover cost accounted for 73% of the
`integrated N-utilization cost on a whole-petal basis at 35 °C.
`The difference in this cost on a dry weight basis between
`25 and 35 °C accounted for 75% of the difference in
`N-utilization cost and 45% of the difference in respiratory
`cost. The cost of nitrate uptake/reduction was high at low
`growth temperatures. We concluded that respiratory cost in
`petals was strongly influenced by protein turnover and
`nitrate uptake/reduction, and on the shoot basis, C invest-
`ment in biomass was highest at 25 °C.
`
`Key-words: nitrate reduction.
`
`INTRODUCTION
`
`including nitrate/
`Nitrogen (N) utilization processes,
`ammonium uptake, nitrate reduction, ammonium assimila-
`tion, amino acid synthesis, protein synthesis, protein
`turnover and amino acid export, incur large respiratory
`costs in plants (Penning de Vries, Brunsting & van Laar
`1974; Zerihun, McKenzie & Morton 1998). Among these
`processes, nitrate reduction and ammonium assimilation
`are thought to consume high respiratory costs. In fact, the
`form of N source (e.g. nitrate or ammonium) greatly influ-
`ences estimates of construction respiration (McDermitt &
`Loomis 1981; Williams et al. 1987). On the other hand,
`
`Correspondence: T. Hachiya. Fax: +81-3-5841-4465;
`takushi@biol.s.u-tokyo.ac.jp
`
`e-mail:
`
`© 2007 The Authors
`Journal compilation © 2007 Blackwell Publishing Ltd
`
`maintenance respiration rates are often correlated with
`protein content, suggesting that protein turnover cost domi-
`nates maintenance respiration (Penning de Vries 1975;
`Amthor 1989; Lambers, Chapin & Pons 1998). The cost of
`amino acid export during senescence is also claimed to be
`substantial (Dangl, Dietrich & Thomas 2000). Because
`these processes consume large respiratory costs, these pro-
`cesses would influence plant growth and production.Thus, it
`is important
`to assess the costs of
`these respective
`N-utilization processes and their responses to environmen-
`tal factors, including growth temperature.
`On the basis of data from the literature, Zerihun et al.
`(1998) calculated the total respiratory cost of N-utilization
`processes (N-utilization cost) in bean leaves.They suggested
`that protein turnover cost dominates the N-utilization cost.
`However, their calculation was based on data on protein
`turnover rates in several plant species grown under different
`conditions. Moreover, these plant materials were at various
`developmental stages, and protein turnover rate is known to
`differ markedly depending on the developmental stage
`(Mae, Makino & Ohira 1983; Barneix et al. 1988; Bouma
`et al. 1994). Therefore, their calculation would have been
`inaccurate, although the approach was valid.
`Growth temperature greatly influences plant respiration.
`This is because growth temperature changes substrate avail-
`ability and/or demand for ATP (Bunce 2004; Atkin, Scheur-
`water & Pons 2006). Generally, the specific maintenance
`respiration rate increases with increasing growth tempera-
`ture, whereas specific construction respiration is affected
`little (Amthor 1989; Marcelis & Baan Hofman-Eijer 1995;
`Van Iersel 2003,2006).Interestingly,the half-life of protein in
`wheat roots decreases with increasing temperature because
`of activity of the ubiquitin proteolytic pathway (Ferguson,
`Guikema & Paulsen 1990). This indicates that the protein
`turnover rate increases with temperature.
`All of these studies indicate that protein turnover cost is
`a major component of the N-utilization cost and is likely to
`increase with growth temperature. Our aims here were to
`estimate the costs of the respective N-utilization processes –
`especially that of protein turnover – and to examine the
`effects of growth temperature on these costs. The quantita-
`tive understanding of these components of respiratory costs
`
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`1270 T. Hachiya et al.
`
`will provide us the information to improve plant growth
`and productivity.
`Growth temperature generally changes not only the spe-
`cific maintenance respiration rate, but the lifespans of plant
`organs (Kikuzawa 1995; Forbes, Black & Hooker 1997).
`Therefore, it is difficult to estimate the protein turnover cost
`from data on one developmental stage of an organ, and
`analyses that cover the whole lifespan of the organ are
`preferable. We estimated each of the N-utilization costs in
`petals of Petunia ¥ hybrida grown at one of three tempera-
`tures throughout their lifespans. Petals are useful materials,
`because they have shorter lifespans than leaves and it is
`easy to determine their developmental stages. Moreover,
`most petals are non-photosynthetic, and therefore ATP and
`reducing equivalents are supplied mainly from the respira-
`tory pathway.
`To calculate the costs of nitrate/ammonium uptake,
`nitrate reduction, ammonium assimilation, amino acid and
`protein syntheses, and amino acid transport, we used the
`specific costs of these respective processes in the literature
`(De Visser, Spitters & Bouma 1992; Boorer & Fischer 1997;
`Zerihun et al. 1998; Amthor 2000; Dangl et al. 2000), and
`multiplied these specific costs by actual changes in the
`amounts of amino acids and proteins. The cost of protein
`turnover was estimated by using cycloheximide (CHI), an
`inhibitor of cytosolic protein synthesis, according to Bouma
`et al. (1994). On the basis of the data obtained, we discuss
`the contribution of protein turnover cost to N-utilization
`cost and respiration rate. We also evaluated the effects of
`different N sources on estimates of N-utilization cost. Fur-
`thermore, we calculated the biomass C, N-utilization cost
`and respiratory cost to the petals on one shoot over 90 d,
`and we discuss the changes in N-utilization cost and in C
`investment in petal biomass with growth temperature. Our
`precise estimations clarified that
`the costs of nitrate
`reduction/uptake and protein turnover were large, and that
`the growth temperature intensively influenced these costs.
`
`MATERIALS AND METHODS
`Plant materials and growth conditions
`
`We used Petunia ¥ hybrida cv. Surfinia ‘White Vein’ (Suntry
`flowers, Tokyo, Japan). We purchased seedlings and
`removed the shoots other than the main one. We then cul-
`tivated the seedlings in vermiculite in pots (diameter,
`10.5 cm; depth, 17.5 cm; one seedling per pot). We placed
`the pots in a growth chamber (Biotron, Nippon Medical &
`Chemical Instruments Co., Osaka, Japan). Light was sup-
`plied by a bank of cool-white fluorescent tubes. The photo-
`synthetically active photon flux density (PPFD) was ca.
`450 mmol m-2 s-1. The air temperature was 20, 25 or 35 °C,
`relative humidity was 55%, and day length was 16 h (from
`1000 to 0200 h). Twice a week, the plants received 200 mL
`per pot of a nutrient solution containing 2 mM KNO3,
`2 mM Ca(NO3)2, 0.75 mM MgSO4, 0.665 mM KH2PO4,
`25 mM ethylenediaminetetraacetic acid (EDTA)-Fe, 5 mM
`MnSO4, 0.5 mM ZnSO4, 0.5 mM CuSO4, 25 mM H3BO3,
`
`0.25 mM Na2MoO4, 50 mM NaCl and 0.1 mM CoSO4. We
`cultivated the plants for approximately 1 month. For mea-
`surements, we used the new petals on shoots newly devel-
`oped in the growth chamber at 20, 25 or 35 °C.We measured
`and sampled the petals between 1400 and 0000 h.
`
`Respiration rate
`
`Respiratory O2 uptake rates of detached petals were mea-
`sured polarographically with a gas-phase oxygen electrode
`system (LD2; Hansatech Instruments Ltd, Kings Lynn,
`Norfolk, UK). The respiratory quotient (RQ) was assumed
`to be 1.
`
`Protein and amino acid contents
`
`Proteins were extracted from samples frozen in liquid N2
`and powdered using a mortar and pestle with a buffer con-
`taining 62.5 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sul-
`phate [w/v], 7.5% glycerol [v/v], 0.01% bromophenol blue
`[w/v], 50 mM 1,4-dithiothreitol, and 1 tablet/50 mL Com-
`plete proteinase inhibitor cocktail (Roche Diagnostics
`GmbH, Mannheim, Germany). The ratio of the sample dry
`weight (DW) to the sample buffer was 10 to 40 mg/mL.
`Extracts were incubated at 100 °C for 5 min and centrifuged
`at 12 000 g. The supernatants were used for protein quanti-
`fication by the modified Lowry method (Peterson 1977).
`Amino acids were extracted from the powdered samples
`with 80% ethanol by the method of Ono, Terashima &
`Watanabe (1996). Amino acids in the supernatant were
`quantified by the modified ninhydrin method (Rosen 1957).
`
`Inhibition of protein synthesis with CHI
`
`To estimate the protein turnover cost, we incubated the
`petals in 40 mM Hepes-KOH buffer (pH 7.2) in the pre-
`sence or absence of 500 mM CHI for 20 min in the dark at
`the growth temperature, and then measured the O2 uptake
`rates.
`
`Definition of petal age and lifespan
`
`Day 0 denoted the day when the petal started to unfold.
`Negative days were those on which the petals were still
`folded. We measured the folded petal
`lengths of
`P. ¥ hybrida everyday at each temperature (n ⱖ 30 on each
`day at each growth temperature) and plotted length against
`the day number. The age of the folded petal (in days) was
`estimated from the folded petal
`length by polynomial
`approximation (R2 ⱖ 0.99). We defined the petal lifespan as
`the number of days for which the size and respiratory activ-
`ity of a single detached petal were sufficient for us to
`measure the respiration rate (see Fig. 1).
`
`Definition of respiratory cost
`
`Respiratory cost was taken to include not only C in the CO2
`emitted by the respiratory pathway to supply ATP and
`
`© 2007 The Authors
`Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 1269–1283
`
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`Respiratory cost to petals at high temperature 1271
`
`(b)
`
`1
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`(g water g FW–1)
`Water content
`
`(a)
`
`20˚C
`25˚C
`
`35˚C
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`(mg DW)
`Dry weight
`
`Figure 1. Changes in dry weight (DW)
`(a), water content on a fresh weight (FW)
`basis (b), respiration rate on a DW basis
`(c) and respiration rate on whole-petal
`basis (d) during development of
`Petunia ¥ hybrida petals at each growth
`temperature (20 °C, circles; 25 °C,
`squares; 35 °C, triangles). Day 0 was the
`first day on which the petal began to
`unfold. Vertical bars represent standard
`error of the mean (SEM) (n ⱖ 4).
`
`0
`–10
`
`–5
`
`0
`
`5
`
`10
`
`15
`
`20
`
`0
`–10
`
`–5
`
`0
`
`5
`
`10
`
`15
`
`20
`
`(d)
`
`300
`
`250
`
`200
`
`150
`
`100
`
`50
`
`(mol O2 petal–1 day–1)
`
`Respiration rate on a whole petal basis
`
`(c)
`
`25
`
`20
`
`15
`
`10
`
`5
`
`(mol O2 g DW–1 day–1)
`
`Respiration rate on a DW basis
`
`0
`–10
`
`–5
`
`0
`
`10
`
`15
`
`20
`
`5
`Day
`
`0
`–10
`
`–5
`
`0
`
`10
`
`15
`
`20
`
`5
`Day
`
`reducing equivalents, but also the C in CO2 emitted in other
`biosynthetic pathways (see Amthor 2000).
`
`2000). The 1 in the denominator is the number of protons
`co-transported with phosphate into the matrix to compen-
`sate for the deficit in phosphate.
`
`Efficiency of respiratory ATP production
`
`Respiratory costs of nitrate/ammonium uptake,
`nitrate reduction, ammonium assimilation,
`amino acid synthesis and protein synthesis
`
`Nitrate and ammonium ions are taken up from the xylem
`into the symplast across the plasma membrane, with
`+, respectively
`stoichiometries of 2H+/NO3
`- and H+/NH4
`(Cannell & Thornley 2000; Kronzucker et al. 2001). We
`assumed that the number of membranes to be crossed was
`one (Kurimoto et al. 2004). The uptake of nitrate and
`ammonium as N sources requires additional specific res-
`piratory costs of proton motive force formation, at 1.24
`and 0.62 mol C mol-1 amino acid residue, respectively
`(Table 1). The processes of unloading of sucrose and
`amide into the petal are assumed to occur only via the
`symplastic pathway and thus require no respiratory costs
`(Cannell & Thornley 2000).
`The respiratory costs of nitrate reduction, ammonium
`assimilation [glutamine (Gln) synthesis], and synthesis of
`amino acids other than Gln were calculated from Zerihun
`et al. (1998) and the AraCyc metabolic map (Zhang et al.
`2005). The elemental composition of amino acid residues
`was considered to be the average of the 20 amino acids, with
`a molecular weight of 118.9 g mol-1 (C5.35H7.95O1.45N1.45S0.10).
`Carbon skeletons and reducing equivalents for amino acid
`synthesis were assumed to be supplied by Gln and sucrose.
`The pathway for each amino acid synthesis (see Appendix)
`was based on the AraCyc metabolic pathway database for
`
`FADH
`
`2
`
`
`
`]) +
`
`(1)
`
`cyt
`
`mit
`
`+N
`
`ATP
`
`ATP production efficiencies from sucrose and reducing
`equivalents were estimated according to Amthor (1994).
`He assumed that oxidation of 1 mol of sucrose via pyruvate
`produces 58 mol of ATP, and that all sucrose is degraded by
`invertase (for details, see Amthor 1994). The number of
`ATP produced via mitochondrial oxidation of reducing
`equivalents (PATP; mol ATP) was estimated as
`[
`] × [
`] +[
`] + [
`(
`NADPH
`]] × [
`]
`ADH
`+[
`]
`)
`H
`
`cyt
`
`I,III,IV
`1
`
`(
`
`+
`
`H
`
`III,IV
`
`P
`ATP
`
`=
`
`NADH
`[
`+
`H
`
`where [H+III,IV] and [H+
`
`I,III,IV] are the numbers of protons
`pumped into the inter-membrane space when a pair of
`electrons flows through Complex III/IV and Complex
`I/III/IV, respectively. We adopted 6 for [H+
`III,IV] and 10 for
`[H+
`(Amthor
`2000).
`I,III,IV]
`[NADHcyt],
`[NADPHcyt],
`[FADH2] and [NADHmit] are the concentrations of the
`corresponding reductants, and the subscripts ‘cyt’ and ‘mit’
`indicate the cytosol and mitochondrial matrix, respec-
`tively, and indicate the compartments where these reduc-
`tants are produced. In the respiratory oxidation of sucrose
`[NADHcyt],
`[NADPHcyt],
`[FADH2] and
`via pyruvate,
`[NADHmit] are given the values 4, 0, 4 and 16, respectively
`(Amthor 2000). [H+
`ATP] is the number of protons moving
`through H+-ATPase per ADP that undergoes phosphory-
`lation; a value of 3 was used for the calculation (Amthor
`
`© 2007 The Authors
`Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 1269–1283
`
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`

`1272 T. Hachiya et al.
`
`Table 1. Specific respiratory costs of nitrate reduction, ammonium assimilation, other amino acid synthesis and nitrate/ammonium
`uptake
`
`Process basis
`
`(1) Nitrate reduction
`
`(2) Ammonium
`assimilation
`
`(3) Other amino
`acid synthesis
`
`(4) NO3
`(5) NH4
`
`- uptake
`+ uptake
`
`Equation
`- + NADHcyt + 3NADPHcyt → NH3 + OH- + H2Oa
`NO3
`(a) 0.05Sucrose → 0.6CO2 + NADHcyt + 0.2FADH2cyt + 0.4ATP
`(b) 0.125Sucrose + 0.25ATP → 1.5CO2 + 3NADPHcyt
`2NH3 + 0.5Sucrose + ATP → L-glutamine + CO2 + NADHcyt + 2NADHmit
`(c) a-ketoglutarate + 2NH3 + 2ATP + NADHcyt → L-glutamine
`(d) 0.5Sucrose → a-ketoglutarate + 2NADHmit + 2NADHcyt + ATP + CO2
`14.5L-glutamine + 9.95Sucrose + 2SO4
`2– + 8NADPHcyt + 19ATPc
`→ 20C5.35H7.95O1.45N1.45S0.10 + D-glyceraldehyde-3-phosphate
`+ 10.5a-ketoglutarate + Succinate + 2Fumarate + 2Acetate + 12.21CO2
`+ 25.5NADHcyt + 7NADHmit
`
`b
`
`Specific cost on amino acid residues
`[mol C mol-1 amino acid residues]
`
`3.05
`
`0.73
`
`0.81
`
`1.24
`0.62
`
`Each specific cost is estimated on the basis of the equations shown.
`aEquations (a) and (b) denote pathways from which NADH and NADPH are supplied, respectively. NADH and NADPH are produced via
`the glycolysis and tricarboxylic acid cycle, and the pentose phosphate pathway, respectively.
`bEquation (c) denotes L-Gln synthesis via glutamine synthetase and glutamine: 2-oxoglutarate amidotransferase reactions. a-ketoglutarate is
`produced from pathway (d). ATP is produced via the oxidation of additional NADHcyt or NADHmit in mitochondrial electron transport.
`cAverage amino acid (C5.35H7.95O1.45N1.45S0.10) is assumed to be the average of 20 amino acids. Pathways for 20 amino acids are summarized in
`the Appendix. NADPH is supplied from the pentose phosphate pathway, as shown in Eqn (b).ATP is produced via the oxidation of additional
`NADHcyt or NADHmit in mitochondrial electron transport.
`
`where DP is the rate of increase in amount of amino acid
`residues in the proteins on a DW basis (mol amino
`acid residues g DW-1 d-1), ES is the specific respiratory
`(mol C mol-1 amino acid
`cost of protein synthesis
`residue) in Table 2 (we used the mean value in Table 2
`for the calculation), and Forg is the amount of protein syn-
`thesis or turnover in the organelles (e.g. mitochondria and
`plastids) as a ratio of that in the whole cell. Forg was
`assumed to be 0%, because (1) fewer than 10% of all
`mitochondrial proteins are encoded by mitochondrial
`DNA and synthesized in the mitochondria and (2) the
`petals of P. ¥ hybrida do not have green tissues and
`include
`little
`ribulose
`1·5-bisphosphate
`carboxylase/
`oxygenase (Rubisco), which accounts for a large pro-
`portion of the proteins synthesized in the leaf chloroplasts.
`Pn is the amount of protein on a whole-petal basis on day
`n (g petal-1). Tn is the sampling time (on day n). DWn–1 is
`the dry weight on day n–1, and 118.9 (g mol-1) is the
`average molecular weight of the amino acid residues as
`noted.
`
`Respiratory cost of protein turnover
`
`From the information provided by De Visser et al. (1992),
`Bouma et al. (1994), Zerihun et al. (1998) and Noguchi et al.
`(2001), we assumed that the total specific respiratory cost of
`protein turnover included the costs of protein biodegrada-
`tion and protein biosynthesis (see Table 2). The expected
`effects of CHI on protein turnover and the specific respira-
`tory costs of these processes are also shown in Table 2.
`The respiratory cost of protein turnover (Rmpro: mol C g
`DW-1 d-1) was as follows:
`
`where An is the amount of amino acid residues in amino
`acids and proteins on a whole-petal basis on day n (mol
`amino acid residues petal-1), Tn is the sampling time (day n),
`and DWn–1 is the dry weight on day n–1.
`The specific respiratory costs of protein synthesis pro-
`cesses are listed in Table 2. The respiratory cost of protein
`synthesis (Rcpro; mol C g DW-1 d-1) was calculated according
`to the following equations:
`
`(3)
`
`(4)
`
`R
`cpro
`
`=
`
`×
`Δ
`P E
`S
`
`×
`
`1
`F
`org
`
`)
`
`−(
`1
`
`and
`
`×
`
`1
`DW
`n
`
`−
`
`1
`
`×
`
`1
`118 9.
`
`)
`)
`
`(
`−
`P P
`n
`n
`(
`−
`T T
`n
`n
`
`1 1
`− −
`
`ΔP
`
`=
`
`© 2007 The Authors
`Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 1269–1283
`
`plant research (Zhang et al. 2005). The specific respiratory
`costs of nitrate reduction, ammonium assimilation and
`other amino acid synthesis are listed in Table 1. The respi-
`ratory cost of pH regulation was neglected, because little
`information was available (Zerihun et al. 1998). To estimate
`the respiratory costs of nitrate/ammonium uptake, nitrate
`reduction, ammonium assimilation and other amino acid
`synthesis, we multiplied the rate of increase in amino acid
`residues on a DW basis (DA; mol amino acid residue g
`DW-1 d-1) by the specific respiratory costs in Table 1. The
`rate of increase in amino acid residues was estimated as
`follows:
`(
`−
`A A
`n
`n
`(
`−
`T T
`n
`n
`
`1
`DW
`n
`
`−
`
`1
`
`(2)
`
`ΔA
`
`=
`
`)
`×−
`1
`)
`−
`1
`
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`Respiratory cost to petals at high temperature 1273
`
`Table 2. Specific respiratory costs for each process of protein synthesis, protein turnover and amino acid export on the basis of amino
`acid residues
`
`Specific cost on an amino acid residues basis [mol C mol-1 amino acid residues]
`
`Process
`
`Protein biodegradation
`(1) Protein biodegradation (Edeg) (a)
`Protein biosynthesis
`(2) Amino acid activation (b)
`(3) Editing for misaminoacylation of tRNA (c)
`(4) Peptide bond formation and translocation (a)
`(5) Signal sequences (c)
`Tool maintenance
`(6) Amino acid turnovera (a,b)
`(7) mRNA turnover (c)
`Amino acid export
`(8) Gln synthesis (Esyn)b (d)
`(9) Membrane transport (Etra) (e)
`Protein synthesis [ES: (2) + (3) + (4) + (5) + (6)]
`Protein turnover [ESP: ES + (7) + (8)]
`Expected CHI effect (ECHI = ES)
`
`Min
`
`0.21
`
`0.41
`0.00
`0.41
`0.04
`
`0.36
`0.03
`
`0.21
`0.21
`0.86
`1.46
`0.86
`
`Max
`
`Mean
`
`CHI effect
`
`0.21
`
`0.41
`0.03
`0.41
`0.21
`
`0.50
`0.07
`
`0.21
`0.21
`1.06
`1.84
`1.06
`
`0.21
`
`0.41
`0.02
`0.41
`0.13
`
`0.43
`0.05
`
`0.21
`0.21
`0.96
`1.65
`0.96
`
`-
`
`+
`+
`+
`+
`
`-
`-
`
`-
`-
`
`‘CHI effect’ denotes the expected effect of cycloheximide (CHI) on the process. It was assumed that oxidation of 1 mol sucrose produced
`58 mol ATP. Letters denote references for estimation of the specific cost of each process (a: Zerihun et al. 1998, b: De Visser et al. 1992, c:
`Amthor 2000, d: Dangl et al. 2000, e: Boorer & Fischer 1997).
`aSpecific respiratory cost of amino acid turnover was estimated on the assumption that 30 to 50% of amino acids are recycled.
`bGln was assumed to be transported across one membrane for its export.
`
`deamination of amino acids, 2 mol of amino acids will be
`required for 1 mol of Gln synthesis. We assumed that Gln is
`exported from the petal via the apoplastic pathway. The
`export of 1 mol of protons is coupled to that of the equiva-
`lent mole of amino acid, in accordance with the stoichiom-
`etry of the Arabidopsis thaliana amino acid transporters
`AAP1 and AAP5 (Boorer & Fischer 1997; Table 2).
`The minimum and maximum respiratory costs (Ramino,min
`and Ramino,max; mol C g DW-1 d-1) of amino acid export from
`the petal were calculated using Eqns 6 and 7. To calculate
`+ was
`the minimum cost, we assumed that half the NH4
`derived from the pool in the same cell and half was derived
`from the deamination of amino acids. To calculate the
`+ was derived from
`maximum cost, we assumed that all NH4
`that pool:
`
`(6)
`
`(7)
`
`and
`
`R
`aminomax
`
`=
`
`×
`
`12
`58
`E
`tra
`
`(
`×
`
`×
`
`E
`deg
`
`+
`NPDR E
`syn
`)
`
`NAER
`max
`
`×
`
`NPDR
`
`+
`
`where Edeg, Esyn and Etra denote the specific respiratory costs
`of protein degradation, Gln synthesis and membrane trans-
`port, respectively (Table 2). NPDR (mol amino acid resi-
`dues g DW-1 d-1) and NAER (mol amino acids g DW-1 d-1)
`
`(
`
`×
`
`×
`
`E
`deg
`
`×
`
`E
`syn
`
`×
`
`NPDR
`
`+
`
`1 2
`
`+
`
`NPDR
`)
`
`×
`
`NAER
`min
`
`R
`amino
`
`min
`
`=
`
`12
`58
`E
`tra
`
`×
`
`1
`F
`org
`
`)
`
`−(
`1
`
`(5)
`
`E E
`
`SP
`
`CHI
`
`R
`mpro
`
`=
`
`(
`
`R
`CHI
`
`−
`
`R
`cpro
`
`) ×
`
`rate
`respiration
`in
`decrease
`the
`is
`where RCHI
`(mol C g DW -1 d-1) in response to CHI application, ESP is
`the specific respiratory cost of protein turnover (mol C-
`mol-1 amino acid residue) and ECHI is the specific respira-
`tory cost of the processes blocked by CHI (mol C mol-1
`amino acid residue). We used the mean value in Table 2 for
`the estimation.
`
`Respiratory cost of amino acid export from
`the petal
`
`We defined amino acid export as a series of processes from
`the biodegradation of proteins into amino acids to the
`loading of amino acids into the phloem. We assumed that
`amino acids were transported in the form of Gln. ATP-
`requiring processes were assumed to be protein biodegra-
`dation, Gln synthesis and transport across the plasma
`membrane.
`In protein biodegradation, amino acids are assumed to be
`converted to Glu by specific transaminases (Dangl et al.
`2000). The specific cost of Gln synthesis from Glu by Gln
`synthetase is 1 mol of ATP per mol Gln produced (Table 2).
`A certain fraction of amino acids from the protein degra-
`+ and a-keto
`dation is considered to be degraded into NH4
`acids by deaminase or Glu dehydrogenase (Dangl et al.
`+ for Gln synthesis is derived from the
`2000). If NH4
`
`© 2007 The Authors
`Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 1269–1283
`
`Regeneron Exhibit 2016
`Page 05 of 15
`
`

`

`expanded. The maximum DW of the petal at 35 °C was half
`that of the petal at 20 °C. At any growth temperature, the
`water content (WC) of the petal on a fresh weight (FW)
`basis gradually increased from about 0.85 to above 0.90 and
`then steeply decreased (Fig. 1b). By the time the petals had
`attained their maximum WC, visible senescence or wilting
`had already occurred. Respiration rates on a DW basis
`consistently declined with time (Fig. 1c). In contrast, respi-
`ration rates on a whole-petal basis increased as the petals
`developed, peaked and then decreased (Fig. 1d).
`
`Changes in the amounts, synthesis, and
`degradation of proteins and amino acids
`
`The amounts of protein on a DW basis decreased with time
`(Fig. 2a) and did not differ markedly among growth tem-
`peratures. At 20 and 25 °C, the amino acid amounts on a
`DW basis also decreased with time (Fig. 2b) and were
`strongly correlated with the protein amounts (at 20 °C,
`r = 0.875, P < 0.0001; at 25 °C, r = 0.941, P < 0.0001). On the
`other hand, at 35 °C, the amount of amino acid started to
`increase just before full petal expansion (Fig. 2b) and by
`day 2 had reached 30% of the amount of protein. The
`amounts of proteins and amino acids are expressed on a
`whole-petal basis in Fig. 2c,d. At 35 °C, the amount of
`amino acid on a whole-petal basis peaked on day 2; this
`peak amount was much higher than the amounts at lower
`temperatures (Fig. 2d).
`The rates of increase in the amounts of protein and
`amino acids in Fig. 3 were calculated from the changes in
`protein and amino acid amounts in Fig. 2c,d (see Materials
`and Methods). Negative values denote decreases in the
`amounts of protein or amino acids. The minimum
`
`1274 T. Hachiya et al.
`
`are the net protein decrease rate and net amino acid export
`rate, respectively. NAER was calculated according to the
`following equations:
`
`(8)
`
`(9)
`
`×
`
`+
`NPDR NADR
`
`1 2
`
`min =
`NAER
`
`and
`
`max =
`NAER
`
`+
`NPDR NADR
`
`where NADR (mol amino acid g DW-1 d-1) is the net amino
`acid decrease rate. We assumed that the amino acids from
`the protein degradation were immediately used for Gln
`synthesis. NPDR and NADR correspond to negative values
`of the rates of increase of proteins and amino acids, respec-
`tively (see Results).
`
`Statistical analyses
`
`All statistical analyses were conducted with STATVIEW
`(ver. 5.0 J; SAS, Cary, NC, USA). The difference between
`respiration rates in the absence and presence of CHI was
`determined using Student’s t-test. For the other empirical
`data, we conducted two-way analysis of variance; the inter-
`action between factors (petal age and growth temperature)
`was statistically significant in all analyses (P < 0.01).
`
`RESULTS
`Effects of growth temperature on growth and
`respiration rate in P. ¥ hybrida petals
`The DW of the P. ¥ hybrida petal increased as the flower
`developed (Fig. 1a). At peak petal DW, the petal was fully
`
`(b)
`
`200
`
`150
`
`100
`
`50
`
`(mol g DW–1)
`
`Figure 2. Changes in amounts of
`protein (a) and amino acids (b) on a dry
`weight (DW) basis, and in amounts of
`protein (c) and amino acids (d) on a
`whole-petal basis during development of
`Petunia ¥ hybrida petals at each growth
`temperature (20 °C, circles; 25 °C,
`squares; 35 °C, triangles). Vertical bars
`represent standard error of the mean
`(SEM) [n ⱖ 3 except on day –3 at 20 °C
`and days 6 and 7 at 35 °C (n = 2)].
`Amounts of protein and amino acids on a
`whole-petal basis were calculated by
`multiplying the amounts of protein and
`amino acids on a DW basis by average
`values of DW.
`
`0
`–10
`
`–5
`
`0
`
`5
`
`10
`
`15
`
`20
`
`(d)
`
`5
`
`4
`
`3
`
`2
`
`1
`
`(mmol amino acid residue petal–1)
`
`0
`–10
`
`–5
`
`0
`
`10
`
`15
`
`20
`
`5
`Day
`
`Amino acid amount on a DW basis
`
`Amino acid amount on a whole-petal basis
`
`20˚C
`25˚C
`
`35˚C
`
`(a)
`
`3
`
`2.5
`
`2
`
`1.5
`
`1
`
`0.5
`
`(mmol amino acid residue g DW–1)
`Protein amount on a DW basis
`
`0
`–10
`
`–5
`
`0
`
`5
`
`10
`
`15
`
`20
`
`–5
`
`0
`
`5
`
`Day
`
`10
`
`15
`
`20
`
`(c)
`
`35
`
`30
`
`25
`
`20
`
`15
`
`10
`
`05
`
`–10
`
`(mol amino acid residue-petal–1)
`
`Protein amount on a whole-petal basis
`
`© 2007 The Authors
`Journal compilation © 2007 Blackwell Publishing Ltd, Plant, Cell and Environment, 30, 1269–1283
`
`Regeneron Exhibit 2016
`Page 06 of 15
`
`

`

`Respiratory cost to petals at high temperature 1275
`
`Figure 3. Changes in protein increase
`rate (a) and amino acid increase rate (b)
`on a whole-petal basis during
`development of Petunia ¥ hybrida petals
`at each growth temperature (20 °C,
`circles; 25 °C, squares; 35 °C, triangles).
`
`(b)
`
`0123
`
`–1
`
`–2
`
`–3
`–10
`
`–5
`
`0
`
`10
`
`15
`
`20
`
`5
`
`Day
`
`(mmol amino acid petal–1 day–1)
`
`Amino acid increase rate on a whole-petal basis
`
`20˚C
`25˚C
`35˚C
`
`10
`
`(a)
`
`5
`
`0
`
`–5
`
`(mmol amino acid residue petal–1 day–1)
`
`Protein increase rate on a whole-petal basis
`
`–10
`
`–5
`
`0
`
`10
`
`15
`
`20
`
`5
`
`Day
`
`(NAERmin) and maximum (NAERmax) net amino acid
`export rates were calculated using these negative values
`(Fig. 3a,b; see Materials and Methods).
`
`Effects of CHI on respiration rates and
`respiratory costs of protein turnover
`
`We measured the effects of CHI on respiration rates
`(Fig. 4). Only FW-based results are shown, because the
`buffer incubation interfered with the measurement of DW.
`At 20 °C, the effects of CHI on respiration rate were greater
`during the initial developmental stages. At 20 °C, the
`decrease in respiration rate in response to CHI ranged from
`9.4% (day 0) to 35.5% (day –3) (Fig. 4a). At 25 °C, the
`decrease in response to CHI treatment ranged from 7.8%
`(day –1) to 32.8% (day –4) (Fig. 4b). In contrast, at 35 °C,
`marked CHI effects were also observed after petal unfold-
`ing (Fig. 4c). For example, CHI treatment decreased the
`respiration rate by 33.3% on day 2 (Fig. 4c).
`The respiratory costs of protein turnover on a DW basis
`were calculated from the data in Fig. 4 and the theoretical
`values in Table 2. Maximum values were observed on day
`–3 at 20 °C and on day –4 at 25 and 35 °C (Fig. 5). At 35 °C,
`there was another peak on day 2. It is noteworthy that the
`cost at 35 °C was approximately twice those at the lower
`temperatures at most times during development.
`
`N-utilization costs and respiratory costs during
`petal development
`
`We summarized the changes in N-utilization costs during
`petal development on a DW basis (Fig. 6a–c) and on a
`whole-petal basis (Fig. 6d–f). The negative values in Fig. 5
`were regarded as zero for the calculation of protein turn-
`over costs. To calculate the costs in Fig. 6, the N source
`was assumed to be solely nitrate. Respiratory costs on a
`DW basis were used for comparisons among growth tem-
`peratures, because in this way petal size did not affect
`the cost estimations. In contrast, costs on a whole-petal
`basis were used to evaluate the contribution of each
`N-utilization cost to the respiratory cost at each growth
`temperature.
`
`+),
`--NH4
`The costs of nitrate uptake/reduction (NO3
`+Gln), other amino acid syn-
`ammonium assimilation (NH4
`thesis (Gln-Amino), protein synthesis (Amino-Protein) and
`protein turnover were large before petal unfolding at all
`temperatures. The contribution of nitrate uptake/reduction
`+) was largest. At 35 °C, the cost of protein turn-
`--NH4
`(NO3
`over accounted for a large part of the cost after petal
`unfolding (Fig. 6c,f).The costs of amino acid export (Amino
`export) arose mainly after petal unfolding and accounted
`for a minimal fraction of the N-utilization cost.
`By using the data in Fig. 6, we integrated the N-utilization
`costs on a DW basis (Fig. 7a) and on a whole-petal basis
`(Fig. 7b) throughout the petal’s whole lifespan. The inte-
`grated N-utilization cost on a DW basis was greatest at
`35 °C, and those at 20 and 25 °C were similar to each other
`(Fig. 7a). The integrated protein turnover cost on a DW
`basis at 35 °C was more than four times that at 25 °C
`(Fig. 7a). The integrated protein turnover cost on a whole-
`petal basis at 35 °C was the largest component of the
`N-utilization cost, followed by the nitrate uptake/reduction
`cost (Fig. 7b). The integrated cost of nitrate uptake/
`reduction was similar to the sum of costs of the subsequent
`three processes from ammonium to protein synthesis at all
`temperatures.
`We calculated the integrated respiratory cost, including
`various processes other than N-utilization processes, by
`integrating the respiration rates throughout the petal’s
`lifespan (Fig. 1c,d).The integrated respiratory cost on a DW
`basis at 35 °C was higher than those at lower temperatures
`(Fig. 7c), but, on a whole-petal basis, the integrated respira-
`tory cost at 35 °C was lowest (Fig. 7d).
`
`Costs of net amino acid export
`
`We assumed that net amino acid export started on the day
`when the value of NAER became positive. The integrated
`costs of amino acid export on a whole-petal basis during
`petal development are shown in Fig. 7b and Table 3. These
`costs accounted for the smallest fraction of the N-u