Differences between the Surface Properties of the PR and PFR Forms of Native Pea Phytochrome, and Their Application to a Simplified Procedure for Purification of the Phytochrome

The ability of phytochrome from etiolated pea shoots (Pisumsativum L. cv. Alaska) to bind to various chromatographic adsorbentsand its mobility during non-denaturing electrophoresis wereexamined with phytochrome in either the red light-absorbingform (PR) or the far-red light-absorbing form (P_FR). Preferentialbinding of P_FR to modified hydrophilic polyvinyl resins, suchas butyl Toyopearl, phenyl Toyopearl, Blue Toyopearl (CibacronBlue F3G-A conjugated) and Red Toyopearl (Procion Red HE-3Bconjugated), was observed. A simplified method for purificationof native phytochrome was developed based on the propertiesof P_R and P_FR. P_FR bound preferentially to the hydrophobic adsorbents,to indicate that the surface of P_FR is more hydrophobic thanthat of P_R. A difference in net surface charges between P_R andP_FR was detected by an analysis based on the different mobilitiesof the two forms during non-denaturing polyacrylamide gel electrophoresisin gels prepared with various concentrations of polyacrylamide.The apparent molecular weights of P_R and P_FR, estimated fromthe analysis, were 378 and 419 kilodaltons, respectively. Thedifference suggests that a significant change in molecular shapeoccurs during the photoconversion. The differences in surfaceproperties of P_R and P_FR are discussed. (Received April 20, 1991; Accepted August 26, 1991)     

Binding Proteins to Phytochrome A in Etiolated Pea Seedlings

In order to detect and characterize a putative receptor(s) fora signal from PhyA, proteins that bind to purified pea PhyAwere searched for in the crude extract of etiolated pea seedlingswith affinity chromatography. PhyA was coupled to the columnsubstrate either in P_R form (P_R column) or in red-irradiatedform (P_FR column). The coupled PhyA of both columns retainsits spectral reversibility between P_R and P_FR, although theirpeptide mapping by trypsin digestion suggests that the C-terminalhalf of PhyA in the P_FR column is partially fixed in P_FR structure.15 polypeptides were detected reproducibly in the elution fromthe P_FR column by silver-staining of SDS-PAGE. These 15 polypeptidesmay form two complexes judging from their elution profiles.Of the 15 polypeptides, the 6 major polypeptides have approximatemol wt of 80, 55, 53, 46, 40 and 35 kDa. On the other hand,only a trace amount of protein, which mainly consists of the46 kDa species, was eluted from P_R column, indicating the presenceof P_FR-specific BPs in the crude extract of etiolated pea seedlings.Of the 6 major polypeptides, the 40 kDa species binds to thePhyA in a photoreversible manner. (Received June 19, 1998; Accepted December 19, 1998)     

Identification of Surface-Exposed Parts of Red-Light- and Far-Red-Light- Absorbing Forms of Native Pea Phytochrome by Limited Proteolysis

Peptide fragments were obtained by limited proteolysis withtrypsin and Staphylococcus aureus V 8 protease from either theP_R or the P_FR form of 121-kDa phytochrome purified from etiolatedpea (Pisum sativum L.) shoots. Patterns of bands after polyacrylamidegel electrophoresis in the presence of SDS of the digests weredifferent, with some bands appearing preferentially when thedigestions were carried out with the P_R or the P_FR form. Amino-terminalsequences of the fragments were analyzed to determine the exactlocations of the amino-termini of the fragments within the aminoacid sequence of the apoprotein of pea phytochrome. The aminoacid compositions of some of the sequenced fragments were determinedin order to confirm the carboxy-terminal amino acids. Threecleavage regions were identified as kinetically favored sitesof cleavage of P_FR (Arg-746 to Lys-752, around Glu-877 and aroundArg-1010), whereas only one was identified for P_R (Glu-38 toArg-62). Regions of Glu-255, Arg-383, Arg-583 to Glu-620 andLys-1093 to Glu-1115 were also identified as potential sitesof proteolytic cleavage in both forms of the phytochrome. Othercleavage sites, the specificities of which have not yet beendetermined, are Glu-404, Glu-695 and Lys-1045. Surface-exposed parts of phytochrome in the P_R and P_FR formsare discussed. (Received June 13, 1992; Accepted October 27, 1992)     

Differential Exposure of Tryptophan Residues in the Red and Far-Red Light Absorbing Forms of Phytochrome, as Revealed by Chemical Modification

The effects of the chemical modification of tryptophan residuesin native pea (Pisum sativum L.) phytochrome by 2-hydroxy-5-nitrobenzylbromide (HNB-Br) were examined. Such treatment had no effecton the spectral properties or on the pattern of tryptic digestionof phytochrome, which indicated that no major conformationalchange in phytochrome had occurred. Amino acid analysis of theHNB-Br-treated phytochrome indicated that the number of modifiedTrp residues after the treatment was dependent on the light-absorbingform. The values were three for P_R and five for P_FR (out ofa total of ten) per monomer. The results indicate that two additionalTrp residues are exposed on the molecular surface of P_FR whenthe photoconversion of P_R to P_FR occurs. The amino acid analysisof a 58-kDa tryptic fragment of phytochrome (a mixture of peptides,residues 63–583 and 66–587) showed that one Trpresidue in the fragment from P_R and two in that from P_FR (outof six) were modified by HNB-Br. In the 56-kDa fragment (a mixtureof peptides, residues 598–1121 and 603–1124), therewere two modified Trp residues in P_R and three in P_FR (out offour). The Trp residue in a 36-kDa fragment (residues 66–383),which includes the tetrapyrrolic chromophore, was not modifiedin the either case. These results indicate that new exposedsites that are generated by the photoconversion of P_R to P_FRare in the region between Trp–456 and Trp–567 andin that between Trp–644 and Trp–787. (Received February 25, 1993; Accepted August 16, 1993)     

Phototransformation of the Red-Light-Absorbing Form to the Far-Red-Light-Absorbing Form of Phytochrome in Pea Epicotyl Tissue Measured by a Multichannel Transient Spectrum Analyser

Phototransformation of the red-light-absorbing form (P_R) tothe far-red-light-absorbing form (P_FR) of phytochrome in 7-day-oldetiolated pea epicotyl hook segments was examined at 0.5C aftera red laser flash excitation using a multichannel transientspectra analyser with electrically gated photomultiplier. Effectsof a red laser pulse on the induction of phototransformationfrom P_R to P_FR were saturated at Ca. 15 mJ for flash wavelengthsof both 640 and 655 nm. The amount of P_FR induced by a saturatinglaser pulse was ca. 50% of that obtained at the photostationaryequilibrium. A difference spectrum measured 15 µs afterthe flash showed an absorbance increase at 697 nm and a decreaseat 663 nm. A difference spectrum determined 200 ms after theflash showed no such major absorbance increase. Kinetic analysisof the rapid absorbance decrease at 700 and 710 nm gave onesimple first-order reaction component having a rate constantof 2,500 s^–1. Kinetics of P_FR appearance measured by absorbanceincrease at 750 nm was resolved into three first-order reactionshaving rate constants of 5, 1.8 and 0.4 s^–1. The secondflash light of 710 nm given 2 µs and 2 ms after the firstred flash irradiation on P_R resulted in the formation of P_Rrather than P_FR. (Received February 8, 1985; Accepted April 11, 1985)     

A Re-evaluation of the Mole Fraction of PFR at the Red-light-induced Photostationary State of Undegraded Rye Phytochrome

Photochemical properties were determined for undegraded phytochromepurified from lyophilized rye seedlings (Secale cereale cv.Cougar). The preparation was shown to be a two-component systemduring phototransformations and dark transformation from red-light-inducedphotostationary state. The mole fraction of P_FR at the 665-nm-light-inducedphotostationary state was 0.84. The ratio of the quantum yieldof photoconversion from P_R to P_FR to that from P_FR to P_R was1.53. ¹ Present address: Division of Biological Regulation, NationalInstitute for Basic Biology, Myodaijicho, Okazaki 444, Japan (Received March 18, 1981; Accepted August 3, 1981)     

Low Temperature Spectrophotometry on Phototransformation of PR in Pelletable Phytochrome of Pea

Phototransformation of P_R to P_FR in a 1,000–7,000 x gpelletable fraction (1–7 KP), which was extracted fromdark-grown pea shoots that had been irradiated by red then far-redlight, was studied by low temperature spectrophotometry. Redlight irradiation of P_R in 1–7 KP at –160°Cinduced an absorption increase at 695–696 nm with a concomitant,small decrease of P_R absorption at 670 nm. These changes werepartially photoreversed when the sample was irradiated subsequentlywith 700-nm light. At –55°C, red light irradiationof P_R resulted mainly in bleaching and consequently in a reductionof the P_R peak, accompanied by minor absorbance increases around695 nm. The intermediates formed at –165°C by 660-nmlight irradiation partly reverted back to P_R or formed a bleachedintermediate (probably the same bleached intermediate describedabove) in the dark, when the pellets were warmed to –60°C.The bleached intermediate was transformed to P_FR in the darkat –10°C or above. These characteristics of P_R transformation observed in the pelletablephytochrome were essentially the same as those observed in invivo or soluble phytochrome. (Received December 24, 1982; Accepted July 28, 1983)     

Effect of Neutral Salts on Spectral Characteristics of Undegraded Phytochrome Partially Purified from Etiolated Pea Shoots

Spectral characteristics of partially purified undegraded peaphytochrome were investigated in different ionic conditions.At the red-light-induced photostationary state in low ionicstrength buffer phytochrome had reduced absorbance in its far-redpeak as reported previously. Elevation of the ionic strengthof the buffer reversibly increased the absorbance in the far-redregion at the photostationary state. It was found that the effectof increase of ionic strength was strengthened secondarily bychaotropicity of salts. It was confirmed that phytochrome preparations of low ionicstrength contained a photosensitive component(s) other thanthe red-light-absorbing form (P_R) and farred- light-absorbingform (P_FR) during photochemical transformation, as well as duringthe first several min in the dark after phototransformation.At high ionic strength, phytochrome became a two-component systemcomposed of only P_R and P_FR at the redlight-induced photostationarystate though a significant accumulation of another component(s)occurred during phototransformations. Increasing ionic strengthalso enhanced A723 of phytochrome at the red-light-induced photostationarystate. The effect could result from either an increased molefraction of P_FR at the photostationary state induced by redlight, or a change in the extinction coefficients of P_FR. ¹ Present address: Division of Biological Regulation, NationalInstitute for Basic Biology, Myodaijicho, Okazaki 444, Japan (Received March 18, 1981; Accepted August 3, 1981)     

Purification and spectral characterization of a 121-kilodalton phytochrome from etiolated pea (Pisum sativum L.) seedlings

Procedures for the purification of native phytochrome from etiolatedpea seedlings without the use of immuno-purification techniquesare described. Phytochrome (in the P_FR form) was purified bypolyethyleneglycol fractionation, adsorption to pentyl agaroseand batch elution, chromatography on DEAE-Sepharose, adsorptionto phenyl Toyopearl and batch elution, and chromatography onRed Toyopearl. The resulting phytochrome had specific absorbanceratios (SAR = A₆₆₆/A₂₈₀ of P_R) that ranged from 0.55 to 0.6.The subsequent chromatography on Sephacryl S-300 yielded verypure phytochrome with a SAR of 0.98. P_R and P_FR peaks in thedifference spectrum of the phytochrome were centered at 665and 730 nm, respectively. The spectral change ratio (Ar/Afr)of the difference spectrum was unchanged after the chromatographyon phenyl Toyopearl, and the value was 1.05–1.08, indicatingthat the spectral properties of this preparation were intact.The absorption spectra indicated that the peak absorbance ofP_FR was at 728–730 nm and that of P_R was at 666–667nm. These peak positions were essentially same as those obtainedwith the undegraded oat phytochrome. Incubation of the samplepurified on Sephacryl S-300 at 25?C for 5 h in either the P_Ror P_FR form did not result in degradation of the molecule. Therate of dark reversion of P_FR observed with the purified peaphytochrome was similar to that observed in vivo. The additionof dithionite had no effect on the reversion rate. ²Present address: Fuji-Gotenba, Research Lab. of Chugai PharmaceuticalCo. Ltd., Gotenba, Shizuoka, 412 Japan (Received February 22, 1990; Accepted May 28, 1990)     

Action Spectra Confirm Two Separate Actions of Phytochrome in the Induction of Flowering in Lemna paucicostata 441

Action spectra studies have shown that in the short day plant(SDP) Lemna paucicostat441 there are at least two actions ofphytochrome in the induction of flowering. At the beginningof the dark period far-red light inhibited flowering, and theaction spectrum corresponded to the absorption spectrum of P_FR,while at the middle of the inductive dark period both red andfar-red light were inhibitory. The action spectrum for the redlight corresponded to that of PR absorption, but there was activityin the region beyond 720 nm which exactly coincided with theabsorption by P_FR observed at the beginning of the dark period,indicating that at the middle of the dark period there was absorptionby both P_R and P_FR. The difference in quantum efficiency betweenthe red and far-red light effects was about 60-fold. These resultsare consistent with there being a stable pool of P_FR necessaryfor the induction of flowering and another pool of phytochromein a different cellular environment which participates in thenight-break reaction as P_R. ¹ Present address: School of Applied Biology, Faculty of Science,Lancashire Polytechnic, Preston PR1 2TQ, U.K. ² 2 Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabemachi, Tsukuba, Ibaraki305, Japan. ³ Present address: Division of Plant Biological Regulation,The Riken Institute for Frontier Research Program, Hirosawa,Wako-shi, 351-01, Japan. (Received December 13, 1986; Accepted July 17, 1987)     

Effect of Monoclonal Antibodies on the in Vitro PFR Dark Reversion of Pea Phytochrome

Extraction as P_FR and immunoaffinity chromatography yieldeda pea phytochrome sample with polypeptide size of 121 kdalton,the same as in a crude extract which was immediately heatedin SDS. A difference spectrum was almost the same as that observedin etiolated pea epicotyls except that A₆₆₆/A₇₃₀ of 1.20 wassignificantly larger. At 10C dark reversion from P_FR occurred,with the decrease in A₇₂₈ being almost equal to the increasein A₆₆₇. The kinetics could be resolved into three first-ordercomponents, the major, slow component accounting for more than90% of the absorbance changes. In the presence of monoclonalanti-pea phytochrome antibodies mAP-1, 3 or 5, which bind awayfrom the chromophore, and mAP-7, which binds near the chromophore,the rate of the major component was reduced at either one orboth wavelengths. None of these antibodies affected the absorptionspectra of phytochrome. In the presence of mAP-9, which is suggestedto bind near the amino-terminus, the absorption at the red-light-inducedphotostationary state was reduced and the rate of dark reversionwas increased, resembling partially degraded phytochrome of114 kdalton, but with no evidence of proteolysis. ¹ Permanent address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan.     

Spectral Properties of Chromophore-Containing Fragments Prepared from Pea Phytochrome by Limited Proteolysis

Five chromophore-containing fragments were prepared from peaphytochrome in PR form (monomer mol wt 114,000) by limited proteolysiswith trypsin, thermolysin or chymotrypsin, and their absorptionand circular dichroism (CD) spectra were determined. The fragmentsof mol wt 62,100 and 56,400 showed photoreversible transformationbetween P_R and P_FR like phytochrome. The smaller fragments ofmol wt 40,300, 39,000 and 33,000 showed an absorption maximumat 657–660 nm (P₆₆₀) which was transformed to a bleachedform (P_BL) after a brief red-light exposure. The phototransformationbetween P₆₆₀ and P_BL was repeatedly reversible. Both P₆₆₀ andP_BL showed a negative CD band in red region like P_R, in contrastwith P_FR which has a positive band in far-red region. The natureof a chromophore domain of phytochrome and spectral propertiesof P_BL are discussed. ¹This study is dedicated to the late Professor J. Ashida. ²Permanent address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received August 7, 1982; Accepted March 26, 1983)     

Flowering of the long-day plant, Lemna gibba, under short-day schedules composed of red and far-red light

Flowering in Lemna gibba, a long-day duckweed, can be inducedunder a short-day condition when the photoperiodic regimes areR₇FR₃ (7 hr red followed by 3 hr far-red), R₅FR₅ and R₃FR₇.This indicates the necessity of a proper balance between redand far-red effects for flowering. The flowering induced bythese regimes is inhibited by a brief exposure to red givenat the start of darkness and this inhibition is reversed bysubsequent exposure to far-red. Thus, the red/far-red reversibleeffect is found only at the beginning of darkness for floweringof L. gibba. However, flowering of L. gibba is promoted by a red light breakgiven near the middle of a 14 hr dark period. The promotiveeffect is not reversed by subsequent exposure to far-red, i.e.,the effect of the red break converts from inhibition to promotionas when given later in the dark period, which suggests the involvementof a timing mechanism. (Received July 21, 1973; )     

Phototransformation of the Far-red Light-absorbing Form of Large Pea Phytochrome by Laser Flash Excitation

Phototransformation of the far-red light absorbing form (P_FR)of large pea phytochrome to the red-light absorbing form (P_R)was examined at 2?C after a 715 nm laser flash excitation usinga custom-built multichannel transient spectra analyzer. Themaximum amount of phototransformation intermediates was producedby a pulse of about 50 mJ, which resulted in ca. 65% of P_R obtainedat the photostationary equilibrium. Some flash-induced intermediateswere assumed to return to P_FR in the dark. A difference spectrummeasured at 10 µsec after the flash showed an absorbanceincrease at 651 nm and a decrease at 724 nm. When the samplewas left in darkness after the flash light irradiation, absorbancein the red and far-red region gradually increased, but thatin the green region rapidly decreased. The decay curve of intermediatesmeasured at 554 nm could be resolved into three reaction componentshaving rate constants of 2,500, 590 and 48 sec^–1, respectively.Difference spectra also indicated that a small but significantincrease in absorbance between 370 and 380 nm and a decreasearound 415 nm took place 10–310 µsec after a flash. (Received February 13, 1982; Accepted April 21, 1982)     

Relationship between Germination and PFR Level in Spores of the Fern Lygodium japonicum

The relationship between germination and P_FR level in sporesof the fern Lygodium japonicum was investigated. Percent P_FRestimated from direct spectrophotometric measurement of sporesincreased with the logarithm of total fluence of 660 nm-light.The transformation from P_R to P_FR was saturated by giving ca.200 Jm^–2 of 660 nm-light and half-saturated by ca. 55J^–2 of 660 nm-light. Clear positive correlation was observedbetween % P_FR levels and germination rates in spores irradiatedwith 660 nm and/or 730 nm-light, or with 686 or 700 nm-light.The P_FR percentage in spores was raised to 16–34% by blue(415 nm) light irradiation. This P_FR level was enough to causesome germination when produced by monochromatic light of redto far-red region, but blue light did not cause any germination. After 660 nm-light irradiation, the P_FR level decreased graduallyin darkness (25±1°C) and P_FR completely disappearedin 8 h, but 730 nm-light given even 16 h after 660 nm-lightirradiation inhibited germination. ⁴Present address: Tropical Botanic Garden and Research Institute,Navaranga Road, Trivandrum 695 011, India. (Received March 15, 1983; Accepted June 4, 1983)     

Effect of pH on Absorption Spectra of Pea 114 and 121 Kilodalton Phytochromes during and after Red-Light Irradiation

Absorption spectra of pea 114 and 121 kDa phytochromes weremeasured at pH 6.8, 7.8 and 8.8 using a custom-made transientmultichannel spectrum analyzer. The absorption spectra of 114kDa phytochrome as P_R and P_FR were least affected by mediumpH. The absorption spectra at photostationary state under redlight, however, were different under the three different pHconditions, and were different from those obtained 55 s afterred-light irradiation, owing to rapid pH-dependent absorbanceincrease in both red and far-red regions in the dark. In contrast,the absorption spectra of 121 kDa phytochrome were significantlyless affected by medium pH. The absorption spectra measuredat the photostationary state showed a lower P_FR peak at higherpH. The absorption spectra obtained 55 s after the irradiationwere similar under the three pH conditions since the rapid absorbanceincrease in the far-red region in the dark was small. Possibleaccumulation of 114 kDa phytochrome population(s) with low absorbanceat red-light-induced photostationary state at pH 8.8, and theprotective role of the 7 kDa polypeptide at the amino terminusagainst the pH effect in 121 kDa phytochrome are discussed. (Received February 1, 1986; Accepted April 1, 1986)     

Photomodulation of Stem Extension in Light-Grown Plants: Evidence for Two Reactions

Internode elongation was measured in plants of Phaseolus vulgarisand Glycine max grown under 8 h photoperiods at 25 W m^–2in white fluorescent light, followed by light-extensions varyingin quality, irradiance and duration. Two distinct responsesto light were observed under these conditions. A reduction in P_FR/P increased elongation, but elongation wasalso modified by a second reaction in which internode lengthincreased with increase in the duration and irradiance of theday-extension. This light-promoted response occurred in bothred and blue light. In the P_FR-inhibition response, light acteddirectly on the expanding internode. The light-promoted response,in contrast, required irradiation of the leaf. The response to a short end-of-day exposure to far-red lightprogressively diminished as successive internodes expanded underthe treatment, whereas the light-promoted response increased.The two processes appeared to interact and, in the later-expandinginternodes, the effect of a reduction in P_FR was greater underlong day-extensions with mixed red and far-red light than inthe end-of-day treatments. ¹ Present address: British Telecom, Brunel House, 2 FitzalanRoad, Cardiff, U.K.     

Time of Flowering of Pea (Pisum sativum L.) as a Function of Leaf Appearance Rate and Node of First Flower

In order to assess the influence of environmental conditionson time of flowering of pea (Pisum sativum L.), a serial sowingtrial was conducted over 2 years at Dijon, France, on two wintercultivars Frisson and Frilene. Time of flowering was analysedaccording to two variables: the leaf appearance rate R_L andthe node of first flower N_I. R_L was linearly related to temperature (r² = 0·94). Thebase temperature was 2°C for both varieties. Growth rateaccounted for the residual variability of R_L . Photoperiod andtemperature acted on N_I in an additive way. Frilene, the latergenotype, was more responsive than Frisson. A model for predicting time of flowering based upon these resultsis proposed. Deviations from this model were related to N nutritionin interaction with the plant water relations. Steps for improvingthe model are then discussed.Copyright 1993, 1999 Academic Press Pisum sativum L., pea, flowering, temperature, photoperiod, phyllochron, model     

Phytochrome in the Fern, Adiantum capillus-veneris L.: Spectrophotometric Detection in Vivo and Partial Purification

Spectrophotometric studies of fern phytochrome were performedusing dark-grown leaves of Adiantum. The absorbance differencespectrum between the red- and far-red-light irradiated sampleshowed a photoreversible absorbance change in the far-red region,with a maximum located at 728–730 nm. The concentrationof phytochrome was highest at the leaf tips and decreased graduallyalong the leaf axis. As in the case of angiosperm phytochrome,the level of fern phytochrome decreased under continuous whitelight, and the level increased again when deetiolated tissuewas transferred back to the dark. When the fern tissue was exposedto a pulse of red light, the dark reversion of P_FR to P_R tookplace with almost no destruction of P_FR. Phytochrome could beextracted from light-grown young leaves of the fern with a slightlyalkaline, aqueous buffer that contained 1 M NaCl. The differencespectrum of the partially purified phytochrome from fern wassimilar to that of partially degraded phytochrome from angio-sperms.A polyclonal antibody raised against phytochrome from etiolatedrye seedlings immuno-stained (albeit weakly) a 110-kDa polypeptideafter fractionation by SDS-polyacrylamide gel electrophoresisof the preparation of fern phytochrome. The band was very probablyfern phytochrome since it emitted zinc-induced fluorescence. (Received July 12, 1990; Accepted October 5, 1990)     

Cold-Tolerant Crop Species Have Greater Temperature Homeostasis of Leaf Respiration and Photosynthesis Than Cold-Sensitive Species

Some plant species show constant rates of respiration and photosynthesismeasured at their respective growth temperatures (temperaturehomeostasis), whereas others do not. However, it is unclearwhat species show such temperature homeostasis and what factorsaffect the temperature homeostasis. To analyze the inherentability of plants to acclimate respiration and photosynthesisto different growth temperatures, we examined 11 herbace-ouscrops with different cold tolerance. Leaf respiration (R_area)and photosynthetic rate (P_area) under high light at 360 µll^–1 CO₂ concentrations were measured in plants grown at15 and 30°C. Cold-tolerant species showed a greater extentof temperature homeostasis of both R_area and P_area than cold-sensitivespecies. The underlying mechanisms which caused differencesin the extent of temperature homeostasis were examined. Theextent of temperature homeostasis of P_area was not determinedby differences in leaf mass and nitrogen content per leaf area,but by differences in photosynthetic nitrogen use efficiency(PNUE). Moreover, differences in PNUE were due to differencesin the maximum catalytic rate of Rubisco, Rubisco contents andamounts of nitrogen invested in Rubisco. These findings indicatedthat the temperature homeostasis of photosynthesis was regulatedby various parameters. On the other hand, the extent of temperaturehomeostasis of R_area was unrelated to the maximum activity ofthe respiratory enzyme (NAD-malic enzyme). The R_area/P_area ratiowas maintained irrespective of the growth temperatures in allthe species, suggesting that the extent of temperature homeostasisof R_area interacted with the photosynthetic rate and/or thehomeostasis of photosynthesis.