Changes in the Chlorophyll Contents of Leaves and in Levels of Cytokinins in Root Exudates during Ripening of Rice Cultivars Nipponbare and Akenohoshi

Changes in fluxes of cytokinins in exudates transported viathe xylem from roots of rice plants cvs. Nipponbare (a standardJapanese cultivar) and Akenohoshi (a slowly senescing cultivar)were measured by mass spectrometry with deuterium-labeled standards.The fluxes of zeatin (Z), trans-ribosylzeatin (trans-RZ), N⁶-isopentenyladenine(iP), and "conjugated Z" (Z in the hydrolysates of highly polarfractions) decreased from heading to the late ripening stagein both cultivars. At the late ripening stage, iP and Z couldno longer be detected, while the flux of N⁶-isopentenyladenosine(iPA) increased slightly. In Akenohoshi, conjugated Z was thepredominant cytokinin from heading to the middle of the ripeningstage. The flux of each of the cytokinins in Akenohoshi washigher than that in Nipponbare at every time point, with theexception of the flux of iPA just after heading. The total concentrationof cytokinins in the xylem exudate of Akenohoshi was higherthan that of Nipponbare after the middle of ripening stage.The chlorophyll content of the third leaves, which were senescingrapidly, was significantly correlated with the flux of totalcytokinins per plant or per unit leaf area. These results suggestthat the larger amounts of cytokinins, in particular conjugatedZ, transported from the roots to the shoots caused the slowsenescence of leaves in Akenohoshi during the ripening stage. (Received May 9, 1994; Accepted July 1, 1995)     

Effects of Light on Degradation of Chlorophyll and Proteins during Senescence of Detached Rice Leaves

Regulatory effects of light on senescence of rice leaves wereinvestigated by measuring degradation of chlorophyll and proteinsin leaf segments which had been kept in the dark or under illuminationwith light of different intensities and colors. When leaveshad been left in total darkness for three days at 30°C,there was an initial long lag that lasted for one whole dayand then chlorophyll was rapidly degraded in the second andthird days. Breakdown of chlorophyll was strongly retarded bycontinuous illumination with white light of intensity as lowas 0.5 µmol photons m^–2 s^–1 but the effectof light decreased at intensities above 10 µmol photonsm^–2 s^–2. The initial lag and subsequent degradationof chlorophyll in the dark were little affected by illuminationwith red or far red light at the beginning of dark treatment.However, a brief illumination with red light at the end of thefirst and/or second day significantly suppressed degradationof chlorophyll during subsequent dark periods and the effectof red light was nullified by a short irradiation with far redlight. Thus, degradation of chlorophyll is regulated by phytochrome.Thylakoid membrane proteins and soluble proteins were also largelydegraded during three days in the dark. Degradation of membraneproteins such as the apoproteins of light-harvesting chlorophylla/b proteins of photosystem II and chlorophyll a-binding proteinsof reaction center complexes showed a long lag and was stronglysuppressed by illumination with weak white light. Thus, theloss of chlorophyll can be correlated with degradation of chlorophyll-carryingmembrane proteins. By contrast, light had only a weak protectingeffect on soluble proteins and ribulose-1,5-bisphosphate carboxylase/oxygenaserapidly disappeared under illumination with weak white light.Thus, breakdown of thylakoid membrane and soluble proteins aredifferently regulated by light. Artifacts which would be introducedby detachment of leaves were also discussed. ¹ Present address: Department of Applied Biology, Faculty ofScience and Technology, Science University of Tokyo, Yamazaki,Noda-shi, Chiba, 278 Japan. ² Present address: Department of Life Science, Faculty of Science,Himeji Institute of Technology, Harima Science Park City, Hyogo,678-12 Japan.     

Effects of Manipulations of Source and Sink on the Carbon Exchange Rate and Some Enzymes of Sucrose Metabolism in Leaves of Soybean [Glycine max (L.) Merr.]

Soybean plants [Glycine max (L.) Merr. cv. AGS129], two andthree weeks after depodding and defoliation, respectively, wereused to examine the possibility of end-product regulation onthe carbon exchange rate and activities of enzymes involvedin sucrose metabolism in leaves. Removal of one and two lateralleaflets per trifoliate leaf reduced the total leaf area by20% and 47%, respectively. Removal of one pod per node reducedthe total pod number by 23% per plant. Dry weights of roots,stems and petioles decreased with reductions in leaf area. Bycontrast, removal of pods resulted in an increase in these parameters.The carbon exchange rate and transpiration rate of leaves increasedwith defoliation and decreased with depodding. The intercellularconcentration of CO₂ in leaves was reduced by defoliation andincreased by depodding. Furthermore, defoliation increased thelevel of leaf chlorophyll in leaves while depodding decreasedit. Removal of pods decreased the activities of sucrose-phosphatesynthase and -amylase but increased that of sucrose synthase.A significant positive correlation was found between the activityof leaf sucrose-phosphate synthase and both the carbon exchangerate and the sucrose content of leaves. Thus, manipulation ofthe sink and source in soybean plants influenced the relationshipbetween sucrose metabolism and the carbon exchange rate in intactleaves. ³Faculty of Agriculture, Okayama University, Tsusimanaka Okayama,700 Japan ⁴Faculty of Agriculture, Saga University, Honjo-machi, Saga,840 Japan ¹Present address: Faculty of Agriculture, Sriwijaya University,J1 Raya Indralaya, OK1 30662, Indonesia ²Present address: Faculty of Agriculture, Saga University, Honjo-machi,Saga, 840 Japan     

Induction of Cytosolic Acetyl-Coenzyme A Carboxylase in Pea Leaves by Ultraviolet-B Irradiation

Levels of subunits of two acetyl-coenzyme A carboxylases werehigh in small leaves of Pisum sativum, decreased with growth,and remained constant in fully expanded leaves. Irradiationof fully expanded leaves induced the cytosolic isozyme only.This result suggests a key role for the cytosolic enzyme inprotection against UV-B. ¹Present address: Laboratory of Molecular Genetics, BiotechnologyInstitute, Akita Prefectural College of Agriculture, 2-2 Minami,Ohgata, Akita, 010-04 Japan ²Present address: Laboratory of Plant Molecular Biology, Schoolof Agricultural Sciences Nagoya University, Nagoya, 464-01 Japan     

Protein synthesis in the embryos of Pinus thunbergii seed I. Influences of imbibition of seeds in the dark

The conditions and requirements of an in vitro protein-synthesizingsystem in the embryos of Pinus thunbergii seeds were studied.Even in the dry seed embryos, the ribosomes retained their syntheticcapacity. Even after imbibition in the dark, the ribosomes didnot show an increase in the activity of protein synthesis. Anincrease in the activity during dark imbibition was found inthe 100,000?g supernatant fraction. The activities of the cell-freesystems prepared from both embryos of dark-imbibed and dry seedswere dependent on the addition of poly U. This suggests thelack or inactivity of messenger RNA in these seed embryos. ¹ Present address: Faculty of Education, Utsunomiya University,Mine-machi, Utsunomiya 320, Japan. (Received July 19, 1976; )     

Effects of Leaf Chilling on Thylakoid Functions, Measured at Room Temperature, in Cucumis sativus L. and Oryza sativa L.

Photosynthetic functions in leaves of cucumber (Cucumis sativusL.) and rice (Oryza sativa L.) were examined before and aftervarious chilling treatments. Cucumber leaves lost the capacityfor the photosynthetic oxygen evolution after chilling at 0°Cin the dark for 48 h. Thyla-koids isolated from such leaveswere not able to reduce dichloroindophenol (DCIP), but the additionof diphenylcarbazide (DPC), an electron donor to PS II, restoredthe ability to reduce DCIP, indicating that the site of damageis in the water-splitting machinery of PS II. In moderate light (500 jumol quanta m^–2s^–1), chillingof cucumber leaves at 5°C for 5 h was sufficient to inducethe complete loss of the capacity for photosynthetic oxygenevolution. Electron transport rates measured in thylakoids wereunaltered, but thylakoids were totally permeable to protons.Since the addition of dicyclohexylcarbodiimide (DCCD) restoredcoupling and the capacity for proton uptake, the primary siteof damage was deduced to be in the ATPase. In rice, both chilling treatments had barely any effect on thylakoidfunctions, although some negative effects was apparent in photosynthesisin leaves. ¹Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan. ²Present address: Department of Botany, Duke University, Durham,NC 27706, U.S.A. (Received January 11, 1989; Accepted June 12, 1989)     

The Photoresponse of S-Adenosylmethionine Decarboxylase Activity in Leaves of Pharbitis nil

S-Adenosylmethionine decarboxylase activity in leaves of Pharbitisnil increased dramatically at lights-on and then gradually decreasedin the light. The enzymatic activity fell dramatically at lights-offthen increased slightly in darkness. Photoinduction was preventedby cycloheximide and the enzymatic activity fell dramaticallyupon treatment with cycloheximide in the light. ¹Present address: Sakuya Konohana Kan, Osaka City Parks Association,Tsurumi-ku, Osaka, 538 Japan     

Changes in Cell Wall Yielding and Stored Growth in Begonia argenteo-guttata L. Leaves during the Development of Water Deficits

Turgor and elongation responses to changes in plant water statuswere investigated in leaves of Begonia plants from which irrigationwas withheld. Withholding irrigation for several days causeddecreases in epidermal cell turgor (P) by ca. 0.25 MPa and leafelongation rates by 66% compared to well watered controls. Rewateringcaused a large and transient increase in the rate of elongation,"stored growth," indicating that during periods at low P therewas an increase in the potential for wall expansion. Subsequently,the ratio of elongation rate/P decreased until both elongationrate and P reached values similar to those observed in leavesof well watered control plants. Tests for significant elasticcontributions or water transport limitations to the elongationresponses were negative: most of the elongation upon rewateringwas caused by irreversible extension and the estimated increasein the water potential gradient between the xylem and the growingcells caused by rewatering was less than 0.02 MPa. Results arediscussed in relation to factors that may contribute to increasethe growth potential at low P. ¹Supported in part by USDA Competitive Grant 8502539 to MAM. ²Present address: Department of Botany/Plant Sciences, Universityof California Riverside, CA 92521, U.S.A.     

Leaf Development in Lolium temulentum: Photosynthesis and Photosynthetic Proteins in Leaves Senescing under Different Irradiances

Changes in carbon fixation rate and the levels of photosyntheticproteins were measured in fourth leaves of Lolium temulentumgrown until full expansion at 360 µmol quanta m^–2s^–1 and subsequently at the same irradiance or shadedto 90 µmol m^–2 s^–1. Ribulose-1,5-bisphosphatecarboxylase/oxygenase (Rubisco), light-harvesting chlorophylla/b protein of photosystem II (LHCII), 65 kDa protein of photosystemI (PSI), cytochrome f (Cytf) and coupling factor 1 (CF₁) declinedsteadily in amount throughout senescence in unshaded leaves.In shaded leaves, however, the decrease in LHCII and the 65kDa protein was delayed until later in senescence whereas theamount of Cyt f protein decreased rapidly following transferto shade and was lower than that of unshaded leaves at the earlyand middle stages of senescence. Decreases in the Rubisco andCF₁ of shaded leaves occurred at slightly reduced rates comparedwith unshaded leaves. These results indicate that chloroplastproteins in fully-expanded leaves are controlled individually,in a direction appropriate to acclimate photosynthesis to agiven irradiance during senescence. (Received August 20, 1992; Accepted January 5, 1993)     

Senescence of Rice Leaves XXIV. Involvement of Calcium and Calmodulin in the Regulation of Senescence

Effects of compounds that influenced calcium uptake and calmodulininhibitors on the senescence of detached rice leaves were examined.Chelators, ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraaceticacid (EGTA) and l,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraaceticacid (BAPTA), significantly promoted senescence of detachedrice leaves in the dark and light. The effect of EGTA can bereversed by treating detached rice leaves with calcium. Verapamil,a calcium channel blocker, and lanthanum chloride, a calciumantagonist, promoted dark-induced, and suppressed BA- and light-retardedsenescence of detached rice leaves. Calcium ionophore A23187 [GenBank] and ruthenium red, believed to raise cytosolic level of Ca²⁺,were quite effective in retarding dark-induced and ABA-promotedsenescence of detached rice leaves. Calmodulin inhibitors, W-7,compound 48/80, chlorpromazine and trifluoperazine, significantlypromoted dark-induced, and suppressed BA- and light-retardedsenescence of detached rice leaves. It is concluded that cytosoliclevel of Ca²⁺ may regulate senescence of detached rice leavesthrough a calmodulin-dependent mechanism. (Received June 13, 1990; Accepted August 3, 1990)     

Senescence of Rice Leaves XVIII. Changes of Stomatal Aperture during Senescence

Changes of stomatal aperture during the course of developmentof rice leaves were directly observed with a scanning electronmicroscope. The stomata reached their maximal aperture sizeafter senescence began in seedling leaves and the flag leafof mature plants. The small stomatal aperture observed priorto senescence seems to be the normal size of stomata in riceleaves, and thus stomata closure does not seem to be the causeof leaf senescence in rice plants. The stomata retain theircapability of movement during senescence, suggesting that guardcells tend to live longer than mesophyll cells. ⁴Present address: Tobacco Taiwan, Republic of China ResearchInstitute, Taichung, Taiwan, Republic of China (Received March 12, 1987; Accepted September 30, 1987)     

Stability of rRNA genes during germination of Vicia faba seeds

Germinating Vicia embryos and the roots of 4-day-old seedlingswere assayed for the proportion of DNA that is complementaryto Vicia rRNA. Hybridization experiments which were performedusing DNA purified by ordinary procedures including RNase digestionshowed a higher level of saturation only for the DNA from theembryos. The embryo DNA showed a Tm of 74?C in 0.1?SSC, whereasthe Tm of the root DNA was 70?C. However, our final conclusionis that there is no gross amplification of rRNA genes for Viciafaba, since the saturation level for the embryo DNA did notexceed that for the root DNA when the DNAs were used after additionalpurification on CsCl gradients. ¹ Present address: Department of Botany, Faculty of Education,Utsunomiya University, Utsunomiya 320, Japan. (Received November 25, 1974; )     

Periplasmic location of dissimilatory nitrate and nitrite reductases in a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroides forma sp. denitrificans

The localization of dissimilatory nitrate and nitrite reductasesof a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroidesforma sp. denitrificans, was investigated. Nitrate and nitritereductases were located in the periplasmic space of the bacteriumgrown anaerobically in the presence of nitrate either in lightor in darkness. Chromatophores showed nitrate and nitrite reductaseactivities when dithionite-reduced benzyl viologen was an electrondonor; this suggests that the enzymes were trapped inside thevesicles. ¹Present address: Japanese Red Cross Central Blood Center, Hiroo4-1-31, Shibuyaku, Tokyo 150, Japan. ²Present address: Plant Growth Laboratory, University of California,Davis, California 95616, U.S.A. (Received November 7, 1979; )     

Effect of Methionine Sulfoximine on Photosynthetic Carbon Metabolism in Wheat Leaves

Methionine sulfoximine (MSO) greatly reduced the carbon dioxideexchange rate (CER) of detached wheat (Triticum aestivvm L.cv Roland) leaves in 21% O₂, but only slightly reduced it in2% O₂. A supply of 50 mM NH₄Cl had little effect on the CERirrespective of the O₂ concentration. A simultaneous additionof glutamine and MSO protected against the inhibition of photosynthesisto a considerable extent and caused the accumulation of moreNH₃ than did the addition of MSO alone. Fixation of ¹⁴CO₂ in wheat leaves was inhibited by MSO treatmentin 22% O₂, and there was decreased incorporation of ¹⁴G intoamino acids and sugars and increased label into acid fractions.The addition of MSO and glutamine together eliminated the effectof MSO on the photosynthetic ¹⁴CO₂ fixation pattern. NH₄Cl stimulatedthe synthesis of amino acids from ¹⁴CO₂, especially the synthesisof serine in 22% O₂. Our observations show that factors other than the uncouplingof photophosphorylation by accumulated NH₃ may be responsiblefor the early stage of photosynthesis inhibition by MSO underphotorespiratory conditions. ¹Present address: Department of Agricultural Chemistry, KyushuUniversity, Fukuoka 812 Japan. ²Also at U.S. Department of Agriculture, Agricultural ResearchService, Urbana, Illionois 61801, U.S.A. (Received September 13, 1983; Accepted February 2, 1984)     

Isolation of a Gene for a Metallothionein-Like Protein from Soybean

Using a synthetic oligonucleotide that corresponded to the consensusnucleotide sequence of the N-terminal region of mammalian metallothioneinas probe, we isolated a cDNA clone from a soybean library. Theclone had an ORF that encode a protein of 79 amino acids whichshowed significant homology to both N- and C-terminal regionsof mammalian and Neurospora crassa metallothioneins ⁴Present address: Department of Biosciences, Teikyo University,Toyosatodai, Utsunomiya, Tochigi, 320 Japan (Received March 13, 1991; Accepted June 17, 1991)     

Changes in Protein Content and in the Structure and Number of Chloroplasts during Leaf Senescence in Rice Seedlings

Two types of experiment were carried out to examine whetheror not the inactivation of photosynthesis is related to lossof chloroplasts during foliar senescence of rice seedlings.Levels of both soluble and insoluble leaf proteins decreasedduring senescence, the loss of the soluble proteins being fasterthan that of the insoluble ones. There was a good positive correlationbetween the rate of oxygen evolution and the level of solubleproteins. The inactivation of photosynthesis was also linearlyrelated to the loss of a major fraction of insoluble proteins.Thus, the loss of photosynthetic ability is ascribable to thedegradation of relevant proteins and enzymes during leaf senescence.Electron microscopy revealed that senescence caused the disorientationof the grana and stroma thylakoids, a decrease in the numberof starch granules, and an increase in the size and number ofplastoglobuli. Large grana consisting 20 to 30 thylakoids appearedin aged leaves. In addition to these changes in ultrastructure,there was a significant decrease in the size of chloroplasts.Furthermore, the number of chloroplasts in mesophyll cells wasalso notably reduced during senescence. Thus, the loss of leafproteins and inactivation of photosynthesis are both relatedto the decrease in the total mass of chloroplasts during senescenceof rice seedlings. ³Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan. (Received January 4, 1989; Accepted April 19, 1989)     

Changes in the Number and Size of Chloroplasts during Senescence of Primary Leaves of Wheat Grown under Different Conditions

Changes in the number and size of chloroplasts in mesophyllcells were investigated in primary leaves of wheat from fullexpansion to yellowing under different growth conditions. Thenumber of chloroplasts per cell decreased slowly, although thedecrease was steady and statistically significant, until thelast stage of leaf senescence, when rapid degradation of chloroplaststook place. Rates of leaf senescence, or the decline in thenumber of chloroplasts, varied greatly among plants grown atdifferent seasons of the year, but about 20% of chloroplastsalways disappeared during the phase when steady loss of chloroplastsoccurred. The area of chloroplast disks also decreased graduallybut significantly, with a rapid decrease late in senescence.Thus, the total quantity of chloroplasts per mesophyll celldecreased substantially during leaf senescence. Yellowed leavescontained numerous structures that resemble oil drops but nochloroplasts. Decreases in rates of photosynthesis that occurduring senescence may, therefore, be largely due to decreasesin the quantity of chloroplasts. However, a better correlationwas found between the decrease in the maximum capacity for photosynthesisand the degradation of RuBP carboxylase. When plants had beengrown with a sufficient supply of nutrients, the number of chloroplastsdecreased steadily but at a reduced rate and the reduction inthe area of chloroplast disks was strongly suppressed. Thus,the quantitative decrease in chloroplasts in senescing leavesappears to be regulated by the requirements for nutrients (nitrogen)of other part of the plant. ³Present address: Department of Biology, Faculty of Science,Toho University, Miyama, Funabashi, Chiba, 274 Japan     

Semi-Quantification of GAX and GA4 in Male-Sterile Anthers of Rice by Radioimmunoassay

The levels of endogenous GA, and GA₄ in the anthers of a standardcultivar and the corresponding male-sterile, single gene mutantof rice, Oryza saliva L. (Japonica), were measured by radioimmunoassayusing GA₄-specific antiserum that showed highly specific immunoreactivitywith GA, and GA₄. The levels of these GAs in the anthers ofthe male-sterile mutant were about one-fifth to one-sixth ofthose in the normal cultivar, suggesting a correlation betweenthe endogenous levels of these GAs and the male sterility. ²Present address: Department of Agricultural Chemistry, UtsunomiyaUniversity, Mine-machi 350, Utsunomiya, 321 Japan (Received August 8, 1990; Accepted March 7, 1991)     

Growth and Sink to Source Transition in Developing Leaves of Sugarbeet

The growth and sink to source transition (cessation of assimilateimport) in light-grown leaves were compared to those of dark-grownleaves. Darkening chambers were placed over sugarbeet (Betavulgaris L.) plants so that new leaves emerged and grew in thedark. New leaves emerged at 1.8 day intervals, regardless ofthe light conditions. The dark-grown leaves were reversiblyretarded in overall growth; they were unable to photosynthesize,but attained photosyn-thetic, vein loading and export capacityafter exposure to sufficient amount and duration of light. Despitethe inability to fix carbon, dark-grown leaves showed dry weightgain. The increase in the dry weight, however, was localizedin the petiole and major veins and not in the laminar tissue.Despite metabolic differences in the two leaf types, sink tosource transition occurred in about a week in both light- anddark-grown leaves; and assimilate importing stopped at about8 days after emergence. While sink to source transition (asdetermined by assimilate import) per se did not appear to belight-regulated, the ability to accumulate sucrose in the veinsfor export out.of leaves was light dependent. This was basedon the observation that post-transitional dark-grown leaveswhich had ceased importing could not export exogenously appliedsucrose unless they were exposed to light for several days.The data indicated that transition is developmentally regulatedand not coupled to photosynthetic capacity. ¹Contribution No. D-15192-1-89 from the New Jersey AgriculturalExperiment Station. This work was funded in part by the BeetSugar Development Foundation and Rutgers Universi ty ResearchCouncil and was submitted as partial fulfillment for M.S. degreeby Lynne H. Pitcher. (Received August 22, 1990; Accepted January 9, 1991)