Photosynthetic Electron Transport During Senescence of the Primary Leaves of Phaseolus vulgaris L.: I. NON-CYCLIC ELECTRON TRANSPORT

Coupled, non-cyclic electron transport was measured for chloroplastsisolated from the primary leaves of Phaseolus vulgaris. Preparationsfrom young, fully expanded leaves gave good rates of electrontransport, but the rates obtained decreased by approximately80% during leaf senescence. Higher rates of electron transportwere recorded for chloroplasts isolated from primary leaveswhich had regreened following removal of the remainder of theshoot. With preparations from leaves of all ages, photophosphorylationwas coupled to electron transport with a mean P/2e ratio ofapproximately 1.3. No evidence was obtained for inactivationof chloroplasts from older leaves during isolation or assay,and it is suggested that the decrease in rate of electron transportover the period of senescence, and its increase during regreening,were consequences of changes in the composition and physicalproperties of the thylakoid membrane which occur in vivo. Thedecrease in rate of non-cyclic electron transport may be importantin limiting the rate of photosynthesis in the senescing leaves.     

Photosynthetic Electron Transport During Senescence of the Primary Leaves of Phaseolus vulgaris L.: III. KINETICS OF CHLOROPHYLL FLUORESCENCE EMISSION FROM INTACT LEAVES

The kinetics of 685 nm chlorophyll fluorescence emission weremeasured at 20 °C following illumination of primary leavesof P. vulgaris. During foliar senescence, a large reductionwas observed in the maximal level of fluorescence emission (P)of the induction curve, normalized with respect to the minimallevel (O), and in the time taken to reach P. This suggests thatfewer plastoquinone (PQ) molecules were able to accept electronsfrom each photosystem two (PS II) reaction centre in older leaves.Measurements of fluorescence emission at 77 °K indicatedthat the primary photochemical quantum yield of the PS II reactioncentres remained constant during senescence. The redox stateof the PQ pool was estimated throughout the induction curveat 20 °C. In both mature and senescent leaves PQ was highlyreduced at P. There followed a reoxidation of PQ in the matureleaves, but in the old leaves the PQ pool remained reduced.This indicates that the rate of electron flow from PQ to photosystemone (PS I) decreased considerably during senescence. Fluorescencewas quenched from P to a steady state level (T) in leaves ofall ages, and this was associated with a redistribution of excitationin favour of PS I. Since, in senescent leaves, changes in theredox state of PQ were absent, it is suggested that quenchingresulted from the generation of proton and ion gradients acrossthe thylakoid membranes, and the synthesis of ATP.     

Photosynthetic Electron Transport During Senescence of the Primary Leaves of Phaseolus vulgaris L.: II. THE ACTIVITY OF PHOTOSYSTEMS ONE AND TWO, AND A NOTE ON THE SITE OF REDUCTION OF FERRICYANIDE

The activity of photosystems one and two (PS I and PS II) wasmeasured in chloroplasts isolated from the primary leaves ofPhaseolus vulgaris. During foliar senescence, the rates of electrontransport through PS I and PS II declined by approximately 25%and 33% respectively. These losses of activity could not accountfor the decrease of 80% in the rate of coupled, non-cyclic electrontransport during senescence. It is therefore suggested thatan impairment of electron flow between the photosystems limitednon-cyclic electron transport in chloroplasts from older leaves.In this study the activity of PS II was measured using oxidizedp-phenylenediamine as the electron acceptor, and trifluralinas an inhibitor of electron transport between PS II and PS I.In chloroplasts from young leaves the reduction of ferricyanidewas a measure of non-cyclic electron transport, but in preparationsfrom older leaves ferricyanide received a large proportion ofelectrons from PS II.     

Starch Accumulation and Photosynthetic Activity in Primary Leaves of Bean (Phaseolus vulgaris L.)

The effects of starch accumulation on photosynthesis and chloroplastultrastructure were studied in primary leaves of bean (Phaseolusvulgaris L. cv. Bulgarian). De-topping the shoot above the primaryleaf node, caused over an 8-day period, a considerable increasein the photosynthetic activity of the primary leaves, despitethe fact that a large quantity of starch had accumulated intheir chloroplasts. The accumulation of starch was greater inthe chloroplasts of spongy cells in comparison with that ofthe palisade cells. Initiation of starch grains was observedmainly in the peripheral part of the chloroplast, distant fromthe cell wall. As a result, most of the starch was accumulatedclose to the inner part of the cell, leaving a considerablemass of the chloroplast near the cell wall free of starch. Theaccumulation of starch was accompanied by the destruction, deformationand disorientation of grana and thylakoids. It is concludedthat the accumulation of starch is not inevitably a limitingfactor in photosynthesis and the results cast doubt on the hypothesisthat starch accumulation or dissipation is the main factor involvedin the regulation of photosynthesis. Phaseolus vulgaris L, bean, photosynthesis, starch accumulation, chloroplast ultrastructure     

Endogenous Regulation of Photosynthetic Rate in Primary Leaves of Bean (Phaseolus vulgaris L.)

The rate of photosynthesis and/or dry matter production wasstudied in fully-expanded primary leaves of bean (Phaseolusvulgaris cv. Bulgarian) plants which had been subjected to varioussurgical and hormonal treatments. Between 30 and 40 per centof the assimilates produced by the primary leaves, over a 4-dayperiod starting with expansion of the first trifoliate leaf,were diverted to the growing shoot above the insertion of theprimary leaves. In detopped plants (i.e. lacking all leaves,stem and buds above insertion of primary leaves), both the rateof net photosynthesis (NP) of the primary leaves 4 days afterdetopping, and the mean net assimilation rate (NAR) over thisinterval, did not differ significantly from those of intactplants. The assimilate normally diverted to the top in intactplants was distributed between the remaining organs of the detoppedplant. When translocation of assimilates from the primary leaveswas stopped by girdling their petioles, both NAR and NP wereas in untreated control plants after a 2-day period. The assimilatesproduced during that period accumulated in the mesophyll chlorenchymain the form of starch granules. Intact plants supplied withGA₃, or IAA, through the primary leaves as well as detoppedplants supplied with IAA through the stump, differed from untreatedcontrol plants in the pattern of distribution of the assimilatesproduced: IAA favoured dry-matter accumulation in the roots,while GA₃ favoured the tops. Nevertheless, neither NP, nor NARdiffered significantly from the corresponding controls.     

Changes in Cell Wall Polysaccharides During the Growth of Phaseolus vulgaris Leaves

The changes in pectic and hemicellulosic polysaccharides, and-cellulose during the expansion growth of the primary leavesof Phaseolus vulgaris L. var. Pinta have been studied. -Celluloseincreased continuously with age, while pectic and water-solublehemicellulose extracted with 4% KOH fractions slightly decreased.The water-soluble hemicelluloses extracted with 24% KOH showedthe most conspicuous changes, increasing until the 8th day,when the absolute growth rate was maximal, and thereafter decreasing.Furthermore, the study of the molecular mass distribution ofpectin, and water-soluble polysaccharides extracted with 4%and 24% KOH, showed an increase in the degree of polymerizationof polyuronic acid and xylan, and an important depolymerizationof galactan and xyloglucan. Accordingly, the mechanism of cellwall loosening in the leaf cell walls is similar to that describedfor plant axes. Key words: Cell wall, growth, leaf     

Ribonuclease in Leaves of Phaseolus vulgaris during Maturation and Senescence

The levels of the enzyme ribonuclease (RNase) were determined in primary leaves of Phaseolus vulgaris in an attempt to correlate changes in RNA with maturation and senescence. RNase, RNA and chlorophyll levels increased in expanding and maturing tissue and subsequently declined in senescing tissue. Senescence of the primary leaves started with the onset of flowering. A simultaneous increase of RNA and RNase in maturing tissue and a decrease during senescence suggests that the enzyme activity is correlated with the rate of‘turnover’of RNA rather than the absolute levels of RNA present in the tissue.     

Dynamics of Imbibition in Phaseolus vulgaris L. in Relation to Initial Seed Moisture Content

The seed moisture level marking the onset of imbibitional injury (breakpoint) was determined for two cultivars of Phaseolus vulgaris L. cvs `Tendercrop' (TC) and `Kinghorn Wax' (KW). At 20°C the breakpoints were 0.15 gram H₂O/gram dry weight (gram per gram) for TC and 0.11 gram per gram for KW. When seeds were imbibed at 5°C, the breakpoints were 0.19 gram per gram (TC) and 0.16 gram per gram (KW). Below the breakpoint germination changed 4.6%/0.01 gram per gram for all treatments. Imbibition rates were maximal at 0.07 gram per gram and 0.33 gram per gram after 20 minutes imbibition. Rates of electrolyte leakage were correlated with the imbibition rate maximum at 0.07 gram per gram but were unaffected by the maximum at 0.33 gram per gram. The transition from tightly bound to semibound water occurred at 0.09 gram per gram and 0.11 gram per gram for KW and TC, respectively. T1 values increased exponentially as seed moisture decreased from 0.47 gram per gram to 0.05 gram per gram. ¹³C-NMR sugar signals increased at moisture levels above 0.14 gram per gram and plateaued at approximately 0.33 gram per gram seed moisture. These results suggest that the breakpoint moisture level for imbibitional damage is a function of temperature while the injury process is similar at both 5 and 20°C. Imbibition and leakage rate maxima reflect transitions in the states of seed water. NMR data support the application of the Water Replacement Hypothesis to seeds. Thus, imbibitional injury may be related to specific, temperature dependent moisture levels that are determined by water binding characteristics in the seed tissue.     

Increase in DNA Content of Primary Leaves of Phaseolus vulgaris upon Decapitation

Changes in DNA content of bean (Phaseolus vulgaris) primaryleaves after decapitation were investigated. When apical budswere removed at 11 d, DNA content per leaf increased by about20% at 15 d and then decreased in parallel with the controls.The RNA and chlorophyll contents, fresh weight, and leaf areaexpressed on a single leaf basis changed in the same manneras the DNA content in response to decapitation. But when bothapical and lateral buds were removed, all these values continuedincreasing during the test period. Thus, growing lateral budsand apical buds have the same effect on the DNA change in primaryleaves as that due to ageing of the leaves. Cell number perleaf was not increased by any treatment, indicating that theobserved increase in the DNA content of primary leaves is ascribableto an increase in DNA per cell. Next, the whole shoots above the nodes of primary leaves wereremoved at various ages. The response of primary leaves to decapitationvaried according to their age. With age, they lost the abilityto increase their fresh weight, area, and chlorophyll contentbut not their DNA and RNA contents in response to decapitation.Decapitation stimulated chloroplast replication only withinthe period in which chloroplasts were replicating in controlleaves, but it induced chloroplast enlargement at any age. Therefore,the increase in DNA content after decapitation may be partiallydue to an increase in the amount of chloroplast DNA. When stems were heat-girdled above the nodes of the primaryleaves, these leaves showed responses similar to but smallerthan those to decapitation. The senescence of primary leavesseems to be controlled by the distribution of substances whichare transported from the roots.     

Transport of 14C-Assimilates in Seedlings of Phaseolus vulgaris L. in Relation to Vascular Anatomy

Patterns of ¹⁴C-photosynthate translocation in bean (Phaseolusvulgaris L.) seedlings have been investigated in relation tovascular-bundle continuity between exporting and importing organsby use of radioassay and tissue-clearing techniques. Assimilatefrom the primary leaves reaches the first trifoliate leaf byan indirect route. There is no direct vascular connection betweenthe primary leaves and distal tissues. Bundles of the primaryleaf petiole connect with an anastomosis at the node, and allbundles which originate from this structure descend the stem.Assimilate from primary leaves moves down the stem, and is thentransferred to an ascending pathway, the bundles of which traversethe anastomosis at the second node. The lateral leaflets ofthe first trifoliate leaf are served differentially by primaryleaves with respect to assimilate supply. Differences are relatedto position, and may be accounted for by differences in vascularcontinuity.     

Effects of Different Inorganic Nitrogen Sources on Photosynthetic Carbon Metabolism in Primary Leaves of Non-nodulated Phaseolus vulgaris L

Young bean plants (Phaseolus vulgaris L. var Saxa) were fed with three different types of inorganic nitrogen, after being grown on nitrogen-free nutrient solution for 8 days. The pattern of ¹⁴CO₂ fixation was investigated in photosynthesizing primary leaf discs of 11-day-old plants (3 days with nitrogen source) and in a pulse-chase experiment in 13-day-old plants (5 days with nitrogen source).

Ammonium caused, in contrast to nitrate nutrition, a higher level of ¹⁴C incorporation into sugar phosphates but a lower incorporation of label into malate, glycolate, glycerate, aspartate, and alanine. The labeling kinetics of glycine and serine were little changed by the nitrogen source. Ammonium feeding also produced an increase in the ratio of extractable activities of ribulose-1,5-bisphosphate carboxylase to phosphoenolpyruvate carboxylase and an increase in dark respiration and the CO₂ compensation concentration. Net photosynthesis was higher in plants assimilating nitrate.

The results point to stimulated turnover of the photosynthetic carbon reduction cycle metabolites, reduced phosphoenolpyruvate carboxylation, and altered turnover rates within the photosynthetic carbon oxidation cycle in ammonium-fed plants. Mechanisms of the regulation of primary carbon metabolism are proposed and discussed.

     

Regulation of Photosynthetic Activity in the Primary Leaves of Bean (Phaseolus vulgaris L.) by Materials Moving in the Water-conducting System

Endogenous factors which determine the photosynthetic capacity of the leaf were studied in the fully expanded, primary leaves of young seedings of bean (cv. Bulgarian). Following removal of the shoot above the primary leaf node and excision of all axillary buds, the primary leaves increased in area and thickness, in chlorophyll content, in levels of soluble protein, and in the specific activity of ribulose-1,5-bisphosphate carboxylase. Plants in which phloem continuity was disrupted by heat-girdling of the stem, between the shoot above the primary leaf node and the organs below, did not exhibit similar increases, whereas the shoot above the girdle continued to grow for several days. Plants in which all developing trifoliate leaves were excised as soon as they became macroscopic exhibited an increase in their photosynthetic activity, area, and thickness, while their main stem and (leafless) branches made considerable growth. Transpiration from the primary leaves was the same in decapitated plants as in the heat-girdled ones, although in the latter it accounted for only about 30% of total transpiration.     

Effects of Benzyladenine on Chlorophyll, DNA, RNA and Protein Content of Attached Young Bean (Phaseolus vulgaris L.) Leaves

N⁶-Benzyladenine (BA) was applied to intact bean (Phaseolusvulgaris L.) primary leaves at 2 and 6 days after imbibition,when they were in the cell division and post-cell division stages,respectively. BA treatment at day 2 temporarily inhibited an increase in chlorophyllcontent in the following day, but stimulated it in later days.No such inhibition by BA was observed for changes with timein DNA, RNA, and protein content and f. wt. On the other hand,BA treatment at day 6 enhanced RNA and protein content, withoutsignificant influence on DNA and chlorophyll content and f.wt. The mode of cytokinin action on greening in leaves during cell-divisiongrowth seems to be different from that in etiolated cotyledons. Phaseolus vulgaris L., bean, greening, benzyladenine, DNA, RNA, protein     

Nitrogen Fixation, Assimilation and Transport During the Initial Growth Stage of Phaseolus vulgaris L

In nodulated common bean (Phaseolus vulgaris L.), there is typicallya period of N stress between 15 and 20 d after emergence (DAE),due to a lack of synchronization between the depletion of Nin the cotyledons and the beginning of N² fixation and transport.Screening trials identified some Rhizobium leguminosarum bv.phaseoli strains with which symptoms of N deficiency were notvisible (‘precocious’ strains). Cultivar Negro Argelwas then inoculated with two ‘traditional’ strains(C-05 and CIAT 727) and two ‘precocious’ strains(CNPAF 146 and CNPAF 512), and plants were harvested from 8to 30 DAE. There were no differences between the two groupsof strains in nodule dry weight or in the acetylene reductionrates between 8 and 16 DAE. However, nodules induced by the‘precocious’ strains showed earlier onset of glutaminesynthetase (GS) (EC 6.3.1.2 [EC] ) and glutamate synthase (GOGAT)(EC 1.4.1.14 [EC] ) activities, and ureide synthesis. The N concentrationin the nodules formed by ‘precocious’ strains variedfrom 4.2 to 4.5%, whereas with the ‘traditional’strains, it increased from 3.2% at 8 DAE to 65% at 18 DAE, atwhich time plants exhibited N-deficiency symptoms. By 21 DAE,GS and GOGAT activities in ‘traditional’ noduleswere increased, as well as the ureide-N-concentration in thexylem sap, nodule N content declined to 4.5% and the leavesbecame green. These results suggest that the N stress with ‘traditional’strains is not a limitation in early N² fixation activity butrather in the rates of expression of the processes of N assimilationand transport. Key words: Glutamate synthase, glutamine synthetase, nitrogen fixation, Phaseolus vulgaris, Rhizobium     

Developmental Features of the Primary Phloem in Phaseolus vulgaris L.

Certain developmental features of the primary phloem were examinedin Phaseolus vulgaris L., chiefly by the use of the pulvinusat the base of the petiole. The cells included in the studywere the sieve element, the companion cell, and the tannin cell.In the sieve element, the sieve plate shows the usual sequenceof conversion of plasmodesmatal canals into pores. The endoplasmicreticulum, which appears as flat cisternae associated with ribosomesin younger cells, later becomes in part stacked and in partaligned parallel with the walls as a network. The stacked ERprecedes the anastornosing parietal ER in time of development,but the parietal ER persists longer. Of the two forms of P-proteincharacteristic of a number of Fabaceae, the crystalline bodyappears considerably in advance of the body composed of tubules.Neither form of P-protein disperses completely in the maturecell, although the crystalline protein may spread out into aggregatesof fine fibrils. The companion cells show the typical denseprotoplasts and branched plasrnodesmatal connections with thesieve elements. The vacuome of these cells is dispersed intonumerous small vacuoles, many of which appear to be concernedwith autophagic digestion of protoplasmic material. The tannincells have large vacuoles in which the tannin material is located.The cells form vertical series in which the end walls becomeperforated.     

Diurnal Geotropic responses of the Primary Leaves of Phaseolus vulgaris

When seedlings of Phaseolus vulgaris with leaves in the daytimeposition (almost horizontal to the ground) were turned upside-downduring the light period, their leaves moved upward away fromthe ground after about 20 min and ceased moving after about1.5 h. But when seedlings with leaves in the night time position(directed downward) were turned upside-down, their leaves moveddownward toward the ground after about 30 min and stopped movingabout 2 h later. Thus, Phaseolus primary leaves showed positiveor negative geotropic responses that correspohded to the darkor light period. This geotropic response of primary leaves was accompanied bythe redistribution of K⁺, Cl^– and NO₃- in the laminarpulvinus. These facts suggest that the circadian endogenousclock that is assumed to exist in Phaseolus vulgaris has atleast two regulation echanisms; one which measures time andanother which determines leaf postition in relation to gravityby changing the ion distribution in the pulvinus (Received February 12, 1983; Accepted May 17, 1983)     

Correlative Growth in Seedlings of Phaseolus vulgaris L. : Inhibition of Stem Growth by the Primary Leaves

Primary leaves of one-week-old seedlings of dwarf beans effectivelyinhibit stem elongation either in natural daylight or in continuousdarkness. Stem elongation is promoted by exogenous gibberellicacid and kinetin and inhibited by indol-3-ylacetic acid (IAA)or abscisic acid, and it is suggested that IAA is the inhibitorysubstance emanating from the primary leaf blades which affectsthe growth of the stem.     

Observations on the Senescence of a Mutant Non-yellowing Genotype of Phaseolus vulgaris L.

A non-yellowing mutant of Phaseolus vulgaris L. was used toinvestigate factors involved in chlorophyll breakdown duringfoliar senescence. The mutant showed physiological changes similarto those of the normal yellowing type during senescence exceptthat leaf chlorophyll did not decline. Transmission electronmicroscope studies did not reveal appreciable differences inchloroplast ultrastructure between the two genotypes, suggestingthat chloroplast membrane integrity was not the factor preventingchlorophyll degradation in the mutant. However, the lack ofplastoglobuli in senescent mutant chloroplasts suggested thatthe lipid environment may be different from that of senescentnormal chloroplasts. Banding patterns of total soluble protein,resolved by sodium dodecyl sulphate-poly aery lamide gel electrophoresisshowed few, if any, differences between mature non-senescentnormal and mutant leaves; however, bands at 14 kD and 58 kDdiminished in senescent normal leaves, but remained in senescentmutant non-yellowing leaves. Key words: Non-yellowing mutant, Phaseolus vulgaris, senescence, chlorophyll degradation     

Transport and Degradation of Auxin in Relation to Geotropism in Roots of Phaseolus vulgaris

The movement of auxin in Phaseolus vulgaris roots has been examined after injection of IAA^?3H into the basal root/hypocotyl region of intact, dark-grown seedlings. Only a portion of the applied IAA^?3H was transported unchanged to the root tip. The major part of the chromatographed, labelled compounds translocated to the roots was indole-3-acetylaspartic acid (IAAsp) and an unidentified compound running near the front in isopropanol, ammonia, water. The velocity of the auxin transport (7.2 mm per hour) was calculated from scintillation countings of methanol extracts from serial sections of the root. An accumulation of radioactive compounds in the extreme root tip, was observed 5 h after the injection of IAA. The influence of exogenous IAA on the geotropical behaviour of the bean seedling roots was examined. Pretreated roots were stimulated for 5 min in the horizontal position and then rotated parallel to the horizontal axis of the klinostat for 60 or 90 min. The resulting geotropic curvature of IAA-injected and control roots showed significantly different patterns of development. When the stimulation was started 5 h after application of the auxin, the geotropic curvature became larger in roots of the injected plants than in the controls. If, however, the translocation period was extended to 20 h the geotropic curvature was significantly smaller in the roots of the injected plants. The auxin injection did not significally affect the rate of root elongation. The change in geotropical behaviour of the roots is interpreted as a result of the influence of the conversion products of the applied IAA on the geotropical responsiveness.     

The Effects of Root Cooling and the Light--Dark Transition on Growth of Primary Leaves of Phaseolus vulgaris L.

Use of Rotary Variable Displacement Transducers attached togrowing primary leaves of Phaseolus vulgaris has shown thatwhen the root systems were rapidly cooled from 23°C to 10°Cleaf extension rate fell to very low values within a few minutes.When the root systems were returned to 23°C leaf extensionincreased almost immediately to overshoot the control rateswithin 5–10 min, before declining to control values overthe next 50–60 min. When lights went off at the end ofthe day cycle there was an immediate and very large increasein leaf extension rate in both root-cooled and control plants;the rate then slowly declined over the next 60 min. This effectwas seen when the photoperiod was artificially shortened orlengthened and was reduced in magnitude when the photoperiodwas ended gradually by dimming the lights. The effect was notattributable to effects on leaf temperature but appears to bethe result of an endogenous rhythm interacting with the endof the photoperiod. At the beginning of the photoperiod therewas a gradual reduction in leaf extension rate occurring over30–45 min. Key words: Phaseolus vulgaris, leaf growth, extension rate, root cooling, wall extensibility, turgor