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The Interaction of Light Intensity and Nitrogen Supplyin the Growth and Metabolism of Grasses andClover (Trifolium repens)IV. Th e Relation of Light Intensity and Nitro gen Supply to the ProteinMetabolism of the Leaves of Grasses

BYG. E. BLACKMAN

ANDW. G. TEMPLEMAN

Departm ent of Botany, Imperial College of Science and Technology, London , and ImperialChemical Industries Research Station, Warfield, Berks.)With 23 Figures in the Text

I N T R O D U C T I O NTHE first of this series of papers (Blackman, 1938) was concerned withthe effects of variations in the light intensity and nitrogen supply on theclover {Trifolium repens) content of a sward. It was shown that in full daylightthe addition of calcium nitrate and more particularly ammpnium sulphatedepressed the clover, whereas at lower light intensities (o-o-c-4 of daylight)the effects of additional nitrogen were completely masked by the diminutionof clover brought about by shading. Subsequently (Blackman and Tem ple-man, 1938) a study was made of the influence of these light and nitrogenfactors on the leaf production of grasses and T. repens when the plants werefrequen tly defoliated. In full daylight it was found that both calcium nitrateand ammonium sulphate increased the leaf production of clover to a smallextent and that of the grasses (Agrostts tenuis and Festuca rubra) by a largeam ount. At lower light levels, more particular ly at an intensity of 0-4 daylight,the grasses and clover reacted very differently to additional nitrogen. In thecase of clover, apart from the very marked effect of shading in lowering leafproduction, neither ammonium sulphate nor calcium nitrate caused anyappreciable change . W hen the grasses were shaded, however, the addition ofnitrogen depressed the production of leaves and the depression was accen-tua ted with each successive defoliation. Moreover, the somewhat surprisingresult was obtained that the diminution caused by calcium nitrate was greaterthan that brought about by ammonium sulphate.In view of the claim of Prianishnikov (1910, 1922) that the accumulationof ammonia nitrogen produces deleterious effects and that such accumulationis associated with carbohydrate deficiency, it seemed clear that the results of[Annals of Botany, N.S . Vol. IV, No. 15, July 1940.]

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534 Blackman and Templeman The Interaction of Light Intensity andEXPERIMENT

IE X P E R I M E N T

Significant _difference =

Control - EXPERIMENT

IffC a ( N 0 i > 2 -

EXPERIMENTm

F i o . i (.tee opposite)

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Nitrogen Supply in the Growth of Grasses and Clover. IV 535these two investigations could not be adequately interpreted withou t a study ofthe nitrogen and carbohydrate constituents of the plants. In the third paper(Blackman and Tem pleman, 1940) the m ethods em ployed for the estimation ofthe various carbohydrate and nitrogen fractions were described. T he prese ntpaper is concerned with the analytical data obtained for the grasses; theresults for T. repens will be given subsequently.

EX PERIMENTAL RESULTST he analyses were carried out on the dried leaves obtained from four of theexperiments of which the growth data have been described in the second paperof this series. In order, however, to interpret with greater clarity the presen tdata the main results for these particular experiments will be recapitulated.Three experiments were conducted in 1935, two with A. tenuis and onewith F. rubra, while in 1936 a single experiment on A. tenuis was undertaken.In each case until the plants were established they were either not cut at allor cut but seldom. During the experimental period they were, however,defoliated some six to eight times at intervals of seven to fourteen days. T herange of light intensity was obtained by shading the plants with screens ofone or more layers of butter muslin while the additional nitrogeneither asammonium sulphate or calcium nitratewas added after each cut.The main effects of light intensity and nitrogen supply on the mean leafproduction are seen in Fig. 1. In all four experiments bo th calcium n itra teand ammonium sulphate at the h ighest light intensity (full daylight) increasedleaf production . At the interm ediate light level (0-61-0-63 of daylight) pro-duction is in part controlled by light, but nevertheless ammonium sulphate,except in experiment I II , brought about an increase. However, in only oneexperiment (expt. I) does the calcium nitrate effect not differ from that ofamm onium sulphate. In experiment I II calcium nitrate has not raised leafproduction, while in experim ent IV it has depressed it. At the lowest lightlevel (0-44-0-37 of daylight) these differences between the ammonium sul-phate and calcium nitra te effects are again noticeable. Moreover, at th islight intensity both calcium nitrate and ammonium sulphate have diminishedleaf production, e.g. experim ent IV . Thu s the experiments fall into a seriesin which the interactions between light intensity and the effect of the twosources of nitrogen are negligible in experiment I and most pronounced inexperiment IV . In the consideration of the analytical data this gradationfrom experiments I to IV will be stressed.

The level of total nitrogen in relation to nitrogen supply and light intensity.In all experiments estimates of the total nitrogen content of the leaves weremade on the samples collected at each cut. The results are shown graphically

FIG. 1. Th e effects of light intensity and of nitrogen supply on the mean leaf production o fgrasses when frequently defoliated. Experiments I, II , and IV, Agrostit tenuis; experiment III,Festuca rubra.

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536 Blackman and TemplemanThe Interaction of Light Intensity and

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037 DayliqhbI I II I I.1st. 2nd. 3nl. 4th. 5th. 6th. 7th. 1st. 2nd. 3rd. 4Ui 5th. 6th.Number of defoliations

FIG. 2. The effects of light intensity and of nitrogen supply on the total nitrogen contentof the leaves ofA. tennis at successive defoliations. (Experiment I, Aug. 14-Nov. 1, 193s;experiment II, May 8-July 9, 1936.)

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Nitrogen Supply in the Growth of Grasses and Clover. IV 53 75-24 84-44 0

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0-61 Daylight0-44Dayiight

1st. 2nd 3rd. 4th. 5th.' 6th 1st 2nd. 3rd 4th . 5th. 6th .Number of defoliationsFIG. 3. Th e effects of light intensity and of nitrogen supply on the total nitrogen content

of the leaves of F. rubra (experiment III) and A. tenuis (experiment IV) at successive defolia-tions. (June 18-Aug. 26, 1935.)in Figs. 2 and 3 and the mean contents given in Tab le I. In the absence ofadditional nitrogen a decrease in the light intensity has led to an increasednitrogen content save in exp eriment I (Fig. 2) where, unlike in th e other th ree

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53 8 Blackman and Templeman The Interaction of Light Intensity andexperiments, the total nitrogen content of the unmanured plants rises rathertha n falls with time. The marked accumulation of nitrogen in the leaves as aresult of manuring (see Figs. 2 and 3) is dependent both on the number ofdefoliations and the light intensity. In full daylight the difference in the

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1st. 2 nd. 3rxt 4th. 5th. 6th . 1st. 2nd 3rd. 4th . 5th. 6th.Number of deFoliationsF ie . 4. Th e effects of light intensity on the protein nitrogen content of the leaves of controlplants (low nitrogen supply) at successive defoliations. (Experiments I, II , and IV, A. temdt;experiment III, F. rubra.) ,

nitrogen content between the control and the manured plants becomes morepronounced with successive cuts in experiments II-IV (see also Figs. 6-8).But in experiment I (Figs. 2 and 5) the difference decreases with time . Atthe lowest light intensity this trend is less evident; in fact, except in experimentI, the gains in nitrogen content over the controls show if anything a negativecorrelation with frequency of cutting.That the effects of high and low light intensity on the accumulation of

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Nitrogen Supply in the Growth of Grasses and Clover. IV 539nitrogen in manured leaves are significantly dissimilar is seen in Table II.For the purpose of statistical analysis regression equations were first fittedto the data shown in Figs. 5-8, i.e. regressions of the difference from the con-trol in nitrogen content against the number of defoliations. Subsequently theanalysis of variance was carried out on the regression coefficients, the resultsfor individual experiments being regarded as replicates.

TABLE IThe Influence of Light Intensity and Nitrogen Supply on the Content and Amount

of Nitrogen in the LeavesTreatments

T *rt1 \JDaylight0-63-0-61Daylight

0-440-37Daylight

(Control(NH4),SO4Icaosro,),(Control(NH4),SO4l(CaNO,),/ Control(NH4),SO4lCa(NOs),

Mean nitrogen contentExpt.4-20

S-i8S-O54-265 2 04-954-O35-124-73

(% ofI II3-855-O7

5 094-185 i 3S1"4-18499466

dry matter)III3-604'734 8 04-224974-804485 004-77

TABLE

IV4-465-325-374 925-525'275-025395 19

II

Total amount of nitrogen(gm. per cut per pot)Expt. I0-0450-1250-1190-0480-0760-0760-0400-0640-066

II0-077O-2OI0-1980-087OI58OI36O-O78OO99OO79

III0-1060-2470-22501230-1730-1460-104o-no0-096

IV01380-2620-2240136017901270-114O-IO2O-O82

The Effects of Light Intensity on the Accumulation of Nitrogen in the Leaves ofNitrogenously Manured Plants

(Statistical analysis based on regressions of difference from the controls innitrogencontent (% of dry matter) against thenumber of defoliations.)Experiments I-IV Nitrogen treatments

(NH4),SO4 Ca(NO,), Mean11-o Daylight 0-065 0-076 0-071

Light treatments(0-44-0-37 Daylight 0-006 0-023 0-014V Mean 0-030 0-027Significant difference between treatments = 0-088,, ,, mean of 2 treatments = 0-062

Experiments I I-IV Nitrogen treatments(NH4),SO4 Ca(NO8), Mean{ i-o Daylight 0133 0154 0144

0-44-0-37 Daylight 0-020 0-005 0-013Mean 0-077 0-080

Significant difference between treatments = 0-027,, means of 2 treatments = 0-013If experiments I-IV are considered together then there is a significant

difference between the effects of additional nitrogen at the two light levels. Indaylight the increased nitrogen content of the leaves from manured plantsrises with the number of cuts, whereas in o-44~o-37 of daylight it tends to fall.

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Fio.5.ExperimentI.Theeffectsoflightintensityandofadditionalnitrogenonthegainsorlosses,relativetothecontrol

changes,inthetotalandproteinnitrogencontentsoftheleavesofA.tettuisatsuccessivedefoliations.

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Nitrogen Supply in the Growth of Grasses and Clover. IV 541There is no appreciable difference in the effects of ammonium sulphate andcalcium nitrate whether or not the data for experiment I are included in theanalyses. Om itting experim ent I does, however, lower the error and accen-tuate the significance of the light effects.mO I 0 D a ^: Protein nitrogen ^2-0Total nitrogen ' '

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542 Blackman and Templeman The Interaction of Light Intensity andProtein level in relation to nitrogen supply and light intensity.

In experiments IIII estimates of the protein nitrogen were made on thesamples collected from the plants growing either in full daylight or o*44-o-37of daylight, while in experiment IV determ inations w ere also carried out at the

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1st i ! i I 0 4 r2ndj3rd;4th , '5 th;6th |

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FIG. 7. Experiment III . The effects of light intensity and of additional nitrogen on thegains or lostes, relative to the control changes, in the total and protein nitrogen contents of theleaves of F. rvbra at successive defoliations.interm ediate ligh t level. The effects of light intensity on the protein contentof the leaves from the unmanured series are seen in Fig. 4 and Table III.T h e general trend is similar to that of the total nitrogen data. Just as loweringthe light intensity increases the total nitrogen content in experiments II-IV,so a redu ction in the light level raises the pro tein con tent. In experiment Ithe trend is again different from experiments II-IV, for in daylight the proteincontent is greatest.Reference has already been made to Figs. 5- 8 ; these, in addition to demon-strating the effects of light intensity and increased nitrogen supply on theaccumulation of nitrogen in the leaves, also show their influence on the con-version of this nitrogen into protein. At the highest light intensity the resultsare very similar in all experiments. T he increase in nitrogen content over thecontrol as a result of the addition of either ammonium sulphate or calciumnitra te is correlated very closely with an increase in protein content. In

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Nitrogen Supply in the Growth of Grasses and Clover. IV 543

Nitrogen(yodru matter)

lab j 2nd! 3rd! 4thI 5th|6bh|Number of defoliations

F I G . 8. Experiment IV. The effects of light intensity and of additional nitrogen on the gainsor losses, relative to the control changes, in the total and protein nitrogen contents of the leavesof A. tenuis at successive defoliations.

TABLE IIIThe Influence of Light Intensity and Nitrogen Supply on the Percentage Con-tent and Amount of Protein Nitrogen in the Leaves

Treatmentsi - o ( Control

ControlI ControlS S & 7 { j * s O '

Mean protein nitrogencontent(% of dry matter)Expt. I

360431421

II III3304344-28

3 1 2397397

3 434-073 77352404368

IV3 8 44-39440 410 433 413

3-69 4163-75 4-24353 396

Total amount of proteinnitrogen(gm. per cut per pot)

E x p t . I II III IV0-039 0-065 0-093 0-1170-103 0-170 0-208 0-2150-098 0-166 0-186 0-184 0-113 0-140 0-102

0-034 0-066 0-085 0-0940-051 0-080 0-083 0-0780-052 0-062 0-073 0-062

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544 Blachman and Templeman The Interaction of Light Intensity andexperiments II-IV (Figs. 6-8) as the difference in nitrogen level from thecon trol rises with successive cuts so does the difference in protein . In experi-m en t I (F ig. 5) where, in con trast, the difference in nitrogen content falls withtime the protein nitrogen also decreases.Although in full daylight the accumulation of nitrogen in the leaves ofmanured plants is coupled with an increase in protein nitrogen, at the lowestlight level increases in nitrogen a re no t always followed by increases in p rotein .This difference in the effects of high and low light is most marked in experi-ments I I I and IV and least in experim ent I. In experiment I und er the lowlight intensity appproximately half the increase in nitrogen can be accountedfor as protein (see Tables I and III), while in daylight approximately three-qua rters of the nitrogen is found as pro tein. In experiment I I this divergenceis again ev iden t; with daylight of 75-85 per cent, of the nitrogen accumulatedis present as protein, whereas in 0-43 daylight the proportion is 33-64 percent. Although in experiments I and I I the increase in nitrogen under thelow light intensity is associated with a gain in protein, the results for experi-ments III and IV demonstrate that an increase in nitrogen may be evennegatively correlated with protein level. Fro m Fig. 7 and Tab le I I I it is seenthat on average the increase in nitrogen content over the control cannot inexperiment I I I be accounted for as protein . On the contrary, especially wherecalcium nitrate is added, the rise in nitrogen is coupled with a loss of protein.In experiment IV (Fig. 8) there is a marked gain in protein in full daylight.At the intermediate light level there is some increase in protein with theammonium sulphate treatment, and on average no change in the case ofcalcium n itrate. Under the lowest light intensity the plants manured withammonium sulphate have gained a little protein but less than at the inter-mediate light level, while as a result of the app lication of calcium nitra te therehas been a loss of protein in the leaves.

Besides the divergent effects of high and low light intensity on the balancebetween total nitrogen and protein, the influence of the frequency of defolia-tion mu st be taken into accoun t. U nd er high light, from the initial to thefinal cut, there is a gradual increase in the protein content of manured plantsover that of the controls except in experimen t I (Fig. 5). But with the lowestlight intensity in all four ex perim ents the re is a fall in the pro tein differencewith time (Figs. 5-8). T ha t these two tren ds under the two light levels aresignificantly different is seen in Table IV, whether the data for experiment Iare included or not in the statistical analysis. On the other hand, neither inhigh or low light are the regressions for ammonium sulphate or calciumnitrate dissimilar.

T h e results for the total amount of protein nitrogen are set out in Table I I I .From the data it is seen that on this basis the effects of light intensity andnitrogen supply are even more accentuated than on the basis of percentagedry matter. In full daylight additional nitrogen has led to a very marked risein pro tein nitrogen. At lower light levels there is a very considerable drop in

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Nitrogen Supply in the Growth of Grasses and Clover. IV 545protein accumulation as a result of manuring. In fact, although in experimentI an increase in protein follows on the application of either ammonium sul-phate or calcium nitrate, yet in experiment IV an increase in the nitrogensupply leads to a decrease in protein.

TABLE IVThe Effects of Light Intensity on the Accumulation of Protein Nitrogen in the

Leaves of Nitrogenously Manured Plants(Statistical analyses based on regressions of difference from controls in proteinnitrogen content (% of drymatter) against numb er of cuts.)

Experiments I-IV Nitrogen treatments(N H 4) ,SO Ca(NO 3 ) t Mean.11 -o Daylight 0-040 0-041 0-040Light treatments!0-4 4-0 -37 Daylight 0-025 0-044 0-035I Mean 0-007 0-002Significant differences between treatments = 0-088 ,, ,, means of 2 treatments = 0-062

LightExperiments

treatments {o-I

I I - IV0 Daylight44-037 Daylight

Mean

(N H 4) ,SOo-ioo 0 - 0 0 70-047

Nitrogen treatments4 Ca(NO 3 ),0-1080-026

0-041

Mean.0 - 1 0 4 0 - 0 1 6

Significant difference between treatments = 0-042 ,, mean of 2 treatments = 0-029Non-protein nitrogen level in relation to nitrogen supply and light intensity.

In order to interpret more fully the changes in the non-protein nitrogen,this fraction was further analysed into nitrate nitrogen and 'organic' non-protein nitrogen. The term 'organic', which is used for th e sake of brevity, isa slight misnomer as it includes any ammonia nitrogen present in the leaves.However, theproportions of ammonia nitrogen found in anumber of samples(see Table IX) are extremely small, and thus this term is likely to be mis-leading only to a very small extent.Nitrate nitrogen. The influence of light intensity and nitrogen supply onthe nitrate nitrogen content of the leaves is even more marked than on thetotal nitrogen andprotein levels. Theeffect of varying light intensity in theabsence of additional nitrogen is seen in Fig. 9 and Table V. In all experi-ments the leaves under the low light intensity contain more nitrate nitrogen,particularly in experiments II-IV. In full daylight the addition of bothammonium sulphate and calcium nitrate has greatly increased the nitratenitrogen level over that of the control, more so in fact than at the lower lightlevels (see Table V).The relationship between the accumulation of total and nitrate nitrogenin the leaves of manured plants is seen in Figs. 9-12. As in the case of the

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546 Blackman and Templeman The Interaction of Light Intensity andTABLE V

The Influence of Light Intensity and Nitrogen Supply on the Content and Am ountof Nitrate Nitrogen in the Leaves

Treatments

/ControlDayl ight (NHJ.SO,lCa(NO s) ,0-63-0-61 //v n- r\ cri

/ C o n t r o lDavliKht 1 ('S^H*)SO

Mean nitrate ni trogen('

Ex p t . I00510-2190-217

0-0710-3310-402

content% ofdry matter)I I0-0270-0870-144

0-1240-2690346

I I I0-0810-2920-274

029305980-696

IV0-1090-22903140199o-4S60-5050-3520-6220-711

Total(gm.Expt. I

0-00060005700053

0-00060-004100053

amount of nitratenitrogen, per cut p er pot)I Io-ooi0-0040-006

O-OO2O-OO5O O O 5

I I IO-OO2O - O I 2O - O I 2

O-OO7O - O I 2O - O I 2

IVO-OO3O- OI4O- OI30-0060-015O-OI20-008O-OIIO-OII

protein accumulation the divergent effects ofhigh and low light intensitiesare most marked in experiments III and IV (Figs. 11 and 12) and least inexperiment I (Fig. 9). Indaylight the increase in total nitrogen content isaccompanied byarelatively small increase in nitrate nitrogen. The averageproportion that the nitrate nitrogen increase bears to the total nitrogen riseranges from 5 to10 per cent, for the ammonium sulphate and calcium nitratetreatments in experiment II to 22 per cent, for both nitrogen treatments inexperiment IV. But at a light intensity of o-44-o-37 of daylight the propor-tions are much larger; in experiment I it is 23 per cent, and 42 per cent, afterthe addition of ammonium sulphate and calcium nitrate respectively, and inexperiment IV 73 per cent, and 210 per cent. Moreover, at the lower lightlevels the accumulation of nitrate nitrogen becomes in each experimentprogressively greater with successive defoliations; thus the ratio of nitrate tototal nitrogen rises steadily with time. Th is is especially evident inFigs. 11and 12where, more particularly with calcium nitrate, thenitrate nitrogenincrease may in the final cuts bemore than four times the increase in totalnitrog en. W ith full daylight there is also a tendency for the nitrate nitrogento rise, but except in experiment I this is linked with an increase in the totalnitrogen. From Table VI it is seen that for experiments I-IV this trend forthe nitrate nitrogen toaccumulate with repeated cutting is not significantlyaffected either by the source of the nitrogen or the light intensity. If experi-ment I is, however, omitted the mean light effect only just fails to reach alevel of significance (see lower half of table).

The absolute amounts of nitrate nitrogen present in the leaves are in linewith the results ona basis of dry matter (see Table V). Indaylight, thenitrate nitrogen following manuring rises at least tofour times and mayreach nine times that in the corresponding control. At the lower light in ten-sities this rise, except inexperiment I, is less than in full daylight.

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U . 2nd. 3nd. 4th 5th. 6th. 1st 2nd 3rd. 4th. Sth. 6th.Number oF defoliations

F I G . 9. The effects of light intensity on the nitrate nitrogen content of the leaves of controlplants (low nitrogen supp ly) at successive defoliations. (Experiments I, II, and IVA. temds;experiment IIIF. rubra.)TABLE VI

The Effects of Light Intensity on the Accumulation of Nitrate Nitrogen in theLeaves of Nitrogenously Manured Plants(Statistical analyses based on regressions of difference from the controls in nitratenitrogen content (% of drymatter) against numb er of cuts.)Experiments I-IV Nitrogen treatments(N H 4) ,SO< Ca(N O ,) , Mean.{ i-o Daylight o-on 0-014 o#oi3

0-43-0-37 Dayl ight 0-013 0-025 0-019Mean 0-012 0-020Significant difference between means = 0-021 ,, means of 2 treatments = 0-015Experiments IIIV/1 -o DaylightLight treatments(0-44-0-37 DaylightI M e a n

Nitrogen treatments. Ca(NO,),0 - 0 1 0 0-012O-O22 O-O280 - 0 1 6 0 - 0 2 0Significant difference between treatments = 0-021 means of 2 treatments = 0-015

966.15 00

Mean,o-on0-025

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548 Blackman and Templeman The Interaction of Light Intensity and'Organic' non-protein nitrogen. Reference to Fig. 13 and Table VII showsthat the effects of shading on the 'organic' non-protein nitrogen content inthe control leaves. Except in experiment I I I the 'organ ic' nitrogen contentin full daylight and in 0-44-0-37 of daylight is on average substantially thesam e. In full daylight the general trends of the changes with successive cutsfollow those in the protein and nitrate nitrogen con tents . At the lower lightintensity the fall with time in 'organic' non-protein nitrogen in experimentsII-IV is more marked than in the case of the nitrate nitrogen changes.

TABLE VIIThe Influence of Light Intensity and Nitrogen Supply on the Content and Am ount

of 'Organic' Non-protein Nitrogen in the Leaves

Treatments

Daylight063Da yl igh t

0-44o-37Da y l igh t

1 Cont ro l( N H , ) , S O

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(WH4)2SO

Ca(NO3)z

IlTotalnitrogen(%drymatter)

E22=Nitratenitrogendrymatter)

=>Organicnon-proteinnitrogen

(drymatter)

4tii.1.5th.]6tti!7th.!

Numberofdefoliations

Fio.10.ExperimentI.Theeffectsoflightintensityandofadditionalnitrogenonthegainsorlosses,relativetothecontrol

changes,inthetotalnitrateandnon-proteinorganicnitrogencontentsoftheleavesofA.tenuitatsuccessivedefoliations. If8-ra.

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550 Blachnan and TemplemanThe Interaction of Light Intensity and

CD Total nitrogenNitrate nitrogenOrganic non-pnjtetn nitrogen

Number defoliationsFio. ii. Experimentll . The effects of light intensity and of additional nitrogen on the gainsor losses, relative to the control changes, inthe total nitrate andnon-protein organic nitrogencontents of the leaves of A. temds at successive defoliations.

TABLE VIIIThe Effects of Light Intensity on the Accumulation of 'Organic' Non-protein

Nitrogen in the Leaves of Nitrogenously Manured Plants(Statistical analyses based on regressions ofdifference from the controls in 'organic'

non-protein nitrogen content (% of dry matter) against number of cuts.)Experiments I-FVf i -o DaylightLight treatments! 0-44-0-37 DaylightI Mean

Nitrogen treatments(NH0,SO4 Ca(NO,), MeanO-Ol6 O-O28 O-O22

O -O O 5 O - O O 9 O - O O 2O - O I I 0-009

Significant difference between treatments = 0-021 means of 2 treatments = 0-015Experiments II-TV

11 -o DaylightLight trea tments ! 0-44-0-37 DaylightV MeanSignificant difference between treatments = 0-026 means of 2, treatments = 0-018

Nitrogen treatments(NH0,SO40-0230-0030013

Ca(NOs),00340-0060-014

Mean0-029o-ooi

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Nitrogen Supply in the Growth of Grasses and Clover. IV 551level is perhaps higher at the lower light intensity. In full daylight the additionof both ammonium sulphate and calcium nitrate has increased to some smallexten t the amino nitrogen. But at the level of 0-44-0-37 of daylight, althoughinitially ammonium sulphate and to a less extent calcium nitrate may haveraised the amino nitrogen content, yet in the final cut the manured leavescontain rather less than the control.

(0oJ?20 J 1

_ Tbtal nitrogm I'O D a y I I 3 h te ratr9 n (%dnj ma tter)Urqanic norvprotem nibroqen^ d n j matter) ^ ^

! \s l :2nd!3rdi4bh!5bh:6bh:2 Number of defo liationsFIG. 12. Experiment II I. Th e effects of light intensity and of additional nitrogen on thegains or losses, relative to the control changes, in the total nitrate and non-protein organicnitrogen contents of the leaves of F. rubra.The data for amide nitrogen show that changes in light intensity andnitrogen supp ly have only minor effects. In th e control plants lowering thelight intensity causes either no change in the amide nitrogen level or increasesit slightly. Similarly, the addition of nitrogen at either light intensity bring sabou t no consistent result. Save for experiment I I I, where manuring hasincreased considerably the amide nitrogen content of the shaded leaves, theamide fraction relative to the control rises and falls irregularly.In the majority of samples the unidentified organic non-protein nitrogenor 'res t' nitrogen equals or exceeds the amino plus amide nitrogen. FromTab le IX it is seen that with the un m anured plants a reduction in the lightintensity in experiment III increases the 'rest' fraction, but in experiment IVthis trend is reversed. The add ition of either amm onium su lphate or calciumnitrate in full daylight, particularly in experiment III, brings about a rise,whereas at 0-44-0-37 of daylight additional nitrogen depresses the 'rest'nitrogen.

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TABLEIX

Day-light fControl(NH4),SO4

tCa(NO,),

0-63/Control

Day-(NH4),SO4

lightlCa(NO,),

InfluenceofLightIntensityandNitrogenSupplyonNon-proteinNitrogenofLeaves.

Non-proteinNitrogenFractions(percentageofdrymatter)

ExperimentIII.Festucarubra

Aminonitrogen.

Amidenitrogen.

'Rest'nitrogen.

Ammonianitrogen.

Cut246Mean

0-0370-0980-1320-089

0-050o-ioo0-1350-095

0-0690-0700-162o-ioo

0-0980-0980-1150-104

CI33O-IO3O-I28O-I2I

00750-09200940-087

Day-light( Control

007200530-0750-067

(NH,),SO40-0840-0950-1390-106

Ca(NO,)to-no0-093o-i2i0-108

0-63IControl0-0740-0900-1160-093

Day-|(NH4),SO40-0960-1150-1020-104

light(Ca(NO1)l0-1030-102o-no0-105

246Mean

0-0560-0400-0130-036

0-0840-0420-1340-087

0-0510-0510-0620-055

0-0710-0410-0370-050

0-054o-na0-1390-102

0-114009701430118246Mean

0-0870-1990-2050-164

02060-3620-2690-279

025104770-3540-361

0-3640-21402590-279

0-3480-3290-2250-301

0-3890-2060-1330243246

Mean

0-0270-0230-0400-030

0-0130-0290-0510-031

0-0090-0260-0350-023

0-0180-017"350-023

0-0380-0650-0420048

0-0330-0350-0300-033

ExperimentIV.Agrostistennis

00790-064

0-0880064

0-0700-099

0-0790-083

0-0960-080

0-0690075 00950-079

0-1300-094

0-0730-081

0-0730-078

0-0070-089

0-081007503450326

O-3590383

03150-309

0-2610-141

0-1410-109

0-2020073 02440305

0-3680-370

O-3SI0-325

03890-264

0-2980-183

0-3100-195O-OO3O-OI2

O-OO2O-OI2

0-0O3O-OO5

O-OO30-003

O-0O5O-OI9

0'004O'OI9 00040-006

O'OI2O-OO9

OOO4O-0O4

0-0120-006

O-OI2O-OI2

O'OI2O'OI2

Nitratenitrogen

46Mean

0-0870-037O-OII0-045

02060-1670-2840-219

0-2510-1990-3780-276

0-3140-1350-3070-252

0-4870-6790-7170-628

0-5140-8250-8520-730

0-1670-0480-0520-089

0-3260-2180-3560-300

0-3510-3210-2530-308

O-3450-3080-4530-369

0-5760-7410-6770-665

0-6490-8850-6390-724

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Nitrogen Supply in the Growth of Grasses and Clover. IV 553There finally remains the ammonia nitrogen which in none of the samplesis found in any appreciable amount. Furth erm ore, neither alterations in thelight intensity nor the nitrogen level have resulted in marked changes. Inthe controls the amm onia content is much the same at both light intensities.The addition of ammonium sulphate has in full daylight only raised the

Ohjaiuc non-fruted ndjogen(Z dry malUr)

^ Number of defo liationsFIG. 13. Experiment IV. Th e effects of light intensity and of additional nitrogen on thegains or losses, relative to the control changes, in the total nitrate and non-protein organic

nitrogen contents of the leaves of A. temdt at successive defoliations.ammonia content to a slight extent, while even at the lower light intensitythere is no pronounced rise as might be expected if the plants were sufferingfrom 'ammonia' poisoning.Considering the 'organic' non-protein nitrogen data in terms of absoluteamount rather than on a percentage basis much the same conclusions arereached. In m anured plants the 'organic' non-protein nitrogen level is similarat the different light intensities (see Table VII ). In daylight additionalnitrogen very considerably increases the 'organic' non-protein level in allexperiments. At the lowest light intensity amm onium sulphate, except inexperiment IV , has raised the 'organic' fraction to a small extent. Calciumnitrate has only brought about a comparable rise in experiment I, while inexperiment IV the 'organic' non-protein nitrogen is depressed.

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554 Blackman and Templeman The Interaction of Light Intensity andWater-soluble carbohydrate level in relation to nitrogen supply and light intensity.

In addition to a study of the nitrogen changes the effects of the experimentalconditions on the w ater-soluble carbohydrates were also investigated. Noattempt has been made for each sample to separate the carbohydrates intosucrose reducing sugars and other components. Analysis of material mainly

g 07* 0-68 0-5 -

J-oO-3 0-7

ocoo

0-50-4

0-3 -

ExptI / \

-Expt.m

\ "* V ^ ?

i i i i i

\o-5

0-70-60-5

/ 0-4

0-3

Expb. E

V''-.Ty bcp t .H

- v 'W /I I 1 1 1 11sb. 2nd. 3rd. 4bh. 5th. 6th . 1st. 2nd. 3 rd 4th. 5th. 6th.

Number of defoliationsFIG. 14 . The effects of light intensity on the non-protein organic nitrogen content of theleaves of control plants (low nitrogen supply) at successive defoliations. (Experiments I, I I,and IV, A. tenuu; experiment III, F. rubra.)

from experiments III and IV has shown that the bulk of the water-solublecarbohydrates consists principally of sucrose and reducing sugars and thatfructosan is only present in small amounts (Blackman and T em plem an, 1939).Some thirty-six additional analyses have demonstrated that the reducingsugar content of the water extract after inversion with invertase is on averageonly slightly less than that obtained with acid inversion (0-2 normal acid).For the leaves grown in full daylight invertase inversion accounts in both




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Nitrogen Supply in the Growth of Grasses and Clover. IV 555experiments III and IV for 92-6 per cent, of the carbohydrates inverted withacid, while the corresponding figures for 0-44 of daylight are 86-5 and 83-8per cent.T he changes in the water-soluble carbohydrate content of the leaves dur ingthe course of each experimen t are shown in Figs. 13 -16. W hile some ofthe trends are common to the four experiments others are divergent. A

TABLE XThe Influence of Light Intensity and Nitrogen Supply on the Content and Amountof' Water-soluble Carbohydrates'

Treatments/ControlDaylight (NH 4),SO 4ICadNTO,),

f.~ t (ControlD a v i h t (N H < ).S ODaylight ( C a ( N o 3 ) i0-43-0-37 /C ontro lof (NH 4) ,SO 4Daylight lCa(NO 3 ),

Mean water-solublecarbohydrates as reduc-ing sugars(% of dry matter)Expt. I II II I IV

8-21 6-67 6-39 6-506-31 6-41 5-69 5-916-78 667 5-30 5-74 4 6 6 4'7S 4 6 17-02 4-95 2-61 3-066-64 5-11 2-70 3-156-20 5-00 2-61 3-14

in the LeavesTotal amount of water-soluble carbohydrates

(gm. per

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9-0

70

5-0

3-0

i^.t.r..T.-A

O = VO Daylight =0-37

1st. 2nd 3rd. 4th.Number oFdefoliations 5th. 6th.FIG. 16. Experiment II. Th e effects of light intensity and of nitrogen supply on the totalwater-soluble carbohydrate content of the leaves of A. tenuis at successive defoliations.

TABLE XIThe Interaction of Light Intensity and Nitrogen Supply on the Water-solubleCarbohydrate Contents of Manured Plants Relative to the Controls

Differences from controls of carbohydrates(reducing sugars as per cent, of dry matter)Manurial treatments(NH),SO 4 Ca(NO,), Mean.f 1-o Daylight 086 082 084Light treatments I 0-44-0-37 Daylight o-oi 0-17Mean o-oi- 0 4 4 0-09050

Significant difference between treatments = 0-44,, ,, means of 2 treatments = 0-31As within each light intensity the nitrogen effects on the carbohydrate con-tent are not pronounced, the total amount of carbohydrate in the leaves islargely dependent on the relative leaf production. From Table X it is seentha t in full daylight additional nitrogen increases the quantity of sugars in eachexperiment, whereas at the lowest light level only the data for experiment I

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558 Blackman and Templeman The Interaction of Light Intensity andshow an increase. In each experiment irrespective of the manurial treatm entsthe production of carbohydrates is considerably greater in full daylight.

90

7 0

s = s - o

IIS 30?I

1st. 2nd. 3rd. 4 th .Number o f de fo l ia t ions

5th. eth.FIG. 17. Experim ent I II . The effects of light intensity and of nitrogen supply on the total

water-soluble carbohydrate content of the leaves of F. rubra at successive defoliations.Total organic acid level in relation to nitrogen supply and light intensity.

Although it was clear from the investigations of Vickery et al. (1935, 1937)that the organic acids might play an imp ortant part in the nitrogen metabolismof the majority of plants, their methods of analysis did not seem applicable tosamples as small as those available in the present experim ents. It was onlywhen the original analytical programme bad been completed in the summerof 1939 that the estimation of the organic acid content of the leaves becamepossible. By this time a micro method for the estimation of the total organicacid content had been tentatively worked out in the B iochemistry Depa rtm entof the Imperial College.1 Although the correction factor for the phosphates

1 Th e me thod of analysis is outlined in Chib nall's recent book (1939, p. 2 07). A fuller andmore detailed account will be published elsewhere.




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Nitrogen Supply in the Growth of Grasses and Clover. IV 559present in the leaves required confirmation, it was decided, in view of theuncertain European situation, to carry out the analyses. Unfortunately, thesedata a re not as com plete as those of previous estimations, for, firstly, the workwas in part interrupted by the outbreak of war and, secondly, with some

8 0CD

Is1A

205oI

JL

1-0 Daylight= 0-61 - 0 4 4

I _L JL _L1st 5th. J6th.2nd. 3rd. 4thNumber oF defoliationsFIG. I 8. Experiment IV. The effects of light intensity and of nitrogen supply on the totalwater-soluble carbohydrate content of the leaves of A. tenuis at successive defoliations.

samples there was no t enough material left for analysis. Neverthe less, suffi-cient estimations were carried out to show the general effects in each experi-ment. The data are given in Figs. 19-22 , where for convenience the contentshave been expressed as if the only acid present was oxalacetic.Although the evidence, more particularly in experiments II-IV, points to areduction in the carbohydrate level as a result of shading, yet in only experi-ment II (Fig. 20) has shading, within each nitrogen treatment, decreased thetotal organic acid content to a marked exten t. In experiment I (F ig. 19) thereis some indication that in cuts III and V a diminution in the Ught intensitycauses a fall in the organic acid level. How ever, in experiment IV (Fig. 22 ),where decreasing the Ught intensity brought about a progressive reductionin the carbohydrate level (Fig. 18), the total organic acid content within eachmanurial treatmen t remains the same at the three Ught intensities. FinaUy,in experiment III (Fig. 21) although initially the shaded leaves contained lessorganic acids than the unshaded, yet subsequently the ir con tent was greater.

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560 Blackman and Templeman The Interaction of Light Intensity andApart, however, from the influence of light intensity on the organic acidsthere is also a nitrogen effect; this is most pronounced in experiment IV(Fig. 22). At all light intensities the plants manured with calcium nitratecontain the highest and the controls the lowest content of organic acids. Th isgain in concentration resulting from the addition of calcium nitrate is also

Control - Ca(N03)z10o

to

-aJ S

3

'a

u"5nfINumber of defoliationsFIG. 19 . Experiment I. The effects of light intensity and of nitrogen supply on the totalorganic acid content of the leaves of A. tenuii at successive defoliations.

seen in experiments I I and I I I (Figs. 20 and 21), but in experiment I (Fig. 19)this effect is not so evident. The results for am monium sulphate are less con-sistent than those for calcium nitrate. Whereas in experiment IV m anuringon the average increases the acid content, yet in experiment I there is adecrease. M oreover, in experiment I I I there is an apparent interactionbetween light intensity and manuring. In daylight the mean acid contentsof the control and ammonium sulphate treatments are the same (571and 579 per cent.), while in 0-44 of daylight organic acids accumulate inthe manured leaves, i.e. a content of 619 as against 546 per cent, in thecontrol.

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Nitrogen Supply in the Growth of Grasses and Clover. IV 561DISCUSSION

Since the pioneer work of Schulze and Prianishnikov many investigatorshave compared thechanges in the nitrogen fractions which take place in thelight in the dark. It hasbeen repeatedly shown that plants growing with an

-I 7s-u (0J= EIf655 cit J - ' S 5

JO ro

G H(0 OO- l/l

1,0

0= 1-0 Daylight = 037

1st. 2nd. 3rd. 4th.Number of defoliations

5th. 6th.F I G . 20. Experiment II. The effects of l ight intensity and of nitrogen supply on the totalorganic acid content of the leaves of A. tennis at successive defoliations.

adequate supply of light and nutrients will elaborate into protein the bulkof the nitrogen absorbed, while in the dark the protein level falls and thereare accumulations of other nitrogen fractions. Yet in spite of these contrastingresults little attention has been paid tothe effects of varying light inten sity onprotein metabolism. It is true tha t anumber of workers have investigated theeffects of long andshort days on the chemical composition of several plants.Their results, however, from the point of view of this investigation, are




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562 Blackman and Templeman The Interaction of Light Intensity andcom plicate d by the fact that wh ereas som e of th e trea tm en ts indu ced floweringoth ers did no t. It is therefore difficult to distingu ish betwe en th e change sdu e to variation s in day-length and the ch ang es due to metab olic differencesin th e vegetative and the flowering pha ses. Ne verth eless, the evidence ofPfeiffer (1926) , Night ingale (1927) , Tincker (1928) , Zimmerman and Hitch-

' a

O =1-0 Daylight = 044

-. o

1st 5th. 6th.'End. 3rd 4th.Number of defoliations

FIG. 2 1 . E xpe rime nt I I I . Th e eflFects of light intens ity and of nitrogen supply on the totalorganic acid content of the leaves of F. rubra at successive defoliations.cock (1929), and Hopkins (1935) indicates that with short days plants containmore nitrogen and there is in particular, when they are fully manured, anaccum ulation of nitrates. T he h igh nitrate concentration is not alwayscorrelated with the stoppage of growth following upon flowering. Hibbardand Grigsby (1934) observed that pea seedlings under short day conditionsaccumulated nitrates, while Nightingale et al. (1930) obtained similar resultsfor the Biloxi soya bean when the day was so short as to inhibit flowering.Associated with this rise in nitrate nitrogen several workers have found thatthere is an increase in the carbohydrates, especially in starch.

Hopkins (1935) investigated not only the effects of short days but also theeffects of shad ing on the soya bean with particular reference to the metabolism




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Nitrogen Supply in the Growth of Grasses and Clover. IV 563of the nitrogen-fixing bacteria in the nodules. He observed that plantsshaded (o-12-0-17 of daylight) for five to seven hours during the middayperiod decreased in carbohydrates and increased in all the nitrogen fractions.These changes, however, cannot solely be attributed to the direct effect ofdecreased light intensity on the plant, the possible indirect effects on thenodules must also be taken into account. T he data of Arthu r et al. (1930)also indicate tha t shading increases the total nitrogen content in a num ber ofplants, while Kraybill (1923) found tha t with th e peach and the apple decreas-ing the light intensity increased the total nitrogen and soluble nitrogen butdepressed the carbohydrates.

The present investigation has shown that the effects of light intensity onthe nitrogen content of the leaves are dependent upon the nitrogen supply.In the unmanured plants the results are in agreement with previous findings,for except in experiment I shading brought about increases in the nitrogencontent. W hen the plants received amm onium sulphate reducing the lightlevel first to o-63-o-6i of daylight caused some increase in the total nitrogen,but a further reduction to 0-44-0-37 of daylight resulted in the shaded leavescontaining the same amount of nitrogen as those grown in full daylight. O nthe other hand, in the calcium nitrate series the nitrogen content declinedprogressively as the light intensity was reduced.

The protein figures to a certain extent follow the same trends as the totalnitrogen data. In full daylight the pa rtition of the total nitrogen is normal,for most of the nitrogen is found in the form of protein, but there is someevidence that with high nitrogen supply the ratio of protein to total nitrogenfalls. W hile in the four experiments the un m anured leaves contain on average85-6-86-3 pe r cent, of their nitrogen as protein only, 63-9-85-1 per cent, of theaccumulated nitrogen due to the addition of ammonium sulphate is found asprotein and only 59-4-79-0 per cent, in the case of calcium nitrate.

In the unmanured series the increases in protein content of the shaded(0-44-0-37 of daylight) over the unshaded leaves follow the correspondingincreases in total nitrogen. T hu s the ratio of protein to total nitrogen remainsrelatively unchangednamely, 82-4-85-1 per ce nt . On the other hand, therises in total nitrogen content due to manuring are not paralleled by similarincreases in protein content. In the case of amm onium sulphate only 64 -2 -11-5 per cen t, of the extra nitrogen found in the leaves is elaborated into p ro -tein. M oreover, the addition of calcium nitrate though it may increase thetotal nitrogen content may at the same time depress the protein level (experi-ments III and IV).

Of the non-p rotein fractions the concentration of nitrates is most susceptibleto changes in bo th manuring and light intensity. In full daylight the u n-manured plants show the low nitrate nitrogen contents characteristic ofnitrogen deficiency. On the other han d, m anured plants, especially whencalcium nitrate is applied, tend to accumulate nitrate nitrogen and the con-centration reached is on occasion more than 0-9 per cent, of the dry matter.

M6.13 P p

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564 BUickman and Templeman The Interaction of Light Intensity andSimilar concentrations following on manuring have been reported for grassesby Eggleton (1935) and Blackman (1936); for tomato by N ightingale, S cher-merhorn, and Robbins (1928), Clark (1936), and Wall (1939), and for pine-apple by Sideris et al. (1938).

Irrespective of the n itrogen supply, shading brings about a marked increasein the nitrate content. In the unm anured p lants where in full daylight theconcentration is low, shading increases the concentration as much as fivetimes, while in the m anured series it is doubled. T he other non -proteinfractions show less marked differences between high and low light intensity.There are no substantial increases in amino or ammonia nitrogen in theshaded leaves, but there is evidence in experiment III that both shading andmanuring may increase the amide content.From the foregoing discussion it is clear that though at all light levels thenitrogen content of the leaves is increased by manuring yet the elaborationof this nitrogen is dependent u pon the light intensity. Some insight into thepossible factors responsible for this variation in the partition of the nitrogencan be obtained from the data concerning the differential effects of amm oniumsulp hate and calcium nitrate. In all four experimen ts while the leaves in fulldaylight gained an equal amount of nitrogen over the controls irrespective ofwhether ammonium sulphate or calcium nitrate was applied, yet at thelowest light intensity the gain was greater when ammonium sulphate wasadded. The extent to which such differences in nitrogen con tent are reflectedin the partition of the accumulated nitrogen is seen in Tab le X II .

TABLE X I IThe Influence of Light Intensity on the Differential Partition of the NitrogenAccumulated in the Leaves of P lants Manured with Amm onium Sulphate andCalcium Nitrate

Mean differences in content of nitrogenfract ions due to ma nurial t rea tme nts(ammonium su lpha te con ten t l es scalcium ni t rate content)(per cent , of dry mat ter)Ex p e r i m en t s I - IV

Nitrogen fractionsT o ta l n itrogen . . . .Protein n i t rogenNitrate n i t rogenOrgan ic non-p ro te in n i t rogen

Dayl igh t 0 - 0 0 2+00370-031- 0 0 0 8

Light intensity0-44-0-37Daylight

+0-283+ 0 2 8 90-084+0-078

Significantdifference(P = o-os)0 1 4 80-0980 0 3 50 0 8 1T he data in Tab le X II indicate tha t in full daylight the composition of theleaves is little affected by the source of nitrogen. In contras t at the lowerlight intensity the addition of ammonium sulphate results in a higher totalnitrogen conten t. At the same time this increase is associated with an equal

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Nitrogen Supply in the Growth of Grasses and Clover. IV 565rise in the protein content w hile the small gain in organic non-p rotein nitrogenis offset by th e greater accumulation of nitrates in the plan ts receiving calciumnitrate. Since to the soils in which the plants were grown ab und ant calciumcarbonate had been added the difference between the ammonium sulphateand calcium nitrate treatments can hardly be attributed to the calcium addedin the latter. I t seems more probable that such differences are associated withthe absorption of the add ed n itrogen, for in the case of calcium nitra te nitrateions only will be absorbed, w hile with the ammonium sulphate treatment bo thammonium and nitrate ions will be taken up since the added ammoniumnitrogen will in part have been converted to nitrates in the soil. It wouldappear from Table X I tha t in full daylight the elaboration of either am moniumor nitrate nitrogen in to organic nitrogen takes place with equal facility. In0-44-0-37 of daylight, however, while ammonium nitrogen is apparentlysynthesized into protein, nitrates tend to accumulate in the tissues un changed.It might be suggested th at if there had been no nitrification in the soil and alarger amount of ammonium ions had been absorbed the organic nitrogenlevel would have been higher and the divergence from the calcium nitra te datastill greater.

Before, however, the part played by light intensity in the reduction ofnitrates can be evaluated some consideration of the other factors responsiblefor nitrate accumulation will be necessary. Nightingale et al. (1930) and W all(1939) found that tomato plants deficient in potassium stored nitrates, whileEckerson (1931a) observed similar high concentrations as a result of phos-phorus starvation. H ibbard and G rigsby (1934) have demonstrated that peaseedlings growing in med ia, low in calcium or potassium, contain considerableamounts of nitrate nitrogen, while Richards and Templeman (1936) state thatlimiting the supply of phosphorus and more particularly potassium, leads tothe accumulation of nitrates in the leaves of barley.

The chain of reactions responsible for the conversion of nitrate to am moniais as yet not fully und erstood . Corbett (1935) has demonstrated th at in theoxidation of ammonia by nitrifying bacteria both hyponitrous acid andhydroxylamine are produced. Chibnall (1939) has suggested tha t in plantcells nitrate reduction proceeds as follows: nitrate, nitrite, hyponitrous acid,hydroxylamine, amm onia. T he presence of nitrites has been detected innum erous p lants including grasses (Eggleton, 1935), and E ckerson has m ade aconsiderable study of the mechanism of nitrate reduction. She first showed(1924) that under carefully controlled conditions it was possible with sapexpressed from plants to convert nitrate to nitrite in vitro and concluded thatthe rate of reduction could be taken as a measure of the activity of enzymereducing system reducase. On the basis of such tests Eckerson (192 4,193 1,1932) and Dittrich (1931) found that roots contained abundant reducase, butthat it was also generally present but to a lesser extent in both stems andleaves. O ther experiments of Eckerson indicated tha t shad ing , and deficienciesof nitrogen, phosphorus, potassium, and possibly the sulphate radicle, all




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566 Blackman and Templeman The Interaction of Light Intensity andcaused a reduction in activity. Nightingale et al. (1930, 1931) also claim thatplan ts deficient in potassium and calcium lack reducase. M oreover, Eckerson(1931a) found that the reducase activity in the apple varies with the seasonand is greatest in the spring.It would thus appear that a surprising large number of factors are respon-sible for limiting either the production or activity of reducase. T ha t all thesefactors are directly concerned in the formation of the enzyme system seemsimprobab le. Rather the results suggest that failure to detect nitrate reductionin the expressed sap does not preclude the possibility that the reduction willtake place in th e living cell. Again, the inability of plants to elaborate nitrateeven in the presence of abundant carbohydrates cannot be ascribed solely tothe absence of reducase, for as Richards and Templeman (1936) and Wall(1939) have pointed out, this could be brought about by mass action due tothe accumulation of intermediate products. M oreover, it has been shown byVickery et al. (1933, 1937), McKee and Lobb (1938), and Pearsall and Billi-moria (1937, 1939) that leaves either originally free from nitrates or suppliedwith amm onium salts will produce nitrates under varying conditions. T henitrate concentrations, therefore, within the p lant will be d ependent upon therelative rates of (i) absorption of nitrates, (ii) reduction of nitrates, and (iii)oxidation of amm onia to nitrate. On this basis it would seem tha t since theunmanured plants in full daylight contained very little nitrate nitrogen, therate of nitrate elaboration was in excess of that of nitrate absorption. T heaccumulation of nitrate in the manured plants, however, indicates thatabsorption took place at a faster rate than conversion. O n the other hand,the markedly higher concentrations of nitrates in o-44-o -37 of daylight, evenin the un m anu red plan ts, points to a decrease in the rate of nitrate conversion,since it is highly improbable that the nitrate absorption was increased byshading. In fact Weissmann (1925) found with cereals tha t the uptake perplant of phosphorus and potassium was greatly decreased by a reduction inlight intensity, while Panchaud (1934) showed that the 'total mineral' uptakeby Raphanus sativus was also depressed by shading. W ith tomatoes grownin a greenhouse Porter (1937) observed that at a quarter of the normal lightthe total salt (ash) uptake was 16 per cent, less than in the unshaded andlarger plan ts. Similar decreases have been obtained by one of us in experi-ments still in progress on the uptake of nitrogen, phosphorus, and potassiumby Scilla nutans grown in the field and in light intensities ranging from full to0-2 daylight.

Although in all the investigations cited m ineral uptake on a plant basis waschecked by shading, the actual content of the tissues was not depressed butrather increased. It is highly unlikely, therefore, tha t in the present experi-ments the effect of shading on the accumulation of nitrates is due to the shadedleaves containing limiting amounts of those elements apparently essential forrapid nitrate reduc tion. Tha t such elements, namely phosph orus, potassium,sulph ur, and calcium were ever deficient is impro bable on othe r grounds. In




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Nitrogen Supply in the Growth of Grasses and Clover. IV 567the first place in full daylight leaf production was largely controlled bynitrogen supply and not by other mineral deficiencies (see Fig . 1). In th esecond place the soil used was a fertile one and in addition very appreciablequantities of calcium carbonate and superphosphate were added initially.Although light intensity and nitrate conversion are apparently directlylinked, in these experiments light does not always appear to be an essentialfactor. Nightingale and Robbins (1928) found that both Narcissus Tassettaand asparagus could convert nitrates into protein in the dark. T hat lighteven of low intensity (850-1150 m.c.) has an accelerating effect is put forwardby Pearsall and Billimoria (1939), who worked w ith daffodil leaves floating ona solution containing amm onium nitrate and glucose. From their observa-tions it would appear that th e acceleration is greatest in the young green tissueand least in the white basal meristematic portion. Tha t nitrate conversion ismost active in the green portions of the pineapple leaf is also concluded bySideris et al. in 1938. Pearsall and Billimoria (1939) also obtained evidencethat the rate of ammonia nitrogen synthesis was greater in the light, whileDastur et al. (1938) claim that the increase in water-soluble nitrogen duringexposure to light is dependent upon the quality of the light source. Th eirresults must, however, be treated with considerable reserve on account ofbo th their experimental procedure and the methods of chemical analysis. Inthe first place whole plants were exposed to the light but only the leaves takenfor analysis (Dastur and Samant, 1933). Secondly the leaves, dried at 55 0 C ,were extracted with cold water and all analyses carried out on this solutiononly. For the total nitrogen estimations no precautions were apparen tlytaken to include nitrates and their technique, for the estimation of amides byconversion to ammonia would not distinguish between amide ammonia andany true amm onia nitrogen present. Moreover, since the plants prior to theexperiment were kept in the dark for seventy-two hours the initial ammoniaconten t may have been considerable. Nevertheless, in spite of these erro rsthe marked variations in the gain in water-soluble nitrogen observed indicatethat light quality may have had some effect on the synthesis of inorganicnitrogen , bu t as to the na ture of this effect the evidence presented is insufficientfor any conclusion to be drawn.

The investigations of Pearsall and Billimoria (1937, 1939) have, however,brough t out another important aspect of nitrate reduction. They found tha twhen leaves were floated on a solution of glucose and salts of inorganicnitrogen the amount of nitrogen absorbed was always greater than that foundin the tissues. Since they observed tha t there was no discrepancy whenorganic sources of nitrogen were used in th e external solution, they concludedthat there was a loss of gaseous nitrogen due to the nitrite, formed during thereduction of nitrate, reacting with the amino acids. Mothes (1938) carryingout similar experiments could, however, detect no loss of nitrogen when theexternal solution contained nitrates, but found that if nitrite was substitutedfor nitrate then the loss was large. He concluded tha t in the nitrate series any

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568 Blackman and Templeman The Interaction of Light Intensity andnitrite formed during the reduction of nitrates m ust be removed so quickly asto prevent reaction with the amino acids. T he reaction cannot always be byany means

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