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Iron distribution in vineleaves with HCO3

‐ inducedchlorosisK. Mengel a , W. Bubl a & H. W. Scherer aa Institute of Plant Nutrition, Justus LiebigUniversity, Giessen, West GermanyPublished online: 21 Nov 2008.

To cite this article: K. Mengel , W. Bubl & H. W. Scherer (1984) Iron distributionin vine leaves with HCO3

‐ induced chlorosis, Journal of Plant Nutrition, 7:1-5,715-724

To link to this article: http://dx.doi.org/10.1080/01904168409363236

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JOURNAL OF PLANT NUTRITION, 7(1-5), 715-724 (1984)

IRON DISTRIBUTION IN VINE LEAVESWITH HCO3

- INDUCED CHLOROSIS

KEY WORDS: Iron chlorosis, iron accumulation, bicarbonate, grapevine plants

K. Mengel, W. Bubl and H. W. Scherer

Institute of Plant NutritionJustus Liebig University

Giessen, West Germany

ABSTRACT

The objective of the investigation was to examine whether ironchlorosis in grape vine grown on calcareous soils was related tothe Fe distribution in the leaf.

Leaf samples collected from three different sites showed inmost cases higher Fe contents in the chlorotic leaves as comparedwith healthy leaves. The solubility of leaf Fe in diluted HC1, how-ever, was lower in chlorotic leaves than in green leaves.

Enzymatic dissolution of leaves into vascular tissue, inter-costal cells and chloroplasts revealed that the Fe content in theintercostal cells of green leaves was significantly higher than inthe intercostal cells of chlorotic leaves. In addition, the inter-costal cells of chlorotic leaves had extremely high Ca and P con-tents.

The P content of green and chlorotic leaves was not related tothe level of available P 1n the soil. It is, therefore, concludedthat the high P content in chlorotic leaves is the sequence and notthe cause of Fe chlorosis.

715

Copyright © 1984 by Marcel Dekker, Inc. 0190-4167/84/0705-0715$3.50/0

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716 MENGEL, BUBL, AND SCHERER

On each of the three sites investigated, higher clay contentswere found under chlorotic grape vine plants than under healthyones. It is assumed that because of this higher clay content, soilcompaction may occur, resulting in an accumulation of C02 and in anincrease of the HCO3

- concentration in the soil solution.

INTRODUCTION

According to recent investigations Fe chlorosis ts occurringvery often in connection with high concentration of HCO3" in thesoil (Boxma , Kovanci et al. 8). Although it has been shown that theuptake and translocation of iron in the plants is affected by HCO3"(Rutland and Bukovac^), to control chlorosis more information isrequired about the process which is leading to the HCO3" inducedchlorosis. Frequently total Fe in chlorotic leaves of plants grownon calcareous soils is higher than the Iron content in green leaves(Bubl3), in contrast to the HC1 soluble Fe which is lower in chlo-rotic leaves as compared with green leaves (Jacobson', Bucher^).

The objective of the investigation presented here was to findout whether HCO3" induced chlorosis in grape vine was related tothe distribution of iron in the leaves. For this purpose chloroticand green leaves of vine plants were sampled and analyzed for dif-ferent Fe fractions: Total Fe, HC1 soluble Fe, Fe of the inter-costal cells (cells between leaf veins), Fe of the main leaf veins,and chloroplast Fe.

MATERIALS AND METHODS

In 1979 leaf samples from chlorotic and healthy vine plantswere collected from the site 'SpieBheim' and in 1980 from the sites'SpieBheim', 'Iphofen' and 'Nierstein1. These sites are susceptibleto Fe chlorosis of vine. On each site leaf samples were taken from20 green and 20 chlorotic vine plants, respectively. From each ofthese plants the 10 youngest leaves of two shoots were collected.The leaf samples were frozen immediately in liquid nitrogen and thenstored in a deep-freezer at -18°C.

Five samples were used for the determination of the mineralcontent of the leaves, while the other 15 samples were used for theenzymic dissolution of the leaves in different fractions,

From the same sites where the leaf samples were collected, in1980 soil samples were taken underneath 10 chlorotic and 10 healthyvine plants, respectively. Each 10 subsamples were combined to onesample. On the sites 'SieBheim' and 'Iphofen1, soil samples weretaken from the depth 0 to 40 cm and 40 to 80 cm, while in (N1erste1n(

samples were taken from the depth 0 to 20 cm and 20 to 60 cm.

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IRON CHLOROSIS 717

Available soil P and K were analyzed by the CAL method, thecommon method used in Germany for the estimation of available P andK. The extractant is consisting of a mixture of Ca lactate, NH4lactate and acetate, pH 4,1 (Schuller^). Iron, Mn, Zn, and Cuwere extracted by DTPA (Lindsay and Norvell9), Carbonate was anal-yzed according to the method of Scheibler^-5 and HCQ3" according tothe method of B ^

The mineral content of the leaves was analyzed after wet ashingof the ground leaf samples by atomic absorption. Phosphate was de-termined according to the vanadate-molybdate method. For the deter-mination of HC1 soluble Fe 0.5 g of the dried and ground leaf mate-rial was extracted with 20 ml of 0.5 N HC1 and 1.0 N HC1, respec-tively, for 30 min, so that two different HC1 soluble fractions wereobtained, one by the extraction with 0.5 N and one by the extractionwith 1.0 N HC1. Iron was determined by atomic absorption.

Enzymic Dissolution of_ the Cells

For the separation of the cells two samples (= 30 leaves total-ly) were combined and thawed slowly in a refrigerator. Then themiddle of the five main leaf veins was isolated. In this fraction,which is called 'leaf veins', Fe, Mn, Zn, Cu, Ca, and P were deter-mined. The rest of the leaves was cut into small pieces and trans-ferred into 10 Erlenmeyer flasks. Then 20 ml of solution No, 1 wasadded.

Soln. 1: 1% mazerozyme (mixture of pectinase, hemicellulaseand cellulase)

2% polyethyleneglycoll 400020 mmolar HEPES buffer0.65 molar mannitpH 5.8

The addition of the buffer was necessary to stabilize the pH,which declined about 0.5 pH units in 2 hours. After 2 h the pH wasreadjusted to pH 5.8 by the addition of 1 N NaOH. The samples wereshaken for 4 hours in a waterbath at 25°C and afterwards poured on a100 urn sieve. After washing with 0.65 M mannit the filtrate wascentrifuged. The sediment consisted mainly of intercostal cells.This fraction, called 'intercostal cells' in the following was ana-lyzed for Fe, Mn, Zn, Cu, Ca, and P. The fraction, which did notpass the sieve, was transferred to Erlenmeyer flasks. To this frac-tion 20 ml of solution No. 2 was added.

Soln. 2: 1% cellulase Onozuka SS*2% polyethyleneglycoll 4Q0Q20% mmolar HEPES buffer0.3 molar mannitpH 5.2

*Firma Welding and Co., Hamburg, West Germany

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718 MENGEL, BUBL, AND SCHEEER

The samples were shaken 1n a water bath at 36°C for 4 hours andthen poured on a 100 urn sieve. The fraction, which passed throughthe sieve, was centrifuged for 10 minutes at 1000 g. The supertnat-ant in the centrifuge tubes was discharged. After adding 20 ml ofsolution No. 2 to the sediment the centrifuge tubes were shaken for1 h at 36°C in a waterbath. By this procedure cells are dissolvedenzymically, whereas the chlorbplasts are not attacked CJacobi6).Chloroplasts were obtained by centrifugation. In the chloroplastfraction Fe, Mn, Zn, Cu, Ca, and P were analyzed. The whole pro-cedure has been described in more detail by bi^

RESULTS

Table 1 is showing the Fe concentration in the various chemicaland anatomical fractions of green and chlorotic leaves. Total ironcontent in chlorotic leaves was as high or even higher than 1n greenleaves; however, the amount of HC1 soluble Fe was higher in the

TABLE 1

Fe c o n c e n t r a t i o n s in the v a r i o u s and ana tomica l f r a c t i o n s o f green

and c h l o r o t i c l e a v e s .

SpieBhein 1979 SpieBheim 1980 Iphofen 1980 Hierstein 1980green ch lorot . green ch lorot . green ch loro t . green ch lo ro t .

Total Fe 76 89(n = 5)

0.5 N HC1 20 18so l . Fe(n = 5)

1 N HC1 22 15*so l . Fe(n = 5)

Leaf veins(n = 7) ,

Intercostal *ce l ls (n = 14) 257 214

288 290

ppn Fe, d.m.

65 64

30 26*

37 31*

58 48

200 139*

538 559Chloroplasts(n = 7)

Comparison between green and chlorot ic leaves:

* p = 5 %, ** p = 1' %, * * * p = 0.1 %

94 97 112 139

37 33 35 32

** *40 36 40 37

72 77 57 60

203 192 331 233

1058 1040 726 619

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IRON CHLOROSIS 719

green leaves as compared with the chlorotic ones. The differencesbetween 1 N HC1 soluble Fe of chlorotic and green leaves were sig-nificant for all samples. The amount of Fe extracted by 0.5 N HC1was nearly as high as that extracted by 1 N HC1. The differencesbetween the Fe contents in the veins of healthy and chlorotic leaveswere small and not significant. The Fe content in the intercostalcells of green leaves was significantly higher than in the inter-costal cells of chlorotic leaves. The Fe contents of the chloro-plasts differed very much between the various sites. There were nomajor differences between the Fe content in chloroplasts of greenand chlorotic leaves. However, the chlorotic leaves contained lesschloroplasts.

As can be seen from Table 2, P concentrations in whole leaveswere significantly higher in the chlorotic samples as compared withthe green ones. The same was true for the P concentrations in theintercostal cells. Also, the Ca concentrations in the chloroticintercostal cells were significantly higher than in the green inter-costal cells. Concentrations of Mn, Zn, and Cu were mainly higherin chlorotic samples than in green samples (Table 3).

TABLE 2

Ca- and P concentrat ions in leaves and l e a f - f r a c t i o n s of green and

ch lo ro t i c leaves.

SpieBheim 1979 SpieBheim I960 Iphofen 1980 Iphofen 1980green chlorot, green chlorot. green chlorot. green chlorot

Whole leaf

Leaf veins

Intercostalcells

Chloroplasts

Whole leaf

Leaf veins

Intercostalcells

1.57 1.51

0.69

1.01

0.22

1.7C

0.93

0.36*

0.13 0.24

% Ca, d.m.

1.16 1.12

1.79 1.67

0.60 1.34

1.25 1.36

% P, d.m.0.20 0.30***

0.11 0.15

0.09 0.17

Chloroplasts 0.85 0.71 C.59 0.56

Comparison between green and.chlorotic leaves:

* p = 5 %, ** p = 1 %, *** p = 0.1 %

1.30 1.50

1.04 1.23*

1.04 1.98*

1.17 1.63

0.19 0.34

0.14 0.17*

0.11 0.16*

0.13 0.15

1.32 1.46

1.43 1.60*

0.76 1.46*

0.99 0.84

0.25 0.38

0.13 0.20*

0.15 0.25

0.35 0.40

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720 MENGEL, BUBL, AND SCHERER

TABLE 3

Mn-, Zn- and Cu concentrations in leaves and leaf-fractions of

green and chlorotic leaves.

Whole leaf

Leaf veins

Intercostalcells

Chloroplasts

Whole leaf

Leaf veins

Intercostalcells

Chloroplasts

Whole leaf

Leaf veins

Intercostalcells

SpieBheim 1979green chlorot.

104

-

16

80

50

-

196

-

7

21

Chloroplasts 61Cnmparison between* **p = 5 %, P = 1

***55

-

24*

65

***138

-

**301

-

***11

21

58green arid

***%, P

SpieGheim 1980green chlorot.

ppm Mn

140

98

190

, d.m.**

192

113**

140

2444 2870

ppm Zn

69

72

91

341

ppm Cu

8

10

16

96chlorotic

= 0.1 %

, d.m.

***139

74

65*

297

, d.m.

***15

14***

24

99leaves:

Iphofen 1980green chlorot.

44

33

13

47

84

72

79

698

13

12

17

76

**57

30

8

31

***137

***112

88

796

12

11***

23

69

Iphofen 1980green chlorot.

144

107

191

2359

55

60

-

-

5

9

16

86

***195

***154

***127

1973

**80

**68

-

-

7*

10***

20

79

The most important soil characteristics and nutrient contentsof the soil samples, which were taken underneath green and chloroticvine plants, are shown in Table 4. There was no relationship be-tween the appearance of chlorosis and the pH, the carbonate content,and the contents of CAL-soluble P and K. Also the Fe, Mn, Zn, andCu content and the HCO3" concentration was not related to the chlo-rosis. However, on each of the three sites the clay contents werehigher underneath the chlorotic vine plants.

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T A B L E 4N u t r i e n t c o n t e n t s and s o i l c h a r a c t e r i s t i c s o f soi l s a m p l e s t a k e n u n d e r c h l o r o t i c and h e a l t h yv i n e p l a n t s . W i t h t h e e x c e p t i o n of H C O 3 " , t h e d a t a r e l a t e t o a i r d r i e d s o i l . S o i l s a m p l e st a k e n 1 9 7 9 w e r e n o t a n a l y z e d f o r h e a v y rietals.

site

SpieR-heim1979

SpieB-heim1980

Ip-hofen1980

Mier-stein1980

soil depth(cm)

0-40

40-80

0-40

40-80

0-40

40-80

0-20

20-60

symptoms

green

chlorot.

green

chlorot.

green

chlorot.

green

chlorot.

green

chlorot.

green

chlorot.

green

chlorot.

green

chlorot.

PH

7.5

7.6

7.6

7.6

7.4

7.4

7.57.5

7.3

7.3

7.3

7.3

7.77.8

7.77.8

HC03"

ppm

418

392

464

493

507

579

610

625

496

524

502

532

299

293

293

299

P205mg/

100 g

40.8

27.8

11.3

5.7

32.4

30.2

11.7

7.0

28.0

40.0

19.9

30.7

62.3

91.7

37.3

35.0

K20 mg/

100 g

89.6

61.4

58.1

32.0

73.2

60.7

52.4

35.3

42.2

55.7

26.7

35.6

68.9

62.3

48.0

39.0

Carbonate

%

26.3

26.0

29.8

30.0

25.5

25.7

28.5

28.9

19.2

18.8

22.5

21.4

10.3

10.0

11.3

9.8

Fe

PPm

-

-

--

10

10

8

8

8

7

9

10

10

10

10

8

Mn

ppm

-

-

-

-

23

24

24

22

9

12

9

11

9

9

9

9

Zn

ppm

-

-

-

-

7

7

9

4

9

14

8

8

30

114

4

Cu

ppm

-

-

--

33

29

12

12

7

13

13

19

29

24

77

clay

%

-

-

-

-

38.7

46.2

45.0

52.4

29.7

34.2

31.1

34.7

31.0

40.9

33.7

34.9

C/3

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722 MENGEL, BUBL, AND SCHERER

DISCUSSION

In carbonate soils high HCO3" concentrations may occur CMengelet al.l') due to the high pH level and the dissolution of carbonates.Chlorosis is then likely to appear. This observation was made byBoxma2 for fruit trees. In our investigations all soils were richin carbonate and the pH was higher than 7. But although we foundhigh HCO3" concentrations on all the sites, there was no relation-ship between iron chlorosis and the HCO3" content. However, meas-uring the HCO3" concentration in a soil sample does not provide areliable information about the concentration of HCO3" in the rhizo-sphere and the HC03" uptake of plants. Because of the excretion ofCO2 and H by roots (Mengel and Malissiovasll), the HCO3" concen-tration in the rhizosphere of carbonate soils may differ consider-ably from the HCO3" concentration of the bulked soil. Enhanced C02production in the vicinity of the roots may lead to high HCO3- con-centrations in the rhizosphere.

Especially HC03" can be accumulated under high soil moistureconditions on soils with a heavy texture. This holds true for ourinvestigations. Higher clay contents were found under chloroticvine plants than under green vine plants. These findings are con-sistent with results of Carter5 who also observed chlorosis on soilswith a high clay content.

Investigations of Bubl^ with H C03 have shown that tPie rootsof vine plants take up HCO3-. It 1s therefore supposed that on cal-careous soil HCO3" is absorbed by vine plants and that HCQ3" directlyor indirectly affects the Fe transport into the intercostal cells.A similar effect has been observed with nitrate nutrition CMengeland Malissiovas^0). Probably an alkaline nutrition has a detri-mental influence on Fe mobility in vine leaves.

The reason why HCO3" is hindering the Fe transport into theintercostal cells is not yet clear. Although the total Fe contentof the chlorotic leaves was at least as high as in green leaves oreven higher. HC1 soluble Fe was significantly lower in chloroticleaves as compared to green leaves. This confirms results of Jacob-son' and Mengel et a.1'.

There was no relationship between the CAL - P content of thesoils and the P content of the leaf samples. But on all sites theP content in the whole leaves and in the intercostal cells, respec-tively, was significantly higher in chlorotic leaves than In thecorresponding samples from green plants. It 1s thus suggested thatFe chlorosis on calcareous soils is not caused by high soil P con-tents. The high P contents 1n the chlorotic leaves are rather theresult of a physiological Fe deficiency. The same holds true forthe high Mn, Zn, and Cu contents of the chlorotic leaves. Also,Bucher* and B00B et al.l found higher P contents in chloroticleaves. Our conclusions that the high P contents 1n chlorotic

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IRON CHLOROSIS 723

leaves are the result and not the cause of the Fe chlorosis areconsistent with the results of Mullner^ and Mengel et al .12,

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2. Boxma, R. 1972. Bicarbonate as the most important soil factorin lime-induced chlorosis in the Netherlands. Plant and Soil37:233-249.

3. Bubl, W. 1981. Eisen-Chlorose bei der Weinrebe - Loslichkeitund Verteilung von Eisen in grunen und chlorotischen Blatternsowie die Bedeutung des Bicarbonates. Ph. D. Thesis, Fach-bereich 19, Fac. Nutritional Sci., Justus Liebig University,Giessen, West Germany.

4. Bucher, R. 1976. Der EinfluB hoher Phosphatgaben im Carbonat-boden auf die Aufnahme einiger fur die Rebe wichtigen Spuren-elemente. Weinberg und Keller 23:257-263.

5. Carter, M. R. 1980. Association of cation and organic anionaccumulation with iron chlorosis of scots pine on parie soils.Plant and Soil 56:293-300.

6. Jacobi, G. 1974. Biochemische Cytologie der Pflanzenzelle.Ein Praktikum. G. Thieme-Verlag, Stuttgart.

7. Jacobson, L. 1945. Iron in the leaves and chloroplasts of someplants in relation to their chlorophyll content. Plant Physi-ol. 20:233-245.

8. Kovanci, I., H. Hakerlerler, and W. Hofner. 1978. Ursachender Chlorosen an Mandarinen (Citrus reticulata bianco) deragaischen Region. Plant and Soil 50:193-205.

9. Lindsay, W. L. and W. A. Norvell. 1978. Development of a DTPAtest for zinc, iron, manganese and copper. Soil Sci. Soc.Amer. J. 42:421-428.

10. Mengel, K. and N. Malissiovas. 1981. Bicarbonat als auslosenderFaktor der Eisenschlorose bei der Weinrebe (Vitis vinifera).Vitis 20:235-243.

11. Mengel, K. and N. Malissiovas. 1982. Light dependent protonexcretion by roots of entire vine plants (Vitis vinifera L.).Z. Pflanzenernahr. Bodenkde. 145:261-267.

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13. Mullner, L. 1979. Ergebnisse eines Chloroseforschungsprojektes,Mitt. Klosterneuburg 29:141-150.

14. Rutland, R. B. and M. J. Bukovac, 1971, The effect of calciumbicarbonate on iron absorption and distribution by Chrysanthe-mum morifolium (Ram,). Plant and Soil 35:225-236,

15. Scheibler, C. 1960. Gasvolumetrische Bestimmung der Kohlenrsaure. In: K. Nehring: Agrikulturchemische Untersuchungs-methoden fur Dunge- und Futternvittel, Boden und Milch, Parey-Verlag, Hamburg und Berlin.

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