Biochemical Relationships in Chlorotic Leaves

Biochemical Relationships in Chlorotic Leaves

of Macadamia

FOREWORD

In 1964 the California Macadamia Society proposed an investigation of the micronutrient requirements of macadamias. The suggestion was made to the University of California Riverside that a graduate student be selected to carry out the study and that it be supported in part by a grant from the society. Thc fortunate selection was Mr. Ian Gilfillan, a graduate student of Dr. W. W. Jones, Prof. of Horticulture.

During the next year Mr. Gilfillan succeeded in establishing relationships between leaf concentrations and deficiency symptoms with iron and manganese. The study also included the relation of each of thc two elements with chlorophyll content in chlorotic leaves. These studies have been reported in detail in the 1966 yearbook.

This art of the investigation provided the background for an attempt to find out exactly what happens chemically in the leaf to cause thc lack of chlorophyll development (chlorosis) when iron or manganese is deficient. The composite of these studies became the thesis for the Ph. D. degree of Mr. Ian Gilfillan.

This work has been of inestimable value to the society and we hope that further projects of this type soon will he started at the university.

Following is the abstract, introduction and summary of Dr. Gilfillan’s dissertation.

Abstract of the Dissertation

BIOCHEMICAL RELATIONSHIPS IN CHLOROTIC LEAVES

OF MACADAMIA

(MACADAMIA TETRAPHYLLA. JOHNSON) UNDER CONDITIONS

OF IRON AND MANGANESE DEFICIENCY

Ian Muir Gilfillan*

Under iron-deficient and manganese-deficient conditions, the Macadamia developed characteristic leaf symptoms. The chlorophyll level in the deficient leaves was found to he closely correlated with their iron or manganese content.

The iron-deficient leaves were shown to have an accumulation of arginine, homoserine, lysine, citric acid, total free amino acids, and Krebs cycle organic acids. The manganese-deficient leaves contained a greater amount of homoserine and a lower level of isocitric acid.

Carbon-14 acetate was fed to excised leaves by way of the transpiration stream, and the fate of the labeled carbonate was followed. In the iron-deficient leaves, the accumulation of citric acid appeared to he associated with the aconitase reaction, while the general build-up of organic acids seemed to be correlated with a lower rate of protein synthesis and an associated accumulation of free amino acids. Evidence was obtained for operation of the glyoxylate cycle under manganese-deficiency conditions.

The pathway leading to chlorophyll biosynthesis was examined for blocks by feeding labeled data-aminolevulinic acid and labeled succinic acid to excised leaves. Both manganese-deficient and iron-deficient leaves showed blocks at points following synthesis of delta-aminolevulinic acid.

BIOCHEMICAL RELATIONSHIPS IN CHLOROTIC LEAVES

OF MACADAMIA

(MACADAMIA TETRAPHYLLA, L. JOHNSON) UNDER CONDITIONS

OF IRON AND MANGANESE DEFICIENCY

INTRODUCTION

Iron chlorosis has been a problem in Macadamia culture in southern California from the time that the first orchards were planted. Young seedlings and nursery trees are particularly affected. Attempts to control iron chlorosis by application of iron compounds to the plant or to the soil have given unreliable results, and a more basic approach to the problem seems necessary. The purpose of this dissertation has been to investigate the biochemical status of iron-deficient Macadamia leaves. In view of the fact that manganese is readily taken up by the Macadamia and reaches high concentration levels in the leaves, and the fact that manganese and iron metabolism have been shown to he intimately linked, manganese deficiency is also examined.

The dissertation is divided into three parts. In Part I, the manganese and iron-deficiency symptoms developed in the leaves are described. The relation between the chlorophyll content of the chlorotic leaves and their content of iron and manganese is determined, and an attempt is made to predict iron and manganese status on the basis of degree of leaf chlorosis. The change in iron and manganese concentration in the leaves with age is traced. In Part II, the organic acid and free amino acid status of the chlorotic leaves is examined. In Part III, various radioactive compounds are fed into excised leaves, and the fate of the labeled carbon is used to attempt an explanation of differences found in the organic acid and amino acid status of the iron- and manganese-deficient leaves.

SUMMARY

1.Leaf symptoms of iron deficiency and manganese deficiency were developed in Macadamia tetraphylla, variety Stephenson. Symptoms of iron deficiency were associated in 3-month-old leaves with concentrations below 20 ppm Fe, while manganese-deficiency symptoms developed below 12 ppm Mn. Concentrations in normal green leaves of this age were 20 to 40 ppm Fe and 100 to 300 ppm Mn.

2.The change in manganese and iron concentrations in the leaf with age was followed over a period of 7 months. Manganese concentration showed a steady increase with age, while iron concentrations dropped over the first 6 weeks, rose sharply to a maximum at about 4 months, and then dropped slowly.

3.The chlorophyll concentration in the leaves was found to be closely related to iron concentration. A quadratic regression curve showed that under the conditions of the experiment the iron concentration in the leaf accounted for 82% of the variability in chlorophyll content. Manganese and chlorophyll concentrations, on the other hand, were not closely linked except when manganese was present in low concentrations. At leaf values below 12 ppm Mn., manganese concentration accounted for 34% of the variability in chlorophyll content.

4.Visual ratings of degree of manganese and iron chlorosis were not reliable indices to the manganese and iron concentrations in the leaves.

5.There was a significant negative correlation between iron and manganese concentrations in 3-year-old leaves. The linear regression equation, however, showed that under the conditions of the experiment, the iron concentration in the leaf accounted for only 5.8% of the variability in manganese concentration.

6.Iron-deficient leaves showed a significantly greater concentration of total free amino acids, significantly greater concentrations of nonprotein arginine, homoserine, and lysine; significantly greater concentrations of the total free organic acids associated with the TCA cycle; and a significantly greater concentration of free citric acid.

7.Manganese-deficient leaves showed a significantly greater concentration of homoserine and a significantly lower concentration of isocitric acid.

8.When 2-C14 acetate was fed by way of the transpiration stream into the tissues of control (normal green) and chlorotic, iron -deficient leaves, considerably more labeled carbon was found in the amino acid and organic acid fractions of the iron-deficient leaves than in the control leaves. The specific activity of citric acid in iron-deficient leaves showed a steady increase, even, after the C14-acetate was replaced by cold 10-5 N HCI. The specific activities of ma lie and succinic acids, on the other hand, were greater in the iron deficient leaves during the period that C14-acetate was supplied but dropped to normal levels once this was removed. The radioactivity in the amino acid fraction was confined over the 3-hour feeding period to glutamic and aspartic acids. The interpretation placed on the data was that the accumulation of citric acid was associated with lower aconitase enzyme activity, while the general build-tip of organic acids was probably associated with a reduced rate of protein synthesis and an associated accumulation of free amino acids.

9.When 2—C14 acetate was fed to manganese-deficient leaves, the behavior of the labeled carbon fitted the pattern that would he expected if the glyoxylate cycle were operating in this tissue. The evidence for the glyoxylate cycle was,

a)No lag period before incorporation, of label in to malic acid of the manganese-deficient leaves, while there was a lag in the control leaves;

h)The lag period before incorporation into glyoxylate followed by a more rapid rate of incorporation of label into this acid in the manganese-deficient leaves;

c) An accumulation of label in isocitric acid in the manganese-deficient leaves, indicating a possible lower activity of Mn+ -dependent isocitric dehydrogenase;

d)A greater specific activity in the malic acid fraction of the manganese-deficient leaves, in comparison to that in the control, when cold 0. IM deficient leaves, in comparison to that in the control, when cold 0. IM succinic acid was fed into the leaves with the 2-C14 acetate. The "pool" of cold succinic acid was thought to trap labeled carbon passing round the TCA cycle, while label entered malic acid directly in the glyoxylate cycle.

e)The presence of the isocitritase enzyme in greater quantity in manganese-deficient leaves.

10.When 4-C14 delta-aminolevulinic acid (ALA) was fed to iron-deficient and manganese-deficient leaves,, there was less label incorporated in the pheophytin extracted in both cases than in the control leaves. This was interpreted as a block or blocks due to deficiency of iron or manganese at some place on the pathway leading to chlorophyll biosynthesis after formation of ALA. The block due to deficiency of iron was more complete than that due to deficiency of manganese.

11.When 2,3-C14 succinic acid was fed to iron-deficient and manganese deficient leaves, there was less label incorporated in the pheophytin extracted in both cases than in the control leaves. This was interpreted as a block at some place on the chlorophyll biosynthetic pathway following condensation of succinyl CoA and glycine to form ALA. It was noted, however, that the relative rate of incorporation of label into chlorophyll from succinate and ALA was nearly identical for the control, iron-deficient, and manganese-deficient leaves. It was concluded, therefore, that the block or blocks occur after formation of ALA.

*Doctor of Philosophy. Graduate Program in Plant Science, University of California, Riverside, August 1967, Professor Winston W. Jones, and Chairman.