The Proof by ¹³C‐NMR Spectroscopy of the Predominance of the C₅ Pathway over the Shemin Pathway in Chlorophyll Biosynthesis in Higher Plants and of the Formation of the Methyl Ester Group of Chlorophyll from Glycine

Abstract

The 13 C‐NMR spectrum of chlorophyll a formed when excised etiolated maize leaves were greened in the presence of [1‐ 13 C]glutamate showed that the four methine bridge carbons (C‐5, C‐10, C‐15 and C‐20) and four pyrrole α‐carbons (C‐4, C‐9, C‐14 and C‐16) were considerably enriched relative to the natural‐abundance spectrum thus providing unequivocal proof of the operation of the C 5 pathway of 5‐aminolaevulinate formation during chlorophyll biosynthesis in maize. [The carbon numbering system used is that recommended by IUB, see Pure Appl. Chem. 51 , 2251–2304 (1979).] The 13 C‐NMR spectrum of chlorophyll a formed during greening in the presence of [2‐ 13 C]glycine showed that little or no enrichment of the above‐mentioned methine bridge or pyrrole α‐carbons had occurred thus indicating that the Shemin pathway makes little or no contribution to chlorophyll formation in greening maize. There was, however, considerable enrichment of the methoxyl carbon of the methoxycarbonyl group attached to isocyclic ring carbon C‐13 2 . This shows that the C‐2 carbon of glycine is used to label the C 1 pool and subsequently the methyl ester carbon of magnesium‐protoporphyrin IX‐monomethyl ester which is an intermediate in chlorophyll biosynthesis. The 13 C‐NMR spectrum of chlorophyll a obtained when greening occurs in the presence of [5‐ 13 C]glutamate showed considerable enrichment of the two carboxyl carbons of chlorophyll a (C‐13 3 and C‐17 3 ). This spectrum also showed that enrichment, equivalent to 15% of that occurring at the carboxyl carbons, occurred at the four methine bridge carbons and four relevant pyrrole α‐carbons. This is consistent with the conversion of [5‐ 13 C]glutamate to [1‐ 13 C]2‐oxoglutarate (or glutamate) via [5‐ 13 C]2‐oxoglutarate and recycling in the tricarboxylic acid cycle: the soformed 1‐ 13 C‐labelled substrate would then be incorporated into chlorophyll by the C 5 pathway. This assessment of the amount of [5‐ 13 C]glutamate incorporated into chlorophyll after recycling as 2‐oxoglutarate in the tricarboxylic acid cycle is used not only to confirm the view that the C 5 pathway is the major, possibly sole, route for chlorophyll biosynthesis in higher plants but is also used to negate previous assertions of the involvement of the Shemin pathway based on comparison of the incorporation of 2‐oxoglutarate 14 C‐labelled at C‐1 or C‐5. In these studies we also found that chlorophyll a purified by silica‐gel thin‐layer chromatography contained allomerization products and was unsuitable for 13 C‐NMR spectroscopy.

Keywords

Affiliated Institutions

• ACT Government
• The University of Sydney AU
• Plant Industry AU
• Bayer (Germany) DE

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