An Arabidopsis Mutant Impaired in Coenzyme A Biosynthesis Is Sugar Dependent for Seedling Establishment

Abstract

Once the plant coenzyme A (CoA) biosynthetic pathway has been elucidated by comparative genomics, it is feasible to analyze the physiological relevance of CoA biosynthesis in plant life. To this end, we have identified and characterized Arabidopsis (Arabidopsis thaliana) T-DNA knockout mutants of two CoA biosynthetic genes, HAL3A and HAL3B. The HAL3A gene encodes a 4#-phosphopantothenoyl-cysteine decarboxilase that generates 4#-phosphopantetheine. A second gene, HAL3B, whose gene product is 86% identical to that of HAL3A, is present in the Arabidopsis genome. HAL3A appears to have a predominant role over HAL3B according to their respective mRNA expression levels. The hal3a-1, hal3a-2, and hal3b mutants were viable and showed a similar growth rate as that in wild-type plants; in contrast, a hal3a-1 hal3b double mutant was embryo lethal. Unexpectedly, seedlings that were null for HAL3A and heterozygous for HAL3B (aaBb genotype) displayed a sucrose (Suc)-dependent phenotype for seedling establishment, which is in common with mutants defective in b-oxidation. This phenotype was genetically complemented in aaBB siblings of the progeny and chemically complemented by pantethine. In contrast, seedling establishment of Aabb plants was not Suc dependent, proving a predominant role of HAL3A over HAL3B at this stage. Total fatty acid and acyl-CoA measurements of 5-d-old aaBb seedlings in medium lacking Suc revealed stalled storage lipid catabolism and impaired CoA biosynthesis; in particular, acetyl-CoA levels were reduced by approximately 80%. Taken together, these results provide in vivo evidence for the function of HAL3A and HAL3B, and they point out the critical role of CoA biosynthesis during early postgerminative growth.

The Coenzyme A Biosynthetic Enzyme Phosphopantetheine Adenylyltransferase Plays a Crucial Role in Plant Growth, Salt/Osmotic Stress Resistance, and Seed Lipid Storage

“…Measurement of the total acyl-CoA pool was performed at 5 d after imbibition (DAI) both in medium lacking or supplemented with Suc as described by Larson and Graham (2001). In agreement with previous results (Rubio et al, 2006), Suc supplementation led to stimulation of acyl-CoA biosynthesis compared to medium lacking Suc (Fig. 6B).…”

“…2, A and B); nevertheless, the phenotype was not so severe as those of aaBb hal3 seedlings in medium lacking Suc (Rubio et al, 2006;Fig. 2C), and, in contrast to aaBb hal3 progeny, seedling establishment was not compromised in ppat-1 (Fig.…”

“…In the next step, 4#-phospho-N-pantothenoylCys decarboxylase (PPCDC; CoaC) catalyzes the generation of 4#-phosphopantetheine. Two Arabidopsis genes, AtHAL3A and AtHAL3B, encode essential PPCDC enzymes, and combined inactivation of both genes aborted embryogenesis at the early globular stage (Rubio et al, 2006). The penultimate step in the CoA biosynthetic pathway is catalyzed by the enzyme 4#-phosphopantetheine adenylyltransferase (PPAT; CoaD), which catalyzes the reversible adenylation of 4#-phosphopantetheine to form 3#-dephospho-CoA (dPCoA) and pyrophosphate (PPi; see Fig.…”

“…Considering the central role of CoA in plant metabolism, the analysis of mutants impaired in CoA biosynthesis has received limited attention (Rubio et al, 2006;Tilton et al, 2006). Recently, we reported that a hal3a-1hal3b-1 double mutant was embryo lethal, whereas seedlings that were null for HAL3A and heterozygous for HAL3B (aaBb hal3 mutant) displayed a Suc-dependent phenotype for seedling establishment (Rubio et al, 2006).…”

“…Recently, we reported that a hal3a-1hal3b-1 double mutant was embryo lethal, whereas seedlings that were null for HAL3A and heterozygous for HAL3B (aaBb hal3 mutant) displayed a Suc-dependent phenotype for seedling establishment (Rubio et al, 2006). In this article, we have focused on the next step of CoA biosynthesis, which is catalyzed by the enzyme PPAT.…”