Phenotypic effects from the expression of a deregulated AtGAD1 transgene and GABA pathway suppression mutants in maize

Abstract

Glutamate decarboxylase (GAD; EC 4.1.1.15) catalyzes the irreversible decarboxylation of glutamate to produce γ-aminobutyric acid (GABA); a ubiquitous non-protein amino acid involved in the regulation of several aspects of plant metabolism and physiology. To study the function of GAD and GABA in maize, we have; 1) introduced native and deregulated forms of AtGAD1 into maize with the intent of increasing the synthesis of GABA and 2) introduced constructs into maize designed to suppress the activity of several GABA shunt, GABA transport and GABA pathway genes. Maize plants expressing the deregulated AtGAD1 exhibit a severe chlorosis and retarded growth phenotype and have high levels of GABA, and Ca++/CaM-independent GAD activity. Plants expressing the suppression constructs for GABA biosynthetic and transport pathway genes had no observable phenotype whereas a knockout of GABA catabolic pathway genes led to growth and developmental defects under standard growth conditions. The implications of this study to our understanding of the action and function of GABA and GAD in crops are discussed.

Fig 1. GABA biosynthesis, transport, and metabolism.

All abbreviations are defined in Table 1 except, OAA, 2-oxaloacetate; NRT2, nitrate transporter.

Fig 2. Growth deformities in AtGAD1-ΔCaMD plant expressed under RTBV promoter A.

A: 30-day-old AtGAD1-ΔCaMD; B: 30-day-old wild-type, C: 150-day-old leaf of wild-type; 150-day-old leaf of AtGAD1-ΔCaMDt; D: 150-day-old tassel of AtGAD1-ΔCaMD; E: 150-day-old root of AtGAD1-ΔCaMD; F: 150-day-old ear of AtGAD1-ΔCaMD plant; G: 150-day-old ear of AtGAD1-ΔCaMD; H: 150-day AtGAD1-ΔCaMD entire plant.

Fig 3. GAD enzyme activity in the leaves of 150-day-old plants over-expressing a truncated GAD with or without 10 μM Ca⁺⁺/10 ng/μL Calmodulin.

Values are the average of 5 events and the error bars represent standard error from the mean. Green bars represent transgenic plants, blue bars represent wild-type plants.

Fig 4. Truncated AtGAD1-ΔCaMD expressing transgenic 150-day-old corn plants show increased accumulation of GABA and other amino acids.

Values are the average of five events. Green bars represent transgenic plants, blue bars represent wild-type plants. An asterisk marks those lines significantly different than wild type (p < 0.05).

Fig 5. GAD activity in the leaves of GABA pathway suppression mutants.

The values represent means of duplicate measurements of 5 separate plants. Green bars represent transgenic plants, blue bars represent wild-type plants. An asterisk marks those lines significantly different than wild type (p < 0.05).

Fig 6. GABA and SSA levels in the leaves of GABA pathway suppression mutants.

The values represent means of duplicate measurements of 5 separate plants. Green bars represent transgenic plants, blue bars represent wild-type plants. An asterisk marks those lines significantly different than wild type (p < 0.05).

Fig 7. Growth deformities upon perturbation of GABA shunt pathway.

A: SSADH 10-day-old seedlings; B: 30-day-old, SSADH on the left, wild-type on the right; C: 60-day-old SSADH plant; D: Wild-type plant on the left, two SCoAL plants on the right.