M. P. Purnell

Tobacco isoenzyme 1 of NAD(H)-dependent glutamate dehydrogenase catabolizes glutamate in vivo

Glutamate (Glu) dehydrogenase (GDH, EC 1.4.1.2–1.4.1.4) catalyzes in vitro the reversible amination of 2-oxoglutarate to Glu. The in vivo direction(s) of the GDH reaction in higher plants and hence the role(s) of this enzyme is unclear, a situation confounded by the existence of isoenzymes comprised totally of either GDH β- (isoenzyme 1) or α- (isoenzyme 7) subunits, as well as another five α-β isoenzyme permutations. To clarify the in vivo direction of the reaction catalyzed by GDH isoenzyme 1, [15N]Glu was supplied to roots of two independent transgenic tobacco (Nicotiana tabacum) lines with increased isoenzyme 1 levels (S4-H and S49-H). The [15N]ammonium (NH4+) accumulation rate in these lines was elevated approximately 65% compared with a null segregant control line, indicating that isoenzyme 1 catabolizes Glu in roots. Leaf glutamine synthetase (GS) was inhibited with a GS-specific herbicide to quantify any contribution by GDH toward photorespiratory NH4+ reassimilation. Transgenic line S49-H did not show enhanced resistance to the herbicide, indicating that the large pool of isoenzyme 1 in S49-H leaves was unable to compensate for GS and suggesting that isoenzyme 1 does not assimilate NH4+ in vivo.

Cloning and characterisation of a glutamate dehydrogenase cDNA from tomato (Lycopersicon esculentum L.)

A full-length cDNA (legdh1) has been cloned encoding glutamate dehydrogenase (GDH) from tomato (Lycopersicon esculentum L.). legdh1 is 1568 bp long and contains an open reading frame encoding a 44.8 kDa polypeptide with a putative mitochondrial-matrix-targeting pre-sequence at its N-terminus. Southern analysis indicates the existence of one copy of legdh1 per haploid genome, and no closely related genes were detected by Southern analysis at low stringency. We hypothesise that in tomato, the two GDH subunits may arise from post-transcriptional modifications of a single gene. Northern analysis reveals high expression of legdh1 in roots, lower levels of expression in stems, flowers and leaves, and no detectable expression in fruits. In general, there was no correlation between steady-state mRNA level and protein activity in the tissues analysed, again suggesting the importance of post-transcriptional events in the regulation of GDH. Comparison of cloned plant GDH proteins reveals a high degree of homology throughout the sequence except for a very specific, highly divergent region.

The N-terminal presequence from F1-ATPase β-subunit of Nicotiana plumbaginifolia efficiently targets green fluorescent fusion protein to the mitochondria in diverse commercial crops

Approximately 10-15% of plant nuclear genes appear to encode mitochondrial proteins that are directed to mitochondria by specific targeting signals. Reports on the heterologous function of these targeting signals are generally limited to one or a few species, with an emphasis on model plants such as tobacco and Arabidopsis. Given their sequence diversity and their insufficient testing in commercially important crops (including monocotyledonous crops), the extent to which these signals can be relied on for biotechnological purposes across species remains to be established. This study provides the experimental verification of a mitochondrial signal that is functional across diverse crop species, including five monocots (sugarcane, wheat, corn, sorghum and onion) and seven dicots (cucumber, cauliflower, tomato, capsicum, pumpkin, coriander and sunflower). In all 12 crops, transient assays following microprojectile bombardment showed that the N-terminal mitochondrial presequence from F1-ATPase β-subunit (ATPase-β) of Nicotiana plumbaginifolia Viv. targeted green fluorescent fusion protein to the mitochondria. The transient assay results in sugarcane were confirmed in stably transformed root cells. The ATPase-β signal should be a useful metabolic engineering tool for directing recombinant proteins to the mitochondrial matrix in diverse plant species of commercial interest.

Non-invasive monitoring of sucrose mobilization from culm storage parenchyma by magnetic resonance spectroscopy

Because sucrose stored in mature stalks (in excess of 40% of stalk dry weight) can be wholly mobilized to supply carbon for the growth of heterotrophic tissues, we propose that sucrose mobilization requires a net sink-to-source transition that acts in toto within sett internode storage parenchyma. Based on our data we propose that mobilization of sucrose from culm storage parenchyma requires minimal investment of metabolic resources, and that the mechanism of sucrose mobilization is metabolically neutral. By magnetic resonance spectroscopy and phloem-specific tracer dyes, strong evidence was found that sucrose is mobilized from sett storage parenchyma via phloem to the growing shoot tissue. An analysis of the enzyme activities involved in sucrose metabolism and glycolysis suggested that sucrose synthase activity is downregulated due to the effects of sucrose mobilization. Overall, metabolism in storage parenchyma shifts from futile cycling to a more quiescent state during sucrose mobilization.

RNAi-mediated abrogation of trehalase expression does not affect trehalase activity in sugarcane

To engineer trehalose metabolism in sugarcane (Saccharum spp. hybrids) two transgenes were introduced to the genome: trehalose-6-phosphate synthase- phosphatase (TPSP), to increase trehalose biosynthesis and an RNAi transgene specific for trehalase, to abrogate trehalose catabolism. In RNAi-expressing lines trehalase expression was abrogated in many plants however no decrease in trehalase activity was observed. In TPSP lines trehalase activity was significantly higher. No events of co-integration of TPSP and RNAi transgenes were observed. We suggest trehalase activity is essential to mitigate embryonic lethal effects of trehalose metabolism and discuss the implications for engineering trehalose metabolism.