Overexpression of mutated forms of aspartate kinase and cystathionine γ‐synthase in tobacco leaves resulted in the high accumulation of methionine and threonine

Abstract

SummaryMethionine and threonine are two essential amino acids, the levels of which limit the nutritional quality of plants. Both amino acids diverge from the same branch of the aspartate family biosynthesis pathway; therefore, their biosynthesis pathways compete for the same carbon/amino substrate. To further elucidate the regulation of methionine biosynthesis and seek ways of increasing the levels of these two amino acids, we crossed transgenic tobacco plants overexpressing the bacterial feedback-insensitive aspartate kinase (bAK), containing a significantly higher threonine level, with plants overexpressing Arabidopsis cystathionine c-synthase (AtCGS), the first unique enzyme of methionine biosynthesis. Plants co-expressing bAK and the full-length AtCGS (F-AtCGS) have significantly higher methionine and threonine levels compared with the levels found in wild-type plants, but the methionine level does not increase beyond that found in plants expressing F-AtCGS alone. This finding can be explained through the feedback inhibition regulation mediated by the methionine metabolite on the transcript level of AtCGS. To test this assumption, plants expressing bAK were crossed with plants expressing two mutated forms of AtCGS in which the domains responsible for the feedback regulation have been deleted. Indeed, significantly higher methionine contents and its metabolites levels accumulated in the newly produced plants, and the levels of threonine were also significantly higher than in the wild-type plants. The transcript level of the two mutated forms of AtCGS significantly increased when there was a high content of threonine in the plants, suggesting that threonine modulates, probably indirectly, the transcript level of AtCGS.

An Essential Role for Tomato Sulfite Oxidase and Enzymes of the Sulfite Network in Maintaining Leaf Sulfite Homeostasis

“…Sulfolipids were separated from the other lipids by two-dimensional thin-layer chromatography in chloroform:methanol:acetic acid:water (73:25:2:4) polar lipid separation solution, followed by quantification on Trace GC Ultra (Thermo Scientific; http://www.thermoscientific.com) as described before (Khozin et al, 1997). Free amino acids and GSH were extracted from frozen leaf samples and detected according to Matityahu et al (2006) and Hacham et al (2008). For protein-bound amino acid determination, total proteins were extracted from 100 mg of leaves using a standard protocol of TCA precipitation (Wang et al, 2006) followed by triple washing the SDS with 100% methanol and 80% acetone.…”

“…For protein-bound amino acid determination, total proteins were extracted from 100 mg of leaves using a standard protocol of TCA precipitation (Wang et al, 2006) followed by triple washing the SDS with 100% methanol and 80% acetone. Extracted proteins were hydrolyzed in constant boiling HCl vapors at 110°C for 22 h under nitrogen (Hacham et al, 2008). Total amino acids were determined by the AccQ$Tag method (Waters; http://www.waters.com/) using a Waters Alliance 2695 HPLC instrument.…”

Improving the Content of Essential Amino Acids in Crop Plants: Goals and Opportunities

“…The major regulatory enzyme of Met biosynthesis is cystathionine g-synthase (CGS), and this activity in Arabidopsis is also regulated by the level of SAM via a compound posttranscriptional control mechanism involving interactions with a highly regulatory multicomponent domain located in the N terminus of the mature CGS polypeptide (Inaba et al, 1994;Chiba et al, 1999;Hacham et al, 2002;Ominato et al, 2002;Hacham et al, 2006). Interestingly, mutations in this region, or its deletion, result in overproduction of Met, which is likely independent of a reduction of SAM synthesis (Chiba et al, 1999;Hacham et al, 2002Hacham et al, , 2006Hacham et al, , 2008Ominato et al, 2002). Moreover, the combination of expression of a mutated form of CGS with a bacterial feedback-insensitive Asp kinase (the first enzyme of the Asp-family pathway) further increases the accumulation of Met (Hacham et al, 2008).…”

“…Interestingly, mutations in this region, or its deletion, result in overproduction of Met, which is likely independent of a reduction of SAM synthesis (Chiba et al, 1999;Hacham et al, 2002Hacham et al, , 2006Hacham et al, , 2008Ominato et al, 2002). Moreover, the combination of expression of a mutated form of CGS with a bacterial feedback-insensitive Asp kinase (the first enzyme of the Asp-family pathway) further increases the accumulation of Met (Hacham et al, 2008). Utilization of CGS enzymes with either deleted or mutated N-terminal domains currently appears as a potentially promising approach to increase the production of free Met with minimal negative effects.…”

Biotechnology for Enhanced Nutritional Quality in Plants

“…Overexpression of the full length CGS and its truncated forms was used in several studies to investigate the regulation of methionine biosynthesis and/or to produce plants with increased levels of methionine (Avraham et al, 2005;Hacham et al, 2008;Dancs et al, 2008). Alfalfa (Medicago sativa L.) is a member of the Fabaceae family that is grown and used worldwide as livestock feed.…”