Synthesis of the Arabidopsis bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase enzyme of lysine catabolism is concertedly regulated by metabolic and stress-associated signals

Abstract

In plants, excess cellular lysine (Lys) is catabolized into glutamic acid and acetyl-coenzyme A; yet, it is still not clear whether this pathway has other functions in addition to balancing Lys levels. To address this issue, we examined the effects of stress-related hormones, abscisic acid (ABA), and jasmonate, as well as various metabolic signals on the production of the mRNA and polypeptide of the bifunctional Lys-ketoglutarate reductase (LKR)/saccharopine dehydrogenase (SDH) enzyme, which contains the first two linked enzymes of Lys catabolism. The level of LKR/SDH was strongly enhanced by ABA, jasmonate, and sugar starvation, whereas excess sugars and nitrogen starvation reduced its level; thus this pathway appears to fulfill multiple functions in stress-related and carbon/nitrogen metabolism. Treatments with combination of hormones and/or metabolites, as well as use of ABA mutants in conjunction with the tester sugars mannose and 3-O-methyl-glucose further supported the idea that the hormonal and metabolic signals apparently operate through different signal transduction cascades. The stimulation of LKR/SDH protein expression by ABA is regulated by a signal transduction cascade that contains the ABI1-1 and ABI2-1 protein phosphatases. By contrast, the stimulation of LKR/SDH protein expression by sugar starvation is regulated by the hexokinase-signaling cascade in a similar manner to the repression of many photosynthetic genes by sugars. These findings suggest a metabolic and mechanistic link between Lys catabolism and photosynthesis-related metabolism in the regulation of carbon/nitrogen partitioning.

Figure 1.

The Lys catabolism pathway and metabolites derived from it. ASD, aminoadipic semialdehyde dehydrogenase. The broken arrow represents several non-specified enzymatic reactions. Glu and acetyl-CoA, the products of this pathway, are boxed.

Figure 2.

Effects of ABA and MeJa on the level of the Arabidopsis bifunctional LKR/SDH mRNA. Nine-day-old Arabidopsis plants were grown in liquid cultures in the absence (control) or presence of increasing concentrations of ABA (A) or MeJa (B) for 24 h. Total RNAs were then reacted in northern blots with the LKR domain of the AtLKR/SDH cDNA as a probe. The different lanes contained comparable amounts of RNA (stained blots in the bottom panels of A and B).

Figure 3.

Effects of ABA and MeJa on the level of the Arabidopsis bifunctional LKR/SDH polypeptide. Nine-day-old Arabidopsis plants were grown in liquid cultures in the absence (control) or presence of increasing concentrations of ABA (A) or MeJa (B) for 24 h. Proteins were then reacted in western blots with monoclonal anti-LKR antibodies (top panels in A and B). The different lanes contained comparable amounts of proteins as depicted by the region of the Rubisco large subunit in the bottom panels of A and B. The relative intensities of the LKR/SDH bands, normalized to controls treated with no hormones (left lane of each section), are indicated between the top and bottom panels. Arrowheads and asterisks on the right indicate the positions of the LKR/SDH and Rubisco small subunit polypeptides, respectively.

Figure 4.

Effects of sugars and sugar starvation on the level of the Arabidopsis bifunctional LKR/SDH mRNA and polypeptide. Nine-day-old Arabidopsis plants grown in a liquid culture were transferred to a new medium containing various levels of different sugars and incubated for different durations before harvesting. The LKR/SDH mRNA (C) and polypeptide (A, B, and D) were detected as described in Figures 2 and 3, respectively. The different lanes contained comparable amounts of RNA (stained blot in the bottom panel of C) as well as proteins (stained blots in the bottom panels of A, B, and D, depicted by the region of the Rubisco large subunit). A, Seedlings were transferred to new media as follows: The first group (lane a) was transferred to a medium containing 2% (w/v) Suc (control level) and grown for 72 h before harvesting. The second group (lane b) was transferred to a Suc-free medium for 72 h before harvesting. The third group (lane c) was first transferred to a medium containing control Suc levels for 48 h and then to a medium containing 5% (w/v) Suc for additional 24 h before harvesting. B, Plants were transferred to media containing 2% or 0% (w/v) Suc, incubated for 48 h and harvested (lanes a and b). In addition, plants exposed to 48 h of sugar starvation (lane b) were further transferred to new media containing 1%, 2%, or 5% (w/v) Suc for additional 24 h before harvesting. C and D, Sucstarved plants were transferred to new media containing 2% (w/v) of the indicated sugars and incubated for 24 h before harvesting. The relative intensities of the LKR/SDH bands, normalized to the controls (left lane in each section), are indicated in between the top and bottom panels.

Figure 5.

Effects of nitrogen, nitrogen starvation, and amide amino acids on the level of the LKR/SDH polypeptide. Nine-day-old Arabidopsis plants grown in a liquid culture were transferred to a new medium containing various levels of different nitrogenous metabolites for different durations before harvesting. Proteins were then reacted in western blots with monoclonal anti-LKR antibodies (top panels in A and B). The different lanes contained comparable amounts of proteins as depicted by the region of the Rubisco large subunit in the bottom panels of A and B. A, Plants were grown in media containing or lacking the regular levels of nitrogen and Suc, as indicated on top, for 72 h and harvested (lanes a–c). In addition, plants were pre-incubated in sugar-free medium for 48 h and then transferred to a new medium lacking sugar and nitrogen for additional 24 h before harvesting (lane d). B, Plants were transferred to new media containing either no added amino acids (C) or 10 mm of any of the amino acids Glu, Gln, Asp, or Asn for 24 h before harvesting (lanes a–e, respectively). The relative intensities of the LKR/SDH bands, normalized to the controls (left lane in each section), are indicated in between the top and bottom panels.

Figure 6.

Effects of combined treatments with ABA and Suc starvation on the level of the LKR/SDH polypeptide. Nine-day-old seedlings grown in a liquid culture were transferred to new media containing or lacking 2% (w/v) Suc for 48 h and then exposed to different concentrations of ABA, as indicated on top, for 24 h before harvesting. Top, Proteins were then reacted in western blot with monoclonal anti-LKR antibodies. The different lanes contained comparable amounts of proteins as depicted by the region of the Rubisco large subunit in the bottom panel. The relative intensities of the LKR/SDH bands, normalized to the control treated with sugar and no ABA (left lane), are indicated in between the top and bottom panels.

Figure 7.

Effects of combined treatments with ABA and MeJa on the level of the LKR/SDH polypeptide. Nine-day-old seedlings grown in a liquid culture were transferred to new media containing different concentrations of MeJa, ABA, or MeJa + ABA, as indicated on top, for 24 h before harvesting. Top, Proteins were then reacted in western blots with monoclonal anti-LKR antibodies. The different lanes contained comparable amounts of proteins as depicted by the region of the Rubisco large subunit in the bottom panel. The relative intensities of the LKR/SDH bands, normalized to the control treated with no hormones (left lane), are indicated in between the top and bottom panels.

Figure 8.

Effects of ABA, MeJa, and sugar starvation on the level of the LKR/SDH polypeptide in different aba mutants. In all experiments, the level of the LKR/SDH polypeptide was monitored by western-blot analysis with monoclonal anti-LKR antibodies. A, Control non-treated nine-day-old seedlings of the wild-type plants (lane a) as well as the aba2-1, abi2-1 and abi1-1 mutants (lanes b–d, respectively) showing the basal levels of the LKR/SDH polypeptide. B, Nine-day-old seedlings of wild type as well as the aba mutants (top to bottom panels) were transferred to new regular media without ABA or MeJa, or with either 50μm ABA or MeJa for 24 h before harvesting (lanes a–c, respectively). Lane d, In addition, plants were transferred to sugar-free medium for 72 h before harvesting. The different lanes contained comparable amounts of proteins as depicted by the region of the Rubisco large subunit in the bottom panels of A to C. The relative intensities of the LKR/SDH bands, normalized to the controls (left lanes), are indicated in between the top and bottom panels of each section.

Figure 9.

Effects of various metabolized and non-metabolized sugars on the level of the LKR/SDH polypeptide. Nine-day-old seedlings grown in a liquid culture were transferred to new media containing different sugars or no sugar, as indicated on top, for 48 h before harvesting. Top, Proteins were then reacted in western blot with monoclonal anti-LKR antibodies. The different lanes contained comparable amounts of proteins as depicted by the region of the Rubisco large subunit in the bottom panel. The relative intensities of the LKR/SDH bands, normalized to the control (left lane), are indicated in between the top and bottom panels.

Figure 10.

Effect of phosphate on the LKR/SDH gene expression. Nine-day-old seedlings of wild-type Arabidopsis grown in a liquid culture were transferred to new media as follows: The first group (lane a) was transferred to a medium containing 1.08 mm phosphate (control level) and grown for 72 h before harvesting. The second group (lane b) was transferred to a phosphate-free medium for 72 h before harvesting. The third group (lane c) was transferred to a medium containing 1.08 mm phosphate for 48 h and then transferred to a medium containing 6.25 mm phosphate for additional 24 h before harvesting. Top, Proteins were then reacted in western blot with monoclonal anti-LKR antibodies. The different lanes contained comparable amounts of proteins as depicted by the region of the Rubisco large subunit in the bottom panel. The relative intensities of the LKR/SDH bands, normalized to the control (left lane), are indicated in between the top and bottom panels.