Glutamate, Ornithine, Arginine, Proline, and Polyamine Metabolic Interactions: The Pathway Is Regulated at the Post-Transcriptional Level

The metabolism of glutamate into ornithine, arginine, proline, and polyamines is a major network of nitrogen-metabolizing pathways in plants, which also produces intermediates like nitric oxide, and γ-aminobutyric acid (GABA) that play critical roles in plant development and stress. While the accumulations of intermediates and the products of this network depend primarily on nitrogen assimilation, the overall regulation of the interacting sub-pathways is not well understood. We tested the hypothesis that diversion of ornithine into polyamine biosynthesis (by transgenic approach) not only plays a role in regulating its own biosynthesis from glutamate but also affects arginine and proline biosynthesis. Using two high putrescine producing lines of Arabidopsis thaliana (containing a transgenic mouse ornithine decarboxylase gene), we studied the: (1) effects of exogenous supply of carbon and nitrogen on polyamines and pools of soluble amino acids; and, (2) expression of genes encoding key enzymes in the interactive pathways of arginine, proline and GABA biosynthesis as well as the catabolism of polyamines. Our findings suggest that: (1) the overall conversion of glutamate to arginine and polyamines is enhanced by increased utilization of ornithine for polyamine biosynthesis by the transgene product; (2) proline and arginine biosynthesis are regulated independently of polyamines and GABA biosynthesis; (3) the expression of most genes (28 that were studied) that encode enzymes of the interacting sub-pathways of arginine and GABA biosynthesis does not change even though overall biosynthesis of Orn from glutamate is increased several fold; and (4) increased polyamine biosynthesis results in increased assimilation of both nitrogen and carbon by the cells.


I.
Concentrations of all amino acids except Ala and Lys were similar in the two genotypes; the former was higher and the latter was lower in the transgenic plants. Most of the amino acids were negatively affected by the absence of N in the medium but not so in the absence of sucrose.

II.
Histidine was a minor amino acid in all cases except in the presence of additional N; in that case both WT and transgenic seedlings had similar concentrations of His (Supplemental Figure 1a).

III.
The absence of N caused a significant reduction in Ser (a major component of the soluble amino acids fraction), the presence of extra N in the medium had no effect and additional sucrose caused a significant increase in Ser only in the transgenic plants (Supplemental Figure 1c).

IV.
In both genotypes, Phe was lower in the absence of N and significantly higher when the medium was supplemented with extra N (Supplemental Figure 1c); 100 mM extra sucrose had a small but significant effect in transgenic plants only.
V. Ala and Val concentrations were positively affected by additional N as well as C, but the response was different in the two genotypes (Supplemental Figures 1e, 1f). While Ala content was significantly higher in the mODC plants than the WT in all treatments; Val showed difference between the two genotypes only in the presence of extra sucrose in the medium. Extra sucrose positively affected Ile in both genotypes (Supplemental Figure  1g). Figure 1i), Lys (Supplemental Figure 1h) and Leu (Supplemental Figure 1d) were generally low in both genotypes, and showed smaller variations in response to the experimental treatments except that Lys was typically lower in the mODC plants. Effects of different concentrations of nitrate and sucrose on PCA soluble amino acids in 12 d old WT and mODC-1-7 plants. *= P≤ 0.05 for significant difference between treatments and control within WT and mODC plants; + represents P≤ 0.05 for significant difference between WT and mODC plants (N= 3; each replicate= 7-8 seedlings).

I.
Whereas the cauline leaves and siliques showed 2 to 3-fold higher concentrations of Put in the transgenic plants (Supplemental Figure 2a), rosette leaves, flower buds and mature flowers did not show significant differences in their Put contents vs. the WT counterparts.

II.
Spermidine concentrations followed a similar trend as Put in various organs and were quite similar in the respective organs of the two genotypes (Supplemental Figure 2b).

III.
Like Put, in most tissues, Spm was higher in the transgenic plants (vs. the WT) in several organs (Supplemental Figure 2c).

IV.
All of the mature organs that were tested contained significant amounts of Cad, which is presumably derived from Lys by the activity of mODC (Supplemental Figure 2d); WT plants had no Cad in any organ.
V. Among the different organs, flower buds and mature flowers typically had the highest concentrations of all four PAs (Supplemental Figure 2e).

I.
In mature plants, the amino acids whose concentrations were significantly higher in transgenic vs. the WT plants (excluding those mentioned in Figure 4) in different organs included His, Leu, Phe, Leu, and Val in the buds (Supplementary Figure 3).

II.
Conversely, Thr in the leaves, Ala in the buds, and His in the siliques were somewhat higher in the WT than in the transgenic plants.

III.
In general, total soluble amino acids were higher in the buds and the flowers in both the transgenic and the WT plants. No significant difference in total soluble amino acids was observed among other organs except for the buds of mODC plants that showed significantly higher amount of total soluble amino acids than the WT buds.

RNA Isolation, cDNA Synthesis and qPCR (Experiment 1; Data for Experiment 2 are presented in the main text)
Previously frozen plant samples (~200 mg FW in liquid nitrogen and stored at -80°C) were used for RNA extraction using ZR Plant RNA MiniPrep TM Kit (Zymo Research, Irvine, CA) following the manufacturer's instructions. Samples were ground in a frozen (in liquid nitrogen) mortar with an autoclaved pestle (also frozen in liquid nitrogen), and mixed with 800 µl of Lysis buffer. Following thawing and centrifugation for 1 min at 12,000 xg, 600 µL of the supernatant was used for RNA purification using Zymo-Spin TM IIIC column. After recommended washes, the column was treated with RNase-Free DNase (Zymo Research, Irvine, CA) at 37°C for 15 min. RNA was eluted in 25 µL of DNase/RNase-free water by centrifugation, quantified using NanoDrop 2000C (Thermo Scientific, Wilmington, DE), and either stored at -80°C or used immediately for cDNA preparation as follows.
For first strand cDNA synthesis, High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Grand Island, NY) was used following the manufacturer's protocol. The final reaction volume of 20 µL contained 10 µL of 2X RT Master Mix and 10 µL of RNA sample (up to 2 µg of total RNA). The reaction was set up in an Eppendorf Mastercycler® thermal cycler (Eppendorf NA, Hauppauge, NY) with the following conditions: 10 min at 25°C, 120 min at 37°C and 5 min at 95°C. The cDNA was stored at -20°C for use in q-PCR analysis. All gene expression analyses reported here were done from the same RNA preparation but two sets of cDNA preparations.
Gene expression was quantified using Fast SYBR® Green Master Mix (Applied Biosystems, Grand Island, NY) following the manufacturer's protocol. Briefly, 5.5 µL of Fast SYBR green, a final concentration of 50 nmol each of gene-specific forward and reverse primers (Suppl. Table  1), and 100 ng of cDNA were mixed in a final reaction volume of 10 µL. The reactions were run in sub-skirted natural 96-well reaction plates (MIDSCI, Valley Park, MO) on the Applied Biosystems 7500 Fast Real-Time PCR thermocycler with the following conditions: initial denaturation at 95°C for 20 sec, and 40 cycles of denaturation at 95°C for 3 sec and annealing for 30 sec at 60°C followed by a dissociation step to confirm the single specific amplicon. A standard curve was generated using 4x serial dilution from 5x dilution of an original cDNA sample mixture. The standard curve was used to determine Ct values for gene expression quantification. The relative quantification to an internal control gene: AtTIP41-At4g34270 (Czechowski et al., 2005;Han et al., 2013) was done using the (Pfaffl, 2001) method. The data represent the results of Experiment 1, which did not have technical replicates. A select group of genes were analyzed using more rigorous approach and two technical replicates per biological replicate; those data are presented in the main body of the publication. The results using the two different approaches were quite consistent in showing that overproduction of Put via induction of mODC in the transgenic plants did not affect the transcriptome associated with the Glu-Orn-Arg-Pro-DAO-PAO and GAD pathway.
Supplementary Figure 5. The results of qPCR for the effects of inducible increase in Put production on changes in the relative expression of various genes of sub-pathways (a) Glu to Orn; (b) Orn to Arg; (c) Glu to Pro; and (d) PA catabolism and GABA biosynthesis in Arabidopsis thaliana using control and mODC induced seedlings collected at 24 after induction with estradiol. The qPCR assays were conducted with three biological replicates per treatment. Data are mean ± SE of three biological replicates. For names of enzymes refer to legend of Figure 1 and Supplemental Table 1. *denotes significant difference (P ≤0.05) between mODC induced seedlings and the corresponding un-induced control plants at the same time of analysis. However, only ≥2 fold differences with their respective controls are indicated as significant for biologically important changes.  Table 1 Primers used for expression analysis (QRT-PCR) of the genes involved in the biosynthesis of Orn, Arg, Pro, and GABA along with the polyamine catabolism genes in Arabidopsis thaliana (1-designed through NCBI using primer design tool; 2-Mattioli et al., 2009;3-Takahashi et al. 2010;4-Marina et al., 2013;5-Page et al., 2007). n.b. P5CR.1 is a splice variant of P5CR.