Evidence for PII with NAGK interaction that regulates Arg synthesis in the microalga Myrmecia incisa in response to nitrogen starvation

To understand why most eukaryotic microalgae accumulate lipids during nitrogen starvation stress, a gene, MiglnB, encoding PII, a signal transduction protein, was cloned from the arachidonic acid-rich microalga Myrmecia incisa Reisigl. Similarly to its homologues, MiPII contains three conserved T-, B-, and C-loops. In the presence of abundant Mg2+, ATP, and Gln, MiPII upregulates Arg biosynthesis by interacting with the rate-limiting enzyme, MiNAGK, as evidenced by yeast two-hybrid, co-immunoprecipitation assays, and kinetics analysis of enzyme-catalyzed reactions. However, this interaction of MiPII with MiNAGK is reversed by addition of 2-oxoglutarate (2-OG). Moreover, this interaction is present in the chloroplasts of M. incisa, as illustrated cytologically by both immunoelectron microscopy and agroinfiltration of Nicotiana benthamiana leaves to determine the subcellular localization of MiPII with MiNAGK. During the process of nitrogen starvation, soluble Arg levels in M. incisa are modulated by a change in MiNAGK enzymatic activity, both of which are significantly correlated (r = 0.854). A model for the manipulation of Arg biosynthesis via MiPII in M. incisa chloroplasts in response to nitrogen starvation is proposed. The ATP and 2-OG saved from Arg biosynthesis is thus suggested to facilitate the accumulation of fatty acids and triacylglycerol in M. incisa during exposure to nitrogen starvation.


Subcellular localization of MiPII and MiNAGK
Immunoelectron microscopy was employed to examine the precise subcellular localization of MiPII in M. incisa using the purified MiPII polyclonal antibody. The freshly collected microalgal cells were subject sequentially to pre-fixation (more than 2 h), post-fixation (3 h) in 2.5% glutaraldehyde, and 1% osmium tetroxide, respectively, in 0.1 M phosphate buffer at pH 7.4 4 . After dehydration in ethanol/acetone series, the fixed samples were then placed in acetone/Spurr's resin (2/1, v/v) overnight, and then incubated in 100% Spurr's resin for 2-3 h at 37°C. The embedded samples were polymerized in a temperature gradient and then thin-sectioned using a LKB 4802 ultrotome ultramicrotome (Leica, Germany).
The ultrathin sections used for immunoelectron microscopy were collected on 200-mesh nickel grids. The grids were washed for 2 min with water and transferred onto drops of balance buffer (BB, containing 50 mM PBS, 1% bovine serum albumin, 0.1 M NaCl, and 0.02% polyethylene glycol 20000) for 30 min at room temperature. Then they were incubated with the purified anti-PII antibody (diluted 1:1000 in BB) at 4C for 48 h. After incubation, the grids were placed onto drops (repeated 3) of water for 3 min and another 30 min incubation with BB. Then the grids were incubated with the secondary antibody, anti-rabbit IgG conjugated to 10 nm gold particles (Sigma, USA) diluted 1:100 with BB at room temperature for 2 h. Following sequential washes in water and dehydration in the air, the sections were stained with 3% uranyl acetate-lead citrate, and observed in a JEOL-1230 Transmission Electron Microscope at 80 kV.
The labeling density was defined as the number of gold particles per area unit (μm 2 ) as described by Bernal 5 , and the area was estimated by Adobe Photoshop software (ver. 3.0). Following counting the gold particles on chloroplasts and the other area calculated by subtracting the total area of chloroplasts in each micrograph, the percentage of particles versus the total number of particles was calculated. The statistical analysis of subcellular distribution of MiPII protein was carried out by using the independent sample test in SPSS Statistics 17.0 program.
Agroinfiltration of tobacco leaves was used as described by Liu et al. 6 for the determination of subcellular localization of MiNAGK and MiPII. The MiargB ORF was amplified from M. incisa using the pair of primers, NAD-F and NAD-R (Table S1), which contains KpnI/XbaI digestion sites. Similarly, the MiglnB ORF was amplified using the pair of primers, PIIGFP-F and PIIGFP-R (Table S1). The PCR product was digested with KpnI and XbaI and subsequently introduced into the KpnI/XbaI-digested pCAMBIA1300-3×GFP (from Prof. Z.-N. Yang) between the CaMV35S promoter and green fluorescent protein (GFP) gene to generate the corresponding fusion vector pC-NAGK-GFP or pC-PII-GFP. This recombinant vector and an empty plasmid, pC1300-GFP, as the negative control, were separately introduced into Agrobacterium tumefaciens GV3101 by electroporation (Bio-Rad, USA) and then infiltrated into the abaxial side of leaves of 4-to 5-week-old Nicotiana benthamiana plants. After agroinfiltration, the transgenic plant was placed in the dark for 48 h. The epidermis cells of the infiltrated tobacco leaves were observed under a confocal laser scanning microscope (Carl Zeiss or Leica, Germany). GFP fluorescence was monitored with a 500-nm to 550-nm band pass emission filter excited at 488 nm, and the autofluorescence of chlorophyll was examined with the same excitation filter as GFP, but with a 650-nm to 750-nm emission filter.

Supplementary References
MiPII PIIGFP-R TGCTCTAGACTAAATGCCGGGCTCCTG The underlined letters in the primers denote the position of restriction enzyme sites. 5 Supplementary Table S2   Table S2. Levels of hydrolyzed amino acids (g/100 g dry weight) in Myrmecia incisa during the culture under nitrogen starvation. Shadowed data denote the minor one in the hydrolyzed amino acids.

Figure S1. Alignment of the deduced amino acid sequences of PII proteins from Myrmecia incisa and other organisms.
Residues highlighted in black or gray backgrounds are identical or conserved in at least 60%, respectively, of all aligned PII proteins. The putative plastid-target sequence is indicated by underlining. Boxes I and II refer to PII signature patterns I and II. The position of the ATPase is indicated by black rhombus. ATP-, NAGK-, 2-OG-binding residues are denoted by black dot, asterisk and empty triangle, respectively.    Residues highlighted in black or gray backgrounds are identical or conserved in at least 60%, respectively, of all aligned BCCP proteins. The putative plastid-target sequence is indicated by underlining. Box denotes the conservative domain. The putative binding sites with biotin carboxylase subunit are denoted by asterisks. Figure S6. Electrophoresis patterns of the prey plasmids pGADT7-argB, pGADT7-accB1 and pGADT7-accB2 constructed for yeast two-hybrid analysis. Lanes 1, 4 and 7, amplified products of MiargB, MiaccB1 and MiaccB2, respectively, ORFs minus signal peptide sequences; Lanes 2, 5 and 8, recombinant plasmids pGADT7-argB, pGADT7-accB1 and pGADT7-accB2, respectively; Lanes 3, 6 and 9, double digested products of plasmids pGADT7-argB, pGADT7-accB1 and pGADT7-accB2, respectively, with EcoRI and XhoI; Lane M, DL 2000 DNA standard marker (Tiangen Biotech Co., Ltd.); and Lane M1, λDNA HindIII DNA marker (Tiangen Biotech Co., Ltd.).

Figure S7. Electrophoresis patterns of the plasmid construction of pET-glnB, heterologous expression profile of MiPII in Escherichia coli (upper panel), purification of the recombinant MiPII, and specificity detection of its preparative polyclonal antibody by Western blot analysis in Myrmecia incisa (lower panel).
Lane 1, amplified product of MiglnB ORF minus signal peptide sequence; Lane 2, recombinant plasmid pET-glnB; Lane 3, double digested products of plasmid pET-glnB with EcoRI and PstI; Lane 4, plasmid pET-28a; Lane 5, double digested products of plasmid pET-28a; Lane 6, expression of recombinant protein MiPII without induction by addition of IPTG; Lane 7, MiPII expression induced by IPTG for 3 h; Lane 8, recombinant MiPII expressed in the supernatant of the transformant after sonication; Lane 9, recombinant MiPII expressed in the precipitate after sonication; Lane 10, Western blot analysis of recombinant MiPII with His-Tag antibody; Lane 11, total proteins isolated from M. incisa; Lanes 12 and 13, Western blot analysis of total proteins (approximately 25 µL and 10 µL, respectively) isolated from M. incisa with the purified MiPII polyclonal antibody; Lane M, DL 2000 DNA standard marker (Tiangen Biotech Co., Ltd.); Lane M1, DNA marker λDNA HindIII (Tiangen Biotech Co., Ltd.); Lane M2, prestained Protein ladder of standard protein (Fermentas); and Lane M3, ladder of standard protein (Fermentas).