Figure 1.
Suggested models of starch biosynthesis in leaves.
(A) The classic model of starch biosynthesis according to which (a) the starch biosynthetic process takes place exclusively in the chloroplast, segregated from the sucrose biosynthetic process taking place in the cytosol, and (b) AGP exclusively catalyzes the synthesis of ADPG. (B) Suggested “additional/alternative” model of starch biosynthesis wherein (a) ADPG is produced in the cytosol by enzyme(s) such as SuSy and then is transported to the chloroplast by the action of an ADPG translocator, and (b) pPGM and AGP play an important role in the scavenging of glucose units derived from starch breakdown. Starch to glucose conversion would involve the coordinated actions of amylases, isoamylase and disproportionating enzyme [21]–[23]. According to this interpretation of transitory starch biosynthesis starch accumulation in leaves is the result of the balance between de novo starch synthesis from ADPG entering the chloroplast and breakdown, and the efficiency by which starch breakdown products are recycled back to starch by means of pPGM and AGP. Thus, this view predicts that the recovery towards starch biosynthesis of the glucose units derived from the starch breakdown will be deficient in pPGM and AGP mutants, resulting in a parallel decline of starch accumulation and enhancement of soluble sugars content since starch breakdown derived products (especially glucose) will leak out the chloroplast through the very active glucose translocator [24]. The enzyme activities involved are numbered as follows: 1, 1′, fructose-1,6-bisphosphate aldolase; 2, 2′, fructose 1,6-bisphosphatase; 3, PPi:fructose-6-phosphate phosphotransferase; 4, 4′, PGI; 5, 5′, PGM; 6, UDPG pyrophosphorylase; 7, sucrose phosphate synthase; 8, sucrose-phosphate-phosphatase; 9, AGP; 10, SS; 11, starch phosphorylase; 12, SuSy; 13, plastidial hexokinase [25], [26]. FBP: fructose bis-phosphate; UDPG: UDP-glucose.
Figure 2.
aps1 and pgm leaves accumulate WT ADPG content.
(A) HPLC-MS/MS detection of ADPG in WT, aps1 and pgm leaves. Upper panels: Total ion chromatograms (TIC) of extracts from the indicated plants in which the selected fragmentation parent ion was 587.8 m/z. Middle panels: Extracted ion chromatograms (EIC) in which the selected ion for fragmentation of the parent ion was 346.1 m/z. Lower panels: Mass spectra (MS2) obtained from fragmentation of parent ion. ADPG was measured using an Agilent 1100 HPLC fitted with a Xbridge C18 column (100×3.0 mm I.D. particle size 3.5 µm) coupled to a MSD-Trap spectrometer (Agilent) (see Materials and Methods for further details). (B) ADPG content in WT, aps1, pgm and aps1/pgm leaves. Plants were simultaneously grown either in soil or solid MS. Leaves from 4-weeks old WT, aps1, pgm and aps1/pgm plants were simultaneously harvested after 10 h of illumination. ADPG was simultaneouly extracted from leaves of WT, aps1, pgm and aps1/pgm plants and content was simultaneously measured by HPLC-MS/MS as described in Materials and Methods. Note that, consistent with [15], leaves of aps1, pgm and aps1/pgm plants accumulated WT ADPG content. Values represent the mean ±SD of determinations on three independent samples.
Figure 3.
Production of WT and ss3/ss4 plants expressing AtADPGP or EcASPP in the plastid.
(A) Western blot of AtADPGP in leaves of WT and ss3/ss4 plants, and leaves of two independent lines each of TP-P541-AtADPGP expressing WT plants and TP-P541-AtADPGP expressing ss3/ss4 plants. (B) ADPGP activity in leaves of WT and ss3/ss4 plants, and leaves of TP-P541-AtADPGP expressing WT plants and TP-P541-AtADPGP expressing ss3/ss4 plants. (C) Western blot of EcASPP in ss3/ss4 leaves, and leaves of two independent lines of TP-P541-EcASPP expressing ss3/ss4 plants. (D) ASPP activity in ss3/ss4 leaves and leaves of two independent TP-P541-EcASPP expressing ss3/ss4 plants. In “A” and “C”, the gels were loaded with 20 µg per lane of protein and AtADPGP and EcASPP were immunodecorated by using antisera specifically raised against AtADPGP and EcASPP.
Figure 4.
Ectopic expression of EcASPP and AtADPGP in the plastid restores the WT growth and partially reverts the ADPG excess phenotype of ss3/ss4 plants.
(A) Time-course of fresh weight of rosettes of WT (▪) and ss3/ss4 (⧫) plants, and rosettes of one representative line each of TP-P541-AtADPGP expressing ss3/ss4 plants and TP-P541-EcASPP expressing ss3/ss4 plants (• and ▴, respectively). Plants were grown under long-day conditions (16 h light/8 h dark, 20°C) and at an irradiance of 90 µmol photons sec−1 m−2. Values represent the mean ±SD of determinations on five independent plants. (B) ADPG content in WT, aps1 and ss3/ss4 leaves, and leaves of plants of two independent TP-P541-EcASPP- and TP-P541-AtADPGP- expressing ss3/ss4 lines. Leaves were harvested after 10 h of illumination. Values represent the mean ±SD of determinations on three independent samples.
Figure 5.
The contribution of the chloroplastic ADPG pool to the total ADPG pool is low in WT and aps1 leaves.
(A) Starch and (B) ADPG contents in leaves of WT and aps1 plants, and leaves of two independent lines each of TP-P541-AtADPGP expressing WT plants, TP-P541-EcASPP expressing WT plants, TP-P541-glgC expressing aps1 plants, and TP-P541-EcASPP expressing aps1 plants. Plants were simultaneously grown and leavesf from 4-weeks old plants were simultaneously harvested after 10 h of illumination. ADPG was simultaneouly extracted and measured by HPLC-MS/MS as described in Materials and Methods. Note that, consistent with [15], leaves of TP-P541-EcASPP expressing WT plants, TP-P541-glgC expressing aps1 plants, and TP-P541-EcASPP expressing aps1 plants accumulated WT ADPG content. Values represent the mean ±SD of determinations on three independent samples.Values represent the mean ±SD of determinations on three independent samples.