Reticulate leaves and stunted roots are independent phenotypes pointing at opposite roles of the phosphoenolpyruvate/phosphate translocator defective in cue1 in the plastids of both organs

Abstract

Phosphoenolpyruvate (PEP) serves not only as a high energy carbon compound in glycolysis, but it acts also as precursor for plastidial anabolic sequences like the shikimate pathway, which produces aromatic amino acids (AAA) and subsequently secondary plant products. After conversion to pyruvate, PEP can also enter de novo fatty acid biosynthesis, the synthesis of branched-chain amino acids, and the non-mevalonate way of isoprenoid production. As PEP cannot be generated by glycolysis in chloroplasts and a variety of non-green plastids, it has to be imported from the cytosol by a phosphate translocator (PT) specific for PEP (PPT). A loss of function of PPT1 in Arabidopsis thaliana results in the chlorophyll a/b binding protein underexpressed1 (cue1) mutant, which is characterized by reticulate leaves and stunted roots. Here we dissect the shoot- and root phenotypes, and also address the question whether or not long distance signaling by metabolites is involved in the perturbed mesophyll development of cue1. Reverse grafting experiments showed that the shoot- and root phenotypes develop independently from each other, ruling out long distance metabolite signaling. The leaf phenotype could be transiently modified even in mature leaves, e.g. by an inducible PPT1RNAi approach or by feeding AAA, the cytokinin trans-zeatin (tZ), or the putative signaling molecule dehydrodiconiferyl alcohol glucoside (DCG). Hormones, such as auxins, abscisic acid, gibberellic acid, ethylene, methyl jasmonate, and salicylic acid did not rescue the cue1 leaf phenotype. The low cell density1 (lcd1) mutant shares the reticulate leaf-, but not the stunted root phenotype with cue1. It could neither be rescued by AAA nor by tZ. In contrast, tZ and AAA further inhibited root growth both in cue1 and wild-type plants. Based on our results, we propose a model that PPT1 acts as a net importer of PEP into chloroplast, but as an overflow valve and hence exporter in root plastids.

Keywords: phosphate translocator; phosphoenolpyruvate; plastids; reticulate mutants; secondary metabolism.

Figure 1

Shoot and root phenotypes of different cue1 alleles and lcd1-1 compared to control plants. Rosettes and leaves of 30 days old pOCA108 (Bensheim) (A), Col-0 (B), lcd1-1 (C), cue1-1 (D), cue1-1/lcd1-1 double mutants (E), and cue1-6 (F), as well as root phenotypes of 21 days old pOCA and cue1-1 (G), Col-0 and cue1-6 (H), or 35 days old Col-0 and cue1-6 (I), and Col-0 and lcd-1 (J). The bars indicate a length of 1 cm referring either to the plants in the pots (A–F) or on agar (G–J).

Figure 2

Development of the cue1-1 shoot and root phenotypes does not require communication between roots and shoots. Different scion|root stock combinations of cue1-1 and pOCA (Bensheim) are shown from two independent grafting experiments. (A) cue1-1|cue1-1(mutant control), (B) cue1-1|pOCA (C), pOCA|pOCA (wild type control), (D) pOCA|cue1-. Pictures were taken 35 days after grafting. The small additional pictures show details of the graft union and/or the leaf phenotype. (E) cue1-1 non-grafted (F), cue1-1|pOCA grafts compared to cue1-1|cue1-1 grafts (G) pOCA non-grafted, (H) pOCA|cue1-1 grafts compared to cue1-1|cue1-1 grafts. The pictures were taken 18 days after grafting. The bars indicate a length of 1 cm.

Figure 3

Rescue of the reticulate leaf phenotype of cue1. The alleles of cue1-1 (A) and cue1-6 (B) were fed with a cocktail of AAA. Four week old plants were grown on ½MS agar and then transferred to ½MS agar in the presence or absence (control) of AAA (2 mM each of Phe, Tyr, and Trp). The bar indicates a length of 1 cm.

Figure 4

Repression of PPT1 expression by a constitutive and inducible RNAi approach. Constructs are shown for a constitutive PPT1RNAi expression driven by the CaMV 35S promoter (p35S) (A) or for the EtOH inducible alcR/alcA system (B). Phenotypic appearance of Col-0 wild type (C), and two lines expressing the PPT1RNAi construct constitutively [(D) Col-0/ RNAi(1):alcA:PPT1; (E) Col-0/RNAi(2):alcA:PPT1]. (F) Time course of PPT1 expression (RT-PCR) after induction of alcA:PPT1RNAi plants with EtOH compared to a wild-type control. The arrows indicate the addition or withdrawal of 0.015 (v/v)% EtOH. Phenotypic appearance of Col-0 (G,I) and Col-0/alcA:PPT1RNAi (H,J) 168 h after induction with EtOH (G,H) or 96 h after withdrawal of EtOH (I,J). The bar in (C–E) indicates a length of 1 cm.

Figure 5

Feeding of cue1-1 with DCG (A) or tZ (B) compared to an unfed control. Plants were grown on ½MS agar for four weeks and were then transferred to a medium containing either 20 μM DCG or 1 μM tZ. DCG rescued the reticulate leaf phenotype only if the roots were excised (-roots) and the rosettes were placed directly with their cut edges on the agar plates, but not intact plants (+roots). The bar indicates a length of 1 cm.

Figure 6

Effect of AAA and tZ on mesophyll cell density in cue1 and lcd1 leaves. Plants were grown for six days on ½MS-agar supplemented with 2 mM each of AAA (B) or 1 μM tZ (C) compared to an unfed control (A). Examples of mesophyll cell density numbers (cells per 0.05 mm²) counted in individual leaves of cue1-6 grown on 1 μM tZ (E) compared to an unfed control of cue1-6 (D). Mesophyll cell density of Col-0, cue1-6 and lcd1-1 six days after feeding a cocktail of AAA compared to an unfed control (F). Each microscopic picture represents an area of 0.05 mm² with a bar indicating a length of 100 μm. Time course of relative mesophyll cell numbers in leaves of Col-0, cue1-6, and lcd1-1 after feeding of AAA (G) or tZ (H). The blue or black symbols represent relative mesophyll cell numbers in the presence or absence of the effectors (AAA or tZ), and the red line represents the ratio of mesophyll cell numbers ± effector. The initial cell density was about 100, 60, or 86 cells per 0.05 mm² in leaves of Col-0, cue1-6, or lcd1-1, respectively. The bar in (C) represents a length of 1 cm. The data in (G) and (H) represent the mean ± SE of n = 10 measurements per time point and condition.

Figure 7

Root phenotypes seven days after feeding of Col-0, pOCA, cue1-1, cue1-6, and lcd1-1 with individual AAA or a cocktail of AAA compared to unfed control plants. The plants were grown for three weeks on ½MS agar and were then transferred to ½MS agar supplemented with the effectors (2 mM each). (A) Phenotypic appearance including root lengths of wild-type/control and mutant plants seven days after start of feeding with AAA (the bar indicates a distance of 1 cm). Relative root lengths of pOCA (B), cue1-1 (C), cue1-3 (D), Col-0 (E), cue1-6 (F), and lcd1-1 (G). The black bars indicate the relative root lengths before transfer of the plants to AAA containing medium, whereas the stacked white or colored bars refer to the increment growth within seven days. Compared to the control treatment of wild-type and control plants all other relative root lengths were significantly different with P-values of at least < 0.01.

Figure 8

Root phenotypes seven days after feeding of Col-0, pOCA, cue1-1, cue1-6 and lcd1-1 with tZ. The plants were grown for three weeks on ½MS agar and were then transferred to ½MS agar supplemented with the effector (1 μM). (A) Phenotypic appearance including root lengths of wild-type/control and mutant plants seven days after start of feeding with AAA (the bar indicates a distance of 1 cm). (B) Relative root lengths of pOCA, cue1-1, cue1-3, Col-0, cue1-6, and lcd1-1. in the absence or presence of tZ. The black bars indicate the relative root lengths before transfer of the plants to AAA containing medium, whereas the stacked white or colored bars refer to the increment growth within seven days. Compared to the control treatments all other relative root lengths were significantly different with P-values of at least <0.01. The data represent the mean ± SE of the mean of n = 18 measurements.

Figure 9

Effect of phytohormone feeding on root growth of cue1 and lcd1 compared to wild-type or control plants. Individual phytohormones (10 μM each) were fed for seven days to Col-0 (A), cue1-6 (B), lcd1-1 (C), pOCA (D), and cue1-1 (E). The relative root lengths are shown compared to the unfed controls (white bars for Col-0 or light gray bars for the other lines). The circles indicate significant differences in root lengths of fed compared to unfed plants with P-values <0.01 (closed circles) and <0.05 (open circles). The data represent the mean ± SE of the mean of n = 9 measurements.

Figure 10

Effect of tZ feeding on the root and leaf phenotypes of cue1 and ppdk1-1 single mutants as well as cue1-1/ppdk double mutants. Plants were grown for three weeks vertically on ½MS agar plates or for four weeks on soil (C,D). Root and shoot phenotypes of Col-0 and ppdk-1 (A) as well as cue1-1 and cue1-1/ppdk-1 double mutants (B). Col-0, cue1-6, pOCA, cue1-1, and the cue1-1/ppdk-1 double mutant fed with 1 μM tZ were compared to an unfed control and the leaf (E) and root (F) phenotypes were analyzed. The data on relative root lengths in (G) represent the mean ± SE of n = 10–24 replicates. The analyses of root lengths also contain BoPPT and FtPPDK overexpressing wild-type and cue1-6 mutant plants. Blue circles indicate significant differences in root length referred to Col-0 (P < 0.01), whereas red circles reflect significant changes in root lengths of BoPPT overexpressors in the cue1-6 background compared to the untransformed mutant or of cue1-1/ppdk1-1 double mutant compared to the cue1-1 single mutant. Closed or open red circles indicate P < 0.01 or P < 0.05, respectively. The scale bars in the individual sub-figures indicate a distance of 1 cm.

Figure 11

Root lengths of cue1 mutants compared to the wild type/control and CKX overexpressing lines. Root (A) and leaf phenotypes (B) of Col-0, cue1-6 and CKX1 and CKX3 overexpressing lines as well as root (C) and leaf phenotypes (D) of stable crosses of cue1-6 with CKX3 (C) or CKX3 with cue1-1 (D) were assessed three weeks after germination. The scale bars indicate a length of 1 cm. The relative root length in (E) is expressed as mean ± SE of n = 10–24 replicates. Blue circles indicate significant difference referred to the wild-type or control plants, whereas the red circle refers to a significant difference compared to cue1-6 (P-value < 0.01).

Figure 12

Heatmap of amino acid spectra in wild-type and mutant plants. The contents of proteogenic amino acids were determined in leaves of wild-type (Col-0) or control plants (pOCA) as well as cue1-1, cue1-6 and lcd1-1 mutants grown either on soil (A,D) or on ½MS agar (B,E). The amino acid spectra of roots were determined from agar grown plants (C,F). The data are expressed as log2 ratios of absolute (A–C) or relative (Mol%) amino acid contents shown in Supplemental Document 1, which also contains a statistical analysis. Amino acid spectra in leaves of wild-type and mutant plants were determined after the plants were fed for seven days either with a cocktail of AAA (H,J) or tZ (I,K) compared to an unfed control (G). For both feeding experiments amino acid ratios were either compared to the wild type (H,J) or the unfed control plant (I,K). The maximum (red) and minimum (green) values as well as intermediate values of the color scale for (A–H) and (G–K) is shown in the figure. Black indicates a value of 0 and in the case of gray shadings no data were available.

Figure 13

Anabolic reaction sequences in chloroplasts (A,B) and root plastids (C,D) of wild-type (A,C) and cue1 mutant plants (B,D). In chloroplasts (A,B) CO₂ is assimilated in the reductive pentose phosphate pathway (RPPP; Calvin-Benson cycle) via ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO;1). Its product, 3-PGA, is then converted to triose phosphates (TP) via the subsequent action of phosphoglycerate kinase and NADP glyceraldehydes 3-phosphate dehydrogenase (PGK/NADP-GAPDH; 2). TPs are exported via the TPT in counter exchange with inorganic phosphate (P_i) and subjected to Suc biosynthesis in the cytosol. Suc is the main transport sugar that is exported to the sinks via the phloem. In chloroplast glycolysis, 3-PGA can only be metabolized to 2-PGA via phosphoglycerate mutase (PGyM; 3), but not further to PEP. Under defined conditions, it can also enter the phospho-serine pathway of Ser biosynthesis. In leaves Ser is, however, mainly produced by photorespiration (PR) or is imported from non-green tissues, such as roots. TPs in the cytosol can undergo the glycolytic conversion to 3-PGA, involving PGK/NAD-GAPDH (5), and further to 2-PGA and PEP by the subsequent action of cytosolic PGyM (6) and enolase (ENO2; 7). PEP has to be imported to the stroma via PPT1 or PPT2 in counter exchange with either 2-PGA or P_i. The latter derive, for instance, from the shikimate pathway, where PEP and erythrose 4-phosphate (E-4-P) serve as precursors. End products of the shikimate pathway are the aromatic amino acids (AAA) Phe and Tyr, which are synthesized from the intermediate chorismate, and Trp, which is synthesized from anthranilate as well as phosphoribosyl pyrophosphate (PRPP) and Ser. AAA are exported via amino acid transporters (AAT). PEP can be further metabolized by plastidial (4) or cytosolic (8) pyruvate kinase (PK) yielding pyruvate. Outside the chloroplasts pyruvate is subjected to mitochondrial respiration. In the chloroplast stroma pyruvate can enter de novo fatty acid biosynthesis, the production of the branched-chain amino acids Leu and Val as well as the MEP pathway of isoprenoid biosynthesis. The third branched-chain amino acid Ile uses Thr as a precursor. In contrast to wild-type chloroplasts (A), chloroplasts from cue1 (B) suffer from a limitation in PEP provision and processes like the production of AAA via the shikimate pathway are impaired (green background) and thus rely on PEP supply by PPT2. Anabolic sequences with pyruvate as precursor would probably be less affected (light blue-green background) as pyruvate might also be supplied by pyruvate transporters (PyrT). In roots (C,D) reducing power and metabolic intermediates are provided by the oxidative pentose phosphate pathway (OPPP) with Glc6P as precursor and TP as end products. Cytosolic Glc6P deriving from the degradation of imported sucrose is provided to the plastid by the GPT in counter exchange with either TP or P_i. Glc6P can be converted to Fru6P by plastidial or cytosolic phosphoglucose isomerise (PGI; 9, 14), activated to Fru1,6P₂ by phosphofructokinase (PFK; 10, 15), and cleaved by aldolase (11, 16) to TP, which are subsequently converted to 3-PGA, 2-PGA, and PEP by plastidial or cytosolic PGK/NAD-GAPDH (5, 12), PGyM (3, 6), and ENO (7, 13). Note that in root plastids ENO1 is present. The fate of PEP in plastidial and cytosolic metabolism of roots is similar as in leaves. However, a blocked PEP transport across the envelope of root plastids due to a knockout of PPT1 cannot be compensated by PPT2 (D), and would lead to an increased production rather than a depletion of products deriving from PEP and pyruvate (light red background). It is likely that processes like the OPPP or the phospho-serine pathway are also increased by feedback regulatory mechanisms.