Pyrimidine Salvage: Physiological Functions and Interaction with Chloroplast Biogenesis

Pyrimidine salvage is mainly driven by uridine/cytidine kinase in the cytosol, and plastidic uracil phorsphoribosyltransferase is needed to establish photosynthesis via its moonlighting activity

Abstract

The synthesis of pyrimidine nucleotides, an essential process in every organism, is accomplished by de novo synthesis or by salvaging pyrimdines from e.g. nucleic acid turnover. Here, we identify two Arabidopsis (Arabidopsis thaliana) uridine/cytidine kinases, UCK1 and UCK2, which are located in the cytosol and are responsible for the majority of pyrimidine salvage activity in vivo. In addition, the chloroplast has an active uracil salvage pathway. Uracil phosphoribosyltransferase (UPP) catalyzes the initial step in this pathway and is required for the establishment of photosynthesis, as revealed by analysis of upp mutants. The upp knockout mutants are unable to grow photoautotrophically, and knockdown mutants exhibit a variegated phenotype, with leaves that have chlorotic pale areas. Moreover, the upp mutants did not show altered expression of chloroplast-encoded genes, but transcript accumulation of the LIGHT HARVESTING COMPLEX B nuclear genes LHCB1.2 and LHCB2.3 was markedly reduced. An active UPP homolog from Escherichia coli failed to complement the upp mutant phenotype when targeted to the chloroplast, suggesting that the catalytic function of UPP is not the important factor for the chloroplast phenotype. Indeed, the expression of catalytically inactive Arabidopsis UPP, generated by introduction of point mutations, did complement the upp chloroplast phenotype. These results suggest that UPP has a vital function in chloroplast biogenesis unrelated to its catalytic activity and driven by a moonlighting function.

Nucleotides are essential molecules in every cell and possess a multitude of functions in all living organisms. Thus, for growth and development, a well-balanced homeostasis among de novo synthesis, salvage, and catabolism is of major importance (Moffatt and Ashihara, 2002; Jung et al., 2009). Pyrimidine nucleotides function in energy metabolism and are the building blocks for nucleic acids; moreover, nucleotide-conjugated sugars act in Suc and cellulose biosynthesis and are involved in glycosylation and lipid metabolism.

Alterations of the pathways for nucleotide metabolism affect many cellular processes. For example, partial inhibition of de novo pyrimidine synthesis in potato (Solanum tuberosum) leads to stimulation of the salvage pathway, improving the synthesis of starch and cell wall components, highlighting the importance of salvage reactions and the interplay with de novo synthesis in plants (Geigenberger et al., 2005). Red blood cells and human parasitic protists like Trypanosoma brucei lack de novo purine synthesis, but salvage pathway activity fully compensates for this lack (Hammond and Gutteridge, 1984; de Koning et al., 2005). For most other organisms, including plants, the relative contributions of one or the other pathway in a given tissue or cell are unknown. Although salvage pathway reactions have been of interest in recent studies, many aspects remain still unknown, and several observations are even conflicting, as pointed out below.

The initial reaction in de novo pyrimidine synthesis is the formation of carbamoyl phosphate by carbamoyl phosphate synthase followed by five reactions that complete the synthesis of uridine monophosphate (UMP). In Arabidopsis (Arabidopsis thaliana), the two initial steps occur in plastids, but the subsequent reactions occur in the cytosol and mitochondria, as shown by analysis of GFP-fusion proteins (Witz et al., 2012). UMP, the first pyrimidine nucleotide resulting from pyrimidine de novo synthesis, appears in the cytosol. How pyrimidines are delivered to the plastid for RNA and DNA synthesis is not clear so far. However, pyrimidine salvage is of the utmost importance for plastid metabolism (Mainguet et al., 2009; Chen and Thelen, 2011), suggesting that precursors in the form of uracil or uridine are imported into plastids.

Present knowledge shows that uracil import is mediated by the PLASTIDIC NUCLEOBASE TRANSPORTER (PLUTO), identified as the sole nucleobase cation symporter1 in Arabidopsis (Witz et al., 2012, 2014). An active uracil phosphoribosyl transferase named UPP (At3g53900) was identified in plastids (Mainguet et al., 2009). In addition, two uridine-kinase-like proteins (UKL1 and UKL2) are localized in plastids (Chen and Thelen, 2011). As UKL1 and UKL2 have cytidine kinase activity in addition to uridine kinase activity, we renamed the corresponding protein family to UCK1 to UCK5 (URIDINE-CYTIDINE KINASE). All five UCK genes present in Arabidopsis consist of two domains, one coding for uracil phosphoribosyltransferase (UPRT), the other for uridine kinase (Islam et al., 2007; Mainguet et al., 2009; Chen and Thelen, 2011). One report found that UCK1 exhibited both UPRT and uridine kinase (UK) activity (Islam et al., 2007); by contrast, another report identified UPP as the sole active UPRT in Arabidopsis, based on activity measurements in corresponding knockout mutants (Mainguet et al., 2009). In addition, recombinant UCK1 was shown to have UK function, but not UPRT function, and UCK2 was shown to exclusively have UK function (Chen and Thelen, 2011).

Mutants in UPP and double mutants of UCK1 and UCK2 exhibit severe growth retardation. It is somewhat astonishing that the two salvage reactions catalyzed by these proteins, both leading to the synthesis of UMP in the same compartment, have such a high impact on development and are not complementary. By contrast, mutants of PLUTO, the only known plastidic uracil transporter, show very little phenotypic alterations (Mourad et al., 2012). To clarify our understanding of this important aspect of plant metabolism and reevaluate the functions of UPP, UCK1, and UCK2, we characterized two other putative bifunctional UPRT/UK genes, UCK3 and UCK4, in Arabidopsis. We confirmed the chloroplast localization and biochemical function of UPP, but according to our results, UCK1 to UCK4 are localized to the cytosol. UCK1 and UCK2 exhibited the highest UK and CK activities, but UCK3 and UCK4 showed low activities and might have other roles in metabolism.

Further studies provided evidence for a predominant function of uridine recycling, whereas uracil salvage seems to be of minor relevance for balancing pyrimidine nucleotide pools. Therefore, we next asked why the knockout of the UPP causes such drastic growth defects in Arabidopsis. Previous observations, showing reduced chloroplast size and less starch, already pointed to a role of UPP in chloroplast development or function (Mainguet et al., 2009). Here, by expression studies of photosynthesis-associated nuclear genes combined with complementation analyses of Arabidopsis lacking UPP, we obtained evidence for a moonlighting function of UPP in the establishment of photosynthesis.

RESULTS

The Main Pyrimidine Salvage Occurs via Uridine/Cytidine

Pyrimidine salvage in Arabidopsis and other plant and nonplant species can take two routes, either via nucleoside kinases or via phosphoribosyltransferases. To identify the main salvage route, pyrimidine de novo synthesis in Arabidopsis seedlings was blocked by N-(Phosphonacetyl)-l-Asp (PALA), a substrate transition state inhibitor of Asp carbamoyltransferase (Chen and Slocum, 2008). By adding the salvage pathway substrates uracil or uridine to PALA-treated seedlings, their potential to rescue these was tested. Addition of 1 mm PALA resulted in a reduction of plant biomass accumulation of 63% (Fig. 1A; Supplemental Fig. S1A). When 1 mm uracil was added, no recovery was observed (fresh weight loss was still 58%). However, when the same amount of uridine (1 mm) was supplied, seedlings recovered to 100% of the control fresh weight (Fig. 1A; Supplemental Fig. S1A). Control experiments, where uracil or uridine were added to otherwise untreated seedlings, showed no effect. In a second approach, we supplied seedlings with the toxic uracil analog 5-fluorouracil (5-FU) or with the uridine analog 5-fluorouridine (5-FD), each at 50 µm concentration. Whereas 5-FU treatment did not affect seedling growth, administration of the same amount of 5-FD inhibited growth beyond the state of germination completely (Fig. 1B; Supplemental Fig. S1B). It has to be mentioned that uracil is efficiently taken up by Arabidopsis seedlings, even exceeding corresponding uptake rates for uridine (Zrenner et al., 2009; Cornelius et al., 2011). Furthermore, sensitivity to 5-FU massively increased when the uracil catabolic enzyme PYD1 was knocked out, indicating that uptake is not a limiting factor for 5-FU incorporation (Cornelius et al., 2011). Nevertheless, uridine appears to be salvaged much more efficiently compared to uracil. Recently, it was proposed that cytidine may become salvaged by bifunctional UCK enzymes (Chen et al., 2016). Therefore, we quantified CK activity in plant extracts together with UK and UPRT activities. The highest UK activity (773 nmol mg⁻¹ Chl; Supplemental Fig. S1C) was measured after 30 min incubation, followed by CK (293 nmol mg⁻¹ Chl; Supplemental Fig. S1C) and UPRT (229 nmol mg⁻¹ Chl; Supplemental Fig. S1C).

Figure 1.

Rescue of PALA-treated plants and properties of UCK1 to UCK4. A and B, Fresh weight of (A) Arabidopsis seedlings treated with PALA (1 mM) supplemented with uracil or uridine and (B) 5-FU- or 5-FD-treated plants. Seedlings were grown on one-half strength Murashige and Skoog (MS) agar plates for 10 d. Data represent the mean from three different experiments on three agar plates with 10 plants each ± se. Different letters indicate significant differences at P < 0.05. C, The root length of uck1-4 was determined on one-half strength MS agar plates without Suc under long-day conditions. Data represent the mean of at least 50 plants per line grown ± se. The asterisks indicate significant differences (***P < 0.001). D, UK and CK activity were determined using radiolabeled substrates in 4-week-old uck1 to uck4 knockout mutants. Data show means and se of three independent experiments. The three asterisks indicate significant differences at P < 0.001. E, N. benthamiana protoplasts were isolated after infection with A. tumefaciens transformed with UCK-GFP constructs. Left, GFP fluorescence of fusion constructs; middle, autofluorescence of chloroplasts; right, merge of GFP and chlorophyll fluorescence; top, control with GFP only. Bars indicate 10 µm. Image below, GFP-positive leaves were analyzed via western blot and GFP-specific antibody to check for the correct size of the GFP-UCK fusion proteins.

UCK1 and UCK2 Represent the Main Pyrimidine Salvage Enzymes and Are Located in the Cytosol

In a next step, we aimed to clarify which proteins are responsible for the observed UK and CK activities. UKs are highly conserved across different kingdoms of life. In Arabidopsis, five genes encode proteins with a conserved UK domain and were renamed UCK1 to UCK5 (previously named UKL1 to UKL5 for URIDINE KINASE LIKE; Islam et al., 2007; Mainguet et al., 2009). UCK1 and UCK2 have been investigated before, but little was known about UCK3 and UCK4 so far. UCK5 exhibits very low expression in photoautotrophic tissues or roots and highest expression in pollen (Genevestigator; Hruz et al., 2008). As this expression pattern differs markedly from UCK1 to UCK4 expression, further work on UCK5 was postponed. UK activity was described for UCK1 and UCK2 as determined using purified, recombinant proteins.

We have repeated and extended this analysis for all four ubiquitously expressed UCK isoforms. All four genes could be expressed heterologously in Escherichia coli, and the recombinant proteins were purified exploiting a 10x-His-Tag. Amount and purity of the proteins were sufficient for a biochemical analysis (Table 1; Supplemental Fig. S2A). UCK1 and UCK2 show highest activities for uridine in a time course experiment, whereas UCK3 and UCK4 activities were barely detectable (Supplemental Fig. S2A). Similar observations were made for CK activity: again UCK1 and UCK2 exhibited much higher (∼10-fold) activities compared to UCK3 and UCK4 (Supplemental Fig. S2B). K_M values for cytidine were 0.33 mM, 0.16 mM, and 3.0 mm for UCK1, UCK2, and UCK3, respectively (Table 1; Supplemental Fig. S2, C–E). UCK4 showed a linear cytidine concentration response without saturation up to 6 mm (Supplemental Fig. S2F). These findings indicate that UCK1 and UCK2 are the main salvage enzymes for uridine and cytidine in Arabidopsis. No UPRT activity was measured for any of the recombinant UCK proteins, although they have a UPRT domain. A previous study proposed that this domain might exert regulatory functions (Mainguet et al., 2009).

Table 1. Biochemical properties of recombinant UCK and UPP.

n.d., not determined, activities were too low for a determination of enzyme properties.

Enzyme	AGI/UniProtKB	Substrate	KM [mM]	vmax [µmol mg−1 protein h−1]
UCK1	At5g40870/Q9FKS0-1	Cytidine	0.33	4.66
UCK2	At3g27190/Q9LK34-1	Cytidine	0.16	2.01
UCK3	At1g55810/Q8VYB2-1	Cytidine	3.00	5.15
UCK4	At4g26510/065583	Cytidine	n.d.	n.d.
UCK1		Uridine	0.34*1	14.8a
UCK2		Uridine	1.18*1	243.9a
UCK3		Uridine	n.d.	n.d.
UCK4		Uridine	n.d.	n.d.
			K0.5 [µm]
UPP	At3g53900/Q9M336-1	Uracil	43.78	13.93

^aChen and Thelen, 2011.

To obtain information on the in vivo function of UCK proteins, individual UCK1 to UCK4 T-DNA insertion mutants (uck1 to uck4) were analyzed. The uck1, uck2, and uck3 mutants have been described previously (Mainguet et al., 2009; Chen and Thelen, 2011), and the UCK4 T-DNA insertion lines were analyzed in the course of this work (Supplemental Fig. S3, A–C; Details are given in the “Materials and Methods” section). When seedlings were grown in the presence of rising concentrations of 5-FD (1 to 50 µM) no differences in seedling fresh weight became apparent (Supplemental Fig. S3D). Root growth tests on vertically oriented agar plates are a sensitive way to monitor plant growth performance. In corresponding analysis, uck1, uck2, and uck3 had 50%–74% shorter roots compared to wild-type controls (Fig. 1C). When UK and CK activities were measured in crude leaf extracts from the same mutants, a 10-fold reduction activity of both reactions was observed for uck1, whereas all other lines showed unchanged activities compared to the control (Fig. 1D).

Next, we investigated the subcellular localization of UCK1 to UCK4. For this, UCK1 to UCK4 GFP-fusion constructs were heterologously expressed in Nicotiana benthamiana protoplasts. In this analysis, UCK1 to UCK4 showed a similar GFP pattern as the GFP-only control (Fig. 1E) indicating all four proteins are present in the cytosol. Activity measurements together with the analysis of the subcellular localization led us to the conclusion that the main salvage occurs in the cytosol of the cell via UCK1 and UCK2.

UPP Is the Sole UPRT in Arabidopsis with a Proposed Moonlighting Function Vital for the Establishment of Photosynthesis Unrelated to Its Catalytic Activity

Although the main pyrimidine salvage occurs via UCKs, there is UPRT activity in plant cells (Supplemental Fig. S1C). The only enzyme responsible for this activity is UPP, as revealed by a complete loss of UPRT activity in UPP T-DNA insertion mutants (upp-1 and upp-2; Mainguet et al., 2009). We could confirm and characterize the biochemical features of recombinant UPP, which exhibits a high affinity for uracil (K_0.5 of 43.78 µm) with a Hill coefficient of 2.168 indicating a positive cooperative mode of action and a V_max of 13.93 µmol mg⁻¹ protein h⁻¹ (Fig. 2, A and B). GTP was shown to activate the UPP homolog from E. coli whereas UMP inhibited activity (Rasmussen et al., 1986; Jensen and Mygind, 1996; Lundegaard and Jensen, 1999). Recombinant Arabidopsis UPP did not respond to GTP in an activity assay but UMP showed a markedly inhibitory effect (Fig. 2B).

Figure 2.

Biochemical analysis of recombinant UPP. A, UPP was heterologously expressed in E. coli and purified via His-Tag with high amount and purity. B, Determination of the substrate affinity was performed by adding different amounts of [¹⁴C]uracil and 0.5 mm PRPP for a reaction time of 2 min. GTP and UMP were added at concentrations of 0.5 mM. Data represent means and se of five independent experiments. C, Mutations in predicted substrate-binding sites were created, and the activity of the respective purified enzymes measured with 0.5 mm uracil and 0.5 mm PRPP. Data represent means and se of three independent experiments. Different letters mark significant differences at P < 0.05. The asterisk indicates a significant difference of P < 0.05 (*) between two mutants.

Arabidopsis UPP is 47% and 54% similar to UPP homologs from Sulfolobus solfataricus (WP_009990487) and E. coli (NP_416993), representing well-characterized UPP proteins. To obtain insights into structure-function relationships, amino acids critical for UPP function in both prokaryotes were mutated in the plant protein. Whereas D220A still exhibits 81% of wild-type activity, in R93A and G224L, activity was reduced to 25% and 38%, respectively. Strongest reduction in activity was observed for P147D, with only 9% residual activity (Fig. 2C). This points to a similar overall fold of Arabidopsis UPP compared to other known UPP proteins. P147 is located in a highly conserved region and is involved in phosphoribosylpyrophosphate (PRPP) binding (Lundegaard and Jensen, 1999). R93 and G224 are located in the predicted PRPP and uracil binding site, respectively, and play a critical role in activity regulation and tetramer formation of S. solfataricus UPP (Arent et al., 2005). A homology model of the tetrameric Arabidopsis UPP build based on the E. coli homolog with 42.3% sequence identity shows the position of the mutated amino acids in its 3D structure (Supplemental Fig. S4).

Whereas UCKs reside in the cytosol, UPP is expressed with an N-terminal extension exhibiting features of a transit peptide. In fact, when this sequence was fused to GFP, targeting to chloroplasts was observed (Mainguet et al., 2009). In line with these results, a fusion of the whole coding sequence, including the transit peptide to GFP at the C terminus, led to a fluorescence signal from chloroplasts when transiently expressed in N. benthamiana leaves (Fig. 3A). To allow for an estimation of a participation of UPP in providing substrate for plastid RNA synthesis, we conducted an RNA labeling experiment. For this, radiolabeled uracil, uridine, or orotate were supplied to young leaves, and after incubation for 24 h, RNA was prepared and separated into a cytosolic ribosomal fraction and a plastidic ribosomal fraction (Supplemental Fig. S5A). If uridine salvage and uracil salvage were strictly separated between cytosol and plastid, respectively, label should only be detected in the corresponding rRNA fraction i.e. cytosol for uridine and orotate, plastid for uracil. However, this was not the case as each provided substrate could label cytosolic and plastidic rRNAs (Supplemental Fig. S5B). To rule out a partial decay of uridine via NUCLEOSIDE HYDROLASE1 (NSH1) and subsequent salvage mediated by UPP as origin for observed plastidic rRNA labeling, we included an NSH1 knockout mutant (Jung et al., 2009) in this analysis, restricting any uridine to uracil conversion. Clearly, incorporation of label in rRNAs increased in this mutant more than 2-fold (Supplemental Fig. S5B). This supports the assumption that exchange of pyrimidine nucleotides between cytosol and plastid occurs.

Figure 3.

Localization of UPP and characterization of upp-1. A, UPP-GFP fusion protein localizes to the chloroplast of N. benthamiana cells. Bar indicates 10 µm. B, upp-1 plants show drastic growth retardation and pale yellow leaves and are unable to survive for more than 3 weeks. Bar indicates 1 mm. Pictures show 2-week-old plants grown on one-half strength MS. C, rRNA content in wild type (WT) and upp-1 was analyzed on a formaldehyde gel, and each line represents a pool of 2-week-old plants. D, Expressional analysis by RT-PCR of UPP and selected genes involved in photosynthesis, based on microarray results (upp-1 compared to wild type). Data represent mean and se of five pools of WT and upp-1 seedlings grown on one-half strength MS agar plates. E and F, Light-dependent expression of (E) UPP in comparison to (F) LHCB1.4 and LHCB2.3. Samples were harvested every hour, and the light phase was from 9 am until 7 pm Data represent mean ± se of four independent experiments.

This raises the question of why UPP T-DNA insertion plants (SALK_086006; upp-1) show such severe growth defects (Fig. 3B; Mainguet et al., 2009) as they are very small, have yellow leaves, and are unable to survive on soil for more than 2 weeks. First, we checked for alterations in the chloroplast rRNA content. However, upp-1 was undistinguishable from control plants in this respect (Fig. 3C). To obtain further insight into why the loss of UPP causes such drastic problems for the plants, we isolated RNA and performed microarray analysis on whole-genome ATH1 Affymetrix chips in duplicate (at Kompetenzzentrum für Fluoreszente Bioanalytik [KFB], Regensburg, Germany). Genes with at least 2-fold increased or decreased expression levels (P value < 0.05, by t test) were considered. For this we used upp1 seedlings grown for 2 weeks on one-half strength MS agar with Suc.

In total, 69 genes were found with an increase in transcript level and 84 with decreased levels. As we assume an important function of UPP for chloroplast development and the establishment of photosynthesis, we first looked at chloroplast encoded genes. Among the 129 chloroplast encoded genes on the ATH1 chip, none was significantly regulated (Supplemental Table S1).

Then we looked at photosynthesis-related nuclear genes. Here, only three genes appeared to be regulated: two encode PSII light harvesting complex proteins LHCB1.4 and LHCB2.3 (At2g34430 and At3g27690), and one encodes the “PSII 5-kD protein” (At1g51400). Transcript levels were reduced 4-fold (LHCB2.3), 2.2-fold (LHCB1.4), and 2.4-fold (At1g51400); corresponding P values were 0.026, 0.11, and 0.15, respectively. (Supplemental Table S1). Reverse transcriptive quantitative PCR (RT-qPCR) analysis supported a marked reduction in transcript levels of the aforementioned LHCB genes and At1g51400 in upp1 relative to wild-type controls (Fig. 3D). In addition, UPP shows light-dependent expression, similar to the well-known strict light-dependent expression of LHCB1.4 and LHCB2.3 (Fig. 3, E and F), supporting the view of functions for all three gene products in light-dependent processes.

In sulfate metabolism, transcript levels for the plastidic sulfate transporter SULTR3.1, the ATP sulfurylase isoform APS3, and the APS kinase isoforms AKN1 and AKN2 were reduced. The corresponding gene products are required for the synthesis of phosphoadenosine phosphosulfate (Supplemental Table S1, highlighted in blue). Phosphoadenosine phosphosulfate is used as substrate in sulfatation, e.g. in the synthesis of glucosinolates. In fact, also several genes of glucosinolate metabolism were found with reduced transcript levels (Supplemental Table S1, highlighted in light blue)

To check for effects of UPP overexpression, corresponding lines where UPP was under control of the constitutive 35S promoter were generated. Several lines with increased transcript levels (in the range of 60- to 100-fold increase) were identified, and one line (#A) was used for further analysis (Fig. 4A). This line was characterized by a 100-fold increase in UPP transcript (Fig. 4B) and a marked increase in UPP protein level (Fig. 4C), as quantified using a specific antiserum raised against the recombinant UPP protein (Fig. 2A). When we tested the sensitivity of seedlings from this overexpression line to toxic 5-FU, they only survived up to a concentration of 25 µm, whereas wild-type plants could still grow up to a concentration of 500 µm (Fig. 4D). No phenotypical alterations under standard growth conditions were observed for this line.

Figure 4.

Analysis of UPP overexpression and cosuppressed plants. A, 35S::UPP#C grows smaller than wild type (WT) and 35S::UPP#A, shows yellow-spotted leaves, and is impaired in photosynthesis (false color presentation for F_v/F_m of 35S::UPP#C shown in the image on the right). B, Expression analysis of selected genes derived from microarray analysis. Three independent experiments were performed. C, Protein levels were detected using polyclonal antiserum raised against recombinant UPP. Thirty micrograms protein (wild type and 35S::UPP#C) and 15 µg protein (35S::UPP#A) was loaded on the gel. D, Sensitivity of mutant seedlings toward 5-flurouracil, grown on agar plates. The plants were grown in the presence of 5-fluorouracil at various concentrations for 10 d. Fresh weight on control plates was set to 1. Data represent means and se of 20 plants. E, Light curve of photosynthetic activity measured with an imaging PAM (Walz, Effeltrich, Germany). Diagram shows mean ± se of at least six plants with four selected leaves each. The asterisks indicate significant differences at *P < 0.05, **P > 0.01, ***P < 0.001; different letters indicate significant differences of P < 0.05.

Among the overexpression lines, one line stuck out because of reduced growth and a variegated leaf coloration, with yellow leaf areas of different size and shape (#C, Fig. 4A). In this line, UPP expression and UPP protein amounts were drastically reduced. Furthermore, this line showed slightly higher resistance to 5-FU (Fig. 4, B–D). When we measured the photosynthetic performance in a light curve experiment, wild-type controls as well as the overexpressor line #A showed a fairly similar behavior. However, “suppressor” line #C was severely reduced in photosynthesis efficiency, especially in yellow leaf areas (Fig. 4, A and E; F_v/F_m was reduced from 0.75 to 0.63). With respect to changes in gene expression, a similarly reduced expression of LHCB2.3 was observed as for upp1 (Fig. 4B).

There is good evidence from the literature that although plastid uracil salvage is active, plants can survive well without or with only little of the corresponding pathway activity (to be explained in detail in “Discussion”). Therefore, we designed a complementation experiment aiming to investigate whether UPRT activity is required for a rescue of the knockout phenotype or whether the protein itself is necessary, e.g. for protein-protein or protein-RNA interactions. First, expression of UPP under control of the medium-strength UBQ10 promoter was performed. These lines (UPP#1 and #2) showed an up-regulation of transcript of up to 3-fold and completely rescued the photoautotrophic lethal upp-1 (Fig. 5, A and B; Supplemental Fig. S5).

Figure 5.

Complementation of upp-1 and analysis of corresponding mutants. A, Complementation of upp-1 was achieved by transformation with an UBQ-UPP construct (UPP#1), mutated UPP variants (R93A, P147D#1), or the coding sequence of the E. coli homolog fused to the plastidic targeting sequence of RBCS (EcUPP). Transformation with UPP, R93A, and P147D can rescue upp1, whereas EcUPP cannot. Bars = 1 mm. B, Expression levels of UPP were checked in the complemented plants with specific UPP primers. Different letters mark significant differences at P < 0.05. C, The protein level of UPP in the complemented plants was analyzed using UPP-specific antibody. CBB, Coomassie Brilliant Blue gel serves as the loading control. Black line indicates deleted empty lane in the western blot. D, Crude extract was incubated with radiolabeled [¹⁴C]uracil, and the formed UMP was measured to determine the UPRT activity in the plants. E, Seedlings were grown on one-half strength MS agar plates with or without 200 µm 5-FU for 10 d, and fresh weight was determined (mean and se are given). Significance was determined and is indicated as * P < 0.05 or n.s. (not significant).

In a second approach, UPP mutant proteins with reduced UPRT activity were generated by making amino acid exchanges at critical positions. Two such mutations were identified with R93A and P147D, and the corresponding recombinant proteins showed activities of 9% and 25% in vitro, relative to unmutated control protein (Fig. 2C). The mutant UPP constructs were then used in our complementation approach. Surprisingly, complementation worked with these nearly catalytically inactive proteins (Fig. 5A). Whereas P147D was undistinguishable from wild-type and complemented lines, R93A was delayed in growth (Supplemental Fig. S6). UPP expression and protein content in complemented lines were lower than wild type in UBQ10::R93A, similar to wild type in UBQ10::UPP, and higher than wild type in UBQ10::P147D (Fig. 5, B and C). Most important, almost no in vivo UPRT activity was detectable in both lines carrying a point mutation (Fig. 5, D and E).

A further approach aimed for a complementation with the well-characterized, highly active UPRT from E. coli (EcUPP). This enzyme clearly did not coevolve with the photosynthetic machinery, so a successful complementation should be purely based on its activity. For this purpose, the chloroplast targeting sequence from the Rubisco (RBCS) small subunit was cloned upstream of EcUPP to obtain a translational fusion with EcUPP. Although EcUPP was transformed successfully as checked by PCR on genomic DNA, no complementation of the phenotype was achieved. On soil, UBQ10::rbcs-EcUPP plants remained dwarf and died after ∼20 d (Fig. 5A). We checked that targeting to the chloroplast worked by expressing the same construct used for complementation, fused to a GFP tag (UBQ10::rbsc-EcUPP-GFP) in N. benthamiana cells (Supplemental Fig. S6A). In addition, UBQ10::rbcs-EcUPP plants (in UPP/upp1 background) showed high sensitivity against 5-FU as well as high UPRT activity in vivo (Fig. 5D). In sum, these results can be interpreted in favor of a protein function of so-far-unknown nature, therefore denominated as moonlighting, not directly linked to UPRT activity but vital for the establishment of photosynthesis.

DISCUSSION

Most organisms rely on two pathways to replenish their nucleotide pools: one is de novo synthesis; the other is salvage. In the pyrimidine salvage pathway, two alternative routes can be taken; one uses uracil and phosphoribosyl pyrophosphate to synthesize UMP, and the other requires uridine and ATP and will lead to the synthesis of the same molecule, UMP.

Pyrimidine salvage differs among eukaryotes. Whereas yeast can use uracil and uridine, protozoan parasites either lack pyrimidine salvage completely or use uracil (Hammond and Gutteridge, 1984; Kurtz et al., 2002; Tiwari and Dubey, 2018). Humans do not employ UPRT and rely on uridine salvage completely (Löffler et al., 2005). Arabidopsis possesses the enzymatic equipment for uridine, cytidine, and uracil salvage. However, only uridine, not uracil, can rescue Arabidopsis seedlings when pyrimidine de novo synthesis is inhibited by PALA. Furthermore, seedlings were more susceptible toward 5-FD compared to 5-FU, indicating higher flux through uridine salvage. The view of a preferential use of uridine as salvage substrate is supported by the finding of increased pyrimidine catabolism when NSH1, converting uridine to uracil, is more active (Jung et al., 2009). Null mutants for the sole uracil importer into chloroplasts, PLUTO, show no phenotypical alterations (Mourad et al., 2012; L. Ohler, S. Niopek-Witz, S.E. Mainguet, and T. Möhlmann, unpublished data), corroborating a minor role of plastidic uracil salvage as UPP; the only Arabidopsis UPRT is a plastid localized enzyme (Mainguet et al., 2009; Fig. 3A). According to our results, (1) all four expressed UCK gene products locate to the cytosol, (2) UCK1 and UCK2 represent the main activities, and (3) besides uridine, cytidine can also enter the salvage via UCK1 and UCK2. Such a dual biochemical function was proposed earlier for UK enzymes (Chen et al., 2016) and was now confirmed in this work. It seems that UCK1 exhibits highest in vivo activities for UK and CK, revealed by analysis of corresponding T-DNA insertion mutants (Fig. 1D). However, UCK2 obviously can compensate when UCK1 is missing, and only in case of the double knockout situation do severe growth restrictions in green tissue become apparent (Chen and Thelen, 2011). This clearly indicates that pyrimidine de novo synthesis alone is not sufficient for growth and development of Arabidopsis plants. Some tissues must rely on uridine/cytidine salvage. Surprisingly, the reduced nucleoside kinase activity of uck1 is not resembled in seedling growth in presence of 5-FD. It is possible that in uck1 plants combined NSH1, UPP, and residual UCK1 activities present sufficient salvage potential to prohibit 5-FD resistance. The exact functions of UCK3 and UCK4 remain unclear so far (see Fig. 6 for overview). However, besides uck1 and uck2, uck3 also showed reduced root growth on orthogonal plates. When investigating eFP-Browser data, these point to expression differences between UCK1, UCK2, and UCK3 in roots (Brady et al., 2007; visualized by eFP Browser, Winter et al., 2007). Whereas UCK1 shows higher expression in developing root phloem and xylem, compared to UCK2, latter gene is higher expressed in pericycle, endodermis, and epidermis cells. UCK3 expression is high in joung and old procambium, responsive to NO₃, and hypoxia (Genevestigator; Hruz et al., 2008). These expression differences in roots might explain the observed differences in root growth; still, this explanation is somehow speculative and requires further experimental substantiation.

Figure 6.

Schematic overview of pyrimidine salvage in Arabidopsis. Pyrimidine nucleotides are synthesized de novo in a pathway distributed between chloroplast and cytosol (dashed line). Nucleosides imported or derived from catabolic processes are salvaged by uridine/cytidine kinases (UCKs) in the cytosol. We postulate exchange of nucleotides (UTP, CTP) between cytosol and chloroplast. Uracil is imported into chloroplasts by PLUTO and subsequently channeled either into catabolism via PYD1 (dihydropyrimidine dehydrogenase1) or salvage via UPP. Resulting UMP can be further metabolized to UTP by PUMPKIN (plastidic UMP kinase) and NDPK2 (nucleoside diphosphate kinase2). Furthermore, UMP is a potent inhibitor of de novo synthesis at the transcriptional as well as enzymatic level (punctate line). Mutants directly involved in chloroplast uracil salvage (marked in red) show strong effects on chloroplast function (NDPK2) and early biogenesis (UPP, PUMPKIN). Other mutant enzymes and transporters around uracil metabolism (marked in blue) show little or no effect on growth and development. CDA1, Cytidine deaminase 1; CTPS, CTP synthase.

The sole Arabidopsis UPRT UPP is an active enzyme and locates to the chloroplast (Mainguet et al., 2009; Figs 2B and 3A). By testing the effect of 5-FU feeding, the functionality of the complete uracil salvage pathway could be shown in agreement with previous results (Cornelius et al., 2011; Mourad et al., 2012). However, besides the functionality of this pathway, a series of experiments question its importance for Arabidopsis development. At least three different mutations will lead to reduced availability of the UPP substrate uracil; these are knockout mutants of NSH1-producing uracil from uridine, overexpression of PYD1 in the uracil degradation pathway, which removes uracil, and knocking out of the uracil importer PLUTO. All of these mutations do not lead to severe growth alterations (Jung et al., 2009; Cornelius et al., 2011; Mourad et al., 2012; Witz et al., 2012; Fig. 6). These observations are in line with our hypothesis of an exchange of pyrimidine nucleotides between cytosol and plastid, which would render plastid salvage nonessential. Therefore, the question comes up why the inactivation (knocking out) of UPP in upp1 has such dramatic consequences like inability of photoautotrophic development, reduced chloroplast size, and the lack of starch synthesis (Mainguet et al., 2009). The UPP knockdown mutant (35S::UPP#C) resembles the upp1 phenotype in a milder form. However, this cosuppression line is also severely reduced in growth, leaf pigmentation, and photosynthetic efficiency. All this, together with a pronounced light dependence of UPP expression (Fig. 3E) and unaffected growth of upp-1 in darkness (Mainguet et al., 2009), point to an important function of UPP for the establishment of photosynthesis.

Gene expression analysis highlights that the mutant phenotype is accompanied by a down-regulation of nuclear encoded LHCB1.4 and LHCB2.3 and genes in the nonreducing branch of sulfur metabolism, leading to sulfatation reactions. LHCB expression is besides others regulated by genomes uncoupled 1 (Susek et al., 1993 ; for review see Kleine and Leister, 2016). As we did not observe altered GUN1 expression in our array analysis and a reduced LHCB expression as consequence of impaired chloroplast function, GUN1 signaling seems to operate normally in our mutant.

Because of altered expression of genes involved in sulfur metabolism, one might think of effects on PAP signaling. Although we cannot rule out this possibility, PAP signaling is mainly functioning under stress conditions such as draft and high light (Estavillo et al., 2011; for review see Kleine and Leister, 2016). However, upp1 develops a clear phenotype under standard growth conditions, making this hypothesis less likely.

rRNA contents were inconspicuous when comparing upp1 with controls, indicating a normal supply of chloroplasts with energy and nucleotide building blocks.

Therefore, one important question remains: Is the UPRT catalytic activity of UPP or a moonlighting function of UPP (exerting a secondary function besides the catalytic activity) essential for the establishment of photosynthesis? To approach this issue, a complementation analysis employing different constructs was done. Complementation with endogenous UPP under control of the UBQ10 promoter lead to a 1.2- to 4-fold increase in UPP transcript relative to wild types and fully rescued the upp-1 phenotype. Resistance against 5-FU was undistinguishable from wild types as well, further indicating full complementation with this construct (Fig. 5). A higher overexpression (up to 95-fold increase in transcript) in wild type background from a 35S promoter-driven Arabidopsis UPP led to higher sensitivity toward 5-FU, but not to any obvious alterations in phenotype (Fig. 4), indicating that a higher UPRT activity is well tolerated by Arabidopsis. In contrast, when we expressed E. coli UPP under control of UBQ10, a recovery of UPRT activity to wild type level was measured. (Fig. 5D). However, no complementation was achieved for soil-grown plants, whereas E. coli UPP-complemented plants survived for few days only when an external carbon source was present. This strongly argues against an essential function of chloroplast UPRT activity for photoautotrophic growth. When Arabidopsis UPP mutant versions with massively reduced activity due to point mutations in critical amino acids were used for complementation, a much better performance compared to E.coli UPP-complemented lines was observed (Fig. 5). R93A-complemented lines show expression and protein level similar to wild-type plants and rescue upp1, although slight growth constraints are visible (Fig. 5). Notwithstanding that P147D-complemented lines exhibit increased expression and UPP protein, activity is drastically reduced but complementation was fully achieved. We conclude from this set of experiments that a minimal amount of UPP protein is required and catalytic activity alone is not sufficient to complement upp1. However, if UPRT activity of UPP was completely dispensable, it would have been lost during evolution due to mutations; this is not the case. Either UPP activity fulfils a role different from chloroplast development, or activity and a moonlighting function act in combination in a so-far-unknown way. A combination of a function in pyrimidine metabolism and a secondary moonlighting function is illustrated by UPP from Bacillus subtilis. Here, UPP, upon binding of UMP, acts as a transcriptional regulator of the pyrimidine synthesis operon (Turnbough and Switzer, 2008). It might also be that the capacity of UMP binding is required and sufficient for full functionality of moonlighting UPP. UMP is known as a potent metabolic inhibitor of transcription of pyrimidine de novo synthesis genes (Brady et al., 2010) as well as an allosteric inhibitor of plastidic CPSase and ATCase activity (O’Neal and Naylor, 1976; Khan et al., 1999). CPSase is synthesizing carbamoyl phosphate, the precursor of Arg and Pyrimidine nucleotides, and plastidic ATCase catalyzes the second step in pyrimidine de novo synthesis. Thus, UMP could act as metabolic integrator, regulating Arg and pyrimidine synthesis and in addition chloroplast biogenesis (Fig. 6).

Interestingly, the next two enzymes in the chloroplast pyrimidine salvage pathway, UMP kinase (PUMPKIN) and nucleoside diphosphate kinase2 (NDPK2) seem to be moonlighting enzymes too. PUMPKIN associates specifically with introns of plastid transcripts from tRNAs (trnG, trnV) and proteins of the photosynthetic machinery (petB, petD, ndhA). Knockout mutants were impaired in growth, plastid translation, and photosynthetic performance (Schmid et al., 2019). NDPK2 is involved in photosynthetic development and oxidative stress management (Dorion and Rivoal, 2015). Thus, enzymes in chloroplast pyrimidine salvage might be nodes in integrating signals from pyrimidine nucleotide metabolism and other central chloroplast pathways (Fig. 6).

PUMPKIN was found to bind RNAs, and as one consequence, corresponding plastid-encoded transcripts of components of the photosynthetic machinery were markedly reduced (Hein et al., 2009; Schmid et al., 2019). A similar moonlighting function of UPP is less likely, as we did not observe such a loss of plastid-encoded transcripts. Protein-protein interactions might be a mechanism by which UPP interacts with photosynthesis. An interaction with the petC protein was found in a yeast two-hybrid analysis (BioGrid, https://thebiogrid.org; Arabidopsis Interactome Mapping Consortium, 2011). Further analysis in this direction will help to unravel the moonlighting function of UPP.

MATERIALS AND METHODS

Plant Growth and Preparation of Leaf Extracts

For DNA isolation, tissue collection, and phenotypic inspection, wild-type and transgenic Arabidopsis (Arabidopsis thaliana) Heynh. plants (ecotype Columbia) were grown in standardized ED73 (Einheitserde und Humuswerke Patzer) soil at 120 μmol quanta m⁻² s⁻¹ in a 10 h light/14 h dark regime (short day) or 14 h light/10 h dark regime (long day), temperature 22°C, humidity 60%. Prior to germination, seeds were incubated for 24 h in the dark at 4°C for imbibition. Nicotiana benthamiana plants were grown under the same conditions (short day).

For growth experiments on sterile agar plates, surface-sterilized seeds were grown on one-half strength MS, supplemented with 1% (w/v) Suc and the substrate analogon PALA (1 mm) or uracil (1 mm) or uridine (1 mm) and the toxic analoga 5-FU and 5-FD at constant (50 µm) or variable concentrations (as indicated in the corresponding figure). For each condition, three plates were prepared. Ten typical seedlings per plate were harvested (without roots) and weighted individually. This experiment was repeated two more times. Seedling fresh weights from all seedlings grown under identical conditions were used to calculate means and se. Analysis of root length was performed in a similar way without Suc added to the medium. Root length was determined with help of ImageJ software on scanned images.

Leaf extract of wild type and mutants was prepared by homogenizing leaf material in extraction buffer (50 mm HEPES-KOH, pH 7.2, 5 mm MgCl₂, 2 mm phenylmethylsulfonyl fluoride [PMSF]) on ice. This homogenous extract was centrifuged for 10 min, 20,000g and 4°C. The supernatant was collected and stored on ice until use.

T-DNA Insertion Lines

T-DNA insertion lines from the Salk and Sail collection were used (Sessions et al., 2002; Alonso et al., 2003). The following lines were studied: uck1, SALK_108486; uck2, SALK_058257; uck3, SAIL_156D06; uck4, SAIL_895_A10; upp1, SALK_086006; and nsh1, SALK_083120. Uck1, uck2, uck3, upp1, and nsh1 have been described previously (Mainguet et al., 2009; Chen and Thelen, 2011; Jung et al., 2011). Two uck4 lines were analyzed (uck4-1, SAIL_895_A10 and uck4-2, SALK_102313). As uck4-2 still shows UCK4 transcript, all work was performed with uck4-1 (named uck4 SAIL_895_A10; Supplemental Fig. S2, A–C). Primers used for PCR testing for presence of the T-DNA insertion and the homozygous state are listed in Supplemental Table S2.

Cloning of UCK and sUPP

Cloning of the four full-length UCKs (UCK1, At5g40870; UCK2, At3g27190; UCK3, At1g55810; and UCK4, At4g26510) from Arabidopsis was carried out using cDNA pools as template. Sequences of gene-specific primers with restriction sites are provided in Supplemental Table S2. Initially, cDNA products were subcloned into pBSK (Stratagene). In the case of UCK1, a silent mutation was added do destroy an internal XhoI site that prevented cloning into the expression vector. After digestion with NdeI and XhoI, the resulting fragments were cloned into pET16 (Novagen) opened with the same enzyme. The resulting constructs contain an N-terminal 10x-His-Tag and were transformed into the Escherichia coli strain BLR(DE3)-pLysS (Studier and Moffatt, 1986). UPP was cloned applying the same procedure.

The binary plasmid used for in planta expression is pXCS-GFP. Therefore, GFP was amplified from pGFP2 vector (Wendt et al., 2000) and cloned into SmaI-digested pXCS (AY457636; Witte et al., 2004) UCKs were digested with PstI and EcoRI and cloned into pXCS-GFP opened with the same enzymes. Fusion constructs were then transformed into Agrobacterium tumefaciens strain GV3101 (pMP90RK; Koncz et al., 1994).

Cloning of the short splice variant of the UPRT (sUPP, At3g53900) from Arabidopsis was also carried out using cDNA pool as template. Initially, cDNA product was subcloned into pBSK (Stratagene) and afterward digested with XhoI/BamHI for further ligation into pET16b (Novagen) opened with the same enzymes. The resulting construct contains an N-terminal 10x-His-Tag and was transformed into the E. coli strain BLR(DE3)-pLysS (Studier and Moffatt, 1986).

The mutated variants were achieved by site-directed mutagenesis using sUPP::pBSK as template. Site-directed mutagenesis was performed by using the Quick-Change II-E site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. Ligation and transformation was carried out as described previously.

For subcellular localization studies, UPP was cloned into the Gateway vector pUBC-eGFP or pUBC-YFP (Grefen et al., 2010) and transformed into A. tumefaciens strain GV3101 (pMP90RK; Koncz et al., 1994). The complementation of the upp-1 plants was done by cloning UPP and the mutated versions into pUBC-Dest (Grefen et al., 2010). The open reading frame of the E. coli UPP homolog was first fused to the signal peptide coding region of the small RBCS subunit (Van den Broeck et al., 1985) and then also cloned into pUBC-Dest. To test the correct localization of the mature EcUPP protein in planta, the fusion construct was also cloned into pUBC-eGFP (Grefen et al., 2010).

UPP overexpressor plants were generated by cloning UPP under the control of the 35S promoter into pGwB2 (INRA), using Gateway technology.

All constructs used for Arabidopsis transformation by floral dip (Narusaka et al., 2010) were previously transformed into A. tumefaciens strain GV3101 (pMP90; Koncz et al., 1994). All primer sequences are provided in Supplemental Table S2.

Homology Modeling of UPP

A three-dimensional model of the short UPP variant was built using Swiss model (Guex et al., 2009; Benkert et al., 2011; Bertoni et al., 2017; Bienert et al., 2017; Waterhouse et al., 2018). The E. coli homolog (2EHJ) was selected as a template. Structural alignment of both models and presentation of the mutated amino acids was done with PYMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger).

UCK and UPP Expression in E. coli and Protein Purification

E. coli BLR cells transformed with HIS₁₀-UCK1–UCK4 or -sUPP pET16b constructs were grown overnight at 37°C in YT medium and used to inoculate 1L Terrific Broth (TB) medium to an OD600 of 0.08. In case of UCK1–UCK4, cells were grown to an OD of 2, transferred to 18°C, and incubated under constant shaking for another hour. Gene expression was induced by 0.5 mm isopropyl-ß-D-thiogalactopyranosid (IPTG) and cultivation proceeded for another 18 to 20 h. Cells for expression of sUPP were grown until an OD₆₀₀ of 0.5, induced with 0.5 mm IPTG, and incubated for another 1.5 h. Then cells were harvested and resuspended in 1.5-fold the pellet volume with lysis buffer (300 mm NaCl, 50 mm Na₂HPO₄, pH 8.0; 20 mm imidazole, 1 mm PMSF, DNase, and RNase). Cells were broken by sonication and debris pelleted at 35,000g for 15 min under cooling. The supernatant was incubated with Ni-sepharose 6 fast flow (GE Healthcare) equilibrated according to the manufacturer’s protocol. After washing, UKL1 to UKL4 were eluted with a buffer (300 mm NaCl, 50 mm Na₂HPO4, pH 8.0) containing 500 mm imidazole. The elutions were desalted in activity buffer and stored a 4°C for further use.

Biochemical Assay for UCK and sUPP

Purified UCK1 to UCK4 (50 µL at 0.2 mg protein mL⁻¹) were incubated in activity buffer (50 mm HEPES-KOH, pH 7.2), 5 mm MgCl₂, 4 mm ATP, 0.1 mm uridine/cytidine, 30 mm NaF, 0.05% (w/v) bovine serum albumin (BSA), and 10⁹ Bq mmol⁻¹ [¹⁴C]uridine/cytidine at 37°C for different time points (modified according to Moffatt et al., 2000). To determine the respective K_M values, different amounts of uridine/cytidine was added and incubated at 37°C for 5 min. For UPRT activity assay (described in Witz et al., 2012), purified sUPP was incubated in activity buffer (50 mm HEPES-KOH, pH 7.2, 5 mm MgCl₂, 10 mm NaN₃, 0.5 mm PRPP and 0.025% [w/v] BSA, uracil as indicated, and [¹⁴C]Uracil at adjusted concentrations) for 2 min. For the analysis of sUPP-mutants 0.5 mm uracil and 0.5 mm PRPP were used as cosubstrate, and the analysis done in a time-dependent manner for 5 min. For stopping and precipitation of the formed UMP/CMP, 50 mm NaAc, 2 mm K₂HPO₄, and 100 mM LaCl₃ (pH 5.0, acetic acid) was added, incubated for 30 min on ice, and terminated by vacuum filtration through a membrane filter (0.45 µm pore size, Whatman). The filters were washed three times with 1 mL ice-cold 50 mm LaCl₃ to remove unincorporated nucleosides, and the radioactivity was quantified in a Packard Tricarb scintillation counter after the addition of 4 mL scintillation cocktail (Roth). For the investigation of salvage activities in Arabidopsis leaf extracts, 10-µL samples were incubated with 90 µL activity buffer and treated as mentioned above.

Transient Expression in N. benthamiana

Transient expression of UCK and UPP fused to GFP or YFP was performed as given in Witte et al. (2004). The transformed A. tumefaciens GV3101 (pMP90RK) strains (OD₆₀₀ 0.4) were incubated with C58C1 (pCH32 35S:p19; OD₆₀₀ 0.1) in 10 mm MES-KOH, pH 5.6, 10 mm MgCl₂, and 150 µM acetosyringone and infiltrated through the lower epidermis of 6-week-old N. benthamiana leaves. After 3 d, leaf protoplasts were isolated (0.45 mm sorbitol, 10 mm CaCl₂, 1% [w/v] cellulase “Onozuka” R-10 [Serva], 0.25% [w/v] Macerozyme R-10 [Serva], and 20 mm MES-KOH, pH 5.7) for 3 h, resuspended in buffer (145 mM NaCl, 125 mm CaCl₂, 5 mm KCl, and 5 mm Glc, pH 5.5 [KOH]) and analyzed for the presence of fluorescence signals with a Leica TCS SP5II microscope (488-nm excitation and 505–540-nm detection of emission for eGFP, 514-nm excitation and 525–582-nm detection of emission for YFP, and 514-nm excitation and a 651– 704-nm emission wavelength for chlorophyll autofluoresence through a HCX PL APO 63 × 1.2 W water immersion objective). Subsequently, correct protein sizes were verified in total protein extracts by immunodetection using an α-eGFP primary antibody and a horseradish peroxidase-coupled secondary antibody (anti-rabbit). Please see also immunoblotting section for additional information.

RT-qPCR and Microarray-Data

Tissue from wild type, upp-1, and 35S::UPP#A and #C was harvested in liquid nitrogen and RNA for RT-qPCR, and micro-array analysis was isolated using the NucleoSpin RNA plant kit (Macherey-Nagel) according to the manufacturer’s instructions. Samples of the day-night cycle were harvested every hour. For this, 4-week-old plants grown under short-day conditions were used. For RT-qPCR, RNA was transcribed into cDNA (qScript cDNA synthesis kit, Quantabio, containing oligo(dT) and random primers) and the PCR performed with CYBR Green. The normalization of the data were performed using geNorm according to Vandesompele et al. (2002). Primers are listed in Supplemental Table S2. For micro-array analysis, RNA was prepared as described above and reverse transcribed using random priming and the procedure of the GeneChip WT PLUS reagent kit (Affymetrix) following the user manual. Hybridization and data analysis was done at Affymetrix Service Provider and Core Facility, KFB-Center of Excellence for Fluorescent Bioanalytics (Regensburg, Germany; www.kfb-regensburg.de).

Immunoblotting

For the detection of UCK-GFP and UPP, 15 µg total protein was separated on SDS PAGE and blotted onto nitrocellulose membrane by wet blotting. Blocking was achieved by incubation in 3% (w/v) milk powder in PBST (137 mm NaCl, 10 mm phosphate, 2.7 mm KCl, pH 7.4, 0.1% [v/v] Tween 20), washings were performed three times in PBST. As primary antibody eGFP monoclonal antibody (MAB 3580, Sigma-Aldrich, PBST + 3% [w/v] milk powder) or anti-HIS ab (SAB 1306085, Sigma-Aldrich, PBST + 3% [w/v] milk powder) were used. For UPP, a polyclonal antiserum against recombinant, purified UPP was raised in rabbit (Eurogentec). As secondary antibody, anti-rabbit-HRP or anti-mouse-HRP (PBST + 3% [w/v] milk powder) were used. Detection of chemiluminescence was performed in a Fusion Solo S6 (Vilber-Lourmat) imager.

Measurements of Photosynthetic Activity

A MINI-IMAGING-PAM fluorometer (Walz Instruments) was used for in vivo chlorophyll A fluorescence assays on intact, 3-week-old dark-adapted Arabidopsis plants. For phenotype quantification, dark-light induction curves and light curves were recorded using standard settings (Schreiber et al., 2007).

Analysis of Radiolabeled RNA

One leaf from 5-week-old Arabidopsis plants was incubated for 24 h at light in 500 µL one-half strength MS medium (without Suc) supplemented with 100 µm uracil, uridine, or orotate, and 5 × 10⁹ Bq mmol⁻¹ [¹⁴C]uracil, uridine, or orotate. Leaves were grinded to measure total amount of incorporated radioactivity, and total RNA was isolated. After separation on a formaldehyde gel, cytosolic and plastidic rRNA was separated and isolated from the gel via heating, and the amount of radiolabeled rRNA was quantified in a Packard Tricarb scintillation counter after the addition of 8 mL scintillation cocktail (Roth).

Statistical Analyses

For statistic evaluation, one-way ANOVA followed by Tukey’s multiple comparison test was performed. Different significance levels of P < 0.0.5, P < 0.01, and P < 0.001 are indicated with one, two, or three asterisks, respectively. Alternatively, different letters represent significant differences of P < 0.05.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers given in Table 1.

Supplemental Data

The following supplemental information is available.

• Supplemental Table S1. Regulated genes upp-1 versus Col-0, results from gene-array experiments.

• Supplemental Table S2. Primers used.

• Supplemental Figure S1. Importance of salvage reaction in vivo.

• Supplemental Figure S2. Biochemical characterization of recombinant UCK1 to UCK4.

• Supplemental Figure S3. Identification of UCK4 T-DNA insertion lines and growth performance of uck1-4 mutant lines.

• Supplemental Figure S4. Model of a UPP homotetramer.

• Supplemental Figure S5. Analysis of nucleotide import into plastids.

• Supplemental Figure S6. Growth performance of wild-type and complemented upp-1 mutant lines.

• Supplemental Figure S7. Localization of RBCS-EcUPP-GFP and UPRT activity in complemented plants.