Nucleoid-enriched proteomes in developing plastids and chloroplasts from maize leaves: a new conceptual framework for nucleoid functions

Abstract

Plastids contain multiple copies of the plastid chromosome, folded together with proteins and RNA into nucleoids. The degree to which components of the plastid gene expression and protein biogenesis machineries are nucleoid associated, and the factors involved in plastid DNA organization, repair, and replication, are poorly understood. To provide a conceptual framework for nucleoid function, we characterized the proteomes of highly enriched nucleoid fractions of proplastids and mature chloroplasts isolated from the maize (Zea mays) leaf base and tip, respectively, using mass spectrometry. Quantitative comparisons with proteomes of unfractionated proplastids and chloroplasts facilitated the determination of nucleoid-enriched proteins. This nucleoid-enriched proteome included proteins involved in DNA replication, organization, and repair as well as transcription, mRNA processing, splicing, and editing. Many proteins of unknown function, including pentatricopeptide repeat (PPR), tetratricopeptide repeat (TPR), DnaJ, and mitochondrial transcription factor (mTERF) domain proteins, were identified. Strikingly, 70S ribosome and ribosome assembly factors were strongly overrepresented in nucleoid fractions, but protein chaperones were not. Our analysis strongly suggests that mRNA processing, splicing, and editing, as well as ribosome assembly, take place in association with the nucleoid, suggesting that these processes occur cotranscriptionally. The plastid developmental state did not dramatically change the nucleoid-enriched proteome but did quantitatively shift the predominating function from RNA metabolism in undeveloped plastids to translation and homeostasis in chloroplasts. This study extends the known maize plastid proteome by hundreds of proteins, including more than 40 PPR and mTERF domain proteins, and provides a resource for targeted studies on plastid gene expression. Details of protein identification and annotation are provided in the Plant Proteome Database.

Figure 1.

Plant material and selection of maize nucleoid preparations. A, Seedlings used for the isolation of nucleoids from the nonphotosynthetic leaf base (above the ligule) and the photosynthetic tip of the third leaf of 9- to 10-d-old seedlings. B, Seedlings used for the isolation of nucleoids from all leaf blades of 7- to 8-d-old seedlings (assigned as young nucleoids). C, SDS-PAGE protein profile of nucleoids from young leaves. The gel (10.5%–14% acrylamide gradient gel) was loaded with 18 μg of protein and was stained with Sypro Ruby. D, Average abundance (as NadjSPC) of the four subunits of the PEP complex in independent nucleoid preparations from base, tip, and young leaf. sd values are indicated (n = 3). [See online article for color version of this figure.]

Figure 2.

Comparison of proteomes of chloroplasts, proplastids, and nucleoids. A, Venn diagram showing the overlap between the identified nucleoid, chloroplast, and proplastid proteomes; in total, 2,460 proteins were identified. B, Comparison of protein abundance (based on NadjSPC) of the different functional groups. Ten different functional groups are defined, namely the photosynthetic electron transport chain (bin 1), the Calvin-Benson cycle and malate shuttle (bin 1), DNA (bin 28), RNA (bin 27), proteins with unknown function and with mTERF, DEAD box, TPR, rhodanese, or DnaJ(-like) domains (bin 26) or pTAC proteins with unknown functions, translation (in bin 29), protein homeostasis (bin 29), transport (bin 34), unknown function (bin 35), other functions (all other bins). C, Dendrogram of the distribution pattern of 771 proteins across the three sample types (chloroplasts, proplastids, average nucleoids) obtained by hierarchical clustering. The 771 proteins were observed in nucleoid fractions, they each had at least an average NadjSPC of 1.10⁻⁵ across the three sample types (chloroplast, proplastid, and nucleoid), and they were not considered extraplastidic (for location assignments or PPDB, see Supplemental Table S1). Red represents values above the mean, black represents the mean, and green represents values below the mean abundance of a protein across the three sample types. D, Protein mass investments in the clusters for chloroplast, proplastid, and nucleoid samples.

Figure 3.

Comparison of protein investments of chloroplasts, proplastids, and nucleoids for the proteins listed in Table I. A, Relative abundance of the proteins in the three main functions as in Table I, namely DNA ([putative] DNA-related functions: DNA replication, DNA organization and quality control, anchoring, and DNA repair), RNA (putative RNA-related functions: transcription, RNA splicing, editing, cleavage, stability, rRNA and tRNA maturation, and other predicted RNA-binding proteins with unknown functions), and Unknown (proteins with unknown functions with TPR, mTERF, DEAD, rhodanese, or DnaJ domains or pTAC proteins with unknown functions). B, Relative mass distribution of proteins within the function DNA. C, Relative abundance of proteins within the function RNA. D, Relative abundance of proteins within the function Unknown. Details are provided in Table I.

Figure 4.

Comparison of the abundance of proteins involved in protein synthesis and homeostasis (bin 29) and of envelope-localized proteins in maize chloroplasts, proplastids, and nucleoids. A, Distribution of different functions within bin 29, namely ribosome biogenesis, 70S ribosomes, tRNA ligases, “synthesis” (translation, elongation, and termination factors), protein folding, posttranslational modifications, protein sorting and translocation, protein assembly, and proteases. B, Distribution of different functions of envelope proteins, namely metabolite transporters, protein translocon of the inner envelope membrane (TIC) and outer envelope membrane (TOC), proteases, metabolic enzymes (mostly fatty acid/lipid synthesis), plastid division and positioning, and other proteins. The inset in B shows the abundance distribution between detected inner and outer envelope membrane proteins.

Figure 5.

Comparison between nucleoid preparations isolated from the leaf base, tip, or young seedlings. A, Venn diagram showing the overlap between the identified nucleoid proteomes; in total, 1,092 proteins were identified. B, Comparison of protein abundance (based on NadjSPC) of the 10 different functional groups defined as for Figure 2B. C, Work flow for determination of the nucleoid core proteome. D, Base-tip ratio plot for the 157 proteins defined in the work flow in C. Data points are color coded based on the 10 functions defined for Figure 2. Identities of a few proteins are indicated. Details for all 157 proteins are provided in Supplemental Table S5.

Figure 6.

Co-IP of nucleoids by anti-Why1 serum and effect of RNase treatment. A, One-dimensional SDS-PAGE gel of the nucleoids prior to co-IP, and nucleoid co-IP by anti-WHY1 with or without RNase treatment. B, Quantitative distribution of protein functions across the three nucleoid samples. The functional groups are as defined in Figure 2. C, Quantitative effect of RNase treatment on the co-IP nucleoids using anti-WHY1 serum. Ribosomal proteins decreased in abundance by the RNase treatment are circled (in red). D, Effect of co-IP and RNase treatment of the quantitative presence of protein in the nucleoid. Cross-correlation of nucleoid protein abundance before and after co-IP and RNase treatment is shown. Proteins quantitatively reduced by RNase are numbered as follows: 1, NP_043018, 30S ribosomal protein S2; 2, GRMZM2G005973_P01, 50S ribosomal protein L1; 3, GRMZM2G377761_P01, 3′ to 5′ PNPase exoribonuclease; 4, GRMZM2G170870_P01, 50S ribosomal protein L6-2; 5, NP_043046, 30S ribosomal protein S18; 6, GRMZM2G105570_P01, ABC and structural maintenance of chromosomes domain protein; 7, GRMZM2G165694_P01, 16S rRNA-processing protein RimM family; 8, GRMZM2G028216_P01, 50S ribosomal protein L29. ^{^} Proteins only found in co-IPs; * proteins lost by co-IP. Detailed information is provided in Supplemental Table S6.

Figure 7.

Summarizing schematic view of nucleoid functions. [See online article for color version of this figure.]