Structural and metabolic transitions of C4 leaf development and differentiation defined by microscopy and quantitative proteomics in maize

Abstract

C(4) grasses, such as maize (Zea mays), have high photosynthetic efficiency through combined biochemical and structural adaptations. C(4) photosynthesis is established along the developmental axis of the leaf blade, leading from an undifferentiated leaf base just above the ligule into highly specialized mesophyll cells (MCs) and bundle sheath cells (BSCs) at the tip. To resolve the kinetics of maize leaf development and C(4) differentiation and to obtain a systems-level understanding of maize leaf formation, the accumulation profiles of proteomes of the leaf and the isolated BSCs with their vascular bundle along the developmental gradient were determined using large-scale mass spectrometry. This was complemented by extensive qualitative and quantitative microscopy analysis of structural features (e.g., Kranz anatomy, plasmodesmata, cell wall, and organelles). More than 4300 proteins were identified and functionally annotated. Developmental protein accumulation profiles and hierarchical cluster analysis then determined the kinetics of organelle biogenesis, formation of cellular structures, metabolism, and coexpression patterns. Two main expression clusters were observed, each divided in subclusters, suggesting that a limited number of developmental regulatory networks organize concerted protein accumulation along the leaf gradient. The coexpression with BSC and MC markers provided strong candidates for further analysis of C(4) specialization, in particular transporters and biogenesis factors. Based on the integrated information, we describe five developmental transitions that provide a conceptual and practical template for further analysis. An online protein expression viewer is provided through the Plant Proteome Database.

Figure 1.

Structural Analysis of the Maize Leaf along the Developmental Gradient. Transverse sections of maize leaves viewed by LM ([A] to [D]) and TEM ([E] to [L], [O], and [P]) at different positions along the developmental gradient. At the leaf base ([A], [E], and [I]), the small veins are immature, each consisting of a cluster of undifferentiated vascular cells (arrows in [A]) surrounded by a ring of undifferentiated BSCs (marked as b) and MCs (marked as m). In (I), many plasmodesmata are seen in transverse section along the cell wall (arrow), and small starch grains are visible in some proplastids (marked as p; asterisk). At 4 to 5 cm above the leaf base ([B], [F], and [J]), leaf tissue has expanded greatly, with prominent vacuoles and airspaces between cells. Arrows in (B) identify veins. In (F), peroxisomes or plastids (arrow) and mitochondria (asterisks) are visible in vascular parenchyma and companion cells, respectively; sieve elements are marked with an s. (J) also shows that thylakoids in BSCs are singular, but those in the MCs have formed small granal stacks (arrow). At 8 to 9 cm above the leaf base ([C], [G], and [K]), the chloroplasts of both MCs and BSCs have enlarged (arrows in [C]). In (G), sieve elements and xylem tracheary elements are indicated with s and x, respectively. Plasmodesmata (indicated by arrows) crossing the cell wall (cw) are visible in (K). Near the leaf tip ([D], [H], and [L]), the MC chloroplasts have continued to enlarge (arrow in [D]). Chloroplasts in the BSCs are distinctly narrowed and curved ([H] and [L]). At the end of the day ([O] and [P]), starch grains (asterisks) are visible in MC chloroplasts, 2 to 3 cm from the leaf base (O) and in BSC chloroplasts near the leaf tip (P). (M) shows morphometric analyses of cell wall thickness (open squares) and plasmodesmata length (closed circles) of the interface between BSC and MC, whereas epidermal cell length is shown by filled triangles. The vertical dotted line in (M) and (N) indicates the 4-cm point, as measured from the ligule. Chloroplast length (squares) and width (triangle) for BSC and MC are plotted in (N). Bars = 25 μm in (A) to (D), 10 μm in (E), 5 μm in (F) to (H), 1.0 μm in (I) to (L), 3 μm in (O), and 6 μm in (P).

Figure 2.

Proteome Analysis of Leaf and BS Strands along the Leaf Developmental Gradient. (A) Cellular protein accumulation patterns along the leaf (left gel, with a representative leaf showing positions of the samples above) and BS strands (right gel) as evidenced by one-dimensional gels and staining with Coomassie blue. A few marker enzymes are indicated. RBCL, Rubisco large subunit; RBCS, Rubisco small subunit; PAL, phenylalanine ammonia lyase; TKL, transketolase. These markers were identified by MS analysis. (B) Protein investment in various cellular functions per leaf section, calculated from NadjSPC per function, as percentage of total NadjSPC. The percentage of protein mass from mitochondrial (mito. %) and plastid proteins (plastid %) in each section are indicated (explanation for the color scheme, see [C]). (C) Dendrogram of the hierachical cluster analysis of protein expression (calculated from NadjSPC) along the developmental gradient of the leaf and BS strand. The two main clusters (I and II) and their assigned subclusters are indicated. The average profile for the accessions in each cluster is indicated, with closed and open circles for leaf and BS strands, respectively. Protein investments in various cellular functions for all accessions in each cluster are indicated, and the percentage of plastid proteins among the identified proteins (p/t %) in each cluster is indicated. N-S-AA refers to nitrogen and sulfur assimilation and amino acid metabolism (Mapman bins 12, 13, and 14). Construction refers to bins 10, 11, 16, 19, 23, and 25 (see also [D]). Catabolism refers to glycolysis, gluconeogenesis/glyoxylate cycle, OPPP, TCA cycle, mitochondrial electron transport, and oxidative phosphorylation (bins 4 to 9), and DNA and RNA (bins 27 and 28). Regulation and signaling includes hormone metabolism, stress response, redox regulation, signaling, and development (bins 17, 20, 21, 30, 31, and 33). Carbon metabolism includes dark reactions of photosynthesis and major and minor carbohydrate metabolism (bins 1 to 3). (D) Distribution across the clusters of functional groups of proteins involved in construction. These functional groups are metabolism of lipid and fatty acid (bin 11), tetrapyrroles (bin 19), nucleotides (bin 23), as well as C1 metabolism (bin 25), secondary metabolism (bin 16), and cell wall biogenesis (bin 10).

Figure 3.

Experimental and Bioinformatics Workflow of the Proteome Analysis.

Figure 4.

Reproducibility between Biological Replicates. Examples of reproducibility for both soluble and integral membrane proteins between biological replicates of the comparative proteome analysis. (A) Accumulation profiles along the leaf gradient for the two individual biological replicates (based on NadjSPC) for MetS (GRMZM2G149751_P01), SuSy (SuSy2-2; GRMZM2G152908_P01), PEPC (GRMZM2G083841_P01), and the D2 integral membrane protein of the PSII complex (NP_043009). Open and closed symbols are used for replicates 1 and 2, respectively. (B) Cross-correlation plot for the four proteins shown in (A). The inset shows the total number of AdjSPC in the leaf gradient samples and the correlation coefficients for each protein.

Figure 5.

Quantitative Protein Expression Analysis of the Light and Dark Reactions of Photosynthesis and the C₄ Shuttle. (A) Expression of proteins (based on NadjSPC) involved in the light (squares) and dark (triangles) reactions of photosynthesis and the C₄ shuttle (asterisks). The inset shows a comparison of the three pathways with NadjSPC values normalized to the maximum value for each pathway to better compare their accumulation kinetics. (B) Accumulation of thylakoid complexes in the developing BS strand distributed over the subclusters. (C) Accumulation pattern of the five thylakoid complexes in the developing BS strand. (D) Accumulation of NDH, PSII, PSI, and cytb6f normalized to proteins of the thylakoid ATP-synthase complex (CF). The inset compares the protein ratio of NDH/PSII along the developmental gradient in the BS strands (open squares) and leaf (closed squares).

Figure 6.

Division of Cluster II into Subclusters and Expression pProfile of MC and BS Strand Marker Proteins. (A) Part of the dendrogram (cluster II) shown in Figure 2C indicating the subclusters for cluster II. The average profile for the accessions in each subcluster is indicated, with closed and open circles for leaf and BS strands, respectively. Protein investments in various cellular functions for all accessions in each cluster are indicated, and the percentage of plastid proteins among the identified proteins in each cluster is indicated. The color coding for the different function is the same as described in Figure 2. The 4-cm point, calculated from the ligule of the leaf base, is marked. (B) Expression profiles of MC and BSC marker proteins in clusters II-3c and II-3e, respectively. Protein names and accession numbers are indicated. Closed symbols and solid lines indicate leaf samples, whereas open symbols and dashed lines indicate BS strand samples. The 4-cm point, calculated from the ligule of the leaf base, is marked.

Figure 7.

Plastid Envelope Transporters Involved in Carbohydrate Balance That Passed the Minimum Threshold for Clustering. (A) Relative molar abundance of plastid envelope transporters in the leaf calculated from the normalized spectral abundance factor, NSAF*1000. Proteins in gray are in cluster I and proteins in black in cluster II. PPT1-1, phosphate/phosphoenolpyruvate translocator1-1 (GRMZM2G174107_P02; PPT1-2 quantification includes closely related GRMZM2G103047_P01 and GRMZM2G047404_P01); MEP1,2,3,4, inner envelope transporter proteins (GRMZM2G071423_P01, GRMZM2G077222_P01, GRMZM2G305851_P01, and GRMZM2G138258_P01, respectively); TPT1, phosphate/triose-phosphate translocator-1 (GRMZM2G070605_P01); GLT1-1, glucose transporter 1-1 (also named GlcT1-1) (GRMZM2G153704_P02); MEX1, maltose exporter (formerly root cap 1 [RCP1]; GRMZM2G156356_P01); PHT4, Pi transporter (also named ANTR1; GRMZM2G088196_P01). (B) Expression of transporter proteins along the developmental gradient in leaves (closed squares and solid lines) and BS strands (open squares and dashed lines). The cluster number is indicated. The 4-cm point, calculated from the ligule of the leaf base, is marked.

Figure 8.

Expression Profiles of Glycolysis, Starch, and Sucrose Metabolism. (A) Cumulative expression of marker proteins involved in sugar synthesis (squares) and degradation (diamonds) along the developmental gradient in leaves (closed symbols and solid lines) and BS strands (open symbols and dashed lines). The signal for sucrose synthesis was multiplied by a factor 30 for better visibility. Marker proteins for sucrose synthesis were sucrose phosphatase (SP1; GRMZM2G055489_P01), sucrose phosphate synthase 2 (SPS-2; GRMZM2G013166_P03 and GRMZM2G140107_P01), sucrose phosphate synthase 3 (SPS-3; GRMZM2G008507_P01), and d-fructose-1,6-bisphosphate 1-phosphohydrolase (F16BPase; GRMZM2G322953_P01). Markers for sucrose degradation were sucrose synthase 1 (SUS1; GRMZM2G089713_P01), sucrose synthase 2-2 (SUS2-2; GRMZM2G152908_P01), sucrose synthase 2-1 (SUS2-1; GRMZM2G060659_P01), fructokinase-1 (FK1; GRMZM2G086845_P01), and fructokinase-2 (FK2; GRMZM2G051677_P01). (B) Comparative analysis of the kinetics of the Calvin-Benson cycle (open squares), starch synthesis (filled squares), starch degradation (filled circles), sucrose synthesis (open diamond), and sucrose gradation (filled diamond) in the BS strands along the developmental gradient. The point of 50% capacity of the various pathways is indicated. NadjSPC for each function are normalized to the maximum value for each function. (C) Cumulative expression of proteins involved in starch synthesis (squares), starch degradation (circles) and β-amylase 5 (BAM5; GRMZM2G058310_P01) (filled triangles) along the developmental gradient in leaves (closed symbols) and BS strands (open symbols). Included in the calculation for starch synthesis were ADP-glucose pyrophosphorylase large subunit 1,2 (GRMZM2G391936_P02 and GRMZM2G027955_P01), ADP-glucose pyrophosphorylase small subunit 1 (APS1; GRMZM2G163437_P01), granule-associated starch synthase (GBSS; GRMZM2G008263_P01), starch synthase I (SSI; GRMZM2G129451_P01), starch synthase IIa (SSIIa; GRMZM2G105791_P01), starch branching enzyme class IIb-2 (BEIIb; GRMZM2G073054_P01), starch synthase IIIb (SSIIIb; GRMZM2G121612_P01), and starch (amylose) binding protein (GRMZM2G042245_P01). Included in starch degradation are α-glucan phosphorylase-2-1,2 (PHS2-1,2; GRMZM2G147770_P01 and GRMZM2G085577_P01), glucan water dikinase (GWD also named Sex1 or R1 protein; GRMZM2G412611_P01), glucan-phosphorylase 1 (PHS1; GRMZM2G074158_P01), phosphoglucan water dikinase (PWD; GRMZM2G040968_P04), α-amylase 3 (AMY3; AC207628.4_FGP006), and dual-specificity protein phosphatase 4 (DSP4 or SEX4; GRMZM2G052546_P03). (D) Cumulative expression of proteins involved in glycolysis (squares) and the irreversible steps of OPPP (triangles) along the developmental gradient in leaves (closed symbols) and BS strands (open symbols). To calculate the profiles for glycolysis and irreversible steps in OPPP, 29 and eight protein accessions were used, respectively. The signal for OPPP was multiplied by a factor 10 for better visibility.

Figure 9.

Sink-Source Relationships in Primary Carbon Metabolism. This figure describes the pathways and identified transporters involved in carbon metabolism that are critical for the sink-source transition along the developing leaf. The expression profiles along the developmental gradient in leaves (closed symbols) and BS strands (open symbols) for key enzymes and transporters are shown in line plots. The figure summarizes carbon metabolism in photosynthetic chloroplasts in the source tissue (toward the tip) and carbon metabolism on nonphotosynthetic, heterotrophic plastids at the base of the leaf, the sink region. For simplicity, we did not show the metabolic exchange and specialization of source BSC and MC chloroplasts; instead, they are collectively summarized under the term “source plastid.” Reduced carbohydrates that are exported from the source plastids are converted into sucrose and then transported via the phloem to the sink region. In the sink region, the sucrose can be transiently stored (e.g., in the vacuoles), and sucrose is then degraded into hexose phosphates (G6P, G1P, and F6P) through two parallel pathways either involving invertase or SuSy. The hexosphosphates are then used in the cytosol or imported into the heterotrophic plastids for glycolysis or the OPPP. Blue arrows indicate the generation of reduced carbohydrates and sucrose in sink tissue. DHAP, dehydroxyacetone phosphate; G1P, glucose-1-phosphate; GAP, glyceraldehyde-3-phosphate; F16BP, fructose-1,6-biphosphate; F26BP, fructose-2,6-biphosphate; Pi, inorganic phosphate; PEP, phosphoenolpyruvate; R5P, ribulose-5-phosphate. The inset (right-hand side) shows a close-up of the expression line plot for MEX1; all other plots have a similar x and y axis, and the vertical dotted line indicates the 4-cm point.

Figure 10.

Induction of Photorespiration and Expression of the Vacuolar V-Type ATPase. (A) Expression patterns of the specific photorespiratory enzymes phosphoglycolate-phosphatase-2 (PGP-2; GRMZM2G018441_P01; squares) and glycolate oxidase 2 (GOX-2; GRMZM2G129246_P01; circles), in plastids and peroxisomes, respectively, along the developmental gradient in leaves (closed symbols) and BS strands (open symbols). (B) Cumulative expression pattern of the (near identical) ammonia transporter homologs in the tonoplast (TIP2; GRMZM2G027098_P01, GRMZM2G121275_P01, and GRMZM2G056908_P01) along the developmental gradient in leaves (closed symbols) and BS strands (open symbols). (C) Cumulative expression pattern of the 11 subunits of the V-type ATPase (20 accession numbers; see Supplemental Data Set 1B online) along the developmental gradient in leaves (closed symbols) and BS strands (open symbols).

Figure 11.

Quantitative Protein Expression of Mitochondrial Respiration and Transporters, Protein Synthesis and Organelle Biogenesis, and Isoprenoid and Tetrapyrrole Metabolism. (A) Cumulative expression of proteins involved in the TCA cycle (circles) and the mitochondrial electron chain and oxidative phosphorylation (squares) along the developmental gradient in leaves (closed symbols) and BS strands (open symbols). The inset shows a comparison of the two pathways with NadjSPC values normalized to the maximum value for each pathway. (B) Cumulative expression of mitochondrial outer membrane porins (circles) and inner membrane transporters (squares) along the developmental gradient in leaves (closed symbols) and BS strands (open symbols). The inset shows a comparison of these groups with NadjSPC values normalized to the maximum value for each group. (C) Cumulative expression of proteins involved in cytosolic translation (squares) and plastid translation (circles) along the developmental gradient in leaves (closed symbols) and BS strands (open symbols). The inset shows a comparison of the two groups with NadjSPC values normalized to the maximum value for each group. (D) Cumulative expression of proteins involved in biogenesis in mitochondria (squares) and plastids (circles) along the developmental gradient in leaves (closed symbols) and BS strands (open symbols). Values for mitochondria are multiplied by 10. (E) Cumulative expression of proteins involved in the plastidic deoxyxylulose phosphate (MEP) pathway (squares), the cytosolic mevalonate (MVA) pathway (triangles), and four plastid enzymes operating immediately downstream of the MEP pathway (post-MEP; circles) along the developmental gradient in leaves (closed symbols and solid lines) and BS strands (open symbols). (F) Cumulative expression of proteins involved in the tetrapyrrole pathway (squares) and of protochlorophyllide reductase A (triangles) along the developmental gradient in leaves (closed symbols) and BS strands (open symbols).

Figure 12.

Expression of Plastid Proteolytic Systems along the Leaf Developmental Gradient. (A) Relative molar abundance of nine different plastid protease systems along the leaf gradient calculated from the NSAF. Color coding is explained in the figure. 5x, The NSAF values are multiplied by 5 to make the bars better visible; AP, aminopeptidase; EP, endopeptidase (B) and (E) to (G) Expression profile of ClpC homologs and ClpPRT subunits (A), thylakoid FtsH proteases (E), stromal PreP1 (F), and stromal DegP2 (G) along the developmental gradient in leaves (closed symbols) and BS strands (open symbols). (C) Accumulation pattern of the total plastid and thylakoid proteomes along the developmental gradient in leaves (closed symbols) and BS strands (open symbols). (D) Expression profile of the ClpC chaperones, ClpPRT subunits, and thylakoid FtsH subunits normalized to the total plastid proteome (based on NadjSPC) along the developmental gradient in leaves.

Figure 13.

Expression of Proteins Involved in ROS Detoxification and Redox Regulation along the Developmental Gradient in Leaves and BS Strands. Leaves, closed symbols; BS strands, open symbols. (A) Expression pattern of plastid Cu,Zn-SOD. (B) Cumulative accumulation of the ascorbate detoxification system within and outside of the plastid. (C) Expression of plastid thioredoxins f2, m2, m4, and plastid thioredoxin reductase, as well as extraplastidic thioredoxin h.

Figure 14.

Expression Patterns in Leaf Sections for Enzymes in Metabolism of Phospholipids, FAs, Wax, Brassinosteroids, and JA. Phospholipids, fatty acids, and wax (A) and brassinosteroids and JA (B). ACCase, acetyl-CoA carboxylase; AOS, allene oxide synthase; AOC, allene oxide cyclase; LOX, lipoxygenase; 5x and 10x, the NadjSPC values are multiplied by 5 or 10 to make the data points better visible.

Figure 15.

Summarizing Overview of the Observed Transients along the Leaf Developmental Gradient and Differentials between the BS Strand and the MCs. The top part of the figure summarizes anatomical and structural features. The bottom part of the figure summarizes observations based on proteomics data. Processes and features in blue are prevalent in the sink part of the leaf, while those in red are prevalent in the source part of the leaf. The vertical dashed line indicates the sink-source transition point. Lines in orange and green refer to processes or proteins, respectively, that are most prominent in the sink (cluster I) and source region (cluster II). *Mitoch./plastid ratio, refers to cross-section ratio of mitochondria/plastids; SAM, S-adenosylmethionine.