Chloroplasts in differentiated bundle sheath (BS) and mesophyll (M) cells of maize (Zea mays) leaves are specialized to accommodate C 4 photosynthesis. This study provides a reconstruction of how metabolic pathways, protein expression, and homeostasis functions are quantitatively distributed across BS and M chloroplasts. This yielded new insights into cellular specialization. The experimental analysis was based on high-accuracy mass spectrometry, protein quantification by spectral counting, and the first maize genome assembly. A bioinformatics workflow was developed to deal with gene models, protein families, and gene duplications related to the polyploidy of maize; this avoided overidentification of proteins and resulted in more accurate protein quantification. A total of 1,105 proteins were assigned as potential chloroplast proteins, annotated for function, and quantified. Nearly complete coverage of primary carbon, starch, and tetrapyrole metabolism, as well as excellent coverage for fatty acid synthesis, isoprenoid, sulfur, nitrogen, and amino acid metabolism, was obtained. This showed, for example, quantitative and qualitative cell type-specific specialization in starch biosynthesis, arginine synthesis, nitrogen assimilation, and initial steps in sulfur assimilation. An extensive overview of BS and M chloroplast protein expression and homeostasis machineries (more than 200 proteins) demonstrated qualitative and quantitative differences between M and BS chloroplasts and BS-enhanced levels of the specialized chaperones ClpB3 and HSP90 that suggest active remodeling of the BS proteome. The reconstructed pathways are presented as detailed flow diagrams including annotation, relative protein abundance, and cell-specific expression pattern. Protein annotation and identification data, and projection of matched peptides on the protein models, are available online through the Plant Proteome Database.Plants can be classified as C 3 or C 4 species based on the primary product of carbon fixation in photosynthesis. The primary product of carbon fixation is a four-carbon compound (oxaloacetate [OAA]) in C 4 plants but a three-carbon compound (3-phosphoglycerate [3PGA]) in C 3 plants. In leaves of C 4 grasses such as maize (Zea mays), photosynthetic activities are partitioned between two anatomically and biochemically distinct bundle sheath (BS) and mesophyll (M) cells. A single ring of BS cells surrounds the vascular bundle, followed by a concentric ring of specialized M cells, creating the classical Kranz anatomy. Active carbon transport (in the form of C 4 organic acids) from M cell to BS cells and specific expression of Rubisco in the BS cells allows Rubisco, the carboxylating enzyme in the Calvin cycle, to operate in a high CO 2 concentration. The high CO 2 concentration suppresses the oxygenation reaction by Rubisco (and the subsequent energy-wasteful photorespiratory pathway), resulting in increased photosynthetic yield and more efficient use of water and nitrogen. The history of C 4 research has been described (Ne...

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.

C4 grasses, such as maize (Zea mays), have high photosynthetic efficiency through combined biochemical and structural adaptations. C4 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 C4 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 C4 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.

We report identification of a new human nucleoside transporter protein by molecular cloning and functional expression of its cDNA. Previously, we used expression selection in Xenopus oocytes to isolate a cDNA from rat jejunal epithelium encoding the pyrimidine-selective Na+-dependent nucleoside transporter rCNT1 (Q.-Q. Huang, S. Y. M. Yao, M. W. L. Ritzel, A. R. P. Paterson, C. E. Cass, and J. D. Young. J. Biol. Chem. 269: 17757-17760, 1994). cDNAs for a human homologue of rCNT1, designated hCNT1, have been isolated from human kidney by hybridization cloning and reverse transcriptase polymerase chain reaction amplification strategies. hCNT1 was 83% identical to rCNT1 in amino acid sequence and exhibited the transport characteristics of an Na+-dependent nucleoside transporter with selectivity for pyrimidine nucleosides and adenosine when expressed in Xenopus oocytes. Deoxyadenosine, which undergoes net renal secretion, and guanosine were poor permeants. hCNT1 did, however, transport 3'-azido-3'-deoxythymidine. This is the first demonstration that members of the CNT family exist in human cells and provides evidence of their involvement in the renal transport of physiological nucleosides and nucleoside drugs. The hCNT1 gene was mapped to chromosome 15q25-26.