Integrating ATAC-seq and RNA-seq to identify differentially expressed genes with chromatin-accessible changes during photosynthetic establishment in Populus leaves.

Leaves are the primary photosynthetic organs, providing essential substances for tree growth. It is important to obtain an anatomical understanding and regulatory network analysis of leaf development. Here, we studied leaf development in Populus Nanlin895 along a development gradient from the newly emerged leaf from the shoot apex to the sixth leaf (L1 to L6) using anatomical observations and RNA-seq analysis. It indicated that mesophyll cells possess obvious vascular, palisade, and spongy tissue with distinct intercellular spaces after L3. Additionally, vacuoles fuse while epidermal cells expand to form pavement cells. RNA-seq analysis indicated that genes highly expressed in L1 and L2 were related to cell division and differentiation, while those highly expressed in L3 were enriched in photosynthesis. Therefore, we selected L1 and L3 to integrate ATAC-seq and RNA-seq and identified 735 differentially expressed genes (DEGs) with changes in chromatin accessibility regions within their promoters, of which 87 were transcription factors (TFs), such as ABI3VP1, AP-EREBP, MYB, NAC, and GRF. Motif enrichment analysis revealed potential regulatory functions for the DEGs through upstream TFs including TCP, bZIP, HD-ZIP, Dof, BBR-BPC, and MYB. Overall, our research provides a potential molecular foundation for regulatory network exploration in leaf development during photosynthesis establishment.

OsTST1, a key tonoplast sugar transporter from source to sink, plays essential roles in affecting yields and height of rice (Oryza sativa L.).

MAIN CONCLUSION: OsTST1 affects yield and development and mediates sugar transportation of plants from source to sink in rice, which influences the accumulation of intermediate metabolites from tricarboxylic acid cycle indirectly. Tonoplast sugar transporters (TSTs) are essential for vacuolar sugar accumulation in plants. Carbohydrate transport across tonoplasts maintains the metabolic balance in plant cells, and carbohydrate distribution is crucial to plant growth and productivity. Large plant vacuoles store high concentrations of sugars to meet plant requirements for energy and other biological processes. The abundance of sugar transporter affects crop biomass and reproductive growth. However, it remains unclear whether the rice (Oryza sativa L.) sugar transport protein OsTST1 affects yield and development. In this study, we found that OsTST1 knockout mutants generated via CRISPR/Cas9 exhibited slower development, smaller seeds, and lower yield than wild type (WT) rice plants. Notably, plants overexpressing OsTST1 showed the opposite effects. Changes in rice leaves at 14 days after germination (DAG) and at 10 days after flowering (DAF) suggested that OsTST1 affected the accumulation of intermediate metabolites from the glycolytic pathway and the tricarboxylic acid (TCA) cycle. The modification of the sugar transport between cytosol and vacuole mediated by OsTST1 induces deregulation of several genes including transcription factors (TFs). In summary, no matter the location of sucrose and sink is, these preliminary results revealed that OsTST1 was important for sugar transport from source to sink tissues, thus affecting plant growth and development.

A dynamic phosphoproteomic analysis provides insight into the C4 plant maize (Zea mays L.) response to natural diurnal changes.

As sessile organisms, plants need to respond to rapid changes in numerous environmental factors, mainly diurnal changes of light, temperature, and humidity. Maize is the world's most grown crop, and as a C4 plant it exhibits high photosynthesis capacity, reaching the highest rate of net photosynthesis at midday; that is, there is no "midday depression." Revealing the physiological responses to diurnal changes and underlying mechanisms will be of great significance for guiding maize improvement efforts. In this study, we collected maize leaf samples and analyzed the proteome and phosphoproteome at nine time points during a single day/night cycle, quantifying 7424 proteins and 5361 phosphosites. The new phosphosites identified in our study increased the total maize phosphoproteome coverage by 8.5%. Kinase-substrate network analysis indicated that 997 potential substrates were phosphorylated by 20 activated kinases. Through analysis of proteins with significant changes in abundance and phosphorylation, we found that the response to a heat stimulus involves a change in the abundance of numerous proteins. By contrast, the high light at noon and rapidly changing light conditions induced changes in the phosphorylation level of proteins involved in processes such as chloroplast movement, photosynthesis, and C4 pathways. Phosphorylation is involved in regulating the activity of large number of enzymes; for example, phosphorylation of S55 significantly enhanced the activity of maize phosphoenolpyruvate carboxykinase1 (ZmPEPCK1). Overall, the database of dynamic protein abundance and phosphorylation we have generated provides a resource for the improvement of C4 crop plants.

OsAPL controls the nutrient transport systems in the leaf of rice (Oryza sativa L.).

MAIN CONCLUSION: OsAPL positively controls the seedling growth and grain size in rice by targeting the plasma membrane H+-ATPase-encoding gene, OsRHA1, as well as drastically affects genes encoding H+-coupled secondary active transporters. Nutrient transport is a key component of both plant growth and environmental adaptation. Photosynthates and nutrients produced in the source organs (e.g., leaves) need to be transported to the sink organs (e.g., seeds). In rice, the unloading of nutrients occurs through apoplastic transport (i.e., across the membrane via transporters) and is dependent on the efficiency and number of transporters embedded in the cell membrane. However, the genetic mechanisms underlying the regulation of these transporters remain to be determined. Here we show that rice (Oryza sativa L., Kitaake) ALTERED PHLOEM DEVELOPMENT (OsAPL), homologous to a MYB family transcription factor promoting phloem development in Arabidopsis thaliana, regulates the number of transporters in rice. Overexpression of OsAPL leads to a 10% increase in grain yield at the heading stage. OsAPL acts as a transcriptional activator of OsRHA1, which encodes a subunit of the plasma membrane H+-ATPase (primary transporter). In addition, OsAPL strongly affects the expression of genes encoding H+-coupled secondary active transporters. Decreased expression of OsAPL leads to a decreased expression level of nutrient transporter genes. Taken together, our findings suggest the involvement of OsAPL in nutrients transport and crop yield accumulation in rice.

PdeHCA2 affects biomass in Populus by regulating plant architecture, the transition from primary to secondary growth, and photosynthesis.

MAIN CONCLUSION: PdeHCA2 regulates the transition from primary to secondary growth, plant architecture, and affects photosynthesis by targeting PdeBRC1 and controlling the anatomy of the mesophyll, and intercellular space, respectively. Branching, secondary growth, and photosynthesis are vital developmental processes of woody plants that determine plant architecture and timber yield. However, the mechanisms underlying these processes are unknown. Here, we report that the Populus transcription factor High Cambium Activity 2 (PdeHCA2) plays a role in the transition from primary to secondary growth, vascular development, and branching. In Populus, PdeHCA2 is expressed in undifferentiated provascular cells during primary growth, in phloem cells during secondary growth, and in leaf veins, which is different from the expression pattern of its homolog in Arabidopsis. Overexpression of PdeHCA2 has pleiotropic effects on shoot and leaf development; overexpression lines showed delayed growth of shoots and leaves, reduced photosynthesis, and abnormal shoot branching. In addition, auxin-, cytokinin-, and photosynthesis-related genes were differentially regulated in these lines. Electrophoretic mobility shift assays and transcriptome analysis indicated that PdeHCA2 directly up-regulates the expression of BRANCHED1 and the MADS-box gene PdeAGL9, which regulate plant architecture, by binding to cis-elements in the promoters of these genes. Taken together, our findings suggest that HCA2 regulates several processes in woody plants including vascular development, photosynthesis, and branching by affecting the proliferation and differentiation of parenchyma cells.

Genome-wide transcriptome and proteome profiles indicate an active role of alternative splicing during de-etiolation of maize seedlings.

MAIN CONCLUSION: AS events affect genes encoding protein domain composition and make the single gene produce more proteins with a certain number of genes to satisfy the establishment of photosynthesis during de-etiolation. The drastic switch from skotomorphogenic to photomorphogenic development is an excellent system to elucidate rapid developmental responses to environmental stimuli in plants. To decipher the effects of different light wavelengths on de-etiolation, we illuminated etiolated maize seedlings with blue, red, blue-red mixed and white light, respectively. We found that blue light alone has the strongest effect on photomorphogenesis and that this effect can be attributed to the higher number and expression levels of photosynthesis and chlorosynthesis proteins. Deep sequencing-based transcriptome analysis revealed gene expression changes under different light treatments and a genome-wide alteration in alternative splicing (AS) profiles. We discovered 41,188 novel transcript isoforms for annotated genes, which increases the percentage of multi-exon genes with AS to 63% in maize. We provide peptide support for all defined types of AS, especially retained introns. Further in silico prediction revealed that 58.2% of retained introns have changes in domains compared with their most similar annotated protein isoform. This suggests that AS acts as a protein function switch allowing rapid light response through the addition or removal of functional domains. The richness of novel transcripts and protein isoforms also demonstrates the potential and importance of integrating proteomics into genome annotation in maize.

A comprehensive analysis of the lysine acetylome reveals diverse functions of acetylated proteins during de-etiolation in Zea mays.

Lysine acetylation is one of the most important post-translational modifications and is involved in multiple cellular processes in plants. There is evidence that acetylation may play an important role in light-induced de-etiolation, a key developmental switch from skotomorphogenesis to photomorphogenesis. During this transition, establishment of photosynthesis is of great significance. However, studies on acetylome dynamics during de-etiolation are limited. Here, we performed the first global lysine acetylome analysis for Zea mays seedlings undergoing de-etiolation, using nano liquid chromatography coupled to tandem mass spectrometry, and identified 814 lysine-acetylated sites on 462 proteins. Bioinformatics analysis of this acetylome showed that most of the lysine-acetylated proteins are predicted to be located in the cytoplasm, nucleus, chloroplast, and mitochondria. In addition, we detected ten lysine acetylation motifs and found that the accumulation of 482 lysine-acetylated peptides corresponding to 289 proteins changed significantly during de-etiolation. These proteins include transcription factors, histones, and proteins involved in chlorophyll synthesis, photosynthesis light reaction, carbon assimilation, glycolysis, the TCA cycle, amino acid metabolism, lipid metabolism, and nucleotide metabolism. Our study provides an in-depth dataset that extends our knowledge of in vivo acetylome dynamics during de-etiolation in monocots. This dataset promotes our understanding of the functional consequences of lysine acetylation in diverse cellular metabolic regulatory processes, and will be a useful toolkit for further investigations of the lysine acetylome and de-etiolation in plants.

Large-scale Proteomic and Phosphoproteomic Analyses of Maize Seedling Leaves During De-etiolation.

De-etiolation consists of a series of developmental and physiological changes that a plant undergoes in response to light. During this process light, an important environmental signal, triggers the inhibition of mesocotyl elongation and the production of photosynthetically active chloroplasts, and etiolated leaves transition from the "sink" stage to the "source" stage. De-etiolation has been extensively studied in maize (Zea mays L.). However, little is known about how this transition is regulated. In this study, we described a quantitative proteomic and phosphoproteomic atlas of the de-etiolation process in maize. We identified 16,420 proteins in proteome, among which 14,168 proteins were quantified. In addition, 8746 phosphorylation sites within 3110 proteins were identified. From the combined proteomic and phosphoproteomic data, we identified a total of 17,436 proteins. Only 7.0% (998/14,168) of proteins significantly changed in abundance during de-etiolation. In contrast, 26.6% of phosphorylated proteins exhibited significant changes in phosphorylation level; these included proteins involved in gene expression and homeostatic pathways and rate-limiting enzymes involved in photosynthetic light and carbon reactions. Based on phosphoproteomic analysis, 34.0% (1057/3110) of phosphorylated proteins identified in this study contained more than 2 phosphorylation sites, and 37 proteins contained more than 16 phosphorylation sites, indicating that multi-phosphorylation is ubiquitous during the de-etiolation process. Our results suggest that plants might preferentially regulate the level of posttranslational modifications (PTMs) rather than protein abundance for adapting to changing environments. The study of PTMs could thus better reveal the regulation of de-etiolation.

Genome-wide transcriptional adaptation to salt stress in Populus.

BACKGROUND: Adaptation to abiotic stresses is crucial for the survival of perennial plants in a natural environment. However, very little is known about the underlying mechanisms. Here, we adopted a liquid culture system to investigate plant adaptation to repeated salt stress in Populus trees. RESULTS: We first evaluated phenotypic responses and found that plants exhibit better stress tolerance after pre-treatment of salt stress. Time-course RNA sequencing (RNA-seq) was then performed to profile changes in gene expression over 12 h of salt treatments. Analysis of differentially expressed genes (DEGs) indicated that significant transcriptional reprogramming and adaptation to repeated salt treatment occurred. Clustering analysis identified two modules of co-expressed genes that were potentially critical for repeated salt stress adaptation, and one key module for salt stress response in general. Gene Ontology (GO) enrichment analysis identified pathways including hormone signaling, cell wall biosynthesis and modification, negative regulation of growth, and epigenetic regulation to be highly enriched in these gene modules. CONCLUSIONS: This study illustrates phenotypic and transcriptional adaptation of Populus trees to salt stress, revealing novel gene modules which are potentially critical for responding and adapting to salt stress.

The developmental dynamics of the Populus stem transcriptome.

The Populus shoot undergoes primary growth (longitudinal growth) followed by secondary growth (radial growth), which produces biomass that is an important source of energy worldwide. We adopted joint PacBio Iso-Seq and RNA-seq analysis to identify differentially expressed transcripts along a developmental gradient from the shoot apex to the fifth internode of Populus Nanlin895. We obtained 87 150 full-length transcripts, including 2081 new isoforms and 62 058 new alternatively spliced isoforms, most of which were produced by intron retention, that were used to update the Populus annotation. Among these novel isoforms, there are 1187 long non-coding RNAs and 356 fusion genes. Using this annotation, we found 15 838 differentially expressed transcripts along the shoot developmental gradient, of which 1216 were transcription factors (TFs). Only a few of these genes were reported previously. The differential expression of these TFs suggests that they may play important roles in primary and secondary growth. AP2, ARF, YABBY and GRF TFs are highly expressed in the apex, whereas NAC, bZIP, PLATZ and HSF TFs are likely to be important for secondary growth. Overall, our findings provide evidence that long-read sequencing can complement short-read sequencing for cataloguing and quantifying eukaryotic transcripts and increase our understanding of the vital and dynamic process of shoot development.

Large-scale Identification and Time-course Quantification of Ubiquitylation Events During Maize Seedling De-etiolation.

The ubiquitin system is crucial for the development and fitness of higher plants. De-etiolation, during which green plants initiate photomorphogenesis and establish autotrophy, is a dramatic and complicated process that is tightly regulated by a massive number of ubiquitylation/de-ubiquitylation events. Here we present site-specific quantitative proteomic data for the ubiquitylomes of de-etiolating seedling leaves of Zea mays L. (exposed to light for 1, 6, or 12 h) achieved through immunoprecipitation-based high-resolution mass spectrometry (MS). Through the integrated analysis of multiple ubiquitylomes, we identified and quantified 1926 unique ubiquitylation sites corresponding to 1053 proteins. We analyzed these sites and found five potential ubiquitylation motifs, KA, AXK, KXG, AK, and TK. Time-course studies revealed that the ubiquitylation levels of 214 sites corresponding to 173 proteins were highly correlated across two replicate MS experiments, and significant alterations in the ubiquitylation levels of 78 sites (fold change >1.5) were detected after de-etiolation for 12 h. The majority of the ubiquitylated sites we identified corresponded to substrates involved in protein and DNA metabolism, such as ribosomes and histones. Meanwhile, multiple ubiquitylation sites were detected in proteins whose functions reflect the major physiological changes that occur during plant de-etiolation, such as hormone synthesis/signaling proteins, key C4 photosynthetic enzymes, and light signaling proteins. This study on the ubiquitylome of the maize seedling leaf is the first attempt ever to study the ubiquitylome of a C4 plant and provides the proteomic basis for elucidating the role of ubiquitylation during plant de-etiolation.

Dynamic N-glycoproteome analysis of maize seedling leaves during de-etiolation using Concanavalin A lectin affinity chromatography and a nano-LC-MS/MS-based iTRAQ approach.

KEY MESSAGE: The identification of N -glycosylated proteins with information about changes in the level of N -glycosylation during de-etiolation provides a database that will aid further research on plant N -glycosylation and de-etiolation. N-glycosylation is one of the most prominent and abundant protein post-translational modifications in all eukaryotes and in plants it plays important roles in development, stress tolerance and immune responses. Because light-induced de-etiolation is one of the most dramatic developmental processes known in plants, seedlings undergoing de-etiolation are an excellent model for investigating dynamic proteomic profiles. Here, we present a comprehensive, quantitative N-glycoproteomic profile of maize seedlings undergoing 12 h of de-etiolation obtained using Concanavalin A (Con A) lectin affinity chromatography enrichment coupled with a nano-LC-MS/MS-based iTRAQ approach. In total, 1084 unique N-glycopeptides carrying 909 N-glycosylation sites and corresponding to 609 proteins were identified and quantified, including 186 N-glycosylation sites from 162 proteins that were significantly regulated over the course of the 12 h de-etiolation period. Based on hierarchical clustering analysis, the significantly regulated N-glycopeptides were divided into seven clusters that showed different N-glycosylation patterns during de-etiolation. We found no obvious difference in the enriched MapMan bincode categories for each cluster, and these clustered significantly regulated N-glycoproteins (SRNPs) are enriched in miscellaneous, protein, cell wall and signaling, indicating that although the N-glycosylation regulation patterns of these SRNPs might differ, they are involved in similar biological processes. Overall, this study represents the first large-scale quantitative N-glycoproteome of the model C4 plant, maize, which is one of the most important cereal and biofuel crops. Our results greatly expand the maize N-glycoproteomic database and also shed light on the potential roles of N-glycosylation modification during the greening of maize leaves.

Histone Acetylation Modifications Affect Tissue-Dependent Expression of Poplar Homologs of C4 Photosynthetic Enzyme Genes.

Histone modifications play important roles in regulating the expression of C4 photosynthetic genes. Given that all enzymes required for the C4 photosynthesis pathway are present in C3 plants, it has been hypothesized that this expression regulatory mechanism has been conserved. However, the relationship between histone modification and the expression of homologs of C4 photosynthetic enzyme genes has not been well determined in C3 plants. In the present study, we cloned nine hybrid poplar (Populus simonii × Populus nigra) homologs of maize (Zea mays) C4 photosynthetic enzyme genes, carbonic anhydrase (CA), pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxykinase (PCK), and phosphoenolpyruvate carboxylase (PEPC), and investigated the correlation between the expression levels of these genes and the levels of promoter histone acetylation modifications in four vegetative tissues. We found that poplar homologs of C4 homologous genes had tissue-dependent expression patterns that were mostly well-correlated with the level of histone acetylation modification (H3K9ac and H4K5ac) determined by chromatin immunoprecipitation assays. Treatment with the histone deacetylase inhibitor trichostatin A further confirmed the role of histone acetylation in the regulation of the nine target genes. Collectively, these results suggest that both H3K9ac and H4K5ac positively regulate the tissue-dependent expression pattern of the PsnCAs, PsnPPDKs, PsnPCKs, and PsnPEPCs genes and that this regulatory mechanism seems to be conserved among the C3 and C4 species. Our findings provide new insight that will aid efforts to modify the expression pattern of these homologs of C4 genes to engineer C4 plants from C3 plants.

Phylogenic and phosphorylation regulation difference of phosphoenolpyruvate carboxykinase of C3 and C4 plants.

In C4 plants, phosphoenolpyruvate carboxykinase (PEPCK) plays a key role in the C4 cycle. PEPCK is also involved in gluconeogenesis and is conserved in both lower and higher organisms, including in animals and plants. A phylogenic tree constructed from PEPCK sequences from bacteria to higher plants indicates that the C4 Poaceae PEPCKs are conserved and have diverged from the PEPCKs of C3 plants. The maximum enzymatic activities of wild-type and phosphorylation mimic PEPCK proteins indicate that there is a significant difference between C3 and C4 plant PEPCKs. The conserved PEPCK phosphorylation sites are regulated differently in C3 and C4 plants. These results suggest that the functions of PEPCK have been conserved, but that sequences have diverged and regulation of PEPCK is important in C4 plants, but not in herbaceous and, in particular, woody C3 plants.

Proteomics analysis reveals the molecular mechanism underlying the transition from primary to secondary growth of poplar.

Wood is the most important natural source of energy and also provides fuel and fiber. Considering the significant role of wood, it is critical to understand how wood is formed. Integration of knowledge about wood development at the cellular and molecular levels will allow more comprehensive understanding of this complex process. In the present study, we used a comparative proteomic approach to investigate the differences in protein profiles between primary and secondary growth in young poplar stems using tandem mass tag (TMT)-labeling. More than 10,816 proteins were identified, and, among these, 3106 proteins were differentially expressed during primary to secondary growth. Proteomic data were validated using a combination of histochemical staining, enzyme activity assays, and quantitative real-time PCR. Bioinformatics analysis revealed that these differentially expressed proteins are related to various metabolic pathways, mainly including signaling, phytohormones, cell cycle, cell wall, secondary metabolism, carbohydrate and energy metabolism, and protein metabolism as well as redox and stress pathways. This large proteomics dataset will be valuable for uncovering the molecular changes occurring during the transition from primary to secondary growth. Further, it provides new and accurate information for tree breeding to modify wood properties.

Analysis of gene expression and histone modification between C4 and non-C4 homologous genes of PPDK and PCK in maize.

More efficient photosynthesis has allowed C4 plants to adapt to more diverse ecosystems (such as hot and arid conditions) than C3 plants. To better understand C4 photosynthesis, we investigated the expression patterns of C4 genes (C4PPDK and PCK1) and their non-C4 homologous genes (CyPPDK1, CyPPDK2, and PCK2) in the different organs of maize (Zea mays). Both C4 genes and non-C4 genes showed organ-dependent expression patterns. The mRNA levels of C4 genes were more abundant in leaf organ than in seeds at 25 days after pollination (DAP), while non-C4 genes were mainly expressed in developing seeds. Further, acetylation of histone H3 lysine 9 (H3K9ac) positively correlates with mRNA levels of C4 genes (C4PPDK and PCK1) in roots, stems, leaves, and seeds at 25 DAP, acetylation of histone H4 lysine 5 (H4K5ac) in the promoter regions of both C4 (C4PPDK and PCK1) and non-C4 genes (CyPPDK1, CyPPDK2, and PCK2) correlated well with their transcripts abundance in stems. In photosynthetic organs (stems and leaves), dimethylation of histone H3 lysine 9 (H3K9me2) negatively correlated with mRNA levels of both C4 and non-C4 genes. Taken together, our data suggest that histone modification was involved in the transcription regulation of both C4 genes and non-C4 genes, which might provide a clue of the functional evolution of C4 genes.

Comparative proteomics of leaves found at different stem positions of maize seedlings.

To better understand the roles of leaves at different stem positions during plant development, we measured the physiological properties of leaves 1-4 on maize seedling stems, and performed a proteomics study to investigate the differences in protein expression in the four leaves using two-dimensional difference gel electrophoresis and tandem mass spectrometry in conjunction with database searching. A total of 167 significantly differentially expressed protein spots were found and identified. Of these, 35% are involved in photosynthesis. By further analysis of the data, we speculated that in leaf 1 the seedling has started to transition from a heterotroph to an autotroph, development of leaf 2 is the time at which the seedling fully transitions from a heterotroph to an autotroph, and leaf maturity was reached only with fully expanded leaves 3 and 4, although there were still some protein expression differences in the two leaves. These results suggest that the different leaves make different contributions to maize seedling growth via modulation of the expression of the photosynthetic proteins. Together, these results provide insight into the roles of the different maize leaves as the plant develops from a heterotroph to an autotroph.

The proteome and phosphoproteome of maize pollen uncovers fertility candidate proteins.

Maize is unique since it is both monoecious and diclinous (separate male and female flowers on the same plant). We investigated the proteome and phosphoproteome of maize pollen containing modified proteins and here we provide a comprehensive pollen proteome and phosphoproteome which contain 100,990 peptides from 6750 proteins and 5292 phosphorylated sites corresponding to 2257 maize phosphoproteins, respectively. Interestingly, among the total 27 overrepresented phosphosite motifs we identified here, 11 were novel motifs, which suggested different modification mechanisms in plants compared to those of animals. Enrichment analysis of pollen phosphoproteins showed that pathways including DNA synthesis/chromatin structure, regulation of RNA transcription, protein modification, cell organization, signal transduction, cell cycle, vesicle transport, transport of ions and metabolisms, which were involved in pollen development, the following germination and pollen tube growth, were regulated by phosphorylation. In this study, we also found 430 kinases and 105 phosphatases in the maize pollen phosphoproteome, among which calcium dependent protein kinases (CDPKs), leucine rich repeat kinase, SNF1 related protein kinases and MAPK family proteins were heavily enriched and further analyzed. From our research, we also uncovered hundreds of male sterility-associated proteins and phosphoproteins that might influence maize productivity and serve as targets for hybrid maize seed production. At last, a putative complex signaling pathway involving CDPKs, MAPKs, ubiquitin ligases and multiple fertility proteins was constructed. Overall, our data provides new insight for further investigation of protein phosphorylation status in mature maize pollen and construction of maize male sterile mutants in the future.

Structural Basis of Reversible Phosphorylation by Maize Pyruvate Orthophosphate Dikinase Regulatory Protein.

Pyruvate orthophosphate dikinase (PPDK) is one of the most important enzymes in C4 photosynthesis. PPDK regulatory protein (PDRP) regulates the inorganic phosphate-dependent activation and ADP-dependent inactivation of PPDK by reversible phosphorylation. PDRP shares no significant sequence similarity with other protein kinases or phosphatases. To investigate the molecular mechanism by which PDRP carries out its dual and competing activities, we determined the crystal structure of PDRP from maize (Zea mays). PDRP forms a compact homo-dimer in which each protomer contains two separate N-terminal (NTD) and C-terminal (CTD) domains. The CTD includes several key elements for performing both phosphorylation and dephosphorylation activities: the phosphate binding loop (P-loop) for binding the ADP and inorganic phosphate substrates, residues Lys-274 and Lys-299 for neutralizing the negative charge, and residue Asp-277 for protonating and deprotonating the target Thr residue of PPDK to promote nucleophilic attack. Surprisingly, the NTD shares the same protein fold as the CTD and also includes a putative P-loop with AMP bound but lacking enzymatic activities. Structural analysis indicated that this loop may participate in the interaction with and regulation of PPDK. The NTD has conserved intramolecular and intermolecular disulfide bonds for PDRP dimerization. Moreover, PDRP is the first structure of the domain of unknown function 299 enzyme family reported. This study provides a structural basis for understanding the catalytic mechanism of PDRP and offers a foundation for the development of selective activators or inhibitors that may regulate photosynthesis.