The BNB-GLID module regulates germline fate determination in Marchantia polymorpha.

Germline fate determination is a critical event in sexual reproduction. Unlike animals, plants specify the germline by reprogramming somatic cells at late stages of their development. However, the genetic basis of germline fate determination and how it has evolved during land plant evolution are still poorly understood. Here, we report that the plant homeodomain (PHD)-finger protein GERMLINE IDENTITY DETERMINANT (GLID) is a key regulator of germline specification in the liverwort Marchantia polymorpha. Loss of MpGLID function causes failure of germline initiation, leading to absence of sperm and egg cells. Remarkably, overexpression of MpGLID in M. polymorpha induces the ectopic formation of cells with male germline cell features exclusively in male thalli. We further show that MpBONOBO (BNB), with an evolutionarily conserved function, can induce the formation of male germ cell-like cells through activation of MpGLID by directly binding to its promoter. The Arabidopsis (Arabidopsis thaliana) MpGLID orthologue, MALE STERILITY1 (AtMS1), fails to replace the germline specification function of MpGLID in M. polymorpha, demonstrating that a derived function of MpGLID orthologues has been restricted to tapetum development in flowering plants. Collectively, our findings suggest the presence of the BNB-GLID module in complex ancestral land plants that has been retained in bryophytes but rewired in flowering plants for male germline fate determination.

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.

The C4 photosynthesis bifunctional enzymes, PDRPs, of maize are co-opted to cytoplasmic viral replication complexes to promote infection of a prevalent potyvirus sugarcane mosaic virus.

In maize, two pyruvate orthophosphate dikinase (PPDK) regulatory proteins, ZmPDRP1 and ZmPDRP2, are respectively specific to the chloroplast of mesophyll cells (MCs) and bundle sheath cells (BSCs). Functionally, ZmPDRP1/2 catalyse both phosphorylation/inactivation and dephosphorylation/activation of ZmPPDK, which is implicated as a major rate-limiting enzyme in C4 photosynthesis of maize. Our study here showed that maize plants lacking ZmPDRP1 or silencing of ZmPDRP1/2 confer resistance to a prevalent potyvirus sugarcane mosaic virus (SCMV). We verified that the C-terminal domain (CTD) of ZmPDRP1 plays a key role in promoting viral infection while independent of enzyme activity. Intriguingly, ZmPDRP1 and ZmPDRP2 re-localize to cytoplasmic viral replication complexes (VRCs) following SCMV infection. We identified that SCMV-encoded cytoplasmic inclusions protein CI targets directly ZmPDRP1 or ZmPDRP2 or their CTDs, leading to their re-localization to cytoplasmic VRCs. Moreover, we found that CI could be degraded by the 26S proteasome system, while ZmPDRP1 and ZmPDRP2 could up-regulate the accumulation level of CI through their CTDs by a yet unknown mechanism. Most importantly, with genetic, cell biological and biochemical approaches, we provide evidence that BSCs-specific ZmPDRP2 could accumulate in MCs of Zmpdrp1 knockout (KO) lines, revealing a unique regulatory mechanism crossing different cell types to maintain balanced ZmPPDK phosphorylation, thereby to keep maize normal growth. Together, our findings uncover the genetic link of the two cell-specific maize PDRPs, both of which are co-opted to VRCs to promote viral protein accumulation for robust virus infection.

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.

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.

Identification and Fine Mapping of the Recessive Gene BK-5, Which Affects Cell Wall Biosynthesis and Plant Brittleness in Maize.

The cellulose of the plant cell wall indirectly affects the cell shape and straw stiffness of the plant. Here, the novel brittleness mutant brittle stalk-5 (bk-5) of the maize inbred line RP125 was characterized. We found that the mutant displayed brittleness of the stalk and even the whole plant, and that the brittleness phenotype existed during the whole growth period from germination to senescence. The compressive strength was reduced, the cell wall was thinner, and the cellulose content was decreased compared to that of the wild type. Genetic analysis and map-based cloning indicated that bk-5 was controlled by a single recessive nuclear gene and that it was located in a 90.2-Kb region on chromosome 3 that covers three open reading frames (ORFs). Sequence analysis revealed a single non-synonymous missense mutation, T-to-A, in the last exon of Zm00001d043477 (B73: version 4, named BK-5) that caused the 951th amino acid to go from leucine to histidine. BK-5 encodes a cellulose synthase catalytic subunit (CesA), which is involved with cellulose synthesis. We found that BK-5 was constitutively expressed in all tissues of the germinating stage and silking stage, and highly expressed in the leaf, auricula, and root of the silking stage and the 2-cm root and bud of the germinating stage. We found that BK-5 mainly localized to the Golgi apparatus, suggesting that the protein might move to the plasma membrane with the aid of Golgi in maize. According to RNA-seq data, bk-5 had more downregulated genes than upregulated genes, and many of the downregulated genes were enzymes and transcription factors related to cellulose, hemicellulose, and lignin biosynthesis of the secondary cell wall. The other differentially expressed genes were related to metabolic and cellular processes, and were significantly enriched in hormone signal transduction, starch and sucrose metabolism, and the plant-pathogen interaction pathway. Taken together, we propose that the mutation of gene BK-5 causes the brittle stalk phenotype and provides important insights into the regulatory mechanism of cellulose biosynthesis and cell wall development in maize.

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.

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.

Characterization of maize leaf pyruvate orthophosphate dikinase using high throughput sequencing.

In C4 photosynthesis, pyruvate orthophosphate dikinase (PPDK) catalyzes the regeneration of phosphoenolpyruvate in the carbon shuttle pathway. Although the biochemical function of PPDK in maize is well characterized, a genetic analysis of PPDK has not been reported. In this study, we use the maize transposable elements Mutator and Ds to generate multiple mutant alleles of PPDK. Loss-of-function mutants are seedling lethal, even when plants were grown under 2% CO2 , and they show very low capacity for CO2 assimilation, indicating C4 photosynthesis is essential in maize. Using RNA-seq and GC-MS technologies, we examined the transcriptional and metabolic responses to a deficiency in PPDK activity. These results indicate loss of PPDK results in downregulation of gene expression of enzymes of the C4 cycle, the Calvin cycle, and components of photochemistry. Furthermore, the loss of PPDK did not change Kranz anatomy, indicating that this metabolic defect in the C4 cycle did not impinge on the morphological differentiation of C4 characters. However, sugar metabolism and nitrogen utilization were altered in the mutants. An interaction between light intensity and genotype was also detected from transcriptome profiling, suggesting altered transcriptional and metabolic responses to environmental and endogenous signals in the PPDK mutants.

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.

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.

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.

Large-scale comparative phosphoprotein analysis of maize seedling leaves during greening.

MAIN CONCLUSION : Large-scale comparative phosphoprotein analysis in maize seedlings reveals a complicated molecular regulation mechanism at the phosphoproteomic level during de-etiolation. In the present study we report a phosphoproteomic study conducted on Zea mays etiolated leaves harvested at three time points during greening (etiolated seedlings and seedlings exposed to light for 6 or 12 h). We identified a total of 2483 phosphopeptides containing 2389 unambiguous phosphosites from 1339 proteins. The abundance of nearly 692 phosphorylated peptides containing 783 phosphosites was reproducible and profiled with high confidence among treatments. Comparisons with other large-scale phosphoproteomic studies revealed that 473 of the phosphosites are novel to this study. Of the 783 phosphosites identified, 171, 79, and 138 were identified in 0, 6, and 12 h samples, respectively, which suggest that regulation of phosphorylation plays important roles during maize seedling de-etiolation. Our experimental methods included enrichment of phosphoproteins, allowing the identification of a great number of low abundance proteins, such as transcription factors, protein kinases, and photoreceptors. Most of the identified phosphoproteins were involved in gene transcription, post-transcriptional regulation, or signal transduction, and only a few were involved in photosynthesis and carbon metabolism. It is noteworthy that tyrosine phosphorylation and calcium signaling pathways might play important roles during maize seedling de-etiolation. Taken together, we have elucidated a new level of complexity in light-induced reversible protein phosphorylation during maize seedling de-etiolation.

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.

Posttranslational Modification of Maize Chloroplast Pyruvate Orthophosphate Dikinase Reveals the Precise Regulatory Mechanism of Its Enzymatic Activity.

In C4 plants, pyruvate orthophosphate dikinase (PPDK) activity is tightly dark/light regulated by reversible phosphorylation of an active-site threonine (Thr) residue; this process is catalyzed by PPDK regulatory protein (PDRP). Phosphorylation and dephosphorylation of PPDK lead to its inactivation and activation, respectively. Here, we show that light intensity rather than the light/dark transition regulates PPDK activity by modulating the reversible phosphorylation at Thr-527 (previously termed Thr-456) of PPDK in maize (Zea mays). The amount of PPDK (unphosphorylated) involved in C4 photosynthesis is indeed strictly controlled by light intensity, despite the high levels of PPDK protein that accumulate in mesophyll chloroplasts. In addition, we identified a transit peptide cleavage site, uncovered partial amino-terminal acetylation, and detected phosphorylation at four serine (Ser)/Thr residues, two of which were previously unknown in maize. In vitro experiments indicated that Thr-527 and Ser-528, but not Thr-309 and Ser-506, are targets of PDRP. Modeling suggests that the two hydrogen bonds between the highly conserved residues Ser-528 and glycine-525 are required for PDRP-mediated phosphorylation of the active-site Thr-527 of PPDK. Taken together, our results suggest that the regulation of maize plastid PPDK isoform (C4PPDK) activity is much more complex than previously reported. These diverse regulatory pathways may work alone or in combination to fine-tune C4PPDK activity in response to changes in lighting.

Comparative proteomics of Thellungiella halophila leaves from plants subjected to salinity reveals the importance of chloroplastic starch and soluble sugars in halophyte salt tolerance.

Thellungiella halophila, a close relative of Arabidopsis, is a model halophyte used to study plant salt tolerance. The proteomic/physiological/transcriptomic analyses of Thellungiella plant leaves subjected to different salinity levels, reported herein, indicate an extraordinary ability of Thellungiella to adapt to large concentrations of exogenous saline by compartmentalizing Na(+) into cell vacuoles and accumulating proline and soluble sugars as organic osmolytes. Salinity stress stimulated the accumulation of starch in chloroplasts, which resulted in a greatly increased content of starch and total sugars in leaves. Comparative proteomics of Thellungiella leaves identified 209 salt-responsive proteins. Among these, the sequences of 108 proteins were strongly homologous to Arabidopsis protein sequences, and 30 had previously been identified as Thellungiella proteins. Functional classification of these proteins into 16 categories indicated that the majority are involved in carbohydrate metabolism, followed by those involved in energy production and conversion, and then those involved in the transport of inorganic ions. Pathway analysis revealed that most of the proteins are involved in starch and sucrose metabolism, carbon fixation, photosynthesis, and glycolysis. Of these processes, the most affected were starch and sucrose metabolism, which might be pivotal for salt tolerance. The gene expression patterns of the 209 salt-responsive proteins revealed through hierarchical clustering of microarray data and the expression patterns of 29 Thellungiella genes evaluated via quantitative RT-PCR were similar to those deduced via proteomic analysis, which underscored the possibility that starch and sucrose metabolism might play pivotal roles in determining the salt tolerance ability of Thellungiella. Our observations enabled us to propose a schematic representation of the systematic salt-tolerance phenotype in Thellungiella and suggested that the increased accumulation of starch, soluble sugars, and proline, as well as subcellular compartmentalization of sodium, might collectively denote important mechanisms for halophyte salt tolerance.

Light-regulated phosphorylation of maize phosphoenolpyruvate carboxykinase plays a vital role in its activity.

Phosphoenolpyruvate carboxykinase (PEPCK)-the major decarboxylase in PEPCK-type C4 plants-is also present in appreciable amounts in the bundle sheath cells of NADP-malic enzyme-type C4 plants, such as maize (Zea mays), where it plays an apparent crucial role during photosynthesis (Wingler et al., in Plant Physiol 120(2):539-546, 1999; Furumoto et al., in Plant Mol Biol 41(3):301-311, 1999). Herein, we describe the use of mass spectrometry to demonstrate phosphorylation of maize PEPCK residues Ser55, Thr58, Thr59, and Thr120. Western blotting indicated that the extent of Ser55 phosphorylation dramatically increases in the leaves of maize seedlings when the seedlings are transferred from darkness to light, and decreases in the leaves of seedlings transferred from light to darkness. The effect of light on phosphorylation of this residue is opposite that of the effect of light on PEPCK activity, with the decarboxylase activity of PEPCK being less in illuminated leaves than in leaves left in the dark. This inverse relationship between PEPCK activity and the extent of phosphorylation suggests that the suppressive effect of light on PEPCK decarboxylation activity might be mediated by reversible phosphorylation of Ser55.