Comparative Ubiquitination Proteomics Revealed the Salt Tolerance Mechanism in Sugar Beet Monomeric Additional Line M14.

Post-translational modifications (PTMs) are important molecular processes that regulate organismal responses to different stresses. Ubiquitination modification is not only involved in human health but also plays crucial roles in plant growth, development, and responses to environmental stresses. In this study, we investigated the ubiquitination proteome changes in the salt-tolerant sugar beet monomeric additional line M14 under salt stress treatments. Based on the expression of the key genes of the ubiquitination system and the ubiquitination-modified proteins before and after salt stress, 30 min of 200 mM NaCl treatment and 6 h of 400 mM NaCl treatment were selected as time points. Through label-free proteomics, 4711 and 3607 proteins were identified in plants treated with 200 mM NaCl and 400 mM NaCl, respectively. Among them, 611 and 380 proteins were ubiquitinated, with 1085 and 625 ubiquitination sites, in the two salt stress conditions, respectively. A quantitative analysis revealed that 70 ubiquitinated proteins increased and 47 ubiquitinated proteins decreased. At the total protein level, 42 were induced and 20 were repressed with 200 mM NaCl, while 28 were induced and 27 were repressed with 400 mM NaCl. Gene ontology, KEGG pathway, protein interaction, and PTM crosstalk analyses were performed using the differentially ubiquitinated proteins. The differentially ubiquitinated proteins were mainly involved in cellular transcription and translation processes, signal transduction, metabolic pathways, and the ubiquitin/26S proteasome pathway. The uncovered ubiquitinated proteins constitute an important resource of the plant stress ubiquitinome, and they provide a theoretical basis for the marker-based molecular breeding of crops for enhanced stress tolerance.

Genome-Wide Identification and Salt Stress Response Analysis of the bZIP Transcription Factor Family in Sugar Beet.

As one of the largest transcription factor families in plants, bZIP transcription factors play important regulatory roles in different biological processes, especially in the process of stress response. Salt stress inhibits the growth and yield of sugar beet. However, bZIP-related studies in sugar beet (Beta vulgaris L.) have not been reported. This study aimed to identify the bZIP transcription factors in sugar beet and analyze their biological functions and response patterns to salt stress. Using bioinformatics, 48 BvbZIP genes were identified in the genome of sugar beet, encoding 77 proteins with large structural differences. Collinearity analysis showed that three pairs of BvbZIP genes were fragment replication genes. The BvbZIP genes were grouped according to the phylogenetic tree topology and conserved structures, and the results are consistent with those reported in Arabidopsis. Under salt stress, the expression levels of most BvbZIP genes were decreased, and only eight genes were up-regulated. GO analysis showed that the BvbZIP genes were mainly negatively regulated in stress response. Protein interaction prediction showed that the BvbZIP genes were mainly involved in light signaling and ABA signal transduction, and also played a certain role in stress responses. In this study, the structures and biological functions of the BvbZIP genes were analyzed to provide foundational data for further mechanistic studies and for facilitating the efforts toward the molecular breeding of stress-resilient sugar beet.

Exploring native Scutellaria species provides insight into differential accumulation of flavones with medicinal properties.

Scutellaria baicalensis is a well-studied medicinal plant belonging to the Lamiaceae family, prized for the unique 4'-deoxyflavones produced in its roots. In this study, three native species to the Americas, S. lateriflora, S. arenicola, and S. integrifolia were identified by DNA barcoding, and phylogenetic relationships were established with other economically important Lamiaceae members. Furthermore, flavone profiles of native species were explored. 4'-deoxyflavones including baicalein, baicalin, wogonin, wogonoside, chrysin and 4'-hydroxyflavones, scutellarein, scutellarin, and apigenin, were quantified from leaves, stems, and roots. Qualitative, and quantitative differences were identified in their flavone profiles along with characteristic tissue-specific accumulation. 4'-deoxyflavones accumulated in relatively high concentrations in root tissues compared to aerial tissues in all species except S. lateriflora. Baicalin, the most abundant 4'-deoxyflavone detected, was localized in the roots of S. baicalensis and leaves of S. lateriflora, indicating differential accumulation patterns between the species. S. arenicola and S. integrifolia are phylogenetically closely related with similar flavone profiles and distribution patterns. Additionally, the S. arenicola leaf flavone profile was dominated by two major unknown peaks, identified using LC-MS/MS to most likely be luteolin-7-O-glucuronide and 5,7,2'-trihydroxy-6-methoxyflavone 7-O-glucuronide. Collectively, results presented in this study suggest an evolutionary divergence of flavonoid metabolic pathway in the Scutellaria genus of Lamiaceae.

Functional Characterization of a Sugar Beet BvbHLH93 Transcription Factor in Salt Stress Tolerance.

The basic/helix-loop-helix (bHLH) transcription factor (TF) plays an important role for plant growth, development, and stress responses. Previously, proteomics of NaCl treated sugar beet leaves revealed that a bHLH TF, BvbHLH93, was significantly increased under salt stress. The BvbHLH93 protein localized in the nucleus and exhibited activation activity. The expression of BvbHLH93 was significantly up-regulated in roots and leaves by salt stress, and the highest expression level in roots and leaves was 24 and 48 h after salt stress, respectively. Furthermore, constitutive expression of BvbHLH93 conferred enhanced salt tolerance in Arabidopsis, as indicated by longer roots and higher content of chlorophyll than wild type. Additionally, the ectopic expression lines accumulated less Na+ and MDA, but more K+ than the WT. Overexpression of the BvBHLH93 enhanced the activities of antioxidant enzymes by positively regulating the expression of antioxidant genes SOD and POD. Compared to WT, the overexpression plants also had low expression levels of RbohD and RbohF, which are involved in reactive oxygen species (ROS) production. These results suggest that BvbHLH93 plays a key role in enhancing salt stress tolerance by enhancing antioxidant enzymes and decreasing ROS generation.

Combined ultraviolet and darkness regulation of medicinal metabolites in Mahonia bealei revealed by proteomics and metabolomics.

Roots of Mahonia bealei have been used as traditional Chinese medicine with antibacterial, antioxidant and anti-inflammatory properties due to its high alkaloid content. Previously, we reported that alkaloid and flavonoid contents in the M. bealei leaves could be increased by the combined ultraviolet B and dark treatment (UV+D). To explore the underlying metabolic pathways and networks, proteomic and metabolomic analyses of the M. bealei leaves were conducted. Proteins related to tricarboxylic acid cycle, transport and signaling varied greatly under the UV + D. Among them, calmodulin involved in calcium signaling and ATP-binding cassette transporter involved in transport of berberine were increased. Significantly changed metabolites were overrepresented in phenylalanine metabolism, nitrogen metabolism, phenylpropanoid, flavonoid and alkaloid biosynthesis. In addition, the levels of salicylic acid and gibberellin decreased in the UV group and increased in the UV + D group. These results indicate that multi-hormone crosstalk may regulate the biosynthesis of flavonoids and alkaloids to alleviate oxidative stress caused by the UV + D treatment. Furthermore, protoberberine alkaloids may be induced through calcium signaling crosstalk with reaction oxygen species and transported to leaves. SIGNIFICANCE: Mahonia bealei root and stem, not leaf, were used as traditional medicine for a long history because of the high contents of active components. In the present study, UV-B combined with dark treatments induced the production of alkaloids and flavonoids in the M. bealei leaf, especially protoberberine alkaloids such as berberine. Multi-omics analyses indicated that multi-hormone crosstalk, enhanced tricarboxylic acid cycle and active calcium signaling were involved. The study informs a strategy for utilization of the leaves, and improves understanding of the functions of secondary metabolites in M. bealei.

Overexpression of a S-Adenosylmethionine Decarboxylase from Sugar Beet M14 Increased Araidopsis Salt Tolerance.

Polyamines play an important role in plant growth and development, and response to abiotic stresses. Previously, differentially expressed proteins in sugar beet M14 (BvM14) under salt stress were identified by iTRAQ-based quantitative proteomics. One of the proteins was an S-adenosylmethionine decarboxylase (SAMDC), a key rate-limiting enzyme involved in the biosynthesis of polyamines. In this study, the BvM14-SAMDC gene was cloned from the sugar beet M14. The full-length BvM14-SAMDC was 1960 bp, and its ORF contained 1119 bp encoding the SAMDC of 372 amino acids. In addition, we expressed the coding sequence of BvM14-SAMDC in Escherichia coli and purified the ~40 kD BvM14-SAMDC with high enzymatic activity. Quantitative real-time PCR analysis revealed that the BvM14-SAMDC was up-regulated in the BvM14 roots and leaves under salt stress. To investigate the functions of the BvM14-SAMDC, it was constitutively expressed in Arabidopsis thaliana. The transgenic plants exhibited greater salt stress tolerance, as evidenced by longer root length and higher fresh weight and chlorophyll content than wild type (WT) under salt treatment. The levels of spermidine (Spd) and spermin (Spm) concentrations were increased in the transgenic plants as compared with the WT. Furthermore, the overexpression plants showed higher activities of antioxidant enzymes and decreased cell membrane damage. Compared with WT, they also had low expression levels of RbohD and RbohF, which are involved in reactive oxygen species (ROS) production. Together, these results suggest that the BvM14-SAMDC mediated biosynthesis of Spm and Spd contributes to plant salt stress tolerance through enhancing antioxidant enzymes and decreasing ROS generation.

Quantitative proteomics and phosphoproteomics of sugar beet monosomic addition line M14 in response to salt stress.

UNLABELLED: Salinity is a major abiotic stress affecting plant growth, development and agriculture productivity. Understanding the molecular mechanisms of salt stress tolerance will provide valuable information for effective crop engineering and breeding. Sugar beet monosomic addition line M14 obtained from the intercross between Beta vulgaris L. and Beta corolliflora Zoss exhibits tolerance to salt stress. In this study, the changes in the M14 proteome and phosphoproteome induced by salt stress were analyzed. We report the characteristics of the M14 plants under 0, 200, and 400mM NaCl using label-free quantitative proteomics approaches. Protein samples were subjected to total proteome profiling using LC-MS/MS and phosphopeptide enrichment to identify phosphopeptides and phosphoproteins. A total of 2182 proteins were identified and 114 proteins showed differential levels under salt stress. Interestingly, 189 phosphoproteins exhibited significant changes at the phosphorylation level under salt stress. Several signaling components associated with salt stress were found, e.g. 14-3-3 and mitogen-activated protein kinases (MAPK). Fifteen differential phosphoproteins and proteins involved in signal transduction were tested at the transcriptional level. The results revealed the short-term salt responsive mechanisms of the special sugar beet M14 line using label-free quantitative phosphoproteomics. BIOLOGICAL SIGNIFICANCE: Sugar beet monosomic addition line M14 is a special germplasm with salt stress tolerance. Analysis of the M14 proteome and phosphoproteome under salt stress has provided insight into specific response mechanisms underlying salt stress tolerance. Reversible protein phosphorylation regulates a wide range of cellular processes such as transmembrane signaling, intracellular amplification of signals, and cell-cycle control. This study has identified significantly changed proteins and phosphoproteins, and determined their potential relevance to salt stress response. The knowledge gained can be potentially applied to improving crop salt tolerance.

Metabolomic Responses of Guard Cells and Mesophyll Cells to Bicarbonate.

Anthropogenic CO2 presently at 400 ppm is expected to reach 550 ppm in 2050, an increment expected to affect plant growth and productivity. Paired stomatal guard cells (GCs) are the gate-way for water, CO2, and pathogen, while mesophyll cells (MCs) represent the bulk cell-type of green leaves mainly for photosynthesis. We used the two different cell types, i.e., GCs and MCs from canola (Brassica napus) to profile metabolomic changes upon increased CO2 through supplementation with bicarbonate (HCO3-). Two metabolomics platforms enabled quantification of 268 metabolites in a time-course study to reveal short-term responses. The HCO3- responsive metabolomes of the cell types differed in their responsiveness. The MCs demonstrated increased amino acids, phenylpropanoids, redox metabolites, auxins and cytokinins, all of which were decreased in GCs in response to HCO3-. In addition, the GCs showed differential increases of primary C-metabolites, N-metabolites (e.g., purines and amino acids), and defense-responsive pathways (e.g., alkaloids, phenolics, and flavonoids) as compared to the MCs, indicating differential C/N homeostasis in the cell-types. The metabolomics results provide insights into plant responses and crop productivity under future climatic changes where elevated CO2 conditions are to take center-stage.

Salt stress response of membrane proteome of sugar beet monosomic addition line M14.

Understanding how plants respond to and tolerate salt stress is important for engineering and breeding effort to boost plant productivity and bioenergy in an ever challenging environment. Sugar beet M14 line is a unique germplasm that contains genetic materials from Beta vulgaris L. and Beta corolliflora Zoss, and it exhibits tolerance to salt stress. Here we report the changes in membrane proteome of the M14 plants in response to salt stress (0, 200, 400mM NaCl) using an iTRAQ two-dimensional LC-MS/MS technology for quantitative proteomic analysis. In total, 274 proteins, mostly membrane proteins, were identified, and 50 proteins exhibited differential protein level changes, with 40 proteins increased and 10 decreased. The proteins were mainly involved in transport, metabolism, protein synthesis, photosynthesis, protein folding and degradation, signal transduction, stress and defense, energy, and cell structure. These results have revealed interesting mechanisms underlying the M14 response and tolerance to salt stress. BIOLOGICAL SIGNIFICANCE: Sugar beet monosomic addition line M14 is a special variety with salt stress tolerance. Analysis of the M14 membrane proteome under salt stress may provide useful information regarding specific adaptive mechanisms underlying salt stress tolerance. Membrane proteins are known to play critical roles in salt stress signaling and adaptation. The purpose of this study was to identify significantly changed membrane proteins and determine their possible relevance to salt tolerance. The proteomic analysis of the M14 line revealed important molecular mechanisms that can be potentially applied to improving crop salt tolerance. This article is part of a Special Issue entitled: Proteomics in India.

Plant single-cell and single-cell-type metabolomics.

In conjunction with genomics, transcriptomics, and proteomics, plant metabolomics is providing large data sets that are paving the way towards a comprehensive and holistic understanding of plant growth, development, defense, and productivity. However, dilution effects from organ- and tissue-based sampling of metabolomes have limited our understanding of the intricate regulation of metabolic pathways and networks at the cellular level. Recent advances in metabolomics methodologies, along with the post-genomic expansion of bioinformatics knowledge and functional genomics tools, have allowed the gathering of enriched information on individual cells and single cell types. Here we review progress, current status, opportunities, and challenges presented by single cell-based metabolomics research in plants.

Proteomic analysis of salt tolerance in sugar beet monosomic addition line M14.

Understanding the mechanisms of plant salinity tolerance can facilitate plant engineering for enhanced salt stress tolerance. Sugar beet monosomic addition line M14 obtained from the intercross between Beta vulgaris L. and Beta corolliflora Zoss exhibits tolerance to salt stress. Here we report the salt-responsive characteristics of the M14 plants under 0, 200, and 400 mM NaCl conditions using quantitative proteomics approaches. Proteins from control and the salt treated M14 plants were separated using 2D-DIGE. Eighty-six protein spots representing 67 unique proteins in leaves and 22 protein spots representing 22 unique proteins in roots were identified. In addition, iTRAQ LC-MS/MS was employed to identify and quantify differentially expressed proteins under salt stress. Seventy-five differentially expressed proteins in leaves and 43 differentially expressed proteins in roots were identified. The proteins were mainly involved in photosynthesis, energy, metabolism, protein folding and degradation, and stress and defense. Moreover, gene transcription data obtained from the same samples were compared to the corresponding protein data. Thirteen proteins in leaves and 12 in roots showed significant correlation in gene expression and protein levels. These results suggest the important processes for the M14 tolerance to salt stress include enhancement of photosynthesis and energy metabolism, accumulation of osmolyte and antioxidant enzymes, and regulation of methionine metabolism and ion uptake/exclusion.

Sugar beet M14 glyoxalase I gene can enhance plant tolerance to abiotic stresses.

Glyoxalase I is the first enzyme of the glyoxalase system that can detoxify methylglyoxal, a cytotoxic compound increased rapidly under stress conditions. Here we report cloning and characterization of a glyoxalase I from sugar beet M14 line (an interspecific hybrid between a wild species Beta corolliflora Zoss and a cultivated species B. vulgaris L). The full-length gene BvM14-glyoxalase I has 1,449 bp in length with an open reading frame of 1,065 bp encoding 354 amino acids. Sequence analysis shows the conserved glyoxalase I domains, metal and glutathione binding sites and secondary structure (α-helixes and ß-sheets). The BvM14-glyoxalase I gene was ubiquitously expressed in different tissues of sugar beet M14 line and up-regulated in response to salt, mannitol and oxidative stresses. Heterologous expression of BvM14-glyoxalase I could increase E. coli tolerance to methylglyoxal. Transgenic tobacco plants constitutively expressing BvM14-glyoxalase I were generated. Both leaf discs and seedlings showed significant tolerance to methylglyoxal, salt, mannitol and H2O2. These results suggest an important role of BvM14-glyoxalase I in cellular detoxification and tolerance to abiotic stresses.

Single-cell-type proteomics: toward a holistic understanding of plant function.

Multicellular organisms such as plants contain different types of cells with specialized functions. Analyzing the protein characteristics of each type of cell will not only reveal specific cell functions, but also enhance understanding of how an organism works. Most plant proteomics studies have focused on using tissues and organs containing a mixture of different cells. Recent single-cell-type proteomics efforts on pollen grains, guard cells, mesophyll cells, root hairs, and trichomes have shown utility. We expect that high resolution proteomic analyses will reveal novel functions in single cells. This review provides an overview of recent developments in plant single-cell-type proteomics. We discuss application of the approach for understanding important cell functions, and we consider the technical challenges of extending the approach to all plant cell types. Finally, we consider the integration of single-cell-type proteomics with transcriptomics and metabolomics with the goal of providing a holistic understanding of plant function.

Cloning of a cystatin gene from sugar beet M14 that can enhance plant salt tolerance.

An open reading frame encoding a cysteine protease inhibitor, cystatin was isolated from the buds of sugar beet monosomic addition line M14 (BvM14) using 5'-/3'-RACE method. It encoded a polypeptide of 104 amino acids with conserved G and PW motifs, the consensus phytocystatin sequence LARFAV and the active site QVVAG. The protein showed significant homology to other plant cystatins. BvM14-cystatin was expressed ubiquitously in roots, stems, leaves and flower tissues with relatively high abundance in developing stems and roots. It was found to be localized in the nucleus, cytoplasm and plasma membrane. Recombinant BvM14-cystatin expressed in Escherichia coli was purified and it exhibited cysteine protease inhibitor activity. Salt-stress treatment induced BvM14-cystatin transcript levels in the M14 seedlings. Homozygous Arabidopsis plants over-expressing BvM14-cystatin showed enhanced salt tolerance. Taken together, these data improved understanding of the functions of BvM14-cystatin and highlighted the possibility of employing the cystatin in engineering plants for enhanced salt tolerance.

Salt stress induced proteome and transcriptome changes in sugar beet monosomic addition line M14.

Sugar beet monosomic addition line M14 displays interesting phenotypes such as apomixis and salt stress tolerance. Here we reported proteomic and transcriptomic analysis of M14 leaves and roots under 500mM NaCl treatment for seven days. Proteins from control and treated samples were extracted and separated using two-dimensional difference gel electrophoresis (2D-DIGE). A total of 40 protein spots from leaf gels and 36 protein spots from root gels exhibited significant changes. Using mass spectrometry and database searching, 38 unique proteins in leaves and 29 unique proteins in roots were identified. The proteins included those involved in metabolism, protein folding, photosynthesis, and protein degradation. In addition, cDNA libraries of differentially expressed genes were constructed using suppression subtractive hybridization (SSH). Fifty-eight unigenes including 14 singletons and 44 contigs were obtained. Some salt-responsive genes were identified to function in metabolism, photosynthesis, stress and defense, energy, protein synthesis and protein degradation. This research has revealed candidate genes and proteins for detailed functional characterization, and set the stage for further investigation of the salt tolerance mechanisms in sugar beet.

Identification of a sugar beet BvM14-MADS box gene through differential gene expression analysis of monosomic addition line M14.

Monosomic addition line M14 carrying an additional chromosome 9 from Beta corolliflora Zosimovic ex Buttler was obtained through hybridization between the wild species B. corolliflora and a cultivated species Beta vulgaris L. var Saccharifera Alef. The M14 line showed diplosporic reproduction and stress tolerance. To identify differentially expressed genes in M14, a subtractive cDNA library was prepared by suppression subtractive hybridization (SSH) between M14 (2n=18+1) and B. vulgaris (2n=18). A total of 190 unique sequences were identified in the library and their putative functions were analyzed using Gene Ontology (GO). One of the genes, designated as BvM14-MADS box, encodes a MADS box transcription factor. It was cloned from M14 and over-expressed in transgenic tobacco plants. Interestingly, this gene was located on chromosome 2 of B. vulgaris, not on the additional chromosome 9. Overexpression of BvM14-MADS box led to significant phenotypic changes in tobacco. The differential expression of BvM14-MADS box gene in M14 may be caused by the interaction between the additional chromosome 9 from B. corolliflora and the B. vulgaris chromosomes in M14.

Functional characterization of Arabidopsis thaliana isopropylmalate dehydrogenases reveals their important roles in gametophyte development.

• Isopropylmalate dehydrogenases (IPMDHs) catalyze the oxidative decarboxylation of 3-isopropylmalate (3-IPM) in leucine biosynthesis in microorganisms. The Arabidopsis thaliana genome contains three putative IPMDH genes. • IPMDH2 and IPMDH3 proteins exhibited significantly higher activity toward 3-IPM than IPMDH1, which is indicative of a pivotal role in leucine biosynthesis. Single mutants of IPMDH2 or IPMDH3 lacked a discernible phenotype. Genetic analysis showed that ipmdh2 ipmdh3 was lethal in male gametophytes and had reduced transmission through female gametophytes. The aborted pollen grains were small, abnormal in cellular structure, and arrested in germination. In addition, half of the double mutant embryo sacs exhibited slowed development. • The IPMDH2/ipmdh2 ipmdh3/ipmdh3 genotype exhibited abnormal vegetative phenotypes, suggesting haplo-insufficiency of IPMDH2 in the ipmdh3 background. This mutant and a triple mutant containing one allele of IPMDH2 or IPMDH3 had decreased leucine biosynthetic enzyme activities and lower free leucine concentrations. The latter mutant showed changes in glucosinolate profiles different from those in the ipmdh1 mutant. • The results demonstrate that IPMDH2 and IPMDH3 primarily function in leucine biosynthesis, are essential for pollen development and are needed for proper embryo sac development.

Proteomic analysis of sugar beet apomictic monosomic addition line M14.

Apomixis in plants holds great promise for agriculture because of its vigor associated with heterozygosity and superior genotype. Despite the significance of apomictic reproductive process, our knowledge of proteins and their functions in apomictic development is limited. Here we report a comparative proteomic and transcriptomic analysis of sexual and apomictic processes in sugar beet. A total of 71 differentially expressed protein spots were successfully identified in the course of apomictic reproductive development using high-resolution 2-DE and MS analysis. The differentially expressed proteins were involved in several processes that might work cooperatively to lead to apomictic reproduction. This study has generated potential protein markers important for apomictic development.

Plant Vacuolar ATP-binding Cassette Transporters That Translocate Folates and Antifolates in Vitro and Contribute to Antifolate Tolerance in Vivo.

The vacuoles of pea (Pisum sativum) leaves and red beet (Beta vulgaris) storage root are major sites for the intracellular compartmentation of folates. In the light of these findings and preliminary experiments indicating that some plant multidrug resistance-associated protein (MRP) subfamily ATP-binding cassette transporters are able to transport compounds of this type, the Arabidopsis thaliana vacuolar MRP, AtMRP1 (AtABCC1), and its functional equivalent(s) in vacuolar membrane vesicles purified from red beet storage root were studied. In so doing, it has been determined that heterologously expressed AtMRP1 and its equivalents in red beet vacuolar membranes are not only competent in the transport of glutathione conjugates but also folate monoglutamates and antifolates as exemplified by pteroyl-l-glutamic acid and methotrexate (MTX), respectively. In agreement with the results of these in vitro transport measurements, analyses of atmrp1 T-DNA insertion mutants of Arabidopsis ecotypes Wassilewskia and Columbia disclose an MTX-hypersensitive phenotype. atmrp1 knock-out mutants are more sensitive than wild-type plants to growth retardation by nanomolar concentrations of MTX, and this is associated with impaired vacuolar antifolate sequestration. The vacuoles of protoplasts isolated from the leaves of Wassilewskia atmrp1 mutants accumulate 50% less [(3)H]MTX than the vacuoles of protoplasts from wild-type plants when incubated in media containing nanomolar concentrations of this antifolate, and vacuolar membrane-enriched vesicles purified from the mutant catalyze MgATP-dependent [(3)H]MTX uptake at only 40% of the capacity of the equivalent membrane fraction from wild-type plants. AtMRP1 and its counterparts in other plant species therefore have the potential for participating in the vacuolar accumulation of folates and related compounds.

Proteomics of pollen development and germination.

In higher plants, pollen grains represent the vestiges of a highly reduced male gametophyte generation. After germination, the pollen tube delivers the sperm cells by tip-growing to the embryo sac for fertilization. Besides the intrinsic importance for sexual reproduction, pollen development and germination serve as an attractive system to address important questions related to cell division, cell differentiation, polar growth, cell-cell interaction, and cell fate. Recently, pollen functional specification has been well-studied using multidisciplinary approaches. Here, we review recent advances in proteomics of pollen development and germination.