Grapevine aquaporins: gating of a tonoplast intrinsic protein (TIP2;1) by cytosolic pH

Grapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops being considered as a fruit ligneous tree model system in which the water status appears crucial for high fruit and wine quality, controlling productivity and alcohol level. V. vinifera genome contains 28 genes coding for aquaporins, which acting in a concerted and regulated manner appear relevant for plant withstanding extremely unfavorable drought conditions essential for the quality of berries and wine. Several Vv aquaporins have been reported to be expressed in roots, shoots, berries and leaves with clear cultivar differences in their expression level, making their in vivo biochemical characterization a difficult task. In this work V. vinifera cv. Touriga nacional VvTnPIP1;1, VvTnPIP2;2 and VvTnTIP2;1 were expressed in yeast and water transport activity was characterized in intact cells of the transformants. The three aquaporins were localized in the yeast plasma membrane but only VvTnTIP2;1 expression enhanced the water permeability with a concomitant decrease of the activation energy of water transport. Acidification of yeast cytosol resulted in loss of VvTnTIP2;1 activity. Sequence analysis revealed the presence of a His(131) residue, unusual in TIPs. By site directed mutagenesis, replacement of this residue by aspartic acid or alanine resulted in loss of pH(in) dependence while replacement by lysine resulted in total loss of activity. In addition to characterization of VvTn aquaporins, these results shed light on the gating of a specific tonoplast aquaporin by cytosolic pH.     

A tonoplast intrinsic protein (TIP) is present in seeds, roots and somatic embryos of Norway spruce (Picea abies)

Many plant species contain a seed-specific tonoplast intrinsic protein (TIP) in their protein storage vacuoles (PSVs). Although the function of the protein is not known, its structure implies it to act as a transporter protein, possibly during storage nutrient accumulation/breakdown or during desiccation/imbibition of seeds. As mature somatic embryos of Picea abies (L.) Karst. (Norway spruce) contain PSVs, we examined the presence of TIP in them. Both the megagametophyte and seed embryo accumulate storage nutrients, but at different times and we therefore studied the temporal accumulation of TIP during seed development. Antiserum against the seed-specific a-TIP of Phaseolus vulgaris recognized an abundant 27 kDa tonoplast protein in mature seeds of P. abies. By immunogold labeling of sectioned mature megagametophytes we localized the protein to the PSV membrane. We also isolated the membranes of the PSVs from mature seeds and purified an integral membrane protein that reacted heavily with the antiserum. A sequence of 11 amino acid residues [AEEATHPDSIR], that was obtained from a polypeptide after in-gel trypsin digestion of the purified membrane protein, showed high local identity to a-TIP of Arabidopsis thaliana and to a-TIP of P. vulgaris. The greatest accumulation of TIP in the megagametophytes occurred at the time of storage protein accumulation. A lower molecular mass band also stained from about the time of fertilization until early embryo development. The staining of this band disappeared as the higher molecular mass (27 kDa) band accumulated in the megagametophyte during seed development. Total protein was also extracted from developing zygotic embryos and from somatic embryos. In zygotic embryos low-levels of TIP were seen at all stages investigated, but stained most at the time of storage protein accumulation. The protein was also present in mature somatic embryos but not in proliferating embryogenic tissues in culture. In addition to the seed tissue material, the antiserum also reacted with proteins present in extracts from roots and hypocotyls but not cotyledons from 13-day-old seedlings.     

Tonoplast-bound protein kinase phosphorylates tonoplast intrinsic protein

Tonoplast intrinsic protein (TIP) is a member of a family of putative membrane channels found in bacteria, animals, and plants. Plants have seed-specific, vegetative/reproductive organ-specific, and water-stress-induced forms of TIP. Here, we report that the seed-specific TIP is a phosphoprotein whose phosphorylation can be monitored in vivo by allowing bean cotyledons to take up [³²P]orthophosphate and in vitro by incubating purified tonoplasts with γ-labeled [³²P]ATP. Characterization of the in vitro phosphorylation of TIP indicates that a membrane-bound protein kinase phosphorylates TIP in a Ca²⁺-dependent manner. The capacity of the isolated tonoplast membranes to phosphorylate TIP declined markedly during seed germination, and this decline occurred well before the development-mediated decrease in TIP occurs. Phosphoamino acid analysis of purified, radiolabeled TIP showed that serine is the major, if not only, phosphorylated residue, and cyanogen bromide cleavage yielded a single radioactive peptide peak on a reverse-phase high-performance liquid chromatogram. Estimation of the molecular mass of the cyanogen bromide phosphopeptide by laser desorption mass spectroscopy led to its identification as the hydrophilic N-terminal domain of TIP. The putative phosphate-accepting serine residue occurs in a consensus phosphorylation site for serine/threonine protein kinases.     

Genome-wide comparative analysis of the codon usage patterns in plants

Codon usage analysis has been a classical area of study for decades and is important for evolution, mRNA translation, and new gene discovery. Recently, genome sequencing has made it possible to perform studies of the entire genome in plant kingdoms. The base composition of the coding sequence, codon usage pattern, codon pairs, and related indicators of relative synonymous codon usage (RSCU), including the Fop, Nc, RSCU, CAI and GC contents, were analyzed. We found that the GC content of single-celled algae is the highest, whereas dicotyledons are the lowest. Moreover, the base composition of plants is similar within the same family. In addition, the GC content of the second base of the codon is lower than the first and third base. In conclusion, the codon usage characteristics are opposite in Gramineae, single-celled algae, fern and dicotyledon, moss, and Pinaceae. Furthermore, the degree of codon usage bias is decreasing with evolution. Therefore, we hypothesize that the lower the plants, the more that they must optimize codons and that higher plants no longer need to optimize codons.     

In vivo imaging of the tonoplast intrinsic protein family in Arabidopsis roots

Background  

Tonoplast intrinsic proteins (TIPs) are widely used as markers for vacuolar compartments in higher plants. Ten TIP isoforms are encoded by the Arabidopsis genome. For several isoforms, the tissue and cell specific pattern of expression are not known.     

Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana

Background

Plant disease resistance (R) genes with the nucleotide binding site (NBS) play an important role in offering resistance to pathogens. The availability of complete genome sequences of Brassica oleracea and Brassica rapa provides an important opportunity for researchers to identify and characterize NBS-encoding R genes in Brassica species and to compare with analogues in Arabidopsis thaliana based on a comparative genomics approach. However, little is known about the evolutionary fate of NBS-encoding genes in the Brassica lineage after split from A. thaliana.

Results

Here we present genome-wide analysis of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. Through the employment of HMM search and manual curation, we identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes, respectively. Phylogenetic analysis among 3 species classified NBS-encoding genes into 6 subgroups. Tandem duplication and whole genome triplication (WGT) analyses revealed that after WGT of the Brassica ancestor, NBS-encoding homologous gene pairs on triplicated regions in Brassica ancestor were deleted or lost quickly, but NBS-encoding genes in Brassica species experienced species-specific gene amplification by tandem duplication after divergence of B. rapa and B. oleracea. Expression profiling of NBS-encoding orthologous gene pairs indicated the differential expression pattern of retained orthologous gene copies in B. oleracea and B. rapa. Furthermore, evolutionary analysis of CNL type NBS-encoding orthologous gene pairs among 3 species suggested that orthologous genes in B. rapa species have undergone stronger negative selection than those in B .oleracea species. But for TNL type, there are no significant differences in the orthologous gene pairs between the two species.

Conclusion

This study is first identification and characterization of NBS-encoding genes in B. rapa and B. oleracea based on whole genome sequences. Through tandem duplication and whole genome triplication analysis in B. oleracea, B. rapa and A. thaliana genomes, our study provides insight into the evolutionary history of NBS-encoding genes after divergence of A. thaliana and the Brassica lineage. These results together with expression pattern analysis of NBS-encoding orthologous genes provide useful resource for functional characterization of these genes and genetic improvement of relevant crops.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-3) contains supplementary material, which is available to authorized users.     

Genome-wide analysis of ACO and ACS genes in pear (Pyrus ussuriensis)

The “Nanguo” pear is a typically climacteric fruit and ethylene is the main factor controlling the ripening process of climacteric fruit. Ethylene biosynthesis has been studied clearly and ACC synthase (ACS) is the rate-limited enzyme. ACO (ACC oxidase) is another important enzyme in ethylene biosynthesis. By exploring the pear genome, we identified 13 ACS genes and 11 ACO genes, respectively, and their expression patterns in fruit and other organs were investigated. Among these genes, 11 ACS and 8ACO genes were expressed in pear fruits. What’s more, 4 ACS and 3ACO genes could be induced by Ethephon and inhibited by 1-MCP treatment. This study is the first time to explore ACS and ACO genes at genome-wide level and will provide new data for research on pear fruit ripening.

     

Sugar starvation induces the central vacuolation with coordinated increase in expression of tonoplast intrinsic protein genes in suspension-cultured rice cells

An efficient monitoring of cytoplasmic vacuolation by fluorescein diacetate (FDA) staining of protoplasts revealed that the major populations of suspension-cultured rice cells undergo a rapid vacuolation upon glucosedepletion. As aquaporin-family proteins, tonoplast intrinsic proteins (TIPs) are known to play in regulating the water balance across the vacuolar membrane. Glucose starvation increased expression of every member of OsTIP family, leading to an enhancement of the total expression by up to 110-fold, which is well matched with an expansion of the vacuolar structure induced by starvation. OsTIPs 1;1, 2;2 and 3;1 are the three most prominently expressed OsTIPs in starved conditions due to their highest responsiveness to sugar deprivation. Feeding experiments with various sugars and glucose analogs indicated that sugar regulated expression of the three major OsTIPs is likely mediated by a hexokinasedependent pathway. Alleviation of sugar-induced suppression of OsTIP expression by co-treatment with the uncoupler of ATP synthesis suggests that sugar signaling for OsTIP regulation is also cross-talked by the energy-deficit conditions. Intriguingly, starvation-induced central vacuolation was effectively prevented by mannose and 2-deoxyglucose, but not by 3-O-methylglucose. These results imply that the hexokinase is able to trigger the signaling to suppress the central vacuolation, independently of fueling the energy metabolism.     

Genome-wide identification and analysis of membrane-bound O-acyltransferase (MBOAT) gene family in plants

Membrane bound O-acyl transferase (MBOAT) family is composed of gene members encoding a variety of acyltransferase enzymes, which play important roles in plant acyl lipid metabolism. Here, we present the first genome-enabled identification and analysis of MBOAT gene models in plants. In total, we identified 136 plant MBOAT sequences from 14 plant species with complete genomes. Phylogenetic relationship analyses suggested the plant MBOAT gene models fell into four major groups, two of which likely encode enzymes of diacylglycerol acyltransferase 1 (DGAT1) and lysophospholipid acyltransferase (LPLAT), respectively, with one–three copies of paralogs present in each of the most plant species. A group of gene sequences, which are homologous to Saccharomyces cerevisiae glycerol uptake proteins (GUP), was identified in plants; copy numbers were conserved, with only one copy represented in each of the most plant species; analyses showed that residues essential for acyltransferases were more prone to be conserved than vertebrate orthologs. Among four groups, one was inferred to emerge in land plants and experience a rapid expansion in genomes of angiosperms, which suggested their important roles in adaptation of plants in lands. Sequence and phylogeny analyses indicated that genes in all four groups encode enzymes with acyltransferases. Comprehensive sequence identification of MBOAT family members and investigation into classification provide a complete picture of the MBOAT gene family in plants, and could shed light into enzymatic functions of different MBOAT genes in plants.     

Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis

The Arabidopsis genome contains approximately 200 genes that encode proteins with similarity to the nucleotide binding site and other domains characteristic of plant resistance proteins. Through a reiterative process of sequence analysis and reannotation, we identified 149 NBS-LRR-encoding genes in the Arabidopsis (ecotype Columbia) genomic sequence. Fifty-six of these genes were corrected from earlier annotations. At least 12 are predicted to be pseudogenes. As described previously, two distinct groups of sequences were identified: those that encoded an N-terminal domain with Toll/Interleukin-1 Receptor homology (TIR-NBS-LRR, or TNL), and those that encoded an N-terminal coiled-coil motif (CC-NBS-LRR, or CNL). The encoded proteins are distinct from the 58 predicted adapter proteins in the previously described TIR-X, TIR-NBS, and CC-NBS groups. Classification based on protein domains, intron positions, sequence conservation, and genome distribution defined four subgroups of CNL proteins, eight subgroups of TNL proteins, and a pair of divergent NL proteins that lack a defined N-terminal motif. CNL proteins generally were encoded in single exons, although two subclasses were identified that contained introns in unique positions. TNL proteins were encoded in modular exons, with conserved intron positions separating distinct protein domains. Conserved motifs were identified in the LRRs of both CNL and TNL proteins. In contrast to CNL proteins, TNL proteins contained large and variable C-terminal domains. The extant distribution and diversity of the NBS-LRR sequences has been generated by extensive duplication and ectopic rearrangements that involved segmental duplications as well as microscale events. The observed diversity of these NBS-LRR proteins indicates the variety of recognition molecules available in an individual genotype to detect diverse biotic challenges.     

Genome-wide identification and analysis of late embryogenesis abundant (LEA) genes in Prunus mume

Late embryogenesis abundant (LEA) proteins play important roles in plant desiccation tolerance. In this study, 30 LEA genes were identified from Chinese plum (Prunus mume) through genome-wide analysis. The PmLEA genes are distributed on all Chinese plum chromosomes except chromosome 3. Twelve (40 %) and five PmLEA genes are arranged in tandem and segmental duplications, respectively. The PmLEA genes could be divided into eight groups (LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, PvLEA18, dehydrin and seed maturation protein). Ten gene conversion events were observed and most of them (70 %) were identified in dehydrin group. Most PmLEA genes were highly expressed in flower (22/30) and up-regulated by ABA treatment (19/30).     

An intrinsic tonoplast protein of protein storage vacuoles in seeds is structurally related to a bacterial solute transporter (GIpF).

The tonoplast mediates the transport of various ions and metabolites between the vacuole and cytosol by mechanisms that remain to be elucidated at the molecular level. The primary structure of only one tonoplast protein, the H(+)-ATPase, has been reported to date. Here we report the primary structure of tonoplast intrinsic protein (TIP), a 27-kilodalton intrinsic membrane protein that occurs widely in the tonoplasts of the protein storage vacuoles (protein bodies) of seeds [Johnson, K.D., et al. (1989). Plant Physiol. 91, 1006-1013]. Hydropathy plots and secondary structure analysis of the polypeptide predict six membrane-spanning domains connected by short loops and hydrophilic, cytoplasmically oriented N- and C-terminal regions. TIP displays significant homology with several other membrane proteins from diverse sources: major intrinsic polypeptide from bovine lens fiber plasma membrane; NOD 26, a peribacteroid membrane protein in the nitrogen-fixing root nodules of soybean; and interestingly, GIpF, the glycerol facilitator transport protein in the cytoplasmic membrane of Escherichia coli. Based on the homology between TIP and GIpF and the knowledge that the protein storage vacuolar membrane and the peribacteroid membrane are active in solute transport, we propose that TIP transports small metabolites between the storage vacuoles and cytoplasm of seed storage tissues.     

Characterization of Leishmania donovani aquaporins shows presence of subcellular aquaporins similar to tonoplast intrinsic proteins of plants

Leishmania donovani, a protozoan parasite, resides in the macrophages of the mammalian host. The aquaporin family of proteins form important components of the parasite-host interface. The parasite-host interface could be a potential target for chemotherapy. Analysis of L. major and L. infantum genomes showed the presence of five aquaporins (AQPs) annotated as AQP9 (230aa), AQP putative (294aa), AQP-like protein (279aa), AQP1 (314aa) and AQP-like protein (596aa). We report here the structural modeling, localization and functional characterization of the AQPs from L. donovani. LdAQP1, LdAQP9, LdAQP2860 and LdAQP2870 have the canonical NPA-NPA motifs, whereas LdAQP putative has a non-canonical NPM-NPA motif. In the carboxyl terminal to the second NPA box of all AQPs except AQP1, a valine/alanine residue was found instead of the arginine. In that respect these four AQPs are similar to tonoplast intrinsic proteins in plants, which are localized to intracellular organelles. Confocal microscopy of L. donovani expressing GFP-tagged AQPs showed an intracellular localization of LdAQP9 and LdAQP2870. Real-time PCR assays showed expression of all aquaporins except LdAQP2860, whose level was undetectable. Three-dimensional homology modeling of the AQPs showed that LdAQP1 structure bears greater topological similarity to the aquaglyceroporin than to aquaporin of E. coli. The pore of LdAQP1 was very different from the rest in shape and size. The cavity of LdAQP2860 was highly irregular and undefined in geometry. For functional characterization, four AQP proteins were heterologously expressed in yeast. In the fps1Δ yeast cells, which lacked the key aquaglyceroporin, LdAQP1 alone displayed an osmosensitive phenotype indicating glycerol transport activity. However, expression of LdAQP1 and LdAQP putative in a yeast gpd1Δ strain, deleted for glycerol production, conferred osmosensitive phenotype indicating water transport activity or aquaporin function. Our analysis for the first time shows the presence of subcellular aquaporins and provides structural and functional characterization of aquaporins in Leishmania donovani.     

Urea transport by nitrogen-regulated tonoplast intrinsic proteins in Arabidopsis

Urea is the major nitrogen (N) form supplied as fertilizer in agricultural plant production and also an important N metabolite in plants. Because urea transport in plants is not well understood, the aim of the present study was to isolate urea transporter genes from the model plant Arabidopsis. Using heterologous complementation of a urea uptake-defective yeast (Saccharomyces cerevisiae) mutant allowed to isolate AtTIP1;1, AtTIP1;2, AtTIP2;1, and AtTIP4;1 from a cDNA library of Arabidopsis. These cDNAs encode channel-like tonoplast intrinsic proteins (TIPs) that belong to the superfamily of major intrinsic proteins (or aquaporins). All four genes conferred growth of a urea uptake-defective yeast mutant on 2 mm urea in a phloretin-sensitive and pH-independent manner. Uptake studies using 14C-labeled urea into AtTIP2;1-expressing Xenopus laevis oocytes demonstrated that AtTIP2;1 facilitated urea transport also in a pH-independent manner and with linear concentration dependency. Expression studies showed that AtTIP1;2, AtTIP2;1, and AtTIP4;1 genes were up-regulated during early germination and under N deficiency in roots but constitutively expressed in shoots. Subcellular localization of green fluorescent protein-fused AtTIPs indicated that AtTIP1;2, AtTIP2;1, and AtTIP4;1 were targeted mainly to the tonoplast and other endomembranes. Thus, in addition to their role as water channels, TIP transporters may play a role in equilibrating urea concentrations between different cellular compartments.     

Genome-wide analysis and environmental response profiling of SOT family genes in rice (Oryza sativa)

Sulphotransferase (SOT) catalyses the transfer of a sulphonate group from 3??-phosphoadenosine 5??-phosphosulphate (PAPS) to an appropriate hydroxyl group of various substrates with the parallel formation of 3??-phosphoadenosine 5??-phosphate (PAP). Although several SOTs have been identified and characterized in mammalian, their role in plant is still unclear. In this study, we report genome-wide comprehensive expression analysis of 35 putative SOT genes in rice. The 35 OsSOTs were tandemly arranged into six clusters. The phylogenetic analysis showed that there were 7 subfamilies of OsSOTs and 11 putatively conserved motifs. Six OsSOTs might be pseudogenes, 25 have the two motifs which were involved in PAPS binding regions I and IV. Microarray data indicated that all the OsSOTs were expressed almost at the same level but with different patterns: most OsSOTs were expressed exclusively in stigma and ovary and induced by IAA and BAP, several genes were induced by tZ and DMSO and 11 OsSOTs were response to abiotic stress. Further analysis showed that these 11 genes contained cis-regulatory elements responding to abiotic stresses.     

Alpha tonoplast intrinsic protein is specifically associated with vacuole membrane involved in an autophagic process

Autophagy in plant cells is induced by nutrient starvation. Initially, double membrane-bound organelles, termed autophagosomes, enclose a portion of cytoplasm, and then fuse with a vacuole or lysosome to give an autolysosome. Autolysosomes can be visualized by incubating cells in the presence of a membrane-permeable cysteine protease inhibitor. The inhibitor presumably decreases proteolytic degradation of the autolysosome contents that are composed of portions of cytoplasm enclosed by the membrane originating from the inner membrane of autophagosomes, and allows them to accumulate. The origin of membranes that give rise to autophagosomes and autolysosomes is unknown. Here we use an acidotropic fluorescent dye, LysoTracker Red, to label autolysosomes specifically. We demonstrate that autolysosome membranes are marked by the presence of alpha-tonoplast intrinsic protein (alpha-TIP) but not by gamma-TIP or delta-TIP. The identification of a TIP specifically associated with membranes derived from an autophagic process may help our understanding of how plant cells generate and maintain functionally distinct types of vacuoles.