Monocot Plant Species Research Articles

Transcriptome analysis of senescence in the flag leaf of wheat (Triticum aestivum L.)

The senescence process in wheat flag leaves was investigated over a time course from ear emergence until 50% yellowing of harvested leaf samples using an in-house fabricated cDNA microarray based on a 9K wheat unigene set. The top 1000 ranked differentially expressed probes were subjected to a cluster analysis and, from these, we selected 140 up-regulated genes with informative annotations. There was a considerable overlap between this list of genes and genes previously observed to be associated with senescence in other species, covering several functional categories involved in the degradation of macromolecules and nutrient remobilization, notably of nitrogen via the metabolism of carboxylic and amino acids. The up-regulation of a number of genes in this metabolism was confirmed by real-time polymerase chain reaction experiments. The data suggest a role for cytosolic/peroxisomal routes in the integration of the degradation of carbohydrates, fatty acids and proteins, leading to the remobilization of nitrogen. Illustrative examples of up-regulated genes comprise cytoplasmic aconitate hydratase and peroxisomal citrate synthase. The data support a protective role of the mitochondria towards oxidative cell damage via the up-regulation of the alternative oxidase, and possibly also involving the up-regulated succinate dehydrogenase. A number of up-regulated regulatory genes were also identified, notably NAC-domain and WRKY transcription factors. These factors have previously been identified as being associated with senescence in other species. The data support the notion that a generic senescence programme exists across monocot and dicot plant species. However, notable differences can also be recognized. We thus found transcriptional up-regulation of the biosynthetic pathway for benzoxazinoids, a group of graminaceous-specific secondary metabolites.

Structural and Functional Insights into the Regulation of Arabidopsis AGC VIIIa Kinases

The AGCVIIIa kinases of Arabidopsis are members of the eukaryotic PKA, PKG, and PKC group of regulatory kinases. One AGCVIIIa kinase, PINOID (PID), plays a fundamental role in the asymmetrical localization of membrane proteins during polar auxin transport. The remaining 16 AGCVIIIa genes have not been associated with single mutant phenotypes, suggesting that the corresponding kinases function redundantly. Consistent with this idea, we find that the genes encoding the Arabidopsis AGCVIIIa kinases have spatially distinct, but overlapping, expression domains. Here we show that the majority of Arabidopsis AGCVIIIa kinases are substrates for the 3-phosphoinositide-dependent kinase 1 (PDK1) and that trans-phosphorylation by PDK1 correlates with activation of substrate AGCVIIIa kinases. Mutational analysis of two conserved regulatory domains was used to demonstrate that sequences located outside of the C-terminal PDK1 interaction (PIF) domain and the activation loop are required for functional interactions between PDK1 and its substrates. A subset of GFP-tagged AGCVIIIa kinases expressed in Saccharomyces cerevisiae and tobacco BY-2 cells were preferentially localized to the cytoplasm (AGC1-7), nucleus (WAG1 and KIPK), and the cell periphery (PID). We present evidence that PID insertion domain sequences are sufficient to direct the observed peripheral localization. We find that PID specifically but non-selectively binds to phosphoinositides and phosphatidic acid, suggesting that PID might directly interact with the plasma membrane through protein-lipid interactions. The initial characterization of the AGCVIIIa kinases presented here provides a framework for elucidating the physiological roles of these kinases in planta.

Molecular and functional characterization of sulfiredoxin homologs from higher plants

By reducing cysteine-sulfinic acid in oxidized peroxiredoxin, sulfiredoxin (Srx) plays an important role in oxidation stress resistance in yeast and human cells. Here, we report the first molecular and functional characterization of Srx homolog from higher plants. Bioinformatic analysis revealed the presence of potential Srx encoding sequences in both monocot and dicot plant species. Putative plant Srx proteins exhibited significant identities to their orthologs from yeast and human, and contained the conserved signature sequence and residues essential for catalysis. However, unlike yeast and human orthologs, plant Srxs were all predicted to possess chloroplast transit peptide in their primary structure. The Srx proteins from Arabidopsis and rice (designated as AtSrx and OsSrx, respectively) complemented functional deficiency of Srx in the SRX1 deletion yeast cells. A GFP fusion protein of AtSrx was targeted to chloroplast in Arabidopsis mesophyll protoplast. AtSrx transcription occurred in both vegetative and reproductive organs, and the highest transcript level was detected in leaves. Under oxidation stress, AtSrx transcript level was substantially increased, which paralleled with enhanced transcription of 2-Cys peroxiredoxins that have been found essential in maintaining chloroplast redox balance. In addition to oxidation stress, osmotic/water deficit or cold treatments also raised AtSrx transcript level. Consistent with above findings, the knock-out mutant of AtSrx was significantly more susceptible to oxidation stress than wild type Arabidopsis plant. Taken together, the results of this work indicate the existence of functional Srx homolog in higher plants that is essential for plants to cope with oxidation stress.

Pattern formation in the vascular system of monocot and dicot plant species.

Plant vascular tissues are organised in continuous strands, the longitudinal and radial patterns of which are intimately linked to the signals that direct plant architecture as a whole. Therefore, understanding the mechanisms underlying vascular tissue patterning is expected to shed light on patterning events beyond those that organise the vascular system, and thus represents a central issue in plant developmental biology. A number of recent advances, reviewed here, are leading to a more precise definition of the signals that control the formation of vascular tissues and their integration into a larger organismal context. Contents Summary 209 I. Introduction 209 II. The plant vascular system 210 III. Ontogeny of the vascular tissues 210 IV. Procambium development 210 V. The organisation of the vascular tissues 212 VI. The regulation of longitudinal vascular pattern formation 214 VII. The regulation of radial vascular pattern formation 220 VIII. Genetic screens for vascular development mutants 231 IX. Genes involved in vascular development identified through reverse genetics approaches 235 X. Conclusions and perspectives 235 Note added at the revision stage 236 Acknowledgements 236 References 236.

RNAi-mediated tocopherol deficiency impairs photoassimilate export in transgenic potato plants.

Tocopherols (vitamin E) are lipophilic antioxidants presumed to play a key role in protecting chloroplast membranes and the photosynthetic apparatus from photooxidative damage. Additional nonantioxidant functions of tocopherols have been proposed after the recent finding that the Suc export defective1 maize (Zea mays) mutant (sxd1) carries a defect in tocopherol cyclase (TC) and thus is devoid of tocopherols. However, the corresponding vitamin E deficient1 Arabidopsis mutant (vte1) lacks a phenotype analogous to sxd1, suggesting differences in tocopherol function between C4 and C3 plants. Therefore, in this study, the potato (Solanum tuberosum) ortholog of SXD1 was isolated and functionally characterized. StSXD1 encoded a protein with high TC activity in vitro, and chloroplastic localization was demonstrated by transient expression of green fluorescent protein-tagged fusion constructs. RNAi-mediated silencing of StSXD1 in transgenic potato plants resulted in the disruption of TC activity and severe tocopherol deficiency similar to the orthologous sxd1 and vte1 mutants. The nearly complete absence of tocopherols caused a characteristic photoassimilate export-defective phenotype comparable to sxd1, which appeared to be a consequence of vascular-specific callose deposition observed in source leaves. CO2 assimilation rates and photosynthetic gene expression were decreased in source leaves in close correlation with excess sugar accumulation, suggesting a carbohydrate-mediated feedback inhibition rather than a direct impact of tocopherol deficiency on photosynthetic capacity. This conclusion is further supported by an increased photosynthetic capacity of young leaves regardless of decreased tocopherol levels. Our data provide evidence that tocopherol deficiency leads to impaired photoassimilate export from source leaves in both monocot and dicot plant species and suggest significant differences among C3 plants in response to tocopherol reduction.

A glycine-rich protein gene family predominantly expressed in tomato roots, but not in leaves and ripe fruit

Plant root is a unique organ conferring various important functions but lacks comprehensive studies. To identify genes that may contribute to root functions, we searched for genes specifically or abundantly expressed in root, but not in fruit tissues. A family of tomato transcripts (designated as LeGRP), sharing a high degree of nucleotide sequence identity in their 3′-regions, was identified. Southern blot analysis revealed the presence of multiple copies of LeGRP homologues in tomato, as well as in other dicot or monocot plant species tested. Comparative genomic Southern and polymerase chain reaction (PCR) analyses indicated that some LeGRP genes could be cultivar-specific. An open-reading frame (ORF) was identified in the sequence of one of the LeGRP genes ( LeGRP2), with the predicted protein sharing good sequence homology with a few known and putative plant structural signal peptide-containing glycine-rich proteins (GRPs). However, the patterns and modulation of LeGRP gene expression were distinct from those of known plant GRP genes identified to date. LeGRP transcripts predominately accumulated in roots at different developmental stages, but not in leaves or ripe fruit tissues, and their levels declined gradually during plant development. Accumulation of LeGRP transcripts was effectively reduced by treatment with hydrogen peroxide and completely suppressed by salinity and application of sodium salicylate, however significantly enhanced with nutrient supplements such as HYPONeX solutions. To our knowledge, this is the first study to report down-regulation of plant GRP expression by salinity and hydrogen peroxide. Possible roles of LeGRP genes in plant growth, development and response to environmental stimuli are discussed.

Some like it sticky: targeting of the rice gene Waxy

For the first time, a gene function can be knocked out by homologous recombination in a monocot plant species. Fertile rice plants with the desired change in their genome can be obtained reproducibly. Although the absolute frequency of targeting does not exceed that of previous experiments in dicots, the use of a negative selectable marker at both extremities of the targeting construct reduces the amount of screening and excludes ectopic targeting, an unwanted by-product of gene targeting.

Patterns of codon usage bias in three dicot and four monocot plant species.

Codon usage in nuclear genes of four monocot and three dicot species was analyzed to find general patterns in codon choice of plant species. Codon bias was correlated with GC content at the third codon position. GC contents were higher in monocot species than in dicot species at all codon positions. The high GC contents of monocot species might be the result of relatively strong mutational bias that occurred in the lineage of the Poaceae species. In both dicot and monocot species, the effective number of codons (ENCs) for most genes was similar to that for the expected ENCs based on the GC content at the third codon positions. G and C ending codons were detected as the "preferred" codons in monocot species, as in Drosophila. Also, many "preferred" codons are the same in dicot species. Pyrimidine (C and T) is used more frequently than purine (G and A) in four-fold degenerate codon groups.

The novel rice (Oryza sativa L.) gene OsSbf1 encodes a putative member of the Na+/bile acid symporter family.

PCR-based differential screening was used to identify ethylene-induced genes in deep-water rice (Oryza sativa L.). One of the isolated cDNAs represented a novel protein, OsSBF1, with high homology to mammalian Na+/bile acid transporters and to sodium-dependent transporters from bacteria. One highly homologous protein and three less conserved homologues were identified in Arabidopsis thaliana indicating that Sbf proteins exist in monocot and dicot plant species. Expression of OsSbf1 in deep-water rice was shown to be elevated by growth-inducing treatments. Since bile acids have not been found in plants to date a possible function of SBF proteins may be in the transport of structurally related sulphonated brassinosteroids.

Molecular distinction between alternative oxidase from monocots and dicots.

The alternative oxidase (Aox) is encoded in two discrete gene subfamilies in higher plants. Aox1 is most widely known for its induction by stress stimuli in many tissues and is present in both monocot and eudicot plant species. Aox2, on the other hand, is usually constitutive or developmentally

Barley stripe mosaic virus-induced gene silencing in a monocot plant.

RNA silencing of endogenous plant genes can be achieved by virus-mediated, transient expression of homologous gene fragments. This powerful, reverse genetic approach, known as virus-induced gene silencing (VIGS), has been demonstrated only in dicot plant species, where it has become an important tool for functional genomics. Barley stripe mosaic virus (BSMV) is a tripartite, positive-sense RNA virus that infects many agriculturally important monocot species including barley, oats, wheat and maize. To demonstrate VIGS in a monocot host, we modified BSMV to express untranslatable foreign inserts downstream of the gammab gene, in either sense or antisense orientations. Phytoene desaturase (PDS) is required for synthesizing carotenoids, compounds that protect chlorophyll from photo-bleaching. A partial PDS cDNA amplified from barley was 90, 88 and 74% identical to PDS cDNAs from rice, maize and Nicotiana benthamiana, respectively. Barley infected with BSMV expressing barley, rice or maize PDS fragments became photo-bleached and accumulated phytoene (the substrate for PDS) in a manner similar to plants treated with the chemical inhibitor of PDS, norflurazon. In contrast, barley infected with wild-type BSMV, or BSMV expressing either N. benthamiana PDS or antisense green fluorescent protein (GFP), did not photo-bleach or accumulate phytoene. Thus BSMV silencing of the endogenous PDS was homology-dependent. Deletion of the coat protein enhanced the ability of BSMV to silence PDS. This is the first demonstration of VIGS in a monocot, and suggests that BSMV can be used for functional genomics and studies of RNA-silencing mechanisms in monocot plant species.

Toward a cytological characterization of the rice genome.

Rice (Oryza sativa L.) will be the first major crop, as well as the first monocot plant species, to be completely sequenced. Integration of DNA sequence-based maps with cytological maps will be essential to fully characterize the rice genome. We have isolated a set of 24 chromosomal arm-specific bacterial artificial chromosomes to facilitate rice chromosome identification. A standardized rice karyotype was constructed using meiotic pachytene chromosomes of O. sativa spp. japonica rice var. Nipponbare. This karyotype is anchored by centromere-specific and chromosomal arm-specific cytological landmarks and is fully integrated with the most saturated rice genetic linkage maps in which Nipponbare was used as one of the mapping parents. An ideogram depicting the distribution of heterochromatin in the rice genome was developed based on the patterns of 4',6-diamidino-2-phenylindole staining of the Nipponbare pachytene chromosomes. The majority of the heterochromatin is distributed in the pericentric regions with some rice chromosomes containing a significantly higher proportion of heterochromatin than other chromosomes. We showed that pachytene chromosome-based fluorescence in situ hybridization analysis is the most effective approach to integrate DNA sequences with euchromatic and heterochromatic features.

The MADS box gene ZmMADS2 is specifically expressed in maize pollen and during maize pollen tube growth

We screened a cDNA library of mature maize pollen for clones encoding transcription factors. Two novel cDNAs (ZmMADS1 and ZmMADS2) encoding MADS box proteins were isolated. To our knowledge, ZmMADS1 and ZmMADS2 represent the first transcription factors isolated from pollen of a monocot plant species. ZmMADS1 can be classified as a member of the TM3 subfamily of MADS box proteins whereas ZmMADS2, which is described here in more detail, belongs to the AGL17 subfamily. Temporal and spatial expression analyses showed that ZmMADS2 expression is restricted to pollen and roots. During pollen development ZmMADS2 is detectable at all stages of pollen development analyzed by RT-PCR but is most abundant in mature pollen after dehiscence. The establishment of a whole-mount RNA in situ hybridization protocol with in-vitro germinated pollen enabled the localization of ZmMADS2 transcripts in pollen tubes. ZmMADS2 transcripts are translocated into pollen tubes immediately upon germination and display a gradient with increasing concentration towards the tube tip. The vegetative nucleus and the sperm cells, putative targets of ZmMADS2, enter the pollen tube about 2 h later.

Isolation and Characterization of a Salt- and Drought-inducible Gene for S-adenosylmethionine Decarboxylase from Wheat ( Triticum aestivum L.)

Summary A full-length cDNA for S-adenosylmethionine decarboxylase (SAMDC), a key enzyme involved in the biosynthesis of spermidine and spermine, was isolated from wheat ( Triticum aestivum L.) by using reverse transcription PCR technique. Sequence analysis of the wheat SAMDC cDNA (designated TaSAMDC ) revealed that it was significantly homologous to SAMDC genes from both monocot and dicot plant species. Alignment of the deduced polypeptide sequence of TaSAMDCwith those of plant, yeast and human SAMDC genes identified several conserved domains, suggesting the structural and functional similarities of SAMDC proteins in eukaryotes. These conserved regions included a proenzyme cleavage site and a putative PEST domain which is characteristic of the rapid protein turnover. Genomic Southern blot indicated that two or three homologous sequences of TaSAMDC gene were present in the wheat genome. Comparative mapping in rice predicts that this gene locates on the long arm of the homologous group 2 of the wheat genome. The expression of TaSAMDC gene under environmental stress conditions was investigated by Northern blot analysis. It was observed that salinity, drought and exogenous ABA can all induce the expression of TaSAMDC , suggesting that this gene is implicated in response of wheat plants to the adverse circumstances.

The wheat poly(A)-binding protein functionally complements pab1 in yeast.

Poly(A)-binding protein (PAB) binds to the poly(A) tail of most eukaryotic mRNAs and influences its translational efficiency as well as its stability. Although the primary structure of PAB is well conserved in eukaryotes, its functional conservation across species has not been extensively investigated. In order to determine whether PAB from a monocot plant species could function in yeast, a protein characterized as having PAB activity was purified from wheat and a cDNA encoding for PAB was isolated from a wheat seedling expression library. Wheat PAB (72 kDa as estimated by SDS/PAGE and a theoretical mass of 70 823 Da as determined from the cDNA) was present in multiple isoforms and exhibited binding characteristics similar to that determined for yeast PAB. Comparison of the wheat PAB protein sequence with PABs from yeast and other species revealed that wheat PAB contained the characteristic features of all PABs, including four RNA binding domains each of which contained the conserved RNP1 and RNP2 sequence motifs. The wheat PAB cDNA functionally complemented a pab1 mutant in yeast suggesting that, although the amino acid sequence of wheat PAB is only 47% conserved from that of yeast PAB, this monocot protein can function in yeast.

Nuclear Matrix Isolated from Plant Cells

Residual nuclear matrices can be successfully obtained from isolated nuclei of different monocot and dicot plant species using either high ionic or low ionic extraction protocols. The protein composition of isolated nuclear matrices depends on the details of isolation protocols. They are stable and present in all cases, a tripartite organization with a lamina, nucleolar matrix, and internal matrix network, and also maintain some of the basic architectural features of intact nuclei. In situ preparations demonstrate the continuity between the nuclear matrix and the plant cytoskeleton. Two-dimensional separation of isolated plant nuclear matrix proteins reveals a heterogeneous polypeptide composition corresponding rather to a complex multicomponent matrix than to a simple nucleoskeletal structure. Immunological identification of some plant nuclear matrix components such as A and B type lamins, topoisomerase II, and some components of the transcription and splicing machineries, internal intermediate filament proteins, and also specific nucleolar proteins like fibrillarin and nucleolin, which associate to specific matrix domains, establish a model of organization for the plant nuclear matrix similar to that of other eukaryotes. Components of the transcription, processing, and DNA-anchoring complexes are associated with a very stable nucleoskeleton. The plant matrix-attached regions share structural and functional characteristics with those of insects, vertebrates, and yeast, and some of them are active in animal cells. In conclusion, the available data support the view that the plant nuclear matrix is basically similar in animal and plant systems, and has been evolutionarily conserved in eukaryotes.

Characterization of four new beta-tubulin genes and their expression during male flower development in maize (Zea mays L.).

Four different beta-tubulin coding sequences were isolated from a cDNA library prepared from RNA from maize seedling shoots. The four genes (designated tub4, tub6, tub7 and tub8) represented by these cDNA clones together with the tub1 and tub2 genes reported previously encode six beta-tubulin isotypes with 90-97.5% amino acid sequence identity. Results from phylogenetic analysis of 17 beta-tubulin genes from monocot and dicot plant species indicated that multiple extant lines of beta-tubulin genes diverged from a single precursor after the appearance of the two major subfamilies of alpha-tubulin genes described previously. Hybridization probes from the 3' non-coding regions of six beta-tubulin clones were used to quantify the levels of corresponding tubulin transcripts in different maize tissues including developing anthers and pollen. The results from these dot blot hybridization experiments showed that all of the beta-tubulin genes were expressed in most tissues examined, although each gene showed a unique pattern of differential transcript accumulation. The tub1 gene showed a high level of transcript accumulation in meristematic tissues and almost no accumulation in the late stages of anther development and in pollen. In contrast, the level of tub4 transcripts was very low during early stages of male flower development but increased markedly (more than 100 times) during the development of anthers and in pollen. Results from RNAse protection assays showed that this increased hybridization signal resulted from expression of transcripts from one or two genes closely related to tub4. The tub4-related transcripts were not present in shoot tissue. Transcripts from the tub2 gene accumulated to very low levels in all tissues examined, but reached the highest levels in young anthers containing microspore mother cells. RNAse protection assays were used to measure the absolute levels of alpha- and beta-tubulin transcripts in seedling shoot and in pollen. The alpha-tubulin gene subfamily I genes (tua1, tua2, tua4) contributed the great majority of alpha-tubulin transcripts in both shoot and pollen. Transcripts from the beta-tubulin genes tub4, tub6, tub7, and tub8 were predominant in shoot, but were much less significant than the tub4-related transcripts in pollen.

Introduction and differential use of various promoters in pollen grains of Nicotiana glutinosa and Lilium longiflorum

As part of our research to develop an alternative system for the transformation of recalcitrant plant species we investigated the use of the male gametophyte as a transformation vector. Therefore the activity of four different promoters (CaMV 35S, LAT52, chiA PA2 and TR2') was analyzed in pollen of a dicot (Nicotiana glutinosa) and a monocot (Lilium longiflorum) plant species. Gene constructs in which the ß-glucuronidase (GUS) gene was placed under the control of these promoters were introduced in pollen using a particle delivery system. No activity of the Cauliflower Mosaic Virus (CaMV) 35S promoter was detected in pollen of both N. glutinosa and L. longiflorum. The promoter of the tomato flower-specific LAT52 gene was highly active in N. glutinosa pollen but remained silent in L. longiflorum pollen. A similar expression pattern was observed for the pollen-specific Chalcone Flavanone Isomerase chiA PA2 promoter originally isolated from petunia. The TR2' mannopine synthase promoter of Agrobacterium tumefaciens, however, was active in pollen from Solanaceous species and also in pollen from the monocot L. longiflorum. This suggests that the TR2' promoter is active in vegetative and sporogenous tissues of dicot and monocot plant species.

CDNA nucleotide sequence and expression of a tobacco cytoplasmic ribosomal protein L2 gene.

The ribosomal protein L2 is an essential component of the ribosomal large subunit by its relation to the peptidyl transferase reaction, subunit association and elongation factor G-GTP binding. We have isolated a 937 nucleotide long cDNA encoding a cytoplasmic ribosomal L2 protein. Its deduced protein contains 260 amino acid residues and shows 65% identity with eucaryotic RL2 but only 32% identity with the chloroplast homologue. In addition, the protein presents the PROSITE signature which matches all the 50S and 60S L2 proteins and the two residues involved in the peptidyl transferase activity. The corresponding mRNA is accumulated in young plant tissues, in growing cell suspension and in germinating seeds but is not detectable in mature plant tissues, stationary cell suspension and in dry seeds. The mRNA accumulation is correlated with the growth process. Southern blot hybridization shows that cytoplasmic ribosomal protein L2 is encoded by two types of gene which could originate from each parent. highly homologous L2 genes were also detected by Southern blots in the genomes of several monocot and dicot plant species.

The distribution of 5-methylcytosine in the nuclear genome of plants.

We have determined the 5-methylcytosine (5mC) content in high molecular weight DNA, from two dicot (tobacco and pea) and two monocot (wheat and maize) plant species, fractionated according to base composition. The results show that the proportion of 5mC in the genomic fractions increases linearly with their guanine + cytosine (G + C) content while the proportion of non-methylated cytosine remains almost constant. This can be interpreted as a consequence of a difference in mutation pressure related to spontaneous deamination of 5mC to thymine between the different compartments of plant genomes.