B1 Plants Research Articles

Identification of rye B chromosome-associated peptides by mass spectrometry.

B chromosomes (Bs) are supernumerary dispensable components of the standard genome (A chromosomes, As) that have been found in many eukaryotes. So far, it is unkown whether the B-derived transcripts translate to proteins or if the host proteome is changed due to the presence of Bs. Comparative mass spectrometry was performed using the protein samples isolated from shoots of rye plants with and without Bs. We aimed to identify B-associated peptides and analyzed the effects of Bs on the total proteome. Our comparative proteome analysis demonstrates that the presence of rye Bs affects the total proteome including different biological function processes. We found 319 of 16776 quantified features in at least three out of five +B plants but not in 0B plants; 31 of 319 features were identified as B-associated peptide features. According to our data mining, one B-specific protein fragment showed similarity to a glycine-rich RNA binding protein which differed from its A-paralogue by two amino acid insertions. Our result represents a milestone in B chromosome research, because this is the first report to demonstrate the existence of Bs changing the proteome of the host.

Misexpression of a transcriptional repressor candidate provides a molecular mechanism for the suppression of awns by Tipped 1 in wheat.

Awns are bristle-like structures formed at the tip of the lemma on the florets of some cereal grasses. Wild-type wheat is awned, but awnletted and awnless variants have been selected and nowadays all forms are cultivated. In this study, we dissected the genetic control underlying variation of this characteristic feature by association mapping in a large panel of 1110 winter wheat cultivars of worldwide origin. We identified the B1 (Tipped 1) locus on chromosome 5A as the major determinant of awnlessness globally. Using a combination of fine-mapping and expression analysis, we identified a putative C2H2 zinc finger protein with an EAR domain, characteristic of transcriptional repressors, as a likely candidate for Tipped 1. This gene was found to be up-regulated in awnless B1 compared with awned b1 plants, indicating that misexpression of this transcriptional regulator may contribute to the reduction of awn length in B1 plants. Taken together, our study provides an entry point towards a better molecular understanding of the evolution of morphological features in cereals through selection and breeding.

De novo transcriptome sequencing and gene expression profiling with/without B-chromosome plants of Lilium amabile.

Supernumerary B chromosomes were found in Lilium amabile (2n = 2x = 24), an endemic Korean lily that grows in the wild throughout the Korean Peninsula. The extra B chromosomes do not affect the host-plant morphology; therefore, whole transcriptome analysis was performed in 0B and 1B plants to identify differentially expressed genes. A total of 154,810 transcripts were obtained from over 10 Gbp data by de novo assembly. By mapping the raw reads to the de novo transcripts, we identified 7,852 differentially expressed genes (log2FC > |10|), in which 4,059 and 3,794 were up-and down-regulated, respectively, in 1B plants compared to 0B plants. Functional enrichment analysis revealed that various differentially expressed genes were involved in cellular processes including the cell cycle, chromosome breakage and repair, and microtubule formation; all of which may be related to the occurrence and maintenance of B chromosomes. Our data provide insight into transcriptomic changes and evolution of plant B chromosomes and deliver an informative database for future study of B chromosome transcriptomes in the Korean lily.

Plant Growth and Photosynthetic Characteristics of Mesembryanthemum crystallinum Grown Aeroponically under Different Blue- and Red-LEDs.

Mesembryanthemum crystallinum is a succulent, facultative crassulacean acid metabolism (CAM) plant. Plant growth and photosynthetic characteristics were studied when M. crystallinum plants were grown indoor under light emitting diodes (LED)-lighting with adequate water supply. Plants were cultured aeroponically for a 16-h photoperiod at an equal photosynthetic photon flux density of 350 μmol m-2 s-1 under different red:blue LED ratios: (1) 100:0 (0B); (2) 90:10 (10B); (3) 80:20 (20B); (4) 70:30 (30B); (5) 50:50 (50B); and (6)100:0 (100B). M. crystallinum grown under 10B condition had the highest shoot and root biomass and shoot/root ratio while those grown under 0B condition exhibited the lowest values. Compared to plants grown under 0B condition, all other plants had similar but higher total chlorophyll (Chl) and carotenoids (Car) contents and higher Chl a/b ratios. However, there were no significant differences in Chl/Car ratio among all plants grown under different red- and blue-LEDs. Photosynthetic light use efficiency measured by photochemical quenching, non-photochemical quenching, and electron transport rate, demonstrated that plants grown under high blue-LED utilized more light energy and had more effective heat dissipation mechanism compared to plants grown under 0B or lower blue-LED. Statistically, there were no differences in photosynthetic O2 evolution rate, light-saturated CO2 assimilation rate (Asat), and light-saturated stomatal conductance (gssat) among plants grown under different combined red- and blue-LEDs but they were significantly higher than those of 0B plants. No statistically differences in total reduced nitrogen content were found among all plants. For the total soluble protein, all plants grown under different combined red- and blue-LEDs had similar values but they were significantly higher than that of plants grown under 0B condition. However, plants grown under higher blue-LEDs had significant higher ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) protein than those plants grown under lower blue-LED. High Asat and gssat but very low CAM acidity of all M. crystallinum plants during light period, imply that this facultative CAM plant performed C3 photosynthesis when supplied with adequate water. Results of this study suggest that compared to red- or blue-LED alone, appropriate combination of red- and blue-LED lighting enhanced plant growth and photosynthetic capacities of M. crystallinum.

Rye (Secale cereale) supernumerary (B) chromosomes associated with heat tolerance during early stages of male sporogenesis.

Rye supernumerary (B) chromosomes have an accumulation mechanism involving the B subtelomeric domain highly enriched in D1100- and E3900-related sequences. In this work, the effects of heat stress during the early stages of male meiosis in 0B and +B plants were studied. In-depth cytological analyses of chromatin structure and behaviour were performed on staged rye meiocytes utilizing DAPI, fluorescence in situ hybridization and 5-methylcytosine immune labelling. Quantitative real-time PCR was used to measure heat effects on the expression of the Hsp101 gene as well as the 3·9- and 2·7-kb E3900 forms in various tissues and meiotic stages. Quantitative real-time PCR established that heat induced equal up-regulation of the Hsp101 gene in 0B and 2B plants, with a marked peak in anthers with meiocytes staged at pachytene. Heat also resulted in significant up-regulation of E3900-related transcripts, especially at pachytene and for the truncated 2·7-kb form of E3900. Cytological heat-induced anomalies in prophase I, measured as the frequency of anomalous meiocytes, were significantly greater in 0B plants. Whereas telomeric sequences were widely distributed in a manner close to normal in the majority of 2B pachytene cells, most 0B meiocytes displayed abnormally clustered telomeres after chromosome pairing had occurred. Relevantly, bioinformatic analysis revealed a significantly high-density heat responsive cis regulatory sequence on E3900, clearly supporting stress-induced response of transcription for the truncated variant. Taken together, these results are the first indication that rye B chromosomes have implications on heat tolerance and may protect meiocytes against heat stress-induced damage.

Bending of protonema cells in a plastid glycolate/glycerate transporter knockout line of Physcomitrella patens.

Arabidopsis LrgB (synonym PLGG1) is a plastid glycolate/glycerate transporter associated with recycling of 2-phosphoglycolate generated via the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We isolated two homologous genes (PpLrgB1 and B2) from the moss Physcomitrella patens. Phylogenetic tree analysis showed that PpLrgB1 was monophyletic with LrgB proteins of land plants, whereas PpLrgB2 was divergent from the green plant lineage. Experiments with PpLrgB–GFP fusion proteins suggested that both PpLrgB1 and B2 proteins were located in chloroplasts. We generated PpLrgB single (∆B1 and ∆B2) and double (∆B1/∆B2)-knockout lines using gene targeting of P. patens. The ∆B1 plants showed decreases in growth and photosynthetic activity, and their protonema cells were bent and accumulated glycolate. However, because ∆B2 and ∆B1/∆B2 plants showed no obvious phenotypic change relative to the wild-type or ∆B1 plants, respectively, the function of PpLrgB2 remains unclear. Arabidopsis LrgB could complement the ∆B1 phenotype, suggesting that the function of PpLrgB1 is the same as that of AtLrgB. When ∆B1 was grown under high-CO2 conditions, all novel phenotypes were suppressed. Moreover, protonema cells of wild-type plants exhibited a bending phenotype when cultured on media containing glycolate or glycerate, suggesting that accumulation of photorespiratory metabolites caused P. patens cells to bend.

Inheritance of changes conditioned by the leaf-reducing gene in Swedish clover (Tnfolium hybridum L.)

A description of a new mutant of <i>Trifolium hybridum</i> with reduced leaf blades is given. The mutants are female sterile due to deficient ovary development. The genetic analysis showed that morphological changes and lowered fertility are caused by one recessive allele of the gene called <i>reductivus</i>. Male infertility in B1 plants depends also on one recessive gene, but located on a different chromosome than is the <i>reductivus</i> gene.

Rye B Chromosomes Influence the Dynamics of Histone H3 Methylation during Microgametogenesis

We have studied the trimethylation dynamics of lysines 4 and 27 of histone H3 in rye with and without B chromosomes (Bs) in root tip mitosis, meiosis, and pollen grain mitosis by immunostaining. In root meristems, H3K4me3 immunolabeling was homogeneous along the chromosome arms of the normal complement (As), with the exception of the pericentromeric and subtelomeric regions which were unlabeled. On the contrary, a signal was observed on the long arm of the B chromosome, in the region where most of the B-specific repeats are located. H3K27me3 immunosignals were observed on the subtelomeric heterochromatic region of the As and the Bs and some interstitial bands of the As. Thus, the terminal region of the Bs showed both signals, whereas the subtelomeric region of the As showed H3K27me3 immunosignals only. During meiosis and first pollen grain mitosis, the immunosignals were observed distributed as in the root tip mitosis in plants with or without Bs. However, we observed remarkable changes in the immunolabeling patterns during the second pollen grain mitosis between 0B and +B plants. In 0B plants, H3K4me3 immunosignals were similarly distributed in the vegetative and generative nuclei. In B-carrying plants, the vegetative nucleus showed a lighter signal than the generative one. In 0B plants, all nuclei of the microgametophyte showed H3K27me3 immunosignals. In B-carrying plants, the generative nucleus and, correspondingly, the second metaphase, anaphase, and sperm nuclei did not show any signal. When the Bs were lost as micronuclei, they did not show any H3K4me3 or H3K27me3 signal. Most remarkably, Bs are able to change the pattern of H3 methylation on K4 and K27 during the second pollen mitosis, resulting in differently labeled sperm nuclei in 0 and +B plants.

Biochemical Evidence for Translational Repression by Arabidopsis MicroRNAs

MicroRNAs (miRNAs) regulate gene expression posttranscriptionally through RNA silencing, a mechanism conserved in eukaryotes. Prevailing models entail most animal miRNAs affecting gene expression by blocking mRNA translation and most plant miRNAs, triggering mRNA cleavage. Here, using polysome fractionation in Arabidopsis thaliana, we found that a portion of mature miRNAs and ARGONAUTE1 (AGO1) is associated with polysomes, likely through their mRNA target. We observed enhanced accumulation of several distinct miRNA targets at both the mRNA and protein levels in an ago1 hypomorphic mutant. By contrast, translational repression, but not cleavage, persisted in transgenic plants expressing the slicing-inhibitor 2b protein from Cucumber mosaic virus. In agreement, we found that the polysome association of miR168 was lost in ago1 but maintained in 2b plants, indicating that translational repression is correlated with the presence of miRNAs and AGO1 in polysomes. This work provides direct biochemical evidence for a translational component in the plant miRNA pathway.

The parasitic effects of rye B chromosomes might be beneficial in the long term

Rye B chromosomes (Bs) have strong parasitic effects on fertility. B carrying plants are less fertile than 0B ones, whereas the Bs have no significant effects on plant vigour. On the other hand, it has been reported that B transmission is under genetic control in such a way that H line plants transmit the Bs at high frequency, whereas the Bs in the low B transmission rate line (L) fail to pair at metaphase I and are frequently lost. In the present work we analyse variables affecting vigour and fertility considering not only the number of Bs of each plant, but also its H or L status and the B number of its maternal parent. Our results show that the Bs not only decrease female fertility of the B carrier, but the fertility of its progeny, with the exception of 0B plants coming from a 4B mother, which are the most fertile. In this way B chromosomes can be considered as a selective factor. Pollen abortion was higher in B carriers, in the progeny of B carriers and in H plants, but 4B plants coming from B carrying mothers produce less aborted pollen, indicating that a high B number is more deleterious if it is transmitted in the pollen grains. A similar result was obtained for endosperm quality estimated as grain weight, because it is negatively influenced by the Bs in 4B plants coming from a 0B mother. H plants were always less fertile than L ones, indicating that alleles increasing the loss of Bs in the L line will be probably selected as a defence of the A genome against the invasive Bs of the H line. Flower number is not affected by the Bs.

Chromosome Instabilities and Programmed Cell Death in Tapetal Cells of Maize With B Chromosomes and Effects on Pollen Viability

Abstract B chromosomes (B’s), knobbed chromosomes, and chromosome 6 (NOR) of maize undergo nondisjunction and micronucleus formation in binucleate tapetal cells. These chromosome instabilities are regular events in the program of tapetal cell death, but the B’s strongly increase A chromosome instability. We studied 1B and 0B plants belonging to selected lines for high or low B transmission rate and their F1 hybrids. These lines are characterized by meiotic conservation or loss of B chromosomes, respectively. The female B transmission (fBtl) allele(s) for low B transmission is dominant, inducing micronucleus formation and B nondisjunction. We hypothesize that the fBtl allele(s) induces knob instability. This instability would be sufficient to produce B loss in both meiocytes and binucleate tapetal cells. B instability could, in turn, produce instabilities in all chromosomes of maize complement. To establish whether the chromosomal instabilities are related to the tapetal programmed cell death (PCD) process, we applied the TUNEL technique. PCD, estimated as the frequency of binucleate tapetal cells with TUNEL label, was significantly correlated with the formation of micronuclei and the frequency of pollen abortion. It can be concluded that the observed chromosome instabilities are important to the PCD process and to the development of microspores to form viable pollen grains.

Chromosome instabilities and programmed cell death in tapetal cells of maize with B chromosomes and effects on pollen viability.

B chromosomes (B's), knobbed chromosomes, and chromosome 6 (NOR) of maize undergo nondisjunction and micronucleus formation in binucleate tapetal cells. These chromosome instabilities are regular events in the program of tapetal cell death, but the B's strongly increase A chromosome instability. We studied 1B and 0B plants belonging to selected lines for high or low B transmission rate and their F1 hybrids. These lines are characterized by meiotic conservation or loss of B chromosomes, respectively. The female B transmission (fBtl) allele(s) for low B transmission is dominant, inducing micronucleus formation and B nondisjunction. We hypothesize that the fBtl allele(s) induces knob instability. This instability would be sufficient to produce B loss in both meiocytes and binucleate tapetal cells. B instability could, in turn, produce instabilities in all chromosomes of maize complement. To establish whether the chromosomal instabilities are related to the tapetal programmed cell death (PCD) process, we applied the TUNEL technique. PCD, estimated as the frequency of binucleate tapetal cells with TUNEL label, was significantly correlated with the formation of micronuclei and the frequency of pollen abortion. It can be concluded that the observed chromosome instabilities are important to the PCD process and to the development of microspores to form viable pollen grains.

Relationship between pachytene synapsis, metaphase I associations, and transmission of 2B and 4B chromosomes in rye

2B rye plants selected for high (H) or low (L) B transmission rate were studied at pachytene and metaphase I of meiosis to determine the relationship between synapsis, bivalents at metaphase I, and B transmission rate. The results show that the 2 B chromosomes (Bs) form bivalents at pachytene in both the H and L lines, whereas the frequency of bivalents at metaphase I is much higher in the H than in the L line. This demonstrates that B transmission is mainly related to the proper association of Bs at metaphase I, as well as that synapsis of the 2 Bs in the L line is normal, but the bivalent is not consolidated by a chiasma in most cases. Crosses were made between 2B plants of the H and L lines in all combinations (H × H, H × L, L × H, and L × L) to obtain 4B plants. Similarly, bivalent formation at pachytene and metaphase I was studied. The results show that 4B plants of the H × H and L × L classes differ significantly at pachytene and metaphase I since the former forms more bivalents. The heterozygous 4 Bs of the H × L and L × H classes show intermediate values. The relation H × H > H × L > L × H > L × L was consistently found for the variables transmission rate, bivalents at pachytene, bivalents at metaphase I, and B mean chiasma frequency. A maternal effect was also found. Our data suggest that there are two separate mechanisms acting upon synapsis and chiasma formation in H and L B chromosomes: (i) there is variable efficiency of the control of synapsis at early stages of meiosis; and (ii) there is variable efficiency of the control of the number of chiasmata.Key words: B chromosomes, synaptonemal complex, Secale cereale.

Relationship between pachytene synapsis, metaphase I associations, and transmission of 2B and 4B chromosomes in rye

2B rye plants selected for high (H) or low (L) B transmission rate were studied at pachytene and metaphase I of meiosis to determine the relationship between synapsis, bivalents at metaphase I, and B transmission rate. The results show that the 2 B chromosomes (Bs) form bivalents at pachytene in both the H and L lines, whereas the frequency of bivalents at metaphase I is much higher in the H than in the L line. This demonstrates that B transmission is mainly related to the proper association of Bs at metaphase I, as well as that synapsis of the 2 Bs in the L line is normal, but the bivalent is not consolidated by a chiasma in most cases. Crosses were made between 2B plants of the H and L lines in all combinations (H x H, H x L, L x H, and L x L) to obtain 4B plants. Similarly, bivalent formation at pachytene and metaphase I was studied. The results show that 4B plants of the H x H and L x L classes differ significantly at pachytene and metaphase I since the former forms more bivalents. The heterozygous 4 Bs of the H x L and L x H classes show intermediate values. The relation H x H > H x L > L x H > L x L was consistently found for the variables transmission rate, bivalents at pachytene, bivalents at metaphase I, and B mean chiasma frequency. A maternal effect was also found. Our data suggest that there are two separate mechanisms acting upon synapsis and chiasma formation in H and L B chromosomes: (i) there is variable efficiency of the control of synapsis at early stages of meiosis; and (ii) there is variable efficiency of the control of the number of chiasmata.

Possibility of Developing a Male Sterile Line of Shallot (Allium cepa L. Aggregatum Group) with Cytoplasm from A. galanthum Kar. et Kir.

In an effort to develop a male sterile line of shallot (Allium cepa L. Aggregatum group), a continuous backcrossing was carried out using A. galanthum Kar. et Kir. as a cytoplasm donor and the shallot as a nucleus donor. Meiosis and fertility in the F1 hybrids, B1, B2, and B3 progenies were examined. The F1 hybrids had low pollen fertility, whereas the B1 plants were pollen sterile or nearly so ; the B2 and B3 plants were completely pollen sterile. Pollen sterility observed in the backcross progenies was attributed to nuclear-cytoplasmic incompatibility because the pollen mother cells in most of these plants underwent normal meiosis. Although the seed fertility varied considerably among the progenies at each backcross generation, it could be improved with the advancement of backcrossing. PCR-RFLP analysis of chloroplast DNA proved that all the backcross progenies had the cytoplasm from A. galanthum. These results demonstrate that there is a realizable possibility of developing a male sterile line of shallot using this genetic approach.

Organisation and origin of a B chromosome centromeric sequence from Brachycome dichromosomatica.

Brachycome dichromosomatica is an Australian native daisy that has two pairs of A chromosomes and up to three B chromosomes in some populations. A putative B-specific tandem repeat DNA sequence (Bd49) was isolated previously. Here we describe further characterisation of this sequence and investigate its possible origin. Southern analysis showed that all individual B chromosomes examined have highly methylated tandem repeats of Bd49 but differences in banding pattern for distinct B isolates suggested that the sequence is in a state of flux. Using in situ hybridisation, the sequence was shown to be located at the centromeric region of the B chromosome. Southern analysis of genomic DNA with Bd49 demonstrated that multiple copies of the sequence exist in the genomes of B. eriogona, B. ciliaris, B. segmentosa and B. multifida (none of which have B chromosomes) whereas other species tested (including 0B plants of B. dichromosomatica and 0B and +B B. curvicarpa and B. dentata) have few or no copies. Genomic clones and Bd49-like sequences derived by the polymerase chain reaction (PCR) were obtained from five species but determination of phylogenetic relationships within the genus and inference as to the possible origin of the B chromosome were problematic because of extensive intragenomic heterogeneity of the sequences.

Genetic control of the rate of transmission of rye B chromosomes. II. 0B × 2B crosses

Twenty ♀ 0B × ♂ 2B crosses were made in which a single male was crossed with four different females, and the character 'mean number of Bs per plant in the progeny' was analysed. The 2B plants used as males and the 0B females belonged either to low or to high transmission rate lines, previously selected. The genetic basis of the difference between the high and low transmission rate lines was statistically tested. The male group (high or low class of the 2B male) has a significant effect on the progeny, while the female group (high or low class of the 0B female) has no significant effect. The genes controlling the transmission rate of B chromosomes affect the proportion of 0B vs. 2B plants in the 0B × 2B cross, but this is not a result of any effect on the nondisjunction mechanism, since the proportion of 1B plants is low in all cases. Structural variants of B chromosomes appeared de novo at a high frequency.

Molecular relationship between the A and B chromosomes of Crepis capillaris

The molecular structure of the B chromosome of Crepis capillaris has been analysed by DNA comparisons of plants with 2B and 0B chromosomes. No additional band, and no differences in the band intensity were detected in 2B plants after digestion with 18 restriction enzymes. One hundred and five clones were obtained from 2B plants using the phenol-emulsion reassociation technique in formamide (F-PERT). Only one (clone pCc21) gave rise to additional hybridization bands in the DNA from 2B plants, indicating that this sequence occurs both in A and B chromosomes. Clone pCc21 contains a 54-bp microsatellite sequence rich in CA dinucleotides, similar to those found in other eukaryotic genomes. We conclude that the B chromosome of C. capillaris carries no specific highly repetitive sequence, but consists of sequences also present in the A chromosomes. The possibility remains that the heterochromatic pericentromeric region of the B has a different organization with respect to standard heterochromatin.

Intergeneric hybridization between Brassica napus and Sinapis pubescens, and the cytology and crossability of their progenies

The cytological possibility of gene transfer from Sinapis pubescens to Brassica napus was investigated. Intergeneric hybrids between Brassica napus (2n = 38) and Sinapis pubescens (2n = 18) were produced through ovary culture. The F1 hybrids were dihaploid and the chromosome configurations were (0-1) III + (2-11) II + (5-24) I . One F2 plant with 38 chromosomes was obtained from open pollination of the F1 hybrid. Thirty-one seeds were obtained from the backcross of the F2 plant with B. napus. Five out of seven plants had 38 chromosomes, and the pollen stainability ranged from 0% to 81.4%. In the B2 plants obtained from the backcross of B1 plants with B. napus, 66.7% of the plants examined had 38 chromosomes. S. pubescens may become a gene source for the improvement of B. napus.

Factors controlling the dynamics of the B chromosome polymorphism in Korean rye

In order to test if a particular rye population has a specific frequency of B chromosomes or, on the contrary, if it can tolerate Bs over a wide range of frequencies, we have studied the transmission of Bs in two Korean varieties which differ in their natural B frequencies. Synthetic populations were made for each variety which carried Bs at both the natural frequency as well as at the frequency of the alternative population. The populations were open pollinated, and the frequencies of the Bs were studied over three generations. The experiment was duplicated in two different environments and the variables of viability and fertility were also compared. Fertility was lower in the 2B than in the 0B plants, and it was much lower again in the 4B ones. Viability was not dependent on the number of Bs. The results were similar for both environments and this finding argues against an adaptive role for B chromosomes. The frequency of Bs in the populations sown at their natural frequencies remained constant over the generations in both environments, while the Bs of both varieties tended to move towards their natural frequencies after they were experimentally changed. There was a strong tendency for the proportion of plants with Bs in the variety with a high natural frequency to increase, while the tendency for the proportion of plants with Bs in the variety with a low natural frequency to decrease was less. These results indicate just how strong are the forces which maintain Bs in populations in the face of their harmful effects upon fertility. In our opinion the genes that control B transmission rates are the main factors that determine the range of frequencies at which a natural population can carry B chromosomes, while the tendency to recover natural frequencies rapidly following a disturbance is due to frequency-dependent selection and to a maternal imprinting effect.