MADS about MOSS

Classic MIKC-type MADS-box genes (MIKC^c) play diverse and crucial roles in angiosperm development, the most studied and best understood of which is the specification of floral organ identities. To shed light on how the flower evolved, phylogenetic and functional analyses of genes involved in its ontogeny, such as the MIKC^c genes, must be undertaken in as broad a selection as possible of plants with disparate ancestries. Since little is known about the functions of these genes in non-seed plants, we investigated the developmental roles of a subset of the MIKC^c genes present in the moss, Physcomitrella patens, which is positioned informatively near the base of the land plant evolutionary tree. We observed that transgenic lines possessing an antisense copy of a MIKC^c gene characteristically displayed knocked-down expression of the corresponding native MIKC^c gene as well as multiple diverse phenotypic alterations to the haploid gametophytic and diploid sporophytic generations of the life cycle.¹ In this addendum, we re-examine our findings in the light of recent pertinent literature and provide additional data concerning the effects of simultaneously knocking out multiple MIKC^c genes in this moss.Key words: Physcomitrella, moss, MADS-box gene functions, gene knock-down, gene knockout, gene expression, evo-devoThe moss, Physcomitrella patens, is the only non-seed plant that is amenable to an investigation of MADS-box gene function comparable to that achieved in angiosperms. P. patens possesses six MIKC^c genes which cluster into two distinct phylogenetic clades.² We recently reported a functional genetic analysis of the three genes (PPM1, PPM2 and PpMADS1) within the PPM2-like clade as an initial contribution towards gaining an understanding of the role(s) of MIKC^c genes in this moss.¹By fusing the respective MIKC^c promoters to a GUS reporter gene, we found that both PPM1 and PpMADS1 exhibited fairly ubiquitous expression patterns in both gametophytic and sporophytic tissues. The levels of PPM1 expression were generally higher than those of PpMADS1, and PpMADS1 was not expressed in antheridia, suggesting subtle differences in the functions of these genes. The observed patterns of widespread expression resemble those characterising the majority of vascular, non-seed plant MIKC^c genes^3–7 and accord with RT-PCR results of Quodt and coworkers.² Our in situ GUS expression and RT-PCR results¹ showed that PPM2 was not expressed or was expressed at levels too low to be detected by these methods. Conversely, the original isolation of PPM2 cDNA⁸ and data from more recent expression studies² indicated that PPM2 is expressed (albeit inconsistently and weakly) ubiquitously with elevated levels of expression sometimes observed in gametangia, sporophytic feet and basal portions of sporophytic setae.² The contradictory expression data for PPM2 may derive from differences between the PPM2-reporter gene constructs used by the respective research groups^1,2 or perhaps from variations in moss culture conditions.We also employed an antisense approach designed to knock down expression of PPM1, and perhaps closely related MIKC^c genes, in order to discern MADS-box gene function in P. patens. Knocked-down strains displayed a complex mutant phenotype comprising delayed gametangia formation and sporophyte production, diminished sporophyte yields, and morphological abnormalities in both leaves and sporophytes, findings that are generally consistent with the ubiquitous expression pattern of PPM1¹ and PPM2''s expression as described by Quodt et al.²The phenotypes of strains with single gene knockouts of PPM1, PPM2 or PpMADS1 appeared to be perfectly normal, not displaying any of the phenotypic alterations observed in PPM1 gene knock-down mutants. While it is possible that subtle, transient or conditional phenotypic changes went unnoticed, it seems more probable that genetic redundancy is responsible for these results since the PPM2-like genes exhibit a very high level of sequence similarity. In an effort to circumvent the problem of functional redundancy, we generated all double knockout combinations for PPM1, PPM2 and PpMADS1. However, the double mutants were also phenotypically unchanged. Finally we attempted to produce triple mutants by co-transforming single PPM2 knockout lines with PPM1 and PpMADS1 linear knockout constructs. Of the 31 stable transformants from two transformation experiments, 55% were shown to be double mutants in which the original PPM2 knockout was accompanied by a second gene knockout in either PPM1 or PpMADS1. However, no triple knockouts were obtained. Given the knockout frequencies generally observed in batch transformation experiments in our laboratory and those of others,⁹ between two and five of the transformants had been expected to be triple mutants. These preliminary data, albeit involving a relatively small sample of transformants, suggest that PPM1, PPM2 and PpMADS1 triple knockouts may be lethal.We have related compelling evidence that functionally redundant PPM2-like MIKC^c genes are involved in several aspects of the moss developmental program. It has been argued that broad expression patterns like theirs represent the ancestral state of MADS-box genes in land plants, and that the sporophytic- and organ-specific expression patterns that characterise many MIKC^c genes in extant spermatophytes, including those that specify floral organ identity, correspond to a derived condition that evolved in the spermatophyte lineage following its separation from lineages that led to bryophytes and ferns and fern allies.¹⁰ Nevertheless, it is the apparent participation of PPM2-like genes in the formation of gametangia (the differentiation of reproductive organs from non-reproductive tissues at the gametophore apex) that is particularly interesting and assumes a special significance because of its analogy to the proposed role for ancestors of seed plant C-function MADS-box genes (identifying those regions of the vegetative SAM that will become reproductive organs).11 Furthermore, expression studies of MIKC^c genes in two charophycean algae, the presumed progenitors of all terrestrial plants,^12–14 suggest that they too are involved in haploid reproductive cell differentiation.¹⁵ While these functional similarities do not infer orthology and may be coincidental, we should not discount yet the admittedly controversial hypothesis that some MIKC^c genes in non-seed plants, for example PPM2-like genes of Physcomitrella, are homologous to spermatophyte class C genes and that the ancient role proposed for ancestral class C genes¹¹ has been conserved, in some form, in all major terrestrial plant taxa.     

Euchromatin and pericentromeric heterochromatin: comparative composition in the tomato genome

Eleven sequenced BACs were annotated and localized via FISH to tomato pachytene chromosomes providing the first global insights into the compositional differences of euchromatin and pericentromeric heterochromatin in this model dicot species. The results indicate that tomato euchromatin has a gene density (6.7 kb/gene) similar to that of Arabidopsis and rice. Thus, while the euchromatin comprises only 25% of the tomato nuclear DNA, it is sufficient to account for approximately 90% of the estimated 38,000 nontransposon genes that compose the tomato genome. Moreover, euchromatic BACs were largely devoid of transposons or other repetitive elements. In contrast, BACs assigned to the pericentromeric heterochromatin had a gene density 10-100 times lower than that of the euchromatin and are heavily populated by retrotransposons preferential to the heterochromatin-the most abundant transposons belonging to the Jinling Ty3/gypsy-like retrotransposon family. Jinling elements are highly methylated and rarely transcribed. Nonetheless, they have spread throughout the pericentromeric heterochromatin in tomato and wild tomato species fairly recently-well after tomato diverged from potato and other related solanaceous species. The implications of these findings on evolution and on sequencing the genomes of tomato and other solanaceous species are discussed.     

Pulse Decomposition Analysis of the digital arterial pulse during hemorrhage simulation

Background

Markers of temporal changes in central blood volume are required to non-invasively detect hemorrhage and the onset of hemorrhagic shock. Recent work suggests that pulse pressure may be such a marker. A new approach to tracking blood pressure, and pulse pressure specifically is presented that is based on a new form of pulse pressure wave analysis called Pulse Decomposition Analysis (PDA). The premise of the PDA model is that the peripheral arterial pressure pulse is a superposition of five individual component pressure pulses, the first of which is due to the left ventricular ejection from the heart while the remaining component pressure pulses are reflections and re-reflections that originate from only two reflection sites within the central arteries. The hypothesis examined here is that the PDA parameter T13, the timing delay between the first and third component pulses, correlates with pulse pressure. T13 was monitored along with blood pressure, as determined by an automatic cuff and another continuous blood pressure monitor, during the course of lower body negative pressure (LBNP) sessions involving four stages, -15 mmHg, -30 mmHg, -45 mmHg, and -60 mmHg, in fifteen subjects (average age: 24.4 years, SD: 3.0 years; average height: 168.6 cm, SD: 8.0 cm; average weight: 64.0 kg, SD: 9.1 kg).

Results

Statistically significant correlations between T13 and pulse pressure as well as the ability of T13 to resolve the effects of different LBNP stages were established. Experimental T13 values were compared with predictions of the PDA model. These interventions resulted in pulse pressure changes of up to 7.8 mmHg (SE = 3.49 mmHg) as determined by the automatic cuff. Corresponding changes in T13 were a shortening by -72 milliseconds (SE = 4.17 milliseconds). In contrast to the other two methodologies, T13 was able to resolve the effects of the two least negative pressure stages with significance set at p < 0.01.

Conclusions

The agreement of observations and measurements provides a preliminary validation of the PDA model regarding the origin of the arterial pressure pulse reflections. The proposed physical picture of the PDA model is attractive because it identifies the contributions of distinct reflecting arterial tree components to the peripheral pressure pulse envelope. Since the importance of arterial pressure reflections to cardiovascular health is well known, the PDA pulse analysis could provide, beyond the tracking of blood pressure, an assessment tool of those reflections as well as the health of the sites that give rise to them.     

NEDD4L Is Downregulated in Colorectal Cancer and Inhibits Canonical WNT Signaling

The NEDD4 family of E3 ubiquitin ligases includes nine members. Each is a modular protein, containing an N-terminal C2 domain for cell localization, two-to-four central WW domains for substrate recognition, and a C-terminal, catalytic HECT domain, which is responsible for catalyzing the ubiquitylation reaction. Members of this family are known to affect pathways central to the pathogenesis of colorectal cancer, including the WNT, TGFβ, EGFR, and p53 pathways. Recently, NEDD4 mRNA was reported to be overexpressed in colorectal cancer, but tumor stage was not considered in the analysis. Expression of the other family members has not been studied in colorectal cancer. Herein, we determined the expression patterns of all nine NEDD4 family members in 256 patients who presented with disease ranging from premalignant adenoma to stage IV colorectal cancer. NEDD4 mRNA was significantly increased in all stages of colorectal cancer. In contrast, NEDD4L mRNA, the closest homolog to NEDD4, was the most highly downregulated family member, and was significantly downregulated in all tumor stages. We also found NEDD4L protein was significantly decreased by western blotting in colorectal cancer samples compared to adjacent normal mucosa. In addition, NEDD4L, but not catalytically inactive NEDD4L, inhibited canonical WNT signaling at or below the level of β-catenin in vitro. These findings suggest that NEDD4L may play a tumor suppressive role in colorectal cancer, possibly through inhibition of canonical WNT signaling.     

Map-based cloning in crop plants: tomato as a model system II. Isolation and characterization of a set of overlapping yeast artificial chromosomes encompassing the jointless locus

A map-based cloning technique for crop plants is being developed using tomato as a model system. The target gene jointless is a recessive mutation that completely suppresses the formation of flower and fruit pedicel abscission zones. Previously, the jointless locus was mapped to a 3 cM interval between the two molecular markers TG523 and RPD158. Physical mapping of the jointless region by pulsed-field gel electrophoresis demonstrated that TG523 and RPD158 reside on a 600 kb SmaI fragment. In this study, TG523 was used as a probe to screen a tomato yeast artificial chromosome (YAC) library. Six tomato YAC (TY) clones were isolated, ranging from 220 to 380 kb in size. Genetic mapping of YAC ends demonstrated that this set of overlapping YACs encompasses the jointless locus. Two YAC ends, TY159L (L indicates left end) and TY143R (R indicates right end), cosegregate with the jointless locus. Only one of the six YACs (TY142) contained single-copy DNA sequences at both ends that could be mapped. The two ends of TY142 were mapped to either side of the jointless locus, indicating that TY142 contains a contiguous 285 kb tomato DNA fragment that probably includes the jointless locus. Physical mapping of the TY142 clone revealed that TY159L and TY143R reside on a 55 kb SalI fragment. Southern blot hybridization analysis of the DNAs of tomato lines nearly isogenic for the jointless mutation has allowed localization of the target locus to a region of less than 50 kb within the TY142 clone.Communicated by H. Saedler     

Molecular mapping of the centromeres of tomato chromosomes 7 and 9

The centromeres of two tomato chromosomes have been precisely localized on the molecular linkage map through dosage analysis of trisomic stocks. To map the centromeres of chromosomes 7 and 9, complementary telo-, secondary, and tertiary trisomic stocks were used to assign DNA markers to their respective chromosome arms and thus to localize the centromere at the junction of the short and long arms. It was found that both centromeres are situated within a cluster of cosegregating markers. In an attempt to order the markers within the centric clusters, genetic maps of the centromeric regions of chromosomes 7 and 9 were constructed from F₂ populations of 1620Lycopersicon esculentum × L. pennellii (E × P) plants and 1640L. esculentum × L. pimpinellifolium (E × PM) plants. Despite the large number of plants analyzed, very few recombination events were detected in the centric regions, indicating a significant suppression of recombination at this region of the chromosome. The fact that recombination suppression is equally strong in crosses between closely related (E × PM) and remotely related (E × P) parents suggests that centromeric suppression is not due to DNA sequence mismatches but to some other mechanism. The greatest number of centromeric markers was resolved in theL. esculentum × L. pennellii F₂ population. The centromere of chromosome 7 is surrounded by eight cosegregating markers: three on the short arm, five on the long arm. Similarly, the centric region of chromosome 9 contains ten cosegregating markers including one short arm marker and nine long arm markers. The localization of centromeres to precise intervals on the molecular linkage map represents the first step towards the characterization and ultimate isolation of tomato centromeres.     

Advanced backcross QTL analysis in a cross between an elite processing line of tomato and its wild relative L. pimpinellifolium

Approximately 170 BC2 plants from a cross between an elite processing inbred (recurrent parent) and the wild species Lycopersicon pimpinellifolium LA1589 (donor parent) were analyzed with segregating molecular markers covering the entire tomato genome. Marker data were used to identify QTLs controlling a battery of horticultural traits measured on BC2F1 and BC3 families derived from the BC2 individuals. Despite its overall inferior appearance, L. pimpinellifolium was shown to possess QTL alleles capable of enhancing most traits important in processing tomato production. QTL-NIL lines, containing specific QTLs modifying fruit size and shape, were subsequently constructed and shown to display the transgressive phenotypes predicted from the original BC2 QTL analysis. The potential of exploiting unadapted and wild germplasm via advanced backcross QTL analysis for the enhancement of elite crop varieties is discussed.     

Genetic analysis of RFLPs, GATA microsatellites and RAPDs in a cross between L. esculentum and L. pimpinellifolium

A population of 257 BC₁ plants was developed from a cross between an elite processing line of tomato (Lycopersicon esculentum cvM82-1-7) and the closely related wild species L. pimpinellifolium (LA1589). The population was used to construct a genetic linkage map suitable for quantitative trait locus (QTL) analysis to be conducted in different backcross generations. The map comprises 115 RFLP, 3 RAPD and 2 morphological markers that span 1279 cM of the tomato genome with an average distance between markers of 10.7 cM. This map is comparable in length to that of the highdensity RFLP map derived from a L. esculentum x L. pennellii F₂ population. The order of the markers in the two maps is also in good agreement, however there are considerable differences in the distribution of recombination along the chromosomes. The segregation of six GATA-containing loci and 47 RAPD markers was also analyzed in subsets of the population. All of the microsatellite loci and 35 (75%) of the RAPDs mapped to clusters associated with centromeric regions.     

Molecular markers locate genes for resistance to the Colorado potato beetle,Leptinotarsa decemlineata,in hybrid Solanum tuberosum x S. berthaultii potato progenies

The wild Bolivian potato, Solanum berthaultii Hawkes, has been used as a source of resistance to the Colorado potato beetle (CPB), Leptinotarsa decemlineata Say, one of the most significant pests of potato. In this study, two reciprocal backcross S. tuberosum x S. berthaultii potato progenies, BCB and BCT, were mapped with RFLP markers and screened for resistance to CPB consumption, oviposition and defoliation. The genotypic and phenotypic data were combined and analysed to locate quantitative trait loci (QTLs) for resistance to CPB. Three QTLs on three chromosomes in BCB, and two QTLs on two chromosomes in BCT influenced resistance. The QTLs were generally additive but one instance of epistasis was noted. Each QTL accounted for 4–12% of the phenotypic variation observed in resistance. In the more resistant BCB population, a three QTL model explained ca. 20% of the variation in CPB oviposition. When alleles at the three QTLs were homozygous S. berthaultii, oviposition was reduced ca. 60% compared to the heterozygotes. The QTLs for resistance to CPB were compared to those previously identified for the type A and B glandular trichomes, which have been implicated in resistance in the same progenies. Generally, the QTLs for resistance to CPB coincided with loci associated with the glandular trichomes confirming the importance of the glandular trichomes in mediating resistance. However, a relatively strong and consistent QTL for insect resistance in both BCB and BCT on chromosome 1 was observed that was not associated with any trichome traits, suggesting the trichomes may not account for all of the resistance observed in these progenies.     

Rapid and reliable screening of a tomato YAC library exclusively based on PCR

An improved procedure is presented to select clones from a tomato yeast artificial chromosome (YAC) library. The procedure is based exlcusively on the polymerase chain reaction (PCR). We combined DNA from approximately 36,000 YAC clones in pools containing 96-single YAC clones from one master plate and further in super pools representing 10 master plates. This pooling strategy allows the selection of single YAC clones homologous to a target sequence after three rounds of PCR using super pools, single pools, and single YAC clones as a template. Single YAC clones were spheroplasted prior to the third PCR round in order to omit the conventional radioactive colony hybridization step. To date, we applied this PCR-based selection strategy to isolate clones homologousto ten different sequence-tagged sites (STS) that are linked to genes targeted for map-based cloning. The selection of YAC clones can be readily accomplished within three days. The PCR-based screening strategy is easy to set up and contributes to a further acceleration of the construction of YAC contigs.