Ancestral and more recently acquired syntenic relationships of MADS-box genes uncovered by the <Emphasis Type="Italic">Physcomitrella patens</Emphasis> pseudochromosomal genome assembly

Key message

The Physcomitrella pseudochromosomal genome assembly revealed previously invisible synteny enabling realisation of the full potential of shared synteny as a tool for probing evolution of this plant’s MADS-box gene family.

Abstract

Assembly of the sequenced genome of Physcomitrella patens into 27 mega-scaffolds (pseudochromosomes) has confirmed the major predictions of our earlier model of expansion of the MADS-box gene family in the Physcomitrella lineage. Additionally, microsynteny has been conserved in the immediate vicinity of some recent duplicates of MADS-box genes. However, comparison of non-syntenic MIKC MADS-box genes and neighbouring genes indicates that chromosomal rearrangements and/or sequence degeneration have destroyed shared synteny over longer distances (macrosynteny) around MADS-box genes despite subsets comprising two or three MIKC genes having remained syntenic. In contrast, half of the type I MADS-box genes have been transposed creating new syntenic relations with MIKC genes. This implies that conservation of ancient ancestral synteny of MIKC genes and of more recently acquired synteny of type I and MIKC genes may be selectively advantageous. Our revised model predicts the birth rate of MIKC genes in Physcomitrella is higher than that of type I genes. However, this difference is attributable to an early tandem duplication and an early segmental duplication of MIKC genes prior to the two polyploidisations that account for most of the expansion of the MADS-box gene family in Physcomitrella. Furthermore, this early segmental duplication spawned two chromosomal lineages: one with a MIKC ^C gene, belonging to the PPM2 clade, in close proximity to one or a pair of MIKC* genes and another with a MIKC ^C gene, belonging to the PpMADS-S clade, characterised by greater separation from syntenic MIKC* genes. Our model has evolutionary implications for the Physcomitrella karyotype.

     

Generation of Chinese cabbage resistant to bacterial soft rot by heterologous expression of <Emphasis Type="Italic">Arabidopsis WRKY75</Emphasis>

Soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) is a serious disease in Chinese cabbage (Brassica rapa L. subsp. pekinensis). To reduce the severity of soft rot symptoms in Chinese cabbage, Arabidopsis AtWRKY75 was introduced into Chinese cabbage by Agrobacterium-mediated transformation, which was previously reported to reduce susceptibility to Pcc infection in Arabidopsis. Three independent Chinese cabbage transgenic lines carrying AtWRKY75 were obtained. The growth phenotypes of AtWRKY75 overexpression (OE) lines were normal. Bacterial soft rot symptoms and Pcc growth were reduced in AtWRKY75-OE Chinese cabbage lines compared with WT plants. In contrast, overexpression of AtWRKY75 had no effect on infection with a hemibiotrophic pathogen, Xanthomonas campestris pv. campestris (Xcc) causing black rot disease. These results are consistent with those observed in the transgenic Arabidopsis. We found that AtWRKY75 activated a subset of Chinese cabbage genes related to defense against Pcc infection, such as Meri15B, BrPR4, and BrPDF1.2 (but not BrPGIP2). Moreover, overexpression of AtWRKY75 caused H₂O₂ production and activation of H₂O₂ scavenge enzyme genes, suggesting that H₂O₂ played a role in AtWRKY75-mediated resistance to Pcc. Together, these results demonstrated that AtWRKY75 decreased the severity of Pcc-caused bacterial soft rot and activated a subset of Pcc infection defense-related genes in Chinese cabbage similar to in Arabidopsis. It is suggested that AtWRKY75 is a candidate gene for use in crop improvement, because it results in reduced severity of disease symptoms without concurrent growth abnormalities.     

Molecular cloning and characterization of a PR-5 like protein gene from <Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis>

Downy mildew caused by Hyaloperonospora parasitica is a serious fungal disease in non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis Makino). Pathogenesis-related 5 (PR-5) genes play an important role in plant resistance to disease invasion. In this study, a gene encoding pathogenesis-related 5-like (PR-5L) protein, named BcPR-5L, was successfully cloned from non-heading Chinese cabbage. The cDNA sequence of BcPR-5L was 747 bp in length. It encoded a protein of molecular mass of 25.78 kDa, an isoelectric point of 4.42, and containing 248 amino acids. Multiple sequence alignment indicated that BcPR-5L protein was highly homologous to other PR-5L proteins identified in 13 different species, with the highest homology to Brassica rapa. We analyzed the subcellular localization of BcPR-5L protein by using onion epidermal cells and found that it was localized in the membrane. Real time quantitative PCR analyses revealed that the expression of BcPR-5L gene was significantly upregulated after H. parasitica infection, and the expression in the resistant cultivar was higher than that in the susceptible cultivar. In summary, our data suggest that BcPR-5L gene may play an important role in the resistance of non-heading Chinese cabbage to H. parasitica infection.     

Metabolism-mediated induction of zinc tolerance in <Emphasis Type="Italic">Brassica rapa</Emphasis> by <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> CS2-1

Brassica rapa (Chinese cabbage) is an essential component of traditional Korean food. However, the crop is often subject to zinc (Zn⁺) toxicity from contaminated irrigation water, which, as a result, compromises plant growth and production, as well as the health of human consumers. The present study investigated the bioaccumulation of Zn⁺ by Burkholderia cepacia CS2-1 and its effect on the heavy metal tolerance of Chinese cabbage. Strain CS2-1 was identified and characterized on the basis of 16S rRNA sequences and phylogenetic analysis. The strain actively produced indole-3-acetic acid (3.08 ± 0.21 μg/ml) and was also able to produce siderophore, solubilize minerals, and tolerate various concentrations of Zn⁺. The heavy metal tolerance of B. rapa plants was enhanced by CS2-1 inoculation, as indicated by growth attributes, Zn⁺ uptake, amino acid synthesis, antioxidant levels, and endogenous hormone (ABA and SA) synthesis. Without inoculation, the application of Zn⁺ negatively affected the growth and physiology of B. rapa plants. However, CS2-1 inoculation improved plant growth, lowered Zn⁺ uptake, altered both amino acid regulation and levels of flavonoids and phenolics, and significantly decreased levels of superoxide dismutase, endogenous abscisic acid, and salicylic acid. These findings indicate that B. cepacia CS2-1 is suitable for bioremediation against Zn⁺-induced oxidative stress.     

Genome-wide analysis and stress-responsive expression of CCCH zinc finger family genes in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Background

Ubiquitous CCCH nucleic acid-binding motif is found in a wide-variety of organisms. CCCH genes are involved in plant developmental processes and biotic and abiotic stress responses. Brassica rapa is a vital economic crop and classical model plant of polyploidy evolution, but the functions of CCCH genes in B. rapa are unclear.

Results

In this study, 103 CCCH genes in B. rapa were identified. A comparative analysis of the chromosomal position, gene structure, domain organization and duplication event between B. rapa and Arabidopsis thaliana were performed. Results showed that CCCH genes could be divided into 18 subfamilies, and segmental duplication might mainly contribute to this family expansion. C-X_7/8-C-X₅-C₃-H was the most commonly found motif, but some novel CCCH motifs were also found, along with some loses of typical CCCH motifs widespread in other plant species. The multifarious gene structures and domain organizations implicated functional diversity of CCCH genes in B. rapa. Evidence also suggested functional redundancy in at least one subfamily due to high conservation between members. Finally, the expression profiles of subfamily-IX genes indicated that they are likely involved in various stress responses.

Conclusion

This study provides the first genome-wide characterization of the CCCH genes in B. rapa. The results suggest that B. rapa CCCH genes are likely functionally divergent, but mostly involved in plant development and stress response. These results are expected to facilitate future functional characterization of this potential RNA-binding protein family in Brassica crops.

     

Preferential retention,expression profile and potential functional diversity analysis of HD-Zip gene family in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Homeodomain-Leu zipper (HD-Zip) gene family performs important biological functions related to organ development, photomorphogenesis and abiotic stress response in higher plants. However, systematic analysis of HD-Zip genes in Brassica rapa has not been performed. In the present study, a bioinformatics approach was used to identify and characterize the BraHD-Zip gene family in B. rapa. A total of 88 members were identified. All putative BraHD-Zip proteins contained a clear HD and LZ combined domain. Eighty-seven BraHD-Zips were non-randomly located on ten chromosomes. This gene family was mainly expanded following the whole genome triplication event and was preferentially over-retained relative to its neighboring genes in B. rapa. On phylogenetic analysis, the BraHD-Zips could be categorized into four distinct major groups (I–IV). Each group exhibited variant gene structures and motif distributions. Some syntenic orthologous gene pairs presented diverse expression profiles, which indicate that these gene pairs may be involved in the development of new functions during evolution. In summary, our analysis provided genome-wide insights into the expansion, preferential retention, expression profiles and functional diversity of BraHD-Zip genes following whole genome triplication in B. rapa.     

Identification of candidate genes of QTLs for seed weight in <Emphasis Type="Italic">Brassica napus</Emphasis> through comparative mapping among <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Brassica</Emphasis>species

Background

Map-based cloning of quantitative trait loci (QTLs) in polyploidy crop species remains a challenge due to the complexity of their genome structures. QTLs for seed weight in B. napus have been identified, but information on candidate genes for identified QTLs of this important trait is still rare.

Results

In this study, a whole genome genetic linkage map for B. napus was constructed using simple sequence repeat (SSR) markers that covered a genetic distance of 2,126.4 cM with an average distance of 5.36 cM between markers. A procedure was developed to establish colinearity of SSR loci on B. napus with its two progenitor diploid species B. rapa and B. oleracea through extensive bioinformatics analysis. With the aid of B. rapa and B. oleracea genome sequences, the 421 homologous colinear loci deduced from the SSR loci of B. napus were shown to correspond to 398 homologous loci in Arabidopsis thaliana. Through comparative mapping of Arabidopsis and the three Brassica species, 227 homologous genes for seed size/weight were mapped on the B. napus genetic map, establishing the genetic bases for the important agronomic trait in this amphidiploid species. Furthermore, 12 candidate genes underlying 8 QTLs for seed weight were identified, and a gene-specific marker for BnAP2 was developed through molecular cloning using the seed weight/size gene distribution map in B. napus.

Conclusions

Our study showed that it is feasible to identify candidate genes of QTLs using a SSR-based B. napus genetic map through comparative mapping among Arabidopsis and B. napus and its two progenitor species B. rapa and B. oleracea. Identification of candidate genes for seed weight in amphidiploid B. napus will accelerate the process of isolating the mapped QTLs for this important trait, and this approach may be useful for QTL identification of other traits of agronomic significance.

     

Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.     

Natural genetic variation in <Emphasis Type="Italic">Brassica</Emphasis> homologs of <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> and characterization of its expression domains

FLOWERING LOCUS T (FT), a major effect gene, regulates flowering time in Arabidopsis. We analyzed evolutionary changes distinguishing two FT homeologous loci in B. rapa, described genetic variation in homologs isolated and reported expression pattern of FT in B. juncea. Synteny analysis confirmed presence of two FT genomic copies in B. rapa ssp. pekinensis and resolved pre-existing anomalies regarding copy number in “AA” genome. Synteny analysis of B. rapa homeologous regions CR1 (129 kb) and CR2 (232 kb) revealed differential gene fractionation and wide-spread re-arrangements. Seven genomic DNA (gDNA) variants (2.1–2.2 kb) and 10 complementary DNA (cDNA) variants (528 bp) were isolated from 6 Brassica species. The gDNA variants shared 72–99 % similarity within Brassica and 58–60 % between Arabidopsis and Brassica. FT cDNA variants shared 92–100 % similarity within Brassica and 87 % between Arabidopsis and Brassica. Phylogenetic analysis of FT gDNA, cDNA and protein sequences revealed two major clades, differentiating homologs derived from species containing shared “BB” and “CC” genomes. Phylogram based on Brassica FT gDNA differentiated homeologs derived from AA-LF (Least fractioned) and AA-MF1 (Moderately fractioned) sub-genomes. Analysis of FT expression pattern in B. juncea revealed increasing levels correlating with attainment of physiological maturity; highest levels were detected in older leaves implying conservation in spatio-temporal expression pattern vis-à-vis Arabidopsis. In conclusion, our study reveals that polyploidy in Brassicas resulted in expansion of FT gene copies with homologs charting independent evolutionary course through accumulation of mutations. However, expression domains of FT remained conserved across Brassicaceae to preserve the critical function of FT in controlling flowering time.     

Genome-wide identification,classification, and expression analysis of the phytocyanin gene family in <Emphasis Type="Italic">Phalaenopsis equestris</Emphasis>

Phytocyanins (PCs) are ancient blue copper-binding proteins in plants that bind to single type I copper atoms and function as electron transporters. PCs play an important role in plant development and stress resistance. Many PCs are considered to be chimeric arabinogalactan proteins (AGPs). Previously, 38, 62, and 84 PC genes were identified in Arabidopsis thaliana, Oryza sativa, and Brassica rapa, respectively. In this study, we identified 30 putative PC genes in the orchid Phalaenopsis equestris through comprehensive bioinformatics analysis. Based on phylogeny and motif constitution, the P. equestris phytocyanins (PePCs) were divided into five subclasses: 10 early nodulin-like proteins, 10 uclacyanin-like proteins, five stellacyanin-like proteins, four plantacyanin-like proteins, and one unknown protein. Structural and glycosylation predictions suggested that 16 PePCs were glycosylphosphatidylinositol-anchored proteins localized to the plasma membrane, 22 PePCs contain N-glycosylation sites, and 14 are chimeric AGPs. Phylogenetic analysis indicated that each subfamily was derived from a common ancestor before the divergence of monocot and dicot lineages and that the expansion of the PC subfamilies occurred after the divergence of orchids and Arabidopsis. The number of exons in PC genes was conserved. Expression analysis in four tissues revealed that nine PC genes were highly expressed in flowers, stems, and roots, suggesting that these genes play important roles in growth and development in P. equestris. The results of this study lay the foundation for further analysis of the functions of this gene family in plants.