Chromosome level genome assembly of colored calla lily (Zantedeschia elliottiana).

The colored calla lily is an ornamental floral plant native to southern Africa, belonging to the Zantedeschia genus of the Araceae family. We generated a high-quality chromosome-level genome of the colored calla lily, with a size of 1,154 Mb and a contig N50 of 42 Mb. We anchored 98.5% of the contigs (1,137 Mb) into 16 pseudo-chromosomes, and identified 60.18% of the sequences (694 Mb) as repetitive sequences. Functional annotations were assigned to 95.1% of the predicted protein-coding genes (36,165). Additionally, we annotated 469 miRNAs, 1,652 tRNAs, 10,033 rRNAs, and 1,677 snRNAs. Furthermore, Gypsy-type LTR retrotransposons insertions in the genome are the primary factor causing significant genome size variation in Araceae species. This high-quality genome assembly provides valuable resources for understanding genome size differences within the Araceae family and advancing genomic research on colored calla lily.

Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Two Species of Calla Lilies (Zantedeschia, Araceae).

In this study, the mitochondrial genomes of two calla species, Zantedeschia aethiopica Spreng. and Zantedeschia odorata Perry., were assembled and compared for the first time. The Z. aethiopica mt genome was assembled into a single circular chromosome, measuring 675,575 bp in length with a 45.85% GC content. In contrast, the Z. odorata mt genome consisted of bicyclic chromosomes (chromosomes 1 and 2), measuring 719,764 bp and exhibiting a 45.79% GC content. Both mitogenomes harbored similar gene compositions, with 56 and 58 genes identified in Z. aethiopica and Z. odorata, respectively. Analyses of codon usage, sequence repeats, gene migration from chloroplast to mitochondrial, and RNA editing were conducted for both Z. aethiopica and Z. odorata mt genomes. Phylogenetic examination based on the mt genomes of these two species and 30 other taxa provided insights into their evolutionary relationships. Additionally, the core genes in the gynoecium, stamens, and mature pollen grains of the Z. aethiopica mt genome were investigated, which revealed maternal mitochondrial inheritance in this species. In summary, this study offers valuable genomic resources for future research on mitogenome evolution and the molecular breeding of calla lily.

Structure-guided identification of antimicrobial peptides in the spathe transcriptome of the non-model plant, arum lily (Zantedeschia aethiopica).

Antimicrobial peptides (AMPs) are small molecules present in all living beings. Despite their huge sequence variability, AMPs present great structural conservation, mainly in cysteine-stabilized families. Moreover, in non-model plants, it is possible to detect cysteine-stabilized AMPs (cs-AMPs) with different sequences not covered by conventional searches. Here, we described a threading application for cs-AMP identification in the non-model arum lily (Zantedeschia aethiopica) plant, exploring the spathe transcriptome. By using the predicted proteins from the Z. aethiopica transcriptome as our primary source of sequences, we have filtered by using structural alignments of 12 putative cs-AMP sequences. The two unreported sequences were submitted to PCR validation, and ZaLTP7 gene was confirmed. By using the structure alignments, we classified ZaLTP7 as an LTP type 2-like. The successful threading application for cs-AMP identification is an important advance in transcriptomic and proteomic data mining. Besides, the same approach could be applied to the use of NGS public data to discover molecules to combat multidrug-resistant bacteria.

Priming of protein expression in the defence response of Zantedeschia aethiopica to Pectobacterium carotovorum.

The defence response of Zantedeschia aethiopica, a natural rhizomatous host of the soft rot bacterium Pectobacterium carotovorum, was studied following the activation of common induced resistance pathways­systemic acquired resistance and induced systemic resistance. Proteomic tools were used, together with in vitro quantification and in situ localization of selected oxidizing enzymes. In total, 527 proteins were analysed by label-free mass spectrometry (MS) and annotated against the National Center for Biotechnology Information (NCBI) nonredundant (nr) protein database of rice (Oryza sativa). Of these, the fore most differentially expressed group comprised 215 proteins that were primed following application of methyl jasmonate (MJ) and subsequent infection with the pathogen. Sixty-five proteins were down-regulated following MJ treatments. The application of benzothiadiazole (BTH) increased the expression of 23 proteins; however, subsequent infection with the pathogen repressed their expression and did not induce priming. The sorting of primed proteins by Gene Ontology protein function category revealed that the primed proteins included nucleic acid-binding proteins, cofactor-binding proteins, ion-binding proteins, transferases, hydrolases and oxidoreductases. In line with the highlighted involvement of oxidoreductases in the defence response, we determined their activities, priming pattern and localization in planta. Increased activities were confined to the area surrounding the pathogen penetration site, associating these enzymes with the induced systemic resistance afforded by the jasmonic acid signalling pathway. The results presented here demonstrate the concerted priming of protein expression following MJ treatment, making it a prominent part of the defence response of Z. aethiopica to P. carotovorum.

Shedding some light over the floral metabolism by arum lily (Zantedeschia aethiopica) spathe de novo transcriptome assembly.

Zantedeschia aethiopica is an evergreen perennial plant cultivated worldwide and commonly used for ornamental and medicinal purposes including the treatment of bacterial infections. However, the current understanding of molecular and physiological mechanisms in this plant is limited, in comparison to other non-model plants. In order to improve understanding of the biology of this botanical species, RNA-Seq technology was used for transcriptome assembly and characterization. Following Z. aethiopica spathe tissue RNA extraction, high-throughput RNA sequencing was performed with the aim of obtaining both abundant and rare transcript data. Functional profiling based on KEGG Orthology (KO) analysis highlighted contigs that were involved predominantly in genetic information (37%) and metabolism (34%) processes. Predicted proteins involved in the plant circadian system, hormone signal transduction, secondary metabolism and basal immunity are described here. In silico screening of the transcriptome data set for antimicrobial peptide (AMP) -encoding sequences was also carried out and three lipid transfer proteins (LTP) were identified as potential AMPs involved in plant defense. Spathe predicted protein maps were drawn, and suggested that major plant efforts are expended in guaranteeing the maintenance of cell homeostasis, characterized by high investment in carbohydrate, amino acid and energy metabolism as well as in genetic information.

A systemic response of geophytes is demonstrated by patterns of protein expression and the accumulation of signal molecules in Zantedeschia aethiopica.

In geophyte plants, such as Zantedeschia, individual leaves are directly connected to a specialized underground storage organ (rhizome/tuber), raising a question regarding systemic resistance as a mechanism of defense. A systemic response requires a transfer of a signal through the storage organ which has been evolutionary adapted to store food, minerals and moisture for seasonal growth and development. We have characterized the nature of induced defense responses in Zantedeschia aethiopica, a rhizomatous (tuber-like) ornamental plant by the application of local elicitation using two well-known defense elicitors, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and methyl jasmonate (MJ). The system consisted leaves in which local responses were directly induced, and systemically responsive leaves in which defense molecules were detected, demonstrating a transported vascular signal. Using anatomical and biochemical tools and local elicitation with MJ, the systemic nature of the response was verified in adjacent leaves by unique protein expression patterns; similarly polyphenol oxidase (PPO) activity was found to increase systemically in all parts of the locally induced plants, including the rhizome, and adjacent leaves; finally, significant accumulation of defense signal molecules such as salicylic and jasmonic acids was recorded in local and systemic leaves following elicitation with BTH. Anatomical sections through the leaves and the rhizome revealed that to be transferred from one leaf to its neighbor, signal molecules must have been transferred through the storage organ. The collected data strongly support our hypothesis that defense signals may and are transferred through the storage organ in monocot geophytes.

Identification and characterization of 43 novel polymorphic EST-SSR markers for arum lily, Zantedeschia aethiopica (Araceae).

PREMISE OF THE STUDY: A new set of microsatellite or simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) was developed for arum lily (Zantedeschia aethiopica), which is one of the most iconic and widely recognized ornamental plants in the world. • METHODS AND RESULTS: Using 2175 unigenes derived from 4283 random ESTs in arum lily, 166 primer pairs were designed and tested for amplification in 24 accessions from Asia, Europe, and Africa. A total of 43 loci were polymorphic, with the number of alleles per locus ranging from two to 10. The observed heterozygosity, expected heterozygosity, and polymorphism information content ranged from 0.2313 to 0.8480, 0.3034 to 0.8648, and 0.1015 to 0.7364, respectively. • CONCLUSIONS: These novel polymorphic EST-SSR markers will facilitate future studies of genetic variation and molecular-assisted breeding systems in arum lily.

Expression and purification of Zantedeschia aethiopica agglutinin in Escherichia coli.

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-beta-D-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.

Structural analysis of catalase from two Musa accessions, FHIA18 and Williams, and from Ravenala madagascariensis.

Nucleotide sequences of catalase were obtained following amplification using specific primers and were blasted against Musa acuminata catalase 2 mRNA from NCBI (157418810). Clustering of the amino acid sequences from NCBI was done using Clustal X. The latter revealed that FHIA18 catalase is more related to Ravenala madagascariensis (Musa relative) catalase while the Williams catalase is more related to a clade containing a Musa acuminata (Musa ancestor) catalase from NCBI. The tertiary structures and the catalase consensus functional sites, based on the Pseudomonas syringae catalase structural template, were obtained for FHIA18, Williams, Ravenala madagascariensis and Musa acuminata catalases. They were found to differ slightly. Using known features of catalase active sites, four pre-requisite criteria were defined to find such sites: (1) Position of tyrosine axial to heme determined by X-ray diffraction, (2) 7 conserved amino acids in the active site found by sequence alignment, (3) favourable docking energy, and (4) presence of an unobstructed long tunnel that leads the ligand to the active site. Two differing potential docking sites were found for both FHIA18 and Williams that fit a maximum number of criteria. In terms of 1D sequence, the region of the docking site for Williams is within the catalase domains as seen upon NCBI blast. The counterpart of FHIA18 for the same region is not. This sequence difference between FHIA18 and Williams affects the best docking site in FHIA18 and Williams in silico.

Production of soft rot resistant calla lily by expressing a ferredoxin-like protein gene (pflp) in transgenic plants.

An efficient protocol for the Agrobacterium tumefaciens-mediated transformation of calla lily (Zantedeschia elliottiana (W. Wats.) Engl. cultivar 'Florex Gold') is described. Shoot basal discs were co-cultivated with A. tumefaciens C58C1 carrying a plasmid containing neomycin phosphotransferase (nptII) and plant ferredoxin-like protein (pflp) genes. After Agrobacterium co-cultivation, the shoot basal discs were exposed to 100 mg l(-1) kanamycin for selection. Twenty-eight out of 260 discs (10.8%) were found to have survived and produced shoot clusters. Twenty-six of these were confirmed to contain the pflp transgene by PCR, ending up in 10% transformation efficiency. The disease resistance investigation revealed that 18 transgenic plants exhibited resistance to soft rot disease caused by Erwinia carotovora subsp. carotovora. The presence of pflp gene was demonstrated by PCR, and its accumulation and activity was confirmed by Western blot and disease resistance assay. This was the first report to show the successful transformation and resistance to a bacterial pathogen in Zantedeschia. The protocol is useful for the quality improvement of calla lily through genetic transformation.

Molecular cloning and characterization of a novel mannose-binding lectin cDNA from Zantedeschia aethiopica.

Using RNA extracted from Zantedeschia aethiopica young leaves and primers designed according to the conservative regions of Araceae lectins, the full-length cDNA of Z. aethiopica agglutinin (ZAA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zaa was 871 bp and contained a 417 bp open reading frame (ORF) encoding a lectin precursor of 138 amino acids. Through comparative analysis of zaa gene and its deduced amino acid sequence with those of other Araceae species, it was found that zaa encoded a precursor lectin with signal peptide. Secondary and three-dimensional structure analyses showed that ZAA had many common characters of mannose-binding lectin superfamily and ZAA was a mannose-binding lectin with three mannose-binding sites. Southern blot analysis of the genomic DNA revealed that zaa belonged to a multi-copy gene family.

Presence of plastid and absence of mitochondrial DNA in male reproductive cells as evidence for cytoplasmic inheritance in Turnera ulmifolia and Zantedeschia aethiopica.

Epifluorescence microscopy of mature pollen grains of Turnera ulmifolia and Zantedeschia aethiopica stained with 4',6-diamidino-2-phenylindole demonstrated the presence of fluorescent cytoplasmic DNA aggregates in the male reproductive cells of both species. Double staining of the cells with 4',6-diamidino-2-phenylindole and 3,3'-dihexyloxacarbocyanine iodide in Technovit resin sections showed that the mitochondria of these cells did not correspond to the fluorescent cytoplasmic DNA aggregates. Electron microscopy studies revealed both plastids and mitochondria in the cells of these species. In addition, immunoelectron microscopy using an anti-DNA monoclonal antibody showed clear labeling of plastids but not mitochondria. These data provide cytological evidence for biparental plastid inheritance and maternal mitochondrial inheritance in these species.

Genomic cloning and characterization of a novel lectin gene from Zantedeschia aethiopica.

A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5' flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3' flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5' flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3'-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49-54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms.