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, 10 (5), e0123474
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A De Novo Floral Transcriptome Reveals Clues Into Phalaenopsis Orchid Flower Development

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A De Novo Floral Transcriptome Reveals Clues Into Phalaenopsis Orchid Flower Development

Jian-Zhi Huang et al. PLoS One.

Abstract

Phalaenopsis has a zygomorphic floral structure, including three outer tepals, two lateral inner tepals and a highly modified inner median tepal called labellum or lip; however, the regulation of its organ development remains unelucidated. We generated RNA-seq reads with the Illumina platform for floral organs of the Phalaenopsis wild-type and peloric mutant with a lip-like petal. A total of 43,552 contigs were obtained after de novo assembly. We used differentially expressed gene profiling to compare the transcriptional changes in floral organs for both the wild-type and peloric mutant. Pair-wise comparison of sepals, petals and labellum between peloric mutant and its wild-type revealed 1,838, 758 and 1,147 contigs, respectively, with significant differential expression. PhAGL6a (CUFF.17763), PhAGL6b (CUFF.17763.1), PhMADS1 (CUFF.36625.1), PhMADS4 (CUFF.25909) and PhMADS5 (CUFF.39479.1) were significantly upregulated in the lip-like petal of the peloric mutant. We used real-time PCR analysis of lip-like petals, lip-like sepals and the big lip of peloric mutants to confirm the five genes' expression patterns. PhAGL6a, PhAGL6b and PhMADS4 were strongly expressed in the labellum and significantly upregulated in lip-like petals and lip-like sepals of peloric-mutant flowers. In addition, PhAGL6b was significantly downregulated in the labellum of the big lip mutant, with no change in expression of PhAGL6a. We provide a comprehensive transcript profile and functional analysis of Phalaenopsis floral organs. PhAGL6a PhAGL6b, and PhMADS4 might play crucial roles in the development of the labellum in Phalaenopsis. Our study provides new insights into how the orchid labellum differs and why the petal or sepal converts to a labellum in Phalaenopsis floral mutants.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Flowers of wild-type and peloric mutant of Phalaenopsis Brother Spring Dancer ‘KHM190’.
(A) Wild-type and (B) peloric mutant flower. Bar = 1 cm. (C) Scanning electron microscopy of petal of floral buds at early developmental stages of (a), (b) wild-type and (c), (d) peloric-mutant flower. Bar = 500 μm.
Fig 2
Fig 2. Characterization of sequence homology of the Phalaenopsis assembled contigs against Nr databases.
(A) E-value distribution of BLASTx hits for the assembled contigs with a cutoff of 1e-5 in the NCBI Nr database. (B) Species distribution of the 25 top BLASTx hits shown as number of contigs of the total homologous sequences with an E-value ≥ 1e-5.
Fig 3
Fig 3. Annotation of the Phalaenopsis transcriptome by gene ontology (GO), KEGG and Pfam classification.
(A) GO classification summarized by three main categories: biological process, cellular component and molecular function. (B) Functional annotation of transcripts based on KEGG classification. (C) Functional characterization of transcripts for enzyme classes. (D) Pfam domains identified in translated Phalaenopsis transcripts.
Fig 4
Fig 4. Changes in gene expression profiles between wild-type and peloric-mutant floral organs.
The number of up- and downregulated genes in peloric sepal (PS) and wild-type sepal (NS), peloric petal (PP) and wild-type petal (NP), and peloric labellum (PL) and wild-type labellum (NL). Six libraries were summarized.
Fig 5
Fig 5. Phalaenopsis flower phenotypes of wild-type and peloric mutant.
(A) Wild-type flower of Phalaenopsis Brother Spring Dancer ‘KHM190’ and its lip-like petal mutant; (B) wild-type flower of Phalaenopsis aphrodite and its lip-like sepal mutant; (C) wild-type flower of Phalaenopsis ‘NPU1458’ and its big lip mutant. Bar = 1 cm.
Fig 6
Fig 6. Real-time PCR analysis of genes expressed in different floral mutants of Phalaenopsis orchid.
Total RNAs isolated from the sepal, petal, lip and column of mature flowers from Fig 5. The wild-type ([A] Phalaenopsis Brother Spring Dancer ‘KHM190’; [B] Phalaenopsis aphrodite and [C] Phalaenopsis ‘NPU1458’) and (N), lip-like petal mutant (P), lip-like sepal mutant (M) and big lip mutant (BP) were used as templates to detect the expression of CUFF.17763(PhAGL6a), CUFF.17763.1 (PhAGL6b), CUFF.25909 (PhMADS4), CUFF.39479.1 (PhMADS5) and CUFF.36625.1(PhMADS1). S, sepal (include upper and lateral sepals); SU, upper sepal; SD, lateral sepal; P, petal; L, lip; C, column.
Fig 7
Fig 7. Possible evolutionary relationships between PhAGL6a, PhAGL6b and PhMADS4 in the regulation of lip formation in Phalaenopsis orchid.

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Grant support

This work was supported by grants from the Council of Agriculture, Agriculture and Food Agency, Taiwan, with the grant numbers 103AS-9.1.1-FD-Z2 and 104AS-9.1.1-FD-Z2.
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