A spontaneous mutant with apetalous flowers was obtained from a hybrid progeny in Brassica napus. The result of genetic analysis showed that the apetalous character was controlled by only one gene locus, petalous flower exhibited incomplete dominance over apetalous flower and that its expression was not affected by cytoplasmic factors. Sixteen agronomically important characteristics of the apetalous line Apet33-10 were compared with those of its petalled near-isogenic line Pet33-10. Results from 4 years of tests indicated that there was no difference between Apet33-10 and Pet33-10 in all tested agronomic characteristics, except for the pod number of main inflorescence and second-order branches at low sclerotinia disease incidence. The Sclerotinia sclerotiorum disease severity index of Apet33-10 was significantly lower than that of Pet33-10, and correspondingly, the plot yield of Apet33-10 was increased obviously in comparison to that of Pet33-10 if sclerotinia disease was serious. The pod number of main inflorescence of Apet33-10 was significantly lower than that of Pet33-10. On the other hand, the pod number of second-order branches of Apet33-10 was significantly higher than that of Pet33-10, hence no overage difference of the pod number per plant was observed between the two lines.
MADS-box transcription factors are important regulators of floral organ identity through their binding to specific motifs, termed CArG, in the promoter of their target genes. Petal initiation and development depend on class A and B genes, but MADS-box genes of the APETALA3 (AP3) clade are key regulators of this process. In the early diverging eudicot Nigella damascena, an apetalous [T] morph is characterized by the lack of expression of the NdAP3-3 gene, with its expression being petal-specific in the wild-type [P] morph. All [T] morph plants are homozygous for an NdAP3-3 allele with a Miniature Inverted-repeat Transposable Element (MITE) insertion in the second intron of the gene. Here, we investigated to which extent the MITE insertion impairs regulation of the NdAP3-3 gene. We found that expression of NdAP3-3 is initiated in the [T] morph, but the MITE insertion prevents its positive self-maintenance by affecting the correct splicing of the mRNA. We also found specific CArG features in the promoter of the NdAP3-3 genes with petal-specific expression. However, they are not sufficient to drive expression only in petals of transgenic Arabidopsis, highlighting the existence of Nigella-specific cis/trans-acting factors in regulating AP3 paralogs.
FIGURE 4. Wild type and an ap2 weak mutant of Arabidopsis transformed with AP2 genes from B. rapa. (A) Wild-type Arabidopsis (Col-0) transformed with AtAP2 (control) and two AP2 genes of B. rapa. Their over-expressed lines p35S:AtAP2/Col, p35S::BrAP2a/Col, and p35S::BrAP2b/Col had normal flowers similar to the wild-type flower. (B) The AP2 mutant of Arabidopsis (ap2-5), which has a mild sepal-to-carpel and petal-to-stamen phenotype, transformed with AtAP2 gene and two B. rapa AP2 genes. All three transgenic plants, p35S:AtAP2/ap2-5, p35S::BrAP2a/ap2-5, and p35S::BrAP2a/ap2-5, rescued the ap2-5 mutant defect and developed normal sepals and petals. (C) The knockdown lines, p35S::AtAP2/ap2-5 KD, p35S::BrAP2a/ap2-5 KD, and p35S::BrAP2b/ap2-5 KD, from three transgenic group of p35S:AtAP2/ap2-5, p35S::BrAP2a/ap2-5, and p35S::BrAP2a/ap2-5. (D) semi-quantitative RT-PCR and Western blot assays to validate the transgenic lines. Bars, 500 μm.
Floral transition and petal onset, as two main aspects of flower development, are crucial to rapeseed evolutionary success and yield formation. Currently, very little is known regarding the genetic architecture that regulates flowering time and petal morphogenesis in Brassica napus. In the present study, a genome-wide transcriptomic analysis was performed with an absolutely apetalous and early flowering line, APL01 and a normally petalled line, PL01, using high-throughput RNA sequencing. In total, 13,205 differential expressed genes were detected, of which 6111 genes were significantly down-regulated, while 7094 genes were significantly up-regulated in the young inflorescences of APL01 compared with PL01. The expression levels of a vast number of genes involved in protein biosynthesis were altered in response to the early flowering and apetalous character. Based on the putative rapeseed flowering genes, an early flowering network, mainly comprised of vernalization and photoperiod pathways, was built. Additionally, 36 putative upstream genes possibly governing the apetalous character of line APL01 were identified and six genes potentially regulating petal origination were obtained by combining with three petal-related quantitative trait loci. These findings will facilitate understanding of the molecular mechanisms underlying floral transition and petal initiation in B. napus.
The emergence of flowers as reproductive units probably contributed substantially to the evolutionary success of flowering plants. In the life cycle of an angiosperm plant, the transition from vegetative to reproductive development is tightly controlled by a complex gene regulatory network. Over the past three decades, work in Arabidopsis thaliana, as well as in several other angiosperm species, including snapdragon (Antirrhinum majus), petunia (Petunia hybrida) and rice (Oryza sativa), has identified a vast number of genes involved in floral transition1,2,3. Recently several reviews provided detailed insights into the gene regulatory network underlying floral transition, which mainly consists of vernalization, photoperiod, gibberellins (GAs), autonomous, ambient temperature and aging pathway1,2,3. The genetic circuits that integrate different signals eventually converge to activate the expression of a group of so-called floral meristem (FM)-associated genes, including LEAFY (LFY) and APETALA1 (AP1)1,2,4,5,6. The floral organ-associated genes are subsequently activated by LFY and AP1, FM develops into distinct domains that give rise to the different types of floral organs7,8.
Apetalous rapeseed with floral organs that are fully developed, except petals, is considered the ideotype of high-yield rapeseed because of its low-energy consumption, high photosynthetic efficiency and superior klendusity to Sclerotinia sclerotiorum27,28,29,30,31,32. Unlike all of the apetalous mutants in Arabidopsis and Antirrhinum, the number and morphology of sepals, stamens and carpels of many apetalous rapeseeds detected in earlier studies are similar to those of the natural variety33,34, seemingly indicating that the genetic mechanism governing petal development of rapeseed is not completely consistent with the model plants at some level. However, the genetic analysis of the apetalous characteristic of B. napus is insufficient because very few stable apetalous mutants are generated. A few studies suggested that the apetalous characteristic in B. napus is governed by recessive genes, possibly by two to four loci35 and identified several associated with QTLs33,34. Only one study suggested that there are two types of AP3 genes in B. napus, B. AP3.a and B. AP3.b36. A knockdown of B. AP3.a led to a deficiency of petals, while natural expression of B. AP3.b ensured normal stamen morphogenesis36. However, the theory failed to explain the determination of the correct number of sepals. Thus, the mechanism underlying the apetalous characteristic of rapeseed appears to be more complex than initially believed. Fortunately, the genome sequence of B. napus was released in 201419 and will contribute to the detection of floral regulatory genes in the whole genome using bioinformatics.
RNA sequencing (RNA-seq) as a revolutionary tool for transcriptomics has been broadly used to explore the molecular basis governing the phenotypic traits of organisms37. In the present study, the rapeseed lines APL01 and PL01, two lines with distinguishable flowering time and petal morphologies, were used for Illumina RNA-seq to study the differential expressed genes (DEGs) in the young inflorescences. In combination with gene ontology (GO)-enrichment analysis and homologous alignments, the discovery of the molecular basis underlying early flowering and apetalous characteristic in line APL01 is expected. Meanwhile, the detection of potential candidate genes regulating the petalous degree (PDgr) of rapeseed is expected to be assisted by coupling RNA-seq with QTL mapping.
Because the variations in flowering time and petal morphogenesis are dominantly based on gene expression changes that occur before the initiation of FMs and petal primordia, young inflorescences only comprised of shoot apical meristem (SAM) and buds at stages 1 to 5 (Based on the summary of stages of flower development in Arabidopsis38), were collected from lines APL01 and PL01 for high-throughput RNA-seq. In total, 56.01 to 69.38 million raw reads for each sample were generated and three biological replicates for each line were performed (Table 1). Subsequently, 55.31 to 68.42 million clean reads were generated by removing low quality regions and adapter-related sequences and were mapped to the B. napus genome using TopHat239 (Table 1).
To understand gene functions related to early flowering and the apetalous character of line APL01, a GO enrichment analysis for the DEGs was performed using the GOseq R package42. The relationships among the significantly enriched GO terms are shown through a directed acyclic graph (DAG) (Supplementary Fig. S3).
To discern the regulatory networks underlying the early flowering of line APL01, 1093 putative homologs of Arabidopsis 282 flowering genes were identified in the B. napus genome through homology alignment (Supplementary Data 3). Based on RNA-seq data, 82 DEGs were possibly involved in the early flowering of line APL01, in which most of the genes functioning in the vernalization and photoperiod pathway were contained (Supplementary Table S2). The possible regulatory network governing the early flowering of line APL01 shown in Fig. 5 is based on the floral transition network in Arabidopsis1 (Supplementary Table S3). 041b061a72