Cytochemical and comparative transcriptome analyses elucidate the formation and ecological adaptation of three types of pollen coat in Zingiberaceae.

BACKGROUND: The pollen ornate surface of flowering plants has long fascinated and puzzled evolutionary biologists for their variety. Each pollen grain is contained within a pollen wall consisting of intine and exine, over which the lipoid pollen coat lies. The cytology and molecular biology of the development of the intine and exine components of the pollen wall are relatively well characterised. However, little is known about the pollen coat, which confers species specificity. We demonstrate three types of pollen coat in Zingiberaceae, a mucilage-like pollen coat and a gum-like pollen coat, along with a pollen coat more typical of angiosperms. The morphological differences between the three types of pollen coat and the related molecular mechanisms of their formation were studied using an integrative approach of cytology, RNA-seq and positive selection analysis. RESULTS: Contrary to the 'typical' pollen coat, in ginger species with a mucilage-like (Caulokaempferia coenobialis, Cco) or gum-like (Hornstedtia hainanensis, Hhn) pollen coat, anther locular fluid was still present at the bicellular pollen (BCP) stage of development. Nevertheless, there were marked differences between these species: there were much lower levels of anther locular fluid in Hhn at the BCP stage and it contained less polysaccharide, but more lipid, than the locular fluid of Cco. The set of specific highly-expressed (SHE) genes in Cco was enriched in the 'polysaccharide metabolic process' annotation term, while 'fatty acid degradation' and 'metabolism of terpenoids and polyketides' were significantly enriched in SHE-Hhn. CONCLUSIONS: Our cytological and comparative transcriptome analysis showed that different types of pollen coat depend on the residual amount and composition of anther locular fluid at the BCP stage. The genes involved in 'polysaccharide metabolism' and 'transport' in the development of a mucilage-like pollen coat and in 'lipid metabolism' and 'transport' in the development of a gum-like pollen coat probably evolved under positive selection in both cases. We suggest that the shift from a typical pollen coat to a gum-like or mucilage-like pollen coat in flowering plants is an adaptation to habitats with high humidity and scarcity of pollinators.

Identification of leucoanthocyanidin reductase and anthocyanidin reductase genes involved in proanthocyanidin biosynthesis in Malus crabapple plants.

Proanthocyanidins (PAs) from plants are a nutritionally valuable component of the human diet and play important roles in defense against pests and diseases. PAs are products of the flavonoid pathway, which also leads to the production of anthocyanins and flavonols. The enzymes leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) are involved in PA biosynthesis. The PA biosynthetic pathway has been characterized in several plant species, but the relationship between its expression and PA accumulation in Malus crabapple remains unclear. Here, we cloned the LAR genes MrLAR1, 2, and the ANR genes MrANR1, 2, from the red leaved Malus crabapple cultivar 'Royalty'. The contents of PAs and the expression levels of the LAR and ANR genes were investigated in different organs of the two crabapple cultivars. The transcript levels of two LAR genes and two ANR genes correlated with the contents of the catechin and epicatechin, which are proanthocyanidin precursors. Over-expression of the MrLAR1, 2 and MrANR1, 2 in tobacco (Nicotiana tabacum) promoted the accumulation of PAs, while transient silencing of their expression in crabapple resulted in reduced PA levels. In addition, a negative correlation between quercetin, anthocyanin, and PA biosynthesis was also found during crabapple leaf and fruit peel development. We also found that MrLAR1 and 2 may contribute to epicatechin biosynthesis. In summary, the LAR and ANR genes are critical factors in PA biosynthesis, and there is competition between the quercetin, anthocyanin, and PA biosynthetic pathways during leaf and fruit peel development in Malus crabapple.

McMYB10 Modulates the Expression of a Ubiquitin Ligase, McCOP1 During Leaf Coloration in Crabapple.

In higher plants, anthocyanins are protective secondary metabolites, which contribute to the color of leaves, stems, flowers, and fruits, and have been found to have an antioxidant role in human health. In this study, we determined the expression of McMYB10 and its specific E3 ubiquitin ligase, McCOP1, in crabapple leaves during the course of a day and in five leaf development stages. Interestingly, the results showed that the transcription level of McCOP1 genes was higher in daylight than at night, and the transcripts of McMYB10 presented a positive correlation with the transcription of McCOP1-1 and McCOP1-2 and anthocyanin accumulation in a crabapple cultivar with red-colored leaves. Several MYB transcription factor (TF) binding sites of the MYBCORE type were found in the McCOP1-1 and McCOP1-2 promoters, and we deduced that there may be a relationship between McMYB10 and McCOP1-1 and McCOP1-2 at the transcriptional level. Yeast one hybrid (Y1H) and electrophoretic mobility shift assays (EMSA) demonstrated that the McMYB10 TF binds specifically to the promoter of McCOP1-1 and McCOP1-2. Furthermore, increased levels of McMYB10 promoted anthocyanin biosynthesis and the expression level of McCOP1-1 and McCOP1-2 in crabapple leaves during continuous light treatments, and overexpression or silencing of McMYB10 in crabapple leaves and apple fruits also result in an increase or decrease, respectively, in the expression of McCOP1-1 and McCOP1-2 and in anthocyanin biosynthesis. Our results reveal a new self-regulation mechanism in where McMYB10 modulates its own expression by activating McCOP1-1 and McCOP1-2 expression to promote ubiquitination of the McMYB10 protein by McCOP1.

Characteristics of dihydroflavonol 4-reductase gene promoters from different leaf colored Malus crabapple cultivars.

Anthocyanins are secondary metabolites in land plants that contribute to the colors of leaves and flowers, and are nutritionally valuable components of the human diet. The DFR gene plays an important role in the anthocyanin biosynthetic pathway. In this study, we investigated the regulation of DFR expression and in different Malus crabapple cultivars that show distinct patterns of leaf coloration, and how it influences leaf anthocyanin accumulation and coloration. Specifically, we studied the ever-red leaved cultivar 'Royalty', the ever-green leaved cultivar 'Flame' and the spring-red leaved cultivar 'Radiant'. RT-PCR analysis showed that the expression of McDFR1 correlated with the expression of a MYB transcription factor, McMYB10, and with anthocyanin accumulation. We isolated five McDFR1 promoter fragments from the three cultivars and identified four different fragments (F1-4) that were present either in several cultivars, or only in one. Yeast one-hybrid and electrophoretic mobility shift assay analyses showed that McMYB10 could bind to all the McDFR1 promoters, except McDFR1-Ra2. The F1, F2 and F3 fragments did not affect McMYB10 binding to the McDFR1 promoters; however, we found evidence that the F4 fragment suppressed binding, and that the MYBGAHV amino-acid sequence maybe an important cis-element for McMYB10 protein binding. This information has potential value for strategies to modify plant color through genetic transformation.