An efficient transformation protocol is a primary requisite to study and utilize the genetic potential of any plant species. A quick transformation system is also crucial for the functional analysis of genes along with the study of proteins and their interactions in vivo. Presently, however, quick and effective transformation systems are still lacking for many plant species including pineapple. This has limited the full exploration of the genetic repository of pineapple as well as the study of its genes, protein localization and protein interactions. To address the above limitations, we have developed an efficient system for protoplast isolation and subcellular localization of desired proteins using pineapple plants derived from tissue culture. A cocktail of 1.5% (W/V) Cellulase R-10 and 0.5% (W/V) Macerozyme R-10 resulted in 51% viable protoplasts with 3 h digestion. Compared to previously reported protocols, our protoplast isolation method is markedly faster (saving 4.5 h), requires only a small quantity of tissue sample (1 g of leaves) and has high yield (6.5 × 105). The quality of the isolated protoplasts was verified using organelle localization in protoplasts with different organelle markers. Additionally, colocalization analysis of two pineapple Mg2+ transporter genes in pineapple protoplasts was consistent with the results in a tobacco transient expression system, confirming that the protoplast isolation method can be used to study subcellular localization. Further findings showed that the system is also suitable for protein–protein interaction studies. Based on our findings, the presently described method is an efficient and effective strategy for pineapple protoplast isolation and transformation; it is convenient and time saving and provides a greater platform for transformation studies.

DNA methylation changes can occur in some loci during callus culture, resulting in somaclonal variations (SVs). In the present study, we applied whole genome bisulfite sequencing to analyze context-specific DNA methylation changes in the pineapple genome between the cutting seedings and 5 SV plants. In general, SV plants exhibited methylation patterns analogous to those of cutting seedlings (CK). A total of 27.98% of the genomic cytosines of CK were methylcytosines, which was higher than that of 5 SV plants. Moreover, mCG and mCHG was hypermethylated, whereas mCHH was hypomethylated among the 5 SV plants genomic when compared with the CK. Most of the variation of DNA methylation was distributed in gene bodies, thus suggesting that phenotypic differences are probably perturbed by genes methylated from callus culture. In addition, the methylated genes were highly enriched for the Gene Ontology (GO) categories of binding and catalytic activity, cell part and organelle, cellular process, abiotic stimulus, and DNA modification. These results suggest that methylation mediates these pathways in the callus culture of pineapple. The results also suggested that the callus culture induced DNA methylation may result in the SV.

DNA methylation plays an important role in somaclonal variation. Cytosine-5 DNA methyltransferases (C5-MTases) are crucial enzymes for maintaining the de novo genome DNA methylation status, and the gene family has been identified and characterized in several species. However, limited information is available about the role of C5-MTases in the somaclonal variation of pineapple. Herein, the characteristic of DNA methylation in the leaf variants of pineapple showed that the methylation rate of variants decreased and the demethylation was mainly. Six AcC5-MTases were identified from pineapple genome. Phylogenetic analysis indicated that the six members could be classified into four subgroups, namely, AcMET, AcCMT, AcDRM, and AcDNMT, demonstrating evolutionary conservation in the C5-MTases across pineapple and other plant species. Cis-elements were presented in AcC5-MTase promoters, including light-responsive, phytohormone-responsive, and plant growth and development elements. Furthermore, we investigated the expression profiles of AcC5-MTases in different tissues and their transcript abundance in the leaves of somaclonal variation. Interestingly, most AcC5-MTase genes were highly expressed in leaf and carpopodium. AcMET, AcCMT2, and AcDRM2 were upregulated in pineapple somaclonal variants, indicating that AcC5-MTases might play an important role in the somaclonal variation of pineapple. Our results will aid in understanding the complex roles of methylation in plants and provide a basis for analyzing the role of methylation modification in the somaclonal variation of pineapple.

Pineapple (Ananas comosus L.) is an important fruit crop in tropical regions, and it requires efficient sugar allocation during fruit development. Sugars Will Eventually be Exported Transporters (SWEETs) are a group of novel sugar transporters which play critical roles in seed and fruit development. However, the function of AcSWEETs remains unknown in the sugar accumulation. Herein, 17 AcSWEETs were isolated and unevenly located in 11 chromosomes. Analysis of a phylogenetic tree indicated that 17 genes were classified into four clades, and the majority of AcSWEETs in each clade shared similar conserved motifs and gene structures. Tissue-specific gene expression showed that expression profiles of AcSWEETs displayed differences in different tissues and five AcSWEETs were strongly expressed during fruit development. AcSWEET11 was highly expressed in the stage of mature fruits in ‘Tainong16’ and ‘Comte de paris’, which indicates that AcSWEET11 was important to fruit development. Subcellular localization analysis showed that AcSWEET11 was located in the cell membrane. Notably, overexpression of AcSWEET11 could improve sugar accumulation in pineapple callus and transgenic tomato, which suggests that AcSWEET11 might positively contribute to sugar accumulation in pineapple fruit development. These results may provide insights to enhance sugar accumulation in fruit, thus improving pineapple quality in the future.

In the original version of this article [1], the spelling of the tenth author name, Moakai Yan, was incorrect. The corrected name is given in this correction article.

Watercore is a physiological disorder in pineapples, which is expressed as fluid deposition in intercellular spaces and presents as water soaked. This disorder affects the fruit quality and decreases storage life, resulting in enormous commercial losses to growers and restricting the development of the pineapple industry in China. However, the molecular mechanism of watercore remains unclear. In order to elucidate the molecular mechanism of pineapple watercore, the transcriptome analyses of watercored and normal fruits were carried out in pineapples for the first time using de novo RNA-seq technology. High-quality reads of 46.66 and 43.71 M were obtained in the transcriptomes of normal and mildly watercored fruits, respectively. Clean reads of 45.50 and 42.79 M were obtained after filtering the original data. These genes are useful resources in subsequent pineapple watercore research. Fifty genes in phenylpropanoid biosynthesis, glucose metabolism, calcium transport, and cell wall metabolism were considerably different between normal and watercored fruits. Among them, the expressions of the AcPME, AcBGLU43, Ac4CL5, AcPER1, and AcPOD genes were upregulated by 7–21 times in watercored fruit, while the expressions of AcSUS7 were downregulated by 16.61 times, and the expressions of other differential genes were upregulated or downregulated by more than 2 times. A total of 38 differentially expressed transcription factors were obtained by screening. Among these transcription factors, WRKY was the most abundant, followed by MYB. The acquisition of these genes is important for the first understanding of the molecular mechanism of this physiological disorder.