The chromodomain helicase DNA-binding family of ATP-dependent chromatin remodeling factors play essential roles during eukaryote growth and development. They are recruited by specific transcription factors and regulate the expression of developmentally important genes. Here, we describe an unexpected role in non-coding RNA-directed DNA methylation in Arabidopsis thaliana. Through forward genetic screens we identified PKL, a gene required for developmental regulation in plants, as a factor promoting transcriptional silencing at the transgenic RD29A promoter. Mutation of PKL results in DNA methylation changes at more than half of the loci that are targeted by RNA-directed DNA methylation (RdDM). A small number of transposable elements and genes had reduced DNA methylation correlated with derepression in the pkl mutant, though for the majority, decreases in DNA methylation are not sufficient to cause release of silencing. The changes in DNA methylation in the pkl mutant are positively correlated with changes in 24-nt siRNA levels. In addition, PKL is required for the accumulation of Pol V-dependent transcripts and for the positioning of Pol V-stabilized nucleosomes at several tested loci, indicating that RNA polymerase V-related functions are impaired in the pkl mutant. PKL is required for transcriptional silencing and has significant effects on RdDM in plants. The changes in DNA methylation in the pkl mutant are correlated with changes in the non-coding RNAs produced by Pol IV and Pol V. We propose that at RdDM target regions, PKL may be required to create a chromatin environment that influences non-coding RNA production, DNA methylation, and transcriptional silencing.

WRKY transcription factors are widely distributed in higher plants and play important roles in many biological processes, including stress resistance. The recently published genome sequence of yellowhorn, an oil tree with robust resistance to cold, drought, heat, salt and alkali, provides an excellent opportunity to identify and characterize the entire yellowhorn WRKY protein family and a basis for the study of abiotic stress resistance of WRKY gene family in forest species. In the present comprehensive analysis of WRKY transcription factors in yellowhorn, 65 WRKY genes were identified and defined based on their location on the chromosome. According to their structure and phylogenetic relationships, XsWRKY genes clustered into WRKY groups I–III. Segmental duplication events played a significant role in the expansion of WRKY gene family. Furthermore, transcriptomic data and real-time quantitative PCR analysis showed that expression of XsWRKY genes responding to salt and drought stresses and a hormone treatment. We also determined structures of the encoded proteins, cis-elements of the promoter region, and expression patterns. These results provide a foundation for the study of the biological function of WRKY transcription factors in yellowhorn.

As essential organs of reproduction in angiosperms, flowers, and the genetic mechanisms of their development have been well characterized in many plant species but not in the woody tree yellowhorn (Xanthoceras sorbifolium). Here, we focused on the double flower phenotype in yellowhorn, which has high ornamental value. We found a candidate C-class gene, AGAMOUS1 (XsAG1), through Bulked Segregant Analysis (BSA) sequencing and genetics analysis with a Long Interpersed Nuclear Elements 1 (LINE1) transposable element fragment (Xsag1-LINE1-1) inserted into its second intron that caused a loss-of-C-function and therefore the double flower phenotype. In situ hybridization of XsAG1 and analysis of the expression levels of other ABC genes were used to identify differences between single- and double-flower development processes. These findings enrich our understanding of double flower formation in yellowhorn and provide evidence that transposon insertions into genes can reshape plant traits in forest trees.

Xanthoceras sorbifolium (yellowhorn) is originated in China and is a unique tree in northern China. Yellowhorn is very popular because of the gradual color change of its flower at different flower developmental stages. During flowering development, the color at the base of yellowhorn flower petals gradually changes from yellow to purple. The mechanism of this miraculous phenomenon is still unclear. Here we show that anthocyanin accumulation during flowering development is the main reason for this color change. RT-PCR results show that the expression level of a variety of anthocyanin biosynthesis genes changes in different flower developmental stages. Realtime results show that the expression changes of these anthocyanin biosynthesis genes are positively regulated by a cluster of R2R3-MYB transcription factor genes, XsMYB113s. Furthermore, the DNA methylation analysis showed that CHH methylation status on the transposon element near the XsMYB113-1 influence its transcript level during flowering development. Our results suggest that dynamic epigenetic regulation of the XsMYB113-1 leads to the accumulation of anthocyanins during yellowhorn flower color change. These findings reemphasize the role of epigenetic regulation in flower development and provide a foundation for further studies of epigenetic regulation in long-lived woody perennials.

Summary: Walnut (Juglans regia L.) is a significant commercial tree, valued both for its timber and nut production and it is extensively cultivated across the globe. We exploit heterografting by using cultivar‐‘Shangsong14’ (Juglans regia; ‘S14’) as scion and hybrid elite‐‘Zhongningqi’ (Juglans hindsii × Juglans regia; ‘Q’) as rootstock and self‐grafting each other as both scion and rootstock to investigate the genetic effects of the rootstock. Heterografting induces vigour and yield in walnut scions through 15 years of continuous observation. Grafting experiment indicated that ‘Q’ as a rootstock caused a reduction of miR396a in scions (S14/Q) compared to self‐grafting (S14/S14), while the transcript levels of JrGRF4b and JrGIF1a were upregulated in S14/Q compared with S14/S14. Moreover, we indicated that JrGRF4b might directly target and enhance JrGATA4 expression, further upregulating JrCycD4;1 and JrCycJ18 transcript abundance and affecting the proliferation of cambium cells. To examine the role of miR396a‐JrGRF4b/JrGIF1a in growth vigour, we produced white birch transgenic lines that overexpressed miR396a, rJrGRF4b and JrGIF1a. Overexpression of rJrGRF4b and JrGIF1a enhanced plant vigour and cambium cell proliferation, while overexpression of miR396a had the opposite effect in white birch. Our findings suggest that Jr‐miR396a was decreased in S14/Q compared to S14/S14, where mRNAs of JrGRF4b were released to enhance the JrGATA4 transcript level, strengthening cell proliferation in scions. The research revealed a novel molecular mechanism about rootstocks enhancing vigour in scions, which have the potential to accelerate breeding rootstocks for perennial woody species.