Balanophora genomes display massively convergent evolution with other extreme holoparasites and provide novel insights into parasite-host interactions.

Parasitic plants have evolved to be subtly or severely dependent on host plants to complete their life cycle. To provide new insights into the biology of parasitic plants in general, we assembled genomes for members of the sandalwood order Santalales, including a stem hemiparasite (Scurrula) and two highly modified root holoparasites (Balanophora) that possess chimaeric host-parasite tubers. Comprehensive genome comparisons reveal that hemiparasitic Scurrula has experienced a relatively minor degree of gene loss compared with autotrophic plants, consistent with its moderate degree of parasitism. Nonetheless, patterns of gene loss appear to be substantially divergent across distantly related lineages of hemiparasites. In contrast, Balanophora has experienced substantial gene loss for the same sets of genes as an independently evolved holoparasite lineage, the endoparasitic Sapria (Malpighiales), and the two holoparasite lineages experienced convergent contraction of large gene families through loss of paralogues. This unprecedented convergence supports the idea that despite their extreme and strikingly divergent life histories and morphology, the evolution of these and other holoparasitic lineages can be shaped by highly predictable modes of genome reduction. We observe substantial evidence of relaxed selection in retained genes for both hemi- and holoparasitic species. Transcriptome data also document unusual and novel interactions between Balanophora and host plants at the host-parasite tuber interface tissues, with evidence of mRNA exchange, substantial and active hormone exchange and immune responses in parasite and host.

An optimized FACS-free single-nucleus RNA sequencing (snRNA-seq) method for plant science research.

Recently, single-cell RNA sequencing (scRNA-seq) provides unprecedented power for accurately understanding gene expression regulatory mechanisms. However, scRNA-seq studies have limitations in plants, due to difficulty in protoplast isolation that requires enzymatic digestion of the cell walls from various plant tissues. Therefore, to overcome this problem, we developed a nuclei isolation approach that does not rely on Fluorescence Activated Cell Sorting (FACS). We validated the robustness of the FACS-free single-nucleus RNA sequencing (snRNA-seq) methodology in mature Arabidopsis plant tissue by comparing it to scRNA-seq results based on protoplasts extracted from the same batch of leaf materials. Sequencing results demonstrated the high quality of snRNA-seq data, as well as its utility in cell type classification and marker gene identification. This approach also showed several advantages, including the ability to use frozen samples, taking less suspension preparation time, and reducing biased cellular coverage and dissociation-induced transcriptional artifacts. Surprisingly, snRNA-seq detected two epidermal pavement cell clusters, while scRNA-seq only had one. Furthermore, we hypothesized that these two epidermal cells represent the top and lower epidermis based on differences in expression patterns of cluster-specific expressed genes. In summary, this study has advanced the application of snRNA-seq in Arabidopsis leaves and confirmed the advantages of snRNA-seq in plant research.

Genome-Wide Analysis of the MADS-Box Gene Family in Holoparasitic Plants (Balanophora subcupularis and Balanophora fungosa var. globosa).

MADS-box is an important transcription factor family that is involved in the regulation of various stages of plant growth and development, especially flowering regulation and flower development. Being a holoparasitic plant, the body structure of Balanophoraceae has changed dramatically over time, and its vegetative and reproductive organs have been extensively modified, with rudimentary flower organs. Meanwhile, extraordinary gene losses have been identified in holoparasitic plants compared with autotrophs. Our study reveals that the MADS-box gene family contracted sharply in Balanophora subcupularis and Balanophora fungosa var. globosa, and some subfamilies were lost, exhibiting reduced redundancy in both. The genes that functioned in the transition from the vegetative to floral production stages suffered a significant loss, but the ABCE model genes remained intact. We further investigated genes related to flowering regulation in B. subcupularis and B. fungosa var. globosa, vernalization and autonomous ways of regulating flowering time remained comparatively integrated, while genes in photoperiod and circadian clock pathways were almost lost. Convergent gene loss in flowering regulation occurred in Balanophora and another holoparasitic plant Sapria himalayana (Rafflesiaceae). The genome-wide analysis of the MADS-box gene family in Balanophora species provides valuable information for understanding the classification, gene loss pattern, and flowering regulation mechanism of MADS-box gene family in parasitic plants.