A subset of genes in plant genomes are labeled with DNA methylation specifically at CG residues. These genes, known as gene-body methylated (gbM), have a number of associated characteristics. They tend to have longer sequences, to be enriched for intermediate expression levels, and to be associated with slower rates of molecular evolution. Most importantly, gbM genes tend to maintain their level of DNA methylation between species, suggesting that this trait is under evolutionary constraint. Given the degree of conservation in gbM, we still know surprisingly little about its function in plant genomes or whether gbM is itself a target of selection. To address these questions, we surveyed DNA methylation across eight grass (Poaceae) species that span a gradient of genome sizes. We first established that genome size correlates with genome-wide DNA methylation levels, but less so for genic levels. We then leveraged genomic data to identify a set of 2,982 putative orthologs among the eight species and examined shifts of methylation status for each ortholog in a phylogenetic context. A total of 55% of orthologs exhibited a shift in gbM, but these shifts occurred predominantly on terminal branches, indicating that shifts in gbM are rarely conveyed over time. Finally, we found that the degree of conservation of gbM across species is associated with increased gene length, reduced rates of molecular evolution, and increased gene expression level, but reduced gene expression variation across species. Overall, these observations suggest a basis for evolutionary pressure to maintain gbM status over evolutionary time.

Plants undergo transcriptome reprograming to adapt to daily and seasonal fluctuations in light and temperature conditions. While most efforts have focused on the role of master transcription factors, the importance of splicing factors modulating these processes is now emerging. Efficient pre-mRNA splicing depends on proper spliceosome assembly, which in plants and animals requires the methylosome complex. Ion Chloride nucleotide-sensitive protein (PICLN) is part of the methylosome complex in both humans and Arabidopsis (Arabidopsis thaliana), and we show here that the human PICLN ortholog rescues phenotypes of Arabidopsis picln mutants. Altered photomorphogenic and photoperiodic responses in Arabidopsis picln mutants are associated with changes in pre-mRNA splicing that partially overlap with those in PROTEIN ARGININE METHYL TRANSFERASE5 (prmt5) mutants. Mammalian PICLN also acts in concert with the Survival Motor Neuron (SMN) complex component GEMIN2 to modulate the late steps of UsnRNP assembly, and many alternative splicing events regulated by PICLN but not PRMT5, the main protein of the methylosome, are controlled by Arabidopsis GEMIN2. As with GEMIN2 and SM PROTEIN E1/PORCUPINE (SME1/PCP), low temperature, which increases PICLN expression, aggravates morphological and molecular defects of picln mutants. Taken together, these results establish a key role for PICLN in the regulation of pre-mRNA splicing and in mediating plant adaptation to daily and seasonal fluctuations in environmental conditions.

Although much is known about the responses of model plants to microbial features, we still lack an understanding of the extent of variation in immune perception across members of a plant family. In this work, we analyzed immune responses in Citrus and wild relatives, surveying 86 Rutaceae genotypes with differing leaf morphologies and disease resistances. We found that responses to microbial features vary both within and between members. Species in two subtribes, the Balsamocitrinae and Clauseninae, can recognize flagellin (flg22), cold shock protein (csp22) and chitin, including one feature from Candidatus Liberibacter species (csp22_CLas), the bacterium associated with Huanglongbing. We investigated differences at the receptor level for the flagellin receptor FLAGELLIN SENSING 2 (FLS2) and the chitin receptor LYSIN MOTIF RECEPTOR KINASE 5 (LYK5) in citrus genotypes. We characterized two genetically linked FLS2 homologs from ‘Frost Lisbon’ lemon (Citrus ×limon, responsive) and ‘Washington navel’ orange (Citrus ×aurantium, non-responsive). Surprisingly, FLS2 homologs from responsive and non-responsive genotypes were expressed in Citrus and functional when transferred to a heterologous system. ‘Washington navel’ orange weakly responded to chitin, whereas ‘Tango’ mandarin (Citrus ×aurantium) exhibited a robust response. LYK5 alleles were identical or nearly identical between the two genotypes and complemented the Arabidopsis (Arabidopsis thaliana) lyk4/lyk5-2 mutant with respect to chitin perception. Collectively, our data indicate that differences in chitin and flg22 perception in these citrus genotypes are not the results of sequence polymorphisms at the receptor level. These findings shed light on the diversity of perception of microbial features and highlight genotypes capable of recognizing polymorphic pathogen features.

Heterosis was the catalyst for the domestication of cultivated strawberry (Fragaria × ananassa), an interspecific hybrid species that originated in the 1700s. The hybrid origin was discovered because the phenotypes of spontaneous hybrids transgressed those of their parent species. The transgressions included fruit yield increases and other genetic gains in the twentieth century that sparked the global expansion of strawberry production. The importance of heterosis to the agricultural success of the hybrid species, however, has remained a mystery. Here we show that heterosis has disappeared (become fixed) among improved hybrids within a population (the California population) that has been under long-term selection for increased fruit yield, weight, and firmness. We found that the highest yielding hybrids are among the most highly inbred (59-79%), which seems counterintuitive for a highly heterozygous, outbreeder carrying heavy genetic loads. Although faint remnants of heterosis were discovered, the between-parent allele frequency differences and dispersed favorable dominant alleles necessary for heterosis have decreased nearly genome-wide within the California population. Conversely, heterosis was prevalent and significant among wide hybrids, especially for fruit count, a significant driver of genetic gains for fruit yield. We attributed the disappearance (fixation) of heterosis within the California population to increased homozygosity of favorable dominant alleles and inbreeding associated with selection, random genetic drift, and selective sweeps. Despite historical inbreeding, the highest yielding hybrids reported to-date are estimated to be heterozygous for 20,370-44,280 of 97,000-108,000 genes in the octoploid genome, the equivalent of an entire diploid genome or more.

Societal Impact Statement: Citrus are intrinsically connected to human health and culture, preventing human diseases like scurvy and inspiring sacred rituals. Citrus fruits come in a stunning number of different sizes and shapes, ranging from small clementines to oversized pummelos, and fruits display a vast diversity of flavors and aromas. These qualities are key in both traditional and modern medicine and in the production of cleaning and perfume products. By quantifying and modeling overall fruit shape and oil gland distribution, we can gain further insight into citrus development and the impacts of domestication and improvement on multiple characteristics of the fruit.Summary: Citrus come in diverse sizes and shapes, and play a key role in world culture and economy. Citrus oil glands in particular contain essential oils which include plant secondary metabolites associated with flavor and aroma. Capturing and analyzing nuanced information behind the citrus fruit shape and its oil gland distribution provide a morphology‐driven path to further our insight into phenotype–genotype interactions. We investigated the shape of citrus fruit of 51 accessions based on 3D X‐ray computed tomography (CT) scan reconstructions. Accessions include members of the three ancestral citrus species as well as related genera, and several interspecific hybrids. We digitally separate and compare the size of fruit endocarp, mesocarp, exocarp, and oil gland tissue. Based on the centers of the oil glands, overall fruit shape is approximated with an ellipsoid. Possible oil gland distributions on this ellipsoid surface are explored using directional statistics. There is a strong allometry along fruit tissues; that is, we observe a strong linear relationship between the logarithmic volume of any pair of major tissues. This suggests that the relative growth of fruit tissues with respect to each other follows a power law. We also observe that on average, glands distance themselves from their nearest neighbor following a square root relationship, which suggests normal diffusion dynamics at play. The observed allometry and square root models point to the existence of biophysical developmental constraints that govern novel relationships between fruit dimensions from both evolutionary and breeding perspectives. Understanding these biophysical interactions prompts an exciting research path on fruit development and breeding.