MethodsMethod S1: Screening of the expressed candidate sex determinants Developing anthers at stage 1-2, which correspond to the differentiation stage of male or female androecium (see Supplementary Figure S1), were sampled from F1 sibling vines derived from an interspecific cross, A. rufa sel. Fuchu × A. chinensis sel. FCM1, named KE population (15), planted on Kagawa University, Japan (N34.28, E134.13), in 10-22 April in 2016-2017. Total RNA was extracted using the Plant RNA Reagent (Invitrogen) and purified by phenol/chloroform extraction. Two micrograms of total RNA were processed in preparation for Illumina Sequencing, according to a previous report (15). The constructed libraries were sequenced on Illumina's HiSeq 4000 sequencer (50-bp single-end or 150-bp paired-end reads). All Illumina sequencing were conducted at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, and the raw sequencing reads were processed using custom Python scripts developed in the Comai laboratory and available online (http://comailab.genomecenter.ucdavis.edu/index.php/), as previously described (9). Male-specific Ychromosomal sequences in kiwifruit, defined MSY contigs, were comprehensively identified in previous study (15). The mRNA-Seq reads from each 5 male and female individuals from the KE population (Supplemental Table S11) (15) were used to identify the genes substantially expressed in developing anthers. The mRNA-Seq reads were aligned to the hypothetical 61 genes located on the 249 MSY contigs (Akagi et al. 2018), using the Burrows-Wheeler Aligner (BWA) (37) allowing up to ca 3% mismatches. The number of reads mapping to each contigs was recorded from the alignment file produced by the Sequence Alignment/Map (SAM) tool (38) (http://samtools.sourceforge.net/). For Friendly Boy (FrBy), which showed male-specific and anther-enriched expression, the expression patterns were further examined using various plant organs and developing anthers (stage 2a, 2b, 3a, and 3b, see Supplementary Figure S1). Method S2: Expression profiling in kiwifruit antherThe described mRNA-Seq reads from each 5 male and female individuals of the KE population were aligned to the whole CDS sequences sets in A. chinensis (27), using BWA with default parameters. The number of reads mapped to each reference sequences was recorded from the alignment file produced by the Sequence Alignment/Map (SAM) tool (38) (http://samtools.sourceforge.net/). The read counts per gene were generated from the aligned SAM files using a custom R script. Differential expression between male and female individuals was analysed in R (version 3.0.1) using the R package DESeq (Anders and Huber, 2010) (version 1.14; http://bioconductor.org/packages/release/bioc/html/DESeq.html). We conducted DESeq analysis using 5 biological replicates from male and female individuals, with the following parameters: method='per-condition' and sharingMode='maximum'. An FDR threshold of 0.1 was used to identify differentially expressed genes. Method S3: in situ RNA hybridizationRNA in ...

The polyphenol profile of apple (Malus × domestica) is dominated by the dihydrochalcone glycoside phloridzin, but its physiological role is yet to be elucidated. Biosynthesis of phloridzin occurs as a side branch of the main phenylpropanoid pathway, with the final step mediated by the phloretin-specific glycosyltransferase UGT88F1. Unexpectedly, given that UGTs are sometimes viewed as 'decorating enzymes', UGT88F1 knockdown lines were severely dwarfed, with greatly reduced internode lengths, narrow lanceolate leaves, and changes in leaf and fruit cellular morphology. These changes suggested that auxin transport had been altered in the knockdown lines, which was confirmed in assays showing that auxin flux from the shoot apex was increased in the transgenic lines. Metabolite analysis revealed no accumulation of the phloretin aglycone, as well as decreases in many non-target phenylpropanoid compounds. This decreased accumulation of metabolites appeared to be mediated by the repression of the phenylpropanoid pathway via a reduction in key transcript levels (e.g. phenylalanine ammonia lyase, PAL) and enzyme activities (PAL and chalcone synthase). Application of exogenous phloridzin to the UGT88F1 knockdown lines in tissue culture enhanced axial leaf growth and partially restored some aspects of 'normal' apple leaf growth. Together, our results strongly implicate dihydrochalcones as critical compounds in modulating phenylpropanoid pathway flux and establishing auxin patterning early in apple development.

26Dioecy, the presence of male and female individuals, has evolved independently in multiple 27 flowering plant lineages. Although theoretical models for the evolution of dioecy, such as the 28 "two-mutation" model, are well established, little is known about the specific genes determining 29 sex and their evolutionary history. Kiwifruit, a major tree crop consumed worldwide, is a 30 dioecious species. In kiwifruit, we had previously identified a Y-encoded sex-determinant 31 candidate gene acting as the suppressor of feminization (SuF), named Shy Girl (SyGI). Here, we 32 identified a second Y-encoded sex-determinant that we named Friendly boy (FrBy), which 33 exhibits strong expression in tapetal cells. Gene-editing and complementation analyses in 34 Arabidopsis thaliana and Nicotiana tabacum indicated that FrBy acts for the maintenance of male 35 (M) functions, independently of SyGI, and that these functions are conserved across angiosperm 36 species. We further characterized the genomic architecture of the small (< 1 Mb) male specific 37 region of the Y-chromosome (MSY), which harbors only two genes significantly expressed in 38 developing gynoecia and androecia, respectively: SyGI and FrBy. Resequencing of the genome 39 of a natural hermaphrodite kiwifruit revealed that this individual is genetically male but carries 40 deletion(s) of parts of the Y-chromosome, including SyGI. Additionally, expression of FrBy in 41 female kiwifruit resulted in hermaphrodite plants. These results clearly indicate that Y-encoded 42SyGI and FrBy act independently as the SuF and M factors in kiwifruit, respectively, and provide 43 insight into the evolutionary path leading to a two-factor sex determination system but also a new 44 breeding approach for dioecious species. 45 46 MAIN TEXT 47

Waxy apple cuticles predominantly accumulate ursane-type triterpenes, but the profile shifts with the induction of skin russeting towards lupane-type triterpenes. We previously characterised several key enzymes in the ursane-type and lupane-type triterpene pathways, but this switch in triterpene metabolism associated with loss of cuticle integrity is not fully understood. To analyse the relationship between triterpene biosynthesis and russeting, we used microscopy, RNA-sequencing and metabolite profiling during apple fruit development. We compared the skin of three genetically-close clones of ‘Golden Delicious’ (with waxy, partially russeted and fully russeted skin). We identified a unique molecular profile for the russet clone, including low transcript abundance of multiple cuticle-specific metabolic pathways in the early stages of fruit development. Using correlation analyses between gene transcription and metabolite concentration we found MYB transcription factors strongly associated with lupane-type triterpene biosynthesis. We showed how their transcription changed with the onset of cuticle cracking followed by russeting and that one factor, MYB66, was able to bind the promoter of the oxidosqualene cyclase OSC5, to drive the production of lupeol derivatives. These results provide insights into the breakdown of cuticle integrity leading to russet and how this drives MYB-regulated changes to triterpene biosynthesis.

The aim of this study was to understand factors affecting oil release from "Hass" avocado cells during the cold-pressed oil extraction process. Early-season "Hass" avocado fruit and pulp were sampled from a commercial extraction process. Light microscopy and electrical impedance spectroscopy were used to examine avocado flesh structure at defined steps during the extraction process (destoning, grinding, malaxing, and decanting). Most parenchyma cells were ruptured during destoning, grinding, and mixing before malaxing. In contrast, the other oil bearing cells, idioblast cells, appeared to remain unruptured and intact during the extraction process. The greatest reduction in electrical resistance was observed after the destoning step. There were also significant differences between the resistances of flesh after destoning, grinding, and prior to malaxing samples. Malaxing for two hours assisted the process of oil aggregation, which led to a greater oil yield. These findings will help to develop methods to enhance oil extraction from early-season "Hass" avocado fruit.