Brassica napus L. (canola) is one of the world's most economically important oilseeds. Despite our growing knowledge of Brassica genetics, we still know little about the genes and gene regulatory networks underlying early seed development. In this work, we use laser microdissection coupled with RNA sequencing to profile gene activity of both the maternal and filial subregions of the globular seed. We find subregions of the chalazal end including the chalazal endosperm, chalazal proliferating tissue, and chalazal seed coat, have unique transcriptome profiles associated with hormone biosynthesis and polysaccharide metabolism. We confirm that the chalazal seed coat is uniquely enriched for sucrose biosynthesis and transport, and that the chalazal endosperm may function as an important regulator of the maternal region through brassinosteroid synthesis. The chalazal proliferating tissue, a poorly understood subregion, was specifically enriched in transcripts associated with megasporogenesis and trehalose biosynthesis, suggesting this ephemeral structure plays an important role in both sporophytic development and carbon nutrient balance, respectively. Finally, compartmentalization of transcription factors and their regulatory circuits has uncovered previously unknown roles for the chalazal pole in early seed development.

Sclerotinia sclerotiorum is a pathogenic fungus that infects hundreds of crop species, causing extensive yield loss every year. Chemical fungicides are used to control this phytopathogen, but with concerns about increasing resistance and impacts on non-target species, there is a need to develop alternative control measures. In the present study, we engineered Brassica napus to constitutively express a hairpin (hp)RNA molecule to silence ABHYRDOLASE-3 in S. sclerotiorum. We demonstrate the potential for Host Induced Gene Silencing (HIGS) to protect B. napus from S. sclerotiorum using leaf, stem and whole plant infection assays. The interaction between the transgenic host plant and invading pathogen was further characterized at the molecular level using dual-RNA sequencing and at the anatomical level through microscopy to understand the processes and possible mechanisms leading to increased tolerance to this damaging necrotroph. We observed significant shifts in the expression of genes relating to plant defense as well as cellular differences in the form of structural barriers around the site of infection in the HIGS-protected plants. Our results provide proof-of-concept that HIGS is an effective means of limiting damage caused by S. sclerotiorum to the plant and demonstrates the utility of this biotechnology in the development of resistance against fungal pathogens.

SUMMARY We profiled the global gene expression landscape across the reproductive lifecycle of Brassica napus. Comparative analysis of this nascent amphidiploid revealed the contribution of each subgenome to plant reproduction. Whole‐genome transcription factor networks identified BZIP11 as a transcriptional regulator of early B. napus seed development. Knockdown of BZIP11 using RNA interference resulted in a similar reduction in gene activity of predicted gene targets, and a reproductive‐lethal phenotype. Global mRNA profiling revealed lower accumulation of Cn subgenome transcripts relative to the An subgenome. Subgenome‐specific transcription factor networks identified distinct transcription factor families enriched in each of the An and Cn subgenomes early in seed development. Analysis of laser‐microdissected seed subregions further reveal subgenome expression dynamics in the embryo, endosperm and seed coat of early stage seeds. Transcription factors predicted to be regulators encoded by the An subgenome are expressed primarily in the seed coat, whereas regulators encoded by the Cn subgenome were expressed primarily in the embryo. Data suggest subgenome bias are characteristic features of the B. napus seed throughout development, and that such bias might not be universal across the embryo, endosperm and seed coat of the developing seed. Transcriptional networks spanning both the An and Cn genomes of the whole B. napus seed can identify valuable targets for seed development research and that ‐omics level approaches to studying gene regulation in B. napus can benefit from both broad and high‐resolution analyses.

One Sentence Summary: 23Global RNA sequencing coupled with laser microdissection provides a critical resource to study 24 subgenome bias in whole seeds and specific tissues of polyploid plants. 25 26 Abstract 27We profiled the gene regulatory landscape of Brassica napus reproductive development using 28 RNA sequencing. Comparative analysis of this nascent amphidiploid across the plant lifecycle 29 revealed the contribution of each subgenome to plant reproduction. Global mRNA profiling 30 revealed lower accumulation of C n subgenome transcripts relative to the A n subgenome. 31Subgenome-specific transcriptional networks identified distinct transcription factor families 32 enriched in each of the A n and C n subgenome early in seed development. Global gene expression 33 profiling of laser-microdissected seed subregions further reveal subgenome expression dynamics 34 in the embryo, endosperm, and seed coat of early stage seeds. Transcription factors predicted to 35 be regulators encoded by the A n subgenome are expressed primarily in the seed coat whereas 36 regulators encoded by the C n subgenome were expressed primarily in the embryo. Data suggest 37 subgenome bias are characteristic features of the B. napus seed throughout development, and that 38 such bias might not be universal across the embryo, endosperm, and seed coat of the developing 39 seed. Whole genome transcription factor networks identified BZIP11 as a transcriptional 40 regulator of early B. napus seed development. Knockdown of BZIP11 using RNA interference 41 resulted in a similar reduction in gene activity of predicted gene targets, and a reproductive-lethal 42 phenotype. Taken together, transcriptional networks spanning both the A n and C n genomes of the 43 B. napus seed can identify valuable targets for seed development research and that-omics level 44 approaches to studying gene regulation in B. napus can benefit from both broad and high-45 resolution analyses. 46 3 47 49Brassica napus (canola) is the second most important oilseed crop in the world with 50 global production of 75 million tons in 2017 (FAOSTAT 2017). It is a relatively recent 51 allopolyploid derived from hybridization between B. rapa and B. oleracea (Nagaharu, 1935). 52The resulting plant is an amphidiploid species which has retained its progenitor subgenomes 53 from B. rapa (A r A r ) and B. oleracea (C o C o ) to form B. napus (A n A n C n C n ). Despite its 54 importance to the global economy, B. napus genome dynamics are poorly understood, especially 55 in the context of gene regulation during seed development. The developmental transition from 56 seed morphogenesis to maturation (Figure 1A) requires a re-orchestration of the genes and gene 57 regulatory networks underpinning seed development. Therefore, B. napus represents an 58 appropriate model for research on transcriptomic changes across seed development of 59 economically important polyploid plants. 60Brassica napus is a nascent amphidiploid which has not yet undergone substantial 61 fractionation, and consequently ha...

Arceuthobium americanum Nutt. ex Engelm., the lodgepole pine dwarf mistletoe, is a dioecious parasitic flowering plant infecting lodgepole pine (Pinus contorta subsp. latifolia (Engelm.) Critchfield) in the Pacific Northwest, compromising timber value by stunting the host’s growth. The plant disperses its seeds by explosive discharge, and thus understanding its reproduction is an integral step toward managing its spread. The life cycle of Arceuthobium americanum occurs over five to six years, and the fruit matures over two consecutive growing seasons, dispersing in the second. Using low-vacuum environmental scanning electron microscopy (eSEM), we examined branching architecture as well as the morphology and anatomy of fruits during their second year of development up to explosive discharge. We found that branching patterns shifted from opposite-decussate to verticillate after several years’ growth. In addition, the perianth parts (sepals), style, and stigma persisted through the season, and the fruit’s abscission zone developed slightly distal to the end of the pedicel. Stomata, found only on the sepals, were located in crypts surrounded by two subsidiary cells, and stomatal density significantly decreased during development. The decline in stomatal density along with the thick pericarp cuticle may function in retaining water inside the fruit to facilitate discharge and (or) provide a heating mechanism through reduced transpiration.