Branching Out: Unravelling the Molecular Control of Communication

The project investigated the molecular mechanisms that regulate cell communication during branching in the model species hybrid aspen (Populus tremula x P. tremuloides). While hormones regulate meristem physiology, cell communication integrates meristem activities. In hybrid aspen, side buds are formed at the shoot tip and gradually develop to maturity before entering an inactive state. Activation results in branch formation, which can be investigated experimentally by removing the shoot tip. It is disputed whether the hormone gibberellin (GA) is promoting or inhibiting branching. To address this, we carried out a comprehensive GA transcript and metabolite analyses. We found that different GA species, GA3/6 and GA1/4, function in sequence to inhibit and promote branching. These opposing effects are based on the crucial feature that the GA-catabolizing GA2oxidase enzymes target GA1/4 but not GA3/6. Our metabolite analyses showed that inactive buds had a high level of GA3/6 and GA2oxidase transcripts, leading to the exclusive degradation of GA1/4. Experimental bud activation diminished expression of GA20oxidase genes, relieving GA1/4 catabolism, and upregulating GA1/4 biosynthesis genes. In short, GA3/6 in buds keeps GA1/4 levels low and prevents outgrowth, whereas activation downregulates GA-catabolising enzymes and promotes GA1/4 signalling and branching. During bud formation lipid bodies (LB) accumulate, which support future growth. LBs are extracted from the endoplasmic reticulum by OLEOSIN proteins. LBs also recruit cytoplasmic proteins and target plasmodesmata, communication channels between meristem cells that integrate meristem function. We investigated how LBs form, what proteins they recruit, and how they reach plasmodesmata to delivering proteins. We discovered that OLEOSIN6 is the gene driving LB production. To study LB transport we genetically transformed Arabidopsis with OLEOSIN6 tagged with fluorescent eGFP (OLE6-eGFP). We showed that LB transport was not due to cytoplasmic streaming, and that LB-OLE6-eGFP reaches plasmodesmata via actin, motorised by myosin XIs motor proteins. Pharmacological inhibition of the actin-myosin transport mechanism arrested LB movement. Moreover, in a triple myosin-XI knockout mutant (xi-k/1/2), transformed with PtOLE6-eGFP, transport of LBs was severely impaired. Together this confirmed that LB movement requires myosin XIs motor proteins. We investigated LB production and analysed the LB body proteome from LBs isolated from mature buds. We found that the dominant OLEOSIN, OLE6, was removed from the LBs before bud completion, while the LBs enlarged. The data indicated that OLEOSINs were removed by a degradation mechanism composed of PUX10, CDC48A, UBPs, and the 26S proteasome, and replaced by cytoplasmically recruited LDIP and LDAP proteins at the enlarged LBs. Reduced molecular crowding at the enlarged LB surfaces likely resulted in an expanded LB proteome. Importantly, the LB proteome also contained the myosin XI-tail-binding RAB-ATPase that binds LBs to the myosin XI-actin assembly, enabling movement on actin. We found that this RAB-ATPase localizes to eGFP-tagged LBs. The proteome contained many proteins that are previously found at LBs or plasmodesmata. Using RNA-sequencing we identified hormonal factors that are early co-responders to decapitation-induced activation. The hormones, strigolactone (SL) and GA together regulate branching. Although SL is considered a branch inhibitor, in hybrid aspen it is not effective in keeping side buds inactive. Activation is mediated by GA4 and requires early downregulation GA degradation genes. In contrast to the consensus view that auxin, SL, cytokinins and abscisic acid are the relevant branching hormones our findings demonstrate the prominent role of GA. Illumina RNA sequencing at the whole genome level combined with analyses of differential gene expression of genes involved in biosynthesis and degradation of the plant hormones corroborate that GA has a key role in promoting bud activation, while SL and ABA maintain inhibition in non-activated buds. Trees display different branching styles, referred to as sylleptic and proleptic. In sylleptic trees, young side buds produce branches in the same season, but in proleptic trees such as hybrid aspen it is the mature side buds that give rise to branches after passage through dormancy. Using a range of methods, we found that ABA downregulates a key GA biosynthesis gene that is required for branching. Syllepsis correlates with accumulation of reserves like lipid bodies, endogenous ABA levels and expression of the branch inhibitor gene BRC1 and 1,3-?-glucanase genes that restore communication between stem and bud. Collectively, the project results indicate that the ABA/GA ratio in inactive side buds determines the branching style by controlling the stem-bud communication paths through production of LBs and recruitment of LB cargo.

The results from this project are a breakthrough in branching research, foremost scientifically as they integrate hormonal, and molecular mechanisms with cellular infrastructure and cell-cell communication and transport. As the project results provide insight into fundamental processes that govern the design of plants, we believe that the work will significantly promote the research field of plant development. The current and the previous projects have trained postdoctoral workers, but unfortunately due to existing national policies the opportunity to maximize return on investment by recruitment of these competent researchers is frustrated.

The proposed approach will contribute to solving one of the most fundamental problems in plant development: 'how do plants regulate shoot architecture' As the number of branches determines quantity and quality of the produce, finding answers will not only benefit production through improved culture practices, but also open up new avenues for breeding and genetic improvement of plants for food production and biofuel industries. The single key event in architectural development, even in a tree, is the activation and outgrowth of axillary buds. The set of known branching hormones auxin and cytokinin was recently expanded to include strigolactone, a hormone that inhibits bud activation by hampering auxin export. Despite this progress, a comprehensive model remains absent. The present project will overcome this be taking a completely novel approach. In this 'plumbers view' the effectiveness of signals to trigger branching depends on the functionality of conduits that bring the signals to their target. We propose that, to form a branch, a bud must establish a functional connection with the stem through plasmodesmata, tiny signal conduits between cells, and sieve tubes, which deliver materials for growth. In addition, we introduce the unorthodox idea that gibberellin is the fourth branching hormone, and that it functions in opening the communication channels, while strigolactone inhibits communication and transport between stem and bud. We are well placed to carry out this work. We pioneered research into plasmodesmata, and identified with collaborators at Helsinki University a class of hormone-regulated genes that control plasmodesmata. Our work is internationally at the forefront, influential, and highly cited, whereas our collaborators at Wageningen University are world leaders in strigolactone research. The project will renew branching-research, and contribute to Norway's national knowledge base to meet future challenges.