CRISPR/Cas9-mediated knockout of Populus BRANCHED1 and BRANCHED2 orthologs reveals a major function in bud outgrowth control

Abstract

The TCP-type transcription factors BRANCHED1 and BRANCHED2 shape plant architecture by suppressing bud outgrowth, with BRANCHED2 only playing a minor role in Arabidopsis. Here, we investigated the function of orthologs of these genes in the model tree Populus. We used CRISPR/Cas9 to generate loss-of-function mutants of previously identified Populus BRANCHED1-1 and BRANCHED2-1 candidate genes. BRANCHED1-1 mutants exhibited strongly enhanced bud outgrowth. BRANCHED2-1 mutants had an extreme bud outgrowth phenotype and possessed two ectopic leaves at each node. While BRANCHED1 function is conserved in poplar, BRANCHED2, in contrast to its Arabidopsis counterpart, plays an even more critical role in bud outgrowth regulation. In addition, we identified a new, not yet reported association of this gene to leaf development.

Genome Editing in Trees: From Multiple Repair Pathways to Long-Term Stability

“…Secondary cell wall synthesis was also compromised by CRISPR-KO of a brassinosteroid biosynthetic gene, supporting a role for brassinosteroids in wood formation ( Shen et al, 2018 ). CRISPR-KO of BRANCHED1-1 ( BRC1-1 ) and BRC1-2 belonging to the TCP family of transcription factors resulted in altered shoot architecture, and revealed an additional role of BRC2 in leaf development not previously reported for its Arabidopsis ortholog ( Muhr et al, 2018 ). A recent study reported successful mutation of essential flowering genes in both male and female poplar genotypes ( Elorriaga et al, 2018 ).…”

“… Wang et al (2017) reported faithful maintenance of PtoMYB115 mutations in tissue culture-propagated Populus tomentosa somaclones, though in one case low frequencies of new mutations not seen in the parent line were detected, indicative of chimeras. Similarly, the CRISPR editing outcomes of BRC1-1 and BRC2-1 were also stable over multiple cycles of vegetative propagation in tissue culture ( Muhr et al, 2018 ). We have maintained a subset of the previously-characterized 717 mutants ( Zhou et al, 2015 ) in the greenhouse for over 4 years by repeatedly cutting back the original transformants and/or propagation using rooted cuttings.…”

Genome-Wide Identification of the TCP Gene Family in Broussonetia papyrifera and Functional Analysis of BpTCP8, 14 and 19 in Shoot Branching

“…It was shown that the BRC1 gene was the focal points for multiple environmental and developmental signals that acted in the axillary buds to inhibit shoot branching. The homologs of AtBRC1 in tomato, pea, chrysanthemum, poplar and cucumber could inhibit lateral bud or branch outgrowth [ 59 , 60 , 61 , 62 , 63 ]. In chrysanthemum, DgBRC1s were mainly expressed in the nodes containing axillary buds and expression of DgBRC1s in Arabidopsis brc1 or wild-type could reduce the number of rosette branches [ 61 ].…”

“…The mutant of PcBRC2 showed an extreme bud outgrowth and had two ectopic leaves at each node, which was different with Arabidopsis . PcBRC2 may have retained or evolved the function of controlling leaf development [ 62 ]. In our work, we validated the function of BpTCP8 , BpTCP14 and BpTCP19 on shoot branching in both Arabidopsis wild-type and brc1 mutant backgrounds.…”

A genetic framework for regulation and seasonal adaptation of shoot architecture in hybrid aspen

“…Whereas branching is well studied in annual models like Arabidopsis and pea, much less is known about control of branching in perennial trees. Changes in gene expression in axillary buds have been described, and the role of a few components including strigolactones, BRC1 orthologs, and flowering-time-related transcription factor RAV1 has been validated in branching in trees (17)(18)(19). However, information on branching control in perennials is fragmented, and there is a significant gap in our knowledge of how branching is controlled and integrated with seasonal growth cycles in perennial trees (20).…”