I was there first: competitive binding sets vascular meristem size

We turn it into building materials, furniture, fuel, and much more: wood. Where does it come from? From the third set of stem cells in the plant body, buried inside the stem. A pool of procambium and cambium cells divide into the precursors to the vasculature: the phloem and xylem, the conductive tissues that transport water, nutrients, and signaling molecules from their site of uptake or biosynthesis to their site of action. These cells also self-renew, although how they achieve balance between division and differentiation remains unclear, in large part due to their location within the stem.

Several clever techniques have been deployed to access the changes in the transcriptome of vascular cells. In this issue of the Plant Cell, Furuya et al. (2021) exploit VISUAL to dissect the role of the transcription factor BEH3 (BRI1-EMS-SUPPRESSOR 1 [BES1]/BRASSINAZOLE-RESISTANT 1 [BZR1] HOMOLOG 3) in secondary vascular development in Arabidopsis (Arabidopsis thaliana).

Only a few cells within the root will be at a given developmental stage at a given time, thus diluting their signal when grinding the entire tissue. One option to increase signal strength is fluorescence-activated cell sorting (FACS) by tagging cells of interest with a fluorescent reporter under the control of a cell-type-specific promoter (Brady et al., 2007). But wouldn’t it be great to have an inducible system that both synchronizes division and differentiation and greatly raises the signal-to-noise ratio?

Enters Vascular cell Induction culture System Using Arabidopsis Leaves (VISUAL; Kondo et al., 2015, 2016). It turns out that vascular development can be induced from Arabidopsis cotyledons by a cocktail of auxin and cytokinin, together with the glycogen synthase kinase (GSK) inhibitor bikinin. Over the span of a few days, mesophyll cells transform into procambium cells before differentiating into xylem or phloem cells in a satisfying temporal sequence easily dissected by transcriptome profiling. Not to be left behind, cell-type-specific FACS datasets were instrumental in validating and annotating the transcriptional sequence of events revealed by VISUAL.

In this study, the authors combined several transcriptome datasets to generate gene regulatory networks that coalesced into four main modules, one of which was cambium-related based on the expression profile of cambium marker genes. The authors focused here on BEH3, a member of a family of six transcription factors involved in brassinosteroid signaling. Multigene families immediately invoke the specter of redundancy, but it is complicated. A change in cambium size indicates a shift in the balance between self-renewal and differentiation. Indeed, loss of BEH3 function resulted in smaller cambium areas on average, with fewer cambium cell layers, but also a wider distribution of cambium sizes, suggesting that BEH3 is important to maintain the pool of cambium stem cells. Of the other BEH3 family members, BES1 is critical for xylem or phloem differentiation from cambial cells, as loss of BES1 function blocks the ectopic development of vascular cells in VISUAL. Surprisingly, the beh3-1 loss-of-function allele responded normally under the same conditions, but is there functional redundancy? Well, loss of BEH3 function in the bes1-1 or the bes1-1 bzr1-2 mutant backgrounds rescued vascular development (see Figure). So clearly some family members are not equal.

Figure Loss of BEH3 function raises the output of other members of the BES/BZR family. Vascular development requires BES1 and BZR1, but the loss of BEH3 can partially compensate for their loss. Scale bars = 100 μm. (Adapted from Furuya et al., [2021], Supplemental Figure S11).

All BES/BZR family members tested showed binding to the same cis-regulatory elements in known target promoters, suggesting that they share the same downstream targets. But what happens once they bind to their target promoters was unexpected: BEH3 did not induce or repress transcription directly, but prevented other BES/BZR members from binding. This observation also helped explain the suppression of the bes1-1 bzr1-2 mutant phenotype by the loss of BEH3: removing BEH3 increased the transcriptional output of the remaining three members, a conclusion that was also validated by mathematical modeling.

Does BEH3 always antagonize the other BES/BZR members? Well, no: a quintuple mutant (with only BEH3) had a smaller vascular area than the hexuple mutant, indicating that BEH3 does repress cambium size when the other members are missing. Redundancy is therefore not trivial in the BES/BZR family. The same principles might apply to other transcription factor families in plants, adding more layers of complexity to the study of gene families.