BRANCHED1 Research Articles

Connective auxin transport contributes to strigolactone-mediated shoot branching control independent of the transcription factor BRC1.

The shoot systems of plants are built by the action of the primary shoot apical meristem, established during embryogenesis. In the axil of each leaf produced by the primary meristem, secondary axillary shoot apical meristems are established. The dynamic regulation of the activity of these axillary meristems gives shoot systems their extraordinary plasticity of form. The ability of plants to activate or repress these axillary meristems appropriately requires communication between meristems that is environmentally sensitive. The transport network of the plant hormone auxin has long been implicated as a central player in this tuneable communication system, with other systemically mobile hormones, such as strigolactone and cytokinin, acting in part by modulating auxin transport. Until recently, the polar auxin transport stream, which provides a high conductance auxin transport route down stems dominated by the auxin export protein PIN-FORMED1 (PIN1), has been the focus for understanding long range auxin transport in the shoot. However, recently additional auxin exporters with important roles in the shoot have been identified, including PIN3, PIN4 and PIN7. These proteins contribute to a wider less polar stem auxin transport regime, which we have termed connective auxin transport (CAT), because of its role in communication across the shoot system. Here we present a genetic analysis of the role of CAT in shoot branching. We demonstrate that in Arabidopsis, CAT plays an important role in strigolactone-mediated shoot branching control, with the triple pin3pin4pin7 mutant able to suppress partially the highly branched phenotype of strigolactone deficient mutants. In contrast, the branchy phenotype of mutants lacking the axillary meristem-expressed transcription factor, BRANCHED1 (BRC1) is unaffected by pin3pin4pin7. We further demonstrate that mutation in the ABCB19 auxin export protein, which like PIN3 PIN4 and PIN7 is widely expressed in stems, has very different effects, implicating ABCB19 in auxin loading at axillary bud apices.

Sugar Transporter Protein 1 (STP1) contributes to regulation of the genes involved in shoot branching via carbon partitioning in Arabidopsis.

We previously demonstrated that alterations in sugar partitioning affect the expression of genes involved in hormone biosynthesis and responses, including BRANCHED1 (BRC1), resulting in enhanced shoot branching in transgenic Arabidopsis plants overexpressing cyanobacterial fructose-1,6-bisphosphatase-II in the cytosol (AcF). The exogenous treatment of wild-type Arabidopsis plants with sugars showed the same transcript characteristics, indicating that sugars act as a signal for branching. We also found that the reductions induced in BRC1 expression levels in wild-type plants by the sugar treatments were suppressed in the knockout mutant of sugar transporter 1 (stp1-1). Intracellular sugar contents were similar in stp1-1 and wild-type plants following the sugar treatments, suggesting that STP1 acts as a factor for the regulation of shoot branching depending on extracellular sugar contents. Abbreviations: BRC1: BRABCHED1; FBP/SBPase: fructose-1,6-/sedoheptulose-1,7-bisphosphatase; Glc: glucose; HXK: hexokinase; SnRK1.1/AKIN10: SNF1-RELATED PROTEIN KINASE 1.1; Suc: sucrose; SnRK1: sucrose non-fermenting 1-related protein kinase; STP: sugar transporter protein.

Transcriptome Profiles Reveal the Crucial Roles of Auxin and Cytokinin in the "Shoot Branching" of Cremastra appendiculata.

Cremastra appendiculata has become endangered due to reproductive difficulties. Specifically, vegetative reproduction is almost its only way to reproduce, and, under natural conditions, it cannot grow branches, resulting in an extremely low reproductive coefficient (reproductive percentage). Here, we performed RNA-Seq and a differentially expressed gene (DEG) analysis of the three stages of lateral bud development in C. appendiculata after decapitation—dormancy (D2), transition (TD2), and emergence (TG2)—and the annual axillary bud natural break (G1) to gain insight into the molecular regulatory network of shoot branching in this plant. Additionally, we applied the auxin transport inhibitors N-1-naphthylphthalamic acid (NPA) and 2,3,5-triiodibenzoic acid (TIBA) to a treated pseudobulb string of C. appendiculata to verify the conclusions obtained by the transcriptome. RNA-Seq provided a wealth of valuable information. Successive pairwise comparative transcriptome analyses revealed 5988 genes as DEGs. GO (Gene Ontology) and KEGG (Kyoto encyclopedia of genes and genomes) analyses of DEGs showed significant enrichments in phytohormone biosynthesis and metabolism, regulation of hormone levels, and a hormone-mediated signaling pathway. qRT-PCR validation showed a highly significant correlation (p < 0.01) with the RNA-Seq generated data. High-performance liquid chromatography (HPLC) and qRT-PCR results showed that, after decapitation, the NPA- and TIBA-induced lateral buds germinated due to rapidly decreasing auxin levels, caused by upregulation of the dioxygenase for auxin oxidation gene (DAO). Decreased auxin levels promoted the expression of isopentenyl transferase (IPT) and cytochrome P450 monooxygenase, family 735, subfamily A (CYP735A) genes and inhibited two carotenoid cleavage dioxygenases (CCD7 and CCD8). Zeatin levels significantly increased after the treatments. The increased cytokinin levels promoted the expression of WUSCHEL (WUS) and inhibited expression of BRANCHED1 (BRC1) in the cytokinin signal transduction pathway and initiated lateral bud outgrowth. Our data suggest that our theories concerning the regulation of shoot branching and apical dominance is really similar to those observed in annual plants. Auxin inhibits bud outgrowth and tends to inhibit cytokinin levels. The pseudobulb in the plant behaves in a similar manner to that of a shoot above the ground.

The TIE1 transcriptional repressor controls shoot branching by directly repressing BRANCHED1 in Arabidopsis

Shoot branching is a major determinant of plant architecture and is regulated by both endogenous and environmental factors. BRANCHED1 (BRC1) is a central local regulator that integrates signals controlling shoot branching. So far, the regulation of BRC1 activity at the protein level is still largely unknown. In this study, we demonstrated that TIE1 (TCP interactor containing EAR motif protein 1), a repressor previously identified as an important factor in the control of leaf development, also regulates shoot branching by repressing BRC1 activity. TIE1 is predominantly expressed in young axillary buds. The gain-of-function mutant tie1-D produced more branches and the overexpression of TIE1 recapitulated the increased branching of tie1-D, while disruption of TIE1 resulted in lower bud activity and fewer branches. We also demonstrated that the TIE1 protein interacts with BRC1 in vitro and in vivo. Expression of BRC1 fused with the C-terminus of the TIE1 protein in wild type caused excessive branching similar to that observed in tie1-D and brc1 loss-of-function mutants. Transcriptome analyses revealed that TIE1 regulated about 30% of the BRC1-dependent genes, including the BRC1 direct targets HB21, HB40 and HB53. These results indicate that TIE1 acts as a positive regulator of shoot branching by directly repressing BRC1 activity. Thus, our results reveal that TIE1 is an important shoot branching regulator, and provide new insights in the post-transcriptional regulation of the TCP transcription factor BRC1.

Largely additive effects of gibberellin and strigolactone on gene expression in Arabidopsis thaliana seedlings.

The phytohormones gibberellin (GA) and strigolactone (SL) are involved in essential processes in plant development. Both GA and SL signal transduction mechanisms employ α/β-hydrolase-derived receptors that confer E3 ubiquitin ligase-mediated protein degradation processes. This suggests a common evolutionary origin of these pathways and possibly a molecular interaction between them. One such indication stems from rice, where the DELLA protein of the GA pathway was reported to interact with the SL receptor. Here, we examine the physiological interaction between both pathways through the analysis of GA (ga1) and SL biosynthesis (max1 and max3) mutants. In ga1 max double mutants, we find indications only for additive interactions when examining several phenotypic readouts. We further identify short-term transcriptional responses to GA and the synthetic SL rac-GR24 through next-generation sequencing of poly-adenylated RNAs (RNA-seq) in ga1 max1. Remarkably, both hormones lead to predominantly additive transcriptional changes of a largely overlapping set of genes. The expression of only a few genes was altered in a synergistic manner but, interestingly, these include the genes encoding the GA catabolic enzyme GA2 OXIDASE2 (GA2ox2) as well as the SL pathway regulators BRANCHED1 (BRC1) and SUPPRESSOR OF max2 1-LIKE8 (SMXL8). We conclude that GA and rac-GR24 signaling in Arabidopsis seedlings converge at the level of transcription of a common gene-set.

BRC1 expression regulates bud activation potential but is not necessary or sufficient for bud growth inhibition in Arabidopsis.

The degree of shoot branching in Arabidopsis is determined by the activation of axillary buds. Bud activity is regulated by diverse environmental and developmental signals, often mediated via plant hormones, including auxin, strigolactone and cytokinin. The transcription factor BRANCHED1 (BRC1) has been proposed to integrate these regulatory signals. This idea is based on increased branching in brc1 mutants, the effects of bud-regulating hormones on BRC1 expression, and a general correlation between BRC1 expression and bud growth inhibition. These data demonstrate the important role of BRC1 in shoot branching, but here we show that in Arabidopsis this correlation can be broken. Buds lacking BRC1 expression can remain inhibited and sensitive to inhibition by strigolactone. Furthermore, buds with high BRC1 transcript levels can be active. Based on these data, we propose that BRC1 regulates bud activation potential in concert with an auxin transport-based mechanism underpinning bud activity. In the context of strigolactone-mediated bud regulation, our data suggest a coherent feed-forward loop in which strigolactone treatment reduces the probability of bud activation by parallel effects on BRC1 transcription and the shoot auxin transport network.

Abscisic acid signaling is controlled by a BRANCHED1/HD-ZIP I cascade in Arabidopsis axillary buds

Shoot-branching patterns determine key aspects of plant life and are important targets for crop breeding. However, we are still largely ignorant of the genetic networks controlling locally the most important decision during branch development: whether the axillary bud, or branch primordium, grows out to give a lateral shoot or remains dormant. Here we show that, inside the buds, the TEOSINTE BRANCHED1, CYCLOIDEA, PCF (TCP) transcription factor BRANCHED1 (BRC1) binds to and positively regulates the transcription of three related Homeodomain leucine zipper protein (HD-ZIP)-encoding genes: HOMEOBOX PROTEIN 21 (HB21), HOMEOBOX PROTEIN 40 (HB40), and HOMEOBOX PROTEIN 53 (HB53). These three genes, together with BRC1, enhance 9-CIS-EPOXICAROTENOID DIOXIGENASE 3 (NCED3) expression, lead to abscisic acid accumulation, and trigger hormone response, thus causing suppression of bud development. This TCP/HD-ZIP genetic module seems to be conserved in dicot and monocotyledonous species to prevent branching under light-limiting conditions.

Long and short photoperiod buds in hybrid aspen share structural development and expression patterns of marker genes.

Tree architecture develops over time through the collective activity of apical and axillary meristems. Although the capacity of both meristems to form buds is crucial for perennial life, a comparative analysis is lacking. As shown here for hybrid aspen, axillary meristems engage in an elaborate process of axillary bud (AXB) formation, while apical dominance prevents outgrowth of branches. Development ceased when AXBs had formed an embryonic shoot (ES) with a predictable number of embryonic leaves at the bud maturation point (BMP). Under short days, terminal buds (TBs) formed an ES similar to that of AXBs, and both the TB and young AXBs above the BMP established dormancy. Quantitative PCR and in situ hybridizations showed that this shared ability and structural similarity was reflected at the molecular level. TBs and AXBs similarly regulated expression of meristem-specific and bud/branching-related genes, including CENTRORADIALIS-LIKE1 (CENL1), BRANCHED1 (BRC1), BRC2, and the strigolactone biosynthesis gene MORE AXILLARY BRANCHES1 (MAX1). Below the BMP, AXBs maintained high CENL1 expression at the rib meristem, suggesting that it serves to maintain poise for growth. In support of this, decapitation initiated outgrowth of CENL1-expressing AXBs, but not of dormant AXBs that had switched CENL1 off. This singles out CENL1 as a rib meristem marker for para-dormancy. BRC1 and MAX1 genes, which may counterbalance CENL1, were down-regulated in decapitation-activated AXBs. The results showed that removal of apical dominance shifted AXB gene expression toward that of apices, while developing TBs adopted the expression pattern of para-dormant AXBs. Bud development thus follows a shared developmental pattern at terminal and axillary positions, despite being triggered by short days and apical dominance, respectively.

Plant Architecture: The Long and the Short of Branching in Potato

A new report details the interaction between two isoforms of an important BRANCHED1 (BRC1) transcription factor gene in potato. The regular long form inhibits lateral branching, like BRC1 in other species, but a modified protein that originates from alternative BRC1 splicing inhibits the long form and promotes lateral branching.

Abscisic Acid Is a General Negative Regulator of Arabidopsis Axillary Bud Growth.

Branching is an important process controlled by intrinsic programs and by environmental signals transduced by a variety of plant hormones. Abscisic acid (ABA) was previously shown to mediate Arabidopsis (Arabidopsis thaliana) branching responses to the ratio of red light (R) to far-red light (FR; an indicator of competition) by suppressing bud outgrowth from lower rosette positions under low R:FR. However, the role of ABA in regulating branching more generally was not investigated. This study shows that ABA restricts lower bud outgrowth and promotes correlative inhibition under both high and low R:FR. ABA was elevated in buds exhibiting delayed outgrowth resulting from bud position and low R:FR and decreased in elongating buds. ABA was reduced in lower buds of hyperbranching mutants deficient in auxin signaling (AUXIN RESISTANT1), MORE AXILLARY BRANCHING (MAX) signaling (MAX2), and BRANCHED1 (BRC1) function, and partial suppression of branch elongation in these mutants by exogenous ABA suggested that ABA may act downstream of these components. Bud BRC1 expression was not altered by exogenous ABA, consistent with a downstream function for ABA. However, the expression of genes encoding the indole-3-acetic acid (IAA) biosynthesis enzyme TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1, the auxin transporter PIN-FORMED1, and the cell cycle genes CYCLIN A2;1 and PROLIFERATING CELL NUCLEAR ANTIGEN1 in buds was suppressed by ABA, suggesting that it may inhibit bud growth in part by suppressing elements of the cell cycle machinery and bud-autonomous IAA biosynthesis and transport. ABA was found to suppress bud IAA accumulation, thus confirming this aspect of its action.

Strigolactones as an auxiliary hormonal defence mechanism against leafy gall syndrome in Arabidopsis thaliana.

Leafy gall syndrome is the consequence of modified plant development in response to a mixture of cytokinins secreted by the biotrophic actinomycete Rhodococcus fascians. The similarity of the induced symptoms with the phenotype of plant mutants defective in strigolactone biosynthesis and signalling prompted an evaluation of the involvement of strigolactones in this pathology. All tested strigolactone-related Arabidopsis thaliana mutants were hypersensitive to R. fascians. Moreover, treatment with the synthetic strigolactone mixture GR24 and with the carotenoid cleavage dioxygenase inhibitor D2 illustrated that strigolactones acted as antagonistic compounds that restricted the morphogenic activity of R. fascians. Transcript profiling of the MORE AXILLARY GROWTH1 (MAX1), MAX2, MAX3, MAX4, and BRANCHED1 (BRC1) genes in the wild-type Columbia-0 accession and in different mutant backgrounds revealed that upregulation of strigolactone biosynthesis genes was triggered indirectly by the bacterial cytokinins via host-derived auxin and led to the activation of BRC1 expression, inhibiting the outgrowth of the newly developing shoots, a typical hallmark of leafy gall syndrome. Taken together, these data support the emerging insight that balances are critical for optimal leafy gall development: the long-lasting biotrophic interaction is possible only because the host activates a set of countermeasures-including the strigolactone response-in reaction to bacterial cytokinins to constrain the activity of R. fascians.

BRANCHED1 Interacts with FLOWERING LOCUS T to Repress the Floral Transition of the Axillary Meristems inArabidopsis

Plant architecture shows a large degree of developmental plasticity. Some of the key determinants are the timing of the floral transition induced by a systemic flowering signal (florigen) and the branching pattern regulated by key factors such as BRANCHED1 (BRC1). Here, we report that BRC1 interacts with the florigen proteins FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) but not with TERMINAL FLOWER1, a floral repressor. FT protein induced in leaves moves into the subtended bud, suggesting that FT protein also plays a role in promotion of the floral transition in the axillary meristem (AM). The brc1-2 mutant shows an earlier floral transition in the axillary shoots compared with the wild type, suggesting that BRC1 plays a role in delaying the floral transition of the AMs. Genetic and gene expression analyses suggest that BRC1 interferes with florigen (FT and TSF) function in the AMs. Consistent with this, BRC1 ectopically expressed in the shoot apical meristem delays the floral transition in the main shoot. These results taken together suggest that BRC1 protein interacts with FT and TSF proteins and modulates florigen activity in the axillary buds to prevent premature floral transition of the AMs.

BRANCHED1 promotes axillary bud dormancy in response to shade in Arabidopsis.

Plants interpret a decrease in the red to far-red light ratio (R:FR) as a sign of impending shading by neighboring vegetation. This triggers a set of developmental responses known as shade avoidance syndrome. One of these responses is reduced branching through suppression of axillary bud outgrowth. The Arabidopsis thaliana gene BRANCHED1 (BRC1), expressed in axillary buds, is required for branch suppression in response to shade. Unlike wild-type plants, brc1 mutants develop several branches after a shade treatment. BRC1 transcription is positively regulated 4 h after exposure to low R:FR. Consistently, BRC1 is negatively regulated by phytochrome B. Transcriptional profiling of wild-type and brc1 buds of plants treated with simulated shade has revealed groups of genes whose mRNA levels are dependent on BRC1, among them a set of upregulated abscisic acid response genes and two networks of cell cycle- and ribosome-related downregulated genes. The downregulated genes have promoters enriched in TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) binding sites, suggesting that they could be transcriptionally regulated by TCP factors. Some of these genes respond to BRC1 in seedlings and buds, supporting their close relationship with BRC1 activity. This response may allow the rapid adaptation of plants to fluctuations in the ratio of R:FR light.

Role of tomato BRANCHED1‐like genes in the control of shoot branching

In angiosperms, shoot branching greatly determines overall plant architecture and affects fundamental aspects of plant life. Branching patterns are determined by genetic pathways conserved widely across angiosperms. In Arabidopsis thaliana (Brassicaceae, Rosidae) BRANCHED1 (BRC1) plays a central role in this process, acting locally to arrest axillary bud growth. In tomato (Solanum lycopersicum, Solanaceae, Asteridae) we have identified two BRC1-like paralogues, SlBRC1a and SlBRC1b. These genes are expressed in arrested axillary buds and both are down-regulated upon bud activation, although SlBRC1a is transcribed at much lower levels than SlBRC1b. Alternative splicing of SlBRC1a renders two transcripts that encode two BRC1-like proteins with different C-t domains due to a 3'-terminal frameshift. The phenotype of loss-of-function lines suggests that SlBRC1b has retained the ancestral role of BRC1 in shoot branch suppression. We have isolated the BRC1a and BRC1b genes of other Solanum species and have studied their evolution rates across the lineages. These studies indicate that, after duplication of an ancestral BRC1-like gene, BRC1b genes continued to evolve under a strong purifying selection that was consistent with the conserved function of SlBRC1b in shoot branching control. In contrast, the coding sequences of Solanum BRC1a genes have evolved at a higher evolution rate. Branch-site tests indicate that this difference does not reflect relaxation but rather positive selective pressure for adaptation.

Phytochrome Regulation of Branching in Arabidopsis

The red light:far-red light ratio perceived by phytochromes controls plastic traits of plant architecture, including branching. Despite the significance of branching for plant fitness and productivity, there is little quantitative and mechanistic information concerning phytochrome control of branching responses in Arabidopsis (Arabidopsis thaliana). Here, we show that in Arabidopsis, the negative effects of the phytochrome B mutation and of low red light:far-red light ratio on branching were largely due to reduced bud outgrowth capacity and an increased degree of correlative inhibition acting on the buds rather than due to a reduced number of leaves and buds available for branching. Phytochrome effects on the degree of correlative inhibition required functional BRANCHED1 (BRC1), BRC2, AXR1, MORE AXILLARY GROWTH2 (MAX2), and MAX4. The analysis of gene expression in selected buds indicated that BRC1 and BRC2 are part of different gene networks. The BRC1 network is linked to the growth capacity of specific buds, while the BRC2 network is associated with coordination of growth among branches. We conclude that the branching integrators BRC1 and BRC2 are necessary for responses to phytochrome, but they contribute differentially to these responses, likely acting through divergent pathways.

Role of TCP Gene BRANCHED1 in the Control of Shoot Branching in Arabidopsis

Branching patterns are major determinants of plant architecture. They depend both on leaf phillotaxy (branch primordia are formed in the axils of leaves) and on the decision of buds to grow out to give a branch or to remain dormant. In Arabidopsis, several genes involved in the long-distance signalling of the control of branch outgrowth have been identified. However, the genes acting inside the buds to cause growth arrest remained unknown until now. In the February issue of Plant Cell we have described the function of BRANCHED1 (BRC1), an Arabidopsis gene coding for a plant-specific transcription factor of the TCP family that is expressed in the buds and prevents their development. Loss of BRC1 function leads to accelerated AM initiation, precocious progression of bud development and excess of shoot branching. BRC1 transcription is affected by endogenous and environmental signals controlling branching and we have shown that BRC1 function mediates the response to these stimuli. Therefore we have proposed that BRC1 function represents the point at which signals controlling branching are integrated within axillary buds.

Arabidopsis BRANCHED1Acts as an Integrator of Branching Signals within Axillary Buds

Shoot branching patterns depend on a key developmental decision: whether axillary buds grow out to give a branch or whether they remain dormant in the axils of leaves. This decision is controlled by endogenous and environmental stimuli mediated by hormonal signals. Although genes involved in the long-distance signaling of this process have been identified, the genes responding inside the buds to cause growth arrest remained unknown in Arabidopsis thaliana. Here, we describe an Arabidopsis gene encoding a TCP transcription factor closely related to teosinte branched1 (tb1) from maize (Zea mays), BRANCHED1 (BRC1), which represents a key point at which signals controlling branching are integrated within axillary buds. BRC1 is expressed in developing buds, where it arrests bud development. BRC1 downregulation leads to branch outgrowth. BRC1 responds to developmental and environmental stimuli controlling branching and mediates the response to these stimuli. Mutant and expression analyses suggest that BRC1 is downstream of the MORE AXILLARY GROWTH pathway and that it is required for auxin-induced apical dominance. Therefore, BRC1 acts inside the buds as an integrator of signals controlling bud outgrowth and translates them into a response of cell growth arrest. The conservation of BRC1/tb1 function among distantly related angiosperm species suggests that a single ancestral mechanism of branching control integration evolved before the radiation of flowering plants.