Plasticity of bud outgrowth varies at cauline and rosette nodes in Arabidopsis thaliana

Abstract

Shoot branching is a complex mechanism in which secondary shoots grow from buds that are initiated from meristems established in leaf axils. The model plant Arabidopsis (Arabidopsis thaliana) has a rosette leaf growth pattern in the vegetative stage. After flowering initiation, the main stem elongates with the top leaf primordia developing into cauline leaves. Meristems in Arabidopsis initiate in the axils of rosette or cauline leaves, giving rise to rosette or cauline buds, respectively. Plasticity in the process of shoot branching is regulated by resource and nutrient availability as well as by plant hormones. However, few studies have attempted to test whether cauline and rosette branching are subject to the same plasticity. Here, we addressed this question by phenotyping cauline and rosette branching in three Arabidopsis ecotypes and several Arabidopsis mutants with varied shoot architectures. Our results showed no negative correlation between cauline and rosette branch numbers in Arabidopsis, demonstrating that there is no tradeoff between cauline and rosette bud outgrowth. Through investigation of the altered branching pattern of flowering pathway mutants and Arabidopsis ecotypes grown in various photoperiods and light regimes, we further elucidated that the number of cauline branches is closely related to flowering time. The number of rosette branches has an enormous plasticity compared with cauline branches and is influenced by genetic background, flowering time, light intensity, and temperature. Our data reveal different levels of plasticity in the regulation of branching at rosette and cauline nodes, and promote a framework for future branching analyses.

Detailed correlative analyses of branching under varied genetic and environmental contexts reveals different plasticity of branching at cauline and rosette nodes of Arabidopsis.

Introduction

Shoot architecture is a highly plastic trait of plants, providing them with enormous flexibility to adapt to their environment and be successful when growing in competition with other plants. In seed plants, the main plant body has a primary apical–basal axis that is established during early embryo development. This main axis is defined by the shoot apical meristem at the shoot apex and the root apical meristem at the root tip. Axillary meristems in the shoot incorporate pluripotent stem cells that, as the name suggests, are located in the axils of leaves. These meristems are surrounded by protective leaf primordia that collectively form an axillary bud.

The shoot of Arabidopsis (Arabidopsis thaliana), which is monopodial, consists of three different metamers described by Schmitz and Theres (1999). Type 1 metamers consist of a very short internode, a leaf, and a bud; these metamers form a rosette. Type 2 metamers consist of an elongated internode, a leaf, and a bud, this node being termed a cauline node. Type 3 metamers consist of an intermediate length internode and a floral bud without a subtending leaf that develops at the top of the main shoot and branches. Branches developing from the rosette axillary buds usually produce all three kinds of metamers, while cauline buds produce only Types 2 and 3 metamers when grown under long-day conditions and lack the rosette-like leaf growth phenotype.

In late-flowering mutants or in wild-type Arabidopsis plants grown in short days, axillary meristems develop first in the axil of older rosette leaves (RLs; Grbić and Bleecker, 2000; Long and Barton, 2000). When these plants start to flower, for example, by shifting them to long-day conditions, the vegetative shoot apical meristem transforms into an inflorescence meristem which now only initiates floral primordia (Smyth et al., 1990; Hempel and Feldman, 1994). After the transition to flowering, leaf primordia are no longer produced at the shoot apical meristem. This also coincides with a switch in axillary meristem formation, with axillary meristems now initiating basipetally in the axil of existing leaf primordia (Hempel and Feldman, 1994; Stirnberg et al., 1999; Grbić and Bleecker, 2000; Long and Barton, 2000; Stirnberg et al., 2002). In long-day grown wild-type Arabidopsis plants, there are no data on the timing of meristem initiation in RLs; however, the initiation seems to happen only after the floral transition takes place (Aguilar-Martínez et al., 2007).

As the growth of axillary buds at cauline nodes is induced in a similar basipetal sequence, it was proposed that rosette buds are activated as part of this sequence (Stirnberg et al., 1999, 2002; Walker and Bennett, 2018) although this has not been examined directly. One perspective of shoot branching is that plants have an optimal number or amount of branches with their outgrowth being regulated by correlative inhibition even if spread across different nodes, rosette, and cauline (Finlayson et al., 2010; Walker and Bennett, 2018). Accordingly, if all branches were considered similar, then Arabidopsis plants that produce fewer cauline branches would tend to allow the release and growth of more rosette branches. If this were the case, then cauline and rosette branch growth should be negatively correlated.

There is genetic variation in the balance of cauline and rosette branch numbers in Arabidopsis. Compared to wild-type plants, several mutants with increased primary rosette (R1) branches do not show differences in the number of primary cauline (C1) branches. These include branched1 (brc1) and brc2 mutants which lack functional transcription factors that belong to the TEOSINTE/CYCLOIDEA/PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR family and that are repressors of branching (Aguilar-Martínez et al., 2007) and the bushy strigolactone synthesis and signaling more axillary growth (max) 1 and max2 mutants (Stirnberg et al., 2002). Particularly in the latter, an acropetal growth pattern was observed in the rosette bud growth after bolting (Stirnberg et al., 2002), contradicting a strictly basipetal activation of branching in Arabidopsis (Hempel and Feldman, 1994; Stirnberg et al., 2002).

The shoot branching pattern of the flowering locus t (ft) mutant is a good example of the potential of plants to differ in the number and position of branches, cauline or rosette. The ft mutant flowers much later than wild-type plants and produces more cauline branches, but has almost no rosette branches (Seale et al., 2017; Fichtner et al., 2021b). So, compared to wild-type plants, the ft mutant would have fewer branches based on rosette branch number, while it would have an increased number of branches based on the sum of cauline and rosette branches (Seale et al., 2017; Fichtner et al., 2021b). The question that remains to be resolved is whether, for any given genotype, branching at cauline nodes negatively impacts branching at rosette nodes, and vice versa (Walker and Bennett, 2018).

This mechanistic and anatomical consideration of branching is important in the context of hormonal and long-distance signaling. In many plants, the shoot tip inhibits the outgrowth of axillary buds by producing a flow of auxin traveling along the main stem, thereby focusing resources on the main shoot (reviewed in Rameau et al., 2015; Barbier et al., 2017). This phenomenon, called apical dominance, can be alleviated by the removal of the shoot tip, allowing dormant buds to grow out into branches. Auxin is produced in the young leaves at the shoot tip and transported downwards in a basipetal manner (reviewed in Domagalska and Leyser, 2011; Brewer et al., 2013; Barbier et al., 2019). Auxin cannot be transported into axillary buds but regulates branching partly via modulating the levels of two other phytohormones—strigolactones and cytokinins—and partly through auxin export from the bud (reviewed in Domagalska and Leyser, 2011; Wang et al., 2018). Auxin is thought to activate the synthesis of strigolactones (Foo et al., 2005; Brewer et al., 2009) that inhibit bud outgrowth (Gomez-Roldan et al., 2008; Umehara et al., 2008), and inhibit the synthesis of cytokinins (Tanaka et al., 2006; Ferguson and Beveridge, 2009; Shimizu-Sato et al., 2009; Müller and Leyser, 2011) that activate bud outgrowth (Sachs and Thimann, 1967; Chatfield et al., 2000). Strigolactones and cytokinins both partially function via regulating the expression of BRC1 (Aguilar-Martínez et al., 2007; Martín-Trillo et al., 2011; Braun et al., 2012; Dun et al., 2012, 2013). Shade and PHYTOCHROME B deficiency both contribute to the inhibition of bud outgrowth in Arabidopsis and grasses (Finlayson et al., 2010; González-Grandío et al., 2013; González-Grandío and Cubas, 2014). Abscisic acid signaling plays an important role in inhibiting bud outgrowth in response to shade, probably acting downstream of BRC1 (González-Grandío et al., 2013, 2017; Reddy et al., 2013; González-Grandío and Cubas, 2014; Yao and Finlayson, 2015). After buds are released from dormancy, they export auxin into the main stem enhancing sustained bud outgrowth (Bennett et al., 2006; Prusinkiewicz et al., 2009; Müller and Leyser, 2011; Brewer et al., 2015; Chabikwa et al., 2019).

Shoot branching is also regulated by resource availability. In addition to affecting auxin levels, the growing shoot tip acts as a strong sink for photoassimilates, suppressing bud outgrowth through sugar deprivation (Mason et al., 2014). Increased sugar availability in buds, for example, due to shoot tip removal, not only provides a source of carbon to sustain growth, but also triggers different signals, thereby releasing buds from dormancy (Barbier et al., 2015, 2021; Fichtner et al., 2017; Patil et al. 2021). Plants have developed different signaling pathways involved in sugar sensing, thus allowing plants to adjust their metabolism, growth, and development to specific environmental conditions (Li and Sheen, 2016; Fichtner et al., 2021a). Some recent work has highlighted that sugar signaling pathways interact with auxin, strigolactone, and cytokinin pathways to promote bud outgrowth (Barbier et al., 2015; Bertheloot et al., 2020; Patil et al., 2021; Salam et al., 2021; Fichtner et al., 2021b).

In this study, we sought to test correlative inhibition between the cauline and rosette regions and did so by investigating the extent to which rosette branching in Arabidopsis is negatively related to cauline branching under varied genetic and environmental contexts. We achieved wide variation in branching and flowering using three different Arabidopsis ecotypes, 25 different Arabidopsis mutants impaired in strigolactone, auxin, or flowering pathways, and a variety of different growth conditions, and we undertook correlation analyses to determine whether cauline and rosette branch numbers were correlated with each other. We further analyzed whether cauline and rosette branch growth are correlated with leaf numbers which represent the number of sites of branch development and may also correlate with resource availability. Our study provides a basis of knowledge for the understanding of shoot architecture regulation in Arabidopsis and offers a framework for future branching analyses.

Results

Cauline branching and rosette branching show differences in plasticity in response to the environment

To determine whether the number of R1 branches depends on the number of C1 branches, we collected phenotypic data obtained in a range of wild-type and mutant Arabidopsis plants with different shoot architectures (Figure 1). These included wild-type and mutant plants grown in long photoperiods (16-h light) at normal and high planting density. For all experiments, R1 and C1 were scored separately along with RL number and cauline leaf (CL) number (Supplemental Table S1). These data were obtained from five different independent laboratories and therefore also span a range of lab-specific conditions (explained in detail in the “Materials and methods” and Supplemental Table S1). We have therefore presented a number of independent experiments, many of which utilize the same genetic material and similar but not identical growth conditions.

Figure 1.

Different variation in cauline and rosette branching occurs in Arabidopsis wild-type and branching mutant plants. A, Schematic representation of the Arabidopsis branching structure and nomenclature of branching and flowering traits. B, Arabidopsis wild-type (Col-0) and max mutant plants were grown at different planting densities in a 16-h photoperiod. C, Arabidopsis wild-type Col-0 and mutant plants were grown under different light intensities. D, Wild-type plants (Col-0 or Ws-4) and branching mutants were grown in 16-h photoperiods. Cauline (yellow, C1) and rosette (green, R1) branch numbers are plotted separately. Letters represent significant differences based on ANOVA with post hoc Fisher's Least Significant Difference testing (P < 0.05). Depicted is the mean ± sem (n = 5–28). Small letters represent significant differences for C1 or R1 branches, respectively. Capital letters represent differences in total branch number (C1 + R1).

A wide variation in shoot architecture was observed across the range of branching mutant and wild-type plants and experimental conditions (Figure 1). The relative differences in R1 number were consistently more varied than the differences in C1 as evidenced from the number of significant differences observed for R1 compared with C1 numbers. Large differences were observed in R1 when wild-type or branching mutant plants are grown in low compared to high densities (Figure 1B). However, there was almost no variation in C1 (Figure 1B). A comparable trend was observed when comparing wild-type and mutant plants that were grown under different light intensities; while there was a lot of variation in R1, C1 and CL numbers did not change (Figure 1C). Similarly, when comparing different mutant plants that show varying degrees of branching grown under normal plant densities and long photoperiods, very little variation in C1 was detected despite a very large variation in R1 (Figure 1D).

To assess the impact of the number of cauline and rosette branches on the interpretation of the overall branching phenotype in each individual experiment, we separately calculated the significant differences for C1 (small letters, top parts) and R1 (small letters, bottom parts) and for the total primary (T1) branches (upper-case letters). In most cases, R1 showed the same trend or outcome as T1; however, there were some clear exceptions. In experiment 1 (Exp 1), for example, brc1-1 plants do not have a significantly different branching phenotype compared to d14-1 and d14-1 htl-3 mutants when T1 is calculated, but do have a significantly different phenotype when branching is scored based solely on R1 (Figure 1D). This is driven by a small, not significant increase in C1 in brc1-1 compared to d14-1 mutants (Figure 1D).

The number of rosette branches does not negatively correlate with the number of cauline branches

The results presented in Figure 1 indicate that cauline and rosette branch numbers are not strongly correlated with each other. To test this, we performed a correlation analysis between the number of C1 and R1 using the data from Figure 1 and additional wild-type and mutant data that were collected across different laboratories (see Supplemental Table S1 for all data). We observed a significant but very weak positive correlation for C1 and R1 (r = 0.17, coefficient of determination [R²] = 0.03) (Figure 2A). When only the wild-types were plotted, Wassilewskija-4 (Ws-4) showed a significant positive correlation between C1 and R1 (r = 0.61, R² = 0.37), while there was no correlation in Columbia-0 (Col-0; Figure 2B). We also analyzed the correlation between C1 and R1 in Landsberg erecta (Ler) wild-type plants and, similar to Col-0, did not detect any correlation between C1 and R1 (Supplemental Figure S1A). The same was also visible when C1 and R1 were plotted for each wild-type and individual experiment sorted in ascending order for the number of C1 branches (Supplemental Figure S2); the number of R1 showed a different trend than the number of C1 further illustrating that there is no negative correlation between C1 and R1 numbers (Supplemental Figure S2). We also plotted the correlation of C1 and R1 for mutants in each ecotype background separately (Figure 2C) and observed a significant positive correlation for mutants in Col-0 (r = 0.36, R² = 0.13) but not for mutants in Ws-4. These results provide no evidence of a negative correlation between C1 and R1 and therefore do not support the hypothesis of correlative inhibition between cauline and rosette regions in intact plants.

Figure 2.

Cauline branch number does not negatively correlate with rosette branch number in Arabidopsis wild-type and mutant plants. A–C, The number of cauline branches (C1) or (D and E) the number of RLs was plotted against the number of rosette branches (R1) and the r, R², and probability (P) were calculated. A and D, The correlation for all data presented in Figure  1 and additional data as indicated in Supplemental Table S1 were used. B and E, The correlation of C1 and R1 for wild-type plants only. C and F, The correlation of C1 and R1 in mutant plants was separated by the corresponding ecotype. All plants were grown in a 16-h photoperiod. Genotypes are indicated by different colors. Each data point represents a single plant. Data points were jittered to avoid overplotting and were alpha blended meaning that regions of high point density appear as areas of high color intensity. Significant correlations (P  < 0.05) are indicated in bold.

Cauline branch number correlates with CL numbers while rosette branch number correlates positively with RL number in Arabidopsis mutants with a highly branched phenotype

As the number of nodes of a given metamer type might influence the number of branches produced of that type, we tested the correlation between the number of cauline branches and rosette branches with the number of CLs and RLs, respectively, in a range of Arabidopsis lines with different shoot architectures. Using the data from Figure 1 and additional data (see Supplemental Table S1 for all data), we observed a strong significant positive correlation (r = 0.98, R² = 0.97) between C1 and CL (Supplemental Figure S3A). This suggests that under long days, C1 strongly depends on the number of cauline nodes produced (CL). This relationship is upheld when the data were separated by the ecotype (Supplemental Figure S3B; Col-0 background: r = 0.99, R² = 0.98; Ws-4 background: r = 1, R² = 1) or when only wild-type plants were used in the correlation analyses (Supplemental Figure S3C; Col-0: r = 0.99, R² = 0.97; Ws-4: r = 0.91, R² = 0.83; Supplemental Figure S1C, Ler: r = 1, R² = 0.99). In contrast to the strong positive correlation of CL and C1, a very weak but significant positive correlation (r = 0.12, R² = 0.014) was observed between R1 and RL for the combined data set (Figure 2D). This weak significant positive correlation is maintained in Col-0 plants (Figure 2E) and branching mutants in the Col-0 ecotype background (Figure 2F) as well as in Ler wild-type plants (Supplemental Figure S1B). Interestingly, wild-type Ws-4 plants (Figure 2E; r = 0.55, R² = 0.31) and mutants in the Ws-4 background (Figure 2F; r = 0.51, R² = 0.26) showed a stronger significant positive correlation. Consequently, the variation in R1 can be only partly explained by the variation in RL, whereas the variation in C1 is completely related to CL. This shows that there is a different plasticity in cauline and rosette branching and supports the hypothesis whereby cauline and rosette branching may be regulated, at least in part, by different regulatory mechanisms or different emphases within the same regulatory mechanism.

We also tested the correlation between C1 and R1 in mutants with different architectures that were grown under at least two different laboratory conditions to ensure enough variability (Figure 3A; Supplemental Table S1). In contrast to the brc1 mutants which did not show a correlation between C1 and R1, a significant strong positive correlation between C1 and R1 was detected in the highly branched strigolactone synthesis and signaling mutants max4 (r = 0.61, R² = 0.38) and max2 (r = 0.54, R² =00.3; Figure 3A). We also plotted the correlation between RL and R1 in the same mutants (Figure 3B). While there was no correlation between RL and R1 in the two brc1 mutants, a significant strong positive correlation between RL and R1 was detected in max4 (r = 0.67, R² = 0.45) and max2 (r = 0.84, R² = 0.71), respectively (Figure 3B). These data show that in highly branched plants like max mutants, RL and R1 strongly correlate and that the number of RL influences the number of R1.

Figure 3.

Cauline branch number correlates with rosette branch number in Arabidopsis strigolactone and ft mutants. A and C, The number of cauline branches (C1) or (B and D) the number of RLs was plotted against the number of rosette branches (R1) and the r, R², and probability (P) were calculated. All mutants were grown in a 16-h photoperiod. (A) and (B) were also plotted as part of Figure  2. All data are presented in the same manner as Figure  2.

The flowering pathway is involved in branch outgrowth under long- and short-day conditions

As the growth of buds into branches in rosette plants is tied to the bolting stage associated with the flowering process, we explored the relationship between branch growth at cauline and rosette nodes of different flowering lines. The late-flowering mutant ft is reported to have a strong reduction in rosette branching (Fichtner et al., 2021b) and was compared with a range of other lines affected in flowering time. In contrast to the other mutants analyzed, ft plants showed a significant negative correlation of C1 and R1 (r = −0.57, R² = 0.33; Figure 3C). We also plotted the correlation of RL and R1 and observed a significant positive correlation in 35S:FT (r = 0.76, R² = 0.58) and a significant negative correlation in ft mutant plants (r = −0.55, R² = 0.3; Figure 3D). Summarizing, C1 and R1 did not correlate in Col-0 wild-type plants or plants with an intermediate branching phenotype (e.g. brc1 mutants), while C1 and R1 positively correlated in highly branched max mutants and negatively correlated in ft plants. Additionally, in highly branched plants, R1 seems to be highly related to RL, while R1 was less well correlated with RL in plants with an intermediate branching phenotype.

Prompted by these observations, we used flowering mutants and photoperiod to test whether cauline branching and rosette branching are impacted by flowering. Under long days, ft and soc1 (suppressor of overexpression of constans 1) mutants have an increase in C1 and a decrease in R1 when compared to Col-0 wild-type plants under the same day length (Figure 4A; three independent experiments from two different laboratories). It was previously suggested that this is a consequence of the negative correlation of C1 and R1 branch numbers (Seale et al., 2017). However, our analyses reveal no negative correlation between C1 and R1 in wild-type plants (Figure 2C). We compared long-day grown ft and soc1 mutants to Col-0 wild-type plants grown in an 8-h photoperiod that show a very similar increase in C1 compared to ft and soc1 mutants (Figure 4A; two independent experiments from two different laboratories, SD1/SD2). We observed increased R1 in late flowering Col-0 wild-type plants grown in an 8-h photoperiod when compared to those grown in a 16-h photoperiod (Figure 4A), which was contrary to our observations of late-flowering ft and soc1 mutants grown in a 16-h photoperiod that showed a decrease in R1 when compared to wild-type plants grown in 16-h as well as 8-h photoperiods. This suggests that increased C1 does not necessarily lead to decreased R1; the decreased R1 observed in ft and soc1 mutants is unlikely to be simply due to an increase in C1. We also grew 35S:FT plants that always have a very early flowering phenotype compared with wild-type plants. These plants have an increase in R1 when compared to Col-0 wild-type plants grown in the same photoperiod (Figure 4A). We performed an additional experiment with late flowering ft and soc1 mutant plants including another late-flowering mutant, fd (flowering locus d), and grew these plants in increased temperatures to induce earlier flowering to potentially further modulate branching (24/21°C compared to 22/18°C day/night; Figure 4B). As observed previously (Figure 4A), all three late-flowering mutants have an increase in C1 and a decrease in R1 when compared to Col-0 wild-type plants (Figure 4B). Interestingly, compared to Col-0 and ft plants grown in the same conditions under standard temperatures (22/18°C), both Col-0 wild-type plants and ft mutants produce more R1 but the same number of C1 when grown under increased temperatures (Figure 4B). To further explore the effect of ft-mediated flowering and branching, we grew Col-0 wild-type plants and ft mutants in short-day conditions where these genotypes produce the same amount of RLs (Col-0 65.8 ± 1.5, ft 67.2 ± 1.6, P > 0.05; 8-h photoperiod; Figure 4C). However as observed in long-day conditions, ft mutants developed more C1 and less R1 branches compared to wild-type plants. Consequently, the ft-mediated flowering pathway influences branching in Arabidopsis under long- and short-day conditions, with high levels of FT promoting rosette branching, and low levels of FT or downstream signaling components (like SOC1) inhibiting it (Figure 4, A–C).

Figure 4.

The flowering pathway affects rosette and cauline branch numbers. A, Arabidopsis Col-0 wild-type and flowering mutant plants were grown in 16- or 8-h photoperiods and the number of C1 (yellow) and R1 (green) branches were determined. B, Col-0 and flowering mutant plants were grown in a 16-h photoperiod under different temperatures and C1 as well as R1 were determined. C, Col-0 and ft mutant plants were grown in an 8-h photoperiod and C1 as well as R1 were determined. Letters represent significant differences based on ANOVA with post hoc Fisher's Least Significant Difference testing (P  < 0.05). Depicted is the mean ± sem (n = 6–19). D, Cluster analysis of branching and flowering traits in Col-0 wild-type and mutant plants in the Col-0 background based on the r. Dendrograms represent clusters using a canberra distance matrix with average-based clustering. E, PCA of branching and flowering traits in Arabidopsis Col-0 wild-type and mutant plants grown in a 16-h photoperiod. Mean values for each trait were used for the analysis. Data points represent a single experiment and were alpha blended meaning that regions of high point density appear as areas of high color intensity. The percentages of total variance represented by PC 1 and PC 2 are shown in parentheses. The loadings of individual traits are indicated (red). Different colors represent the different genotypes. bolting, days to bolting.

Cauline branch number clusters with flowering traits in Arabidopsis wild-type and mutant plants

To further investigate the relationship between cauline branching, rosette branching, and flowering, as well as to highlight potential mechanisms regulating these processes, a cluster analysis was performed (Figure 4D). We used the Pearson correlation coefficient (r) of all data available for mutants in the Col-0 background and Col-0 wild-type plants with the following variables: days to bolting (bolting), CL number, RL number, cauline branches (C1), rosette branches (R1), total branches (T1, the sum of C1 and R1), and R1 divided by RL (R1/RL) (Figure 4D, see Supplemental Table S1 for data set). Hierarchical clustering of the Pearson’s r of these variables led to the formation of two main clusters, with the first cluster comprising CL, C1, RL, days to bolting and T1, and the second cluster comprising R1 and R1/RL (Figure 4D). Additionally, principal component analysis (PCA) was performed based on the averages of all variables for mutants in the Col-0 background and Col-0 wild-type plants grown in long-day conditions (Figure 4E; see Supplemental Table S1 for all data). Here, the strigolactone mutants, the brc1 mutants, and the 35S:FT line seemed to separate from the Col-0 wild-types. Similarly, the late-flowering mutants also diverged away from Col-0 wild-type plants (Figure 4E). Again, these divergences support the notion of independent genetic regulation of the values of flowering-related traits (RL, CL, and C1) compared with the values of the rosette branching (R1) and related traits T1 and R1/RL. As in the previous cluster analysis based on the Pearson’s r (Figure 4D), C1, CL, and RL were tightly aligned in the PCA as represented by their loading (i.e. the weight they have in the analysis; red arrows on the horizontal axis, Figure 4D) driving the data along principal component (PC1) (56.1% of variation). This is suggesting these traits are highly correlated. Interestingly, the loading for T1 was in the middle of the R1 traits and the highly connected C1/CL/RL group (Figure 4D). T1 and R1 largely drove data separation along PC2, which accounted for most (36.9%) of the remaining variation (Figure 4D). In conclusion, both approaches (Figure 4) support the results of the visual inspection (Figure 1) as well as correlation analyses (Figures 2 and 3) that C1 does not negatively correlate with R1. In addition, the clustering of C1 with flowering-dependent traits like leaf number and days to bolting indicates that C1 might be connected to flowering time.

Interestingly, dividing R1 by RL further separated the data in the PCA. Consequently, R1/RL may be useful to account for variation in the branching phenotype among individuals and between genotypes with altered flowering time and/or leaf number. This may be particularly useful where variation in branching due to environmental effects on flowering is to be minimized.

There is a significant weak positive correlation of RL and R1 in long-day grown wild-type plants, a strong positive correlation of RL and R1 in strigolactone mutants, and a range of individual 35S:FT plants, and a strong negative correlation in ft mutants (Figure 3). This indicates that the number of R1 is somewhat related to RL and thus rosette node numbers in Arabidopsis. Therefore, we replotted the data for Figure 4, A and B based on R1/RL (Figure 5, A and B). This highlighted that, relative to their RL, ft and soc1 plants branch much less when compared to Col-0 plants (grown in either long or short days) independent of the growth temperature and that 35S:FT plants have a strong increase in branching at rosette nodes (Figure 5, A and B). Interestingly, 35S:FT plants seem to produce >1 R1 per RL, indicating that, similar to strigolactone mutant plants, they are likely limited in branching by the number of leaves/nodes developed. This would also explain why 35S:FT mutants seemed to cluster with strigolactone mutants in the PCA (Figure 4D). In order to compare branching in 35S:FT and max mutants, we subsequently plotted R1/RL for all available experiments with max4 and max2 plants and compared them to available experiments with 35S:FT plants. While only a minor increase in R1 was detected in 35S:FT plants compared to wild-type controls (Figure 4A), from the perspective of R1/RL, 35S:FT plants actually branch to a similar degree as the max4 and max2 mutants (Figure 5C).

Figure 5.

Exemplar where RL number can be used to normalize rosette branch number in highly branched plants. A and B, Data from Figure  4, A and B were replotted based on the number of rosette branches per RLs (R1/RL). C, The number of R1/RL in highly branched (green) and Col-0 wild-type plants (gray) are shown. Letters represent significant differences based on ANOVA with post hoc Fisher's Least Significant Difference testing (P < 0.05). Depicted is the mean ± sem (n = 6–19).

Cauline branch number consistently correlated with flowering time in different Arabidopsis ecotypes and photoperiods

To further investigate the relationship between C1, R1, and flowering time measures, we sought to increase the variability in C1, R1, CL, and RL numbers by investigating three Arabidopsis ecotypes grown in a variety of photoperiods and light intensities (Figure 6A). In Col-0, Ler, and Ws-4 wild-type plants, C1, CL, and RL consistently increased in shorter photoperiods (a single experiment is given as an example in Supplemental Figure S4, A–C; all data can be found in Supplemental Table S1). In contrast, R1 was less related to photoperiod and more related to light regime (Supplemental Figure S4, A–C). This further supports the hypothesis that cauline and rosette buds have a different response to environmental and endogenous signals and are therefore not regulated equivalently.

Figure 6.

Cauline branch number is not correlated with rosette branch number in Arabidopsis wild-type plants grown under different photoperiods. A, Arabidopsis ecotypes (Col-0 [circles]; Ler [squares]; Ws-4 [diamonds]) were grown in a variety of photoperiods. Different shades of yellow to black represent different light intensities; bright yellow for 150 µmol m⁻² s⁻¹ (NL), to decreasing light intensities of 75 µmol m⁻² s⁻¹ (LL), 40 µmol m⁻² s⁻¹ (LLL) and 5 µmol m⁻² s⁻¹ (LLLL) with all plants experiencing at least 8 h of complete darkness (shown in black). B, Correlation analyses of the number of C1 and R1 branches, respectively, and leaf numbers (CL and RL, respectively). The r, R² and probability (P) were calculated. Each data point represents a single plant. Data points were jittered to avoid overplotting and are alpha blended. Significant correlations (P < 0.05) are indicated in bold.

Correlation analysis using the combined available data from these different ecotypes (including data from Figures 1–4) confirmed our previous results: no correlation was obtained between the number of C1 and R1, but a significant positive correlation was observed for the number of C1 and CL (Figure 6B). No correlation between RL and R1 was detected (Figure 6B). However, a strong positive correlation between C1 and RL was detected in all three ecotypes (Figure 6B), as these traits largely depend on flowering time. The same relationships were detected when the data were correlated based on the mean values for each individual experiment (Supplemental Figure S4D).

When examining the correlation of C1 and CL, Ler and Ws-4 ecotypes grown under short-day conditions diverted from the linear relationship of C1 and CL (Figure 6B; Supplemental Figure S5D), implying that not all CL axillary buds elongated in these ecotypes under this photoperiod. Interestingly, under short day or short-day to long-day shift conditions, lower node cauline branches could elongate before upper node cauline branches (Supplemental Figure S5, A–B) and, in some instances, rosette branches grew out before upper node cauline branches were activated (Supplemental Figure S5, C and D).

To investigate if these effects are simply due to photoperiod effects, we also performed correlation analyses for all three ecotypes in long-day grown plants only (Supplemental Figure S6A). The results are very similar to the analyses done on the combined photoperiod data set: high positive correlations between CL and C1 as well as RL and C1; no correlation between RL and R1 (Supplemental Figure S6A). In long-day conditions, however, a significant positive correlation between C1 and R1 was obtained, although with a very low R² of 0.01 indicative of a very weak and potentially not biologically relevant correlation (Supplemental Figure S6A).

To investigate the relationships between C1, R1, days to bolting, CL, and RL in highly branched plants, a cluster analysis based on Pearson’s r was performed on the data from Figure 6 (Figure 7A). The results were remarkably reminiscent of those obtained using the set of mutants grown in long-day conditions (Figure 4D). Hierarchical clustering led to the formation of two main clusters: R1 and R1/RL formed one cluster, and days to bolting, RL, C1, CL, and T1 formed the other cluster (Figure 7A). The same clustering was obtained when only the experiments of long-day grown ecotypes were analyzed (Supplemental Figure S6B). In all analyses, T1 clustered separately from R1 and R1/RL. We also performed PC analyses using the mean of each experiment, which again gave very similar results (Figure 7B). C1, RL, and CL drove the data in the same direction along PC 1 (63.6% of the total variation). R1 on the other hand drove the data along PC 2 (29.3% of the total variation). The loading of T1 was between R1 and the highly linked group of C1, CL, and RL. In contrast, R1/RL drove the data in the other direction in an orthogonal way, further separating it (Figure 7B). These analyses in different Arabidopsis ecotypes (Figure 7B) and different mutants (Figure 4E) suggest that dependent on the biological question, using T1 (R1 + C1) as a parameter for branching may be inappropriate, especially in plants that flower at different times (Figure 8). In contrast, R1/RL facilitates some correction of the data for differences in flowering time that are tightly linked to differences in leaf number (Figure 8).

Figure 7.

Cauline branch number clusters with flowering traits. A, Cluster analysis of branching and flowering traits based on the r in different Arabidopsis ecotypes. Dendrograms represent clusters using a canberra distance matrix with average-based clustering. B, PCA of branching and flowering traits in different Arabidopsis ecotypes (Col-0 [circles]; Ler [squares]; Ws-4 [triangles]). Mean values for each trait were used for the analysis. Data points represent a single experiment and were alpha blended (regions of high point density show up as areas of high color intensity). The percentages of total variance represented by PC 1 and PC 2 are shown in parentheses. The loadings of individual traits are indicated (red). Different colors represent the different photoperiods as indicated in Figure  6A. bolting, days to bolting.

Figure 8.

Representation of the architectural plasticity of Arabidopsis shoot branching and scenarios showing implications for analysis of rosette branching in Arabidopsis. A, Environmental factors influence flowering time which in turn directly influences CL and RL numbers and therefore influences the respective node numbers. There is low plasticity in C1 branch outgrowth as the number of CL directly influences the amount of cauline branches (C1) (correlation ≈ 1). There is high plasticity in R1 branch outgrowth as the number of RL only partially influences the number of rosette branches (R1) in wild-type plants. Both C1 and R1 numbers determine the final shoot architecture. The dashed line represents partial dependency. B, Scenario 1 compares Arabidopsis plants that are grown in the same photoperiod and that have the same flowering time and the same RL but are not extremely bushy. In this scenario, R1 can be counted as a representation of the rosette branching phenotype. Scenario 2 is the same as Scenario 1, but includes plants that are close to their maximum branching capacity (i.e. close to 1 R1 branch per RL or node). In this scenario, R1, as well as R1/RL, should be analyzed due to the impact of any variation in leaf number on R1. For Scenario 3, where plants differ in flowering time and therefore RL, again both R1, as well as R1/RL, should be analyzed, and attention given to the impact of flowering on RL and cauline branch number. Images are intended as schematic representations.

Discussion

Branching at cauline and rosette nodes are independent variables subject to different developmental plasticity

In this study, we showed that R1 and C1 are not negatively correlated in Arabidopsis wild-type and mutant plants providing little evidence for correlative inhibition between the cauline and rosette regions in intact plants. As there is no negative tradeoff between these variables, branching at rosette and cauline nodes highlights potential differences in, for example, gene regulatory, hormonal, and/or environmental variables during ontogenetic development in Arabidopsis. As such, C1 and R1 should be treated separately. We showed that C1 is highly correlated with the number of cauline nodes (CL) produced across our wide experimental range (Figure 6; Supplemental Figures S1, S3, S4, and S6). Our study highlights that, contrary to cauline branching, the variation in the number of rosette nodes only partly affects the number of rosette branches in wild-type plants (Figure 8A). So, while there is only limited plasticity of branch outgrowth at cauline nodes, there is an enormous plasticity at rosette nodes, suggesting that there must be certain differences in their outgrowth regulation. However, in highly branched mutants (max4 and max2), the number of rosette branches is highly correlated with the number of RLs. A significant correlation was also observed in plants overexpressing FT and which are very early flowering. Using clustering analyses, we demonstrated that C1 clusters with traits related to flowering time (RL and bolting date). This explains the positive correlation of R1 and C1 as well as of RL and R1 in mutants that branch at their maximum capacity (R1/RL is close to 1 in max4, max2, and 35S:FT, Figure 5) where the limiting factor of branching is the number of nodes produced (reflected by the number of RLs). When highly branched plants flower late, they develop more RL and CL, leading to the formation of more R1 and C1. As these plants develop branches at almost every node, this would in turn cause the positive correlation between C1 and R1 in these genotypes. Thus, when comparing the branching phenotype of plants affected in flowering and/or in the number of nodes produced, dividing R1 by RL partially accounts for differences in RL and therefore highlights significant differences in phenotype (Figure 8B). Moreover, as C1 and R1 are shown here to be not negatively correlated and probably not part of the same/dependent activation sequence, the total number of primary branches obfuscates the branching phenotype. This is also highlighted by how T1 influenced the PCs (Figures 4E and 7B) and highlights a value in analyzing R1 and C1 separately (Figure 8B; Aguilar-Martínez et al., 2007; González-Grandío et al., 2013, 2017; Chevalier et al., 2014; Brewer et al., 2016; Barbier et al., 2021; Fichtner et al., 2021b). This would be particularly important when plants flower at different time points or are grown under different photoperiods.

We also detected a weak but significant positive correlation of RL and R1 in Col-0, Ws-4, and Ler wild-type plants (Figure 2F; Supplemental Figure S1B). This suggests that, in wild-type Arabidopsis, part of the differences in R1 depends on RL number. The correlation between leaf number and branching in long-day conditions might be a consequence of increased sugar supply as more leaves would usually produce more total photoassimilates. Evidence that carbon/sugar availability influences R1 development in Arabidopsis has been obtained with plants grown in low light conditions or exposed to a night extension. Barbier et al. (2021) quantified the very early rosette bud growth that occurs after the floral transition but before bolting in long days, and observed less growth in plants with less photosynthetically active light. Recent advances in shoot branching research have illustrated that the release of bud dormancy and outgrowth into a new branch are dependent on sugar availability and involve sugar signaling pathways, notably mediated by trehalose 6-phosphate (Tre6P) and HEXOKINASE1, which interact with the hormones controlling branching (Mason et al., 2014; Barbier et al., 2015; Fichtner et al., 2017; Tarancón et al., 2017; Bertheloot et al., 2020; Barbier et al., 2021; Patil et al., 2021; Salam et al., 2021; Fichtner et al., 2021b).

The FT-mediated flowering pathway is involved in rosette branch regulation in Arabidopsis

In Arabidopsis, FT is synthesized in phloem companion cells in leaves under inductive long-day conditions and moves in the phloem sieve elements to the shoot apical meristem, where it interacts with the FD protein to promote floral transition (Turck et al., 2008). There is a growing body of evidence suggesting that FT is an important regulator of branching, based on studies in rice (Oryza sativa; Tsuji et al., 2015), tomato (Solanum lycopersicum; Weng et al., 2016), tobacco (Nicotiana tabacum; Li et al., 2015), and pea (Pisum sativum; Beveridge and Murfet, 1996; Hecht et al., 2011). Flowering in Arabidopsis is dependent on Tre6P synthesis (Schluepmann et al., 2003; Wahl et al., 2013; Fichtner et al., 2020), a sucrose-specific signaling metabolite in plants (Fichtner and Lunn, 2021). FT transcription is also a target of Tre6P signaling (Fichtner et al., 2021b). Plants with higher Tre6P in the vasculature have an early flowering and an increased branching phenotype, and this coincides with upregulation of FT (Fichtner et al., 2021b). Stimulation of branching by increased Tre6P in the vasculature was abolished in an ft mutant background (Fichtner et al., 2021b), further implicating FT in the regulation of branching in Arabidopsis.

Here, we showed that wild-type Col-0 plants that have an increase in C1 similar to the level observed in ft plants, still initiate R1 and do not have a decreased R1, unlike ft plants. However, in contrast to wild-type plants, C1 and R1 were negatively correlated in ft mutants as were RL and R1 (Figure 3, C and D). This affirms two of the observations we made previously, that C1 branch number is tightly related to flowering and that the flowering pathway is also involved in R1 branch number regulation. Triggering earlier flowering via, for example, an increase in temperature also increased the number of R1 in wild-type and ft mutant plants (Figure 4B). However, the number of R1 in ft mutants was always lower than the respective number in wild-type plants grown under the same temperatures, demonstrating that the FT-mediated flowering pathway is involved in regulating rosette branching in Arabidopsis. We further showed that the FT-mediated flowering pathway also seems to be important for rosette branch outgrowth regulation in short-day conditions as ft mutants also produce less R1 branches compared to Col-0 wild-type plants in short days (Figure 4C). It was demonstrated previously that there is detectable FT expression in short days especially under elevated ambient temperatures, although much lower when compared to long-day conditions (Yamaguchi et al., 2005; Balasubramanian et al., 2006; Lee et al., 2007; Kim et al., 2012). This builds on our speculation that FT has a role for bud outgrowth in short- and long-day conditions.

It has been demonstrated that FT can move not only to the shoot apical meristem but also to axillary meristems, and promote their elongation and development (Niwa et al., 2013; Tsuji et al., 2015; Dixon et al., 2018). FT in Arabidopsis, wheat (Triticum aestivum), and hybrid aspen (Populus tremula × tremuloides) has been shown to interact directly with BRC1, and this interaction leads to a reciprocal repressive effect between the two proteins (Niwa et al., 2013; Dixon et al., 2018; Maurya et al., 2020). 35S:FT plants developed more than one R1 per RL (Figure 4C). This is very similar to the phenotype of brc1 mutants that overexpress a Tre6P synthase in the vasculature resulting in an increase in Tre6P (Fichtner et al., 2021b). We speculated that this phenotype in brc1 plants with high levels of Tre6P might be a consequence of higher levels of FT and the loss of BRC1 having a strong additive effect on branching (Fichtner et al., 2021b). This would also be a plausible explanation for the branching phenotype of the 35S:FT plants that have potentially a very strong and relatively constitutive overexpression of FT, so potentially a complete inhibition of BRC1 activity, resulting in bud release.

Shoot branching regulation in Arabidopsis rosette and cauline nodes is influenced differently by photoperiod and light intensity

By growing different Arabidopsis ecotypes in a wide variety of different light regimes and photoperiods, we demonstrated that C1 is influenced by flowering time, while R1 seemed to be more related to the light regime and intensity (Figures 6, 7, and 8, A). While we detected significant positive correlations between RL and R1 in all three ecotypes when the relationship was analyzed in long-day conditions and separated by ecotype, there was no correlation when data from different photoperiods were combined (Figure 6B) or when data from long days and all three ecotypes were merged (Supplemental Figure S6A). This provides evidence of genetic regulation of the relation of RL to R1. Interestingly, the correlation between RL and R1 seems to be stronger in ecotypes that develop less RLs as the correlation in Ws-4 is much stronger compared to Col-0 (Figure 2F). This is in line with sugars having an important role in rosette branching. It would be interesting to analyze the relationship of RL and R1 in additional Arabidopsis ecotypes and genotypes to test further how leaf number affects branching in Arabidopsis. Future research should also address the role of the FT-mediated and other flowering pathways on branching and how sugar and Tre6P signaling might interact with the flowering pathway during this process.

The correlation analyses of the different ecotypes grown in different photoperiods confirmed the strong correlation (close to R² = 1) of C1 and CL (Figures 6 and 7; Supplemental Figures S4 and S6). This highlights that there is almost no plasticity in cauline branching per se, with every CL giving rise to one cauline branch (Figure 8A). This is in stark contrast to rosette branching which correlated only weakly with RL number when long-day grown ecotypes were analyzed separately (Figure 2D; Supplemental Figure S1) showing that branching at rosette nodes is not simply a consequence of leaf number and is potentially regulated by the integration of many other endogenous and exogenous signals (Figure 8A).

In contrast to rosette buds, cauline buds might receive different signals because of their location on the main stem. Due to this position, they are continuously exposed to red light potentially resulting in very low levels of BRC1 and abscisic acid (González-Grandío et al., 2013; Reddy et al., 2013; Yao and Finlayson, 2015; González-Grandío et al., 2017; Holalu and Finlayson, 2017). This might be the cause of the strong activation of cauline branches and might be a potential reason why cauline buds behave differently from rosette buds in terms of activation and outgrowth. Future studies should aim at addressing these differences in cauline and rosette bud outgrowth in detail and would also benefit from determining the extent to which axillary buds may form different numbers of leaves prior to rapid elongation into a mature branch (Ferguson and Beveridge, 2009; Barbier et al., 2019).

Impacts for shoot branching across cauline and rosette nodes

We show that C1 and R1 are rarely negatively correlated in Arabidopsis and therefore highlight that the underlying biological control of C1 and R1 differs somewhat in network components and/or their regulation (Figure 8B). Cauline branching is highly correlated to the number of cauline nodes produced, which in turn is related, to a large extent, to flowering time. We highlight here that the mechanism controlling rosette branching involves not only hormonal and nutrient (including sugar) signaling pathways, but also involves flowering regulation, light signaling, and potentially further unknown signaling pathways. In highly branched strigolactone mutants, RL and R1 are highly correlated variables. RL, as well as R1/RL, are helpful to distinguish small genetic and environmental effects on shoot branching as well as independent effects on branching in plants that flower differently (Figure 8B).

Materials and methods

Plant material and growth

Branching and flowering data from different laboratories working on branching in Arabidopsis (Arabidopsis thaliana) were collected and used in this study. A. thaliana Col-0, Ler or Ws-4 ecotypes and mutants in these backgrounds were used. Some parts of the data were published previously, including Figure 1 Exp 3 (Aguilar-Martínez et al., 2007), Figure 1 Exp 5 (Brewer et al., 2016), and Figures 4, A/5, A Exp 1 and Exp 2 (Fichtner et al., 2021b). Arabidopsis plants were all grown in a 16-h photoperiod unless otherwise stated according to the following light and temperature conditions: All plants from condition A were grown on UQ23 potting mix (70% composted pine bark 0–5 mm, 30% coco-peat) supplemented with dolomite and osmocote, using light intensities of 120–150 µmol m⁻² s⁻¹ (unless otherwise stated) and a temperature of 22°C day/18°C night (except for experiment in higher temperature in Figure 4B). All plants from condition B were grown in a 1:1 mixture of soil (Stender, Papenburg, Germany) and vermiculite using light intensities of 150 µmol m⁻² s⁻¹ and a temperature of 22°C day/18°C night. All plants from condition C were grown in Seed & Cutting Premium Germinating mix (Debco, Vaughan, Canada) at 23°C constant temperature and 75 µmol m⁻² s⁻¹ (C.1) or 120 µmol m⁻² s⁻¹ (C.2) light intensity. All plants from condition D were grown as described in Aguilar-Martínez et al. (2007) using light intensities of 120 µmol m⁻² s⁻¹ and a temperature of 20°C. All plants from condition E were grown at different densities (1, 3, or 10 plants per 33 cm² cell) on a mixture of three parts seed and modular compost plus sand (Scott Levington) to one part vermiculite for horticultural use (Sinclair), at a light intensity of 240 µmol m⁻² s⁻¹ and a temperature of 23°C. In the case of Supplemental Figure S4A, the different Arabidopsis ecotypes were all grown in the same cabinets using the same soil type (condition A) but under a large variety of different photoperiods and light regimes. Different photoperiods and light regimes included: black, 16-h photoperiod with 150 µmol m⁻² s⁻¹ light intensity; gray, 16-h photoperiod with 4 weeks of 150 µmol m⁻² s⁻¹ and a subsequent shift to 40 µmol m⁻² s⁻¹ light intensity; blue, 16-h photoperiod with 75 µmol m⁻² s⁻¹ light intensity; green, 4 weeks in an 8-h photoperiod then shift to a 16-h photoperiod (150 µmol m⁻² s⁻¹ light intensity each); yellow, 8-h photoperiod with 150 µmol m⁻² s⁻¹ light intensity; red, 8 h of 150 µmol m⁻² s⁻¹ light intensity followed by 8 h of 5 µmol m⁻² s⁻¹ light intensity.

Arabidopsis mutant lines

All Arabidopsis mutant lines used in this study were described earlier: brc1 mutants (Aguilar-Martínez et al., 2007); lbo-10 (lbo-1 mutation backcrossed 6 times to Col-0, thus termed here lbo-10) and lbo-1, max4-9, and lbo-1 max4-9 (Ws-4 background; Rasmussen et al., 2012; Brewer et al., 2016); d27-1 (Waters et al., 2012); max1-1 and max2-1 (Stirnberg et al., 2002); max3-9 (Booker et al., 2004); max4-1 (Sorefan et al., 2003); d14-1 (Chevalier et al., 2014); htl-3 (a kai2 allele isolated in Col-0; Toh et al., 2014); smxl678 (smxl6-4,7-3,8-1) with the max2-1 mutation crossed out (Soundappan et al., 2015); 35S:YUCCA1 (also referred to as yuc1D; Zhao et al., 2001); ft-10 and 35S:FT (Yoo et al., 2005); soc1-6 (Wang et al., 2009); fd-3 (Searle et al., 2006).

Phenotyping

RLs and CLs were counted separately to give RL and CL numbers. R1 and C1 branches (shoots ≥ 0.5 cm) were counted and R1 and C1 were added to give the T1 branch number. RL and R1 were used to determine R1 branch number per leaf (R1/RL). In the growth conditions A, B, D, and E, branch numbers were assessed when plants started to senesce (i.e. the rosette turned brown and started to die, >4 weeks after bolting). In condition C branching was assessed when the first siliques turned brown and full branching was achieved.

Statistical analysis and data visualization

Data analyses and plotting were performed using R Studio version 1.4.1717 (www.rstudio.com) with R version 4.1.0 (https://cran.r-project.org/) and the packages ggplot2, stats, and agricolae using Pearson’s correlation or an analysis of variance (ANOVA)-based post hoc comparison of means test (Fisher’s Least Significant Difference test). Heatmap analyses were performed with the heatmap.2 function (R package heatmaply) using the agglomeration method “average” for the hierarchical cluster analysis of genotypes, correlation-based clustering of traits, and the distance measure “canberra” for the computation of the distance matrix. PCAs were done using the R package factoextra. Figures were compiled using Adobe Illustrator 2021.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: BRC1, AT3G18550; LBO, AT3G21420; D27, AT3G21420; MAX1, AT2G26170; MAX2, AT2G26170; MAX3, AT2G44990; MAX4, Locus: AT4G32810; D14, AT4G32810; HTL/KAI2, AT4G37470; SMXL6, AT1G07200; SMXL7, AT2G29970; SMXL8, AT2G40130; YUCCA1, AT4G32540; FT, AT1G65480; SOC1, AT2G45660; FD, AT2G45660.

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Correlation analysis of Ler wild-type plants.

Supplemental Figure S2. The variation in cauline and rosette branching is different in three Arabidopsis ecotypes.

Supplemental Figure S3. Correlation analysis of the number of CLs and C1 in Arabidopsis wild-type and mutant plants.

Supplemental Figure S4. Different Arabidopsis ecotypes (Col-0 [circles]; Ler [squares]; Ws-4 [diamonds]) were grown in a variety of photoperiods as indicated in Figure 6A.

Supplemental Figure S5. Branch skipping phenomenon in different Arabidopsis ecotypes observed in short day or short-day to long-day shift conditions.

Supplemental Figure S6. Different Arabidopsis ecotypes were grown in long-day photoperiods.

Supplemental Table S1. Raw data presented in this study.

 

F.F. performed data analyses, collated the data, and prepared the figures. C.D. performed experiments and helped with data collection. F.F., F.F.B., S.C.K., and P.B.B. designed the experiments, collected the phenotypic data, and helped with data interpretation. All authors contributed to the experiment design, conceived the project, and interpreted the data. F.F. wrote the article, with contributions from all the authors; C.A.B. supervised and completed the writing. All authors have read and approved the final version of the manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is Franziska Fichtner (f.fichtner@uq.edu.au).