Prolonged cold exposure to Arabidopsis juvenile seedlings extends vegetative growth and increases the number of shoot branches

ABSTRACT

Environmental factors such as photoperiod, temperature, phytohormones, sugars, and soil nutrients can affect the development of axillary meristems and emergence of shoot branches in plants. We investigated how an extended period of cold exposure to Arabidopsis plants before and after inflorescence meristem differentiation would affect plant growth and shoot branching. The number of rosette leaves and shoot branches increased when wild type (WT) juvenile seedlings, but not adult plants, were subjected to a prolonged cold exposure (10/7°C day/night cycle). As the duration of cold exposure to WT juvenile seedlings increased, so too did the rosette area, number of leaves, and rosette branches revealing an extended period of vegetative growth. The prolonged cold treatment also increased the primary inflorescence stem height and number of cauline branches in WT plants revealing a delay in reproductive development that could be altered by early (set domain group 8; sdg8) and late (methyltransferase 1; met1) flowering mutants. The axillary buds/leaf and rosette branches/leaf ratios declined significantly in WT, yet were enhanced in the loss-of-function of carotenoid cleavage dioxygenase 7 (ccd7) and teosinte branched 1 (brc1) hyper-branched mutants. This indicated that axillary meristem differentiation continued during the cold exposure, which did not directly impact axillary bud formation or shoot branching. We conclude that a prolonged cold exposure to juvenile seedlings prior to inflorescence meristem development extended vegetative growth and delayed the reproductive phase to allow additional leaf primordia and axillary meristems to differentiate that enhanced the number of shoot branches in Arabidopsis.

Introduction

Shoot branching is a highly plastic developmental trait of plants that determines plant architecture, foliar coverage, photosynthetic area, and fruit and seed production. An intricate interaction between the environment (e.g. light, temperature, and nutrients), phytohormones, and sugars control the development of axillary meristems, shoot bud formation, and branching architecture during plant development.^1,2 The shoot tip of Arabidopsis juvenile seedlings develops leaf primordia in the periphery of the shoot apical meristem (SAM). Development of an axillary meristem within the axil of leaf initiates the formation of an axillary bud.^3,4 The differentiation of the inflorescence meristem (IM) during the transition from vegetative to reproductive development ceases the formation of axillary meristems.⁵ Axillary buds do not develop during vegetative development in Arabidopsis, except when plants are grown under a shorter photoperiod. Under a longer photoperiod, axillary buds are formed after flowering.^3,6,7

Environmental conditions show pronounced impacts on the shoot branching habit of plants. In Arabidopsis, a long photoperiod promotes early flowering and a rapid life cycle in comparison to plants grown under a short photoperiod.⁸ The vegetative phase of a long-day grown plant ends 10–12 days after germination after the SAM transitions into an IM.⁴ A shorter photoperiod delays IM development and extends the vegetative phase leading to an increase in the number of rosette leaves, branches, and plant biomass.^6,8-10 The bolting transition and cauline leaf numbers can be an indicator of a reproductive phase change in that an extended cauline zone correlates with an increased number of days to flowering.^11,12 A change in temperature can alter the vegetative to reproductive transition. A warmer temperature can induce early flowering and shorten the life span in Arabidopsis.^13,14 The winter-annual accessions of Arabidopsis that contain FRIGIDA require juvenile seedlings (or seeds) to be exposed to a prolonged period of cold exposure (30–40 days), a process called vernalization that promotes early flowering.¹⁵ The vernalization-induced induction of flowering in winter-annual Arabidopsis is under epigenetic control.¹⁶ Whether a duration of cold exposure (e.g. one or more weeks) to juvenile and adult plants can affect their shoot branching habit in Arabidopsis remained unclear.

The differential regulation of the biosynthesis of carotenoid-derived apocarotenoids affects shoot branching in Arabidopsis. The β-carotene derived apocarotenoid strigolactone (SL) is a phytohormone that inhibits lateral shoot bud outgrowth to control shoot branching in plants.^17,18 The CAROTENOID ISOMERASE (CRTISO) catalyses the isomerization of 7,9,9′,7′-tetra-cis-lycopene to all-trans-lycopene, that undergoes cyclization to generate β-carotene.¹⁹ CAROTENOID CLEAVAGE DIOXYGENASE (CCD) enzymes (CCD7 and CCD8) cleave β-carotene in the early steps of SL biosynthesis.²⁰ The loss-of-function in CCD7 (ccd7 or more axillary branching 3; max3) or CCD8 (ccd8 or max4) blocks SL biosynthesis leading to an increase in the number of rosette shoot branches.^17,21 crtiso mutants also show an increased number of shoot branches in rice and Arabidopsis, presumably due to a perturbation in SL biosynthesis.^22,23

The TEOSINTE BRANCHED 1 (BRC1) encodes a transcription factor proposed to regulate shoot architecture by integrating phytohormones, developmental and environmental signals that suppress axillary bud outgrowth.^24-27 BRC1 suppresses auxin efflux and auxin accumulation in axillary buds; inhibiting lateral bud outgrowth such as in cucumber (Cucumis sativus L.).²⁸ The interaction of BRC1 with FLOWERING LOCUS T (FT) delays the floral transition in axillary meristems and extends the vegetative phase of development.²⁹ BRC1 acts as a signaling hub downstream of the strigolactone signaling pathway to arrest bud outgrowth.²⁴

Cold exposure can induce selective modifications in histone methylation patterns; subsequently, altering the gene expression landscapes and phenotypes in different plants.^30-32 A prolonged cold treatment (e.g. vernalization) of juvenile Arabidopsis seedlings induces epigenetic silencing of the transcriptional repressor of flowering time, FLOWERING LOCUS C (FLC), and promotes early flowering under warmer growth conditions.^16,33 The loss-of-function in SET DOMAIN GROUP8 (SDG8) mutant (sdg8 or early flowering in short days; efs, or chloroplast and carotenoid regulation 1; ccr1) has a shorter juvenile phase and flowers early independent of vernalization by repressing FLC expression.^23,34 SDG8 promotes histone-3-lysine-4 (H3K4) and lysine-36 (H3K36) trimethylation surrounding the ATG start codon of FLC and has other pleiotropic effects on plant growth and metabolism.³⁵ For example, SDG8 promotes CRTISO gene expression and regulates carotenoid composition in a tissue-specific manner.³⁶ sdg8/ccr1 mutant alleles show an enhanced number of rosette and cauline branches despite having a shorter juvenile phase that causes early flowering in Arabidopsis Col-0.^23,37

Epigenetic modifications such as DNA methylation at CG, CHG, and CHH (H is A, C, or T) sequences, can alter the plant’s response to a prolonged cold exposure.³² In trees such as hybrid poplar (Populus tremula × alba), chilling-dependent induction of DEMETER-LIKE 10 (PtaDML10) activated progressive demethylation of genomic DNA and shoot bud outgrowth, revealing that DNA methylation is required to mediate axillary bud differentiation in response to winter chilling.³⁸ In Arabidopsis, DNA METHYLTRANSFERASE 1 (MET1) functions to establish and maintain CG context-dependent DNA methylation.³⁹ MET1 represses shoot regeneration by inhibiting the homeobox transcription factor WUSCHEL (WUS).⁴⁰ WUS is expressed in the organizing center of the SAM, axillary meristems, floral meristems, and adventitious shoot meristems where it maintains an undifferentiated stem cell niche.^41-43 MET1-dependent methylation of the WUS promoter represses WUS expression, whereas demethylation activates WUS expression and induces axillary bud differentiation.⁴⁰ The stronger met1-1 mutant allele shows a delay in Arabidopsis (Col-0) flowering time and juvenile-to-adult phase transition that is accompanied by the production of additional rosette leaves and aerial cauline leaves prior to elongation of the primary inflorescence stem.^44,45

Here we reveal how shoot branching can be enhanced by exposing juvenile (vegetative), but not adult (reproductive) Arabidopsis plants to a prolonged duration of cold. Arabidopsis mutants that display hyper-branching (crtiso; ccd7 and brc1), early flowering (sdg8), and extended juvenility (met1) were used to explore the impacts of prolonged cold exposure on branching phenotypes. All genotypes showed an enhanced number of rosette leaves and rosette branches. Our findings reveal that a prolonged cold exposure to juvenile seedlings extended vegetative growth and delayed the reproductive phase thereby enhancing shoot branching in Arabidopsis.

Materials and methods

Germplasm and plant growth conditions

The Arabidopsis thaliana (ecotype Col-0) and mutants crtiso (ccr2.1),⁴⁶ sdg8 (ccr1.1),²³ ccd7 (max3-11),²³ brc1-1 (SALK_091920 C), and met1-7 (SALK_076522) were used this study. Mutants were confirmed by carotenoid profiling, phenotyping, and/or genotyping. Arabidopsis plants were grown in a Climatron Star700 growth cabinets (Thermoline Scientific, Australia). Seeds were sown onto soil (Debco Seed Raising and Superior germinating soil mixture supplemented with 3% Osmocote) and stratified for 3 days at 4°C in the dark. Seeds were germinated and plants grown under normal growth conditions (16/8 h light/dark photoperiod; 130–150 µmolm⁻² sec⁻¹ cool fluorescent lighting; 22/18°C day/night temperatures) unless stated otherwise.

Cold treatments and plant phenotyping

Arabidopsis plants; 7, 14, and 21 days after stratification (DAS), were subject to a cold treatment (10/7°C day/night cycle, 16/8 h light/dark photoperiod; 130–150 µmolm⁻²sec⁻¹ cool fluorescent lighting) for a period of between 4 weeks depending upon the experiment (Figure 1a). After the period of cold treatment, plants were grown under normal growth conditions until reproductive maturity. Juvenile Arabidopsis seedlings (1-week old; 7DAS) were exposed to the cold for 1–5 weeks, after which they were transferred back to normal growth conditions (22/18°C day/night cycle) and grown to reproductive maturity (Figure 1b).

Figure 1.

Schematic representation of cold treatments and phenotype characterization of plants.

a) Time schedule used to identify plant developmental stages (from juvenile to adult) at which a four-week prolonged cold exposure can affect shoot branching. Plants were transferred after 1, 2 and 3 weeks of normal growth (22/18°C day/night cycle) and exposed 4 weeks of cold (10/7°C day/night cycle). b) Duration scheme to determine the effect of increasing cold exposure on shoot branching. Juvenile seedlings (7 days after stratification; DAS) growing under normal conditions (22/18°C day/night cycle) were exposed to 1, 2, 3, 4, or 5 weeks of cold (10/7°C day/night cycle). The solid green lines represent normal growth period and dashed blue lines represent cold treatment period in weekly intervals.

The rosette area, number of rosette leaves, and rosette biomass were measured when the primary inflorescence stem emerged (<5 mm). Mutants included in this study had different flowering times and therefore inflorescence stem height (to the first silique), the number of shoot branches, and shoot biomass for each genotype was determined when 3–5 basal siliques of the primary floral stem started yellowing and/or had opened to disperse seeds. The rosette area was measured by analyzing rosette pictures using ImageJ, an open-source Java-based image processing computer program.⁴⁷ The dry weight of rosettes was determined by drying for 48 h at 40°C. Each experiment was repeated at least twice to minimize the experimental variations assuring reproducibility of the data.

Results

Prolonged cold treatment to juvenile seedlings increases the number of rosette branches

Arabidopsis wild type (WT) and crtiso mutant plants were grown at normal temperatures (22/18°C day/night cycle) and exposed to 4 weeks of prolonged cold (10/7°C day/night cycle) at different developmental stages (7, 14, and 21 DAS); after which plants were returned to normal growth temperatures to quantify shoot branching (Figure 1a). Exposure of both WT and crtiso mutant juvenile seedlings (7 DAS) to 4 weeks of cold significantly increased the number of rosette and cauline branches, while branching was not significantly altered in plants transferred to the cold on 14 or 21 DAS (Figure 2a-b). The number of rosette branches were two-fold higher in crtiso compared to WT (Figure 2a), keeping consistent with a previous report showing that crtiso mutants can develop additional rosette branches under certain environmental conditions.²³ Therefore, a prolonged period of cold exposure to juvenile seedlings prior to IM development (before 10–12 DAS) can cause a greater number of rosette shoot branches to develop in Arabidopsis.

Figure 2.

Prolonged cold exposure to juvenile seedlings enhances plant growth traits and shoot branching.

a) Rosette (R) and b) Cauline (C) branching traits in juvenile (7 DAS) and adult (14 DAS and 21 DAS) wild type and crtiso plants treated to 4 weeks of cold exposure (10/7°C day/night cycle) and allowed to grow until reproductive maturity. c) Rosette (R) and Cauline (C) branching traits, d) Number of rosette leaves, rosette area (cm²), primary inflorescence stem (St) height (cm), and e) Rosette branches per rosette leaf in mature wild type plants that were exposed to cold (10/7°C day/night cycle) for 1, 2, 3, 4 and 5 weeks at 7 DAS. f) Shoot biomass (dry weight g/plant), seed yield/plant and harvest index in mature wild type plants that were exposed to cold (10/7°C day/night cycle) for 5 weeks at 7 DAS. Plant growth periods for control; stratification 3 days, final harvest after 49 day’s normal growth at 22°C (7 weeks total). Plant growth periods for prolonged cold; stratification for 3 days, pre-cold growth at 22°C for 7 days, cold treatment for 35 days, final harvest after 35 day’s post-cold growth at 22°C (11 weeks total). Final harvest was done on 35^th day after cold treatment for prolonged cold and 49 DAS for control. Shoot Biomass is shoot dry weight (g) at the final harvest. Harvest Index is calculated as shoot dry weight/seed yield. g and h) Wild type rosettes from control (G) and prolonged cold (5 weeks) exposed (h) plants at the stage of primary inflorescence stem emergence. I to L) Shoot branching architecture of control (i and k) and prolonged cold exposed (j and l) wild type plants at reproductive stage showing rosette and cauline branch habits. M to N) Wild type rosette leaves (juvenile to adult phase) from control (M) and prolonged cold (5 weeks) exposed wild type plants at the time of primary inflorescence stem emergence. The white bar in figures G-N scales a measure of 5 cm. Mean data values show standard error of the means (A, B; n = 10), (C to F; n = 20) from a single dataset representative of two independent experiments. Letter codes in plots indicate the level of statistical variation (p < .05) in growth attributes across the treatment groups determined by a Two-Way ANOVA (a and b) and One-Way ANOVA (c-f) using Holm-Sidak post hoc multiple comparison test.

Next, we examined what effect extending the duration of cold exposure might have on various plant growth traits. WT juvenile seedlings were grown under normal temperature conditions (22/18°C day/night cycle) for 7 DAS and transferred to the cold (10/7°C day/night cycle) for 0, 1, 2, 3, 4, and 5 weeks; after which they were returned to normal temperatures (22/18°C day/night cycle) (Figure 1b). The number of rosette leaves, rosette area, rosette branches, cauline branches and primary inflorescence stem height increased with a longer duration of cold exposure (Figure 2c-d). The shoot biomass and seed yield were also significantly enhanced when juvenile seedlings were exposed to 5-weeks of cold treatment (Figure 2f).

The increased number of cauline branches and primary inflorescence stem (primary bolt) height indicated a possible delay in flowering time (Figure 2c-d,i-l). Plants exposed to 5-weeks of cold exposure showed a fivefold larger rosette area (Figure 2g-h) and 2.3- to 2-fold increase in the number of rosette leaves (Figure 2d,m-n) and rosette shoot branches (Figure 2c,i-l), respectively. Intriguingly, the number of rosette branches to leaf ratio significantly declined in cold exposed plants, revealing either a reduction in axillary meristem formation or increase leaf primordia development (Figure 2e). Taken together, a prolonged period of cold exposure to juvenile seedlings for 5 weeks can significantly enhance vegetative and reproductive growth traits, leading to a branchy phenotype in Arabidopsis.

Prolonged cold enhances rosette shoot branching in hyper-branched mutants

We next tested if the prolonged cold exposure affected axillary meristem formation using mutants impaired in strigolactone biosynthesis (ccd7/max3) and axillary bud development (brc1). Under normal growth conditions (control), the mutants showed a similar number of rosette leaves and cauline branches, and only a slight shorter primary inflorescence stem in comparison to WT (Figure 3a,d-e). ccd7 and brc1 produced few rosette buds (<5 mm) and showed a substantially higher number of rosette branches (>5 mm) relative to WT (Figure 3b-c). In response to the prolonged cold exposure, there was a significant increase in the number of rosette leaves, rosette buds, rosette branches, cauline branches, and primary inflorescence stem height (vegetative part below the first silique) in all germplasm (Figure 3a-e). The increase in shoot branching was highly pronounced in ccd7 and brc1 hyper-branched mutants as expected (Figure 3c). Therefore, the prolonged cold treatment of juvenile seedlings most likely extended vegetative development providing time to further increase the development of additional rosette branches in ccd7 and brc1 hyper-branched mutants.

Figure 3.

Prolonged cold exposure of juvenile plants enhanced growth and branching traits in mutants displaying a hyper-branched phenotype and stem cell maintenance of the shoot apical meristem. Juvenile wild type (WT) and mutants (carotenoid cleavage dioxygenase 7; ccd7, branched 1; brc1, set domain group 8; sdg8, and DNA methyl transferase 1; met1 were grown for 7 DAS under normal conditions and then transferred to the cold (10/7°C day/night cycle) for 5-weeks, after which they were returned to normal growth conditions until reproductive maturity. Growth traits measured include; a) number of rosette leaves, b) number of emerged rosette shoot buds (<5 mm), c) number of rosette branches (>5 mm), d) number of cauline branches, e) primary inflorescence stem height to the first silique, F) number of shoot buds per rosette leaf, g) number of branches per rosette leaf, H) number of cauline branches per cm stem height. Mean values display standard error of the means (n = 15). Denoted letters indicate statistical variation (p < .05) in growth attributes within treatment groups and across genotypes as determined by a Two-Way ANOVA using a Holm-Sidak post hoc multiple comparison.

We next examined if prolonged cold affected axillary meristem formation. The significant increase in the ratio of cauline branches relative to the height of the primary inflorescence stem is consistent with the increased number of rosette leaves reflecting delayed flowering (Figure 3h). The ratio of the number of axillary buds relative to the rosette leaves remained unchanged in WT, yet increased in ccd7, and was substantially higher in brc1 following cold treatment, revealing axillary meristem formation continued under prolonged cold treatment (Figure 3f). The ratio of the number of rosette branches to leaves was decreased in WT, remained unchanged in brc1 and was considerably higher in ccd7 (Figure 3g). The rosette branch to leaf ratio of approximately one in ccd7 reflected an equal number of leaves and rosette branches developed during the cold treatment. Therefore, the prolonged cold does not impact axillary meristem formation, since the hyper-branched mutants ccd7 and brc1 were able to develop additional axillary buds and rosette branches.

Prolonged cold enhances the number of shoot branches by extending vegetative growth

sdg8 and met1 mutants flower early and late, respectively.^23,44 Under normal growth conditions, the number of rosette leaves as well as the primary inflorescence stem height of sdg8 and met1 mutant plants significantly decreased and increased respectively, compared to WT (Figure 3a,e). The substantially higher inflorescence stem height allowed additional cauline branches to develop in met1 (Figure 3d). These phenotypes displayed by sdg8 and met1 were consistent with mutants that flower early and late, respectively. Exposure of sdg8 and met1 juvenile seedlings to prolonged cold significantly increased the number of rosette leaves, albeit to a slightly lesser extent compared to WT (Figure 3a). Therefore, SDG8 and MET1 activities were not required for the formation of additional leaf primordia during the extended vegetative phase. In contrast, the primary inflorescence stem height and cauline branch number in sdg8 and met1 were not enhanced by the cold treatment (Figure 3d-e), revealing that SDG8 and MET1 activities can affect the spatial organization of the cauline leaf zone between the rosette and the inflorescence.

sdg8 and met1 mutants developed additional and fewer rosette branches, respectively, to WT under normal growth conditions (Figure 3c). Compared to WT, sdg8 mutants displayed a similar axillary bud/leaf ratio and higher rosette branch/leaf ratio, exemplifying that sdg8 develops a hyper-branched phenotype under normal growth conditions. The substantially higher number of axillary buds and relatively low rosette branch/leaf ratio in met1 revealed that MET1 may function in controlling dormancy or outgrowth of the axillary bud into a rosette branch (Figure 3b). Exposure of sdg8 and met1 juvenile seedlings to prolonged cold did not change the number of axillary buds, and like WT, there was a significant decrease in the ratio of axillary buds relative to the number of rosette leaves (Figure 3b,f). Prolonged cold exposure of sdg8 and met1 juvenile seedlings significantly increased the number of rosette branches, and yet did not reduce the rosette branch/leaf ratio like that observed in WT (Figure 3c,g). These findings revealed that the cold-induced increase in the number of rosette branches could be a consequence of additional leaf primordia developing under the extended period of vegetative development.

Discussion

Prolonged cold treatment of juvenile seedlings extends vegetative development

Shoot branching contributes to foliage cover, leaf photosynthetic area, shoot biomass, and fruit and seed production in plants.⁴⁸ A shorter photoperiod during the winter season can enhance the number of rosette leaves and shoot branches in plants.¹⁰ How a prolonged period of cold exposure to plants growing under a longer photoperiod impacts shoot branching in Arabidopsis remained unclear. We demonstrate that exposing juvenile WT and crtiso mutant seedlings (7 DAS) to 4 weeks of prolonged cold enhanced the number of rosette and cauline branches. The significantly higher number of rosette branches displayed by crtiso mutants subjected to cold exposure is consistent with a previous report describing that crtiso mutant alleles can generate additional branches depending upon the growth environment.²³ The adult WT and crtiso mutant plants (2-weeks or older in age) were unresponsive to the cold treatment and displayed a similar branching habit to the untreated control plants grown under normal growth temperatures (Figure 2) revealing an age-dependent effect of prolonged cold exposure on shoot branching. A longer duration of cold exposure to WT correlated with an increased number of rosette leaves, rosette area, and enhanced branching habit, that after 5 weeks collectively enhanced plant biomass, seed yield, and the harvest index (Figure 2). These phenotypes were consistent to when Arabidopsis plants are grown under a shorter photoperiod, revealing that the prolonged cold exposure may have extended vegetative growth (Figure 4).

Figure 4.

Model showing how exposure of juvenile seedlings to prolonged cold enhances shoot branching in Arabidopsis. DNA METHYLTRANSFERASE 1 (MET1) negatively regulates WUSCHEL (WUS), which maintains the stem cell niche of the shoot apical meristem (SAM) in an undifferentiated state. The met1 mutant impairs CG context-dependent DNA methylation, enhancing WUS expression in the SAM that enhances the formation of axillary buds.^40,49 Leaf primordia and axillary bud differentiation in the axillary meristem (AM) is programmed during juvenile seedling development (<12 days after stratification; DAS), after which the SAM transitions into a inflorescence meristem (IM; >14 DAS).⁴ Upon flowering the primary inflorescence stem (FS; floral bolt) emerges from the IM. A cauline branch (CB) and leaf emerges at nodes along the primary inflorescence stem. SET DOMAIN GROUP 8 (SDG8) maintains CAROTENOID ISOMERASE (CRTISO) gene expression and accumulation of β-carotene, a substrate required for biosynthesis of strigolactone (SL).²³ SL regulates BRANCHED 1 (BRC1), which represses axillary bud outgrowth, that would otherwise emerge into a rosette branch (RB).^24,50 sdg8 (ccr1) and crtiso (ccr2) mutants may impair SL biosynthesis,⁵¹ while the loss-of-function in CAROTENOID CLEAVAGE DIOXYGENASE 7 mutant (ccd7) prevents SL biosynthesis.²¹ Negative and positive regulation are indicated by solid red and green arrowed lines, respectively. Black arrowed lines indicate carotenoid biosynthesis steps in pathway. The red dashed lines indicate a presumed function of SDG8 in controlling histone methylation in the SAM.

In Arabidopsis, the transition from vegetative to reproductive development is affected by day length, a critical checkpoint in determining the number of rosette leaves and branching habit.^10,52,53 Transcriptomic analysis of temporal changes in the SAM from long-day grown Arabidopsis plants demonstrated that the vegetative to reproductive transition occurs between 10 and 12 days after germination.⁴ Therefore, we reason that moving adult plants to the cold after differentiation of the IM (>14 DAS) would be restricted from differentiating additional leaf primordia under the long photoperiod due to the reproductive transition (Figure 2). A shorter photoperiod can extend vegetative growth and delay flowering to allow the differentiation of additional leaf primordia, rosette leaves, and shoot branches.^6,10 We conclude that the prolonged cold treatment of juvenile seedlings extended vegetative growth and delayed the transition of the SAM into an IM, thereby increasing the number of leaves, axillary meristems, and rosette branches per plant (Figure 4).

Prolonged cold delayed the reproductive phase to enhance the cauline zone

The SAM in juvenile seedlings would transition into an IM during the 5 weeks of prolonged cold exposure to extend the reproductive phase. Arabidopsis Col-0 will generally, flower within 3–4 weeks after sowing, with 6–8 rosette leaves and 2–3 cauline leaves, when normal growth conditions and a long-day photoperiod are provided. The prolonged cold caused a substantial increase in cauline leaf number and a taller primary inflorescence stem height (Figure 3). This was indicative of an enhanced cauline leaf zone between the rosette and true inflorescence and hence delayed flowering.^11,12 This prompted us to explore if mutants that disrupt flowering time could impair the cold-induced increase in the cauline zone. DNA and histone methylation can affect the timing of flowering in Arabidopsis. MET1 controls context-dependent CG DNA methylation and the loss-of-function met1 mutant alters shoot meristem identity to delay the reproductive phase in Arabidopsis (Figure 4).^44,45 Consistent with these reports, met1 plants flowered late producing a greater number of rosette leaves and cauline branches as well as a taller primary inflorescence stem height causing a higher ratio in the number of cauline branches/stem height compared to WT.

SDG8 maintains permissive histone-3-lysine-4/36 trimethylation surrounding key genes associated with the developmental programming of the SAM (Figure 4), and sdg8 mutant plants transition sooner to the reproductive phase.^23,35,54,55 Consistent with these reports, sdg8 (Col-O ecotype) mutants flowered early generating fewer rosette leaves and a shorter inflorescence stem height per plant in comparison to WT. sdg8 mutant plants generated a slightly higher number of cauline branches, due to pleiotropic effects associated with the reduction in H3K4 tri-methylation at specific genomic loci⁵⁶ and/or the global reduction in H3K36 tri-methylation⁵⁴ that causes the formation of two cauline branches per node of the primary inflorescence stem.²³ As such the ratio of cauline branches to leaves was higher in sdg8 relative to WT when grown under normal growth temperatures.

The met1 and sdg8 inflorescence phenotypes are consistent with delayed and early flowering, respectively. Interestingly, both met1 and sdg8 impaired the cold-induced increase in primary inflorescence stem height and cauline branch number per plant that was characteristically observed in WT (Figure 3). This was further exemplified by a modest increase in the cauline branches/inflorescence stem height ratio compared to WT and hyper-branched mutants. However, met1 and sdg8 like WT plants produced additional rosette leaves in response to prolonged cold. Therefore, the increased primary inflorescence stem height and cauline branch number triggered by the prolonged cold treatment of WT Arabidopsis seedlings could be due to an extended reproductive phase that delayed flowering and increased the cauline leaf zone between the rosette and the primary inflorescence (Figure 4).

Axillary meristem differentiation and bud outgrowth continue under prolonged cold

Prolonged cold exposure of juvenile seedlings increased the number of axillary buds and shoot branches per plant, yet also increased the number of rosette leaves and hence leaf primoridia differentiation. Cold exposure to plants during vegetative growth can increase the level of sucrose to facilitate stress acclimation.^57–59 Soluble sugars such as sucrose and trehalose-6 phosphate can regulate axillary bud outgrowth.^17,60,61 Increased sucrose suppresses the auxin-induced strigolactone pathway to trigger bud outgrowth.⁶² To further investigate the effects of a prolonged cold exposure on axillary meristem formation we assessed ratios in the number of axillary buds/leaf as well as rosette branches/leaf. Contrary to the effect of prolonged cold increasing the number of rosette branches, a slightly lower ratio in the number of axillary buds/leaf and rosette branches/leaf indicated that either the differentiation of axillary meristems and/or leaf primordia were lower and/or greater, respectively (Figure 3).

We first considered if the cold exposure to juvenile Arabidopsis plants affected axillary meristem differentiation or bud outgrowth. The ccd7 and brc1 hyper-branched mutants show enhanced axillary bud outgrowth, in concert with ccd7 being reported to have a twofold higher rosette branch/leaf ratio in Arabidopsis.^24,63 Under normal growth conditions, the number of rosette branches/leaf ratio was greater than two-fold higher in ccd7 and brc1 keeping consistent with previous reports.^63,64 This was further supported by a lower ratio in the number of axillary buds/leaf, indicating that axillary bud outgrowth was considerably higher in the hyper-branched mutants. The prolonged period of cold-induced a higher ratio in the number of axillary buds/leaf in the hyper-branched mutants, revealing that axillary meristem formation can proceed in the absence of CCD7 and BRC1 activities. The rosette branches/leaf ratio was further increased in ccd7 following cold exposure indicating that bud outgrowth could be further enhanced in the absence of strigolactone. The extraordinarily higher number of axillary buds and axillary bud/leaf ratio displayed by met1 remained higher than WT following cold exposure, further evidencing that axillary meristem differentiation and bud formation were not impacted by prolonged cold treatment. Collectively, these evidences highlight the progression of axillary meristem differentiation and rosette bud outgrowth into a rosette branch that can continue during prolonged cold treatment of Arabidopsis juvenile plants.

Rather, our evidence revealed that the prolonged cold exposure dramatically enhanced the number of leaf primordia, irrespective of the genotype being hyper-branched, early or late in flowering time. That is, WT, hyper-branched (ccd7/max3, brc1), early flowering (sdg8), and late flowering (met1) mutants all generated additional rosette leaves during the extended cold exposure. This would be in part due to the extended period of vegetative development allowing additional leaf primordia to develop. Given that axillary meristem differentiation in the leaf axils was not affected by the cold treatment, and that WT showed a relatively lower rosette branch/leaf ratio, the cold treatment more likely caused a subtle increase in leaf primordia differentiation in the SAM. The increased differentiation of leaf primordia could explain why the brc1 and sdg8 mutants did not show a higher rosette branch/leaf ratio like that observed for ccd7/max3. It can also explain why early (sdg8) and late (met1) flowering mutants, as well as WT, all showed a lower axillary bud/leaf ratio after cold exposure. Therefore, the cold treatment may have slightly enhanced leaf primordia development to, thereby, lower the axillary bud/leaf and rosette branch/leaf ratios.

We have demonstrated that a prolonged period of cold exposure to Arabidopsis juvenile seedlings extended vegetative development to allow additional leaf primordia with axillary meristems to develop extra shoot branches, and delayed flowering to allow a larger inflorescence stem height with additional cauline branches to form that collectively enhanced plant biomass and seed yield (Figure 4). In Arabidopsis, previous reports demonstrated that gene-specific differential DNA methylation can affect shoot branching via MET1-dependent transcriptional regulation of WUS (Figure 4). The MET1-mediated methylation repressed WUS expression inhibiting shoot regeneration, while demethylation of the WUS promoter as in met1 mutants can activate WUS expression and promote axillary bud formation.^40,49 In concert with the phenotypes of loss-in-function in MET1 and CCD7, axillary meristem formation and axillary bud outgrowth can proceed during a prolonged cold exposure of Arabidopsis juvenile seedlings. The juvenility of the plant and duration of cold exposure (1–5 weeks) are key factors that underpin the Arabidopsis growth response to the prolonged period of cold exposure. The application of prolonged cold exposure to juvenile seedlings of winter-annual species could be harnessed to enhance plant biomass and yield within a protected cropping environment.

Funding Statement

This work was supported by the Australian Research Council (AU) [DP130102593].

Author contribution

CIC and ND conceived ideas. ND performed experiments, data collection and analyses and prepared figures. ND wrote the manuscript with input from CIC. CIC supervised ND.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.