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

Figures & data

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.

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.

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.

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.

Liu H, Zhang H, Dong YX, Hao YJ, Zhang XS. DNA METHYLTRANSFERASE1-mediated shoot regeneration is regulated by cytokinin-induced cell cycle in Arabidopsis. New Phytol. 2017;217:219–232. doi:10.1111/nph.14814.

 PubMed Web of Science ®Google Scholar

Li W, Liu H, Cheng ZJ, Su YH, Han HN, Zhang Y, Zhang XS. DNA methylation and histone modifications regulate de novo shoot regeneration in Arabidopsis by modulating WUSCHEL expression and auxin signaling. PLoS Genet. 2011;7(8):e1002243. doi:10.1371/journal.pgen.1002243.

 PubMed Web of Science ®Google Scholar

Klepikova AV, Logacheva MD, Dmitriev SE, Penin AA. RNA-seq analysis of an apical meristem time series reveals a critical point in Arabidopsis thaliana flower initiation. BMC Genomics. 2015;16(1):466. doi:10.1186/s12864-015-1688-9.

 PubMedGoogle Scholar

Cazzonelli CI, Cuttriss AJ, Cossetto SB, Pye W, Crisp P, Whelan J, Finnegan EJ, Turnbull C, Pogson BJ. Regulation of carotenoid composition and shoot branching in Arabidopsis by a chromatin modifying histone methyltransferase, SDG8. Plant Cell. 2009;21(1):39–53. doi:10.1105/tpc.108.063131.

 PubMed Web of Science ®Google Scholar

Aguilar-Martinez JA, Poza-Carrion C, Cubas P. Arabidopsis BRANCHED1 acts as an integrator of branching signals within axillary buds. Plant Cell. 2007;19(2):458–472. doi:10.1105/tpc.106.048934.

 PubMed Web of Science ®Google Scholar

Braun N, de Saint Germain A, Pillot J-P, Boutet-Mercey S, Dalmais M, Antoniadi I, Li X, Maia-Grondard A, Le Signor C, Bouteiller N, et al. The pea TCP transcription factor PsBRC1 acts downstream of Strigolactones to control shoot branching. Plant Physiol. 2012;158(1):225–238. doi:10.1104/pp.111.182725.

 PubMed Web of Science ®Google Scholar

Cazzonelli CI, Yin K, Pogson BJ. Potential implications for epigenetic regulation of carotenoid biosynthesis during root and shoot development. Plant Signal Behav. 2009;4(4):339–341. doi:10.4161/psb.4.4.8193.

 PubMedGoogle Scholar

Booker J, Auldridge M, Wills S, McCarty D, Klee H, Leyser O. MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule. Current Biology: CB. 2004;14(14):1232–1238. doi:10.1016/j.cub.2004.06.061.

 PubMed Web of Science ®Google Scholar