Vernalization Alters Sink and Source Identities and Reverses Phloem Translocation from Taproots to Shoots in Sugar Beet

Abstract

During their first year of growth, overwintering biennial plants transport Suc through the phloem from photosynthetic source tissues to storage tissues. In their second year, they mobilize carbon from these storage tissues to fuel new growth and reproduction. However, both the mechanisms driving this shift and the link to reproductive growth remain unclear. During vegetative growth, biennial sugar beet (Beta vulgaris) maintains a steep Suc concentration gradient between the shoot (source) and the taproot (sink). To shift from vegetative to generative growth, they require a chilling phase known as vernalization. We studied sugar beet sink-source dynamics upon vernalization and showed that before flowering, the taproot underwent a reversal from a sink to a source of carbohydrates. This transition was induced by transcriptomic and functional reprogramming of sugar beet tissue, resulting in a reversal of flux direction in the phloem. In this transition, the vacuolar Suc importers and exporters TONOPLAST SUGAR TRANSPORTER2;1 and SUCROSE TRANSPORTER4 were oppositely regulated, leading to the mobilization of sugars from taproot storage vacuoles. Concomitant changes in the expression of floral regulator genes suggest that these processes are a prerequisite for bolting. Our data will help both to dissect the metabolic and developmental triggers for bolting and to identify potential targets for genome editing and breeding.

Figure 1.

Biomass and Sugar Contents in Cold-Treated Sugar Beet Shoots and Taproots. (A) to (C) Response of shoots and taproots to cold temperatures using three different sugar beet genotypes. Recording of biomass and sugar accumulation started concurrently with the 4°C treatment. Fresh weight (FW; [A]), dry weight (DW; [B]), and water content (C) of shoots and roots are shown. (D) to (G) Contents of glucose (D), Fru (E), Suc (F), and starch (G) during the course of the chilling (4°C) period in shoots and taproots. Data points show means from n = 6 to 10 plants ± sd. Significant changes relative to the control condition (first data point) were calculated using one-way ANOVA with posthoc Tukey’s HSD (P < 0.05; Supplemental File 1).

Figure 2.

Photosynthetic Parameters, CO₂ Assimilation, and Expression Data of Sugar Beet Leaves after Cold Exposure. (A) PAM measurements using leaves of the three different genotypes. (B) Gas-exchange measurements. Intercellular leaf CO₂ concentration (C_i), CO₂ assimilation rate (A), and transpiration rate (E) are shown. Data points in (A) and (B) show means from four independent plants used throughout the entire time period. Temperature intervals are highlighted in light orange (12°C) or light blue (4°C). Asterisks indicate significant changes to the 20°C condition (0 days after transfer) according to Students t-test (P < 0.05) (C) Percentage of RNA-seq reads annotated as genes coding for photosynthesis (PS)-related proteins. Pie charts represent averaged means from three different genotypes at 20°C (control) and after 14 d at 4°C. (D) Expression of sugar beet RCA (Bv2_025300_tzou.t1), RBCS (Bv2_026840_jycs.t1), CAB (Bv_002570_dmif.t1), and PC (Bv_004160_hgjn.t1). Data represent mean normalized cpm values of three independent RNA-seq analyses per genotype and temperature condition ± sd. Significant changes relative to the control condition (20°C) were calculated using Student’s t test (*, P < 0.05; Supplemental File 1).

Figure 3.

Changes in Major Carbohydrate Metabolism and Energy State in Response to Cold. (A) Respiration (CO₂ production) from leaf tissue of three sugar beet genotypes under control conditions (20°C; yellow) or 1 week after transfer to 4°C (blue). (B) Respiration (CO₂ production) of different taproot regions from GT2 under control conditions (20°C; yellow) or 1 week after transfer to 4°C (blue). FW, fresh weight. (C) ATP, ATP/ADP ratio, and energy charge ([ATP] + 0.5 [ADP]/[ATP] + [ADP] + [AMP]). (D) Heat-map analysis of grouped expression values extracted from RNA-seq data. Unit variance scaling was applied to rows. Rows use Manhattan distance and average linkage. (E) Expression values for two sugar beet SPS genes (SPSA1 and SPSA2) and two SUS genes (SUS1 and SUS2) extracted from RNA-seq data. For (A), (B), (C), and (E), data are means of at least four individual plants ± sd for (A) and (B) or of three pools each consisting of four plants for (C) to (E). Asterisks represent P < 0.05 (Supplemental File 1) using a double-sided t test in comparison with the values at the control condition (20°C).

Figure 4.

Distribution of [¹⁴C]Suc and Esculin in Leaves. (A) to (D) Autoradiography of [¹⁴C]Suc in leaves. (A) Schematic depiction of the experiment: a source leaf from a representative plant grown for 1 week at 4°C. (B) Blackening of veins indicates radioactivity incorporated and distributed into leaf tissue after the injection of radiolabeled Suc into taproots. mv, middle vein; p, petiole; 1°, first order lateral vein; 2°, second order lateral vein. (C) Source leaf from a representative control plant grown at 20°C. (D) Radioactivity in cpm was measured in isolated petioles from plants grown at either 4 or 20°C. Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots; crosses represent sample means; n = 16 sample points. The asterisk indicates a significant difference between the 20 and 4°C treatments using a t test (*, P < 0.05; Supplemental File 1). DW, dry weight. (E) to (K) Esculin loadings. Yellow fluorescence indicates lignified xylem vessels; blue fluorescence indicates esculin trafficking. (E) Schematic depiction of the experiment. (F) and (G) Sections through a petiole of a source leaf not loaded with esculin. Cross sections of petioles from 20°C are shown as a bright-field image (F) and a UV light fluorescence image (G). (H) and (I) Cross sections of petioles from 4°C are shown as a bright-field image (H) and a UV light fluorescence image (I). ph, phloem; xy, xylem. (J) and (K) Longitudinal sections of a petiole from 4°C are shown as a bright-field image (J) and a UV light fluorescence image (K). Bars = 1 cm in (B) and (C), 100 µm in (F) to (I), and 100 µm in (J) and (K).

Figure 5.

Cold-Dependent Accumulation of TST2;1 and SUT4 in Three Different Sugar Beet Genotypes. (A) Illustration of cold-induced processes. The top image shows cold-dependent sugar relocations from taproots to shoots. The middle image shows a schematic of taproot vacuolar transport processes and factors. A vacuolar ATPase (V-H⁺-ATPase) establishes a proton motif force across the vacuolar membrane; TST2;1 acts as a proton/Suc antiporter using the proton-motive force to drive Suc import into vacuoles. SUT4 acts as a proton/Suc symporter using the proton-motive force for vacuolar Suc export. The bottom image shows reciprocal cold-induced regulation of TST2;1 and SUT4 mRNA levels in taproots. (B) Transcript abundance of TST2;1 based on RNA-seq reads. (C) Subcellular localization of SUT4-GFP in leaf mesophyll protoplasts of Arabidopsis or sugar beet. Single optical sections are shown in all images. Green color, GFP signal; red color, chlorophyll autofluorescence. Arrowheads point toward the vacuolar membrane (tonoplast). Bars = 5 µm. (D) Transcript abundance of SUT4 based on RNA-seq reads. Values in (B) and (D) represent means from n = 3 biological replicates ± se. Asterisks indicate significant differences between the 20 and 4°C treatments using a t test (*, P < 0.05; Supplemental File 1).

Figure 6.

Expression of Floral Regulator Genes. Transcript abundances of sugar beet BBX19 (Bv9_216430_rwmw.t1), BTC1 (Bv2_045920_gycn.t1), FT1 (Bv9_214250_miuf.t1), and FT2 (Bv4_074700_eewx.t1) based on RNA-seq reads in shoots and taproots of three different genotypes. Values represent means from n = 3 biological replicates ± se. Asterisks indicate P < 0.05 using a double-sided t test (Supplemental File 1).

Figure 7.

Schematic Illustration of Cold-Induced Sink-to-Source Transition. Leaf and taproot tissues of sugar beet are reprogrammed and source and sink identities are shifted upon cold. Shoots adopt sink identity during cold treatment. Biomass and sugar concentration in the shoot increase (A) despite reduced photosynthetic activity and inactivation of carbon assimilation (B). Concomitantly, shoot respiration increases (C) and cellular starch pools decrease (D). By contrast, taproots show a decrease of Suc levels (E) but lower respiration rate (F) as well as increased Suc biosynthesis (G). Taproot sugar is remobilized in the cold due to opposite regulation of the activities of taproot-specific vacuolar Suc importer (TST2;1) and exporter (SUT4; [H]). Together, this results in a reversal of the phloem translocation stream (I) triggered by a reprogramming of source and sink identities, which might correlate with inflorescence initiation.CC, companion cell; PS, photosynthesis; RuBP, ribulose-1,5-bisphosphate SE: sieve element.