Early-stage sugar beet taproot development is characterized by three distinct physiological phases

Abstract

Despite the agronomic importance of sugar beet (Beta vulgaris L.), the early-stage development of its taproot has only been poorly investigated. Thus, the mechanisms that determine growth and sugar accumulation in sugar beet are largely unknown. In the presented study, a physiological characterization of early-stage sugar beet taproot development was conducted. Activities were analyzed for fourteen key enzymes of carbohydrate metabolism in developing taproots over the first 80 days after sowing. In addition, we performed in situ localizations of selected carbohydrate-metabolic enzyme activities, anatomical investigations, and quantifications of soluble carbohydrates, hexose phosphates, and phytohormones. Based on the accumulation dynamics of biomass and sucrose, as well as on anatomical parameters, the early phase of taproot development could be subdivided into three stages-prestorage, transition, secondary growth and sucrose accumulation stage-each of which was characterized by distinct metabolic and phytohormonal signatures. The enzyme activity signatures corresponding to these stages were also shown to be robustly reproducible in experiments conducted in two additional locations. The results from this physiological phenotyping approach contribute to the identification of the key regulators of sugar beet taproot development and open up new perspectives for sugar beet crop improvement concerning both physiological marker-based breeding and biotechnological approaches.

Keywords: assimilate partitioning; carbohydrate metabolism; developmental regulation; physiological phenotyping; sucrose accumulation; taproot development.

FIGURE 1

Biomass in developing sugar beet taproots over 80 days after sowing (main set of experiments, location A, Graz, Austria; means of 15 individuals ± SD). Beets grew exponentially throughout the period of investigation in all experiments, and fresh weight at 80 das was strongly dependent on the growing season. (a) experiment A1‐es. (b) experiment A2‐s. (c) experiment A3‐a. (d) experiment A4‐sp. (e) experiment A5‐es. (f) experiment A6‐sp

FIGURE 2

Anatomy (a–g) of developing sugar beet taproots at 10 (a), 15 (b), 20 (c), 30 (d), 40 (e), 60 (f), and 80 (g) days after sowing (das), and schematic representation (h) of central cylinder development up to 40 das. c, cortex; ca, cambium; cr, cambial ring (consisting of supernumerary cambium plus weakly differentiated phloem and xylem); e, endodermis; p, pericycle; pa, storage parenchyma; pe, periderm; ph, phloem; xy, xylem; arrows = initial cell divisions for new cambia; bar = 100 µm (a–e) or 500 µm (f,g)

FIGURE 3

Soluble carbohydrates (Glc, Fru, Suc) and hexose‐to‐Suc ratio in developing sugar beet taproots over 80 days after sowing (main set of experiments, location A, Graz, Austria). (a–c) experiment A1‐es (means of two parallel extracts ± SD). (d–f) experiment A2‐s (means of two technical replicates ± SD). (g–i) experiment A4‐sp (values from single extract). (j–l) experiment A5‐es (values from single extract). Experiment A6‐sp is not shown (no data for Glc and Fru due to technical problems with the quantification of hexoses; for Suc data, see Dataset S1)

FIGURE 4

Hexose phosphates in developing sugar beet taproots over 80 days after sowing (main set of experiments, location a, Graz, Austria; means of two parallel extracts ± SD)—Glc‐6‐phosphate (G6P), Glc‐1‐phosphate (G1P), and Fru‐6‐phosphate (F6P). (a–c) experiment A4‐sp. (d–f) experiment A5‐es

FIGURE 5

Carbohydrate‐metabolic enzyme activities in developing sugar beet taproots over 80 days after sowing (main set of experiments, location a, Graz, Austria; means of two parallel extracts ± SD). Part 1—vacuolar, cytoplasmic, and cell wall‐bound invertase isoenzymes (vacInv, cytInv, cwInv), sucrose synthase (SuSy), and hexose‐phosphorylating enzymes hexokinase (HXK) and fructokinase (FK). (a–c) experiment A1‐es. (d–f) experiment A2‐s. (g–i) experiment A3‐a. (j–l) experiment A4‐sp. (m–o) experiment A5‐es. (p–r) experiment A6‐sp

FIGURE 6

Carbohydrate‐metabolic enzyme activities in developing sugar beet taproots over 80 days after sowing (main set of experiments, location a, Graz, Austria; means of two parallel extracts ± SD). Part 2—hexose phosphate interconverting enzymes phosphoglucose isomerase (PGI) and phosphoglucomutase (PGM), glycolytic enzymes aldolase (Ald) and phosphofructokinase (PFK), UDP‐glucose pyrophosphorylase (UGPase), and glucose‐6‐phosphate dehydrogenase (G6PDH). (a–c) experiment A1‐es. (d–f) experiment A2‐s. (g–i) experiment A3‐a. (j–l) experiment A4‐sp. (m–o) experiment A5‐es. (p–r) experiment A6‐sp

FIGURE 7

Overview of carbohydrate metabolism in developing sugar beet taproots. Color intensity of boxes and thickness of lines indicate the activity levels of enzymes or the abundance of metabolites. Dashed grey lines indicate that enzyme activities of the respective pathways or levels of the respective metabolites were not analyzed in the presented study. At the prestorage stage (a), sucrose is cleaved into its hexose monomers by invertases. As metabolic enzyme activities are low, relatively low amounts of carbohydrates appear to be used for metabolic processes—hexoses and hexose phosphates are assumed to accumulate and to act as sugar signals and as an osmotic driving force for growth. At the secondary growth and sucrose storage stage (b), sucrose synthase is the main sucrolytic enzyme. High activities of various carbohydrate‐metabolic enzymes suggest that sucrose is constantly partitioned between sucrose storage and metabolism. ADPGlc, ADP‐glucose; AGPase, ADP‐glucose pyrophosphorylase; F6P, fructose‐6‐phosphate; FK, fructokinase; Fru, fructose; G1P, glucose‐1‐phosphate; G6P, glucose‐6‐phosphate; G6PDH, glucose‐6‐phosphate dehydrogenase; Glc, glucose; HXK, hexokinase; Inv, invertase isoenzymes; PFK, phosphofructokinase; PGI, phosphoglucoisomerase; PGM, phosphoglucomutase; SPS, sucrose‐phosphate synthase; Suc, sucrose; UDPGlc, UDP‐glucose; UGPase, UDP‐glucose pyrophosphorylase

FIGURE 8

Histochemical localization of enzyme activities in developing sugar beet taproots. (a–c) acid invertases (a, 10 das; B 30 das; C, 80 das). (d–f) SuSy (d, 10 das; e, 30 das; f, 60 das). (g–i) PGI (g, 10 das; E, 30 das; I, 80 das). (j–l) PGM (j, 10 das; k, 30 das; l, 80 das). (m–o) G6PDH (m, 10 das; n, 30 das; o, 80 das). Controls without substrates were free from staining (see Figures S1–S5). Bar = 1 mm (a–c) or 100 µm (d–o)

FIGURE 9

Phytohormones in developing sugar beet taproots over 80 days after sowing (main set of experiments, location A, Graz, Austria)—cytokinins (CK), indole‐acetic acid (IAA), abscisic acid (ABA), and phytohormone ratios. (a–c) experiment A4‐sp (means of two parallel extracts ± SD). (d–f) experiment A5‐es (values from single extract). (g–i) experiment A6‐sp (values from single extract). Experiment A1‐es is not shown (no data for total storage CK, total CK, and active‐to‐storage CK ratio due to technical problems with the quantification of ZOG; for the full phytohormone dataset, see Table S3 and Dataset S3)