Whole-Transcriptome Analysis Unveils the Synchronized Activities of Genes for Fructans in Developing Tubers of the Jerusalem Artichoke

Abstract

Helianthus tuberosus L., known as the Jerusalem artichoke, is a hexaploid plant species, adapted to low-nutrient soils, that accumulates high levels of inulin in its tubers. Inulin is a fructose-based polysaccharide used either as dietary fiber or for the production of bioethanol. Key enzymes involved in inulin biosynthesis are well known. However, the gene networks underpinning tuber development and inulin accumulation in H. tuberous remain elusive. To fill this gap, we selected 6,365 expressed sequence tags (ESTs) from an H. tuberosus library to set up a microarray platform and record their expression across three tuber developmental stages, when rhizomes start enlarging (T₀), at maximum tuber elongation rate (T₃), and at tuber physiological maturity (T_m), in "VR" and "K8-HS142"clones. The former was selected as an early tuberizing and the latter as a late-tuberizing clone. We quantified inulin and starch levels, and qRT-PCR confirmed the expression of critical genes accounting for inulin biosynthesis. The microarray analysis revealed that the differences in morphological and physiological traits between tubers of the two clones are genetically determined since T₀ and that is relatively low the number of differentially expressed ESTs across the stages shared between the clones (93). The expression of ESTs for sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT), the two critical genes for fructans polymerization, resulted to be temporarily synchronized and mirror the progress of inulin accumulation and stretching. The expression of ESTs for starch biosynthesis was insignificant throughout the developmental stages of the clones in line with the negligible level of starch into their mature tubers, where inulin was the dominant polysaccharide. Overall, our study disclosed candidate genes underpinning the development and storage of carbohydrates in the tubers of two H. tuberosus clones. A model according to which the steady-state levels of 1-SST and 1-FFT transcripts are developmentally controlled and might represent a limiting factor for inulin accumulation has been provided. Our finding may have significant repercussions for breeding clones with improved levels of inulin for food and chemical industry.

Keywords: Helianthus tuberosus; expressed sequence tags; fructosyltransferases; inulin; microarray; qRT-PCR; tuber phenology.

Figure 1

Morphological comparison of the rhizomes of Helianthus tuberosus “VR” and “K8HS142” clones during the tuber development. The white arrows point to the initial part of tuber enlargement. The sizes at the three stages are on scale. The number of nodes is reported.

Figure 2

Tuber traits in Helianthus tuberosus “VR” and “K8-HS142” clones. (A) Phases of tuber development. Bars show the duration of each of the three tuber developmental stages, from the onset of tuberization to physiological maturation. The scale starts from the day of planting. Data are from the average of three plants per clone. (B) Tuber weight. Data are from tubers collected from four different plants per clone. (C) Tuber shape. Ratio between length and width in tubers of “VR” and “K8-HS142” clones, collected from four different plants. According to Pas'ko (1973) tubers from “VR” and “K8-HS142” were classified as short pear-shaped and spindle-shaped, respectively (Figure 1; Figure S1). (D) Temporal variation in the length of the main axis (mm) of “VR” (red line) and “K8-HS142” (blue line) tubers. *: Significances (p < 0.05). The bar indicates ±1 standard error.

Figure 3

Inulin content and degree of polymerization (DP) in tubers of Helianthus tuberosus “VR” and “K8-HS142” clones. Significantly different values (p < 0.05) at different stages in the same clone are labeled with different letters (lowercase for “VR,” uppercase for “K8-HS142”) and those between clones at the same stage with *. The bar indicates ±1 standard error.

Figure 4

Temporal expression of the expressed sequence tags (ESTs) involved in the metabolism of storage carbohydrates in Helianthus tuberosus “VR” and “K8-HS142” clones. The relative emission levels of ESTs implied in the metabolism of sucrose, fructans, and starch at T₃ or T_m compared to T₀ reference stage (when tuberization begins), both in “VR” and “K8-HS142,” are displayed through a heat map. Magnitudes and statistical relevance of each EST contest are in the map. The statistical significance of the differences were tested according to the LIMMA. Key to genes. Simple sugars metabolism. SuSym, Sucrose H⁺/symporter; SuP, Sucrose phosphatase; SuS, Sucrose synthase; INV, Neutral/alkaline invertase; GCK, Glucokinase; PGM, Phosphoglucomutase; UDPGP, UDP-glucose pyrosphorilase; ERD, Early Response to Dehydration 6 - H⁺/Glucose symporter. Fructan polymerization. 1-SST, Sucrose:sucrose 1-fructosyltransferase; 1-FFT, Fructan:fructan 1-fructosyltransferase. Starch metabolism and polymerization. G6PT, Glucose 6-phosphate translocator; SS, Starch synthase. Ht, Helianthus tuberosus; St, Solanum tuberosum; Ci, Chicorium intybus; As, Allium sativum.

Figure 5

qRT- PCR analysis of key genes ruling the metabolism of storage carbohydrates in developing tubers of Helianthus tuberosus “VR” and “K8-HS142” clones. The temporal expression profiles by means of qRT- PCR at three specific tuber developmental stages (T₀, T₃, and T_m) of genes implied in the cytoplasmatic metabolism of hexoses such as SuS and PGM, and fructan polymerization, 1-SST and 1-FFT, which were differentially expressed upon microarray analysis, are shown. The relative expression of each gene is calculated using the (2^-ΔCt) algorithm. Statistically significant differences were assessed according to a two-way ANOVA between T₀, T₃, and T_m stages in “VR” and “K8-HS142” (indicated as a, b, c and A, B, C, respectively). Asterisks **, *** indicate the significance of the differences of transcript levels between clones at a given tuber stage (0.01 ≤ p < 0.01 and p < 0.001, respectively). The bars indicate ±1 standard deviation.

Figure 6

Model of inulin accumulation and cell growth in developing tubers of Helianthus tuberosus. A scheme is proposed to display the metabolic pathways of sucrose breakdown in the cytoplasm, fructan polymerization in the vacuole, and starch biosynthesis in the amyloplast from when rhizome starts enlarging (T₀) until the stages of tuberous maximum growth rate (T₃) and final tuber maturation (T_m). While cell at T₀, regarded as the reference stage in tuber development process, is displayed in the foreground, cells at the two next stages, when inulin deposition takes place moderately (T₃) and massively (T_m), are depicted smaller near the T₀ cell. The main enzymes affecting the metabolism of carbohydrates as well as cell wall assembling and the overall cell growth are shown and placed in the cell compartments of their action. Emphasized arrows point to massive metabolic pathways. Enzymes whose genes are differentially expressed (DE) between T₀ and T_m are reported in bold blue (if DE in both clones) or in red (if DE in one clone). The enzymes indicated in orange have genes not investigated in this study.