Helianthus tuberosus genetic resources: from the plant phenotype to the gene expression analyses during tuber development for studying carbohydrate biosynthesis, tuber biomass productivity, and ancestors of the species
Author(s)
Bizzarri, Marco
Date Issued
June 11, 2013
Type
Doctoral Thesis
Abstract
The global warming scenario and the urgent public demand of energy, require most effective solutions in order to mitigate the actual emission levels of greenhouse gases, mainly carbon dioxide, and provide non-polluting and inexpensive energy. Among the alternative sources of energy, biomasses represent the most valid means for achieving the mentioned purposes, providing carbon-negative electricity, heat, biogas and mostly biofuels.
The most attractive next generation biofuel systems are algae and few additional plant species such as the tuber-producing Helianthus tuberosus L. (2n=6x=102, Ht). Ht grow rapidly, shows high carbohydrate content, mainly fructans, and is able to produce 2-3 times more biomass per unit land area than any other annual cropping system as well as a higher bioethanol yield. The high amount of fructans stored in stalks and tubers of Ht leads to the production of bio-ethanol and other energy-storage chemicals upon microbial hydrolysis and fermentation. In addition, Ht has a high nitrogen and water use efficiency, utilize nutrient-rich waste water, and is resistant/tolerant to pathogens and pests.
Integrating phenomic, transcriptomic, metabolomic, and genomic approaches into a breeding program aimed to enhance Ht tuber biomass production is the most efficient strategy that, currently, convey efforts towards the establishment of a research-based platform for the production of carbon-negative biofuels, and for this reason this strategy has been adopted for this dissertation.
By carrying out a phenomic study, the phenotypic variability extant among 67 different clones belonging to sub-primary both wild and cultivated gene pool grown in the Experimental Farm of University of Tuscia, were evaluated for morphological (tuber shape, plant architecture and height) and physiological (date of beginning of tuber enlargement, flowering time, resistance to biotic and abiotic stimuli) traits related to the tuber biomass production.
‘K8-HS142’, selected by a half-sib progeny of the cultivated ‘K8’ clone and the cultivated ‘Violet de Rennes’ (‘VR’) were the best Ht clones detected to be suitable for biomass production. The monostem ‘K8-HS142’ forming spindle-shaped tubers and the semi-brushy and branched ‘VR’ forming short-pear shaped tubers resulted the most attractive clones because of their highest production in rainfed (18 ton ha-1) and water supply (20 ton ha-1) conditions, respectively, as well as for other phenotypic traits such as drought resistance and precocity of flowering (‘K8-HS142’) and powdery mildew resistance and precocity of tuberization (‘VR’).
The fructan content during tuber development at the three crucial stages of initial tuberization (T0), active growth (T3) and final maturation (Tm) was determined by carrying out a metabolomic study and a maximum content of 425 and 450 g kg-1 of dry matter was ascertained for ‘K8-HS142’ and ‘VR’ at T3 when water supply conditions occurred.
The analysis regarding the tuber content of starch evidenced substantial lacking of that polysaccharide in the mature tuber from ‘K8-HS142’ and ‘VR’ clones.
Because of their different i) tuber biomass yields and tuber carbohydrate content from both rainfed and irrigated conditions, ii) tuber shape and precocity of tuberization and iii) plant architecture fitting high (‘K8-HS142’) and low plant density (‘VR’) cropping systems, the most productive ‘K8-HS142’ in rainfed condition and the ‘VR’ (used as control), were chosen in order to carry out a transcriptomic study focused on the expression analysis regarding the set of genes encoding for enzymatic and structural proteins involved in carbohydrate biosynthesis and storage during the tuber development.
The expression level of a set of 6,365 ESTs selected from the CHT(LMS)_library of 41,000 Ht EST sequences annotated to genes included in pathways for tuber carbohydrate biosynthesis and storage using Blast2GO® bioinformatic tool were used for the microarray analysis based on the CombiMatrix© technology performed at the developmental stages of cessation of rhizome elongation-beginning of apical meristem differentiation for tuberization (T0), active tuber development (T3) and tuber maturation (Tm).
A total of 123 and 11 ESTs were differentially expressed between the two Ht clones at T0 and T3, respectively, whereas no ESTs were differentially expressed at Tm. In both clones, 11 and 127 different ESTs were discovered differentially expressed between T0 and T3 and T0 and Tm respectively, whereas no ESTs displayed a differential expression between T3 and Tm. Those results reasonably suggest that the highest gene expression for carbohydrate biosynthesis occur during the initial tuberization stage T0 - T3 and that a peculiar peak expression occur for 11 genes at the T3 stage.
Furthermore, 12 ESTs of genes specifically involved in both fructan and starch biosynthesis were significantly expressed at high level during tuber development. Those ESTs encode for enzymes implied in fructan polymerization into the vacuole (Sucrose:sucrose 1-fructosyltransferase [1SST], Fructan:fructan 1-fructosyltransferase [1FFT], Sucrose H+/symporter [SuSym]) and starch biosynthesis into the amyloplast (Starch synthase [SS], Sucrose phosphate phosphatase [SuPP], Glucose 6P/P translocator [G6PT], -invertase [INV], Phosphoglucomutase [PGM], Sucrose synthase [SuSy], UDPG pyrophosphorilase [UDPGP]).
The ESTs for 1SST and 1FFT were found up to 3 times more expressed than SS, suggesting that the fructan polymerization prevailed over starch polymerization and explain why fructans are the major tuber storage carbohydrate. However the magnitude of expression produced by genes dealing with carbohydrate biosynthesis allowed to possibly suppose their activity in the starch biosynthetic network.
The expression pattern of the 1SST-ESTs was in line with the expected function of the encoded 1SST enzyme. In fact, the fructan biosynthesis starts with the intervention of the 1SST enzyme which catalyze the synthesis of the triose 1-kestose from two sucrose molecules, which in turn is used by 1FFT as starting building-block for the further polymerization of fructans by adding fructose monomers to the growing fructan chain.
Two other 1SST-ESTs with 73% and 100% sequence identity to the 1SST-EST from Allium sativum (As) and Cichorium intybus (Ci), respectively, were also highly expressed during the T0-T3 tuber developmental stage. The presence in Ht of additional genes orthologous to genes from other plant species encoding for 1SST suggests the pivotal role of 1SST in fructan accumulation in organs for vegetative propagation in both Monocotyledons and Dicotyledons and the allopolyploid origin of the hexaploid Ht from diploid with common ancestry to other Asterceae. That is in line with the evidence that Helianthus genus is one of the most evolutionary diverse groups of organisms whose biodiversity represents a rich resource for bioprospecting orthologous genes related not only to fructan biosynthesis, but also to other genes expressed in heterotrophic tissues of carbohydrate storage organs.
Performing the genomic study, the polymorphism for amplicons from genes above mentioned involved in carbohydrate biosynthesis (except SuSym, UDPGP, SuPP) was determined in diploid-non-tuberizing (H. annuus, H. argophyllus, H. niveus, 2n=2x=34), diploid-tuberizing (H. maximilianii, H. angustifolius, H. decapetalus and H. nuttalli, 2n=2x=34), tetraploid-tuberizing (H. hirsutus, H. strumosus, 2n=4x=68) and hexaploid-tuberizing (H. schweinitzii, Ht, 2n=2x=102) Helianthus species. The amplicon lengths were used to explore the average similarity among species using the Dice index (DSI) with respect to ESTs for 1SST and SS in order to depict a reliable
genealogy of Ht, and prepare a bioprospection of amplicons to clone new homeoalleles for improving Ht ability to produce tuber biomass.
A striking monomorhpism was detected for amplicons from 1FFT, SuS, PGM, SuSym, while a strong and light polymorphism was evidenced for amplicons from 1SST, SS and INV, G6PT, respectively. Non-tuberizing diploid had a larger polymorphism than tuberizing diploid, which indicate that the higher polymorphism in tetraploid and hexaploid Helianthus species might be the a signature of a parental non-tuberizing diploid species in the pedigree of polyploid Helianthus.
The non-tuberizing annual diploid H. annuus had highest average similarity (DSI=0.50) to Ht which make it a relevant species for exploring the genome of the putative diploid ancestor of Ht.
The tetraploid-tuberizing species H. hirsutus had also a high average similarity to Ht (DSI=0.46) which make it a robust candidate as tetraploid ancestor of Ht.
The genomic tools have also been used for setting the stage for identification and exploration of the genome sequence of the regulatory regions in Ht 1SST and 1FFT genes.
A representative subset of 55,296 clones of a sunflower BAC library prepared from the nuclear genome of the Helianthus annuus (Ha) ‘line 89’ was screened to detect cloned DNA inserts encoding the 1SST and 1FFT enzymes using the 1SST and 1FFT amplicons from Ht as probes. The BAC library covering 5 times the sunflower haploid genome was screened by using a pool of 10 different 32P-labelled probes was prepared from the selected amplicons after PCR of the DNA from the Ht clones ‘CSR’ (a feral Ht-type from Latium, Italy) and ‘CU-3B’ (a cultivated clone from Hungary) and the Ha ‘line 89’ taken as control.
A total of 23 different positive BAC clones were detected, paving the way to next sequencing and identification of promoter regions useful for exploring polymorphism extant among Helianthus species for those DNA sequences.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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