Enhancing grain size in durum wheat using RNAi to knockdown GW2 genes

Sestili F., Pagliarello R., Zega A., Saletti R., Pucci A., Botticella E., Masci S., Tundo S., Moscetti I., Foti S., Lafiandra D. 2019 Enhancing grain size in durum wheat using RNAi to knock-down GW2 genes. Theoretical and Applied Genetics, 132(2): 419-429 https://doi.org/10.1007/s00122-018-3229-9. Abstract Key message Knocking down GW2 enhances grain size by regulating genes encoding the synthesis of cytokinin, gibberellin, starch and cell wall. Abstract Raising crop yield is a priority task in the light of the continuing growth of the world’s population and the inexorable loss of arable land to urbanization. Here, the RNAi approach was taken to reduce the abundance of Grain Weight 2 (GW2) transcript in the durum wheat cultivar Svevo. The effect of the knockdown was to increase the grains’ starch content by 10–40%, their width by 4–13% and their surface area by 3–5%. Transcriptomic profiling, based on a quantitative real-time PCR platform, revealed that the transcript abundance of genes encoding both cytokinin dehydrogenase 1 and the large subunit of ADP-glucose pyrophosphorylase was markedly increased in the transgenic lines, whereas that of the genes encoding cytokinin dehydrogenase 2 and gibberellin 3-oxidase was reduced. A proteomic analysis of the non-storage fraction extracted from mature grains detected that eleven proteins were differentially represented in the transgenic compared to wild-type grain: some of these were involved, or at least potentially involved, in cell wall development, suggesting a role of GW2 in the regulation of cell division in the wheat grain

The silencing of TdIPK1 genes enhances micronutrient concentration in durum wheat grain

Phytic acid (PA) is the main storage form of phosphorus in kernel and is considered an anti-nutritional compound because of its ability to bind to essential minerals such as iron (Fe), zinc (Zn), potassium (K), calcium (Ca) and magnesium (Mg), thus limiting their availability, especially for populations whose diet is largely based on staple crops. This study reports a promising nutrient biofortification approach of durum wheat. The approach was based on the silencing of the gene encoding the inositol pentakisphosphate 2- kinase 1 (IPK1), involved in the last step of the PA biosynthetic pathway, through a Targeting Induced Local Lesions IN Genomes (TILLING) approach. Single knockout mutants for the IPK1 homeoalleles were identified and crossed to pyramid the two mutations. Although an elevated number of plants (F2 and F3 progenies) were analysed, no genotypes lacking both the homeoalleles were recovered, suggesting that the expression of IPK1 is crucial for seed formation in the spike and/or for plant germination and development. The characterization of the single null genotypes highlighted that the partial TdIPK1-B1- mutants showed a lower accumulation of PA in the kernel along with a higher content of essential microelements (Fe, Mn, Zn) compared to the control wild-type. The pattern of mineral accumulation was different for the TdIPK1-A1- mutants which only presented a greater accumulation of K

The suppression of TdMRP3 genes reduces the phytic acid and increases the nutrient accumulation in durum wheat grain

Micronutrient malnutrition affects more than half of the world population. Reduced bioavailability of microelements in the raw materials is considered one of the main causes of mineral deficiency in populations whose diet is largely based on the consumption of staple crops. In this context, the production of low phytic acid (lpa) cereals is a main goal of the breeding programs, as phytic acid (PA) binds essential mineral cations such as iron (Fe), zinc (Zn), manganese (Mn), potassium (K), calcium (Ca) and magnesium (Mg) precipitating in the form of phytate salts poorly digested by monogastric animals, including humans, due to the lack of phytases in the digestive tract. Since PA limits the bioavailability of microelements, it is widely recognized as an anti-nutritional compound. A Targeting Induced Local Lesions IN Genomes (TILLING) approach has been undertaken to silence the genes encoding the TdABCC13 proteins, known as Multidrug-Resistance associated Proteins 3 (TdMRP3), transporters involved in the accumulation of PA inside the vacuole in durum wheat. The TdMRP3 complete null genotypes showed a significant reduction in the content of PA and were able to accumulate a higher amount of essential micronutrients (Fe, Zn, Mn) compared to the control. The number of spikelets and seeds per spike, traits associated with the agronomic performances, were reduced compared to the control, but the negative effect was in part balanced by the increased grain weight. The TdMRP3 mutant lines showed morphological differences in the root apparatus such as a significant decrease in the number of root tips, root length, volume and surface area and an increase in root average diameter compared to the control plants. These materials represent a promising basis for obtaining new commercial durum wheats with higher nutritional value

Mutagenesis for the development of novel wheats: modification of starch composition and improvement of nutritional value

Il presente progetto di dottorato si inserisce in una linea di ricerca che punta al miglioramento delle caratteristiche tecnologiche e nutrizionali della farina di frumento attraverso la manipolazione genetica della composizione chimica dell’amido. L’amido è costituito da due polimeri glucanici, l’amilosio e l’amilopectina, che differiscono nella lunghezza delle catene e nel grado di ramificazione. La manipolazione del rapporto amilosio-amilopectina consente la produzione di amidi con nuove proprietà chimico-fisiche. É stato dimostrato che l’aumento della percentuale di amido resistente, riscontrata negli “amidi ad alto contenuto di amilosio” (HAS), apporta notevoli benefici alla salute umana , riducendo il rischio di importanti malattie quali il cancro, il diabete, l’osteoporosi. La strategia utilizzata per l’ottenimento di HAS consiste nella manipolazione delle vie biosintetiche dei due polimeri che costituiscono l’amido, l’amilosio e l’amilopectina. I due polimeri sono sintetizzati attraverso vie biosintetiche distinte che condividono lo stesso substrato; la mancanza di attività di due classi di enzimi coinvolte nella sintesi dell’amilopectina, SSII e SBEIIa, è stato associato, in frumento, a genotipi caratterizzati da un notevole incremento della percentuale di amilosio. In particolare il silenziamento dell’enzima SBEIIa, attraverso RNA interference, ha permesso di ottenere linee di frumento con amidi costituiti per il 70% da amilosio rispetto a circa il 25 % dei genotipi normali. Nel presente progetto di dottorato sono state generate nuove linee di frumento tenero caratterizzate dalla perdita di espressione degli alleli SBEIIa attraverso un approccio di genetica inversa basato sul TILLING. La prima parte di questo lavoro è consistito nell’isolamento e sequenziamento dei due omoeoalleli SBEIIa-A e SBEIIa-B di frumento tenero localizzati rispettivamente sul braccio corto dei cromosomi 2AS e 2BS. L’allineamento delle sequenze genomiche dei tre alleli ha inoltre evidenziato la presenza di diverse inserzioni transposon like; sono stati condotti esperimenti finalizzati allo studio del grado di conservazione di tali elementi in diversi frumenti diploidi e poliploidi. Mediante il confronto delle sequenze nucleotidiche dei tre omeoalleli sono state identificate regioni geniche adatte al disegno di oligonucleotidi genoma specifici utilizzati nelle amplificazioni PCR degli esperimenti di TILLING. L’analisi di una popolazione mutagenizzata di frumento tenero, cv Cadenza, ha portato all’identificazione di numerose varianti alleliche per ciascuno dei tre geni. In totale sono stati identificati 59, 14 e 49 nuove varianti alleliche , rispettivamente per gli alleli SBEIIa-A, SBEIIaB e SBEIIa-D. In particolare, per ciascuno dei tre omeologhi, sono state identificate due mutazioni deleterie responsabili delle perdita di espressione genica. Sono stati generati due genotipi caratterizzati dalla presenza di un codone di stop all’interno della regione codificante dell’allele SBEIIa-A; una mutazione in un sito di splicing e una non senso sono state identificate per ciascun dei due alleli SBEIIa-B e SBEIIa-D. L’analisi dei trascritti dell’allele del genoma A, nei due genotipi singoli nulli SBEIIa-A-, ha evidenziato una drastica riduzione dell’espressione del gene rispetto al genotipo wild type indicando l’intervento di un meccanismo di degradazione dell’RNA messaggero noto come NMD (Nonsense- Mediated RNA Decay). L’analisi della progenie M3 dei genotipi nulli SBEIIa, effettuata attraverso un saggio di tipo PCR-MAS (Marker Assisted Selection), ha permesso l’identificazione e la selezione di genotipi mutanti omozigoti. È stato avviato un programma di incrocio tra le linee nulli SBEIIa per la realizzazione di una “seria fenotipica” caratterizzata dalla riduzione o mancanza dell’enzima target e da diversi livelli di incremento del contenuto di amilosio. Inoltre sono stati ottimizzati i protocolli di diverse metodiche di analisi delle proprietà chimico fisiche dell’amido utilizzando linee di frumento ad alto e a basso contenuto di amilosio. Tali metodiche saranno utilizzate per l’analisi dell’amido dei genotipi nulli completi e parziali SBEIIa generati in questo lavoro.Reserve starch represents the main component in wheat endosperm whose composition strongly influences the quality and nutritional value of wheat-based food products. A new challenge to develop novel wheat varieties with improved quality characteristics has recently been identified through the manipulation of starch composition. Starch is composed by two glucan polymers different in chains length and branching grade.The modulation of amylose/amylopectin ratio greatly affects wheat flour nutritional and processing properties. Currently many efforts focus on the objective of increasing the amylose fraction in cereal starches. Higher amylose content correlates with an increased amount of “resistant starch”, a particular starch fraction, considered to have beneficial effects on human health lowering the risk of important diseases. Amylose and amylopectin are synthesized from a common substrate, ADP-glucose, by two distinct pathways. Amylose production involves one enzyme – Granule Bound Starch Synthase I (GBSSI)-whereas three classes of enzyme, known as starch synthases (SSs), branching (SBEs) and debranching enzymes (DBEs) take part to the synthesis of amylopectin. Starch branching enzymes IIa are one of the main targets to increase amylose content in cereals. Recent studies, in bread and durum wheat, have shown that the RNA interference technology can result efficiently in the loss of SBEIIa functionality, leading to a drastic change of the amylose/amylopectin ratio. The TILLING-Targeting Induced Lesions In Genomes-represents an effective reverse genetics tool for the production of novel allelic variants in valuable traits. In TILLING the combination of traditional chemical mutagenesis with high-throughput detection methods of point mutations has originated an attractive strategy both for functional genomics and breeding applications overcoming limits associated with transgenic issues. In this work an EMS mutagenised population of bread wheat has been analyzed for the identification of SNPs (single nucleotide polymorphisms) of interest in the three homoeoalleles SBEIIa located in A, B and D genomes by a TILLING approach. The two genes SBEIIa-A and SBEIIa-B have been isolated and sequenced and polymorphic regions have been selected in order to design allele specific primer pairs to be used in TILLING. The comparison of the three genes have led to the identification of several transposon like insertions. These elements have also been investigated in diploid and polyploid wheats in order to establish phylogenetic relationship. Several targeted regions of the three SBEIIa genes have been analyzed in approximately two thousand wheat lines by High Resolution Melting technology, based on the comparison of the melting behaviours of the amplified PCR fragments. Fifty nine mutant genotypes have been characterized for SBEIIa-A, fourteen for SBEIIa-B and forty nine for SBEIIa-D confirming a mutation frequency of 1 SNP for each 36kb associated to the TILLING library. Most of the mutations described are localized in the coding region. Worthy of note two deleterious mutations have been identified in each of the three SBEIIa homoeoalleles. Two nonsense mutation localized, respectively, in the exon IX and exon XII, have been identified for SBEIIa-A gene; one nonsense mutation and one affecting a splicing site, have been found for each of the two alleles SBEIIa-B and SBEIIa-D. For all three homoeologues different missense mutations, causing the change of conserved residues located in catalytic domains of SBEIIa enzymes, have been identified. Transcripts analysis of the two SBEIIa-A nonsense mutants revealed a drastic reduction of the gene expression compared to wild type genotype, indicating mRNA degradation by a NMD (Nonsense Mediated Decay) mechanism. SBEIIa-single null genotypes have been selected in the M3 progenies by a PCR-Marker Assisted Selection assay based on the development of dCAPS primers. Electrophoretic analyses carried out in the SBEIIa single null mutants of A and B genomes have not detected any changes in the starch granule proteins profile. Moreover in this work, methods for the characterization chemical physical properties of starch have been optimized using wheat lines with an altered amylose/amylopectin ratio. These methods will be used to characterize the SBEIIa of complete and partial null genotypes.Dottorato di ricerca in Biotecnologie vegetal

The Triple Jags of Dietary Fibers in Cereals: How Biotechnology Is Longing for High Fiber Grains

Cereals represent an important source of beneficial compounds for human health, such as macro- and micronutrients, vitamins, and bioactive molecules. Generally, the consumption of whole-grain products is associated with significant health benefits, due to the elevated amount of dietary fiber (DF). However, the consumption of whole-grain foods is still modest compared to more refined products. In this sense, it is worth focusing on the increase of DF fractions inside the inner compartment of the seed, the endosperm, which represents the main part of the derived flour. The main components of the grain fiber are arabinoxylan (AX), β-glucan (βG), and resistant starch (RS). These three components are differently distributed in grains, however, all of them are represented in the endosperm. AX and βG, classified as non-starch polysaccharides (NSP), are in cell walls, whereas, RS is in the endosperm, being a starch fraction. As the chemical structure of DFs influences their digestibility, the identification of key actors involved in their metabolism can pave the way to improve their function in human health. Here, we reviewed the main achievements of plant biotechnologies in DFs manipulation in cereals, highlighting new genetic targets to be exploited, and main issues to face to increase the potential of cereals in fighting malnutrition.n

Can Manipulation of Durum Wheat Amylose Content Reduce the Glycaemic Index of Spaghetti?

Resistant starch (RS) in foods has positive benefits for potentially alleviating lifestyle diseases. RS is correlated positively with starch amylose content. This study aimed to see what level of amylose in durum wheat is needed to lower pasta GI. The silencing of starch synthases IIa (SSIIa) and starch branching enzymes IIa (SBEIIa), key genes involved in starch biosynthesis, in durum wheat cultivar Svevo was performed and spaghetti was prepared and evaluated. The SSIIa and SBEIIa mutants have a 28% and 74% increase in amylose and a 2.8- and 35-fold increase in RS, respectively. Cooked pasta was softer, with higher cooking loss but lower stickiness compared to Svevo spaghetti, and with acceptable appearance and colour. In vitro starch digestion extent (area under the digestion curve) was decreased in both mutants, but much more in SBEIIa, while in vivo GI was only significantly reduced from 50 to 38 in SBEIIa. This is the first study of the glycaemic response of spaghetti prepared from SBEIIa and SSIIa durum wheat mutants. Overall pasta quality was acceptable in both mutants but the SBEIIa mutation provides a clear glycaemic benefit and would be much more appealing than wholemeal spaghetti. We suggest a minimum RS content in spaghetti of ~7% is needed to lower GI which corresponded to an amylose content of ~58%