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

Structural Characterization and in vitro Lipid Binding Studies of Non-specific Lipid Transfer Protein 1 (nsLTP1) from Fennel (Foeniculum vulgare) Seeds

Non-specific lipid transfer proteins (nsLTPs) are cationic proteins involved in intracellular lipid shuttling in growth and reproduction, as well as in defense against pathogenic microbes. Even though the primary and spatial structures of some nsLTPs from different plants indicate their similar features, they exhibit distinct lipid-binding specificities signifying their various biological roles that dictate further structural study. The present study determined the complete amino acid sequence, in silico 3D structure modeling, and the antiproliferative activity of nsLTP1 from fennel (Foeniculum vulgare) seeds. Fennel is a member of the family Umbelliferae (Apiaceae) native to southern Europe and the Mediterranean region. It is used as a spice medicine and fresh vegetable. Fennel nsLTP1 was purified using the combination of gel filtration and reverse-phase high-performance liquid chromatography (RP-HPLC). Its homogeneity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. The purified nsLTP1 was treated with 4-vinyl pyridine, and the modified protein was then digested with trypsin. The complete amino acid sequence of nsLTP1 established by intact protein sequence up to 28 residues, overlapping tryptic peptides, and cyanogen bromide (CNBr) peptides. Hence, it is confirmed that fennel nsLTP1 is a 9433 Da single polypeptide chain consisting of 91 amino acids with eight conserved cysteines. Moreover, the 3D structure is predicted to have four α-helices interlinked by three loops and a long C-terminal tail. The lipid-binding property of fennel nsLTP1 is examined in vitro using fluorescent 2-p-toluidinonaphthalene-6-sulfonate (TNS) and validated using a molecular docking study with AutoDock Vina. Both of the binding studies confirmed the order of binding efficiency among the four studied fatty acids linoleic acid \u3e linolenic acid \u3e Stearic acid \u3e Palmitic acid. A preliminary screening of fennel nsLTP1 suppressed the growth of MCF-7 human breast cancer cells in a dose-dependent manner with an IC50 value of 6.98 µM after 48 h treatment

Genome-Wide Transcriptional Changes and Lipid Profile Modifications Induced by Medicago truncatula N5 Overexpression at an Early Stage of the Symbiotic Interaction with Sinorhizobium meliloti.

Plant lipid-transfer proteins (LTPs) are small basic secreted proteins, which are characterized by lipid-binding capacity and are putatively involved in lipid trafficking. LTPs play a role in several biological processes, including the root nodule symbiosis. The Medicago truncatula nodulin 5 (MtN5) LTP has been proved to positively regulate the nodulation capacity, controlling rhizobial infection and nodule primordia invasion. To better define the lipid transfer protein MtN5 function during the symbiosis, we produced MtN5-downregulated and -overexpressing plants, and we analysed the transcriptomic changes occurring in the roots at an early stage of Sinorhizobium meliloti infection. We also carried out the lipid profile analysis of wild type (WT) and MtN5-overexpressing roots after rhizobia infection. The downregulation of MtN5 increased the root hair curling, an early event of rhizobia infection, and concomitantly induced changes in the expression of defence-related genes. On the other hand, MtN5 overexpression favoured the invasion of the nodules by rhizobia and determined in the roots the modulation of genes that are involved in lipid transport and metabolism as well as an increased content of lipids, especially galactolipids that characterize the symbiosome membranes. Our findings suggest the potential participation of LTPs in the synthesis and rearrangement of membranes occurring during the formation of the infection threads and the symbiosome membrane

Biosynthesis and transport of terpenes

Terpenoids are the largest class of natural product that are produced by plants, with functions that range from a role in plant development to direct defence against pathogens and indirect defence against insects through the attraction of natural enemies. While terpene biosynthesis genes have been well studied, there is still only limited knowledge on how terpenes are transported within the cell and from the cell to the apoplast. In this thesis, different aspects of transport of terpenes in plants were addressed. Firstly, the issue of intermediate transport between enzymes was studied, focussing on the regulation of intermediate flux between two different biosynthesis enzymes (CYP71AV1 and DBR2) that determines the resulting Artemisia annua, low or high artemisinin, chemotype. We also investigated the role of Lipid Transfer Proteins and vesicles in the transport of terpenes. Some LTP mutants were indeed shown to emit lower terpene levels, but the exact mechanism could not be resolved. Inhibition of vesicle transport increased terpene levels, most likely due to an effect on protein stability. I conclude that there are multiple transport mechanisms involved in terpene transport which complicates the analysis of a single transport pathway.</p

Μελέτη της δράσης φωσφολιπασών D κατά την απόκριση του φυτού Gossypium hirsutum σε περιβαλλοντικές καταπονήσεις

Οι φωσφολιπάσες D (PLD, E.C. 3.1.4.4) είναι υδρολυτικά ένζυμα που δρουν σε φωσφολιπίδια των μεμβρανών και απαντώνται σε όλους τους οργανισμούς. Οι φυτικές PLD εμφανίζουν τη μεγαλύτερη ποικιλία ισομορφών, που κατατάσσονται σε έξι τάξεις (α-ζ). Από αυτές, οι α και δ είναι οι πιο διαδεδομένες. Η δράση των PLD στα φυτά συνδέεται με αποκρίσεις σε περιβαλλοντικές καταπονήσεις μέσω του παραγόμενου φωσφατιδικού οξέος (PtdOH), που δρα ως ενδοκυτταρικό σήμα. Στην περίπτωση παρουσίας πρωτοταγούς αλκοόλης, αναστέλλεται μερικώς η υδρολυτική δράση των PLD και καταλύεται η χαρακτηριστική για το ένζυμο αντίδραση μεταφωσφατιδυλίωσης. Στο καλλιεργούμενο βαμβάκι (Gossypium hirsutum) εντοπίστηκε για πρώτη φορά δραστικότητα PLD στο κλάσμα των μικροσωμάτων. Μετά από μηχανικό τραυματισμό -που προσομοιάζει με βλάβη στον ιστό προερχόμενη είτε από βιοτικούς είτε από αβιοτικούς παράγοντες- παρατηρήθηκε άμεση αύξηση της δραστικότητας της ισομορφής α της PLD στα φύλλα, τοπικά και συστημικά. Ο προσδιορισμός των ενδογενών επιπέδων PtdOH αποκάλυψε ανάλογη, ραγδαία αύξηση της συγκέντρωσης του λιπιδίου τοπικά, ενώ διπλασιασμός των επιπέδων του παρατηρήθηκε ως ύστερη, τόσο τοπική όσο και συστημική, απόκριση στον τραυματισμό. Επιπλέον, βρέθηκε πως η φωσφατιδυλοαιθανολαμίνη είναι το προτιμώμενο υπόστρωμα της PLDα και, επίσης, καταναλώνεται σε προτεραιότητα κατά τα πρώτα στάδια μετά τον τραυματισμό. Μετά από 30 min, όμως, βρέθηκε μετατόπιση της προτίμησης προς τη φωσφατιδυλοχολίνη, πιθανώς λόγω της δράσης άλλων ισομορφών και, κυρίως, της δ. Έλεγχος των επιπέδων RNA των ισομορφών PLDα και δ έδειξε πως τοπικά η έκφραση και των δύο ισομορφών επάγεται 30 min μετά τον τραυματισμό, ενώ συστημικά επάγεται η έκφραση μόνο της δ. Αυτό υποδεικνύει πως η PLDδ είναι κυρίως υπεύθυνη για τη συστημική απόκριση στον τραυματισμό, όπως επιβεβαιώνεται και από in vitro έλεγχο της δραστικότητάς της. Τέλος, πειράματα έκφρασης έδειξαν πως η λιποξυγενάση, η οξειδάση του NADPH και η συνθάση της κυτταρίνης επάγονται, επίσης, από τον τραυματισμό. Η in vivo επίδραση 1-βουτανόλης, που αναστέλλει την παραγωγή PtdOH, ανέστειλε και την επαγωγή των τριών γονιδίων, υποδεικνύοντας ότι αυτά είναι πιθανώς καθοδικοί στόχοι της δράσης της PLD κατά την καταπόνηση του φυτού.Phospholipases D (PLD, E.C. 3.1.4.4) hydrolyze membrane phospholipids and are present in all organisms studied. Plant PLDs exist in a wide range of isoforms, classified into six classes (α-ζ). Among them, α and δ are the most common. PLD activity in plants is involved in environmental stress responses via its product, phosphatidic acid (PtdOH), a signalling molecule. In the presence of a primary alcohol, PLD hydrolytic activity is partially inhibited, as PLD transphosphatidylation reaction is preferentially catalyzed. In this study, we have found for the first time PLD activity in microsomal fraction of cultivated cotton (Gossypium hirsutum). Upon mechanical wounding -resembling tissue damage caused by biotic or abiotic factors- acute increase of PLDα isoform activity was observed in leaves, both locally and systemically. Endogenous PtdOH determination revealed analogous, rapid increase of its concentration locally, while duplication in PtdOH levels was observed as a late, local and systemic, response to wounding. Moreover, it was found that phosphatidylethanolamine is the prefered PLDα substrate and it is also preferentially consumed during early stages after wounding. However, after 30 min, a shift in selectivity towards phosphatidylcholine was observed, possibly due to other PLD isoforms’ activity, mainly δ. Expression analysis of PLDα and δ isoforms showed mRNA accumulation of both isoforms in the wounded tissue, but only PLDδ was induced in systemic leaves, suggesting that PLDδ is mainly responsible for systemic response to wounding. This was confirmed by PLDδ activity assay. Finally, expression experiments showed that lipoxygenase, NADPH oxidase and cellulose synthase are wound-induced, as well. In vivo effect of 1-butanol, that suppresses PLD-derived PtdOH production, inhibits the induction of all three genes tested, implying that they are possible downstream PLD targets during G. hirsutum stress

Construction and analysis of a plant non-specific lipid transfer protein database (nsLTPDB)

<p>Abstract</p> <p>Background</p> <p>Plant non-specific lipid transfer proteins (nsLTPs) are small and basic proteins. Recently, nsLTPs have been reported involved in many physiological functions such as mediating phospholipid transfer, participating in plant defence activity against bacterial and fungal pathogens, and enhancing cell wall extension in tobacco. However, the lipid transfer mechanism of nsLTPs is still unclear, and comprehensive information of nsLTPs is difficult to obtain.</p> <p>Methods</p> <p>In this study, we identified 595 nsLTPs from 121 different species and constructed an nsLTPs database -- nsLTPDB -- which comprises the sequence information, structures, relevant literatures, and biological data of all plant nsLTPs <url>http://nsltpdb.life.nthu.edu.tw/</url>.</p> <p>Results</p> <p>Meanwhile, bioinformatics and statistics methods were implemented to develop a classification method for nsLTPs based on the patterns of the eight highly-conserved cysteine residues, and to suggest strict Prosite-styled patterns for Type I and Type II nsLTPs. The pattern of Type I is C X₂V X_5-7C [V, L, I] × Y [L, A, V] X_8-13CC × G X₁₂D × [Q, K, R] X₂CXC X_16-21P X₂C X_13-15C, and that of Type II is C X₄L X₂C X_9-11P [S, T] X₂CC X₅Q X_2-4C[L, F]C X₂[A, L, I] × [D, N] P X_10-12[K, R] X_4-5C X_3-4P X_0-2C. Moreover, we referred the Prosite-styled patterns to the experimental mutagenesis data that previously established by our group, and found that the residues with higher conservation played an important role in the structural stability or lipid binding ability of nsLTPs.</p> <p>Conclusions</p> <p>Taken together, this research has suggested potential residues that might be essential to modulate the structural and functional properties of plant nsLTPs. Finally, we proposed some biologically important sites of the nsLTPs, which are described by using a new Prosite-styled pattern that we defined.</p