WO2020157164A1 - Modified plant with improved rubisco activity - Google Patents

Abstract

The present invention concerns the improvement of the RuBisCO activity in a plant by the expression of a transcription elongation factor SPT5 that is mutated in its NGN domain.

Description

MODIFIED PLANT WITH IMPROVED RUBISCO ACTIVITY

FIELD OF THE INVENTION

The present invention concerns the improvement of the RuBisCO activity in a plant, particularly for improving starch, oil, and/or protein production in a cultivated or commercial crop plant.

STATE OF THE ART

Global agricultural demand is rapidly increasing as the world’s population reaches 9 billion humans by the middle of the century (FAOSTAT 2016). According to projections, agricultural production will have to increase by 70 to 100% to meet this demand (Ray et al., 2013; Tilman et al., 201 1), even while available arable land is stagnating or even decreasing. Selective cultivation and increased inputs (fertilizers) have allowed global agricultural production to double, while improving yield potential and resistance to environmental and biotic stresses. Improving the efficiency of photosynthesis has played only a minor role in improving yield potential (Ort et ai, 2015; Ray et ai, 2013; Zhu et ai, 2010). Although it is necessary to improve crop plants and cropping systems to meet the challenge of doubling production, improving yield potential must play a central role for which improving the efficiency of photosynthesis should be a central focus.

In addition, carbon dioxide poses several environmental problems in modern society. Carbon dioxide is an important component of the Earth’s atmosphere because it allows visible light to pass through the atmosphere while trapping some of the long-wave infrared radiation that is reflected and radiated as heat from the Earth’s surface. Unfortunately, modern processes release huge amounts of carbon dioxide into the atmosphere, resulting in unremitting global warming. It is feared that such warming could lead to an imbalance that would alter the ecosystem. Consequently, the amount of carbon dioxide entering the atmosphere must be reduced.

There are two solutions for reducing the amount of carbon dioxide entering the atmosphere. The first alternative consists in reducing the amount of carbon dioxide emitted by industrial processes. The second alternative involves recycling the carbon dioxide in Earth’s atmosphere. The environmental problems associated with carbon dioxide pollution can be solved by recycling carbon dioxide by absorption by plants.

RuBisCO is the enzyme that binds carbon dioxide from the Calvin cycle in photosynthetic organisms. This protein, its various forms, and their sequences, structures and activities have been widely studied and are well known to the skilled person (Parry et ai., 2013).

Ribulose-1 ,5-bisphosphate carboxylase / oxygenase (RuBisCO, E.C. 4.1.1.39) is the most abundant and possibly the most important enzyme on Earth. It catalyses the first limiting step of carbon fixation by photosynthesis, namely the transfer of atmospheric CO2 to the five- carbon acceptor, ribulose-1 ,5-bisphosphate (RuBP) to generate two 3-phosphoglycerate (3- PGA) molecules. Due to its key position in biomass production, RuBisCO is important for agriculture. For several reasons, it is widely accepted that improving RuBisCO activity will lead to a significant increase in crop productivity. First, the reaction catalysed by RuBisCO limits the growth rate of plants under optimal growth conditions (high temperature and light intensity, abundant nitrogen). Second, compared with many other enzymes, RuBisCO appears to be an ineffective catalyst that leaves much room for optimization (Whitney et a!., 2011).

An increase in RuBisCO activity in a genetically modified plant includes, but is not limited to, an increase in photosynthesis rate and/or plant productivity.

Improving RuBisCO activity in photosynthetic organisms, and in particular plants, has the potential to lead to a significant improvement in phenotypic traits such as plant growth, plant biomass, crop yield, increased cell proliferation, increased organ or cell size and increased total plant mass.

Yield is normally defined as the measurable product of the economic value of a crop. This can be defined in terms of amount and/or quality. Yield depends directly on several traits, such as organelle number and size, plant architecture (for example, number of branches), seed production, leaf senescence, root development, nutrient absorption, stress tolerance, photosynthetic carbon uptake rates and early vigour can also be important traits in determining yield. Optimizing and improving the above-mentioned traits can therefore help to increase crop yields. Yield increase can be characterized by increasing the plant’s yield under stress-free conditions or increasing the plant’s yield under one or more environmental stress conditions, including, but not limited to, water stress, cold stress, heat stress, high salinity stress, shade stress and stress due to low nitrogen availability. In another aspect of the present invention, the genetically modified plants have improved traits, such as improved plant development, plant morphology, plant physiology or seed composition compared with a corresponding trait of a control plant. The various aspects of this invention are particularly useful for genetically modified seeds and genetically modified plants with improved traits in maize, soybean, cotton, canola, rapeseed, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruits and vegetables, and turf.

An increase in seed yield is a particularly important trait because the seeds of many plants are important for human and animal consumption. Crops such as maize, rice, wheat, canola, rapeseed and soybean account for more than half of total human caloric intake, whether through direct consumption of seeds or through the consumption of meat products raised on processed seeds. They are also a source of sugars, oils and many types of metabolites used in industrial processes. Seeds contain an embryo (source of new shoots and roots) and an endosperm (source of nutrients for embryo growth during germination and early seedling growth). Seed development involves many genes and requires the transfer of metabolites from the roots, leaves and stems to the growing seed. The endosperm, in particular, assimilates the metabolic precursors of carbohydrates, oils and proteins and synthesizes them into storage macromolecules to fill the seed. An increase in plant biomass is important for forage crops such as alfalfa, silage maize and hay. Many genes are involved in the metabolic pathways that contribute to plant growth and development. Modulating the expression of one or more of these genes in a plant can produce a plant with improved growth and development compared with a control plant but can often produce a plant whose growth and development are altered relative to a control plant. Consequently, methods for improving plant growth and development are needed. The inventors have identified genetic modifications to be made in plants in order to improve RuBisCO activity.

DISCLOSURE OF THE INVENTION

The invention thus concerns a plant genetically modified at the level of a transcription elongation factor to improve RuBisCO activity.

The modified plants according to the invention are more particularly crop plants, selected notably from rice, wheat, sugar cane, maize, sorghum, rye, barley, millet, rapeseed, soybean, sunflower, cotton, tomato, legume, tobacco, camelina, aubergine, chilli, pepper, potato, peanut, broad bean, bean, lentil, alfalfa, chickpea, clover, squash, cucumber, marrow, melon, pumpkin.

The invention also concerns parts of the modified plants, notably the roots, leaves, fruits or seeds of these plants, or manufactured products containing parts of these modified plants according to the invention.

The invention concerns a process for growing plants according to the invention.

DESCRIPTION OF THE FIGURES

Figure 1 shows a 3D representation of the superposition of the NGN domains of SPT5 from Arabidopsis thaliana and maize (Zea mays). The secondary structural elements of the NGN domain of SPT5 from A. thaliana are shown in black. Alanine 231 is represented as a black stick. The secondary structural elements of the NGN domain of SPT5 from Z. mays have been superimposed on those of the NGN domain of SPT5 from A. thaliana and are shown in light grey. The side chain of alanine 237 structurally equivalent to alanine 231 is shown as a light grey stick.

Figure 2 shows the consensus sequence identified in plant SPT5 protein sequences by alignment of protein 3D representations (the relative size of the letters representing an amino acid at a given position is associated with the number of occurrences where the same amino acid is found at the same position in an SPT5 gene).

DEFINITIONS

The terms “genetically modified plant” or“modified plant” are used interchangeably herein and refer to plants that have been genetically modified to express or overexpress endogenous nucleotide sequences, to express heterologous nucleotide sequences, or that have an altered expression of an endogenous gene.

“Alteration” means that the expression of the gene, or level of an RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or the activity of one or more polypeptides or polypeptide subunits, is regulated so that the expression, level or activity is higher or lower than that observed in the absence of modification.

It is understood that the terms“genetically modified plant’ or“modified plant’ refer not only to a particular genetically modified plant, but to the progeny or the potential progeny of such a plant. As certain modifications may occur in subsequent generations, due to mutation or environmental influences, these offspring may not be identical to the parent organism, but they are still included within the scope of the term as used herein.

The term “exogenous" as used herein in reference to various molecules (nucleotide sequences, peptides, enzymes, etc.) refers to molecules that are not normally or naturally found in and/or produced by the plant in question. Conversely, the term“ endogenous” or “native” in reference to various molecules (nucleotide sequences, peptides, enzymes, etc.) refers to molecules that are normally or naturally found in and/or produced by the organism concerned.

“Genetically modified’ means that the plant genome has been modified to incorporate a nucleic sequence either to replace a native nucleic sequence, to modify a native nucleic sequence, to delete a native nucleic sequence, or to add a new nucleic sequence. Said nucleic sequence may have been introduced into the genome of said plant or of one of its ancestors by means of any suitable molecular cloning method. In the context of the invention, the plant genome refers to all genetic material contained in the plant, including extrachromosomal genetic material contained for example in plasmids, episomes, synthetic chromosomes, etc. The introduced nucleic sequence may be a heterologous sequence, i.e. one that does not naturally exist in said organism, or a homologous sequence. The nucleic sequence may be a gene fragment introduced to replace the native gene or a fragment of the latter, in particular to replace a coding sequence, in whole or in part, or to delete the native coding sequence, in whole or in part. To introduce a new coding sequence, a transcription unit containing the nucleic sequence of interest, under the control of one or more promoters, is introduced into the organism’s genome. Such a transcription unit also includes, advantageously, common sequences such as transcription terminators and, if need be, other transcription regulatory elements.

It is understood that a“homologous gene” has a substantially marked sequence similarity with another gene present in the chromosome.“Homologous genes” are all derived from the same ancestral gene.

“Yield” or“crop yield” refers to the measurement of photosynthesis, the measurement of the amount of a crop that has been harvested per unit area of land. Crop yield is the measure often used for cereals and generally corresponds to the amount of plants harvested per unit area over a given period, i.e. in metric tonnes per hectare or in kilograms per hectare. Crop yield can also refer to the seed or biomass produced or generated by the plant.

“Trait improvement” includes, but is not limited to, increasing yield, notably increasing yield under non-stress conditions and increasing yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold exposure, heat exposure, reduced availability of nitrogen nutrients, phosphorus and high plant densities. Many agronomic traits can influence“yield”, notably plant height, stem strength, root strength, stem diameter, stem volume, wood density, stem dry weight, bark dry weight, average internode length, internode number, vegetative growth, biomass production, seed production, pod number, pod position on the plant, the incidence of pod shatter, seed size, nodulation and nitrogen fixation efficiency, nutrient uptake efficiency, resistance to biotic and abiotic stress, carbon uptake, plant architecture, lodging resistance, seed germination percentage, seedling vigour and juvenile traits. The other characteristics that may affect yield are notably germination efficiency (including in harsh conditions), growth rate (including growth rate in harsh conditions), number of ears, number of seeds per ear, seed size, seed composition (starch, oil, protein, moisture content) and seed filling characteristics. The generation of transgenic plants with desirable phenotypic properties, which may or may not confer an increase in overall plant yield, is also of interest. These properties include improved plant morphology, plant physiology or improved components of the mature seed harvested from the genetically modified plant.

“Trait improvement” or“trait optimization” refers to a detectable and desirable difference in a characteristic of a genetically modified plant compared with a control plant or with a reference. In some cases, trait improvement is measured quantitatively. For example, improving the trait may involve at least one desirable difference of 2% in an observed trait, at least one desirable difference of 5%, at least one desirable difference of about 10%, at least one desirable difference of about 20%, at least about 30% desirable difference, at least about 50% desirable difference, at least about 70% desirable difference, or at least about 100% difference or an even more desirable difference. In other cases, the improvement of the trait is measured only qualitatively. It is known that there are natural variations in a trait. Consequently, the observed trait improvement implies a change in the normal distribution of traits in the genetically modified plant relative to the distribution of traits observed in a control plant or a reference, which is evaluated by statistical methods.

The“yield improvement” of a genetically modified plant of this invention is demonstrated and measured in different ways, including photosynthetic yield, specific weight, number of seeds per plant, seed weight, number of seeds per unit area (i.e. seeds or seed weight per square meter), bushels per square meter, tonnes per square meter, kilos per hectare. The genetic modification described in this invention improves RuBisCO activity, thus improving photosynthesis, resulting in genetically modified plants with improved growth and development, and ultimately increased yield.

“Crop plant” refers to plants that are grown on farms.

“Commercial plant” or“commercial variety” refers to plants or plant varieties that have suitable agronomic properties to be grown on farms.

A "cultivated plant" is understood within the scope of the invention to refer to a plant that is no longer in the natural state but has been developed by human care and for human use and/or consumption. "Cultivated plants" are further understood to exclude those wild-type species which comprise the trait being subject of this invention as a natural trait and/or part of their natural genetics.

DETAILED DESCRIPTION OF THE INVENTION

The invention concerns a plant that is genetically modified to improve RuBisCO activity.

The plants include, but are not limited to, monocotyledonous or dicotyledonous plants, and more particularly crop plants. Monocotyledonous crops include rice, wheat, sugar cane, maize, sorghum, rye, barley and millet. Dicotyledonous crops include rapeseed, soybean, sunflower, cotton, tomato, legume, tobacco, camelina, Arabidopsis thaliana, eggplant, chilli, pepper, potato, peanut, broad bean, bean, lentil, alfalfa, chickpea, clover, squash, cucumber, marrow, melon, pumpkin.

Plants have different ways of fixing CO2 during photosynthesis. The type of photosynthesis of a plant is determined by the number of carbon atoms of the first organic molecule formed during CO2 fixation. These mechanisms differ in the efficiency of this carboxylation step. C3 plants convert CO2 into a 3-carbon compound (phosphoglyceric acid, or PGA) with ribulose-1 ,5-bisphosphate carboxylase / oxygenase (RuBisCO). C4 plants and CAM plants convert CO2 into a 4-carbon intermediate (oxaloacetate, or OAA) with phosphoenolpyruvate carboxylase (PEPC). CAM plants differ from C4 plants because CAM plants fixC0₂at night to store it as a 4-carbon intermediate (malic acid).

C3 plants include Arabidopsis thaliana, tobacco, tomato, rice, wheat, soybean, sunflower, rapeseed, cucumber and alfalfa. C4 plants include maize, sorghum, millet and sugar cane. CAM plants include pineapple and agave.

The modification of the plant genome occurs in the genome of all of its constituent cells.

The invention thus concerns a plant genetically modified at the level of a transcription elongation factor to improve RuBisCO activity.

Transcription elongation factors are known to the skilled person, in particular the elongation factor SPT5, a transcription elongation factor that assists in DNA-templated RNA synthesis by cellular RNA polymerases (RNAP) (Yakhnin and Babitzke, 2015). The modular domain composition of SPT5 and the way it binds to RNAP are conserved in all three domains of life. SPT5 closes RNAP around a DNA binding channel, thereby modulating transcription processivity (Hartzog et al., 1998; Wada et al., 1998). Recruitment of additional factors to elongating RNAP may be another conserved function of this ubiquitous protein. SPT5, present in all eukaryotes and archaea, couples RNA processing and chromatin modification to transcription elongation (Blythe et al., 2016). SPT5 is important in the stability of RNAP elongation machinery.

The table below lists, by way of example, the sequences encoding an elongation factor SPT5.

Table 1 : Examples of sequences encoding SPT5

The corresponding proteins in the plants are also known or identifiable by comparing sequences with the SPT5 sequences above, in particular by comparing the 3D structures to identify the secondary structures common to the proteins and in particular the domains equivalent to the SPT5 NGN domain (Figure 1) (Guo et al., 2008; Yakhnin and Babitzke, 2015).

The SPT5 NGN domain consists of a four-stranded antiparallel beta sheet (b1 , b2, b3 and b4) flanked by three alpha helices (a1 , a2 and a3) (Guo et al., 2008) (Figure 1). This domain can be identified by sequence alignment and by molecular modelling of the protein 3D structures by superimposing the 3D representations as shown in Figure 1. The skilled person is familiar with protein 3D representation methods, notably with the PyMOL software (The PyMOL Molecular Graphics System (2002), see www.pymol.org). The procedures for aligning a sequence comparison are well known to the skilled person. Thus, a mathematical algorithm can be used to determine the percentage of sequence identity of two sequences. Below are several non-limiting examples of algorithms: the mathematical algorithm of Myers and Miller (Myers and Miller, 1988), the CABIOS algorithm (Grice et al., 1997; Wheeler and Hughey, 2000), the global alignment method of Needleman and Wunsch (Needleman and Wunsch, 1970; Pearson and Lipman, 1988), the local alignment method (Karlin and Altschul, 1993, 1990). The computer versions of these mathematical algorithms can be used for sequence comparison to determine sequence identity. These include, but are not limited to, the Geneious® program purchased from Biomatters (New Zealand), the MultiAlin online program (Corpet, 1988) (http://multalin.toulouse.inra.fr/multalin/), the T-Coffee online program (http://tcoffee.crg.cat/) which is a multiple sequence alignment package. T-Coffee can be used to align sequences or to combine the output of alignment methods (Clustal, Mafft, Probcons, Muscle, etc.) into a single alignment (M-Coffee). The default settings to use are the alignments using programs from the following examples: CLUSTAL program (Corpet, 1988; Higgins and Sharp, 1989, 1988; Huang et al., 1992; Pearson, 1994), the program of Myers and Miller (Myers and Miller, 1988) and the BLASTP BLAST protein search program (score = 100, word length = 12), to obtain protein sequences homologous to a protein sequence of the present invention, in order to obtain split alignments (for comparison purposes), such as those described by Altschul et al., 1997 (see blast.ncbi.nlm.nih.gov).

This sequence can be identified by sequence alignment and molecular modelling of protein 3D structures by superimposing the 3D representations as shown in Figure 1. The skilled person is familiar with protein sequence alignment and 3D representation methods.

According to the invention, elongation factor SPT5 refers in particular to any protein containing an NGN domain which includes an NGN sequence having at least 30% identity with the following NGN sequence of SPT5 from Arabidopsis t ha liana· PKLWMVKCAI GREREVAVCL MQKFIDRGAD LQIRSVVALD HLKNFIYVEA DKEAHVKEAI KGMRNIYANQ KILLVPIREM TDVLSVE (SEQ ID NO 1). Preferably, the NGN domain has at least 35% identity with the Arabidopsis thaliana SPT5 NGN sequence, more preferably at least 40% identity.

More particularly, elongation factor SPT5 refers to a protein containing an NGN domain that includes a 12-amino-acid consensus sequence as shown in Figure 2.

This consensus sequence consists of a first non-polar amino acid which will mainly be leucine or valine, a second amino acid with a polar and/or bulky side chain (lysine, glutamine, arginine, etc.), a third amino acid which may be either asparagine or glycine, a fourth amino acid whose side chain has large steric hindrance and/or is polar (tyrosine, phenylalanine, serine, etc.), a fifth non-polar amino acid (leucine, isoleucine, valine, etc.), a sixth amino acid whose side chain has large steric hindrance and/or is polar (tyrosine, phenylalanine, etc.), a seventh non-polar amino acid (isoleucine, valine, etc.), an eighth amino acid which will be glutamic acid, a ninth amino acid which will be either alanine or serine or valine, a tenth and an eleventh amino acid having a polar and/or bulky side chain, a twelfth amino acid being mainly an amino acid having a polar side chain (glutamic acid, aspartic acid, etc.) but which may also be a non-polar amino acid (alanine, etc.).

The plant protein consensus sequences are given in Table 2 below.

Table 2: Exemplary 12-amino-acid consensus sequences in the SPT5 NGN domain

The genetic modification consists in expressing, in the plant according to the invention, a transcription elongation factor SPT5 that is mutated in its NGN domain.

More specifically, the genetic modification consists in expressing, in the plant according to the invention, a factor SPT5 mutated in the C-terminus of the b3 sheet (Figure 1).

More specifically, the mutation will be introduced into the 12-amino-acid consensus sequence shown in Figure 2.

According to a preferred embodiment, the mutated protein expressed in the plant according to the invention contains a transposition of the ninth amino acid of the consensus sequence in Figure 2, alanine, to any other amino acid. More specifically, the mutation consists of a transposition of the ninth amino acid, alanine, to proline. This will make it easy to identify the structural equivalence of the position of the A9P mutation in the SPT5 NGN domain consensus sequence in the corresponding plant proteins as identified above.

According to a first embodiment of the invention, the genetically modified plant contains a heterologous gene encoding a mutated protein as defined above, whether it is a plant protein or a microorganism protein. In particular, a heterologous gene that encodes the modified protein of a plant of the same species will be expressed. Advantageously, the coding sequence of the heterologous gene will be optimized at the codon level for expression in the genetically modified plant, particularly when a mutated protein derived from a microorganism is expressed in the plant, or when a mutated protein of monocotyledonous plant origin is expressed in a dicotyledonous plant, and vice versa. Codon optimization methods are well known to the skilled person.

The regulatory elements of the introduced heterologous gene are well known to the skilled person. Advantageously, the regulatory elements will be selected to promote its expression in the aerial parts of the plant and more particularly the leaves. This is particularly the case for constitutive regulatory elements, in particular the usual constitutive promoters used for expressing genes of interest in plants, such as cauliflower mosaic virus promoters, such as the CaMV35S and CaMV19S promoters, the nopaline synthase promoter, the alfalfa mosaic virus promoters, the plant actin gene promoters, notably the rice or maize actin gene promoters, and the plant histone gene promoters, particularly the rice and maize histone gene promoters.

According to a second, preferred, embodiment of the invention, the plant is genetically modified by introducing a mutation into the coding sequence of the native gene encoding the transcription elongation factor.

This mutation may be introduced, for example, by replacing all or part of the coding sequence of the native gene, notably using known homologous recombination techniques. Particular mention may be made of targeted gene modification techniques, notably for introducing point mutations, which are also known to the skilled person, notably described in reference works such as Advances in New Technology for Targeted Modification of Plant Genomes (Zhang et al., 2015) and Site-directed insertion of transgenes (Renault and Duchateau, 2012).

Particular mention may be made of the methods described in patent applications WO 91/02070, WO 2009/006297, WO 2010/009147, WO 2011/064750, WO 20110/64736, WO 2013/026740, WO 2013/102875, WO 2013/160230, WO 2014/093768, WO 2014/161821.

This mutation may also be a point mutation introduced by replacing the codon of the amino acid to be transposed with the codon of the amino acid that replaces it in the protein sequence.

The methods for replacing codons in plant genes are well known to the skilled person, notably by the use of precise genome editing technologies to modify the endogenous sequence. These methods include, but are not limited to, meganucleases designed against the plant genomic sequence of interest (D’Halluin et al., 2013b), CRISPR-Cas9 (Jaganathan et al., 2018; Jiang et al., 2013), CRISPR-Cpf1 (Li et al., 2018; Zaidi et al., 2017; Zetsche et al., 2016), TALEN and other technologies for precise genome editing (D’Halluin et a!., 2013a; Feng et al., 2013; Podevin et al., 2013; Qi et al., 2016; Zetsche et al., 2015), Argonaute- mediated DNA insertion (Gao et al., 2016), Cre-lox site-specific recombination (Albert et al., 1995; Lyznik et ai, 2003), FLP-FRT recombination (Li et ai, 2009); Bxbl-mediated integration (Yau et al., 2011), zinc finger induced integration (Cai et al., 2009; Wright et al., 2005) and homologous recombination (Lieberman-Lazarovich and Levy, 2011 ; Puchta, 2002). Mention may also be made of the patents and patent applications US 9,840,699, US 2013/0321210, EP 3 216 687 and the publication by Nishida et ai., 2016.

According to a preferred embodiment of the invention, the plant is genetically modified by the expression of a native gene modified specifically so as to express a transcription elongation factor mutated at the 9^th amino acid of the consensus sequence defined above.

In the case where the genetically modified plant has several homologous genes encoding a transcription elongation factor, the genetic modification may be introduced into a single homologous gene, into several homologous genes, or into all homologous genes of the genetically modified plant. The preparation of a plant genetically modified by the expression of a heterologous gene or by the expression of a mutated native sequence requires the introduction of nucleic acid molecules into the genome of said plants. Plant transformation methods are well known to the skilled person, notably described in reference works such as Plant Transformation Technologies (Stewart et al., 201 1), Recent Advances in Plant Biotechnology and Its Applications (Neumann et al., 2008), Transgenic Plants: Methods and Protocols (Pena, 2005), Handbook of Molecular and Cellular Methods in Biology and Medicine (Cseke et al., 201 1). Suitable methods include, but are not limited to, microinjection (Crossway, 1989), electroporation (D’Halluin et al., 1992; Riggs and Bates, 1986), Agrobacterium-mediated transformation (US Patents No. 5,563,055 and No. 5,981 ,840), direct gene transfer (Paszkowski et al., 1984), microprojectile bombardment (US Patents No. 4945050, No. 5,879,918, No. 5,886,244 and No. 5,932,782; (Gamborg and Phillips, 1995; McCabe et al., 1988)) and Led transformation (WO 00/28058). Other examples are also available depending on the target plant, for example for onion (T M Klein et al., 1988; Sanford et al., 1987; Weising et al., 1988); for soybean (Christou et al., 1988; Finer and McMullen, 1991 ; Singh et al., 1998); for rice (Christou and Ford, 1995; Datta et al., 1990; Li et al., 1993); for maize (Fromm et al., 1990; Ishida et al., 2007, 1996; Klein et al., 1989; Theodore M. Klein et al., 1988) and US patents No. 5,240,855; No. 5,322,783; for cereals (Hooykaas-Van Slogteren et al., 1984) and US patent No. 5,736,369 for lily.

The present invention may be used to transform any plant species, including, but not limited to, monocots and dicots. Exemplary plant species of interest include, but are not limited to Arabidopsis thaliana, tobacco ( Nicotiana tabacum), maize (Zea mays), tomato ( Solanum lycopersicum), rice ( Oryza sativa), wheat ( Triticum aestivum), soybean ( Glycine max), cucumber ( Cucumis sativus), sunflower ( Helianthus annuus), rapeseed, canola, Brassica sp. (for example, B. napus, B. rapa, B. juncea), in particular the Brassica species useful as sources of seed oil, alfalfa ( Medicago sativa), barrel medic ( Medicago truncatula), rye ( Secale cereale), sorghum ( Sorghum bicolor, Sorghum vulgare), millet (e.g., millet ( Pennisetum glaucum), common millet ( Panicum miliaceum), foxtail millet ( Setaria italica), finger millet ( Eleusine coracana), saffron ( Carthamus tinctorius), potato ( Solanum tuberosum), peanut ( Arachis hypogaea), camelina ( Camelina sativa), cotton ( Gossypium barbadense, Gossypium hirsutum), sweet potato ( Ipomoea batatas), cassava (Manihot esculenta), coffee ( Coffea spp.), coconut ( Cocos nucifera), pineapple ( Ananas comosus), citrus fruits ( Citrus spp.), cocoa ( Theobroma cacao), tea ( Camellia sinensis), banana ( Musa spp.), avocado ( Persea americana), fig ( Ficus carica), guava (Psidium guajava), mango ( Mangifera indica), olive (O/ea europaea), papaya ( Carica papaya), cashew nut ( Anacardium occidentale), macadamia nut ( Macadamia integrifolia), almond ( Prunus amygdalus), sugar beet ( Beta vulgaris), sugar cane ( Saccharum spp.), pigeon pea ( Cajanus cajan), chickpea ( Cicer arietinum), Cucurbitaceae (Cucurbita sp.) such as squash ( Cucurbita pepo L.) and pumpkin ( Cucurbita maxima subsp. maxima), brown mustard ( Brassica juncea ), ryegrass ( Lolium sp.), beans ( Phaseolus spp.), oil palm ( Elaeis guineensis Jacq.), apple ( Malus domestica ), vine (for example Vitis vinifera ), onion ( Allium cepa ), oat, barley ( Hordeum vulgare), legume, glasswort ( Salicornia sp.), fruits and vegetables, such as blackberry, blueberry, strawberry and raspberry, cantaloupe, carrot, coffee, cucumber, eggplant, grape, lettuce, mango, melon, papaya, peppers, spinach, woody species such as pine, poplar and eucalyptus, mint, rubber tree, ornamental plants and conifers.

The plants according to the invention advantageous have an improved yield compared with a corresponding plant that does not have an SPT5 gene mutation. In particular, for plants grown for starch production, such as cereals, soybean and maize, this improved yield is found in a higher amount of starch relative to biomass dry weight produced than that obtained with a plant that does not include the SPT5 gene mutation. For plants grown for oil production, oilseeds such as soybean, rapeseed, sunflower in particular, this improved yield is found in a higher amount of fat relative to biomass dry weight produced than that obtained with a plant that does not include the SPT5 gene mutation. The improved yield is also found in an improved seed protein and/or starch content compared with a corresponding plant that does not have an SPT5 gene mutation.

The genetically modified plants according to the invention, in particular Arabidopsis thaliana, poplars or eucalyptus, have an improved change in plant height and/or stem diameter and/or stem volume and/or wood density and/or stem dry weight and/or bark dry weight and/or average internode length and/or internode number and/or vegetative growth and/or biomass production and/or seed production and/or seed lipid content compared with a corresponding plant that does not have an SPT5 gene mutation.

In particular, the genetically modified tomatoes according to the invention have an improved fruit yield compared with a corresponding plant that does not have an SPT5 gene mutation.

The genetically modified plants according to the invention, in particular tobacco, have an improvement in leaf size and/or leaf angle and/or concentration of chemical components such as nicotine, total alkaloids or reducing sugars compared with a corresponding plant that does not have an SPT5 gene mutation.

The genetically modified plants according to the invention, in particular rice, have an improvement in grain yield and/or grain quality compared with a corresponding plant that does not have an SPT5 gene mutation.

The genetically modified plants according on the invention may also be modified with genes of interest in order to give them agronomic properties of interest, such as resistance to pests or diseases, resistance to certain stresses such as water stress, increased fruit or seed size, etc. According to the preferred embodiments of the invention, the plants modified by introduction of a mutation into the coding sequence of the native gene encoding the transcription elongation factor are selected from the following plants: Arabidopsis thaliana, tobacco ( Nicotiana tabacum ), maize (Zea mays), tomato ( Solanum lycopersicum), rice ( Oryza sativa), wheat ( Triticum aestivum ), soybean ( Glycine max), cucumber ( Cucumis sativus), sunflower ( Helianthus annuus), rapeseed, canola, Brassica sp. (for example, B. napus, B. rapa, B. juncea), cotton ( Gossypium barbadense, Gossypium hirsutum), plantain ( Plantago sp.), yam ( Dioscorea sp.), barley ( Hordeum vulgare), sorghum ( Sorghum bicolor, Sorghum vulgare), beans ( Phaseolus spp.), sweet potato ( Ipomoea batatas), cassava ( Manihot esculenta), potato ( Solanum tuberosum), sugar cane ( Saccharum spp.), banana ( Musa spp.), onion ( Allium cepa), apple ( Malus domestica), vine (for example, Vitis vinifera), oil palm ( Elaeis guineensis Jacq.), peanut ( Peanut hypogaea) and barrel medic ( Medicago truncatula).

According to a preferred embodiment, the SPT5 protein expressed in the genetically modified plant according to the invention has a mutation consisting of a transposition of the ninth amino acid, alanine, to any amino acid other than the amino acid present in the native consensus sequence.

More preferentially, the mutation consisting of a transposition of the ninth amino acid of the consensus sequence, alanine, to proline is introduced.

The skilled person will be able to identify the structural equivalence of the position of the mutation.

To generate the mutation of alanine to proline, the first base, guanine (G), of the alanine codon must be changed to cytosine (C). A major limitation of cytidine deaminase-mediated base editing is its inability to induce other forms of base conversion beyond the transition cytosine (C) to adenine (A) and guanine (G) to thymine (T). However, adenine base editors have been adapted for plant applications (Hua et al., 2018; Yan et al., 2018). The combination of adenine and cytosine base editors can now generate the four base transition mutations. For example, the modification of cytosine to guanine has already been obtained (Ma et al., 2016).

In order to generate the mutation of alanine to proline, base and/or gene editing tools will have to be developed specifically for each plant as a function of the nucleotide sequence of the gene. The change from G to C may be carried out with, but is not limited to, Cas9 where a guide RNA targeting the non-coding DNA strand is required. To perform editing with Cpf1 , for example, a guide RNA targeting the coding DNA strand is required. Different endonucleases will have to be evaluated each time, such as, but not limited to, SpCas9 (Hua et al., 2018), Cpf1 (Li et al., 2018), SaCas9 (Jaganathan et al., 2018; Jia et al., 2017) and xCas9 (Hu et al., 2018). Each endonuclease recognizes different protospacer adjacent motif (PAM) sequences that will be critical for binding the endonuclease to the target DNA. For example, SpCas9 recognizes an NGG PAM sequence, xCas9 recognizes an NG PAM sequence, Cpf1 requires a thymine-rich PAM sequence, SaCas9 requires an NGG PAM sequence.

In Arabidopsis thaliana, for example, the amino acid at position 9 of the consensus sequence corresponds to alanine at position 231 in the SPT5 (Gl: 826391) protein and to alanine at position 218 in the SPT5 (Gl: 817982) protein. The mutation advantageously consists of a substitution of alanine 231 in the SPT5 (Gl: 826391) protein and/or alanine at position 218 in the SPT5 (Gl: 817982) protein by any amino acid other than alanine. More preferentially, the mutation consists of a substitution of alanine 231 in the SPT5 (Gl: 826391) protein and/or alanine at position 218 in the SPT5 (Gl: 817982) protein by proline. In the case of the substitution of alanine 231 of the SPT5 (Gl: 826391) protein by proline, the mutation will be performed using, by way of non-limiting example, Cpf1 or SaCas9 endonuclease. In the case of the substitution of alanine at position 218 in the SPT5 (Gl: 817982) protein by proline, the mutation will be performed using, by way of non-limiting example, xCas9 or SaCas9 endonucleases.

In tobacco ( Nicotiana tabacum ), for example, the amino acid at position 9 of the consensus sequence corresponds to alanine at position 178 in the SPT5 (Gl: 107774556) protein and to alanine at position 228 in the SPT5 (Gl: 107770981) protein. The mutation advantageously consists of a substitution of alanine at position 178 in the SPT5 (Gl: 107774556) protein and/or alanine at position 228 in the SPT5 (Gl: 107770981) protein by any amino acid other than alanine. More preferentially, the mutation consists of a substitution of alanine at position 178 in the SPT5 (Gl: 107774556) protein and/or alanine at position 228 in the SPT5 (Gl: 107770981) protein by proline. In the case of the substitution of alanine at position 178 in the SPT5 (Gl: 107774556) protein by proline, the mutation will be performed using, by way of non-limiting example, xCas9 endonuclease. In the case of the substitution of alanine at position 228 in the SPT5 (Gl: 107770981) protein by proline, the mutation will be performed using, by way of non-limiting example, Cpf1 endonuclease.

In maize (Zea mays), for example, the amino acid at position 9 of the consensus sequence corresponds to alanine at position 237 in the SPT5 (Gl: Zm00001 d044971) protein and to alanine at position 237 in the SPT5 (Gl: Zm00001 d037142) protein. The mutation advantageously consists of a substitution of alanine at position 237 in the SPT5 (Gl: Zm00001 d044971) protein and/or alanine at position 237 in the SPT5 (Gl: Zm00001 d037142) protein by any amino acid other than alanine. More preferentially, the mutation consists of a substitution of alanine at position 237 in the SPT5 (Gl: Zm00001 d044971) protein and/or alanine at position 237 in the SPT5 (Gl: Zm00001d037142) protein by proline. In the case of the substitution of alanine at position 237 in the SPT5 (Gl: Zm00001d044971) protein and/or alanine at position 237 in the SPT5 (Gl: Zm00001d037142) protein by proline, the mutation will be performed in both cases using, by way of non-limiting example, saCas9 or Cpf1 endonucleases. In tomato ( Solanum lycopersicum), for example, the amino acid at position 9 of the consensus sequence corresponds to alanine at position 217 in the SPT5 (Gl: 101260813) protein. The mutation advantageously consists of a substitution of alanine at position 217 in the SPT5 (Gl: 101260813) protein by any amino acid other than alanine. More preferentially, the mutation consists of a substitution of alanine at position 217 in the SPT5 (Gl: 101260813) protein by proline. In the case of the substitution of alanine at position 217 in the SPT5 (Gl: 101260813) protein by proline, the mutation will be performed using, by way of non-limiting example, xCas9 or Cpf1 endonucleases.

In rice ( Oryza sativa subsp. Japonica ), for example, the amino acid at position 9 of the consensus sequence corresponds to alanine at position 230 in the SPT5 (Gl: OJ1611_C08.10) protein, alanine at position 247 in the SPT5 (Gl: OsJ_20538) protein and alanine at position 230 in the SPT5 (Gl: 0s02g0772000) protein. The mutation advantageously consists of a substitution of alanine at position 230 in the SPT5 (Gl: OJ1611_C08.10) protein and/or alanine at position 247 in the SPT5 (Gl: OsJ_20538) protein and/or alanine at position 230 in the SPT5 (Gl: 0s02g0772000) protein by any amino acid other than alanine. More preferentially, the mutation consists of a substitution of alanine at position 230 in the SPT5 (Gl: OJ1611_C08.10) protein and/or alanine at position 247 in the SPT5 (Gl: OsJ_20538) protein and/or alanine at position 230 in the SPT5 (Gl: 0s02g0772000) protein by proline. In the case of the substitution of alanine at position 230 in the SPT5 (Gl: OJ1611_C08.10) protein by proline, the mutation will be performed using, by way of non-limiting example, Cpf1 endonuclease. In the case of substitution of alanine at position 247 in the SPT5 (Gl: OsJ_20538) protein by proline, the mutation will be performed using, by way of non-limiting example, Cpf1 or SaCas9 endonucleases. In the case of the substitution of alanine at position 230 in the SPT5 (Gl: 0s02g0772000) protein by proline, the mutation will be performed using, by way of non limiting example, Cpf1 or SaCas9 endonucleases.

In wheat ( Triticum aestivum ), for example, the amino acid at position 9 of the consensus sequence corresponds to alanine at position 94 in the SPT5 (Gl: TAE22507G002) protein, alanine at position 240 in the SPT5 (Gl: TAE56554G006) protein, alanine at position 242 in the SPT5 (Gl: TAE37492G003) protein, alanine at position 237 in the SPT5 (Gl: TAE34328G001) protein and alanine at position 240 in the SPT5 (Gl: TAE29648G001) protein. The mutation advantageously consists of a substitution of alanine at position 94 in the SPT5 (Gl: TAE22507G002) protein and/or alanine at position 240 in the SPT5 (Gl: TAE56554G006) protein and/or alanine at position 242 in the SPT5 (Gl: TAE37492G003) protein and/or alanine at position 237 in the SPT5 (Gl: TAE34328G001) protein and/or alanine at position 240 in the SPT5 (Gl: TAE29648G001) protein by any amino acid other than alanine. More preferentially, the mutation consists of a substitution of alanine at position 94 in the SPT5 (Gl: TAE22507G002) protein and/or alanine at position 240 in the SPT5 (Gl: TAE56554G006) protein and/or alanine at position 242 in the SPT5 (Gl: TAE37492G003) protein and/or alanine at position 237 in the SPT5 (Gl: TAE34328G001) protein and/or alanine at position 240 in the SPT5 (Gl: TAE29648G001) protein by proline. In the case of the substitution of alanine at position 94 in the SPT5 (Gl: TAE22507G002) protein by proline, the mutation will be performed using, by way of non-limiting example, Cpf1 endonuclease. In the case of the substitution of alanine at position 240 in the SPT5 (Gl: TAE56554G006) protein by proline, the mutation will be performed using, by way of non-limiting example, SaCas9 endonuclease. In the case of the substitution of alanine at position 242 in the SPT5 (Gl: TAE37492G003) protein by proline, the mutation will be performed using, by way of non-limiting example, SaCas9 endonuclease. In the case of alanine at position 237 in the SPT5 (Gl: TAE34328G001) protein by proline, the mutation will be performed using, by way of non-limiting example, Cpf1 endonuclease. In the case of the substitution of alanine at position 240 in the SPT5 (Gl: TAE29648G001) protein by proline, the mutation will be performed using, by way of non-limiting example, SaCas9 endonuclease.

In soybean ( Glycine max), for example, the amino acid at position 9 of the consensus sequence corresponds to alanine at position 233 in the SPT5 (Gl: 100801380) protein and to alanine at position 244 in the SPT5 (Gl: 100784916) protein. The mutation advantageously consists of a substitution of alanine at position 233 in the SPT5 (Gl: 100801380) protein and/or alanine at position 244 in the SPT5 (Gl: 100784916) protein by any amino acid other than alanine. More preferentially, the mutation consists of a substitution of alanine at position 233 in the SPT5 (Gl: 100801380) protein and/or alanine at position 244 in the SPT5 (Gl: 100784916) protein by proline. In the case of the substitution of alanine at position 233 in the SPT5 (Gl: 100801380) protein by proline, the mutation will be performed using, by way of non limiting example, xCas9 endonuclease. In the case of the substitution of alanine at position 244 in the SPT5 (Gl: 100784916) protein by proline, the mutation will be performed using, by way of non-limiting example, xCas9 endonuclease.

In cucumber ( Cucumis sativus), for example, the amino acid at position 9 of the consensus sequence corresponds to alanine at position 21 1 in the SPT5 (Gl: KGN47486.1) protein. The mutation advantageously consists of a substitution of alanine at position 211 in the SPT5 (Gl: KGN47486.1) protein by any amino acid other than alanine. More preferentially, the mutation consists of a substitution of alanine at position 211 in the SPT5 (Gl: KGN47486.1) protein by proline. In the case of the substitution of alanine at position 21 1 in the SPT5 (Gl: KGN47486.1) protein by proline, the mutation will be performed using, by way of non-limiting example, xCas9 or Cpf1 or SaCas9 endonucleases.

In sunflower ( Helianthus annuus ), for example, the amino acid at position 9 of the consensus sequence corresponds to alanine at position 238 in the SPT5 (Gl: OTG29706.1) protein. The mutation advantageously consists of a substitution of alanine at position 238 in the SPT5 (Gl: OTG29706.1) protein by any amino acid other than alanine. More preferentially, the mutation consists of a substitution of alanine at position 238 in the SPT5 (Gl: OTG29706.1) protein by proline. In the case of the substitution of alanine at position 238 in the SPT5 (Gl: OTG29706.1) protein by proline, the mutation will be performed using, by way of non-limiting example, xCas9 endonuclease.

In rapeseed ( Brassica napus), for example, the amino acid at position 9 of the consensus sequence corresponds to alanine at position 230 in the SPT5 (Gl: CDY34005.1) protein and to alanine at position 200 in the SPT5 (Gl: CDY61565.1) protein. The mutation advantageously consists of a substitution of alanine at position 230 in the SPT5 (Gl: CDY34005.1) protein and/or alanine at position 200 in the SPT5 (Gl: CDY61565.1) protein by any amino acid other than alanine. More preferentially, the mutation consists of a substitution of alanine at position 230 in the SPT5 (Gl: CDY34005.1) protein and/or alanine at position 200 in the SPT5 (Gl: CDY61565.1) protein by proline. In the case of the substitution of alanine at position 230 in the SPT5 (Gl: CDY34005.1) protein by proline, the mutation will be performed using, by way of non-limiting example, xCas9 endonuclease. In the case of the substitution of alanine at position 200 in the SPT5 (Gl: CDY61565.1) protein by proline, the mutation will be performed using, by way of non-limiting example, xCas9 or Cpf1 or SaCas9 endonucleases.

In barrel medic ( Medicago truncatula), for example, the amino acid at position 9 of the consensus sequence corresponds to alanine at position 233 in the SPT5 (Gl: AES72378.1) protein. The mutation advantageously consists of a substitution of alanine at position 233 in the SPT5 (Gl: AES72378.1) protein by any amino acid other than alanine. More preferentially, the mutation consists of a substitution of alanine at position 233 in the SPT5 (Gl: AES72378.1) protein by proline. In the case of the substitution of alanine at position 233 in the SPT5 (Gl: AES72378.1) protein by proline, the mutation will be performed using, by way of non-limiting example, xCas9 endonuclease.

The invention also concerns the parts of the modified plants, notably the roots, leaves, fruits or seeds of these plants, which contain cells whose genome has been modified as defined above.

The invention thus also concerns a process for growing a genetically modified plant according to the invention, as defined above and in the examples, which includes growing the organism on/in a growth medium suitable for its growth and the production of biomass in the presence of carbon dioxide and light.

For the plants, the growth medium may be earth (soil) for conventional crops, or artificial growth media for soil-less crops or hydroponics or aquaponics, for example.

Light may be provided with or without artificial lighting, the lighting generally following a cycle of periods of darkness and lighting, advantageously alternating day and night over 24 hours. Biomass is produced by growing plants which are then harvested at an appropriate developmental stage depending on the type of crop.

The invention thus also concerns the parts of plants according to the invention harvested after cultivation, whether they are roots, leaves, flowers, fruits or seeds.

The invention also concerns seeds, a set of seeds, of genetically modified plants, intended to be used for the production of plants by the above-mentioned cultivation methods. These seeds may come from the cultivation of a previous generation of plants according to the invention and be stored for later cultivation. They may also be hybrid seeds, at least one of whose parents is a genetically modified plant according to the invention. Advantageously, both parents of the seeds according to the invention are genetically modified plants containing the transcription elongation factor modification.

The invention also concerns manufactured products that contain parts of these modified plants according to the invention. These manufactured products include in particular oilcake obtained after pressing oilseeds, such as soybean cake intended for animal feed.

In view of the above teaching, the skilled person may seek to identify wild plants or plants that have undergone random mutations under the action of mutagenic agents that have the SPT5 gene mutation, in particular the mutation described above.

The genetically modified plants as described in the present application may be derived, by way of non-limiting example, from crosses with TILLING® plant lines. TILLING® is a molecular biology method that allows directed identification of mutations in a specific gene. TILLING® was introduced in 2000 on the model plant Arabidopsis thaliana. TILLING® has since been used as a reverse-genetics method in other organisms such as zebrafish, maize, wheat, rice, soybean, tomato and lettuce.

The process combines a standard and efficient technique of mutagenesis using a chemical mutagen (for example, ethyl methanesulphonate (EMS)) with a sensitive DNA- screening technique that identifies single base mutations (also called point mutations) in a target gene. EcoTILLING is a method that uses TILLING® techniques to search for natural mutations in individuals, usually for population genetic analysis (Comai et al., 2004; Gilchrist et al., 2006; Mejlhede et al., 2006; Nieto et al., 2007). DEcoTILLING is a modification of TILLING® and EcoTILLING that uses a low-cost method to identify fragments (Garvin and Gharrett, 2007). The TILLING® method relies on the formation of heteroduplexes that are formed when multiple alleles (which may come from a heterozygote or from a pool of multiple homozygotes and heterozygotes) are amplified by PCR and then heated and slowly cooled. A “bubble” forms between the two DNA strands (the mutation induced in TILLING® or the natural mutation or SNP in EcoTILLING), which is then cleaved by single strand nucleases. The products are then separated by size on several different platforms.

There are several TILLING® centres around the world that focus on agriculturally important species: UC Davis (United States), focused on rice; Purdue University (United States), focused on maize; University of British Columbia (Canada), focused on Brassica napus ; John Innes Centre (United Kingdom), focused on Brassica rapa, Lotus and Medicago ; Fred Hutchinson Cancer Research, focused on Arabidopsis ; Southern Illinois University (United States), focused on soybean, and INRA (France), focused on pea and tomato.

A more detailed description of TILLING® methods and compositions can be found in patents U.S. Pat. No. 5,994,075, US 2004/0053236 A1 , WO 2005/055704 and WO 2005/048692.

Thus, in certain embodiments, the selection procedures of the present description comprise selection with one or more TILLING® plant lines with one or more identified mutations.

There are libraries of plant genetic material (germplasm) of different plants, whether they are plant cells or plants, especially their seeds or plant parts. For simplicity,“plant” will refer to all the forms contained in libraries, whether they are cells, plant parts or whole plants. These libraries may collect genetic material of local varieties adapted to a particular climate and territory, such as indigenous varieties, or wild plants close to or related to commercial crop varieties that can be combined under the name“commercial plant genetic material”. They may also be libraries of plant genetic material that have undergone mutations under the action of different mutagens (chemical mutagens or radiation in particular).

The skilled person may seek to identify plants that have an SPT5 gene mutation, in particular the mutation described above, more particularly in the 12-amino-acid consensus sequence as shown in Figure 2, and preferably with proline for the ninth amino acid in this consensus sequence.

The invention thus also concerns a process for identifying a plant containing an SPT5 gene mutation which comprises the steps of

i) providing a library of plant genetic material,

ii) screening this library to identify the presence of an SPT5 gene mutation as described above and

iii) selecting the plants that have an SPT5 gene mutation.

The process may also include a step of analysing the RuBisCO content produced by the plants containing said mutation in order to select those with the highest RuBisCO contents.

Preferably, the identification process will be used to identify the presence of the mutation in crop plants, or in varieties related to crop plants.

After selecting these plants, the skilled person will be able to cross-breed them with commercial varieties so as to ultimately obtain plants homozygous for the SPT5 gene mutation.

The invention thus also concerns the use of a genetically modified plant according to the invention or a plant selected from a library by the above process in a variety selection programme for the production of commercial plants containing the SPT5 gene mutation described above, and notably for the preparation of hybrid plants.

Selection methods are well known to the skilled person, as are methods for identifying the presence of a mutation in a particular genetic sequence of a plant or plant cell. The skilled person will notably be able to use all methods of DNA sequence detection by PCR by selecting the appropriate primers for amplification of the SPT5 gene necessary for its sequencing.

EXAMPLES

EXAMPLE 1 : Introduction of a mutation into coding sequence of native gene encoding transcription elongation factor SPT5 from Arabidopsis thaliana leading to a substitution of Alanine 218 and Alanine 231 to Proline.

Genes encoding for transcription elongation factor SPT5 from Arabidopsis thaliana

In Arabidopsis thaliana two genes, AT2G34210 and AT4G08350 code transcription elongation factor SPT5. In gene AT2G34210, the codon GCG encodes for Alanine 218. The substitution of G to C is performed by base editing using SaCas9 or xCas9 endonuclease. In gene AT4G08350, the codon GCA encodes for Alanine 231. The substitution of the G to C is performed by base editing using SaCas9 endonuclease.

Base editing of AT2G3421 gene encoding for transcription elongation factor SPT5 from Arabidopsis thaliana

To generate the pGG-C-AtxCas9nickase-D module, a synthetic fragment is made (SGI DNA) to generate six of the seven SNPs to convert standard SpCas9 (Addgene) into xCas9 (Hu et al., 2018). Three PCR fragments are amplified from an Arabidopsis thaliana SpCas9 template to mutate the seventh position and assemble the xCas9 sequence. The synthetic fragment and the three PCR products are used in a Gibson assembly (NEBuilder, New England Biolabs) reaction with Bsal-digested pGGCOOO (Lampropoulos et al., 2013). The assembled product is transformed into DH5a E. coli cells. A clone with the correct sequence is identified by Sanger sequencing. This generates pGG-C-AtxCas9-D. To make the nickase variant, an oligo with the D10A mutation is used to PCR amplify with the pGG-C-AtxCas9-D plasmid as template. The PCR product is inserted into pGGCOOO. A clone with the correct sequence is verified by Sanger sequencing.

To generate the base editing (BE) destination vector 1 for Arabidopsis thaliana transformation pFASTRK-AtxCas9BE, pGG-A-PcUbi-B, pGG-B-APOBEC1-C, pGG-C-AtxCas9nickase-D, pGG-D-GFP-linkerNLS-E, pGG-E-G7T-F, and pGG-F-AtU6-26-Aarl-scaffold-G are GoldenGate assembled into the vector pFASTRK-A-G. A clone is validated by diagnostic digest and selective Sanger sequencing across the expression cassettes.

Destination vector 1 is further modified by replacing the Aarl restriction sites in the gRNA module with a Bsal-ccdB/CMR-Bsal fragment. This greatly streamlines downstream cloning. Clones are verified by diagnostic digest.

To add single gRNA (ATCCGCTTCAATATATACAT), an annealed oligo cloning strategy (Fauser et al., 2014) is used. Briefly, 1 mI_ of each 100 mM oligo is added to 48 mI_ of MQ water followed by incubation with a slow-cooling program on the thermal cycler e.g.: 5 minutes at 95°C; 95-85°C, -2°C/second; 85-25°C, -0.1 °C/second. The annealed oligos are cloned in the destination vector via a Golden Gate reaction. The final plasmid 1 is validated by Sanger sequencing and diagnostic digest.

Plasmid 1 is transformed into Agrobacterium tumefaciens for floral-dip transformation into Arabidopsis thaliana plants (Clough and Bent, 1998) using red-fluorescent seed as the selection system (Shimada et al., 2010).

The plants are subsequently placed for 18 hours into a humid chamber. Thereafter, the pots are returned to the greenhouse for the plants to continue growing. The plants remain in the greenhouse for another 10 weeks until the seeds are ready for harvesting.

Depending on the resistance marker used for the selection of the transformed plants the harvested seeds are planted in the greenhouse and subjected to a spray selection or else first sterilized and then grown on agar plates supplemented with the respective selection agent. Since the vector contains the bar gene as the resistance marker, plantlets are sprayed four times at an interval of 2 to 3 days with 0.02% BAST A® and transformed plants are allowed to set seeds.

The seeds of the transgenic A. thaliana plants are stored in the freezer (at -20°C).

Base editing of AT4G08350 gene encoding for transcription elongation factor SPT5 from Arabidopsis thaliana

To generate pGG-C-SaCas9nickase-D, the SaCas9 sequence is amplified from MSP1830 (Addgene), digested with Bsal and ligated to a Bsal-digested pGGCOOO. A clone with the correct sequence is identified by Sanger sequencing. To make the nickase variant, an oligo with the D10A mutation is used to PCR amplify with the pGG-C-SaCas9-D plasmid as template. The PCR product is inserted into pGGCOOO. A clone with the correct sequence is verified by Sanger sequencing.

To generate the base editing (BE) destination vector 2 for Arabidopsis thaliana transformation pFASTRK-SaCas9BE, pGG-A-PcUbi-B, pGG-B-APOBEC1-C, pGG-C-SaCas9nickases-D, pGG-D-GFP-linkerNLS-E, pGG-E-G7T-F, and pGG-F-AtU6-26-Aarl-scaffold-G are GoldenGate assembled into the vector pFASTRK-A-G. A clone is validated by diagnostic digest.

Destination vector 2 is further modified by replacing the Aarl restriction sites in the gRNA module with a Bsal-ccdB/CMR-Bsal fragment. This greatly streamlines downstream cloning. Clones are verified by diagnostic digest.

To add single gRNA (CTTGTCTGCTTCAACATATA), an annealed oligo cloning strategy (Fauser et al., 2014) is used. Briefly, 1 mI_ of each 100 mM oligo is added to 48 mI_ of MQ water followed by incubation with a slow-cooling program on the thermal cycler e.g.: 5 minutes at 95°C; 95-85°C, -2°C/second; 85-25°C, -0.1 °C/second. The annealed oligos are cloned in the destination vector via a Golden Gate reaction. The final plasmid 2 is validated by Sanger sequencing and diagnostic digest.

Plasmid 2 is transformed into Agrobacterium tumefaciens for floral-dip transformation into Arabidopsis thaliana plants (Clough and Bent, 1998) using red-fluorescent seed as the selection system (Shimada et al., 2010).

The seeds of the transgenic A. thaliana plants are produced as described in Example 1 and are stored in the freezer (at -20°C).

Base editing of AT2G3421 and AT4G08350 genes encoding for transcription elongation factor SPT5 from Arabidopsis thaliana

To generate pGG-C-SaCas9nickase-D, the SaCas9 sequence is amplified from MSP1830 (Addgene), digested with Bsal and ligated to a Bsal-digested pGGCOOO. A clone with the correct sequence is identified by Sanger sequencing. To make the nickase variant, an oligo with the D10A mutation is used to PCR amplify with the pGG-C-SaCas9-D plasmid as template. The PCR product is inserted into pGGCOOO. A clone with the correct sequence is verified by Sanger sequencing.

To generate pGG-A-AtU6-26AarlSascaffold-B, overlapping oligos containing the Sascaffold sequence are generated and used to PCR amplify with pGG-A-AtU6-26Aarlscaffold-B as template. This is inserted into pGGAOOO and pGGFOOO. The pGG-A-Sascaffold-B entry with pGG-B-linkerll-C, pGG-C-AtU6-26scaffold-D and pGG-D-linkerll-G are Golden Gate assembled into the entry vector pEN-L1-A-G-L2. This clone serves as a template for PCR to generate paired gRNA vectors.

To generate the base editing (BE) destination vector 3 for Arabidopsis thaliana transformation pFASTRK-SaCas9BE, pGG-A-PcUbi-B, pGG-B-APOBEC1-C, pGG-C-SaCas9nickases-D, pGG-D-GFP-linkerNLS-E, pGG-E-G7T-F, and pGG-F-AtU6-26-Aarl-scaffold-G are GoldenGate assembled into the vector pFASTRK-A-G. A clone is validated by diagnostic digest.

Destination vector 3 is further modified by replacing the Aarl restriction sites in the gRNA module with a Bsal-ccdB/CMR-Bsal fragment. This greatly streamlines downstream cloning. Clones are verified by diagnostic digest.

To add paired gRNAs (CAT ATCCGCTTCAAT ATATA; CTTGTCTGCTT C A AC AT ATA) , a strategy similar to a previously-published (Xing et al., 2014) one is used. Briefly, two primers containing Bsal tails, the gRNA target sites are used to amplify from the paired gRNA template plasmids. A Golden Gate reaction is then used to insert the PCR products into the destination vectors. The final plasmids are validated by Sanger sequencing and diagnostic digest. Plasmid 3 is transformed into Agrobacterium tumefaciens for floral-dip transformation into Arabidopsis thaliana plants (Clough and Bent, 1998) using red-fluorescent seed as the selection system (Shimada et al., 2010).

The seeds of the transgenic A. thaliana plants are produced as described in Example 1 and are stored in the freezer (at -20°C).

EXAMPLE 2: Introduction of a mutation into coding sequence of native gene encoding transcription elongation factor SPT5 from maize leading to a substitution of Alanine 237 to Proline

Genes encoding transcription elongation factor SPT5 from maize

In maize (Zea mays) two genes, Zm00001 d044971 and Zm00001d037142 encode transcription elongation factor SPT5 from maize. In genes, Zm00001d044971 and Zm00001 d037142, the codon GCT encodes for Alanine 237. The substitution of the G to C is performed by base editing using SaCas9 endonuclease.

Base editing of genes Zm00001 d044971 and Zm00001d037142 encoding transcription elongation factor SPT5 from maize

To generate pGG-C-SaCas9nickase-D, the SaCas9 sequence is amplified from MSP1830 (Addgene), digested with Bsal and ligated to a Bsal-digested pGGCOOO. A clone with the correct sequence is identified by Sanger sequencing. To make the nickase variant, an oligo with the D10A mutation is used to PCR amplify with the pGG-C-SaCas9-D plasmid as template. The PCR product is inserted into pGGCOOO. A clone with the correct sequence is verified by Sanger sequencing.

To generate pGG-A-OsU3-26AarlSascaffold-B, overlapping oligos containing the Sascaffold sequence are generated and used to PCR amplify with pGG-A-OsU3-26Aarlscaffold-B as template. This is inserted into pGGAOOO and pGGFOOO. The pGG-A-Sascaffold-B entry with pGG-B-linkerll-C, pGG-C-TaU3-scaffold-D, and pGG-D-linkerll-G are Golden Gate assembled into the entry vector pEN-L1-A-G-L2. This clone serves as a template for PCR to generate paired gRNA vectors.

To generate the base editing (BE) destination vector 4 for maize transformation pBb7- SaCas9BE, pGG-A-ZmUbi-B, pGG-B-APOBEC1-C, pGG-C-SaCas9nickases-D, pGG-D- GSyellow-linkerNLS-E, pGG-E-nosT-F, and pGG-F-OsU3-26-Aarl-scaffold-G are Golden Gate assembled into the vector pBb7-A-G. A clone is validated by diagnostic digest.

Destination vector 4 is further modified by replacing the Aarl restriction sites in the gRNA module with a Bsal-ccdB/CMR-Bsal fragment. This greatly streamlines downstream cloning. Clones are verified by diagnostic digest.

To add paired gRNAs (CTTCTCAGCTT C A AC AT AAA ; CTTTT C AG CTT C A AC AT AAA) , a strategy similar to a previously-published (Xing et al., 2014) one is used. Briefly, two primers containing Bsal tails, the gRNA target sites are used to amplify from the paired gRNA template plasmids. A Golden Gate reaction is then used to insert the PCR products into the destination vectors. The final plasmid 4 is validated by Sanger sequencing and diagnostic digest.

Plasmid 4 is transformed into Agrobacterium tumefaciens for transformation into maize plants (Coussens et a!., 2012) using glufosinate ammonium.

The seeds of the transgenic maize plants are produced as described in Example 1 and are stored in the freezer (at -20°C).

EXAMPLE 3: Introduction of a mutation into coding sequence of native gene encoding transcription elongation factor SPT5 from tobacco ( Nicotians tabacum) leading to a substitution of Alanine 178 and Alanine 228 to Proline

Genes encoding transcription elongation factor SPT5 from tobacco

In tobacco, two genes, Nitab4.5_0000445g0150 and Nitab4.5_0004987g0010 encode transcription elongation factor SPT5. In gene Nitab4.5_0000445g0150, the codon GCA encodes for Alanine 178. The substitution of G to C is performed by base editing using xCas9 endonuclease. In gene Nitab4.5_0004987g0010, the codon GCT encodes for the Alanine 228. The substitution of the G to C is performed by base editing using Cpf1 endonuclease.

Base editing of gene Nitab4.5_0000445g0150 encoding transcription elongation factor SPT5 from tobacco

To generate the pGG-C-AtxCas9nickase-D module, a synthetic fragment is made (SGI DNA) to generate six of the seven SNPs to convert standard SpCas9 (Addgene) into xCas9 (Hu et al., 2018). Three PCR fragments are amplified from an AtSpCas9 template to mutate the seventh position and assemble the xCas9 sequence. The synthetic fragment and the three PCR products are used in a Gibson assembly (NEBuilder, New England Biolabs) reaction with Bsal-digested pGGCOOO (Lampropoulos et al., 2013). The assembled product is transformed into DH5a E. coli cells. A clone with the correct sequence is identified by Sanger sequencing. This generated pGG-C-AtxCas9-D. To make the nickase variant, an oligo with the D10A mutation is used to PCR amplify with the pGG-C-AtxCas9-D plasmid as template. The PCR product is inserted into pGGCOOO. A clone with the correct sequence is verified by Sanger sequencing.

To generate the base editing (BE) destination vector 5 for tobacco transformation pFASTRK- AtxCas9BE, pGG-A-PcUbi-B, pGG-B-APOBEC1-C, pGG-C-AtxCas9nickase-D, pGG-D-GFP- linkerNLS-E, pGG-E-G7T-F, and pGG-F-AtU6-26-Aarl-scaffold-G are GoldenGate assembled into the vector pFASTRK-A-G. A clone is validated by diagnostic digest and selective Sanger sequencing across the expression cassettes.

Destination vector 5 is further modified by replacing the Aarl restriction sites in the gRNA module with a Bsal-ccdB/CMR-Bsal fragment. This greatly streamlines downstream cloning. Clones are verified by diagnostic digest.

To add single gRNA (GTCTGCTTCTATATATATAT), an annealed oligo cloning strategy (Fauser et al., 2014) is used. Briefly, 1 mI_ of each 100 mM oligo is added to 48 mI_ of MQ water followed by incubation with a slow-cooling program on the thermal cycler e.g.: 5 minutes at 95°C; 95-85°C, -2°C/second; 85-25°C, -0.1 °C/second. The annealed oligos are cloned in the destination vector via a Golden Gate reaction. The final plasmid 5 is validated by Sanger sequencing and diagnostic digest with primer forward TCAGGGATCCTAAGCTGTGGA and primer reverse AGCAGATAGGCAGCGTACAA.

Plasmid 5 is transformed into Agrobacterium tumefaciens for transformation into tobacco plants using kanamycine as the selection system as described in Hirohata et al., 2018.

The seeds of the transgenic tobacco plants are produced as described in Example 1 and are stored in the freezer (at -20°C).

Base editing of gene Nitab4.5_0004987g0010 encoding transcription elongation factor SPT5 from tobacco

To generate pGG-C-LbdCas12a-D, the LbCas12a sequence is amplified from pYPQ230 (Addgene), digested with Bsal and ligated to a Bsal-digested pGGCOOO. A clone with the correct sequence is identified by Sanger sequencing. To make the nuclease-dead variant, oligos with the R1 138A mutations are used to PCR amplify with the pGG-C-SaCas9-D plasmid as template. The PCR product is inserted into pGGCOOO. A clone with the correct sequence is verified by Sanger sequencing.

To generate the base editing (BE) destination vector 6 for tobacco transformation pFASTRK- Cas12a9BE, pGG-A-PcUbi-B, pGG-B-APOBEC1-C, pGG-C-LbdCas12a-D, pGG-D-GFP- linkerNLS-E, pGG-E-G7T-F, and pGG-F-SacBAarl-linker-G are GoldenGate assembled into the vector pFASTRK-A-G. A clone is validated by diagnostic digest.

To generate the crRNA modules, the crRNA (TATTGAGGCTGACAAACAAT) is cloned into pGG-B-LbCpf1-ccdB-C using an annealed oligo cloning strategy (Fauser et al., 2014). Briefly, 1 mI_ of each 100 mM oligo is added to 48 mI_ of MQ water followed by incubation with a slow- cooling program on the thermal cycler e.g.: 5 minutes at 95°C; 95-85°C, -2°C/second; 85-25°C, -0.1 °C/second. The annealed oligos are cloned in the crRNA entry vector via a Golden Gate reaction. The final plasmid is validated by Sanger sequencing.

The crRNA module together with pGG-A-Rps5a-B, pGG-C-linker-D, and D-pea3AT-G are Golden Gate assembled into pFASTRK-Cas12a9BE. The final plasmid 6 is validated by diagnostic digest.

Plasmid 6 is transformed into Agrobacterium tumefaciens for transformation into tobacco plants using kanamycine as the selection system as described in Hirohata et al., 2018.

The seeds of the transgenic tobacco plants are produced as described in Example 1 and are stored in the freezer (at -20°C). EXAMPLE 4: Introduction of a mutation into coding sequence of native gene encoding transcription elongation factor SPT5 from tomato leading to a substitution of Alanine 217 to Proline

Genes encoding transcription elongation factor SPT5

In tomato, one gene, Solyc04g064700 encodes transcription elongation factor SPT5. In gene Solyc04g064700, the codon GCG encodes for Alanine 217. The substitution of G to C is performed by base editing using xCas9 endonuclease.

Base editing of Solyc04g064700 gene encoding transcription elongation factor SPT5 from tomato using xCas9 endonuclease

To generate the pGG-C-AtxCas9nickase-D module, a synthetic fragment is made (SGI DNA) to generate six of the seven SNPs to convert standard SpCas9 into xCas9 (Hu et al., 2018). Three PCR fragments are amplified from an AtSpCas9 template to mutate the seventh position and assemble the xCas9 sequence. The synthetic fragment and the three PCR products are used in a Gibson assembly (NEBuilder, New England Biolabs) reaction with Bsal-digested pGGCOOO (Lampropoulos et al., 2013). The assembled product is transformed into DH5a E. coli cells. A clone with the correct sequence is identified by Sanger sequencing. This generated pGG-C-AtxCas9-D. To make the nickase variant, an oligo with the D10A mutation is used to PCR amplify with the pGG-C-AtxCas9-D plasmid as template. The PCR product is inserted into pGGCOOO. A clone with the correct sequence is verified by Sanger sequencing.

To generate the base editing (BE) destination vector 7 for tomato transformation pFASTRK- AtxCas9BE, pGG-A-PcUbi-B, pGG-B-APOBEC1-C, pGG-C-AtxCas9nickase-D, pGG-D-GFP- linkerNLS-E, pGG-E-G7T-F, and pGG-F-AtU6-26-Aarl-scaffold-G are GoldenGate assembled into the vector pFASTRK-A-G. A clone is validated by diagnostic digest and selective Sanger sequencing across the expression cassettes.

Destination vector 7 is further modified by replacing the Aarl restriction sites in the gRNA module with a Bsal-ccdB/CMR-Bsal fragment. This greatly streamlines downstream cloning. Clones are verified by diagnostic digest.

To add single gRNA (GTCCGCTTCTATGTATATATAG), an annealed oligo cloning strategy (Fauser et al., 2014) is used. Briefly, 1 pL of each 100 mM oligo is added to 48 pL of MQ water followed by incubation with a slow-cooling program on the thermal cycler e.g.: 5 minutes at 95°C; 95-85°C, -2°C/second; 85-25°C, -0.1 °C/second. The annealed oligos are cloned in the destination vector via a Golden Gate reaction. The final plasmid 7 is validated by Sanger sequencing and diagnostic digest with primer forward ACTCCCTTGTGTGCTTAGGC and primer reverse AGGCATATATATTGCGCATACCCT.

Plasmid 7 is transformed into Agrobacterium tumefaciens for transformation into tomato plants using kanamycine as the selection system as described (Van Eck et al., 2019). The seeds of the transgenic tomato plants are produced as described in Example 1 and are stored in the freezer (at -20°C).

Base editing of Solyc04g064700 gene encoding transcription elongation factor SPT5 from tomato using saCas9 endonuclease

To generate pGG-C-SaCas9nickase-D, the SaCas9 sequence is amplified from MSP1830 (Addgene), digested with Bsal and ligated to a Bsal-digested pGGCOOO. A clone with the correct sequence is identified by Sanger sequencing. To make the nickase variant, an oligo with the D10A mutation is used to PCR amplify with the pGG-C-SaCas9-D plasmid as template. The PCR product is inserted into pGGCOOO. A clone with the correct sequence is verified by Sanger sequencing.

To generate the base editing (BE) destination vector 8 for tomato transformation pFASTRK- SaCas9BE, pGG-A-PcUbi-B, pGG-B-APOBEC1-C, pGG-C-SaCas9nickases-D, pGG-D-GFP- linkerNLS-E, pGG-E-G7T-F, and pGG-F-AtU6-26-Aarl-scaffold-G are GoldenGate assembled into the vector pFASTRK-A-G. A clone is validated by diagnostic digest.

Destination vector 7 is further modified by replacing the Aarl restriction sites in the gRNA module with a Bsal-ccdB/CMR-Bsal fragment. This greatly streamlines downstream cloning. Clones are verified by diagnostic digest.

To add single gRNA (TTTGTCCGCTTCTATGTATA), an annealed oligo cloning strategy (Fauser et al., 2014) is used. Briefly, 1 pl_ of each 100 mM oligo is added to 48 mI_ of MQ water followed by incubation with a slow-cooling program on the thermal cycler e.g.: 5 minutes at 95°C; 95-85°C, -2°C/second; 85-25°C, -0.1°C/second. The annealed oligos are cloned in the destination vector via a Golden Gate reaction. The final plasmid 8 is validated by Sanger sequencing and diagnostic digest.

Plasmid 8 is transformed into Agrobacterium tumefaciens for transformation into tomato plants using kanamycine as the selection system as described Van Eck et al., 2019.

The seeds of the transgenic tomato plants are produced as described in Example 1 and are stored in the freezer (at -20°C).

EXAMPLE 5: Effect of substitution of Alanine 218 and Alanine 231 to Proline in transcription elongation factor SPT5 of Arabidopsis thaliana

Arabidopsis thaliana biomass yield increases under standardized conditions

In this experiment, a plant screening for biomass yield increase under standardized growth conditions in the absence of substantial abiotic stress and biotic stress is performed. In a standard experiment soil is prepared as 3.5:1 (v:v) mixture of nutrient rich soil (GS90, Tantau, Wansdorf, Germany) and quartz sand. Alternatively, plants are sown on nutrient rich soil (GS90, Tantau, Germany). Pots are filled with soil mixture and placed into trays. Water is added to the trays to let the soil mixture take up appropriate amount of water for the sowing procedure. The seeds for transgenic A. thaliana plants (Example 1) and their non-transgenic wild-type controls are sown in pots (6 cm diameter). Then the filled tray is covered with a transparent lid and transferred into a precooled (4°C-5°C) and darkened growth chamber. Stratification is established for a period of 3-4 days in the dark at 4°C-5°C Germination of seeds and growth is initiated at a growth condition of 20°C, 60% relative humidity, 16 h photoperiod and illumination with fluorescent light at approximately 200 pmol/m²s. Covers are removed 7- 8 days after sowing. BASTA selection is done at day 10 or day 1 1 (9 or 10 days after sowing) by spraying pots with plantlets from the top. In the standard experiment, a 0.07% (v:v) solution of BASTA concentrate (183 g/L glufosinate-ammonium) in tap water is sprayed once or, alternatively, a 0.02% (v:v) solution of BASTA is sprayed three times. The wild-type control plants are sprayed with tap water only (instead of spraying with BASTA dissolved in tap water) but are otherwise treated identically. Plants are individualized 13-14 days after sowing by removing the surplus of seedlings and leaving one seedling in soil. Transgenic events and wild-type control plants are evenly distributed over the chamber. Watering is carried out as needed, every two days after removing the covers in a standard experiment or, alternatively, every day. For measuring biomass performance, plant fresh weight is determined at harvest time (24-29 days after sowing) by cutting shoots and weighing them. Plants are in the stage prior to flowering and prior to growth of inflorescence when harvested. Transgenic plants are compared to the non-transgenic wild-type control plants. Per transgenic construct up to five events, with up to 60 plants per event, are tested in up to four experimental levels.

Biomass production is measured by harvesting and weighing plant rosettes. Biomass increase is calculated as ratio of average weight of transgenic plants compared to average weight of wild-type control plants from the same experiment. The transgenic Arabidopsis expressing a transcription elongation factor SPT5 with a substitution of Alanine 218 and Alanine 231 to Proline produces more biomass than non-transgenic control plants.

Leaf elongation rates (LER)

Leaf elongation rates are measured using a HR4000 Linear Variable Displacement Transducer (LVDT). After emergence of the fifth leaf, plants are transferred to a cabinet containing the LVDT unit and randomly assigned to one of the eight measurement stations. The fifth leaf is clipped to the apparatus and growth is measured for a 24-h period. Plants are grown at 22°C for the initial 12 h of growth in the dark followed by 28°C for the first 4 h of the light period. The cabinet temperature is then raised to 45°C for 4 h and returned to 28°C for the final 4 h of the day period. Leaf length is measured and logged every 6 mins using the software program VuGrowth, version 1 .0 (Applied Measurement, Oakleigh, Victoria, Australia). The ambient temperature of the cabinet is logged ever 10 min by a portable data logger (Onset HOBO, Massachusetts, USA) placed next to the plants. Leaf lengths are converted to leaf elongation rate (LER) by subtracting the leaf length at any given time by the leaf length 1 h prior to that measurement. The experiment is repeated on six occasions

Leaf elongation rate is measured and the transgenic Arabidopsis expressing a transcription elongation factor SPT5 with a substitution of Alanine 218 and Alanine 231 to Proline elongates faster than non-transgenic control plants.

Leaf gas exchange

Leaf gas exchange measurements are carried out. The following parameters are recorded with an open infrared gas analyser system LI-6400XT (LI-COR Inc., Lincoln, NE, USA):

a) Net photosynthesis rates (A_n) under saturating light of 1500 PAR (integrated light source of the LI-6400XT leaf chamber) and at 400 ppm CO2 and 45°C (block temperature setting), b) maximum carboxylation rates of Rubisco (V_cmax), derived from the slope of A_n vs. Q (internal sub-stomatal CO2 concentration) curves that are recorded by stepwise changing CO2 concentration around the leaf inside the leaf chamber,

c) the rate of A_n induction during dark-light transitions, subjecting plants to low light (ca. 50 PAR) for about 30min, then illuminating the measured leaf at 1500 PAR while following the rise of A_n over time (ca. 15min).

The initial slope is taken as surrogate for induction kinetics of photosynthesis and hence of Rubisco activation (Carmo-Silva and Salvucci, 2013; Yamori et al. , 2012).

The transgenic Arabidopsis expressing a transcription elongation factor SPT5 with a substitution of Alanine 218 and Alanine 231 to Proline shows higher values of A_n and V_cmax than non-transgenic control plants.

The rate of induction of Rubisco activity (slope A_n min^"1) is significantly higher in the transgenic Arabidopsis than in non-transgenic control plants, indicating a beneficial effect of the substitution of Alanine 218 and Alanine 231 to Proline in the transcription elongation factor SPT5 of Arabidopsis on the activity of RuBisCO and so contributes to a better photosynthetic performance.

Seed yield

The days to flowering are recorded for every plant. After first plant starts to flower temperature will be reduced to 28-29°C (control conditions) in order to avoid pollen sterility. All plants are harvested at full maturity and number of tillers, panicles and seeds as well as vigor (visual score 1 to 6), and seed set (%) are taken. Seed weight is recorded after drying seeds (kg/ha). The transgenic Arabidopsis expressing a transcription elongation factor SPT5 with a substitution of Alanine 218 and Alanine 231 to Proline shows a seed weight higher than non- transgenic control plants.

Tiller development

Tiller numbers are counted for plants in each block after a week (six-week-old plants) as a measure of shoot development. Tiller numbers are re-counted after seven weeks. The transgenic Arabidopsis expressing a transcription elongation factor SPT5 with a substitution of Alanine 218 and Alanine 231 to Proline has significantly more tillers than the control (WT).

EXAMPLE 6: Effect of substitution of Alanine 237 to Proline in transcription elongation factor SPT5 from maize.

Zea mays biomass yield increases under standardized conditions

Engineering corn plants are obtained as described in Example 2. Excised embryos are grown on callus induction medium, then maize regeneration medium, containing a selection agent. The Petri plates are incubated in the light at 25° C for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to maize rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse.

T1 or T2 generation plants are produced and are grown under standardized conditions described in Example 5. The transgenic maize expressing a transcription elongation factor SPT5 with a substitution of Alanine 237 to Proline produces more biomass than non-transgenic control plants.

Leaf elongation rates (LER)

Leaf elongation rates of maize are measured as described in Example 5 using a HR4000 Linear Variable Displacement Transducer (LVDT).

Leaf elongation rate is measured and the transgenic maize expressing a transcription elongation factor SPT5 with a substitution of Alanine 237 to Proline elongates faster than non- transgenic control plants.

Leaf gas exchange

Leaf gas exchange measurements of maize are carried out as described in Example 5. The following parameters are recorded with an open infrared gas analyser system LI-6400XT (Ll- COR Inc., Lincoln, NE, USA).

The transgenic maize expressing a transcription elongation factor SPT5 with a substitution of Alanine 237 to Proline shows higher values of A_n and Vc,max than non-transgenic control plants.

The rate of induction of Rubisco activity (slope A_n min^"1) is significantly higher in the transgenic maize than in non-transgenic control plants, indicating a beneficial effect of the substitution of Alanine 237 to Proline in the transcription elongation factor SPT5 of maize on the activity of RuBisCO and so apparently to a better photosynthetic performance.

Seed yield

Seed weight are determined as described in Example 5.

The transgenic maize expressing a transcription elongation factor SPT5 with a substitution of Alanine 237 to Proline shows a seed weight higher than non-transgenic control plants. Tiller development

Tiller numbers are counted for maize as described in Example 5. The transgenic maize expressing a transcription elongation factor SPT5 with a substitution of Alanine 237 to Proline has significantly more tillers than the control (WT).

EXAMPLE 7: Effects of substitution of Alanine 178 and Alanine 228 to Proline of transcription elongation factor SPT5 from tobacco (Nicotians tabacum).

Chlorophyll Fluorescence Measurements

Transgenic tobacco seeds (Example 3) are germinated under ambient air conditions on Murashige and Skoog (MS) plates with essential vitamins in a controlled environment chamber (Environmental Growth Chambers, Chagrin Falls, Ohio, USA) with 14 h day (25°C)/10 h night (22°C) and light intensity of 500 pmol nr² s ¹. Eight days after germination, seedling plates are transferred to a custom assembled low CO2 chamber inside the controlled environment growth chamber. The light levels are increased to 1200 pmol nr² s ¹ for 24 hours and CO2 concentration is maintained below 35 pbar. Fv’/Fm’ is determined on each plate using the CF Imager Technologica (www.technologica.co.uk). Maximum flash intensity is 6800 pmol-rn ² s ¹ for 800 milliseconds. Image values are obtained for each individual plant by detecting colonies within the fluorimager software program defining each position as has been previously described (South et al., 2017).

The transgenic Nicotiana tabacum expressing a transcription elongation factor SPT5 with a substitution of Alanine 178 and Alanine 228 to Proline presents an improvement of Chlorophyll fluorescence compared to non-transgenic control plants (WT).

Growth Analysis (Greenhouse)

To determine if the substitution of Alanine 178 and Alanine 228 to Proline of transcription elongation factor SPT5 from tobacco would result in increased growth capacity and growth rate, stem height, and dry weight biomass is determined. Single insert T2 seeds are germinated on LC1 sunshine mix (Sun Gro 202 Horticulture, Agawam, MA, USA). 10 days after germination seedlings are transferred to 4L pots (400C, Hummert International, Earth City, MO, USA) with LC1 sunshine mix supplemented with slow release fertilizer (Osmocote Plus 15/9/12, The Scotts Company LLC, Marysville, OH, USA). Pots are randomized within the greenhouse and positions are changed before each watering. Light intensity within the greenhouse is measured using a quantum sensor (LI-190R, LI-COR, Lincoln, Nebraska, USA). Air temperature, relative humidity and [C02] are measured using a combined temperature and humidity sensor (HMP60-L, Vaisala Oyj, Helsinki, Finland) and an infrared gas analyzer (SBA- 5, PPsystems, Amesbury, MA, USA). All climate data is logged using a data logger (CR1000, Campbell Scientific Inc, Logan, UT, USA). Greenhouse growth conditions utilized are similar to those previously reported in the literature (Kromdijk and Long, 2016). Above ground biomass is harvested at seven weeks after determination of stem height and dried for 2 weeks and dry weight is determined for each fraction.

The transgenic Nicotiana tabacum expressing a transcription elongation factor SPT5 with a substitution of Alanine 178 and Alanine 228 to Proline produces more biomass than non- transgenic control plants (WT).

Field Experiment

As a proof of concept experiment, the effect of the substitution of Alanine 178 and Alanine 228 to Proline of transcription elongation factor SPT5 from tobacco under field conditions. Five independent transformation events of tobacco are compared with two wild type (WT) and two empty vector (EV) controls are planted in a randomized block design. Homozygous single insert T2 seeds are germinated in pots containing soil mix (Sun Gro 202 Horticulture, Agawam, MA, USA) and grown for seven days then transferred to floating trays as previously described (Kromdijk and Long, 2016). Each block is 6 x 6 spaced 30 cm apart. The internal 16 plants per block are the indicated transgenic plant lines surrounded by a WT border. An additional two row border of WT plants surrounded the experiment. Watering is provided as needed from six water towers placed within the plot. Weather data, including Light intensity, air temperature, and precipitation are measured. Apparent quantum efficiency of photosynthesis (0a) including the light saturated level of photosynthesis at ambient 400 pbar and low 100 pbar CO2 concentrations is measured on the youngest fully expanded leaf 14- 20 days after transplanting to the field. 0a is determined from assimilation measurements in response to light levels at the indicated [CO2]. Gas exchange measurements are performed using a LI-COR 6400XT with a 2 cm² fluorescence measuring cuvette with gasket leaks corrected for as outlined in the manual (LI-COR Biosciences, Lincoln, NE, USA). Measurements of CO2 assimilation are done at light intensities of 1200, 380, 120, 65, 40, 30, 25, 18, and 10 pmol-m ^s ¹ , assimilation is recorded after a minimum of 120 seconds at each light level. 0a is calculated from the slope of the initial response of assimilation at low light levels. The saturating level of assimilation (A_sat) is determined from the 1200 pmol- f^s^-1 measurement at the indicated [CO2]. Stem height, leaf and stem biomass is determined for 8 plants per plot at 7 weeks post planting. After stem height is assessed, above ground biomass is harvested and separated into leaf and stem fractions. Plant material is dried for a minimum of 2 weeks prior to biomass measurements. The transgenic Nicotiana tabacum expressing a transcription elongation factor SPT5 with a substitution of Alanine 178 and Alanine 228 to Proline increases in the quantum efficiency of photosynthesis (0a), photosynthetic efficiency and plant productivity under agricultural conditions. The combined fluorescence screen, greenhouse and field experiment studies show that the substitution of Alanine 178 and Alanine 228 to Proline in the transcription elongation factor SPT5 is able to outperform WT and EV in total plant growth.

Gas exchange To determine the net photosynthetic assimilation rate from a CO2 dose response the fifth leaf from the base of seven-week-old N. tabacum plants are clamped into the fluorescence cuvette of a LI-COR 6800 infrared gas analyzer (Li-Cor Biosciences, Lincoln, NE, USA) with leaf temperature controlled at 25°C and light intensity set at 1500 pmol nr² s ¹. Leaves are acclimated at 400 pmol mol ¹ to achieve a steady state. The CO2 concentration of the response curve is set at 400, 200, 100, 50, 30, 400, 600, 800, 1000, 1500, 2000 pmol mol ¹ and measurements are taken when assimilation reached a steady state. To determine the maximum rate of carboxylation (V_cmax) , maximum electron transport rate (Jmax) and mitochondrial respiration rate a model for leaf photosynthesis with temperature corrections is used assuming infinite mesophyll conductance from the collected CO2 response curves. I^~ _* and Rd measurements using the common intersection method Gas exchange is performed using a LI-COR 6800 (LI- COR Biosciences) using a fluorescence chamber. G_* is measured using the common intersection method by measuring the CO2 response of photosynthesis under various sub-saturating irradiances. The common intersection is determined using slope-intercept regression to produce more accurate and consistent values of Ci* and R_d (Walker et al., 2016). Plants are acclimated under 250 pmol rrf² s^-1 light at 150 pBar CO2 until photosynthesis reached steady and measured at 150, 120, 90, 70, 50, and 30 pBar CO2 under irradiances of 250, 165, 120, 80, and 50 pmol rrf² s ¹. The x-intersection point is converted to G_* as previously reported (Walker et al., 2016).

The transgenic Nicotiana tabacum expressing a transcription elongation factor SPT5 with a substitution of Alanine 178 and Alanine 228 to Proline demonstrates increases in V_cmax and in Jmax suggesting more efficient photosynthesis at lower [CO2] than wild type Nicotiana tabacum.

EXAMPLE 8: Effect of substitution of Alanine 217 to Proline in transcription elongation factor SPT5 from tomato.

Tomato biomass yield increases under standardized conditions

Engineering tomato plants are obtained as described in Example 4.

T1 or T2 generation plants are produced and are grown under standardized conditions described in Example 5. The transgenic tomato expressing a transcription elongation factor SPT5 with a substitution of Alanine 217 to Proline produces more biomass than non-transgenic control plants.

Leaf elongation rates (LER)

Leaf elongation rates of tomato are measured as described in Example 5 using a H R4000 Linear Variable Displacement Transducer (LVDT).

Leaf elongation rate is measured and the transgenic tomato expressing a transcription elongation factor SPT5 with a substitution of Alanine 217 to Proline elongates faster than non- transgenic control plants. Leaf gas exchange

Leaf gas exchange measurements of tomato are carried out as described in Example 5. The following parameters are recorded with an open infrared gas analyser system LI-6400XT (Ll- COR Inc., Lincoln, NE, USA).

The transgenic tomato expressing a transcription elongation factor SPT5 with a substitution of Alanine 217 to Proline shows higher values of A_n and V_cmax than non-transgenic control plants. The rate of induction of Rubisco activity (slope A_n min^"1) is significantly higher in the transgenic tomato than in non-transgenic control plants, indicating a beneficial effect of the substitution of Alanine 217 to Proline in the transcription elongation factor SPT5 of tomato on the activity of RuBisCO and so contributes to a better photosynthetic performance.

Seed yield

Seed weight are determined as described in Example 5.

The transgenic tomato expressing a transcription elongation factor SPT5 with a substitution of Alanine 217 to Proline shows a seed weight higher than non-transgenic control plants.

Tiller development

Tiller numbers are counted for maize as described in Example 5. The transgenic tomato expressing a transcription elongation factor SPT5 with a substitution of Alanine 217 to Proline has significantly more tillers than the control (WT).

BIBLIOGRAPHY

Albert, H., Dale, E.C., Lee, E., Ow, D.W., 1995. Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J. 7, 649-59.

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389-402.

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Claims

1. A genetically modified plant characterized in that it expresses a transcription elongation factor SPT5 that is mutated in its NGN domain.

2. The genetically modified plant according to claim 1 , characterized in that the SPT5 NGN domain comprises the 12-amino-acid consensus sequence shown in Figure 2.

3. The genetically modified plant according to one of claims 1 or 2, characterized in that the mutation is in the C-terminus of the b3 sheet of the NGN domain.

4. The genetically modified plant according to one of claims 2 or 3, characterized in that the mutation consists of a transposition of the ninth amino acid of the consensus sequence in Figure 2 to any other amino acid.

5. The genetically modified plant according to claim 4, characterized in that the mutation consists of a transposition of the ninth amino acid of the consensus sequence in Figure 2 to an amino acid selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, valine, proline and glycine, preferably proline.

6. The genetically modified plant according to one of claims 1 to 5, characterized in that the mutated transcription elongation factor SPT5 is encoded by the native gene of said plant wherein the genetic modification consists in a mutation introduced into its coding sequence.

7. The genetically modified plant according to one of claims 1 to 6, characterized in that the mutation is a mutation introduced by genome editing using a CRISPR/Cas enzyme.

8. The genetically modified plant according to one of claims 1 to 5, characterized in that it comprises a heterologous gene encoding the mutated transcription elongation factor SPT5.

9. The genetically modified plant according to one of claims 1 to 6, characterized in that it is a cultivated or commercial crop plant.

10. The genetically modified plant according to claim 9, characterized in that it is selected from C3 plants.

1 1. The genetically modified plant according to claim 10, characterized in that it is selected from tobacco, tomato, rice, wheat, soybean, sunflower, rapeseed, cucumber and alfalfa.

12. The genetically modified plant according to claim 9, characterized in that it is selected from C4 plants.

13. The genetically modified plant according to claim 12, characterized in that it is selected from maize, sorghum, millet and sugar cane.

14. Manufactured products, characterized in that they contain all or part of the genetically modified plants according to one of claims 1 to 13.

15. A method for identifying a plant having a SPT5 gene mutation described in claims 1 to 5 which comprises the steps of

i) providing a library of plant genetic material,

ii) screening this library to identify the presence of an SPT5 gene mutation described in claims 1 to 5 and

iii) selecting the plants that have an SPT5 gene mutation.

16. A method of producing a cultivated or commercial crop plant having a SPT5 gene mutation described in claims 1 to 5 which comprises the steps of

i) selecting a plant from a library identified according to claim 15, and

ii) propagating or multiplying said plant to obtain a cultivated or commercial crop plant having the said SPT5 gene mutation.

17. A method for increasing the content of at least one energy storage component of a plant said method comprising the steps of

i) screening a population of plants for the presence of an SPT5 gene mutation described in claims 1 to 5 or introducing such mutation into a plant, ii) selecting the plants which have the said mutation and an increased energy storage component content, and

iii) further propagating or multiplying said plant.

18. The method of claim 17, where in the energy storage component is selected from the group consisting of starch, sugar, oil, and protein.

19. The method of any claim 15 to 18, characterized in that it is a cultivated or commercial crop plant as identified in any of claims 9 to 13.

20. A plant cultivated / commercial crop having a SPT5 gene comprising the consensus sequence of Figure 2, characterized in that ninth amino acid of the said consensus sequence of Figure 2 is a proline.

21. Seeds of a plant as defined in one of claims 1 to 13 and 20 or obtained by the method of one of claims 16 to 18.

22. Use of a genetically modified plant according to one of claims 1 to 13 or 20 or a plant selected from a library by the process of claim 16 in a variety selection programme for the production of commercial plants containing the SPT5 gene mutation according to one of claims 1 to 5.