Intron-mediated Enhancement Research Articles

Intron-mediated enhancement of DIACYLGLYCEROL ACYLTRANSFERASE1 expression in energycane promotes a step change for lipid accumulation in vegetative tissues

BackgroundMetabolic engineering for hyperaccumulation of lipids in vegetative tissues is a novel strategy for enhancing energy density and biofuel production from biomass crops. Energycane is a prime feedstock for this approach due to its high biomass production and resilience under marginal conditions. DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) catalyzes the last and only committed step in the biosynthesis of triacylglycerol (TAG) and can be a rate-limiting enzyme for the production of TAG.ResultsIn this study, we explored the effect of intron-mediated enhancement (IME) on the expression of DGAT1 and resulting accumulation of TAG and total fatty acid (TFA) in leaf and stem tissues of energycane. To maximize lipid accumulation these evaluations were carried out by co-expressing the lipogenic transcription factor WRINKLED1 (WRI1) and the TAG protect factor oleosin (OLE1). Including an intron in the codon-optimized TmDGAT1 elevated the accumulation of its transcript in leaves by seven times on average based on 5 transgenic lines for each construct. Plants with WRI1 (W), DGAT1 with intron (Di), and OLE1 (O) expression (WDiO) accumulated TAG up to a 3.85% of leaf dry weight (DW), a 192-fold increase compared to non-modified energycane (WT) and a 3.8-fold increase compared to the highest accumulation under the intron-less gene combination (WDO). This corresponded to TFA accumulation of up to 8.4% of leaf dry weight, a 2.8-fold or 6.1-fold increase compared to WDO or WT, respectively. Co-expression of WDiO resulted in stem accumulations of TAG up to 1.14% of DW or TFA up to 2.08% of DW that exceeded WT by 57-fold or 12-fold and WDO more than twofold, respectively. Constitutive expression of these lipogenic “push pull and protect” factors correlated with biomass reduction.ConclusionsIntron-mediated enhancement (IME) of the expression of DGAT resulted in a step change in lipid accumulation of energycane and confirmed that under our experimental conditions it is rate limiting for lipid accumulation. IME should be applied to other lipogenic factors and metabolic engineering strategies. The findings from this study may be valuable in developing a high biomass feedstock for commercial production of lipids and advanced biofuels.Graphical abstract

Mechanisms of Intron-Mediated Enhancement of Expression: Welcome to the Hotel California

Mechanisms of Intron-Mediated Enhancement of Expression: Welcome to the Hotel California

The presence of an intron relieves gene repression caused by promoter-proximal four-bp specific sequences in yeast

Introns can enhance gene expression in eukaryotic cells in a process called intron-mediated enhancement (IME). The levels of enhancement are affected not only by the intron sequence but also by coding sequences (CDSs). However, the parts of CDSs responsible for mediating IME have not yet been identified. In this study, we identified an IME-mediating sequence by analyzing three pairs of IME-sensitive and -insensitive CDSs in Saccharomyces cerevisiae. Expression of the CDSs yCLuc, yRoGLU1, and KmBGA1 was enhanced by the presence of an intron (i.e., they were IME sensitive), but the expression of each corresponding codon-changed CDS, which encoded the identical amino acid sequence, was not enhanced (i.e., they were IME insensitive). Interestingly, the IME-insensitive CDSs showed higher expression levels that were like intron-enhanced expression of IME-sensitive CDSs, suggesting that expression of IME-sensitive CDSs was repressed. A four-nucleotide sequence (TCTT) located in the promoter-proximal position of either the untranslated or coding region was found to be responsible for repression in IME-sensitive CDSs, and repression caused by the TCTT sequence was relieved by the presence of an intron. Further, it was found that the expression of intron-containing yeast-native genes, UBC4 and MPT5, was repressed by TCTT in the CDS but relieved by the introns. These results indicate that TCTT sequences in promoter-proximal positions repress gene expression and that introns play a role in relieving gene repression caused by sequences such as TCTT.

Effect of human β-Globin second intron on transient gene expression in mammalian cell lines

Exogenous protein expression in mammalian cells is necessary to produce therapeutic proteins and modern medical applications like developing DNA vaccines and gene therapy. This study examines the human-Globin (hBG) second intron's capacity for intron-mediated enhancement (IME) in various mammalian cell lines. Our study's main aim is to investigate the effect of the incorporation and arrangement of the second intron of the human Beta-globin gene into the pVAX-1 expression cassette on improving the expression of foreign genes. Two plasmids were constructed, one with the hBG second intron positioned upstream and the other downstream in the expression cassette. EGFP expression was evaluated at the mRNA and protein levels after transfection using Lipofectamine 2000 using One-way ANOVA analysis. Results showed that the pVAX-1 harbouring the hBG second intron did not lead to enhanced transient EGFP expression and did not exhibit Intron Mediated Enhancement (IME) in tested mammalian cell lines. Further investigations are necessary to understand factors contributing to the lack of enhancement and explore alternative intron options for optimizing foreign gene expression in cell lines.

Impact of Intron and Retransformation on Transgene Expression in Leaf and Fruit Tissues of Field-Grown Pear Trees.

Stable and high expression of introduced genes is a prerequisite for using transgenic trees. Transgene stacking enables combining several valuable traits, but repeated transformation increases the risk of unintended effects. This work studied the stability and intron-mediated enhancement of uidA gene expression in leaves and different anatomical parts of pear fruits during field trials over 14 years. The stability of reporter and herbicide resistance transgenes in retransformed pear plants, as well as possible unintended effects using high-throughput phenotyping tools, were also investigated. The activity of β-glucuronidase (GUS) varied depending on the year, but silencing did not occur. The uidA gene was expressed to a maximum in seeds, slightly less in the peel and peduncles, and much less in the pulp of pear fruits. The intron in the uidA gene stably increased expression in leaves and fruits by approximately twofold. Retransformants with the bar gene showed long-term herbicide resistance and exhibited no consistent changes in leaf size and shape. The transgenic pear was used as rootstock and scion, but grafted plants showed no transport of the GUS protein through the graft in the greenhouse and field. This longest field trial of transgenic fruit trees demonstrates stable expression under varying environmental conditions, the expression-enhancing effect of intron and the absence of unintended effects in single- and double-transformed woody plants.

Promoters and introns as key drivers for enhanced gene expression in Saccharomyces cerevisiae.

The transcription of genes in the yeast Saccharomyces cerevisiae is governed by multiple layers of regulatory elements and proteins, cooperating to ensure optimum expression of the final protein product based on the cellular requirements. Promoters have always been regarded as the most important determinant of gene transcription, but introns also play a key role in the expression of intron-encoding genes. Some introns can enhance transcription when introduced either promoter-proximal or embedded in the open reading frame of genes. However, the outcome is seldom predictable, with some introns increasing or decreasing transcription depending on the promoter and reporter gene employed. This chapter provides an overview of the general structure and function of promoters and introns and how they may cooperate during transcription to allow intron-mediated enhancement of gene expression. Since S. cerevisiae is a suitable host for recombinant protein production on a commercial level, stronger and more controllable promoters are in high demand. Enhanced gene expression can be achieved via promoter engineering, which may include introns that increase the efficacy of recombinant expression cassettes. Different models for the role of introns in transcription are briefly discussed to show how these intervening sequences can actively interact with the transcription machinery. Furthermore, recent examples of improved protein production via the introduction of promoter-proximal introns are highlighted to showcase the potential value of intron-mediated enhancement of gene expression.

Establishment of an Efficient Genome Editing System in Lettuce Without Sacrificing Specificity.

The efficiency of the CRISPR/Cas9 genome editing system remains limited in many crops. Utilizing strong promoters to boost the expression level of Cas9 are commonly used to improve the editing efficiency. However, these strategies also increase the risk of off-target mutation. Here, we developed a new strategy to utilize intron-mediated enhancement (IME)-assisted 35S promoter to drive Cas9 and sgRNA in a single transcript, which escalates the editing efficiency by moderately enhancing the expression of both Cas9 and sgRNA. In addition, we developed another strategy to enrich cells highly expressing Cas9/sgRNA by co-expressing the developmental regulator gene GRF5, which has been proved to ameliorate the transformation efficiency, and the transgenic plants from these cells also exhibited enhanced editing efficiency. This system elevated the genome editing efficiency from 14–28% to 54–81% on three targets tested in lettuce (Lactuca sativa) without increasing the off-target editing efficiency. Thus, we established a new genome editing system with highly improved on-target editing efficiency and without obvious increasement in off-target effects, which can be used to characterize genes of interest in lettuce and other crops.

Identification of essential intron sequences that enhance gene expression independently of splicing in the yeast Saccharomyces cerevisiae

Gene expression in eukaryotes is enhanced by the presence of introns in a process known as intron-mediated enhancement (IME), but its mechanism remains unclear. In Saccharomyces cerevisiae, sequences at the 5′-splice sites (SS) and branch point sites (BPS) are highly conserved compared with other higher eukaryotes. Here, the minimum intron sequence essential for IME was investigated using various short introns and a yeast codon-optimized luciferase gene as an IME model. Mutations at the 5′-SS conserved sequence and branch point in the QCR10 intron caused splicing deficiency with either a complete loss or a marked decrease in IME. By contrast, however, the 3′-AG to tG mutant was spliced and retained IME function. Moreover, heterologous introns, which did not show IME in S. cerevisiae, gained splicing competency and IME ability by substitutions to the S. cerevisiae-type 5′-SS and BPS sequences. Intriguingly, several deletion mutants between the 5′-SS and BPS in introns exhibited high levels of IME despite a loss in splicing competency. In most cases, further deletions or substitutions did not recover splicing competency and were found to decrease IME. However, a 16-nt variant consisting of the conserved 5′-SS and BPS sequences and 3′-CAG showed an IME level comparable with that of the wild-type intron. These results indicate that IME can be independent of splicing in S. cerevisiae while intron sequences at the 5′-SS and BPS play an essential role in IME.

Expression of an Antiviral Gene GmRUN1 from Soybean Is Regulated via Intron-Mediated Enhancement (IME).

Most of R (resistance) genes encode the protein containing NBS-LRR (nucleotide binding site and leucine-rich repeat) domains. Here, N. benthamiana plants were used for transient expression assays at 3–4 weeks of age. We identified a TNL (TIR-NBS-LRR) encoding gene GmRUN1 that was resistant to both soybean mosaic virus (SMV) and tobacco mosaic virus (TMV). Truncation analysis indicated the importance of all three canonical domains for GmRUN1-mediated antiviral activity. Promoter-GUS analysis showed that GmRUN1 expression is inducible by both salicylic acid (SA) and a transcription factor GmDREB3 via the cis-elements as-1 and ERE (ethylene response element), which are present in its promoter region. Interestingly, GmRUN1 gDNA (genomic DNA) shows higher viral resistance than its cDNA (complementary DNA), indicating the existence of intron-mediated enhancement (IME) for GmRUN1 regulation. We provided evidence that intron2 of GmRUN1 increased the mRNA level of native gene GmRUN1, a soybean antiviral gene SRC7 and also a reporter gene Luciferase, indicating the general transcriptional enhancement of intron2 in different genes. In summary, we identified an antiviral TNL type soybean gene GmRUN1, expression of which was regulated at different layers. The investigation of GmRUN1 gene regulatory network would help to explore the mechanism underlying soybean-SMV interactions.

Identification of cis-regulatory motifs in first introns and the prediction of intron-mediated enhancement of gene expression in Arabidopsis thaliana

BackgroundIntron mediated enhancement (IME) is the potential of introns to enhance the expression of its respective gene. This essential function of introns has been observed in a wide range of species, including fungi, plants, and animals. However, the mechanisms underlying the enhancement are as of yet poorly understood. The goal of this study was to identify potential IME-related sequence motifs and genomic features in first introns of genes in Arabidopsis thaliana.ResultsBased on the rationale that functional sequence motifs are evolutionarily conserved, we exploited the deep sequencing information available for Arabidopsis thaliana, covering more than one thousand Arabidopsis accessions, and identified 81 candidate hexamer motifs with increased conservation across all accessions that also exhibit positional occurrence preferences. Of those, 71 were found associated with increased correlation of gene expression of genes harboring them, suggesting a cis-regulatory role. Filtering further for effect on gene expression correlation yielded a set of 16 hexamer motifs, corresponding to five consensus motifs. While all five motifs represent new motif definitions, two are similar to the two previously reported IME-motifs, whereas three are altogether novel. Both consensus and hexamer motifs were found associated with higher expression of alleles harboring them as compared to alleles containing mutated motif variants as found in naturally occurring Arabidopsis accessions. To identify additional IME-related genomic features, Random Forest models were trained for the classification of gene expression level based on an array of sequence-related features. The results indicate that introns contain information with regard to gene expression level and suggest sequence-compositional features as most informative, while position-related features, thought to be of central importance before, were found with lower than expected relevance.ConclusionsExploiting deep sequencing and broad gene expression information and on a genome-wide scale, this study confirmed the regulatory role on first-introns, characterized their intra-species conservation, and identified a set of novel sequence motifs located in first introns of genes in the genome of the plant Arabidopsis thaliana that may play a role in inducing high and correlated gene expression of the genes harboring them.

Ectopic expression of VRT-A2 underlies the origin of Triticum polonicum and Triticum petropavlovskyi with long outer glumes and grains

Polish wheat (Triticum polonicum) is a unique tetraploid wheat species characterized by an elongated outer glume. The genetic control of the long-glume trait by a single semi-dominant locus, P1 (from Polish wheat), was established more than 100 years ago, but the underlying causal gene and molecular nature remain elusive. Here, we report the isolation of VRT-A2, encoding an SVP-clade MADS-box transcription factor, as the P1 candidate gene. Genetic evidence suggests that in T. polonicum, a naturally occurring sequence rearrangement in the intron-1 region of VRT-A2 leads to ectopic expression of VRT-A2 in floral organs where the long-glume phenotype appears. Interestingly, we found that the intron-1 region is a key ON/OFF molecular switch for VRT-A2 expression, not only because it recruits transcriptional repressors, but also because it confers intron-mediated transcriptional enhancement. Genotypic analyses using wheat accessions indicated that the P1 locus is likely derived from a single natural mutation in tetraploid wheat, which was subsequently inherited by hexaploid T. petropavlovskyi. Taken together, our findings highlight the promoter-proximal intron variation as a molecular basis for phenotypic differentiation, and thus species formation in Triticum plants.

Intron-mediated enhancement of transgene expression in the oleaginous diatom Fistulifera solaris towards bisabolene production

Microalgae have been widely recognized as promising hosts for production of various valuable compounds. However, low expression or silencing of transgenes in most microalgae species have hindered their broader implementation in biotechnological applications. In many eukaryotes, intron mediated enhancement (IME) of gene expression by inclusion of introns in heterologous sequences has resulted in a significant increase in transgene expression. This phenomenon has been found in a broad range of eukaryotic organisms including animals, plants, yeasts, and more recently green microalgae. However, no investigation has been conducted in diatoms. Here, we present findings of IME in the oleaginous diatom, Fistulifera solaris. Inclusion of a native intron found in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene into a bleomycin-resistance gene (shble) resulted in enhancement of the mRNA level of shble, leading to increase in resistance and selection. We tested this IME in a heterologous plant sesquiterpene synthase gene construct (AgBS), leading to expression approximately double that of non-intron containing cassettes. The heterologous AgBS lead to accumulation of the sesquiterpene (E)-α-bisabolene, indicating that native precursors were amenable to manipulation and available in the cytoplasm of this diatom. To the best of our knowledge, this is the first study to investigate the effect of IME in diatoms.

Gene Architecture Facilitates Intron-Mediated Enhancement of Transcription

Introns impact several vital aspects of eukaryotic organisms like proteomic plasticity, genomic stability, stress response and gene expression. A role for introns in the regulation of gene expression at the level of transcription has been known for more than thirty years. The molecular basis underlying the phenomenon, however, is still not entirely clear. An important clue came from studies performed in budding yeast that indicate that the presence of an intron within a gene results in formation of a multi-looped gene architecture. When looping is defective, these interactions are abolished, and there is no enhancement of transcription despite normal splicing. In this review, we highlight several potential mechanisms through which looping interactions may enhance transcription. The promoter-5′ splice site interaction can facilitate initiation of transcription, the terminator-3′ splice site interaction can enable efficient termination of transcription, while the promoter-terminator interaction can enhance promoter directionality and expedite reinitiation of transcription. Like yeast, mammalian genes also exhibit an intragenic interaction of the promoter with the gene body, especially exons. Such promoter-exon interactions may be responsible for splicing-dependent transcriptional regulation. Thus, the splicing-facilitated changes in gene architecture may play a critical role in regulation of transcription in yeast as well as in higher eukaryotes.

Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii.

Efficient nuclear transgene expression in the green microalga Chlamydomonas reinhardtii is generally hindered by low transcription rates. Introns can increase transcript abundance by a process called Intron-Mediated Enhancement (IME) in this alga and has been broadly observed in other eukaryotes. However, the mechanisms of IME in microalgae are poorly understood. Here, we identified 33 native introns from highly expressed genes in C. reinhardtii selected from transcriptome studies as well as 13 non-native introns. We investigated their IME capacities and probed the mechanism of action by modification of splice sites, internal sequence motifs, and position within transgenes. Several introns were found to elicit strong IME and found to be broadly applicable in different expression constructs. We determined that IME in C. reinhardtii exclusively occurs from introns within transcribed ORFs regardless of the promoter and is not induced by traditional enhancers of transcription. Our results elucidate some mechanistic details of IME in C. reinhardtii, which are similar to those observed in higher plants yet underly distinctly different induction processes. Our findings narrow the focus of targets responsible for algal IME and provides evidence that introns are underestimated regulators of C. reinhardtii nuclear gene expression.

First Come, First Served: Sui Generis Features of the First Intron.

Most of the transcribed genes in eukaryotic cells are interrupted by intervening sequences called introns that are co-transcriptionally removed from nascent messenger RNA through the process of splicing. In Arabidopsis, 79% of genes contain introns and more than 60% of intron-containing genes undergo alternative splicing (AS), which ostensibly is considered to increase protein diversity as one of the intrinsic mechanisms for fitness to the varying environment or the internal developmental program. In addition, recent findings have prevailed in terms of overlooked intron functions. Here, we review recent progress in the underlying mechanisms of intron function, in particular by focusing on unique features of the first intron that is located in close proximity to the transcription start site. The distinct deposition of epigenetic marks and nucleosome density on the first intronic DNA sequence, the impact of the first intron on determining the transcription start site and elongation of its own expression (called intron-mediated enhancement, IME), translation control in 5′-UTR, and the new mechanism of the trans-acting function of the first intron in regulating gene expression at the post-transcriptional level are summarized.

Conserved exitrons of FLAGELLIN-SENSING 2 (FLS2) across dicot plants and their functions

The alternative splicing of pattern recognition receptor genes regulates immune signalling in mammals, but in plants its role is still unknown. Here, we detected alternatively spliced introns (exitrons) in the first annotated exons of FLAGELLIN-SENSING 2 (FLS2) genes in all the examined dicot plants across nine families. The 5′ splice site (SS) regions were conserved and with rare synonymous substitutions. Point mutations and gene swaps indicated that the position and efficiency of exitron splicing primarily depended on the nucleotide sequences of FLS2 genes. Single-nucleotide mutations in the invariable codon carrying 5′ SS dramatically altered the accumulation of poplar and tomato FLS2 transcripts, indicating the 5′-proximal exitrons of FLS2 function as stimulatory introns on gene expression. The 3′ SSs of exitrons are diverse and can be changed by 1–2 nucleotide mutations in Salicaceae FLS2. The alternative transcripts (ATs) of poplar and tobacco FLS2, which encode small secreted proteins, were specifically induced by flg22, and one such AT from tobacco FLS2 suppressed flg22-induced response. Our results indicated that the exitrons of FLS2 genes regulate the accumulation of transcripts by an intron mediated enhancement (IME) mechanism and some ATs have the potential to encode suppressors for FLS2 pathway.

Association Study of the 5'UTR Intron of the FAD2-2 Gene With Oleic and Linoleic Acid Content in Olea europaea L.

Cultivated olive (Olea europaea L. subsp. europaea var. europaea) is the most ancient and spread tree crop in the Mediterranean basin. An important quality trait for the extra virgin olive oil is the fatty acid composition. In particular, a high content of oleic acid and low of linoleic, linolenic, and palmitic acid is considered very relevant in the health properties of the olive oil. The oleate desaturase enzyme encoding-gene (FAD2-2) is the main responsible for the linoleic acid content in the olive fruit mesocarp and, therefore, in the olive oil revealing to be the most important candidate gene for the linoleic acid biosynthesis. In this study, an in silico and structural analysis of the 5′UTR intron of the FAD2-2 gene was conducted with the aim to explore the natural sequence variability and its role in the gene expression regulation. In order to identify functional allele variants, the 5′UTR intron was isolated and partially sequenced in 97 olive cultivars. The sequence analysis allowed to find a 117-bp insertion including two long duplications never found before in FAD2-2 genes in olive and the existence of many intron-mediated enhancement (IME) elements. The sequence polymorphism analysis led to detect 39 SNPs. The candidate gene association study conducted for oleic and linoleic acids content revealed seven SNPs and one indel significantly associated able to explain a phenotypic variation ranging from 7% to 16% among the years. Our study highlighted new structural variants within the FAD2-2 gene in olive, putatively involved in the regulation mechanisms of gene expression associated with the variation of the content of oleic and linoleic acid.

A universal approach to investigate circRNA protein coding function

Circular RNAs (circRNAs) are an emerging class of RNA molecules that have been linked to human diseases and important regulatory pathways. Their functional roles are still under investigation, often hampered by inefficient circRNA formation in and ex vivo. We generated an intron-mediated enhancement (IME) system that—in comparison to previously published methods—increases circRNA formation up to 5-fold. This strategy also revealed previously undetected translation of circRNA, e.g., circRtn4. Substantiated by Western blots and mass spectrometry we showed that in mammalian cells, translation of circRtn4 containing a potential “infinite” circular reading frame resulted in “monomers” and extended proteins, presumably “multimer” tandem repeats. In order to achieve high levels of circRNA formation and translation of other natural or recombinant circRNAs, we constructed a versatile circRNA expression vector—pCircRNA-DMo. We demonstrated the general applicability of this method by efficiently generating two additional circRNAs exhibiting high expression levels. The circRNA expression vector will be an important tool to investigate different aspects of circRNA biogenesis and to gain insights into mechanisms of circular RNA translation.

In vivo measurements reveal a single 5\u2032-intron is sufficient to increase protein expression level in Caenorhabditis elegans

Introns can increase gene expression levels using a variety of mechanisms collectively referred to as Intron Mediated Enhancement (IME). IME has been measured in cell culture and plant models by quantifying expression of intronless and intron-bearing reporter genes in vitro. We developed hardware and software to implement microfluidic chip-based gene expression quantification in vivo. We altered position, number and sequence of introns in reporter genes controlled by the hsp-90 promoter. Consistent with plant and mammalian studies, we determined a single, natural or synthetic, 5′-intron is sufficient for the full IME effect conferred by three synthetic introns, while a 3′-intron is not. We found coding sequence can affect IME; the same three synthetic introns that increase mcherry protein concentration by approximately 50%, increase mEGFP by 80%. We determined IME effect size is not greatly affected by the stronger vit-2 promoter. Our microfluidic imaging approach should facilitate screens for factors affecting IME and other intron-dependent processes.

Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots.

SummaryPlant synthetic biology and cereal engineering depend on the controlled expression of transgenes of interest. Most engineering in plant species to date has relied heavily on the use of a few, well‐established constitutive promoters to achieve high levels of expression; however, the levels of transgene expression can also be influenced by the use of codon optimization, intron‐mediated enhancement and varying terminator sequences. Most of these alternative approaches for regulating transgene expression have only been tested in small‐scale experiments, typically testing a single gene of interest. It is therefore difficult to interpret the relative importance of these approaches and to design engineering strategies that are likely to succeed in different plant species, particularly if engineering multigenic traits where the expression of each transgene needs to be precisely regulated. Here, we present data on the characterization of 46 promoters and 10 terminators in Medicago truncatula, Lotus japonicus, Nicotiana benthamiana and Hordeum vulgare, as well as the effects of codon optimization and intron‐mediated enhancement on the expression of two transgenes in H. vulgare. We have identified a core set of promoters and terminators of relevance to researchers engineering novel traits in plant roots. In addition, we have shown that combining codon optimization and intron‐mediated enhancement increases transgene expression and protein levels in barley. Based on our study, we recommend a core set of promoters and terminators for broad use and also propose a general set of principles and guidelines for those engineering cereal species.