KEY MESSAGE: This study highlights the changes in umami-related nucleotide and glutamate levels when the AMP deaminase gene was elevated in transgenic tomato. Taste is perceived as one of a combination of five sensations, sweet, sour, bitter, salty, and umami. The umami taste is best known as a savoury sensation and plays a central role in food flavour, palatability, and eating satisfaction. Umami flavour can be imparted by the presence of glutamate and is greatly enhanced by the addition of ribonucleotides, such as inosine monophosphate (IMP) and guanosine monophosphate (GMP). The production of IMP is regulated by the enzyme adenosine monophosphate (AMP) deaminase which functions to convert AMP into IMP. We have generated transgenic tomato (Solanum lycopersicum) lines over expressing AMP deaminase under the control of a fruit-specific promoter. The transgenic lines showed substantially enhanced levels of AMP deaminase expression in comparison to the wild-type control. Elevated AMP deaminase levels resulted in the reduced accumulation of glutamate and increased levels of the umami nucleotide GMP. AMP concentrations were unchanged. The effects on the levels of glutamate and GMP were unexpected and are discussed in relation to the metabolite flux within this pathway.
KEY MESSAGE: Several hypomethylated sites within the Karma region of EgDEF1 and hotspot regions in chromosomes 1, 2, 3, and 5 may be associated with mantling. One of the main challenges faced by the oil palm industry is fruit abnormalities, such as the "mantled" phenotype that can lead to reduced yields. This clonal abnormality is an epigenetic phenomenon and has been linked to the hypomethylation of a transposable element within the EgDEF1 gene. To understand the epigenome changes in clones, methylomes of clonal oil palms were compared to methylomes of seedling-derived oil palms. Whole-genome bisulfite sequencing data from seedlings, normal, and mantled clones were analyzed to determine and compare the context-specific DNA methylomes. In seedlings, coding and regulatory regions are generally hypomethylated while introns and repeats are extensively methylated. Genes with a low number of guanines and cytosines in the third position of codons (GC3-poor genes) were increasingly methylated towards their 3' region, while GC3-rich genes remain demethylated, similar to patterns in other eukaryotic species. Predicted promoter regions were generally hypomethylated in seedlings. In clones, CG, CHG, and CHH methylation levels generally decreased in functionally important regions, such as promoters, 5' UTRs, and coding regions. Although random regions were found to be hypomethylated in clonal genomes, hypomethylation of certain hotspot regions may be associated with the clonal mantling phenotype. Our findings, therefore, suggest other hypomethylated CHG sites within the Karma of EgDEF1 and hypomethylated hotspot regions in chromosomes 1, 2, 3 and 5, are associated with mantling.
KEY MESSAGE: Transcript profiling during the early induction phase of oil palm tissue culture and RNAi studies in a model somatic embryogenesis system showed that EgENOD93 expression is essential for somatic embryogenesis. Micropropagation of oil palm through tissue culture is vital for the generation of superior and uniform elite planting materials. Studies were carried out to identify genes to distinguish between leaf explants with the potential to develop into embryogenic or non-embryogenic callus. Oil palm cDNA microarrays were co-hybridized with cDNA probes of reference tissue, separately with embryo forming (media T527) and non-embryo (media T694) forming leaf explants sampled at Day 7, Day 14 and Day 21. Analysis of the normalized datasets has identified 77, 115 and 127 significantly differentially expressed genes at Day 7, Day 14, and Day 21, respectively. An early nodulin 93 protein gene (ENOD93), was highly expressed at Day 7, Day 14, and Day 21 and in callus (media T527), as assessed by RT-qPCR. Validation of EgENOD93 across tissue culture lines of different genetic background and media composition showed the potential of this gene as an embryogenic marker. In situ RNA hybridization and functional characterization in Medicago truncatula provided additional evidence that ENOD93 is essential for somatic embryogenesis. This study supports the suitability of EgENOD93 as a marker to predict the potential of leaf explants to produce embryogenic callus. Crosstalk among stresses, auxin, and Nod-factor like signalling molecules likely induces the expression of EgENOD93 for embryogenic callus formation.
KEY MESSAGE: Potentially embryogenic oil palms can be identified through leaf transcriptomic signatures. Differential expression of genes involved in flowering time, and stress and light responses may associate with somatic embryogenesis potential. Clonal propagation is an attractive approach for the mass propagation of high yielding oil palms. A major issue hampering the effectiveness of oil palm tissue culture is the low somatic embryogenesis rate. Previous studies have identified numerous genes involved in oil palm somatic embryogenesis, but their association with embryogenic potential has not been determined. In this study, differential expression analysis of leaf transcriptomes from embryogenic and non-embryogenic mother palms revealed that transcriptome profiles from non- and poor embryogenic mother palms were more similar than highly embryogenic palms. A total of 171 genes exhibiting differential expression in non- and low embryogenesis groups could also discriminate high from poor embryogenesis groups of another tissue culture agency. Genes related to flowering time or transition such as FTIP, FRIGIDA-LIKE, and NF-YA were up-regulated in embryogenic ortets, suggesting that reproduction timing of the plant may associate with somatic embryogenesis potential. Several light response or photosynthesis-related genes were down-regulated in embryogenic ortets, suggesting a link between photosynthesis activity and embryogenic potential. As expression profiles of the differentially expressed genes are very similar between non- and low embryogenic groups, machine learning approaches with several candidate genes may generate a more sensitive model to better discriminate non-embryogenic from embryogenic ortets.
We applied an integrative approach using multiple methods to verify cytosine methylation in the chloroplast DNA of the multicellular brown alga Saccharina japonica. Cytosine DNA methylation is a heritable process which plays important roles in regulating development throughout the life cycle of an organism. Although methylation of nuclear DNA has been studied extensively, little is known about the state and role of DNA methylation in chloroplast genomes, especially in marine algae. Here, we have applied an integrated approach encompassing whole-genome bisulfite sequencing, methylated DNA immunoprecipitation, gene co-expression networks and photophysiological analyses to provide evidence for the role of chloroplast DNA methylation in a marine alga, the multicellular brown alga Saccharina japonica. Although the overall methylation level was relatively low in the chloroplast genome of S. japonica, gametophytes exhibited higher methylation levels than sporophytes. Gene-specific bisulfite-cloning sequencing provided additional evidence for the methylation of key photosynthetic genes. Many of them were highly expressed in sporophytes whereas genes involved in transcription, translation and biosynthesis were strongly expressed in gametophytes. Nucleus-encoded photosynthesis genes were co-expressed with their chloroplast-encoded counterparts potentially contributing to the higher photosynthetic performance in sporophytes compared to gametophytes where these co-expression networks were less pronounced. A nucleus-encoded DNA methyltransferase of the DNMT2 family is assumed to be responsible for the methylation of the chloroplast genome because it is predicted to possess a plastid transit peptide.
Anthocyanins and volatile phenylpropenes (isoeugenol and eugenol) in petunia (Petunia hybrida) flowers have the precursor 4-coumaryl coenzyme A (CoA) in common. These phenolics are produced at different stages during flower development. Anthocyanins are synthesized during early stages of flower development and sequestered in vacuoles during the lifespan of the flowers. The production of isoeugenol and eugenol starts when flowers open and peaks after anthesis. To elucidate additional biochemical steps toward (iso)eugenol production, we cloned and characterized a caffeoyl-coenzyme A O-methyltransferase (PhCCoAOMT1) from the petals of the fragrant petunia 'Mitchell'. Recombinant PhCCoAOMT1 indeed catalyzed the methylation of caffeoyl-CoA to produce feruloyl CoA. Silencing of PhCCoAOMT1 resulted in a reduction of eugenol production but not of isoeugenol. Unexpectedly, the transgenic plants had purple-colored leaves and pink flowers, despite the fact that cv Mitchell lacks the functional R2R3-MYB master regulator ANTHOCYANIN2 and has normally white flowers. Our results indicate that down-regulation of PhCCoAOMT1 activated the anthocyanin pathway through the R2R3-MYBs PURPLE HAZE (PHZ) and DEEP PURPLE, with predominantly petunidin accumulating. Feeding cv Mitchell flowers with caffeic acid induced PHZ expression, suggesting that the metabolic perturbation of the phenylpropanoid pathway underlies the activation of the anthocyanin pathway. Our results demonstrate a role for PhCCoAOMT1 in phenylpropene production and reveal a link between PhCCoAOMT1 and anthocyanin production.
Seed storage reserves mainly consist of starch, triacylglycerols, and storage proteins. They not only provide energy for seed germination and seedling establishment, but also supply essential dietary nutrients for human beings and animals. So far, the regulatory networks that govern the accumulation of seed storage reserves in plants are still largely unknown. Here, we show that TRANSPARENT TESTA GLABRA1 (TTG1), which encodes a WD40 repeat transcription factor involved in many aspects of plant development, plays an important role in mediating the accumulation of seed storage reserves in Arabidopsis (Arabidopsis thaliana). The dry weight of ttg1-1 embryos significantly increases compared with that of wild-type embryos, which is accompanied by an increase in the contents of starch, total protein, and fatty acids in ttg1-1 seeds. FUSCA3 (FUS3), a master regulator of seed maturation, binds directly to the TTG1 genomic region and suppresses TTG1 expression in developing seeds. TTG1 negatively regulates the accumulation of seed storage proteins partially through transcriptional repression of 2S3, a gene encoding a 2S albumin precursor. TTG1 also indirectly suppresses the expression of genes involved in either seed development or synthesis/modification of fatty acids in developing seeds. In addition, we demonstrate that the maternal allele of the TTG1 gene suppresses the accumulation of storage proteins and fatty acids in seeds. Our results suggest that TTG1 is a direct target of FUS3 in the framework of the regulatory hierarchy controlling seed filling and regulates the accumulation of seed storage proteins and fatty acids during the seed maturation process.
The spontaneous occurrence of resistance to the herbicide glyphosate in weed species has been an extremely infrequent event, despite over 20 years of extensive use. Recently, a glyphosate-resistant biotype of goosegrass (Eleusine indica) was identified in Malaysia exhibiting an LD(50) value approximately 2- to 4-fold greater than the sensitive biotype collected from the same region. A comparison of the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity by glyphosate in extracts prepared from the resistant (R) and sensitive (S) biotypes revealed an approximately 5-fold higher IC(50)(glyphosate) for the (R) biotype. Sequence comparisons of the predicted EPSPS mature protein coding regions from both biotypes revealed four single-nucleotide differences, two of which result in amino acid changes. One of these changes, a proline to serine substitution at position 106 in the (R) biotype, corresponds to a substitution previously identified in a glyphosate-insensitive EPSPS enzyme from Salmonella typhimurium. Kinetic data generated for the recombinant enzymes suggests that the second substitution identified in the (R) EPSPS does not contribute significantly to its reduced glyphosate sensitivity. Escherichia coli aroA- (EPSPS deficient) strains expressing the mature EPSPS enzyme from the (R) biotype exhibited an approximately 3-fold increase in glyphosate tolerance relative to strains expressing the mature EPSPS from the (S) biotype. These results provide the first evidence for an altered EPSPS enzyme as an underlying component of evolved glyphosate resistance in any plant species.
Tomato (Solanum lycopersicum) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals, and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodeling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin-degrading enzymes are involved in cell wall remodeling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin-degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a), and β-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL, PG2a, and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to β-1,4-galactan, and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a, and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines.
Dehydration-responsive element binding (DREB) transcription factor plays an important role in controlling the expression of abiotic stress responsive genes. An intronless oil palm EgDREB1 was isolated and confirmed to be a nuclear localized protein. Electrophoretic mobility shift and yeast one-hybrid assays validated its ability to interact with DRE/CRT motif. Its close evolutionary relation to the dicot NtDREB2 suggests a universal regulatory role. In order to determine its involvement in abiotic stress response, functional characterization was performed in oil palm seedlings subjected to different levels of drought severity and in EgDREB1 transgenic tomato seedlings treated by abiotic stresses. Its expression in roots and leaves was compared with several antioxidant genes using quantitative real-time PCR. Early accumulation of EgDREB1 in oil palm roots under mild drought suggests possible involvement in the initiation of signaling communication from root to shoot. Ectopic expression of EgDREB1 in T1 transgenic tomato seedlings enhanced expression of DRE/CRT and non-DRE/CRT containing genes, including tomato peroxidase (LePOD), ascorbate peroxidase (LeAPX), catalase (LeCAT), superoxide dismutase (LeSOD), glutathione reductase (LeGR), glutathione peroxidase (LeGP), heat shock protein 70 (LeHSP70), late embryogenesis abundant (LeLEA), metallothionine type 2 (LeMET2), delta 1-pyrroline-5- carboxylate synthetase (LePCS), ABA-aldehyde oxidase (LeAAO) and 9-cis- Epoxycarotenoid dioxygenase (LeECD) under PEG treatment and cold stress (4 °C). Altogether, these findings suggest that EgDREB1 is a functional regulator in enhancing tolerance to drought and cold stress.
We have characterized an oil palm (Elaeis guineensis Jacq.) constitutive promoter that is derived from a translationally control tumor protein (TCTP) gene. The TCTP promoter was fused transcriptionally with the gusA reporter gene and transferred to monocot and dicot systems in order to study its regulatory role in a transient expression study. It was found that the 5' region of TCTP was capable of driving the gusA expression in all the oil palm tissues tested, including immature embryo, embryogenic callus, embryoid, young leaflet from mature palm, green leaf, mesocarp and stem. It could also be used in dicot systems as it was also capable of driving gusA expression in tobacco leaves. The results indicate that the TCTP promoter could be used for the production of recombinant proteins that require constitutive expression in the plant system.
KEY MESSAGE: Transcriptomes generated by laser capture microdissected abnormal staminodes revealed adoption of carpel programming during organ initiation with decreased expression of numerousHSPs,EgDEF1, EgGLO1but increasedLEAFYexpression. The abnormal mantled phenotype in oil palm involves a feminization of the male staminodes into pseudocarpels in pistillate inflorescences. Previous studies on oil palm flowering utilized entire inflorescences or spikelets, which comprised not only the male and female floral organs, but the surrounding tissues as well. Laser capture microdissection coupled with RNA sequencing was conducted to investigate the specific transcriptomes of male and female floral organs from normal and mantled female inflorescences. A higher number of differentially expressed genes (DEGs) were identified in abnormal versus normal male organs compared with abnormal versus normal female organs. In addition, the abnormal male organ transcriptome closely mimics the transcriptome of abnormal female organ. While the transcriptome of abnormal female organ was relatively similar to the normal female organ, a substantial amount of female DEGs encode HEAT SHOCK PROTEIN genes (HSPs). A similar high amount (20%) of male DEGs encode HSPs as well. As these genes exhibited decreased expression in abnormal floral organs, mantled floral organ development may be associated with lower stress indicators. Stamen identity genes EgDEF1 and EgGLO1 were the main floral regulatory genes with decreased expression in abnormal male organs or pseudocarpel initials. Expression of several floral transcription factors was elevated in pseudocarpel initials, notably LEAFY, FIL and DL orthologs, substantiating the carpel specification programming of abnormal staminodes. Specific transcriptomes thus obtained through this approach revealed a host of differentially regulated genes in pseudocarpel initials compared to normal male staminodes.
Plant cells contain a diverse repertoire of RNA-binding proteins (RBPs) that coordinate a network of post-transcriptional regulation. RBPs govern diverse developmental processes by modulating the gene expression of specific transcripts. Recent gene annotation and RNA sequencing clearly showed that heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins which form a family of RBPs, are also expressed in higher plants and serve specific plant functions. In addition to their involvement in post-transcriptional regulation from mRNA capping to translation, they are also involved in telomere regulation, gene silencing and regulation in chloroplast. Here, we review the involvement of plant hnRNP-like proteins in post-transcription regulation of RNA processes and their functional roles in control of plant developmental processes especially plant-specific functions including flowering, chloroplastic-specific mRNA regulation, long-distance phloem transportation and plant responses to environmental stresses.
Abiotic stress reduces plant growth and crop productivity. However, the mechanism underlying posttranscriptional regulations of stress response remains elusive. Herein, we report the posttranscriptional mechanism of nucleocytoplasmic RNA transport of stress-responsive transcripts mediated by EgRBP42, a heterogeneous nuclear ribonucleoprotein-like RNA-binding protein from oil palm, which could be necessary for rapid protein translation to confer abiotic stress tolerance in plants. Transgenic Arabidopsis overexpressing EgRBP42 showed early flowering through alteration of gene expression of flowering regulators and exhibited tolerance towards heat, cold, drought, flood, and salinity stresses with enhanced poststress recovery response by increasing the expression of its target stress-responsive genes. EgRBP42 harbours nucleocytoplasmic shuttling activity mediated by the nuclear localization signal and the M9-like domain of EgRBP42 and interacts directly with regulators in the nucleus, membrane, and the cytoplasm. EgRBP42 regulates the nucleocytoplasmic RNA transport of target stress-responsive transcripts through direct binding to their AG-rich motifs. Additionally, EgRBP42 transcript and protein induction by environmental stimuli are regulated at the transcriptional and posttranscriptional levels. Taken together, the posttranscriptional regulation of RNA transport mediated by EgRBP42 may change the stress-responsive protein profiles under abiotic stress conditions leading to a better adaptation of plants to environmental changes.
Phenylalanine ammonia lyase (PAL) plays a major role in plant growth, development and adaptation. In Arabidopsis thaliana, the enzyme is encoded by four genes, namely PAL1, PAL2, PAL3, and PAL4 with PAL1 and PAL2 being closely related phylogenetically and functionally. PAL1 promoter activities are associated with plant development and are inducible by various stress agents. However, PAL2 promoter activities have not been functionally analysed. Here, we show that the PAL2 promoter activities are associated with the structural development of a plant and its organs. This function was inducible in an organ-specific manner by the avirulent strain of Pseudomonas syringae pv. tomato (JL1065). The PAL2 promoter was active throughout the course of the plant development particularly in the root, rosette leaf, and inflorescence stem that provide the plant with structural support. In aerial organs, the levels of PAL2 promoter activities were negatively correlated with relative positions of the organs to the rosette leaves. The promoter was inducible in the root following an inoculation by JL1065 in the leaf suggesting PAL2 to be part of an induced defence system. Our results demonstrate how the PAL2 promoter activities are being coordinated and synchronised for the structural development of the plant and its organs based on the developmental programme. Under certain stress conditions the activity may be induced in favour of certain organs.
The 1,053-bp promoter of the oil palm metallothionein gene (so-called MSP1) and its 5' deletions were fused to the GUS reporter gene, and analysed in transiently transformed oil palm tissues. The full length promoter showed sevenfold higher activity in the mesocarp than in leaves and 1.5-fold more activity than the CaMV35S promoter in the mesocarp. The 1,053-bp region containing the 5' untranslated region (UTR) gave the highest activity in the mesocarp, while the 148-bp region was required for minimal promoter activity. Two positive regulatory regions were identified at nucleotides (nt) -953 to -619 and -420 to -256 regions. Fine-tune deletion of the -619 to -420 nt region led to the identification of a 21-bp negative regulatory sequence in the -598 to -577 nt region, which is involved in mesocarp-specific expression. Gel mobility shift assay revealed a strong interaction of the leaf nuclear extract with the 21-bp region. An AGTTAGG core-sequence within this region was identified as a novel negative regulatory element controlling fruit-specificity of the MSP1 promoter. Abscisic acid (ABA) and copper (Cu(2+)) induced the activity of the promoter and its 5' deletions more effectively than methyl jasmonate (MeJa) and ethylene. In the mesocarp, the full length promoter showed stronger inducibility in response to ABA and Cu(2+) than its 5' deletions, while in leaves, the -420 nt fragment was the most inducible by ABA and Cu(2+). These results suggest that the MSP1 promoter and its regulatory regions are potentially useful for engineering fruit-specific and inducible gene expression in oil palm.
Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.
One of the targets in oil palm genetic engineering programme is the production of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) in the oil palm leaf tissues. Production of PHB requires the use of phbA (beta-ketothiolase type A), phbB (acetoacetyl-CoA reductase) and phbC (PHB synthase) genes of Ralstonia eutropha, whereas bktB (beta-ketothiolase type B), phbB, phbC genes of R. eutropha and tdcB (threonine dehydratase) gene of Escherichia coli were used for PHBV production. Each of these genes was fused with a transit peptide (Tp) of oil palm acyl-carrier-protein (ACP) gene, driven by an oil palm leaf-specific promoter (LSP1) to genetically engineer the PHB/PHBV pathway to the plastids of the leaf tissues. In total, four transformation vectors, designated pLSP15 (PHB) and pLSP20 (PHBV), and pLSP13 (PHB) and pLSP23 (PHBV), were constructed for transformation in Arabidopsis thaliana and oil palm, respectively. The phosphinothricin acetyltransferase gene (bar) driven by CaMV35S promoter in pLSP15 and pLSP20, and ubiquitin promoter in pLSP13 and pLSP23 were used as the plant selectable markers. Matrix attachment region of tobacco (RB7MAR) was also included in the vectors to stabilize the transgene expression and to minimize silencing due to positional effect. Restriction digestion, PCR amplification and/or sequencing were carried out to ensure sequence integrity and orientation.
The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels.
A high leaf vein density is both an essential feature of C4 photosynthesis and a foundation trait to C4 evolution, ensuring the optimal proportion and proximity of mesophyll and bundle sheath cells for permitting the rapid exchange of photosynthates. Two rice mutant populations, a deletion mutant library with a cv. IR64 background (12,470 lines) and a T-DNA insertion mutant library with a cv. Tainung 67 background (10,830 lines), were screened for increases in vein density. A high throughput method with handheld microscopes was developed and its accuracy was supported by more rigorous microscopy analysis. Eight lines with significantly increased leaf vein densities were identified to be used as genetic stock for the global C4 Rice Consortium. The candidate population was shown to include both shared and independent mutations and so more than one gene controlled the high vein density phenotype. The high vein density trait was found to be linked to a narrow leaf width trait but the linkage was incomplete. The more genetically robust narrow leaf width trait was proposed to be used as a reliable phenotypic marker for finding high vein density variants in rice in future screens.