De novo transcriptome assemblies of C3 and C4 non-model grass species reveal key differences in leaf development
BMC Genomics volume 24, Article number: 64 (2023)
C4 photosynthesis is a mechanism that plants have evolved to reduce the rate of photorespiration during the carbon fixation process. The C4 pathway allows plants to adapt to high temperatures and light while more efficiently using resources, such as water and nitrogen. Despite decades of studies, the evolution of the C4 pathway from a C3 ancestor remains a biological enigma. Interestingly, species with C3-C4 intermediates photosynthesis are usually found closely related to the C4 lineages. Indeed, current models indicate that the assembly of C4 photosynthesis was a gradual process that included the relocalization of photorespiratory enzymes, and the establishment of intermediate photosynthesis subtypes. More than a third of the C4 origins occurred within the grass family (Poaceae). In particular, the Otachyriinae subtribe (Paspaleae tribe) includes 35 American species from C3, C4, and intermediates taxa making it an interesting lineage to answer questions about the evolution of photosynthesis.
To explore the molecular mechanisms that underpin the evolution of C4 photosynthesis, the transcriptomic dynamics along four different leaf segments, that capture different stages of development, were compared among Otachyriinae non-model species. For this, leaf transcriptomes were sequenced, de novo assembled, and annotated. Gene expression patterns of key pathways along the leaf segments showed distinct differences between photosynthetic subtypes. In addition, genes associated with photorespiration and the C4 cycle were differentially expressed between C4 and C3 species, but their expression patterns were well preserved throughout leaf development.
New, high-confidence, protein-coding leaf transcriptomes were generated using high-throughput short-read sequencing. These transcriptomes expand what is currently known about gene expression in leaves of non-model grass species. We found conserved expression patterns of C4 cycle and photorespiratory genes among C3, intermediate, and C4 species, suggesting a prerequisite for the evolution of C4 photosynthesis. This dataset represents a valuable contribution to the existing genomic resources and provides new tools for future investigation of photosynthesis evolution.
C4 photosynthesis repeatedly, and in some cases rapidly, evolved as a mechanism that reduces the rate of photosynthesis in some environments (reviewed by [1, 2]). This adaptation became increasingly more advantageous in the past 30 million years, as CO2 levels declined and O2 levels increased in the atmosphere . Although only about 3% of all angiosperms use the C4 pathway, C4 species are among the most economically important crops of the world (maize, sugar cane, sorghum, and millet), and C4 grasslands cover nearly 25% of the earth’s land surface [4,5,6]. Thus, C4 plants have a disproportionate influence on natural ecosystems and the world’s food supply.
Despite decades of studies, there are still many open questions about the genetic changes that occurred during evolution of C4 photosynthesis from a C3 ancestor. The C4 pathway requires changes in plant anatomy, cell structure, biochemistry, and physiology, and appears to involve dozens of genes. The leading hypothesis suggests that the assembly of C4 photosynthesis requires a series of evolutionary changes, which includes the relocalization of photorespiratory enzymes and the establishment of the intermediate C2 pathway [3, 7,8,9]. The C2 pathway represents a low-efficiency version of a photosynthetic carbon concentration mechanism in which the two-carbon compound glycine serves as a transport metabolite. Indeed, the C2 pathway is thought to be a major driver to the evolution of C4 photosynthesis because it initiates a shuttle of metabolites between the mesophyll and bundle sheath cells.
Surprisingly, the C4 pathway has evolved independently nearly 70 times in the angiosperms, with more than a third of the origins occurring in the grass family (Poaceae) [3, 10, 11]. Interestingly, few C3-C4 intermediate grass species do exist. In particular, the Paspaleae subtribe Otachyriinae presents a unique opportunity to investigate the origins of C4 photosynthesis in grasses. The Otachyriinae lineage includes 35 species distributed in the Americas, and has been the subject of recent phylogenetic and taxonomic work [12,13,14] (Additional file 1: Fig. S1). The current phylogeny shows multiple independent origins of species with intermediate anatomical and physiological characteristics that reduce levels of photorespiration (C3 Proto-Kranz [PK], and C2 photosynthesis), and at least one origin of C4 photosynthesis. The photosynthetic diversity present at Otachyriinae make this group a useful model to study the evolution of photosynthesis in closely related species.
Recent advancements in high-throughput sequencing technologies have enabled the use of genomic approaches to study the evolution of photosynthesis and the transcriptomic dynamics along a leaf development in model grass species [15,16,17,18,19]. Such results are an exceptional framework to advance in the understanding of photosynthesis on non-model species. In this work, we sought to investigate leaf gene expression of C3 (Hymenachne amplexicaulis), PK (Rugoloa pilosa), and C4 (Antaenanthia lanata) species of Otachyriinae subtribe from an evolutionary perspective. For each species, transcriptomes of four leaf segments, that capture different moments of development, were sequenced. Based on that, de novo assemblies were generated and annotated. Then, transcriptional profiles were comparatively investigated among segments and species.
Leaf maturation in selected Otachyriinae species
Four different leaf cross sections that capture different moments of development of C3, Hymenachne amplexicaulis (Rudge) Nees; PK, Rugoloa pilosa (Sw.) Zuloaga; C4, Anthaenantia lanata (Kunth) Benth  were studied (Fig. 1a). Using light microscopy we observed that leaf segments of both C3 and PK species presented a typical C3 grass leaf anatomy with horizontally extended primary and secondary vascular bundles (VB) surrounded by two layers of bundle sheath cells without chloroplasts: the smaller Inner Bundle Sheath or mestome sheath (IBS) and the larger Outer Bundle Sheath (OBS) (Fig. 1b). The OBS of consecutive VB are separated by more than two mesophyll cell (M) layers. In particular, in H. amplexicaulis, we observed the presence of aerenchymatous tissue between VB as an adaptation to wetlands. By contrast, leaf segments of the C4 species A. lanata presented a tri-dimensional pattern of distribution of secondary VB. One layer of larger bundle sheath cells with chloroplasts, conventionally named IBS, surrounds both primary and secondary VB (Fig. 1b). In addition, one or two mesophyll cell layers separate the IBS from VB in A. lanata (Fig. 1b). Under light microscopy, we observed that segments 1 and 2 (S1, S2; leaf base) of each species are underdeveloped (in terms of present and distribution of chloroplast and vein development) in comparison with segments 5 and 7 (S5, S7; leaf blade) (Additional file 1: Fig. S2).
The datasets generated and analyzed during the current study are available at NCBI Sequence Read with the accession N° PRJNA813546 (https://dataview.ncbi.nlm.nih.gov/object/PRJNA813546?reviewer=6vmhgdsbsobv6pd6u6tr9rp4oe). De novo transcriptome assembly was carried out using the Trinity package [20, 21]. Many transcript isoforms were detected after assembly. Indeed, we found 55.9, 136.9, and 112.2% more transcripts than genes for the C3, PK and C4 transcriptomes respectively (Additional file 1: TableS1). Although a high number of isoforms may be a possibility due to the complexity of grass genomes, lowly expressed isoforms could represent chimeric transcripts generated by the assembler. Therefore, only the most expressed isoforms were kept for downstream analyses. Coding sequences were identified using Transdecoder after redundant sequences were removed with CD-Hit [21, 22]. Transcript assemblies were then filtered by selecting the “single best” open reading frame (ORF) per transcript using Transdecoder  (Table 1; Additional file 1: Table S1).
To evaluate the level of duplicate sequences and transcriptome completeness, a BUSCO search against the monocots database was performed  (Table 1). The results showed a high level of completeness for the de novo transcriptome assemblies of each species (85.7, 83.4 and 82.8% for C3, PK and C4 species respectively), and a low level of duplicated sequences (1.8, 1.7 and 1.8% for C3, PK and C4 species respectively) for all transcriptomes. Quality information for the final transcriptomes is shown in Table 1. The total number of protein coding transcripts varied widely between species: 56,064 transcripts for C3 H. amplexicaulis, 29,370 for PK R. pilosa, and 50,890 for C4 A. lanata.
Transcript annotation and ortholog inference
For transcript annotation and ortholog identification, Orthofinder was used with six reference proteomes from Otachyriinae subtribe species Steinchisma hians, Steinchisma laxa, and four grass outgroups, Zea mays, Sorghum bicolor, Setaria viridis and Oryza sativa. The species tree reconstructed by orthofinder is consistent with the species phylogeny reported in the literature (Fig. 2a; Additional file 1: Fig. S1). Around 81% of transcripts were placed in orthogroups (OG) and 2246 single-copy OG were detected between the nine transcriptomes. When S. bicolor sequences were present in an OG we used S. bicolor sequence information to infer the annotation for the Otachyriinae transcripts. In the OG where S. bicolor sequences were absent, we used annotations from Z. mays or S. viridis. A total of 25,134 (44.8%), 21,710 (73.9%), and 27,147 (53.3%) transcripts were annotated for C3 H. amplexicaulis, PK R. pilosa and C4 A. lanata respectively (Fig. 2b; Additional file 1: Table S2). Despite the differences in transcriptome sizes, the number of annotated transcripts were similar. To further explore the annotation rate differences, transcripts were filtered based on expression (less than 1 cpm in 3 replicate libraries), and the proportion assigned to OG was assessed. The results showed that 91.6, 90.3, and 79.7% of the transcripts that were retained after filtering LET were annotated transcripts belonging to OG for C3, PK and C4 respectively (Fig. 2b; Additional file 1: Table S2). Altogether, this suggests that transcripts annotated from OG represent a core set of high-quality transcripts.
A Principal Component Analysis (PCA) of samples, using expression values from 13,373 OG present in all three species, showed replicates grouping together (Fig. 2c). We observed that PC1 explains 26.4% of the variation and discriminates samples by developmental stage and PC2 explains 20.4% of variation and discriminates samples by species. In order to understand the biological processes represented by the principal components, we calculated the load factors of each OG for both components, and identified gene ontology terms enriched in the OG with the top and bottom 5% load factors  (Additional File 2: Supplementary datasheet 1). Results indicated that PC1 represented processes related to cellular development such as protein folding and transport, cytokinesis, photosynthesis and sucrose metabolism. The load factors from PC2 were negatively associated with autophagy, regulation of transcription and RNA metabolism. Positively associated terms were related to C4 metabolism such as mitochondria pyruvate transport, carbon fixation and organic hidroxy compound biosynthesis. We also found that PC3 discriminates PK samples from other species explaining 15.9% of variation and PC4 correlated with sink-source transition zone samples and explains 10% of variance (Additional File 1: Fig. S3).
Differentially expressed genes
Defining sink and sources zones
To link our anatomical observations with gene expression along the species leaf segments, we investigated the transcript levels of genes that are known markers of sink and source zones [15, 16, 25, 26]. Sink markers, such as cell cycle modulators Cyclin (CycD4 and CycD6) and sucrose synthase (SUS), decrease in expression from S1-S3 to S5-S7, indicating that S1 and S3 belong to the leaf sink zone. By contrast, the expression level of the source markers sucrose transporters (SWEET and STP1) and nitrate reductase (NR1) are low in segment S1 and S3 but have increased expression in S5 and S7, indicating that S5 and S7 correspond to the source zone of the leaves (Additional file 1: Fig. S4).
Transcriptome dynamics of developing leaves
We studied the dynamics of the transcriptomes along the leaf development for each of the three species. Pairwise comparisons within species were done between consecutive segments to evaluate the number of differentially expressed genes (DEG) (Fig. 3a). The transition between S1 and S3 resulted in the most DEG in all species, and the number of DEG tends to decrease during leaf maturation. Overall, when the number of DEG in adjacent segments were compared between species, we observed that the C3 leaf showed a lower number of DEG than the C4 leaf. The PK presented intermediate numbers of DEG, similar to the C4 maximum in S1-S3 transitions and the C3 minimum in S3-S5 and S5-S7 transitions. These results imply a more dynamic leaf transcriptome in C4 species in terms of the amount of DEG along the leaf segments. In terms of total DEG, we observed that the PK transcriptomic profile resembles that of the C4 leaf more than that of the C3 for every leaf segment (Fig. 3b).
A Gene Ontology (GO) analysis was performed with TopGO to obtain information about the biological processes enriched in the DEG in each transition. Overall, the three species showed a well-preserved developmental progression, with differences in the onset and length of several processes (Additional file 3: Supplementary datasheet 2). All species showed GO terms related to photosynthesis (such as light harvesting, photosystems I and II, thylakoid membrane, etc.), enriched in upregulated DEG in S3 compared to S1. In particular, the C4 species, GO terms related to photosynthesis continue to be present in the upregulated DEG of S5 compared to S3. All species showed GO terms associated with cell growth and development (DNA replication, modification of the cell wall, development and cellular division processes) in down regulated DEG in S3 compared to S1. In addition, the C4 species included terms related to translation and ribosome biogenesis in S3 compared to S1 (Additional file 3: Supplementary datasheet 2). In the later stages of leaf development, comparisons between S3 and S5 of all species showed DEG enriched in the categories of oxide-reduction, carbohydrate metabolism, and response to oxidative stress. In particular, in S5 of C3, transcripts involved in protein phosphorylation and defense response processes were upregulated. Finally, in all species, terms such as cellulose biosynthetic process, cell wall modification, and transmembrane transport were detected late in development, both in comparisons between S3 and S5 as well as S5 and S7 (Additional file 3: Supplementary datasheet 2).
Gene expression comparisons at successive stages of leaf development between species
Between species, comparisons of differential gene expression among segments of the same developmental stage were analyzed using orthologs inferred from Orthofinder (Fig. 3b and c). In most cases OG were made of single copy orthologs (70.3, 78.8 and 62.8% in C4, PK and C3, respectively). In the case of multicopy OG, the sum of the expression values of the transcripts was used and summarized the annotation information. As a result, 13,373 OG were tested to find Differentially Expressed OG (DEO).
The number of DEO between species were 8264, 7087, and 6518 for C4-C3, C4-PK and PK-C3, respectively. The proportion of TF within these DEO (6.6, 6.1, and 6.6% respectively for each comparison) is in line with the overall proportion of TF in the total of OG studied (6.3%, or 851 out of 13,373 OG). Overall, the number of DEO between C4 and the other species increased from base (immature sink segments) to tip (mature source segments). In contrast, the number of DEO between C3 and PK remains relatively constant across segments (Fig. 3b). The number of DEO between C4 and PK showed a stepwise increase from immature (S1, 2617 DEO) to mature segments (S7, 4723 DEO). However, the number of DEO between C4 and C3 showed a substantial difference in S1 (4197 DEO), which increases only moderately in mature segments (S7, 5108 DEO) (Fig. 3c). In terms of annotated transcription factors, we observed that the number of transcription factors belonging to DEO between C4 and the other species, doubles from base to tip (70 to 151 from S1 to S7) when species are compared. Meanwhile, the proportion of TF to total DEO has a soft increase from 4.5% (70/1534) to 5.5% (151/2757) between S1 and S7) (Fig. 3c).
Differentially expressed orthogroups involved in photosynthetic processes along leaf development
To study the processes related to photosynthesis, OG lists were constructed according to GO terms and genes classified in previous studies [27,28,29] (Additional File 4: Supplementary datasheet 3). For each list, the percentage of DEO between species was estimated according to the stage of development (Fig. 4). In terms of DEO, the differences between C3 and PK species were smaller than those observed between these species and the C4. We observed differences in OG related to photorespiration and Calvin-Benson-Bassham cycle (CBB cycle) from base to tip between C4 species and PK and C3 species (Fig. 4A-B). In particular, 36% and 24% of the OG involved in photorespiration were upregulated in S1 of C3 and PK, respectively. Similarly, 52% and 38% of the OG included in the CBB cycle were upregulated in S1 of C3 and PK, respectively. Overall, the proportion of DEO related to photorespiration increased from base to tip between C4 and the other species; however, DEO related to CBB decreased in source segments. The number of upregulated OG in the C4 cycle group increases from base to tip for the C4 species, reaching 46.2% and 42.3% in S7 for C4 vs C3 and C4 vs PK respectively (Fig. 4c). Also, we observed that, as leaves mature, the differences in expression in the C4 cycle between C4 and C3 species are slightly higher than compared to PK species.
Additionally, a heatmap is presented for each group showing the relative expression of each OG along the leaf segments and by species (Fig. 4; Additional File 1: Fig. S5A-C). We observed that: (1) most OG present an ascending pattern of expression from base to tip and, (2) the differences in the relative expression of each OG between C4 and the other species tend to be magnified, also, from base to tip. Overall, these results may suggest that photosynthetic machinery is activated later in leaf development of the C4 than in the C3 and PK species.
Differential expression of orthogroups in vasculature regulation and suberin biosynthesis processes along leaf development
Our results documented a delay in the expression of transcripts related to photosynthesis of the C4 leaf in comparison to leaves of C3 and PK species. To investigate whether the delay correlates with the existence of a longer period of anatomical development that precedes photosynthesis maturation, we looked for markers of leaf anatomical development. In particular, we analyzed the expression of genes related to vascular bundle formation and suberin biosynthesis (Additional file 4: Supplementary datasheet 3). Indeed, OG related to the regulation of venation were upregulated in C4 species compared to C3 and PK species (Fig. 5a). This is particularly evident from S3 to S7, in line with the ascendant pattern of vascular density in the C4 leaf during development. Based on the heat maps for these OG, we noted that many OG related to regulation of venation are highly expressed at the leaf base (S1) of C3 and PK species and to a lesser extent in C4 species (Fig. 5a; Additional File 1: Fig. S5D). Subsequently, OG expression declines rapidly in C3 species, whereas in PK species this decline appears to be more gradual (Fig. 5a). On the other hand, we observed an increase in OG expression in S7 of C4 species (Fig. 5a).
We also observed contrasting patterns of expression when suberin biosynthesis OG were compared between C4 and C3/PK species (Fig. 5b; Additional File 1: Fig. S5E). While suberin biosynthesis OG are expressed at S1 in C3 species, in PK and C4 species the maximum of expression is reached in S3 for most of the OG.
In vasculature regulation and suberin biosynthesis processes, we observed a shift of DEO from S3 to S7 when C3/PK and C4 were compared. This may suggest that these processes are activated early (S1) in leaf development of C3/PK species, while they are turned on later (S3-S7) in C4 leaf species.
Expression patterns and levels of expression for C4 cycle and photorespiration OG
To investigate the dynamic regulation signatures of the C4 cycle, the expression patterns and transcript levels of OG that encode for enzymes and transporters in the C4 pathway were compared across the leaf segments (Fig. 6). In order to be able to compare the expression patterns across species, a z-score normalization of the OG expression was performed individually. We found that in 17 of the 26 OG related to the C4 cycle, the expression pattern along the leaf segments were preserved among species despite having different photosynthetic pathways (Fig. 6a). Overall, most of the key enzymes of the C4 cycle show an ascending pattern as the leaves develop.
Although the expression patterns were preserved for most of OG, the levels of expression varied depending on the species and leaf segment analyzed. Meanwhile only three OG showed similar levels of expression in all species (CA, CUE1-b and BASS-b); the expression of most of the OG was substantially higher in C4 mature segments (Fig. 6b). The expression level of the OG associated with PEP-CK and AMK were similar in the C4 and PK species, but higher in the C3 species (Fig. 6b). In mature segments, decarboxylating enzymes associated with the subtypes NAD-ME and PEP-CK are less expressed in C4 than in C3 species. The OG involved in photorespiration behaved similarly as the C4 cycle. All of the OG sampled here have a conserved expression pattern among species, but only a few had similar levels of expression (Additional file 1: Fig. S6 and Fig. S7).
Closely related non-model species with different carbon assimilation pathways represent an underutilized resource for understanding the genetic changes that drive photosynthetic evolution. Here we present high-confidence protein-coding leaf transcriptomes from three species in the Otachyriinae subtribe. Transcriptomes were assembled across four different leaf segments that capture different stages of development. This dataset represents a valuable contribution to the existing genomic resources and provides new tools for future investigation of photosynthesis evolution.
The de novo assembly of the transcriptomes contained a large number of genes and transcripts. These preliminary results seem inflated compared to estimates of gene and transcript numbers in other grass species . The large number of genes and transcripts observed in our dataset compared with the known Z. mays, S. bicolor, and S. viridis transcriptomes may be explained by one or a combination of three possible phenomena: (a) the presence of a large number of the isoforms in our transcriptomes, (b) the product of sequencing errors or incomplete transcripts in poorly expressed genes and/or, (c) the presence of pre mRNA in highly expressed transcripts [31, 32]. Isoform filtering resulted in a reduction in the number of transcripts by approximately 50%, while the mapping rate had a minor reduction from 95 to 97% to 87–91%. This hypothesis, like many others related to the quality of transcriptomes, can be verified when the genomes of these species are sequenced.
Additionally, a substantial reduction in the number of genes was observed when sequences without a complete or partial ORF were filtered from the datasets. In this step, two-thirds of the C3 and C4 sequences and half of the PK sequences were removed. However, this resulted in a decrease in the mapping rate from 87 to 91% to 56–63%. Part of this reduction is likely due to the removal of UTR segments from transcripts by Transdecoder. Besides spurious transcript assemblies, filtered sequences may belong to non-coding RNA and transposable elements given that libraries represent enrichments of mRNA from a total RNA sample. Similar results were obtained when the maize leaf transcriptome was analyzed . Indeed, Li et al. (2010) found that 84% of the reads mapped to protein-coding genes, while the rest belonged to introns, intergenic regions, transposable elements, and splice junctions between exons. Finally, we confirmed that most of the sequences that were removed because they were very poorly expressed did not belong to any OG. In addition, after filtering by expression values, both the sizes of the transcriptomes and the percentage of the annotated sequences were remarkably homogeneous (Additional file 1: Table S1 and S2).
The quality analysis of the transcriptomes performed with BUSCO found two interesting results. First, the number of duplicated sequences is very low (1.7 to 1.8%), which implies little redundancy in the transcriptomes. Second, the completeness of the de novo transcriptome assemblies was higher than expected given that they were derived from a single tissue type. When using only leaf tissue samples, it would be expected to find a limited number of transcripts involved in the germination processes, root development or flower development, resulting in moderate levels of completeness. Indeed, we found a relatively low number of missing sequences (11.0 to 12.7%). The high transcriptome completeness presented could be due to the variety of leaf developmental stages used. Alternatively, the total number of reads used in the assembly (around 420 million reads per species) increased our ability to detect lowly expressed transcripts.
Divergent leaf transcriptome dynamics among species
An analysis of DEG found that immature segments showed a higher transcriptional dynamic, estimated as the number of DEG, whereas in the mature segments the dynamics substantially decreased. This pattern is observed in C4 and PK leaves; however, the trend of decreasing DEG is reduced in the C3 leaf segments. These results are in agreement with the leaf developmental gradients previously reported in several monocot species [15,16,17, 19].
Interestingly, among species studied in this work, only A. lanata maintains high levels of DEG until S5 (Fig. 3). Gene ontology analysis confirmed that genes involved in leaf anatomy differentiation are overrepresented until S5 suggesting a longer time of leaf differentiation in the C4 leaf in comparison with leaves of C3 and PK species. This result suggests that the C4 leaf takes more time to complete the necessary anatomical differentiation prior to the activation of the photosynthetic machinery. Thus, expression of photosynthetic genes is delayed to more mature segments of the leaf, in comparison with C3 and PK species. Interestingly, the longer time of leaf differentiation and the resultant delay in the activation of the photosynthesis machinery may be a peculiarity of the unique leaf anatomy of A. lanata as has also been shown for Cleomaceae . In fact, A. lanata showed a tri-dimensional pattern of distribution of secondary VB in contrast with the horizontally extended primary and secondary VB of most of the grasses. Indeed, neither maize, sorghum, rice nor Dichanthelium presented such delay in the activation of the photosynthesis machinery [15, 17,18,19]. Notably, R. pilosa presents an intermediate development pattern between the two species, with an increase in the dynamics of expression in the first developmental transition and a marked decrease in the following transitions. Interestingly, phylogenetic studies in Otachyriinae place H. amplexicaulis and R. pilosa as close relatives but, in terms of DEG, the leaf transcriptome of PK is more similar to that of C4 in the immature segments of the leaf. This may suggest commonalities in the dynamic of the expression at the sink zone of PK and C4 leaves.
Conserved expression pattern and diverse level of expression
To analyze the degree of conservation of C4 photosynthesis related processes, we analyzed the expression patterns of key OG. A conserved pattern was observed for enzymes associated with the NADP-ME subtype of the C4 pathway along the leaf segments for the C3, PK, and C4 studied species. Conservation of the expression patterns between C3 and C4 species have also been described in other taxonomic groups . In particular, Xu et al. (2016) found that 7 out of a total of 15 genes that encode for C4 enzymes showed conversed expression patterns during the process of rice and corn leaf de-etiolation. Interestingly, we find 14 of the 15 genes previously studied by Xu et al. (2016) have highly conserved expression patterns along the leaf segments of the C3, PK, and C4 species studied here. This result suggests a higher degree of conservation of the C4 pathway in Otachyriinae, likely due to the evolutionary close proximity of the studied species.
Although we observed a uniform expression pattern in C4 OG among the different leaf types, we confirmed differences in the levels of expression of key photosynthetic enzymes and photorespiratory markers between the leaf of the C4 species and C3/PK species. Overall, the leaf of A. lanata showed that OG encoding for C4 enzymes are highly expressed in mature segments of the leaves compared to the studied C3/PK species. In addition, most of the OG that codes for photorespiratory pathway presented an ascending pattern of expression from base to tip for all species; however, the difference lies in the level of expression in the source segments, where more than 50% of them are up regulated in C3/PK vs C4 species.
In summary, transcriptome analysis shows the conservation of expression patterns of C4 cycle genes and photorespiratory cycle in C3/PK/C4 species suggesting that the developmental program precedes the evolution of C4 photosynthesis. In this scenario, installing new pathways in C3 species would require fewer changes in expression patterns during the development and an increase of the expression activity of key genes at the right moment of leaf development.
New transcriptomes presented here expand the leaf development of closely related non-model grass species C3, intermediate and C4 leaves. We found commonalities and key differences on the leaf transcriptome performance among the three studied species as well as with other grass species. Overall, we found that genes associated with photorespiration and the C4 cycle are differentially expressed between C4 and C3 species, whereas their expression patterns are well preserved throughout leaf development. Indeed, similar trends were documented for rice and maize . Interestingly, the C3-PK transcriptomic profile is more similar to C4 than C3 in early stages of development suggesting that R. pilosa and A. lanata have commonalities in the transcription activity during the anatomical setup but are different in the biochemistry of photosynthesis. Finally, the analysis of the transcriptomes showed some peculiarities of the gene expression along the leaf segments of A. lanata leaf, which may correlate with its unique foliar anatomy among grasses.
This dataset represents a valuable contribution to the existing genomic resources and provides new tools for future investigation of photosynthesis evolution.
Plant material and growth conditions
Plant specimens were deposited at “Arturo E. Ragonese” Herbarium (SF) (Facultad de Ingeniería Agronómica, Universidad Nacional del Litoral, Esperanza, Santa Fe, Argentina). The detailed voucher numbers are listed in Additional file 1 (Fig. S1). Individuals of H. amplexicaulis (C3), R. pilosa (C3-Proto Kranz) and A. lanata (C4- NADP-ME) were grown in a grow chamber at 27 °C under long day conditions (16 hours of light and 8 hours of darkness). For each species, the 5th young leaf of 90 individuals was collected when it reached the length of the 4th leaf. Each leaf was divided into two sections, taking the sink-source transition zone as a reference. The sections were then divided into four leaf segments of equal length and labeled S1 to S8 from the base to the tip of the leaf. For transcriptomic sequencing, segments 1, 3, 5 and 7 from 90 leaves were collected in 9 pools of 10 individuals each, which were randomized to create 3 replicates of 30 individuals. For histological analysis segments 1, 3, 5 and 7 from 10 leaves were collected. The replicates are paired samples.
Fresh leaf segments were arranged in molds with 5% low melting point agarose in PBS 0.05 M at 50 °C and left to harden at 4 °C for 1 hour. A Leica VT1000S (Leica Biosystems, Germany) vibratome was used to obtain 100 μm thick sections of plant material. Sections were mounted onto microscope slides and covered with PBS 0.05 M to be studied under light microscopy (Nikon Eclipse E200).
Transcriptome sequencing and assembly
Total RNA was extracted with Tripure reagent (Sigma) following the manufacturer’s protocol. An additional purification of the RNA was carried out using LiCL precipitation . Libraries were prepared by the Roy J. Carver Biotechnology Center at the University of Illinois using the standard Illumina TruSeq mRNA sample prep kit. Thirty-two libraries were pooled and sequenced using an Illumina NovaSeq SP flowcell lane that produced 22 to 45 million 250 bp paired-end reads per library.
The selected pipeline for de novo transcriptome assembly was based on Carruthers et al. (2018) and Moreno-Santillan et al. (2019) [35, 36] The raw read quality of each paired-end library was examined using the bioinformatics tool FastQC v0.11.5 . Low quality reads were trimmed and filtered with trimmomatic . After this, between 3 and 7% of the reads were discarded. De novo transcriptome assembly was carried out using Trinity v.2.8.5 . The proportion of reads mapped to the assembly was assessed with Bowtie2 v2.3.2 .
To reduce the probability of obtaining spurious transcripts and attenuate transcript redundancy, the contigs were filtered using three methods. First, weakly expressed isoforms were removed based on their expression values. For that, TPM values were obtained by SALMON v0.14.1 , and weakly expressed isoforms were removed using the Trinity script filter_low_expr_transcripts.pl with “–highest_iso_only” parameter. Second, a set of non-redundant representative transcripts was generated using the CD-Hit package v4.6.6  with an identity threshold of 95%. Finally, transdecoder v5.1.0  was used to identify all likely coding regions within our assembled transcripts, and then filtered by selecting the single best ORF per transcript. Any transcripts with ORFs less than 100 bp in length were removed before performing further analyses. Transcriptome completeness and redundancy was assessed using the bioinformatics tool BUSCO v.3.0.1 (Benchmarking Universal Single-Copy Orthologs) to obtain the percentage of single-copy orthologs represented in the monocots dataset .
Orthofinder v2.3.7  was used for orthologues identification using six reference proteomes. Two transcriptomes were from the same subtribe, Steinchisma hians and Steinchisma laxa (Studer unpublished data), and four grass outgroups, Zea mays RefGen_V4, Sorghum bicolor v3.1.1, Setaria viridis v2.1 and Oryza sativa v7.0 (obtained from Phytozome v. 12.1.6 ). Orthologs were used to retrieve functional annotations from S. bicolor, S. viridis and Z. mays downloaded from the Phytozome database.
Gene expression analysis
To quantify transcript abundance the align_and_estimate_abundance Perl script of the Trinity package was applied, by mapping the reads of each biological replicate against the respective assembled transcriptome. In this analysis, SALMON was used as the abundance estimation method and quality check for biological replicates was assessed with the PtR utility from trinity. The Gene Expression Matrices were built using the abundance_estimates_to_matrix.pl script. Counts matrices were imported to R and the EdgeR package (v. 3.38.1)  was used for the Differential expression analysis. P-values were FDR corrected and genes with an FDR < 0.01 and a log2 fold change > 1 were considered as significant.
Gene ontology enrichment analysis
The Gene Ontology (GO) enrichment analysis was performed using the R package TopGO v2.42 . GO terms for each transcript were obtained from S. bicolor, Z. mays and S. viridis genomes from the phytozome database. P-values were adjusted using the “elim” algorithm. A term was considered significant if it had an adjusted p value < 0.01.
Availability of data and materials
The Illumina RNA-Seq reads and transcriptomes assembly supporting the conclusions of this article are available from the NCBI Sequence Read Archive (PRJNA813546, https://dataview.ncbi.nlm.nih.gov/object/PRJNA813546?reviewer=6vmhgdsbsobv6pd6u6tr9rp4oe).
Differentially Expressed Genes
Differentially Expressed Orthogroups
Inner Bundle Sheath Cell
Outer Bundle Sheath Cell
Open Reading Frame
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We thank members of the Development and Evolution lab (LED, IAL) and the Instituto de Agrobiotecnología del Litoral (UNL-CONICET) for helpful discussions. We also thank Juan Manuel Acosta for providing plant material. A special thanks to the FULBRIGHT program and the Ministry of Education of Argentina that made possible the realization of this work. We are grateful to anonymous reviewers for critically reading the manuscript.
This study was funded by the Universidad Nacional del Litoral (CAID+D 2020 - 50620190100039LI) to RR and a seed grant from the University of Illinois to AJS.
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Prochetto, S., Studer, A.J. & Reinheimer, R. De novo transcriptome assemblies of C3 and C4 non-model grass species reveal key differences in leaf development. BMC Genomics 24, 64 (2023). https://doi.org/10.1186/s12864-022-08995-7
- Differentially expressed genes
- Differentially expressed orthogroups