3.1. Effects of elevated light intensities on photosynthetic performance and molecular response in tomato seedlings
Commonly employed non-invasive assessments of a plant's physiological state rely on measuring chlorophyll a fluorescence. In this study, we aimed to investigate the combined reaction to high light intensity and nematode infection therefore the conditions typically used in tomato/potato cyst nematode experiments were slightly modified (Dąbrowska-Bronk et al., 2015; Święcicka et al., 2017). Tomato seedlings were exposed to elevated light intensities being placed on a medium in a plastic Petri dish sealed with permeable medical adhesive tape. The shoots remained uncut, while the roots were shielded with a black envelope. The maximum light intensity was adjusted to a level that did not cause temperature shifts. The impact of transferring tomato plants from low light (LL) to high light (HL) conditions on photosystem II (PSII) photochemistry was assessed using various chlorophyll fluorescence-related parameters, including maximum quantum efficiency of PSII (Fv/Fm), non-photochemical quenching (NPQ), photochemical fluorescence quenching (qP), PSII quantum yield (ΦPSII), PSII quantum yield in light-adapted leaves (Fv’/Fm’), and plant vitality (Rfd) (Baker, 2008). Measurements were taken at two time points: 1 day post-transfer (dpt) and 3 dpt, with appropriate LL controls on two-week-old tomato seedlings. Two parameters exhibited statistically significant increases after transferring plants to elevated light conditions at both time points: PSII quantum yield and photochemical fluorescence quenching (Fig. 1C and E). The other parameters showed insignificant fluctuations (Fig. 1A, B, D and F). These results indicate that seedlings cultured under higher light intensities had greater efficiency of PSII, consistent with previous literature (Takagi et al., 2019).
To track the activation of signaling pathways, we evaluated the expression of several stress-related marker genes using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The expression was checked in leaves (organs subjected to HL stimuli) and roots (remote, shaded organs) at 1 and 3 dpt. Cyst nematodes migrate along the roots to find the initial syncytial cell (ISC) before inducing the feeding structure. Upon initial penetration, the root cells undergo localized damage, triggering the plant's defense response. Additionally, cyst nematodes secrete effector proteins into the plant root to manipulate host cell functions and modulate plant hormone levels to promote syncytium formation and alter root development. Therefore, the first few days are crucial for parasite success (Matuszkiewicz & Sobczak, 2023). The selected genes played a role in the interaction of other nematode/host species, as well as in response to light stress (Huang et al., 2019).
In leaves, the strongest up-regulation at 1 dpt was observed in the ACO1, NPR1, Pr1a4, APX1, and DHAR genes, while down-regulation was observed for ISC, HY5, PHYA, and PHYB2 transcripts (Fig. 2A). Changes in gene expression at 3 dpt were less widespread, with up-regulation of NPR1, APX1, and DHAR and down-regulation of ACCase and HY5. The observed changes in leaves may be attributed to the activation of defense and antioxidant pathways to counteract oxidative stress and damage caused by excessive light. Simultaneously, down-regulation of ACCase, ICS, and HY5 may aim to conserve energy, possibly due to ACCase's role in lipid metabolism and fatty acid synthesis, while also reducing susceptibility to light-induced stress.
Interestingly, the tomato root system was more sensitive to applied conditions, even when protected from light. We observed down-regulation of RBOHD and RBOHF and up-regulation of APX1, SOD, and DHAR among oxidative stress-sensitive genes. Genes involved in SA, JA, and ET signaling were up-regulated. Notably, both PHYA and PHYB2 were strongly down-regulated in roots (Fig. 2B). At 1 dpt, all the mentioned gene expression changes were observed, while at 3 dpt, only NCED1 (involved in ABA biosynthesis) exhibited statistically significant alterations in roots, suggesting effective adaptation to unfavorable conditions and moderation of stress-related responses.
3.2. Higher light intensities modify PSII photochemistry during G. rostochiensis parasitism
The introduction of a second stress factor can significantly alter the plant's response across various dimensions, including physiological, biochemical, and molecular aspects. Therefore, our objective was to investigate how changes in light conditions might impact the susceptibility of tomatoes and whether these alterations would affect the efficiency of the photosynthetic apparatus. Among the six parameters measured, three showed differences associated with the combination of nematode parasitism and the transfer of plants to different light regimes: PSII yield, qP, and Rfd. In plants infected with G. rostochiensis and cultivated in low light (LL) conditions, the PSII yield decreased. However, when infected plants were transferred to high light (HL) conditions, their PSII yield levels were similar to those of both control groups of plants. A similar trend was observed for photochemical fluorescence quenching and the parameter representing plant vitality. Infected plants acclimated to LL conditions exhibited a photoinhibition-like response, as evidenced by a decrease in the aforementioned parameters (see Fig. 3).
3.3. Light intensity variation is irrelevant to nematodes
The interplay between plant roots and cyst nematodes is a highly complex and extended process involving developmental and metabolic changes of plant cells, along with responses to damages, molecular patterns, and effectors associated with parasite activity (Matuszkiewicz & Sobczak, 2023). The observed changes in photosynthetic efficiency and gene expression in non-infected plants may translate into the susceptibility level of the tomato (see Figs. 1 and 2). To test this hypothesis, we quantified the number of induced syncytia on tomato root systems in plants that were either continuously grown in low light (LL) conditions or transferred to high light (HL) intensities, and we did not observe any differences (see Fig. 4). It is worth mentioning that we did not observe changes in the morphology of the root system in both comparisons, which could potentially interfere with the level of susceptibility.
3.4. Transcriptome analysis reveals DEGs in response to different light conditions during G. rostochiensis parasitism in tomato
Transcriptome profiling has become the fundamental diagnostic tool for monitoring plant reactions to stress, with RNA-seq being increasingly favoured over microarrays, systematic RT-qPCR, SAGE, or cDNA-AFLP due to its advantages. Despite the importance of the tomato/PCN interaction, there is still a lack of RNA-seq perspective. In this study, we employed RNA-seq to investigate the regulatory networks active in nematode-infected roots subject to varying light conditions. Our investigation involved analyzing RNA-seq data derived from plants persistently grown under both low light (LL) and high light (HL) conditions, as well as plants subjected to a transition from LL to HL intensities. This comparative analysis aimed to elucidate the complexity of the response to dual stimuli. We also included non-infected plants subjected to an increase in light intensity, characterizing this scenario as a “light response”.
An initial noteworthy observation was that, despite the absence of variances in tomato susceptibility to G. rostochiensis across distinct light conditions, we detected substantial alterations in the abundance of differentially expressed transcripts. The smallest number of DEGs, 173, was observed when comparing transcriptomes of infected LL-grown roots to control samples. In HL conditions, this comparison yielded nearly twice as many DEGs (303). The highest number of DEGs emerged in the double stimuli comparison – 2979, while the light response alone resulted in 1746 DEGs (Fig. 5A). In all comparisons, up-regulated DEGs constituted the predominant group, with the exception of the LL comparison, where down-regulated genes accounted for 68% of the total DEGs (Fig. 5A). This indicates that, despite relatively small differences observed in DEGs between LL and HL conditions, light exerts a dominant influence on the regulation of gene expression. Moreover, light and nematode response synergistically interact, yielding more DEGs than the sum of individual stimuli.
The overlap of the aforementioned groups of DEGs would indicate more general mechanisms of plant reaction to biotic and abiotic stimuli (Fig. 5B). For example, in the group common for all four comparisons (9 DEGs), we found genes such as Zinc finger protein (Solyc08g006470.5), Peroxidase (Solyc10g078890.2), Glutathione S-transferase (Solyc09g011540.2), and Defensin protein (Solyc07g007750.3). Interestingly, in the common pool of DEGs for nematode-related response (11 DEGs), genes involved in secondary metabolism such as O-methyltransferase (Solyc06g064510.2), Flavin-containing monooxygenase (Solyc08g068160.2), Glycosyltransferase (Solyc11g007460.1),
2-oxoglutarate (Solyc11g072110.2), and ABA 8'-hydroxylase (Solyc04g078900.3) were present. Additionally, the analysis of DEGs revealed transcripts unique for each treatment, indicating specific processes related to the tested variables and their synergy. The smallest number of exclusive DEGs was found in the LL comparison, while the highest number was observed under double stress conditions, totalling 1641 DEGs. Here, we found genes involved in defense response, hormone homeostasis, ROS signalling, and regulation of primary and secondary metabolic processes (Fig. 5B).
Our analysis of DEGs uncovered a remarkable dynamic range of FC-value for several genes throughout the comparisons. However, as usual, the highest changes were detected for genes with very low expression. For a more detailed description, see Supplementary Table 3.
3.5. Categorization of Differentially Expressed Genes
Screening large datasets of DEGs encounters problems with drawing more general conclusions; therefore, we employed ShinyGO v.0.77 software for functional categorization of DEGs and gene ontology (GO) enrichment analysis. Genes were classified accordingly to four groups of GO terms: KEGG pathways, biological process (BP), molecular function (MF), and cellular component (CC) (see Fig. 6 and Supplementary Table 4). This approach allowed us to dissect processes being preferentially targeted upon potato cyst nematode parasitism in combination with environmental stimuli.
In all analyzed DEG groups, the most general category, “Metabolic Pathways” and also quite capacious “Biosynthesis of Secondary Metabolites” were consistently enriched. Specifically, among the LL-inf DEGs, notable enrichment was observed in “Valine, Leucine, and Isoleucine Degradation” as well as the “MAPK Signaling Pathway” categories (when KEGG pathways define the category) (see Fig. 6). Among other categories, the highest enrichment was evident in “Glutamine Family Amino Acid Catabolic Process” and ”Negative Regulation of Hydrolase Activity” categorized accordingly to BP, and in “Fatty Acid Binding” and “Water Channel Activity” when MF determines functional category (refer to Supplementary Table 4). As expected, the HL-inf DEGs were enriched in pathways associated with “Photosynthesis”. Moreover, two additional categories, “Sulfur Metabolism” and “Phenylpropanoid Biosynthesis” were highly enriched (see Fig. 6). These findings were consistently supported across both GO terms for BP and MF (refer to Supplementary Table 4). The most pronounced KEGG pathway overrepresentation among the double stimuli DEGs was “Photosynthesis” followed by “Alanine, Aspartate, and Glutamate Metabolism”. Notably, the “Valine, Leucine, and Isoleucine Degradation” pathway also showed enrichment among double stimuli DEGs, highlighting the significance of amino acid metabolism in plants exposed to a complex environment (see Fig. 6). Additionally, “Gamma-Aminobutyric Acid Metabolic Process” emerged as a highly enriched category for BP, while “Cytidine Triphosphate (CTP) Synthase Activity” stood out for MF. Moreover, in both category groups – BP and MF, we found expected functional enrichments related to plant-nematode interaction, such as stress response, phytohormone regulation, defense response, cell wall remodelling, and ROS signalling.
The association of a gene product with a gene ontology term does not always proportionally reflect its engagement in a given molecular function, cellular component, or biological process. Therefore, dissecting domains from complex multidomain arrangements and conducting enrichment analysis could provide valuable supplementary insights needed for understanding large candidate lists. We found the Analysis of Motif Enrichment (AME; McLeay & Bailey, 2010) particularly helpful in interpreting our DEGs lists (refer to Table 1). Among LL-inf DEGs, five significantly enriched motifs were identified. Among them, three were unique for LL-inf: “Hydroxymethylglutaryl-coenzyme A reductases signature 2” (PS00318), “cysteine-rich secretory proteins - CRISP family” (PS01009), and “nitrite and sulfite reductases iron-sulfur/siroheme-binding site” (PS00365). The Hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase is a key enzyme in the mevalonate pathway, responsible for biosynthesizing isoprenoids including sterols (Friesen & Rodwell, 2004). Both LL-inf and HL-inf DEGs share the enriched motif named “Soybean trypsin inhibitor (Kunitz) protease inhibitors family” (PS00283). Three DEGs groups, the LL-inf, double stimuli, and light response share the enriched signature “Zinc finger RING-type” (PS00518), describing a conserved RING domain pivotal in the ubiquitination pathway. The potential role of proteins with this motif in stress responses could be linked to modulating protein abundance or turnover via ubiquitin-mediated protein degradation (Sun et al., 2019). Among HL-inf DEGs, the “Cytochrome P450 cysteine heme-iron ligand signature” (PS00086) is enriched, while among the double stimuli DEGs, three significantly enriched motifs were found: “Eukaryotic and viral aspartyl proteases active site” (PS00141), “Serine/Threonine protein kinases active-site” (PS00108), and “2Fe-2S ferredoxin-type iron-sulfur binding region” (PS00197). These results were partially confirmed by a similar, recently published tool - Simple Enrichment Analysis (SEA; Bailey & Grant, 2021; refer to Supplementary Table 5). The presence of the aforementioned motifs within protein sequences may be linked to particular condition-specific functions and regulatory roles in plant-biotic interactions.
Table 1
The regulatory protein motifs enriched in analyzed datasets. The Analysis of Motif Enrichment (AME) method was employed to identify over-represented motifs within the proteins encoded by DEGs from G. rostochiensis attacked tomato roots under different light conditions.
DEG group | motif ID (prosite) | motif alternative ID | consensus | p-value | adj_p-value | E-value |
LL-inf | PS00318 | HMG_COA_REDUCTASE_2 | LGXLGGGT | 5.01e-4 | 2.50e-3 | 2.45e0 |
PS01009 | CRISP_1 | GRFSALLWXXS | 1.44e-3 | 2.89e-3 | 2.82e0 |
PS00365 | NIR_SIR | SGCXXXCXXXXXXELGL | 1.44e-3 | 2.89e-3 | 2.82e0 |
PS00283 | SOYBEAN_KUNITZ | LXDXNGKXLXXXXXYXL | 1.44e-3 | 2.89e-3 | 2.82e0 |
PS00518 | ZF_RING_1 | CXHXLCXXCL | 1.44e-3 | 4.33e-3 | 4.23e0 |
HL-inf | PS00283 | SOYBEAN_KUNITZ | LXDXEGKXLXXXXXYXL | 2.34e-4 | 7.03e-4 | 6.88e-1 |
PS00086 | CYTOCHROME_P450 | FSXGXKXCLG | 3.85e-3 | 7.69e-3 | 7.52e0 |
double stimuli | PS00518 | ZF_RING_1 | CXHXLCXXCL | 1.75e-6 | 2.27e-5 | 2.22e-2 |
PS00141 | ASP_PROTEASE | LLSDSGSSXSXL | 1.48e-4 | 1.18e-3 | 1.16e0 |
PS00108 | PROTEIN_KINASE_ST | LXYXDLKXXNLLL | 1.48e-4 | 1.48e-3 | 1.44e0 |
PS00197 | 2FE2S_FER_1 | CXXGXCSSC | 1.48e-4 | 1.62e-3 | 1.59e0 |
light response | PS00518 | ZF_RING_1 | CXHXLCXXCL | 7.42e-6 | 9.64e-5 | 9.43e-2 |
PS00198 | 4FE4S_FER_1 | CXXCXXCXXXCG | 9.56e-4 | 4.77e-3 | 4.67e0 |
3.6. Consistency of RNA-seq results with other transcriptomic studies
Meta-analyses of transcriptomic results obtained by different methods help identify strong candidates for further research. Despite our RNA-seq data reflecting gene expression changes in whole nematode-infected root systems 14 days post inoculation (since syncytia initiation was not synchronized, root samples contained 10–14 days old feeding structures), we compared the DEGs list to earlier studies on the same species, where cDNA-AFLP was used to monitor transcriptome changes at 1, 3, 7 and 14 days post infection (dpi) with dissected syncytia (Swiecicka et al., 2009; Święcicka et al., 2017). Thirty-four DEGs overlapped between these two approaches − 19 up-regulated, 13 down-regulated, and 2 were stable in the RNA-seq study (refer to Fig. 7; Supplementary Table 6). Notably, 65% of the DEGs found in both analyses (22 genes) confirmed expression trends (up- or down-regulation upon nematode infection at any time point), while 35% (12 DEGs) demonstrated an inconsistent expression pattern. Among transcripts with a consistent expression pattern, there are several intriguing candidates for future research. The highly up-regulated gene, Solyc11g021060.2, encodes the TOMARPIX proteinase inhibitor (with a 3.09 log2FC in the double stress comparison). Conversely, a strongly down-regulated gene observed in the double stress response was Solyc08g014130.3, which encodes Isopropylmalate synthase (with a -1.44 log2FC).