Abstract

   Cold stress poses a significant threat to the quality and productivity
   of lychee (Litchi chinensis Sonn.). While previous research has
   extensively explored the genomic and transcriptomic responses to cold
   stress in lychee, the translatome has not been thoroughly investigated.
   This study delves into the translatomic landscape of the 'Xiangjinfeng'
   cultivar under both control and low-temperature conditions using RNA
   sequencing and ribosome profiling. We uncovered a significant
   divergence between the transcriptomic and translatomic responses to
   cold exposure. Additionally, bioinformatics analyses underscored the
   crucial role of codon occupancy in lychee's cold tolerance mechanisms.
   Our findings reveal that the modulation of translation via codon
   occupancy is a vital strategy to abiotic stress. Specifically, the
   study identifies ribosome stalling, particularly at the E site AAU
   codon, as a key element of the translation machinery in lychee's
   response to cold stress. This work enhances our understanding of the
   molecular dynamics of lychee's reaction to cold stress and emphasizes
   the essential role of translational regulation in the plant's
   environmental adaptability.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12864-024-10591-w.

   Keywords: Lychee, Ribosome profiling, Cold stress, Codon occupancy

Background

   Lychee (Litchi chinensis Sonn.) is a key subtropical fruit known for
   its economic value, nutritional profile, exotic flavor, and visual
   appeal [[35]1–[36]5]. However, climate change, characterized by global
   warming and extreme temperatures, poses significant challenges and
   increases the incidence of abiotic stressors. These changes have
   profoundly affected global crop production [[37]2, [38]6, [39]7]. Among
   these challenges, cold stress is particularly detrimental and affects
   both the survival and flavor quality of lychee [[40]8, [41]9].

   Cold stress can have several detrimental effects on lychee seedlings.
   Prolonged exposure to low temperatures can hinder the growth and
   vitality of lychee seedlings, leading to stunted development and
   increased susceptibility to diseases [[42]10, [43]11]. Extreme or
   prolonged cold can damage tissues, impair physiological functions, and
   disrupt metabolic processes in lychee seedlings, resulting in overall
   reduced vitality. At the molecular level, cold stress can alter gene
   expression in lychee, leading to the accumulation of reactive oxygen
   species (ROS) and oxidative damage [[44]12]. It can also affect the
   stability and function of cellular membranes and proteins in lychee
   seedlings [[45]13]. Insufficient cold accumulation due to unusually
   high winter temperatures can result in inadequate floral initiation and
   poor flowering in lychee, ultimately affecting fruit yield and quality
   [[46]14]. Additionally, the variability in cold requirements among
   different lychee varieties complicates effective management [[47]15,
   [48]16]. Balancing the right amount of cold stress is crucial, as
   excessive cold exposure pose significant challenges to the health and
   productivity of lychee seedlings [[49]17–[50]19]. Therefore,
   understanding the molecular mechanisms underlying these responses in
   lychee is essential for developing strategies to mitigate the negative
   impacts of cold stress.

   Gene regulatory networks for lychee's stress responses have been
   studied using high-throughput sequencing and bioinformatics tools, but
   the molecular response to low-temperature stress remains unclear
   [[51]5, [52]8, [53]20]. The lack of precise genome annotation has
   further hindered the study of lychee's transcriptome and translatome.
   Plant adaptation to stress involves complex regulation, including gene
   expression, post-transcriptional processes, post-translational
   modifications, and metabolite feedback mechanisms [[54]7, [55]9,
   [56]20–[57]22]. Ribosome profiling, a high-resolution deep-sequencing
   technique, is crucial for analyzing RNA translation dynamics in lychee
   (Litchi chinensis) [[58]23–[59]26]. This method involves quantifies
   ribosome-protected mRNA fragments (RPFs) after RNase treatment,
   allowing detailed analysis of translation. Ribosome profiling has
   revealed shifts in translation dynamics under low-temperature stress,
   providing insights into ribosome coverage, translation efficiency, and
   codon occupancy [[60]23–[61]36]. High-quality data exhibit distinct
   3-nt periodicity, essential for confirming the accurate translation
   measurement [[62]23–[63]35, [64]37, [65]38]. Applying ribosome
   profiling to lychee enables detailed investigation of translational
   mechanisms contributing to stress resilience. Comprehensive sequencing
   of the lychee genome provides an opportunity to study lychee's response
   to cold stress with precision [[66]39]. Utilizing this genomic
   resource, our study employs ribosome profiling and RNA-seq technologies
   to survey the translational landscape of lychee. The methodologies
   advance our understanding of stress responses in higher plants and
   highlight the critical role of translational regulation in lychee's
   adaptation to a changing climate. This study, supported by the recent
   lychee genome sequence, allows for an in-depth investigation of
   lychee's response to low-temperature stress [[67]39]. Leveraging this
   genomic blueprint, we undertake research to scrutinize lychee’s
   translational landscape, extending our understanding of stress
   responses and emphasizing the pivotal role of translational regulation
   in lychee's adaption to shifting climatic conditions.

   In summary, lychee faces escalating challenges from climate change,
   with cold stress being a significant threat. By exploring lychee's
   translatome, we aim to understand its response to cold stress, offering
   valuable insights for crop protection and enhancement. This study
   underscores the importance of using ribosome profiling and
   translational regulation in understanding lychee's adaptation to a
   changing environment.

Results

Library preparation and assessment of ribosome-protected footprints in lychee
leaves

   To elucidate the translatome landscape of Litchi chinensis under
   low-temperature stress, we conducted a comprehensive ribosome profiling
   study focusing on lychee leaves in the absence of low-temperature
   stress treatment (Fig. [68]1A). Employing rigorous experimental
   standards, we performed two replicates for each treatment condition. To
   identify translational differences, we employed polysome profiling
   (n = 3) to compare ribosome distribution between control and samples
   subjected to low-temperature stress (Fig. S1). As expected, translation
   was modestly suppressed under low-temperature conditions, resulting in
   a reduced polysome fraction (Fig. S1), confirming the temperature's
   impact on translation regulation. Ribosome profiling was excuted to
   ensure data quality and treatment efficacy, including the assessment of
   read lengths within the 29–31 nt range (Fig. [69]1B-D) and the presence
   of a 3-nt periodicity through metagene plots (Figs. [70]1E, [71]2A).

Fig. 1.

   [72]Fig. 1
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   Evaluation of ribosome profiling libraries from lychee leaves. A
   Schematic representation of the ribosome profiling procedure. B
   Distribution of ribosome protected footprint lengths across the entire
   sequencing dataset. C Footprint length distribution near the start
   codon. D Comparative analysis of read length distribution at the start
   codon versus the entire transcript length. E Metagene plot depicting
   P-site frequency along the transcript

Fig. 2.

   [74]Fig. 2
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   Comparison of global transcriptional and translational change. A P-site
   signal accumulation along footprint length in the regions of 5' UTR,
   CDS, and 3' UTR under normal conditions (CK). B P-site signal
   accumulation along footprint length in the regions of 5' UTR, CDS, and
   3' UTR under cold stress (LT). C P-site signal distribution in the
   regions of 5' UTR, CDS, and 3' UTR under normal conditions (CK). D
   P-site signal distribution in the regions of 5' UTR, CDS, and 3' UTR
   under cold stress (LT). E Global changes in the transcriptome. F Global
   changes in the translatome

   Preliminary processing of deep sequencing data demonstrated high
   reproducibility for single replicates (Fig. S1B, C). Analysis of the
   length distribution of ribosome-protected footprints (RPFs) in our
   samples revealed a predominant range of 28 to 31 nt (Fig. [76]1B, D).
   Notably, the characteristic RPF length was observed at 29 nt,
   indicative of monosome-protected fragments. Metagene plots of our
   samples also displayed periodic peaks spaced at 3-nt intervals
   (Fig. [77]1E). Previous research has confirmed that the bulk of
   translation footprints predominantly manifest within coding regions.
   However, an accumulation of queuing ribosomes is typically anticipated
   preceding translation initiation, along with instances of stalling near
   start and stop codons (Fig. [78]1E). This phenomenon was corroborated
   by the relatively heightened peaks proximate to the start and stop
   codons in our metagene plots. Collectively, these outcomes validate the
   creation of robust libraries for lychee samples, which were suitable
   for downstream analyses.

Comparative analysis of lychee transcriptome and translatome landscapes under
cold stress

   To explore translational initiation responses across different
   translational features, we analyzed P-site positioning from ribosome
   profiling data. A strong translational signal was observed within the
   CDS region in both control and low-temperature groups (Fig. [79]2C, D),
   consistent with protein synthesis initiation within the coding region
   of RNA. Furthermore, heatmap analysis (Fig. [80]2A, B) showed superior
   periodicity of the P-site signal in the CDS compared to the 5' UTR and
   3' UTR. We also compared P-site signals between control and cold stress
   groups. No significant differences were found, indicating that
   low-temperature treatment did not significantly affect global
   translation initiation.

   In addition, we compared transcriptome fold changes using RNA-seq data
   with two replicates alongside ribosome profiling (Fig. S2A, B). With a
   significance threshold of P-value < 0.05, we identified genes
   differentially expressed at transcriptional and translational levels.
   Surprisingly, the transcriptome was more affected by low-temperature
   stress than the translatome (Fig. [81]2E, F). RT-qPCR validation
   confirmed that the three most down-regulated genes (highlighted as
   purple in Fig. [82]2E) had significantly lower expression than controls
   (Fig. S2D). This suggests that translation dynamics are less affected
   by low-temperature stress in lychee. Our findings indicate a potential
   disparity between transcriptome and translatome responses to cold
   stress, warranting further investigation into regulatory mechanisms.
   Gene Ontology (GO) analysis on transcriptome data revealed a
   significant enrichment of transaltion-related pathways in samples
   exposed to low-temperature stress (Fig. S3A), including "mRNA cap
   binding complex" and "RNA cap binding complex". This suggests that cold
   stress broadly impacts translation processes, particularly affecting
   mRNA cap binding complexes. Kyoto Encyclopedia of Genes and Genomes
   (KEGG) pathway analysis revealed perturbations in energy supply
   pathways, such as the Tricarboxylic Acid (TCA) cycle (Fig. S3B),
   suggesting altered energy metabolism under cold stress. These insights
   into translation and energy metabolism pathways highlight the
   multifaceted molecular impact of low-temperature stress, providing a
   deeper understanding of the biological response to environmental
   challenges.

Effects of cold stress on coding features in lychee

   Understanding plants’ responses to abiotic stresses through coding
   feature dynamics is vital for comprehending their adaptive strategies.
   This study focused on coding events in lychee under cold stress, a
   significant environmental factor. We analyzed P-site signal
   metaheatmaps to examine translational activity across mRNA transcripts
   (Fig. [83]3A). The metaheatmaps showed uniform signal patterns with
   distinct three-nucleotide periodicity, confirming the accuracy of our
   ribosome profiling data and providing a solid foundation for predictive
   modeling of translation events. Our analysis highlighted significant
   trends in coding localization, predominantly in the coding sequence
   (CDS) region, emphasizing its role in protein synthesis. In contrast,
   the 5' untranslated region (5' UTR) displayed the fewest coding events,
   suggesting it primarily contains regulatory elements. This insight
   underscores the complexity of translational control in Litchi
   chinensis. We also examined the independent coding distribution for
   each treatment, revealing a strong alignment between our prediction
   model and calculated values (Fig. [84]3B, C). This consistency
   underscored the robustness of our approach, Interestingly, no
   significant alterations in coding features were observed under of cold
   stress, which indicated the remarkable resilience and adaptability of
   lychee to environmental challenges.

Fig. 3.

   [85]Fig. 3
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   Comparison of coding features. A Metaheatmap of P-site signal
   concentrated on start and stop codon. B P-sites distribution along
   coding features evaluated from predicted model (RNAs) and transcriptome
   data under CK. C P-sites distribution along coding features evaluated
   from the predicted model (RNAs) and transcriptome data under
   low-temperature stress. D GO analysis for translationally affected
   genes under cold stress. E KEGG analysis for translationally affected
   genes under cold stress

   Although no significant coding changes were found under cold stress, we
   thoroughly examined translation efficiency for potential genes and
   pathways. that may have been affected. Unlike transcriptome, only a few
   genes were impacted at the translational level (Fig. S2C). Gene
   Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
   analyses (Fig. [87]3D, E) revealed significant perturbations in
   ribosome function under low-temperature stress, particularly in
   substructures like the 'peribosome', '90S peribosome', and 'nuclear
   dicing body'. This raises questions about how low-temperature stress
   regulates ribosomes, possibly due to ribosome stalling on specific
   codons. In summary, our examination of coding features in lychee under
   low-temperature stress highlights the plant's translational responses
   and adaptive strategies. The consistency of our findings and the
   absence of significant coding events alterations underscore the plant's
   robustness and ability to withstand environmental stressors, revealing
   intriguing aspects of its adaptability.

Codon usage analysis in Litchi chinesis coding sequences

   We investigated codon utilization patterns using ribosome profiling
   data to understand how cold stress influences codon usage.
   Ribosome-protected reads were aligned to the lychee coding sequences,
   allowing direct comparisons of codon usage preferences. The diversity