Exploration of the truncated cytosolic Hsp70 in plants - unveiling the diverse T1 lineage and the conserved T2 lineage

Abstract

The 70-kDa heat shock proteins (Hsp70s) are chaperone proteins involved in protein folding processes. Truncated Hsp70 (Hsp70T) refers to the variant lacking a conserved C-terminal motif, which is crucial for co-chaperone interactions or protein retention. Despite their significance, the characteristics of Hsp70Ts in plants remain largely unexplored. In this study, we performed a comprehensive genome-wide analysis of 192 sequenced plant and green algae genomes to investigate the distribution and features of Hsp70Ts. Our findings unveil the widespread occurrence of Hsp70Ts across all four Hsp70 forms, including cytosolic, endoplasmic reticulum, mitochondrial, and chloroplast Hsp70s, with cytosolic Hsp70T being the most prevalent and abundant subtype. Cytosolic Hsp70T is characterized by two distinct lineages, referred to as T1 and T2. Among the investigated plant and green algae species, T1 genes were identified in approximately 60% of cases, showcasing a variable gene count ranging from one to several dozens. In contrast, T2 genes were prevalent across the majority of plant genomes, usually occurring in fewer than five gene copies per species. Sequence analysis highlights that the putative T1 proteins exhibit higher similarity to full-length cytosolic Hsp70s in comparison to T2 proteins. Intriguingly, the T2 lineage demonstrates a higher level of conservation within their protein sequences, whereas the T1 lineage presents a diverse range in the C-terminal and SBDα region, leading to categorization into four distinct subtypes. Furthermore, we have observed that T1-rich species characterized by the possession of 15 or more T1 genes exhibit an expansion of T1 genes into tandem gene clusters. The T1 gene clusters identified within the Laurales order display synteny with clusters found in a species of the Chloranthales order and another species within basal angiosperms, suggesting a conserved evolutionary relationship of T1 gene clusters among these plants. Additionally, T2 genes demonstrate distinct expression patterns in seeds and under heat stress, implying their potential roles in seed development and stress response.

Methodology and Implementation Steps

This study established a large-scale genomic analysis pipeline to thoroughly investigate the function and evolution of plant truncated Heat Shock Protein 70 (Hsp70T).
The research methodology encompassed the following key steps:

1. Large-scale genome database construction and search:
   Our study involved a comprehensive collection and search for Hsp70T sequences across the whole genomes of 192 sequenced plant and green algal species. This extensive database encompasses diverse plant lineages, ranging from algae, bryophytes, and ferns to gymnosperms and angiosperms, ensuring the broad scope and representativeness of our analysis.
2. Protein sequence analysis and gene classification:
   We performed multiple sequence alignments, specific sequence motif searches, and phylogenetic tree analyses on the collected Hsp70T protein sequences. This allowed for a detailed classification of Hsp70T subtypes, specifically differentiating between the T1 and T2 subtypes of cytosolic Hsp70T, and analyzing their similarities and sequence variations compared to typical Hsp70s.
3. Gene synteny analysis:
   To explore the expansion mechanisms of Hsp70T genes within plant genomes, particularly their proliferation as gene clusters, we conducted synteny analyses. This identified homologous Hsp70T gene clusters and their evolutionary relationships across different species, with a particular focus on the expansion patterns of T1 type gene clusters in Lauraceae plants.
4. Gene expression analysis:
   Utilizing public RNA-seq databases, we assessed the expression levels of Hsp70T in various plant tissues, developmental stages, and under different stress conditions. This allowed us to infer their potential functional locations and physiological roles, especially distinguishing the expression patterns of T1 and T2 subtypes.
5. Cross-species distribution and gene family expansion analysis:
   We quantified the numbers of each Hsp70T type across diverse plant species to study their gene family expansion and evolutionary pathways. This analysis was crucial for understanding the evolutionary context of Hsp70T in the adaptation of plants to terrestrial environments. By integrating bioinformatics and comparative genomics approaches, this study not only significantly accelerated the process of plant gene research but also provided a robust technical framework for future studies in plant diversity.

Innovation and Cross-Disciplinary Collaboration

The innovativeness of this study is multifaceted. Firstly, we for the first time confirmed that plant Hsp70T is not a pseudogene but a gene family with important functions, moving beyond the limitations of previous research that relied solely on mutant studies in model organisms. Secondly, this study provides new evidence for the evolutionary association between Hsp70T and plant adaptation to terrestrial environments, supporting the APG IV phylogenetic hypothesis for plant classification and addressing previous controversies regarding Magnoliids evolution. Most importantly, we established a large-scale comparative analysis method for plant genomes, encompassing nearly 200 model and non-model species, providing a more comprehensive genomic analysis pipeline and evolutionary information. This method has already been successfully applied to the identification of other plant functional genes. Regarding cross-domain collaboration, this research received support from the Taiwan Comprehensive University System's inter-university research program. We collaborated with laboratories at National Cheng Kung University and National Yang Ming Chiao Tung University to complete the study and publish our findings. This cross-institutional collaboration model brings together expertise from diverse fields, enhancing the depth and breadth of the research.

Expected Results and Contributions

The results of this study are expected to make significant contributions in several aspects:

1. Promoting sustainable agriculture and biodiversity conservation:
   By thoroughly analyzing the role of plant Hsp70T in stress resistance, our findings lay the groundwork for developing novel plant protection strategies. This has the potential to reduce pesticide use and lessen agriculture's ecological impact, thereby contributing to the conservation of Taiwan's endemic plant resources and providing a scientific basis for sustainable agricultural development, in line with the United Nations Sustainable Development Goals (SDGs).
2. Deepening understanding of plant evolution:
   Investigating the unique characteristics of Hsp70T in Lauraceae plants and its synteny with Chloranthaceae and Nymphaeales offers a new perspective for re-examining the evolutionary relationships within Lauraceae. This also supports important plant evolutionary hypotheses, such as the APG IV system.
3. Advancing plant biotechnology:
   The established large-scale comparative genomic analysis method can be broadly applied to crop improvement, aiding in the identification of key genes related to stress resistance, yield, and quality. The high flexibility of this method allows for future integration with emerging technologies, enhancing the accuracy of gene function prediction and providing innovative solutions for global agricultural challenges. This research provides scientific evidence for plant adaptation to land and offers research directions for the function of plant Hsp70T.