Chitin is the predominant polysaccharide in the diatom cell wall, and its various functions permit diatoms to adjust to the changeable ocean environment. Chitinase, the enzyme that degrades chitin into more valuable derivatives with shorter sugar chains and lower molecular weights, therefore deserves more attention in diatoms. Although the chitinase gene family has been widely investigated in insects, higher plants, and fungi [26, 27, 51, 52], there is little information about this gene family in Stramenopiles, and a systematic investigation has yet to be undertaken. The completeness of the T. pseudonana genome sequence enables the identification of putative diatom chitinase genes [16]. In the present study, T. pseudonana chitinase genes were identified from genome sequences, and their phylogenetic relationships within Thalassiosira, gene structures, conserved motifs and domains, scaffold locations, duplication events, stress-related cis-acting elements, and transcriptional expression profiles and enzymatic activities under abiotic stress were investigated in an integrated manner.
Diversity in structure and intron abundance may generate chitinases with various functions in T. pseudonana
In total, 24 chitinase genes were retrieved from the T. pseudonana genome and verified based on the identification of conserved chitinase domains. Similar numbers of chitinase genes have been reported previously in T. pseudonana (more than 20 chitinase-encoding genes) [53] and Cyclotella cryptica (22 chitinases) [17]. Members of the T. pseudonana chitinase family displayed different secondary and 3D structures (Tables S8, S9), suggesting that they may function in multiple biological processes, such as stress-related responses, growth, and developmental operations. Intron gain and loss amplifies the complexity of gene organization [54], and we found diverse exon-intron organization in the T. pseudonana chitinase genes (Fig. 1) that may have contributed to their structural diversity during evolution. Importantly, the TpChis generally possessed a large number of introns. Twenty-five percent had more than 10 introns, over half had 7 or more introns (14, 58%), and members of GroupV had the most abundant introns (Fig. 1). When abundant introns are embedded in gene sequences, alternative splicing (AS) is likely to occur during gene transcription. In eukaryotes, AS spatially and temporally regulates gene expression by selectively preserving or removing some exons, thereby producing multiple mRNAs [55]. The many possible AS variants of the TpChis may therefore enlarge the diversity of the T. pseudonana chitinase transcriptome and proteome.
Tandem duplication and genetic diversification may co-affect the evolution of the TpChi gene family
T. pseudonana chitinase genes were classified into eight groups (I–Ⅷ) based on a phylogenetic tree that also included proteins from two additional Thalassiosira species. Chitinases in each group displayed similar domain architectures, motif compositions (Fig. 2), and conserved secondary (Fig. 5) and 3D structure (Table. S9), suggesting that they may have a closer evolutionary history and similar cellular functions.
Over the course of evolution, segmental and tandem duplications have propelled the expansion of gene families [56]. Here, we found two tandemly duplicated pairs of T. pseudonana chitinase genes (TpChi11 and TpChi12, TpChi21 and TpChi22) and no segmental duplication events, showing that tandem duplication has played a more important role in the expansion of the T. pseudonana chitinase gene family. TpChi21 and TpChi22 were located close together on Chr19a_19; they were classified into GroupⅢ (Figs. 2 and Fig. 3), and exhibited conserved domains and motif compositions (Fig. 1), as well as similar secondary and 3D structures (Fig. 5 and Table. S9). The other pair, TpChi11 and TpChi12, were distantly located on Chr_7 (362 kb) and were classified into different phylogenetic groups. TpChi11 and TpChi12 contained a common Glyco_hydro_18 domain. However, TpChi12 also possessed a Glyco_hydro_19 domain and a longer sequence length. Genetic and functional divergence of duplicated genes are known to occur during evolution [57]. TpChi12 was identified as a tandem duplicate of TpChi11, although it had an additional chitin-degrading domain. These observations suggest either the acquisition of a Glyco_hydro_19 domain by TpChi12 or its loss in TpChi11. Furthermore, the exon-intron structures and the gene expression profiles under silica limitation differed between TpChi11 and TpChi12, indicating that this gene pair has undergone functional divergence. Based on these results, we speculate that the chitinase gene family may have undergone duplication first, then experienced genetic diversification that drove further functional diversification.
Several subcellular locations are predicted for TpChi proteins
Chitin fibrils are located between the silica frustule and the cell membrane of chitin-producing diatoms [58], but the locations of chitin degradation sites are unknown. We therefore investigated the subcellular locations of the T. pseudonana chitinases. HECTAR predictions indicated that half of the chitinase family members are involved in the secretory pathway, consistent with the finding that a majority of chitinase genes in B. rapa and Arabidopsis thaliana are secretory [24, 27]. This suggests that chitinases have extracellular activity and important in vitro applications. The remaining chitinases, however, were predicted to reside on the chloroplast or mitochondrial membranes. Given the secondary endosymbiosis exhibited by diatoms [59], T. pseudonana chitinases anchored to the chloroplast or mitochondrial membranes may be encoded by genes originally acquired from red algae or non-photosynthetic exosymbionts.
Different chitinase expression patterns may support chitin metabolism and other physiological needs of T. pseudonana in changeable environments
Twenty-six types of stress-related cis-regulatory elements were found in the promoter regions of the 24 TpChi genes (Fig. 4), indicating that they may undergo complex transcriptional regulation. A total of 199 light-responsive cis-acting elements were unevenly distributed in the TpChi gene promoters, suggesting that the expression of most chitinase genes was indispensably induced by light. Consistent with this observation, LTR, TC-rich repeat, and MBS elements are also important for the induction of chitinase genes in cucumber, B. rapa, B. juncea, and Camelina sativa [27, 28, 51].
Multiple studies have demonstrated variations in the transcription of chitinase family genes during biotic disease resistance in plants [27, 28, 51]. Cao & Tan (2019) investigated the expression of tomato chitinase genes in response to the pathogen Sclerotinia sclerotiorum and under low temperature, high temperature, drought, and salt stresses [60]. They observed that the expression of chitinase genes was broadly elevated under these stress conditions. The present study demonstrated a similar pattern of overall upregulation of TpChi genes in response to low irradiance, low temperature, and silica depletion. The different transcriptional profiles of T. pseudonana chitinase genes under different abiotic stress conditions suggest that they have various response and regulation mechanisms and may be involved in a variety of physiological processes.
No significant change in in vivo chitinase activity was detected in response to abiotic stresses, despite the increase in chitinase gene transcripts. This can probably be ascribed to the restriction in enzymatic activity or a decrease in the translation of chitinase proteins in adverse environments. The inconsistency between gene transcription and enzyme activity may support the proposal that T. pseudonana chitinase genes are involved in other cellular processes apart from chitin metabolism, or it may represent a compensatory adjustment of chitin metabolism in shifting environments. The inconsistent results also suggest the possibility of post-transcriptional regulation during chitin degradation in T. pseudonana. By contrast, the expression of seabuckthorn chitinase genes was elevated at both the transcriptional [61] and protein activity levels [62] under cold stress. Moreover, chitinase activity of cucumber and tobacco also increased after treatment with biotic or abiotic stress inducers [63]. The results presented here suggest that there may be functional differences in the regulation and activity of chitinases in diatoms compared to those of higher plants.
Durkin et al. (2009) discovered that silica deposition and chitin exposure on the T. pseudonana cell wall were inversely correlated [53]. They suggested that chitin biosynthesis is enhanced in stressed cells that are unable to precipitate silica. In addition, co-regulation of transcript quantity by silicic acid and iron depletion is reported to be associated with cell wall processes [64]. In the present study, chitinase transcripts accumulated while enzymatic activity remained steady under silica depletion. Therefore, stable chitinase activity and higher chitin accumulation may allow chitin to serve as a substitute cell wall material when silica cannot be deposited. Moreover, as silica is required for diatom cell growth, chitin metabolism may also be affected by the abnormal growth of cells, further affecting cycles of carbon and other nutrients in the ocean [65]. However, further investigation of other genes involved in chitin metabolism (e.g., chitin synthases and chitin deacetylases) is required.
Auxiliary domains may assist in chitin binding by the T. pseudonana chitinases
Most chitinases contain a catalytic domain and additional auxiliary domains such as carbohydrate-binding modules (CBMs) [66], which are classified into 87 families at the CAZy database (http://www.cazy.org/). The CBMs anchor the enzyme to the substrate and disrupt its crystalline structure, creating free chain ends and thereby enhancing the enzyme’s catalytic activity towards insoluble substrates [66, 67]. Chitin binding has been demonstrated for a protein containing only two CBM_14 domains [68]. In the present study, the CBM_14, Chitin_bind_1, and LMPO_10 domains, which are typically found in chitin-binding proteins, were identified in the protein sequences of TpChi3, TpChi5, TpChi8, TpChi11,TpChi16, TpChi17, TpChi21, TpChi22, TpChi23, and TpChi24 (41.7%), five of which possessed two or more CBM_14 domains (TpChi3, TpChi21,TpChi22, TpChi23, and TpChi24) (Fig. 1), suggesting chitin-binding ability of T. pseudonana chitinases. Moreover, seven chitinases (TpChi3, TpChi11, TpChi16, TpChi21, TpChi22, TpChi23, and TpChi24) that contained the CBM_14 domain were predicted to be extracellular (containing a signal peptide) or located in the chloroplast or mitochondrial membrane (Table S6). This finding suggests more subcellular location of diatom chitinases containing the CBM_14 domain than the space between the plasma membrane and the silica frustule of diatoms as Traller et al. (2016) discussed [17].