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Regulation of Heat Stress in Physcomitrium (Physcomitrella) patens Provides Novel Insight into the Functions of Plant RNase H1s

RNase H1s are associated with growth and development in both plants and animals, while the roles of RNase H1s in bryophytes have been rarely reported. Our previous data found that PpRNH1A, a member of the RNase H1 family, could regulate the development of Physcomitrium (Physcomitrella) patens by regulating the auxin. In this study, we further investigated the biological functions of PpRNH1A and found PpRNH1A may participate in response to heat stress by affecting the numbers and the mobilization of lipid droplets and regulating the expression of heat-related genes. The expression level of PpRNH1A was induced by heat stress (HS), and we found that the PpRNH1A overexpression plants (A-OE) were more sensitive to HS. At the same time, A-OE plants have a higher number of lipid droplets but with less mobility in cells. Consistent with the HS sensitivity phenotype in A-OE plants, transcriptomic analysis results indicated that PpRNH1A is involved in the regulation of expression of heat-related genes such as DNAJ and DNAJC. Taken together, these results provide novel insight into the functions of RNase H1s.


Introduction
With global warming, heat stress (HS) has gradually become a major limiting factor affecting plant growth, plant geographic distribution, crop yield, and quality [1][2][3]. Research analysis showed that the negative impact of high temperature on crop yield is becoming more severe [4]. Plants do not move and have evolved a series of self-protective mechanisms against external stresses to adapt to the environment [5]. Heat shock proteins (HSPs) and molecular chaperones are commonly found to be involved in plants' responses to HS [6,7]. DnaJ (HSP40) proteins are important molecular chaperones involved in signal transduction, cellular proteostasis, and tolerance to various stresses in plants [8,9]. The expression levels of these genes are up-regulated to improve heat tolerance in plants [4,6]. For example, GmDNJ1 (a major HSP40) is highly induced at high temperature, and Gmdnj1knockout mutants have more severe browning and lower chlorophyll content, and higher reactive oxygen species (ROS) content under HS, which suggest that GmDNJ1 plays an important role in response to heat stress in soybean [10].
Cytosolic lipid droplets play important roles in plant growth and responses to stress. Lipid droplets (or so-called lipid bodies in some cases) are subcellular structures that are found localized in both nucleus and cytosol, which suggested a species-specific role of RNH1A in P. patens. In this research, we further explore the function of PpRNH1A and found that overexpression of PpRNH1A (A-OE) plants were more sensitive to heat stress by regulation of heat-related gene expressions, such as DNAJ and DNAJC. Chloroplast development but not pigment synthesis was affected by overexpression of PpRNH1A. Ultrastructural and surface structural observations show growth defects in A-OE plants, and abnormal accumulation and mobilization of lipid droplets were found in the cytosol, which is associated with the sensitivity of A-OE plants to heat stress.

Phylogenetic Analysis of RNases H1s
In our previous study, we identified two family members of RNase H1s in P. patent, named PpRNH1A (Pp3c4_14290) and PpRNH1B (Pp3c2_25170), which contain the RNase H catalytic domain [45]. To investigate the evolutionary relationship of RNase H1s in plants, we used the full-length protein sequence of PpRNH1A from the Phytozome database to search for the RNase H1s homologs in representative species such as algae, mosses, and higher terrestrial plant groups. Public database Phytozome, Ensembl plants, NCBI, and the klebsormidium genome project were used for BLAST analysis. The phylogenetic tree ( Figure 1) showed that three main branches were identified according to their full-length protein sequences, and the development process of RNase H1s was consistent with the evolutionary history of organisms. PpRNH1A and PpRNH1B were grouped together and showed a close evolutionary relationship to Chlamydomonas reinhardii and Klebsormidium nitens. One to three family members of RNase H1s were identified in different plant species, and some of the proteins in the same species were assigned to different sub-branches, which suggested that they may have differentiated functions.

PpRNH1A Is Involved in Growth Development and Stress Tolerance
To explore the features of the PpRNH1A (Pp3c4_14290), we first investigated the expression pattern of the PpRNH1A in wild type throughout the growth cycle of P. patens. Expression data were downloaded from the public database Physcomitrella eFP Browser

PpRNH1A Is Involved in Growth Development and Stress Tolerance
To explore the features of the PpRNH1A (Pp3c4_14290), we first investigated the expression pattern of the PpRNH1A in wild type throughout the growth cycle of P. patens. Expression data were downloaded from the public database Physcomitrella eFP Browser (BAR, http://bar.utoronto.ca/, data shown in Figure 2 were accessed on 19 February 2022). The higher expression levels of PpRNH1A were found in spores, rhizoids, archegonia, and at the sporophyte S1 stage, with relatively lower expression levels in chloronema, gametophores, and at the S2, S3, and M stages of sporophyte development (Figure 2A,B). This result suggests that PpRNH1A might be involved in P. patens growth and development. , data are presented as means ± SEM of three replicates; t-test was used for statistics; asterisks indicate the significant difference between treatment group and the control group, * p < 0.05, *** p < 0.001.

Creation of Stable Transgenic P. Patens Line Overexpressing PpRNH1A
To detect the functions of PpRNH1A, we obtained the CDS of the PpRNH1A gene by PCR amplification. The CDS fragment without the stop codon was integrated into the overexpression vector ( Figure 3A) under the promoter of PpEF1a followed by a PEG-mediated P. patens protoplast transformation. Positive PpRNH1A-overexpression plants were presented as means ± SEM of three replicates; t-test was used for statistics; asterisks indicate the significant difference between treatment group and the control group, * p < 0.05, *** p < 0.001. We next examined the expression levels of PpRNH1A under abiotic stresses. The wildtype gametophores of P. patens were exposed to heat stress (HS) at 40 • C for 18 h and then recovered for 4 days, and samples at three timepoints (control, heated, and recovered) were taken to determine the relative expression level of the PpRNH1A. Our results showed that HS significantly induced the expression of the PpRNH1A (3.91 times induction compared to the control) with a continuous induction (6.33 times induction compared to the control) during the recovery period ( Figure 2C). Furthermore, PpRNH1A expression levels under dehydration stress treatment were extracted from our previous dehydrated transcriptome data [46]. The result showed that PpRNH1A was slightly induced (1.47 times induction compared to the control) by dehydration stress treatment and then returned to a normal level after recovery ( Figure 2D). Our results showed that PpRNH1A participates in both development and stress tolerance in P. patens.

Creation of Stable Transgenic P. patens Line Overexpressing PpRNH1A
To detect the functions of PpRNH1A, we obtained the CDS of the PpRNH1A gene by PCR amplification. The CDS fragment without the stop codon was integrated into the overexpression vector ( Figure 3A) under the promoter of PpEF1a followed by a PEGmediated P. patens protoplast transformation. Positive PpRNH1A-overexpression plants were selected using the hygromycin resistant marker and confirmed at both DNA and RNA levels ( Figure 3B,C). Seven positive transformants at the DNA level were obtained. RNA was taken from each line and followed by quantitative real-time polymerase chain reaction (qRT-PCR) to confirm the expression levels of PpRNH1A-overexpressing lines (A-OE). One A-OE with a three times overexpression level compared to wild-type plants was shown in Figure 3C. The pigment contents, including Chlorophyll A (Chl a), Chl b, total Chls, and carotenoids in WT and PpRNH1A-overexpression plants were measured under normal growth conditions, and the result showed that no significant changes were found between WT and A-OE in pigment contents ( Figure 3D).

Overexpression of PpRNH1A Affects Lipid Droplets Metabolism and Mobilization
The intracellular ultrastructure of A-OE plants was observed by transmission electron microscopy (TEM). We found that the cell walls of the overexpression plants were much thinner than the WT ( Figure S1A). Interesting, there was no clear laminal structure of the chloroplast stroma observed in A-OE plants ( Figure S1B), although the level of pigments in A-OE seems not to have been affected. Furthermore, in A-OE plants, more plastoglobuli were observed in chloroplasts ( Figure S1A,B). The surface of the plant was then observed by scanning electron microscopy (SEM). The results showed that there were protrusions like glandular hairs on the surface of the A-OE plants ( Figure S1C). These results lead us to hypothesize that the lipid metabolite was affected in A-OE plants, since the plant cuticle is a lipid membrane covering plant surfaces, and the plastoglobuli is a kind of lipid droplets within the chloroplasts.
To confirm whether the overexpression plants could affect the accumulation of cytosolic lipid droplets, the dye Nile red was used to stain the A-OE plants and wild-type plants to observe the cytosolic lipid droplets under the confocal microscope. As shown in Figure 4, the yellow fluorescence represented the accumulation of cytosolic lipid droplets by Nile red. Chloroplast autofluorescence was marked with red fluorescence in both wild-type and A-OE plants ( Figure 4). The results showed that the lipid droplets were hardly seen in the gametophore of wild-type plants under normal growth conditions, however, in contrast, abundant of cytosolic lipid droplets around chloroplasts were observed in A-OE plants ( Figure 4). In addition, a proportion of the lipid droplets observed in the overexpression plants had irregular morphology ( Figure 4). Although additional experimental evidence is needed, these results revealed that overexpression of PpRNH1A may be associated with the metabolism and mobilization of lipid droplets. , data are presented as means ± SEM of three replicates; t-test was used for statistics; asterisks indicate the significant difference between WT and A-OE, *** p < 0.001.

Overexpression of PpRNH1A Affects Lipid Droplets Metabolism and Mobilization
The intracellular ultrastructure of A-OE plants was observed by transmission electron microscopy (TEM). We found that the cell walls of the overexpression plants were much thinner than the WT ( Figure S1A). Interesting, there was no clear laminal structure of the chloroplast stroma observed in A-OE plants ( Figure S1B), although the level of pigments in A-OE seems not to have been affected. Furthermore, in A-OE plants, more plastoglobuli were observed in chloroplasts ( Figure S1A,B). The surface of the plant was then observed by scanning electron microscopy (SEM). The results showed that there were protrusions like glandular hairs on the surface of the A-OE plants ( Figure S1C). These results lead us to hypothesize that the lipid metabolite was affected in A-OE plants, since the plant cuticle is a lipid membrane covering plant surfaces, and the plastoglobuli is a kind of lipid droplets within the chloroplasts.
To confirm whether the overexpression plants could affect the accumulation of cytosolic lipid droplets, the dye Nile red was used to stain the A-OE plants and wild-type , data are presented as means ± SEM of three replicates; t-test was used for statistics; asterisks indicate the significant difference between WT and A-OE, *** p < 0.001.

PpRNH1A-Overexpression Line Is More Sensitive to Heat Stress
To detect the function of the protein deduced by PpRNH1A, plants were exposed to heat stress. Thirty days old gametophores of the wild-type plants and A-OE plants were treated with 40°C for 18 h. Followed by a recovery at normal temperature (25 • C) for 5 days. Results showed that A-OE plants were more sensitive to heat stress and could not recover from the HS ( Figure 5A). Photosynthetic parameters, including the maximal efficiency of PSII photochemistry (Fv/Fm), non-photochemical energy dissipation (NPQ), and electron transport rate (ETR) were measured at the timepoints of before heat stress (the control), after heat stress, and recovered for 4 days. Similar Fv/Fm, NPQ, and ETR levels were observed between A-OE plants and WT before HS. Heat stress severely affects the function of the photosystem in both A-OE plants and WT, whereas the Fv/Fm and NPQ levels in A-OE plants were significantly lower than in WT plants, both under heat stress and after recovery ( Figure 5B-D). These results revealed that the overexpression of PpRNH1A resulted in decreasing tolerance to heat stress in P. patens plants.
plants to observe the cytosolic lipid droplets under the confocal microscope. As shown in Figure 4, the yellow fluorescence represented the accumulation of cytosolic lipid droplets by Nile red. Chloroplast autofluorescence was marked with red fluorescence in both wildtype and A-OE plants (Figure 4). The results showed that the lipid droplets were hardly seen in the gametophore of wild-type plants under normal growth conditions, however, in contrast, abundant of cytosolic lipid droplets around chloroplasts were observed in A-OE plants (Figure 4). In addition, a proportion of the lipid droplets observed in the overexpression plants had irregular morphology (Figure 4). Although additional experimental evidence is needed, these results revealed that overexpression of PpRNH1A may be associated with the metabolism and mobilization of lipid droplets.

PpRNH1A-Overexpression Line Is More Sensitive to Heat Stress
To detect the function of the protein deduced by PpRNH1A, plants were exposed to heat stress. Thirty days old gametophores of the wild-type plants and A-OE plants were treated with 40 ℃ for 18 h. Followed by a recovery at normal temperature (25 °C ) for 5 days. Results showed that A-OE plants were more sensitive to heat stress and could not recover from the HS ( Figure 5A). Photosynthetic parameters, including the maximal efficiency of PSII photochemistry (Fv/Fm), non-photochemical energy dissipation (NPQ), and electron transport rate (ETR) were measured at the timepoints of before heat stress (the control), after heat stress, and recovered for 4 days. Similar Fv/Fm, NPQ, and ETR levels were observed between A-OE plants and WT before HS. Heat stress severely affects the function of the photosystem in both A-OE plants and WT, whereas the Fv/Fm and NPQ levels in A-OE plants were significantly lower than in WT plants, both under heat stress and after recovery ( Figure 5B-D). These results revealed that the overexpression of PpRNH1A resulted in decreasing tolerance to heat stress in P. patens plants.

PpRNH1A Regulates the Expression of Heat-Responsive Genes including DNAJ and DNAJC
Considering that overexpression plants are more sensitive to heat stress, we then tested if PpRNH1A regulates the heat-related genes. Transcriptomic profiles of WT plants before HS (control) and 18 h after HS were compared to identify the heat-responsive genes. In total, 10,538 DEGs (Fold change > 1.5 or 0 < Fold change < 0.67, p-value < 0.05) were found responding to heat stress in wild-type plants (collection: WT-vs-WT_H). To identify the genes regulated by PpRNH1A, 3768 DEGs were identified from the comparison between WT and A-OE plants under normal growth conditions (collection: WT-vs-A-OE). The overlap of these two collections of DEGs showed that 2292 heat-related genes were regulated by PpRNH1A ( Figure 5E). Representative heat-related genes [47] such as Pp3c27_7640 (molecular chaperone DNAJ, HSP40) and Pp3c21_19830 (DNAJ homolog subfamily C member DNAJC) were selected for further analysis. Heat stress significantly induced the expression level of DNAJ and DNAJC in WT plants. However, in A-OE plants, DNAJC was not induced by heat ( Figure 5F). The fold change of DNAJ induced by HS in A-OE plants was much less than that in the WT plants. These results suggest that PpRNH1A regulates the expression of heat-responsive genes, including DNAJ and DNAJC ( Figure 5F).

Discussion
RNase H1s are ribonucleases widely present in organisms [26], and most studies on RNase H1s are related to growth and development. In Arabidopsis, AtRNH1B and AtRNH1C play important roles in maintaining the normal development of embryos, and AtRNH1C deletion mutants showed severe growth-defective phenotypes [39]. Our recent progress on P. patens revealed that PpRNH1A regulates the transcription of auxin-related genes by controlling the formation of R-loops, further regulating the development [45]. In this study, we found novel biological functions of PpRNH1A, which may also participate in heat stress responses, possibly by affecting the numbers and the mobilization of lipid droplets and regulating the expression of heat-related genes.
Phylogeny analysis results in this study suggest that there is functional differentiation of RNase H1s within and among plant species (Figure 1), which confirmed our previous finding that there may be functional differentiation between AtRNH1A and PpRNH1A [45]. Previously, we found that the development of gametophores of the pprnh1a mutant was affected through the modulation of R-loop formation on auxin-related genes. Overexpression plants of PpRNH1A were obtained ( Figure 3A) in this study, and we found that the chloroplasts development was affected and no clear lamellar structure was observed ( Figure S1B). Interestingly, although the chloroplast development was impaired, the pigments did not show any difference in A-OE plants compared to WT plants ( Figure 3D). Abnormal development of lamellar structure and more plastoglobuli were found in the chloroplasts of the A-OE plants ( Figure S1A,B). Furthermore, protrusions like glandular hairs were found on the surface of the A-OE plants as well. In addition, abundant lipid droplets with irregular morphology were accumulated in the cytosol of A-OE plants (Figure 4). These results suggest that overexpression of PpRNH1A resulted in disordered lipid metabolism and mobilization.
TAGs are the primary constituents of lipid droplets, which do not accumulate in vegetative tissues of plants under normal growth conditions but accumulate significantly under stress conditions, such as drought, high temperature, low temperature, and nutrient starvation, especially in leaves [48,49]. Correspondingly, lipid droplets were reported to be closely associated with plant biotic and abiotic stress as well. Pseudomonas-infected leaves caused an accumulation of lipid droplets and induced hypersensitivity reactions in Arabidopsis [14]. Seeds with irregular lipid droplets morphology were found susceptible to chilling injury during germination [50,51]. Consistent with the above, we found that PpRNH1A is involved in plant responses to heat stress, which provided a novel function of RNase H1s in plants. In our study, the expression levels of PpRNH1A were found to be significantly induced by abiotic stresses such as heat ( Figure 2C) and drought treatment ( Figure 2D). Moreover, A-OE plants were sensitive to heat stress ( Figure 5A), further confirmed by the levels of photosynthetic parameters ( Figure 5B-D). This is coincident with the findings in sunflower (Helianthus annuus L.), where stress-sensitive lines showed longer retention of the lipid droplets membrane under salt stress, thus exhibiting higher lipid accumulation and faster mobilization than that of stress-tolerant lines [22]. As a note, the lipid droplets mentioned here in this study are not what had been described as "oil body" in liverworts. Lipid droplets are present in almost all green plants accumulating compounds that are not soluble in the aqueous phase, and oil bodies are present only in liverwort, storing large quantities of toxic sesquiterpenoids [17,[52][53][54][55]. Lipid droplets are subcellular organelles of monolayer membranes with a diameter of about 0.5-2 µm, while oil bodies are an endocrine structure surrounded by a lipid bilayer membrane [13,55]. The size of small oil bodies is 2-5 × 3-9 µm on average, and the diameter of large oil bodies can reach 70 µm [56,57]. Although both mosses and liverworts are bryophytes, lipid droplets were found in our experiment.
HS affects all aspects of plant growth and elicits responses in a range of genes, including the accumulation of heat shock proteins (HSPs). We found that 60.8% of the genes regulated by overexpression of PpRNH1A are responsive to heat stress ( Figure 5E), which is one of the explanations for why A-OE plants were more sensitive to HS than wild-type plants ( Figure 5A-D). Heat responsive genes play critical roles in regulating the resistance/tolerance of plants to heat stress. GmDNJ1 (a major HSP40) was induced by heat stress and was responsible for enhanced heat tolerance in soybean [10]. In tomatoes, the expression levels of LeCDJ1 of Lycopersicon esculentum and SlDnaJ20 of Solanum lycopersicum were induced by heat stress, and overexpression of LeCDJ1 or SlDnaJ20 could improve the heat tolerance of plants [9,58]. In alfalfa (Medicago sativa), the DnaJ-like protein (MsDJLP) gene was induced by heat stress, and ectopic expression of MsDJLP in tobacco enhances the heat tolerance of tobacco [59]. All of these indicate that DNAJ could be used as a representative heat responsive marker and its expression level is closely related to heat stress tolerant ability. Our results showed that both DNAJ and DNAJC were induced by heat stress in WT plants, however, on the contrary, the induction folds in the A-OE plants were not as strong as that in the wild type, especially DNAJC, whose induction was totally hampered under heat stress. This is corroborated by the result that our A-OE plants is more susceptible to heat stress.  [60]. Multiple sequence alignments of these amino acid sequences were conducted with the ClustalW of MEGA-X, and the phylogenetic tree construction was performed with MEGA-X using the maximum likelihood (ML) method and 1000 bootstrap [61]. The best-fit model we selected was JTT + G + I [62].

Plant Materials and Growth Conditions
Gransden 2004 (Courtesy Prof. Mitsuyasu Hasebe) was the wild-type (WT) Physcomitrium (Physcomitrella) patens genetic material we used. All plant materials were grown on BCD medium supplemented with 5 mM ammonium tartrate and 1 mM CaCl 2 . Plants were grown at 25°C under 16 h light photoperiod per 8 h dark photoperiod with light intensity 60-80 µmol photons m −2 s −1 .

Protoplast Transformation
Protoplast of P. patens were prepared from protonema which were continuously disrupted with an electric stirrer every week. The overexpression vector pPOG1-A was linearized using the restriction enzyme MssI (Thermo Fisher Scientific, Waltham, MA, USA). The transformants were obtained by transferring the linearized vector into wild-type using polyethylene glycol (PEG)-mediated protoplast transformation [63].

PCR and Real-Time qRT-PCR Characterization of Overexpression Plants
Stable transgenetic lines were identified by PCR and qRT-PCR at both DNA and RNA level. The transformants were initially screened at the DNA level using primers "F" and "R". Total RNA from P. patens tissues was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the instructions. cDNA synthesis was performed using a PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer's instructions. qRT-PCR was performed using SYBR Premix Ex Taq II (Takara, Dalian, China) and carried out on Bio-Rad CFX96. The primer sequences for PCR and qRT-PCR are listed in Supplementary Table S1.

Analysis of Gene Expression Patterns and Analysis of Expression by Stress Treatment
Expression pattern data were retrieved and visualized from the public database Physcomitrella eFP Browser of BAR (http://bar.utoronto.ca/, accessed on 19 February 2022).

Observation of Cell Ultrastructure
Leaves were cut into 1 mm 2 with a blade and immediately fixed with 3% glutaraldehyde overnight at 4 • C. Rinse the material by adding 0.1 M phosphate buffer (PBS buffer, pH 7.2) every 30 min for 3 times. Subsequently, 1% osmium tetroxide was added for sample fixation at 4 • C for 2 h. Continue rinsing three times for 20 min each. The materials were continuous rinsed for 3 times and dehydrated in a serial ethanol gradient. After embedded in Epon 812 resin, materials were sectioned and stained with 2% uranyl acetate and lead citrate under EM UC7 ultramicrotome (Leica, Weztlar, Germany). The cellular ultrastructure was observed under JEM-1400Plus transmission electron microscopy (JEOL, Tokyo, Japan).

Observation of Cell Surface Structure
Leaves of the material were cut into 9 mm 2 with a blade and immediately fixed with 3% glutaraldehyde overnight at 4 • C. The rinsing, fixation, and dehydration procedures are the same as previous mentioned. Samples were dried with a CO 2 critical-point drier. The dried materials were mounted on aluminium stubs using tweezer and sputter-coated with gold. Cell surface structure was observed by Zeiss Sigma 300 scanning electron microscopy (Zeiss, Oberkochen, Germany).

Nile Red Staining
Gametophytes grown for 30 days were taken and placed in a certain volume of Nile red working solution for staining in dark for 5 mins as previously described, with appropriate modifications [64]. Lipid droplets were observed under Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan). The emission filters for Nile red were 493-636 nm.

Heat Stress Assay
Thirty-days old gametophytes of P. patens were used to perform a heat stress assay. The wild-type plants and overexpression plants were treated at 40 • C for 18 h, followed by a recovery at 25 • C for 4-5 days. The plants to be tested were placed in the dark for 30 min. Relevant photosynthetic parameters were determined using IMAGING-PAM (Walz, Effeltrich, Germany) and the Imaging Win software (Walz, Effeltrich, Germany) as described previously [65]. Pigment content was determined using the N, N-dimethylformamide (DMF) method, as described previously [65].

Bioinformatics and Data Analysis
Wild-type and A-OE plants before and after heat treatment were used to extract RNA. Samples for RNA sequencing (RNA-seq) were treated as previous description [66]. Differentially expressed genes (DEGs) between two samples were identified with the criteria of "Fold change > 1.5" and "p value < 0.05". Differentially expressed genes were visualized with TBtools [67]. The dehydrated transcriptome data used in this study were obtained from Dong et al. [46].

Statistical Analysis
All experiments were performed with three biological replicates. Student's t-test was used for hypothesis testing in statistics between two samples. Significant differences were defined and indicated by asterisks *, **, and ***, corresponding to p-values < 0.05, <0.01, and <0.001, respectively.

Conclusions
In conclusion, we revealed that PpRNH1A not only participates in the regulation of growth development of P. patens plants, it also plays a crucial role in plant response and tolerance to abiotic stresses such as heat, possibly by regulating the expression of heat-related genes and causing the abnormal accumulation and the mobilization of lipid droplets in the cytosol. Our data highlights the important role played by PpRNH1A in plant heat stress response, providing a novel insight into the function of RNase H1s.