Cold tolerance of ScCBL6 is associated with photosynthesis and tonoplast transporters in Arabidopsis

Plant adapted in the fragile zone offer enormous opportunity to understand the stress biology under ecological scenario . Stipa capillacea is widely distributed in the frigid and arid region of Tibet Plateau, but its signal system has never been investigated yet. In this study, we isolated a signal transduction gene, ScCBL6 , in Stipa capillacea , to characterize its cold tolerance capacity by ectopic expression in Arabidopsis . The results suggested that full length ScCBL6 encodes 227 amino acids, and phylogenetically clustered with CBL6 protein in Stipa purpurea and Oryza sativa . In comparison with wild type (WT) plants, ScCBL6 overexpressing plants ( ScCBL6- OXP) are tolerant to cold stress but not drought stress, which attested by the higher photosynthetic capacity (Fv/Fm) and survival rate of ScCBL6- OXP under cold stress. We further compared their cold-responsive transcriptome profiles through RNA-Seq. Totally, 3931 genes were differentially expressed by introduction of ScCBL6. They are participated in multiple processes like immune system, lipid catabolic, secondary metabolic and mainly enriched in plant hormone signal transduction and biomacro-molecule metabolism as regard to KEGG pathway. Differential expressed genes (DEGs) were predicted to locate in chloroplast, mitochondrion, vacuole, and so on, suggesting multitudinous function of ScCBL6. Based on the integrated analysis of ScCBL6-OXP, we inferred that ScCBL6 improve plant cold stress tolerance via regulate photosynthesis redox and vacuole metabolites transport in Arabidopsis .


Background
Sessile plants are confronted with ubiquitous environmental stimulus which shaping their geographical distribution [1,2]. In the long-term biology-environment interaction, plant signal system elaborately orchestrates with functional protein to confer stress defense. Ca 2+ is regarded as life in cell and developmental biology for its constituent role and flexible oscillation within cell parts [3], thus act as central hubs of plant signaling networks to coordinate plant growth [4]. Under the natural condition, Ca 2+ keep nanomole range in cytoplasm and millimole range in cellular compartment [5,6]; while cell organelles supported by double membrane (such as chloroplasts) promoted Ca 2+ wave upon stress initiation [7]. However, as a signaling messenger in plant cell, Ca 2+ is decoded by several kinds of proteins like CDPKs, CaMs and CBLs [8,9], so that well connected the stress signal with tolerance response.
Unlike CaMs and CDPKs exert regulations in nucleus [10], the CBLs protein could be membrane anchored by myristylation and acylation but gain flexible regulations roundly through interact with CBL-interacting protein kinase (shorted as CIPK). Structurally, CBLs proteins characterized by conical EF-hand motif and share sequence similarity with yeast calcineurin B subunit of yeast and animal neuronal calcium sensor [11][12][13]. There are 10 family members in Arabidopsis and Oryza sativa.
AtCBL1 was differentially responsive to multiple stress including salt, drought and cold stress [14].
AtCBL7 was documented to modulates plant low nitrate response [15]. Other CBLs proteins of Arabidopsis were also supported to mediate physiological response like osmotic regulation and ion homeostasis [16,17], and also seed development [18]. However, gene clone and functional characterization of CBL6 and CBL8 are still at a juvenile stage, both for model and non-model plants.
Cold stress is one of the environmental factors that cause physiological disorder and impair plant growth [17]. Low temperature lead to mechanical strain on cell wall and membrane rigidification by the lamellar to hexagonal II phase transitions lesion [19][20][21]. As responses, plants on one hand accumulate solutes like malate, fumarate and proline to enhance freezing tolerance [22], and one the other hand adjust gene expression to reprogramme the cell structures and redress physiological metabolism [23]. Unraveling corresponding cold responsive gene and network are of benefits to crop improvement engineering. However, ICE-CBF is the best-know cold stress tolerance cascade [24], more other components need to be investigated.
Stipa capillacea is a constructive species and important forage that tolerant to multiple abiotic stressors. Because of the long-established domestication in alpine climate of Tibet Plateau, S. capillacea provided a well system to resolve cold stress tolerance mechanism. In the previous study, SpCBL6 of its sibling species, Stipa purpurea, was found to enhance cold tolerance in Arabidopsis. In this study, we isolated its homologous CBL6-like protein from cold-hardy species Stipa capillacea, to study whether ScCBL6 share the same functionality and explore the extent to which the integrated regulation and cold tolerance was conditioned by CBL6 protein. By ectopic transformation in Arabidopsis, comparative transcriptomic analysis was employed to unravel the molecular mechanism of ScCBL6 at transcriptional level.  1A). All these proteins share four classical EF-hand in the motif scan analysis. ScCBL6 clustered with OsCBL6 in the phylogenetic analysis (Fig. 1B). The subcellular location result found ScCBL6 protein mainly resided at tonoplast and plasma membrane (Fig. 1C).

ScCBL6 Overexpressed Arabidopsis enhanced cold tolerance
To explore the biological function of ScCBL6 in vivo, we generated transgenic Arabidopsis carrying ScCBL6 under the promoter of cauliflower mosaic virus 35S. Three independent transgenic lines were obtained by gene-specific PCR (Fig. S1). Under the natural greenhouse condition, ScCBL6 overexpressed plants (short as ScCBL6-OXP) did not show much growth difference from wild type (WT) Arabidopsis, which also applied to the drought treatment as revealed by survival rate and Fv/Fm in ScCBL6-OXP retrieve normal growth gradually, indicating the better tolerance of ScCBL6-OXP than WT in response to cold stress. We also characterized the water loss rate of cold treated plants, and found that there is no significant difference between ScCBL6-OXP than WT (Fig. 3).

Comparative transcriptome analysis revealed transcriptional regulation of ScCBL6
To further investigate the molecular regulation excited by ScCBL6, we compared transcriptome variation between ScCBL6-OXP and WT for both cold and normal condition. In total, we sought out ScCBL6-OXP and WT irrespective of stress treatment. 4582 DEGs were found to be regulated by cold treatment in Col background (WT), however, 2185 DEGs were purely modified by the over expression of ScCBL6. In terms of cold response, ScCBL6-OXP share 43.64% (1300) overlaps with WT responders, the remaining 1679 (933+746) showed ScCBL6 dependency (Fig. 4). Among the 1300 overlaps, only 23.08% DEGs could be activated without cold treatment, suggesting the capability of ScCBL6 in response to cold stress.
For all the ScCBL6 regulated DEGs, the metabolic process, membrane and transferase activity Prospective cold tolerance mechanism mediated by ScCBL6 As documented in Zhang et al [25] and results of subcellular location of ScCBL6 (Fig. 1C), we inferred that ScCBL6 mediated cold tolerance by targeting of organelle located proteins. Therefore, ScCBL6 regulated DEGs were sequence extracted and searched against signal peptide database. The prediction of subcellular location indicated that most of the annotated DEGs located in chloroplast (266) and engaged in secretory pathway (472), only 12 proteins located at tonoplast (Fig. 6).
However, most of the tonoplast resided genes showed down-regulated expression, only two protein, i.e., polyamine choline transporter CAT2 and carbohydrate transporter TMT1 up-regulated their expression in response to cold treatment (Table 1). On the other hand, better chlorophyll fluorescence was observed in Fig. 2. We then further ranked the chloroplast located DEGs by |log2FC|, and plotted their expression profiles, 70% of top chloroplast DEGs up-regulated their expression levels (Fig. 7).
Two up-regulated genes among the top 20 coresets, vicinal oxygen chelate (VOC) superfamily member members AT1G80160 and glutathione transferase GSTU3, pertains to the redox regulation.
As indicated by the GR enzyme activity assays, antioxidant ability of ScCBL6-OXP overexpression lines were indeed improved (Fig. 8).

Discussion
The signal transduction is of great importance to the well domestication of alpine plants. Ca 2+ mediated signal transduction has been widely documented in plant kingdom [26]. CBL protein convey a broad range of stimulus signal via reversible Ca 2+ docking in its EF-hand domain [27,28], and expanded from one member in chlorophytes Ostreococcus lucimarinus to more ten members in higher plant like Arabidopsis [29]. Stipa capillacea has long distributed in Qinghai-tibet plateau, and its signal transduction has not been investigated ever. Based on transcriptome data, we use homologybased method to isolate one CBL gene from Stipa capillacea successfully. Sequence analysis found that ScCBL6 protein was very conservative, at least among grass types (Fig.1A). Phylogenetically, ScCBL6 showed paralogous relationship with CBL2 and CBL3 in both Oryza sativa and Arabidopsis thaliana, suggesting earlier gene divergence before speciation. However, CBL2 and CBL3 are wellknown chloroplast located protein [18], lending support to the tonoplast target of CBL6 in Arabidopsis [25]. CBL2 and CBL3 proved to affect seed size and embryonic development by transgenic operation in Arabidopsis [18]. However, up to date, functional characterization of CBL6 merely reported in Stipa purpurea and Triticum dicoccoides [30], all of which provided novel insight but just catch a little glimpse of its talents. In this study, we enforced functional analysis for ScCBL6 by ectopic expression in Arabidopsis, followed by comparative transcriptome analysis between its wild type (WT) and transgenic lines as regard to cold stress response.
Gain-of-function mutants, ScCBL6-OXP, enhanced cold tolerance but not drought tolerance in Arabidopsis (Fig. 2), which is highly consistent with the performance in transgenic SpCBL6 plants [31].
ScCBL6 share 96.77% similarity with SpCBL6 at the nucleotide level, and only 2 aa different at protein level. The two sites were not thought to functional divergent, which supported by the KaKs calculator results (⍵<1, p<0.05). As cold stress impose damage on plant body mainly through membrane damage and dehydration [23,32,33], we speculated that ScCBL6 confer the cold tolerance mainly through membrane functions, as we did not find significant difference for water loss between WT and transgenic lines (Fig. 3).
In the transcriptome analysis, ScCBL6 totally regulated the expression levels of 3931 genes (Fig. 4).
They are widely enriched in plant hormone signal transduction, secondary metabolism, fatty acid and protein regulation (Fig. 5C), indicating the miscellaneous role of ScCBL6 in plant in vivo. However, after cold stress, 31.37% (1233) of 3931 differently expressed genes (DEGs) were further regulated and 746 DEGs act as the ScCBL6 specific cold-inductive responder, they should constitute the core repertoire of cold tolerance mechanism imparted by ScCBL6. They execute holistic regulation across tissues like chloroplast, mitochondrion and cytoplasm, and so on (Fig. 6). However, only 12 genes were thought as tonoplast resided, the up-regulated gene CAT2 could interact with glutathione metabolism protein (OXP1), and tyrosine catabolism protein (HGO) in STRING network (Fig. S2).
Additionally, 266 genes were predicted to chloroplast resided, and they are involved in enzyme upregulation. GULLO2 encodes a homolog of rat L-gulono-1,4-lactone (L-GulL) oxidase, which involved in the biosynthesis of L-ascorbic acid. BT2 encode telomerase component, serve as scaffold protein and commonly regulated by a bZIP-mediated SnRK-dependent pathway [34]. IRX7 participate in secondary cell wall biosynthesis, is required for proper anchoring of seed coat mucilage to reduced xylan to form high molecular weight polymer [35]. Together with the membrane positioning of ScCBL6, it is reasonable to postulate that ScCBL6 enhanced cold tolerance through redox regulation and structure maintenance.
Direct interactions of ScCBL6 with these targets were not expected, because they are most probably subject to phosphorylation by CIPKs. In this study, CIPK12, CIPK16, CIPK17, CIPK22 and CIPK25 were documented to up-regulated by cold stress in WT, and all of them except CIPK12 are the same for cold-treated ScCBL6-OXP. Especially, CIPK16 and CIPK25 increased 27.33 and 17.02 times in coldtreated ScCBL6-OXP, indicating their potential interaction with ScCBL6. However, for the untreated comparison of WT and ScCBL6-OXP, no CIPK was significantly up-regulated, this result well correlated with the temperature dependence of CBL6/CIPK complex formation documented in Zhang et al [25]. In total, for cold stress response conferred by ScCBL6, many genes like GULLO2 and PCAP2 were potential targets to improve cold tolerance in other crop plant, however, for the lack of transformation system for Stipa capillacea, their underlying functional mechanism and pros and cons plasticity in other plant need more experimental investigation.

Conclusions
This study provided a well paradigm for the complexity and polyfunctionality of environmental signal transduction system. In this study, ScCBL6 isolated from Stipa capillacea caught variations in nearly whole cell part. In ScCBL6 overexpressing transgenic Arabidopsis, cold stress tolerance was verified by higher survival rate and better photosynthetic capacity. Further RNA-Seq revealed that genes related to metabolites transporter in tonoplast and genes related to redox regulation in chloroplast showed preferential up-regulation in cold treated ScCBL6-OXP. That is also confirmed by the enzyme of GR. Vacuole as the largest plant cell compartment, ScCBL6 as a membrane anchored protein, its controlled functions are important for the environmental adaptation.

Plant preparation and controlled stress
S. capillacea seeds used for experiment were collected from Qinghai-Tibet Plateau (33°26´56.5" N, 79°48´58.4" E). As S. capillacea is not endangered and distributed widely in semi-arid hillside the of alpine steppe (Fig. S4)

Isolation and sequence analysis of ScCBL6
ScCBL6 was isolated from Stipa capillacea by the homology-based cloning methods. Total RNA was extracted by kit as referenced in Zhou et al [36]. Primers of forward (5´-ATGGTGGATTTCCCGGAAGG-3 ) and reverse (5´-TCAAGCGTCCTCAACCTGAG-3´) were used to amplify the complete coding sequence. The PCR reaction amplified with Q5 and recycled as follows: 94℃ for 30s, 52℃ for 30s and 1 min at 72℃ for extension. PCR products was purified and ligated to pMD18-T vector (Takara) and

Plasmid construction and transgenic plants manipulation
To obtain overexpressing construct of ScCBL6, its coding sequence was amplified with extended primers: ScCBL6-F (5-GTCGACCCCGGGggtaccATGGTGGATTTCCCGGAAG-3) and ScCBL6-R . The 5´end of primers are sticky to plant binary vector pRI101-GFP, which could initiate the expression of chimeric protein contains target gene fused with GFP sequence by CaMV-35S promoter. After Kpn I digestion of binary vector, PCR products was sub-cloned into vector pRI101-GFP by recombinase. Resultant construct was introduced into Agrobacterium tumefaciens strain GV3101 and infected wild type Arabidopsis ()) by floral-dip method [37]. Transgenic seeds were screened by kanamycin and positive transformants were identified by gene-specific primers. Two lines of ScCBL6 overexpressing Arabidopsis were used for further analysis.

Subcellular location of ScCBL6
Agrobacterium tumefacien carrying the recombinant vector pRI101-ScCBL6GFP was inoculated into LB solution and propagated overnight. Agrobacterium tumefaciens were introduced into leaf abaxial epidermis of Nicotiana tabacum by syringe injection. Green fluorescence excited by GFP protein was imaged in Olympus FV1000 laser confocal microscopy (Olymplus, Japan).  [38].

Water loss assay
To explore the water loss rate under dehydration conditions, leaves of 4-week-old wild type and transformant Arabidopsis plants were cut off and allowed to dry on Whatman paper at room temperature, and net weights were documented at the 0.5, 1, 2, 4, 6 h, respectively. Water loss rate was calculated as weight loss per unit time.
Antioxidative enzyme activity assays The enzymatic activity was assayed according to [39]  Note: Transcript id were the assembly outputs in cufflinks, and corresponding gene id and locus were obtained from reads mapping against gff files. Log 2 FC were calculated from FPKM ratio of before and after cold treated ScCBL6-OXP.   Veen diagram showed the overlap and specialty of DEGs affiliated to different condition.

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