The sHSPs, especially those in plants, are a large and complex family of proteins [35]. The plant sHSPs are divided into 12 subfamilies, of which chloroplast-localized sHSPs family has been identified in diverse higher plant species [59, 60]. This family of sHSPs have a Met-rich domain, a unique amphipathic domain at their N-terminus, not present in other sHSPs. In this study, AsHSP26.8a possesses both a Met-rich domain and a chloroplast transit peptide at its N-terminus (Fig. S2). Phylogenetic analysis and subcellular localization further support the classification of the AsHSP26.8a protein as a member of the chloroplast-localized sHSPs family (Fig.S3 and Fig. 1).
AsHSP26.8a-mediated HSP/HSF pathway and plant abiotic stress tolerance
The major abiotic stresses such as drought, high salinity, extreme temperature, negatively influence plant survival, growth and productivity. As sessile organisms, plants are unable to change their sites to escape from the unfavorable environmental adversities, but have developed a great degree of resilience to conditions that would be considered harmful to many other organisms. A network of interconnected cellular stress response systems is essential for plant survival and productivity [61]. Within the complex stress response network, transcription factors (TFs) play a core role in the conversion of stress signal perception to stress-responsive gene expression by interacting with the promoter regions of various target stress-responsive genes, thus activating the whole network of genes to act together in enhancing plant tolerance to the harsh environmental conditions [62]. Heat shock transcription factors (HSFs) are the central regulators in plant cellular response to various abiotic stresses, especially to heat stress [63, 64]. Class B HSF and Class C HSF have been implicated in plant response to heat stress. HSFB1 and HSFB2b repress the expression of HSFs, but positively impact the acquired thermotolerance [65]. Capsicum annuum HSFB2a forms a transcriptional cascade with CaWRKY6 and CaWRKY40 to positively regulate the response to high temperature and high humidity [66]. Guan et al. [67] found that regulation of heat stress-responsive genes including HSFC1 and other HSFs by RCF2 and its interacting partner NAC transcription factor NAC019 is critical for thermotolerance in Arabidopsis. A recent study showed that HSFC1b from tall fescue (Festuca arundinacea) plays a positive role in plant tolerance to heat stress in association with the induction and upregulation of heat-protective genes [68]. Our results showed that overexpression of AsHSP26.8a alters plant heat stress response (Fig. 4) and results in down-regulated expression of the two HSFs, HSFB2a and HSFC1 in transgenic plant (Table 1), suggesting that AsHSP26.8a may function to repress HSF gene expression, modulating heat-responsive genes and thus attenuating plant response to heat stress.
AsHSP26.8a modulates ABA-dependent stress signaling and plant abiotic stress response
ABA, commonly known as the “stress hormone”, responds to an array of biotic and abiotic stresses [69]. Under osmotic stress condition such as drought and high salinity stress, a number of genes functioning in stress response and tolerance are induced, and ABA is accumulated [56, 70, 71]. The expression of stress-responsive genes is regulated by ABA-dependent and ABA-independent pathways [71]. These genes encode the late embryogenesis abundant (LEA) proteins, enzymes, transcription factors, protein kinases et al. LEA accumulation is a functional adaptation of plants in gaining tolerance against osmotic as well as oxidative stresses [72]. Overexpression of genes encoding LEA proteins can improve the stress tolerance of transgenic plants [73-76]. MYB transcription factors are a large group of proteins identified in eukaryotes and widely distributed in plants [77-79]. Some of the MYB protein family members are involved in ABA-dependent signaling pathways regulating stress adaption and conferring plant stress tolerance [78, 80-82]. In this study, overexpression of AsHSP26.8a alters plant development and plant response to ABA and salt stress (Fig. 5 and 6) and leads to significantly reduced expression of several genes encoding LEA and MYB proteins (Table 1) as well as some stress-responsive transcription factors, such as WRKY transcription factors, involved in ABA-dependent signaling [83]. Abiotic stresses such as heat, high salinity and drought also induce the WRKY genes and trigger a cascade of signaling pathways for improved plant stress tolerance [84, 85]. Many studies showed that overexpression of a WRKY family gene confers abiotic stress tolerance in transgenic plants. For example, AtWRKY25 and AtWRKY26 overexpression enhanced plant heat tolerance in transgenic Arabidopsis [86]. Transgenic Arabidopsis overexpressing a wheat WRKY transcription factor, TaWRKY33 exhibited enhanced heat tolerance [87]. In cotton, GhWRKY17 overexpression increased plant sensitivity to drought and salt stress as well as ABA-mediated seed germination and root growth by reducing the levels of ABA and transcripts of ABA-inducible genes including NbAREB1 (ABA-responsive element binding), NbDREB (dehydration-responsive element binding), NbNCED (9-cis-epoxycarote-noid dioxygenase), NbERD (early responsive to dehydration) and LEA protein, NbLEA [88]. These data suggest that AsHSP26.8a functions as a chaperone protein, contributing to ABA-dependent signaling in plant abiotic stress responses.
AsHSP26.8a modulates ABA-independent stress signaling and plant abiotic stress response
Our results also showed that AsHSP26.8a modulates stress-related transcription factor gene expression in the ABA-independent signaling pathways. The ABA-independent stress-responsive gene expression is regulated by DREB proteins. DREBs belong to Ethylene Response Factor (ERF)/AP2 family and consist of two subclasses, DREB1/CBF and DREB2 induced by cold and dehydration/high salinity, respectively [71, 89-96]. Arabidopsis DREB1A overexpression was reported to enhance LEA protein levels and therefore abiotic stress tolerance in Arabidopsis [97, 98] and various crops including rice, soybean, peanut and wheat [99-102]. Heterologous expression of AtDREB1B in Salvia miltiorrhiza enhanced plant drought tolerance by activating different downstream DREB/CBF genes [103]. Moreover, AtHSFA3 is a transcription factor that is transcriptionally induced during heat stress by DREB2A and in turn regulates the expression of HSP-encoding genes [104]. Overexpressing AtDREB2A in Arabidopsis plants induces not only drought- and salt-responsive genes but also heat-shock-related genes. Thermotolerance was significantly increased in plants overexpressing DREB2A and decreased in DREB2A knockout plants [92]. The ERF (ethylene-responsive element binding factor) is another subfamily of the AP2/ERF family of TFs and plays vital roles in the regulation of biotic and abiotic stress responses [105-107]. Overexpression of a tomato ERF transcription factor, SlERF84 in Arabidopsis endows transgenic plants with ABA hypersensitivity and enhanced tolerance to drought and salt stress [108]. Overexpression of CmERF053 of chrysanthemum could enhance drought tolerance [109]. In Tamarix hispida, constitutive expression of an ERF transcription factor, ThCRF1, increased biosynthesis of trehalose and proline and the activities of SOD and POD, resulting in an altered osmotic potential and an enhanced reactive oxygen species (ROS) scavenging, and therefore significantly improved salt tolerance in transgenic plants. On the contrary, suppression of ThCRF1 led to decreased plant salt tolerance [110]. In this study, transgenic plants overexpressing AsHSP26.8a displayed significant expression changes in ten ERF/AP2 family genes (Table 1), for example, compared to the wild type, DREB1B and ERF 105 expression in AsHSP26.8a transgenic plants was down-regulated over sixty-fold and thirty-fold, respectively (Table 1). These results suggest that AsHSP26.8a-modulated expression of the genes in the ABA-independent signaling pathways may also contribute to plant response to various abiotic stresses.
Other stress signaling pathways mediated by AsHSP26.8a and plant abiotic stress response
Calcium (Ca2+) is the most widely accepted second massager and involved in plant stress responses and cytoplasmic Ca2+ signal is recognized by Ca2+ sensors including calmodulins (CaM), calmodulin-like proteins (CMLs), calcium dependent protein kinases (CDPKs) and calcineurin B-like proteins (CBLs) [111-118]. Overexpression of AtCML24 enhances transgenic Arabidopsis tolerance to various ions including Co2+, Zn2+ and Mg2+ [119]. CML18 directly interacts with Na+/H+ antiporter NHX1 to regulate plant salinity tolerance [120]. CML9 is suggested to negatively regulate ABA-dependent salinity tolerance [121]. OsANN1, a calcium-binding protein of rice modulates antioxidant accumulation under abiotic stress to confer abiotic stress tolerance. OsANN1-knockdown led to increased plant sensitivity to heat and drought stresses, whereas OsANN1 overexpression resulted in improved plant growth with higher expression of OsANN1 under abiotic stress [122]. Receptor-like protein kinases (RLKs), a class of single-pass transmembrane proteins located in the plasma membrane, sense and transmit a variety of signals to regulate plant growth and development [123, 124]. Many RLKs have been implicated in abiotic stress responses, including the abscisic acid response, calcium signaling and antioxidant defense. Upon drought stress, the Arabidopsis LRK10L1.2 responds to directly or indirectly regulate stomata closure via ABA-mediated signaling [125], while a Glycine soja ABA-responsive receptor-like cytoplasmic kinase (RLCK), GsRLCK, responds to modulate ABA sensitivity in plants by regulating the expression of ABA-responsive genes [126]. In Pisum sativum, salinity induced lectin receptor-like kinase (PsLecRLK) gene expression and overexpression of PsLecRLKs led to improved plants salt tolerance due to enhanced ROS-scavenging [127]. In Medicago spp., the LRR-RLK gene, SRLK has been shown to regulate the root response to salt stress [128]. Large families of zinc finger transcription factors are abundant in plants and have diverse functions including DNA binding and transcriptional regulation [129]. Cys2/His2-type (C2H2) zinc finger proteins are implicated in plant response to a variety of adversities including low-temperatures, salt, drought, oxidative stress, excessive light and silique shattering [130-132]. One example is ZAT6 in Arabidopsis that positively regulates cadmium tolerance via the glutathione-dependent pathway [133]. Another example is the C2H2 zinc finger protein gene, Zat7 whose constitutive expression suppressed growth and enhanced salt tolerance in transgenic Arabidopsis plants [134]. Moreover, transgenic analysis in Arabidopsis points to the involvement of ZAT10 or STZ (salt tolerance zinc finger) in determining plant tolerance to drought, salt, osmotic, heat, photo-inhibitory light and oxidative stresses [135-138]. In this study, AsHSP26.8 overexpression led to significantly reduced expression of the genes encoding four CMLs, fifteen RLKs and nine zinc finger proteins in transgenic Arabidopsis (Table 1). For example, ZAT11 expression in AsHSP26.8a TG Arabidopsis plants was down-regulated over thirty-fold and ZAT12, ZAT7, CML38 and CML30 were down-regulated over sixteen-fold compared to wild type controls (Table 1). These results suggest that other than the ABA-dependent and -independent signaling pathways, AsHSP26.8a may also participate in other signaling pathways responding to abiotic stresses.
A few studies about the negative effect of HSPs on plant response to abiotic stress have previously been reported. Song et al. [139] found that overexpression of cytosolic and organellar AtHSP90s transgenic Arabidopsis overexpressing cytosolic and organellar AtHSP90s exhibited suppressed expression of stress-responsive genes and consequently reduced salt and drought tolerance. Our previous study also showed that overexpression of AsHSP17, a creeping bentgrass sHSP, attenuates plant response to abiotic stress by modulating plant photosynthesis and ABA-dependent and -independent signaling [15]. Moreover, Wang and Luthe [9] were unable to amplify ApHSP26.8 from the heat-tolerant variant selected bentgrass, which was regenerated from callus that survived selection at 40℃ for 1 week, but they were able to amplify the gene from the heat-sensitive variant non-selected bentgrass, which was not subjected to heat stress. The result suggests that similar to AsHSP17, as a chaperone protein, AsHSP26.8a is regulated to maintain an appropriate level in protecting a stressed plant. The excessive amount of AsHSP26.8a in transgenic plants may negatively impact the stress response regulatory network, compromising on plant stress tolerance. Alternatively, AsHSP26.8a may need to stay inactive under normal condition bot to become a stress itself. Research on impact of the regulated expression of AsHSP26.8a in transgenic creeping bentgrass itself is being conducted in order to shed light on molecular mechanisms of AsHSP26.8a-mediated plant development and stress response.