In MAPK signal-plant pathway, we found that many pathways generated by signaling molecules and stress-induced were affected by Cd stress. However, most of genes involved in these pathways were down-regulated by Cd, for example, FLS2 involved in fg22-mediated disease resistance, NDPK2 mediated by H₂O₂, CAM4 mediated by wounding ( Figures 2A, B ). Whereas, Cd activated genes encoding serine/threonine-protein kinase SRK2 (SnRK2) involved in ABA signaling pathway ( Figures 2A, B ). Moreover, the expression of downstream genes encoding ABA responsive element binding factor (ABF) and MKK3 was enhanced by 1.1-1.4 folds and 1.2-1.5 folds, respectively by Cd ( Supplementary Table S3 ). Furthermore, expression of key genes encoding 15-cis-phytoene desaturase (PDS), prolycopene isomerase (crtISO) and zeaxanthin epoxidase (ZEP) involved in ABA biosynthesis pathway was also induced by Cd ( Figure 2C ; Supplementary Table S3 ).

As the enhanced expression of genes related to the ABA synthesis pathway was observed in G. lemaneiformis after Cd stress, ABA content quantified 1 day post-stress using UPLC-MS. The MS data and retention times of ABA are detailed in Supplementary Table S2 . Notably, the presence of Cd led to a 34.5% remarkable increase in ABA content ( Figure 2D ).

Considering the content of hormones and expression profiles, we conducted that ABA might be play a positive role in response to Cd stress in G. lemaneiformis. To further verify the effect of ABA on growth of G. lemaneiformis under Cd stress, different concentrations of ABA were applied to G. lemaneiformis under 75 µM Cd stress. At 10 µM, ABA reduced the inhibition of Cd on the growth of G. lemaneiformis and increased the contents of PE and PC ( Figures 2E–G ). A number of reports have demonstrated that ABA is closely related to perception and mediation of Cd stress (Rasafi et al., 2020). For instance, in rice seedlings and lettuce, there is a rapid enhancement of ABA content under Cd stress (Hsu and Kao, 2003; Tang et al., 2020), and in Arabidopsis thaliana exogenous application of ABA has been shown to significantly decreased Cd content and alleviated Cd toxicity (Meng et al., 2022). Wang et al. (2022) reported that gene involved in ABA signaling pathway can enhance Cd resistance ability of T. hispida by regulating the production of ROS under Cd stress. Our results revealed that ABA and ABA signal may be involved in the induction of protective mechanisms against Cd toxicity in G. lemaneiformis.

GO enrichment analysis revealed that “antioxidant activity” and “cellular oxidant detoxification” was involved in the response to Cd. The antioxidant system may play key roles in Cd stress in plants (Zhang and Reynolds, 2019). In plants, the antioxidant defense system mainly consists of superoxide mutase (SOD), which converts oxygen radicals (O²⁻) into hydrogen peroxide (H₂O₂), and L-ascorbate peroxidase (APX), peroxidase (POD), and catalase (CAT), these three enzymes remove toxic H₂O₂ (Zhang and Reynolds, 2019). Furthermore, the sulfur-containing antioxidant including thioredoxin (Trx), ascorbate (AsA) -GSH cycle and glutaredoxin cycle, etc., may also involve in the detoxification of Cd (Smiri et al., 2011; Zhang et al., 2020; Zhang et al., 2023a). A total of 6 (3 upregulated and 3 downregulated) and 12 (8 upregulated and 4 downregulated) DEGs associated with antioxidant activity were identified at 1 and 3d after Cd stress, respectively ( Figure 3 ). Among them, expression of 2 SOD encoding genes (SOD3 and EC-SOD) and 2 of 3 CAT encoding genes (CAT-B, CAT) was down-regulated, while APX encoding gene (APX4) and two thioredoxin (Trx) encoding genes (TRX2.1 and TRX2.2) were up-regulated ( Figures 3A, C ). Trxs are disulfide reductases containing a conserved active redox center thioredoxin domain (WCGPC), which also exist in GlTRX2.1 and GlTRX2.2 ( Supplementary Table S4 ). Moreover, expression of three genes encoding thioredoxin-like disulfide reductases (TRLs) were up-regulated at 3 d after Cd stress ( Figures 3A, C ). GlTRLs possess a thioredoxin domain and TPR-like_helical domain ( Supplementary Table S4 ), which catalyze the transfer of electrons from a reduced substrate to the disulfide bridge of Trx ( Figure 3A ). Trxs distribute the reductant to selected targets via a dithiol-disulfide exchange, resulting in the structural and functional modification of a target protein (Balsera and Buchanan, 2019). Zhang et al. reported that the thioredoxin-like protein CDSP32 improves cadmium tolerance by alleviating Cd-induced photosynthetic inhibition (Zhang et al., 2020) and modulating antioxidant system in tobacco leaves (Zhang et al., 2023b).

The Glutaredoxin (Grx) cycle, comprised of Grxs, GSH, and glutathione reductase (GR), is crucial for maintaining intracellular redox homeostasis (Ogata et al., 2021). Grxs are a multifunctional family of glutathione-dependent disulfide oxidoreductases. In G. lemaneiformis, expression of Grx encoding gene (GLX2) was upregulated at 3 d after Cd stress ( Figures 3B, C ). The ascorbate-glutathione cycle is acknowledged as a cellular redox buffer which donates electrons to various oxidized compounds in plants (Decros et al., 2023). Expression of genes encoding L-galactose dehydrogenase (GLDH) and glutathione synthetase (GS) was upregulated; these enzymes catalyze the conversion of L-galactose to ascorbate and γ-glutamylcysteine to GSH, respectively. Expression levels of APX, which detoxifies excess toxic ROS H₂O₂ to H₂O, were also upregulated ( Figure 3B ). These data suggested that the Grx cycle and ascorbate-glutathione cycle alleviates Cd toxicity by scavenging ROS. Glutathione have been proposed as Cd-binding chelator (Sears, 2013). The upregulation of these genes encoding Trxs, Grxs and GSS showed that sulfur antioxidants may play important roles in controlling oxidative stress in G. lemaneiformis exposed to Cd.

In plants, Cd transporters mainly include natural resistance-associated macrophage protein (Nramp), heavy metal-transporting ATPase (HMA), iron regulated transporter protein (IRT), zinc transporter protein (ZIP), ATP-binding cassette (ABC) and yellow stripe-like (YSL) families (Hu et al., 2022; Tao and Lu, 2022). Up to now, Nramps, ZIP5/6/9 and IRT1/2 are found to be involved in the uptake of Cd from the soil to roots in plants (Tao and Lu, 2022). In G. lemaneiformis, YSL transporters do not exist, and the expression of GlNramps and GlHMA was not altered by Cd. Similarly, Nramps were not differentially expressed under Cd stress in algae C. reinhardti (Zhang et al., 2023a). In G. lemaneiformis, the expression of transporter genes GlIRT1 and GlZIP6 was significantly enhanced by Cd ( Figure 3 ). This suggested that G. lemaneiformis actively engages in the uptake and transportation of Cd upon exposure.

Plants possess several detoxification mechanisms to mitigate Cd toxicity including efflux, chelation binding to metallothioneins/PCs and compartmentation to vacuole. In rice, ZIP1, ABCG36 and HMA9 mediate Cd efflux, and ABCG8 mediates Cd efflux in Arabidopsis thaliana (Tao and Lu, 2022). In G. lemaneiformis, Cd induced the expression of genes encoding GlZIP1 and GlABCG22 for Cd efflux ( Figure 4 ). Particularly, expression of GlZIP1 was upregulated more than 2-fold at both 1 and 3 d after Cd stress ( Figure 4 ), and that in C. reinhardti increased by more than 5 times (Zhang et al., 2023a). However, expression of genes coding for phytochelatins or metallothioneins did not elevated by Cd. HMA3 and ABCC subfamily transported Cd into vacuole for sequestration in plants (Tao and Lu, 2022). In G. lemaneiformis, the expression of multidrug resistance protein 7 (MRP 7, ABCC subfamily) was significantly upregulated at 3 d after Cd stress ( Figure 4 ). Cd is also can bind the dimer of GSH (Zhang et al., 2022b), which is consistent with the increase of GSH content in our result, and then transported to vacuole in form of chelates. These results indicated that G. lemaneiformis through synthesis of GSH, then combined Cd transport into the vacuole and improve Cd efflux to reduce the Cd damage.

KEGG analysis revealed an enrichment of energy metabolism processes, including photosynthesis - antenna proteins and nitrogen metabolism under Cd stress. In the process of photosynthesis - antenna proteins, the genes encoding allophycocyanin (APC), phycocyanin (PC) and light-harvesting chlorophyll protein complex (LHC) were affected under Cd stress ( Supplementary Table S5 ). In total, 27 and 22 transcripts were differentially expressed at 1 and 3 d, separately. Of these, 27 and 21 transcripts encoding ApcB-C, CpcA-C, Lhca1 and Lhca4 were down-regulated at 1 and 3 d after Cd stress, respectively ( Table 3 ; Supplementary Table S5 ). This is consistent with a decrease in photosynthetic pigments PE, PC and Chl a content. In the nitrogen metabolism pathway, 9 (1 up and 8 down) and 13 (4 up and 9 down) transcripts encoding nitrate/nitrite transporter (NRT), nitrate reductase (NAD(P)H), ferredoxin-nitrite reductase, glutamine synthetase, glutamate synthase (ferredoxin) and glutamate dehydrogenase (NAD(P)+) were differentially expressed at 1 and 3 d, respectively ( Supplementary Table S5 ). In addition, Cd destroys chloroplast structures, such as the photosynthetic membrane ( Table 3 ; Supplementary Table S5 ), which is consistent with results reported in plants (Parmar et al., 2013). These findings indicated that Cd downregulated the expression of genes involved in the energy metabolism pathway to inhibit the growth of G. lemaneiformis.

Many studies have shown that transcription factors are involved in the response to Cd in plants. In G. lemaneiformis, most DEGs related to the “transcription factor” including MYB, TBP-binding protein (TBP) and SNF7 were significantly upregulated under Cd stress, suggesting that TFs participate in response to Cd ( Figure 5A ; Supplementary Table S6 ). Among them, 3 transcripts encoding MYB transcription factors were significantly upregulated by Cd at both 1 and 3 d. The transcription factor MYBs is one of the most widely distributed families in plants and participate in responses to stresses by binding to the cis-elements of MYB in the promoter of target genes (Wang et al., 2021). Overexpression of AtMYB4 and SbMYB15 resulted in increased tolerance to Cd (Sapara et al., 2019; Agarwal et al., 2020). In addition, “G-Protein” was also significantly upregulated under Cd stress ( Supplementary Table S7 ). G proteins function as molecular switches to participate in the transmission of various extracellular stimulus signals and responses to abiotic stresses in plants (Zhang et al., 2021). Studies have shown that overexpression of AGG3 can increase tolerance to Cd (Alvarez et al., 2015).

HSPs act as molecular chaperones for a variety of target proteins in response to heavy metal stress and play a crucial role in maintenance of protein homeostasis (Fragkostefanakis et al., 2015). In our results, expression of transcripts encoding HSPs including 6 sHsp, 4 Hsp 40, 1 Hsp 90-5 and 1 Hsp 101 was upregulated by Cd ( Figure 5B ). Particularly, sHSP was greatly induced by Cd, and the multiple was generally between 3-6 times. SHSPs, the first line of stress defense (Haslbeck and Vierling, 2015), can bind denatured proteins, stabilize them, and prevent an aggregating of denatured proteins without depending on ATP, then the denatured proteins are refolded by either the HSP70 system or the HSP40/HSP70 system ( Figure 5B ). The highly conserved C-terminal region of sHsp is the α-crystallin domain (ACD domain) (Haslbeck and Vierling, 2015), which present in all these DEGs of sHsp ( Supplementary Table S8 ). In Closterium ehrenbergii, expression of sHSP10 and sHSP17.1 was significantly upregulated under heavy metal stress (Abassi et al., 2019). Expression of small heat shock protein OsMSR3 in Arabidopsis enhanced tolerance to Cd stress (Cui et al., 2013). HSP40s together with HSP70s constitute a versatile chaperone machinery for stress responses (Qiu et al., 2006). However, HSP70s were not induced by Cd in G. lemaneiformis. Hsp40 proteins contain the J domain through which they bind to Hsp70s (Qiu et al., 2006), and the J domain exist in the DEGs of GlHsp40s ( Supplementary Table S8 ). In Venerupis philippinarum, the expression of VpHSP40 induced by Cd (Li and Dewey, 2011) and the Hsp40-like protein provided tolerance to Cd toxicity (Thakur et al., 2021). Together, these results indicated that HSPs might improve the tolerance to Cd in G. lemaneiformis.