4.1 The germination and morphological alteration of V. bonariensis under differential Cd stress
During germinal and individual development, seeds was sensitive to environmental stress [30]. Therefore, the study on this stage reflected the tolerance to these stress in plants. Previous studies have demonstrated that 10 mg/L Cd concentration severely affected the germination of Medicago Sativa [31]. Coreopsis drummondii and Impatiens walleriana Hook. f. seeds, compared with the controlling group, the germination rate of experimental group was reduced by about 50% [32]. Our results showed that the threshold Cd concentration on V. bonariensis germination was about 50 mg/L. Cd solutions within 20 mg/L concentration promoted the germination and growth on seedlings.
The growth and morphology alteration served as the basic adaptation mechanisms. The roots were suffered primarily from HMs in soil sites. Botanical growth was hindered, pigmentation, lateral root numbers, root activity were lessened. The absorption of water and nutrient utilization were disturbed [33]. With the HM ions shifted to shoot, the symptoms of toxicity altered: plant dwarfism, leaf chlorosis, reduced biomass, inhibited photosynthesis occurred, eventually death happened [34]. Under Cd stress, these changes were present in V. bonariensis (Fig.3). Under Cd stress the roots elongation was severer inhibited than in the aboveground part of V. bonariensis, which was consistent with studies of Pinus sylvestris L and hyperaccumulator S. nigrum [35, 36]. Petiole was the transportation channel of water and nutrient from leaf to stem [37]. By speeding up the transportation of water and nutrients, the shorten petiole of V. bonariensis elevated the resistance to Cd stress. For leaf chlorosis, there existed two possible reasons: one was that the certain amount of Cd in the leaves rendered chlorophyll destruction and leaves chlorosis; the other was that due to the serious affliction to the root system and the malfunction of water transportation system, water shortage occurred in leaves. The above speculation was supported by the decrease of chlorophyll content, petiole length, leaf area, root length and number in V. bonariensis (Fig.3c).
4.2 Cd accumulation and transportation in V. bonariensis
Typically, most positively charged HM ions tended to bind negative-charged compounds in tissues. Consequently, these ions accumulated in roots [38]. In our results, Cd accumulation in roots was significantly higher than that in aboveground parts, for the retention on Cd2+ in root system. Through Cd enrichment in root, Cd2+ were prevented from interrupting photosynthesis and metabolism in plants. Consequently, botanical survival under stress could be possible. The biomass of V. bonariensis were significantly reduced in 100 mg/kg Cd solution. This very consistency was significantly higher than the critical concentration of S. nigrum, Cd stress over 25 mg/kg inhibited the growth of S.nigrum and decreased its biomass (Additional file 2: Figure S1) [39]. BCF indicated the transportation difficulty of HM elements in soil plant system [39]. The transportation and accumulation level of HMs from plant roots to the upper part of the plant were assessed by the BTF. For a hyperaccumulator, the BCF and BTF should be greater than 1 (Fig.2a, b). The results proved that V. bonariensis showed no sign of hyperaccumulator. The absorption amount of Cd was 31.66 ug/pot in V. bonariensis (Fig.2c). By contrast, Cd hyperaccumulator Bidens pilosa L. was only 17.92 ug/pot [39].
Based on the research results, V. bonariensis did not meet the standard of Cd hyperaccumulator. However, it had strong tolerance and absorption ability to Cd. A large amount of Cd was accumulated in roots of V. bonariensis under Cd stress. Consequently, the reduced amount of Cd in leaves and other sensitive organs cast lighter toxic effects on plants. This was consistent with the results of the study that Lonicera Japanica Thunb [40] and Helianthus annuus [41]. In brief, with rapid growth capability, large biomass, strong Cd tolerance and absorption ability, V. bonariensis possessed potential application value in the remediation of Cd pollution.
4.3 Effects of Cd stress on cell wall and cell membrane of V. bonariensis
The cell wall weighed significantly in botanical HM defense and detoxification [42]. As the first HMs barrier, it was firstly affected by Cd2+. The cell wall and carbohydrates protected Cd from entering roots by bounding it to the pectin site, which prevents HM ions from entering the protoplasm of the cell and protecting it from harm [43]. When exposed to HMs, the cell wall could activate hundreds of specific stress-responsive signaling proteins to protect the cell from crashing into the protoplast on susceptible sites. The lignin had a strong adsorption capacity for HM ions because it means a lot of radical groups, such as oxhydryl, methoxy and carbonyl group. The particle size of lignin was small, which was beneficial to the exposure of more radical groups and more HM ions could be adsorbed [44]. In our results, there were 7 GO entries with cell wall tissue correlation, which suggested that V. bonariensis might increase its tolerance to HMs by combining the root cell wall with Cd2+. The lignin relating to phenylpropanoid pathway could reinforce specialized cell walls [45]. All 18 DEGs associated with lignin synthesis was up-regulated. The content of lignin under Cd stress was significantly higher than that of the control. This indicated that the cell wall of V. bonariensis might be reinforced and substantial Cd2+ in soil be absorbed under Cd stress.
The cell membrane served as the second barrier against trespassing of HMs. Cd was an important mutagen of plasma membrane peroxidation. MDA was induced by more ROS produced under Cd stress, causing membrane lipid peroxidation as well as destroying membrane ion channel structure [46]. The membrane lipid peroxidation in the cell of V. bonariensis was demonstrated by the significant elevation of MDA in leaves and roots under Cd stress.
4.4 Effects of Cd stress on ROS scavenging system in V. bonariensis
Previous studies have shown that HM may injure plants by two biological pathways [47, 48]. On one side, the HM stress oxidation inhibited the activity of protective enzyme. The main biological macromolecules such as proteins and nucleic acids were destroyed by the induced free radicals. On the other side, when absorbed into the plant, HM ion not only combined with nucleic acids, proteins, enzymes and other substances, but also supplant some specific elements exercising the function of enzymes and proteins, which make the related enzymes and proteins denature or reduce their activities. Under Cd stress, the ROS scavenging system played a vital role in plants. As the primary defense enzyme purging ROS in cell, SOD converted O2- disproportionation into H2O2 and eliminated -OH by catalyzing the Fenton reaction [49]. Cd stress is thought to elevate SOD activity in plants, but this promotion to SOD activity vary with HM treatment concentration and duration, plant species, and plant size [50]. In our study, the SOD activity in leaves and roots decreased under Cd treatment, it was speculated that excessive Cd2+ or stress time could inhibit the activity of SOD. Under Cd stress, the activities of POD, CAT and APX elevated in leaves of V. bonariensis. However, the results were opposite in roots. For its contact with soil, the roots were primarily susceptible to HM. Consequently, the stress level in roots was higher than that in leaves. When antioxidant enzyme activities in the root were hampered, the very activities in leaves continued coping with Cd stress. In the up-regulated GO enrichment categories relating to oxidative reactions, the enrichment degree of ‘oxidation-reduction process’, ‘oxidoreductase activity’ and ‘catalytic activity’ were high. The result showed that the oxidative reactions might be activated in response to Cd stress. By gearing the antioxidant system up, V. bonariens refrained from HM damage.
4.5 Effects of Cd stress on chelating reaction in V. bonariensis
Upon exposure to HMs, plants synthesized diverse metabolites, particularly specific amino acids, such as PRO and histidine, peptides (glutathione and phytochelatins (PC) etc), and organic acids [51]. These matters mentioned above interacted with Cd2+ to form chelates, such compounds reduced the concentration of Cd2+ in soil. Furthermore, direct contact between Cd2+ and organelles were eliminated. Thereby the toxicity of Cd was reduced in soil.
Amino acid, as one of the plant’s fundamental metabolites, counted great deal in the alleviation of HM stress. It served as integral part of the involved coenzyme and ligand in the metal complexation [52]. Cd stress resulted in a significant increase in the content of some amino acids, which might be a plant specific genetic trait. PRO regulated plant osmotic/redox reactions and participated in the metal complexation. In our study, Cd stress increased the accumulation of PRO in aboveground part by 29.76%, whereas in roots the percentage was 4.68%. Similarly, the amount of PRO in leaves was higher than that in roots of Bacopamonnieri under Cd stress [53].
For the great affinity to HMs, PCs chelate various HMs to deactivation [54]. When the Cd2+ entered the cytoplasm through the cell wall and cell membrane, it combined with PC to form LMW complex, which was transferred into vacuole under the action of htm1 membrane transportation protein. Then HMW complexes were synthesized by LMW and Cd, eventually immobilized in vacuole. The HMW complexes were less toxic to plants. PCs was a sulfhydryl polypeptide composed of cysteine, glutamic acid and glycine. As the precursor of PC synthesis, GSH composed some sulfur-containing compounds in root cells and Cd2+ to form stable chelates [55]. In our study, 76% of the DEGs involved in the ‘glutathione metabolism’ pathway was up-regulated. GSH content increased (Fig. 6). In this result, the promotion of PC content was predictable.
The organic acids of plants, such as oxalic acid, malic acid and citric acid, could be transformed the toxic Cd into low toxic or non-toxic form by chelating, promoting the tolerance of plants [56]. The pathways of organic acids in V. bonariensis were significant up-regulated in our results. It was estimated that the efficiency of organic acid synthesis was elevated. This promoted the binding of Cd2+ to organic acids in the cytoplasm or vacuoles, and alleviated the damage of HMs to V. bonariensis. The organic acids secretion capacity in Cd-tolerant plants such as Rorippaglobosa was far greater than that in non-tolerant plants Rorippa [57]. The improvement of organic acid raised the soil acidity of the rhizosphere as well as reducing the Cd uptake by plants. Exposed to low concentration of Cd, Bechmerianivea could secrete organic acids in its rhizosphere. with Cd chelating, the consistency of Cd2+ around the rhizosphere of Bechmerianivea decreased [58]. In transcriptome data of V. bonariensis under Cd stress, there were three pathways relating to organic acid metabolism among top-ten up-regulated pathway, including ‘Citrate cycle’, ‘Glyoxylate and dicarboxylate metabolism’ and ‘alpha-Linolenic acid metabolism’. The result proved the significant function of organic acid metabolism in V. bonariensis under Cd stress.
4.6 Effects of Cd stress on Secondary metabolites of V. bonariensis
Although minor to plant growth and development, secondary metabolites were often involved in environmental stress [59]. The phenolic metabolism was an important process in plants’ secondary metabolism. Under Abiotic stresses, a large number of phenolic compounds was induced to form mechanical barriers in order to prevent osmotic stress, or to remove excessive amounts of ROS in cells [60]. Most of the phenolic compounds were composed of flavonoids, simple phenols and quinones. The flavonoids, as an important botanical antioxidant, played a key role in resistance to stress [61]. The synthesis efficiency of flavonoids could be improved by the activation of peroxidase under Cd stress [62]. CHS and ANS relating to flavonoid biosynthesis belonged to the family of oxidoreductases. CHS was the first enzyme to spur phenylpropane metabolic pathway to conduct flavonoids synthesis. It served as a natural defense enzyme as well as a synthetic intermediate in plants [63]. Anthocyanins was a strong antioxidant, it can alleviate the toxicity of oxygen free radicals in plant cells. In our results, only one gene down-regulated in 5 CHS and 9 ANS genes, respectively. The activity of CHS and ANS were significantly elevated. The results showed that CHS and ANS genes may play an important role in V. bonariensis in response to Cd stress.
The phenylpropanoid biosynthesis has been demonstrated to contribute to various aspects of plant biotic and abiotic responses [64]. The improvements of phenolic compound content under abiotic stress, particularly with respect to phenylpropanoid, have been extensively described [65]. In Lupinus luteus L., the phenylpropanoid pathway metabolites elevated Pb tolerance in its roots [66]. Occupied the third place in up-regulated pathway, the ‘Phenylpropanoid biosynthesis’ was essential under Cd stress in V. bonariensis (Table 4).
4.7 Effects of Cd stress on transpiration and photosynthesis in V. bonariensis
Under Cd stress, the Tr of V. bonariensis decreased. The entrance of Cd2+ in guard cells through Ca2+ ion channel might induce stomatal closure through ABA pathway and inhibit transpiration in plants. These elements disturbed stomatal opening. In addition, Cd stress reduced the length and number of roots, limiting water intake (Fig.3a). Therefore, the leaf area of V. bonariensis decreased to maintain water in cell. Similarly, Tr and leaf area of Brassica juncea were hampered under Cd stress [67].
Cd2+ damaged nucleoli in the cell of root tip, precluding the synthesis of RNA and the activities of RNAase, ribonuclease and proton pump. This process decreased nitrate reductase activity, reduced the uptake and transportation of nitrate from the root to the aboveground part. With the HM ions shifted to the upper part of plants, dwarfism and decreased biomass occurred. The upward transportation of nutrients was forestalled by the factors mentioned above. The lack of nutrients hindered photosynthesis and the growth of the plants. This action decreased photosynthetic rate, destroyed photosynthetic organs, damaged photosynthetic systems, disturbed carbon dioxide fixation, and even death [68].
In our experiment, Pn and Gs decreased whereas CO2 concentration (Ci) increased (Fig.6). Stomatal and non-stomatal components were closely related to the Pn decrease [69]. Besides, as a non-stomatal limitation, chlorophyll decomposition accounted for the decline of Pn. The results illustrated that under Cd stress photosynthesis in V. bonariensis leaves were abated. As a result of Gs decline, CO2 supply decreased. The non-stomatal factors that hindered the utilization of CO2, resulted in the accumulation of intercellular CO2. Non-stomatal factors took a great to injure the chloroplast of V. bonariensis under stress and decrease the photosynthetic cell activity.
In ‘Photosynthesis-antenna proteins’ pathway, only one gene encoding LHC was up-regulated. As a peripheral antenna system, antenna proteins in LHC elevated the efficiency of absorption of light energy [70]. Most of the DEGs associated with the ‘Photosynthesis’ were down regulated, indicating that Cd stress arose disorders in photosynthetic responses. Cd stress prevented light harvesting, electron transportation and carbon assimilation efficiency during photosynthesis in V. bonariensis. This was consistent with previous studies on the response of Maize to Pb [71]. These physiological and molecular changes suggested that down-regulation of the photosynthetic pathway might be a responsive step in V. bonariensis under Cd stress.