Demand for the application of nano-materials in the field of biology, medicine and food sciences has been recently increased (Azizi et al. 2020; Salama et al. 2021; Panahirad et al. 2021). Many reports revealed that nano-materials could directly influence the biological systems as promoter. In fact, nano-particles could somewhat act as carriers for conjugated substances by which target delivery could be possible. In this way, probability of degradation of conjugated substance would be considerably decreased. The main challenge in producing substance-anchored nanoparticle, would be adopting the appropriate synthetizing procedure at which the considered substance is mounted onto the special nanoparticle. Many studies have demonstrated the amplified effects of substance anchored nanoparticles compared to individual treatment of substance or nanoparticle.
As mentioned earlier, features of the MWCNT-Put-Fl were characterized by various methodologies including FT-IR, EDX, SEM and TEM analysis. Obviously, EDX analysis indicated the characteristic peak only for carbon, oxygen and nitrogen approving the modification of MWCNTs surface by putrescine and fluorescein. It means high purity of the synthetized nanotube and removal of the unreacted elements from the nanotube during the synthesis. Our results are in conformity with the opinions of the researchers which had been used EDX analysis for elucidation of nanoparticle purity (Ramachandran et al. 2013; Gohari et al. 2020a). Likewise, XRD is known as valuable tool for demonstrating the microstructure of synthetized nanotubes (Das et al. 2014). In the case of the MWCNT-Put-Fl, XRD pattern indicated that the microstructure of MWCNT remain unchanged during oxidation and functionalizing with putrescine and fluorescein. Also, the robust diffraction peaks indicates that the MWCNT-Put-Fl possess proper crystallinity which is in agreement with the results reported by other researchers (Ramachandran et al. 2013; Subbenaik 2016; Yang et al. 2018; Azizi et al. 2020). Also, line widening of the XRD peaks indicates that the size of the MWCNT-Put-Fl reduces in the domain of nano range (Ramachandran et al. 2013). So, we presumed that MWCNT retains own functionality while acts as carrier for putrescine. Alternatively, FT-IR spectra have been successfully utilized determining the presence of conjugated groups along with chemical modification of MWCNTs (Gohari et al. 2020b, a). In the FT-IR spectra of the MWCNT-Put, peak of C = O at 1698 cm− 1 depicts bonding of putrescine to MWCNT via amide formation. In this way, another broad peak could be seen at 3170–3551 cm− 1, which is due to O―H stretching vibration of fluorescein. (Fig. 2B). The results confirmed the successful linkage of putrescine and fluorescein on the surface of MWCNTs. Current study is in accordance with the works indicating the potential of FT-IR in detection of chemical groups onto the nanoparticles (Jelvehgari et al. 2014; Azizi et al. 2020).
Supplemented to chemical analysis, SEM images show the irregular aggregation and agglomeration of MWCNTs (Fig. 3A). Furthermore, TEM images demonstrate an interwoven network of carbon nanotubes (Fig. 3B). Both SEM and TEM images depicted that the structure of MWCNTs was remained unchanged during acidic oxidation and modification.
In this study we described an ordinary methodology for production of putrescine anchored MWCNT for the first time. The results obtained from various tests, including electron microscopy and chemical analysis confirmed the successful linkage of putrescine on the surface of MWCNTs.
Bio-imaging and localization of the MWCNT-Put-Fl
Divers methods are used for bio-imaging and localization of nanoparticles in tissue or cells (Hauser et al. 2017). Epi-fluorescence microscopy (EFLM) is a good choice for this purpose, because it known as low cost and easy to use in respect to other procedures (Peighambardoust et al. 2010). The technique has been successfully utilized for detecting nanoparticles in plants. The approach for indirectly detection of non-fluorescence nanoparticles created based on the fact that they could be visible if their background, i.e., tissue or cell, stained with suitable fluorchrome (Ghafariyan et al. 2013). Our results indicated that labeling the nanotube with fluorescein as a fluorescence dye, provides opportunity for direct detection of nanotube using easy-to-use conventional fluorescence microscopy.
Effects of MWCNT-Put on the growth and development of plantlets
Leaf formation and plastochron are good indicators for assessment of apple growth and development (Foster et al. 2003; Dadpour et al. 2011). The rate of leaf initiation is in close relation with the organogenesis of shoot apical meristem (SAM) and its activity. Leaf primordia are formed at the peripheral zone of SAM subsequent to generous cell division. Therefore, leaf formation is known as dependent process to cell division from organogenesis point of view (Fleming 2005). Also, plastochron which defines the leaf formation as the function of time is directly tied with the rate of cell division (Dadpour et al. 2011). Many reports indicated that cell growth and division is promoted by nanotubes (Khodakovskaya et al. 2012; Khorrami et al. 2020; Samadi et al. 2020). Alternatively, polyamines could enhance cell division (Maki et al. 1991), act as effective regulator for shoot morphogenesis (Walden et al. 1997; Yadav and Rajam 1997; Zhu and Chen 2005) and increase seedling height (Farooq et al. 2009). As well, polyamines has decisive role in regard to cell structure by effect on actin formation and microtubules assembling (Oriol-Audit 1978; McCormack et al. 1994; Savarin et al. 2010; Sánchez-Elordi et al. 2019). In the current study, enhanced growth and development of the in-vitro plantlets could be associated with stimulatory effects of both components of the MWCNT-Put, i.e., MWCNT and putrescine, which is confirmed by the above mentioned literatures. Furthermore, well organized microtubules in treated cells by MWCNT-Put which were appeared in microscopic studies (Fig. 5A), could elucidate the effectiveness of putrescine in this regard. Ability of the putrescine in adjusting the water contents of a cell could attributes to plant growth and performance (Farooq et al. 2009). Similarly, evidence indicated MWCNTs can trigger the expression of PIP1 and PIP2 (water carrier proteins) which enhance plant growth and development through improving the water status of tissue (Khodakovskaya et al. 2012). In the current study, inimitable growth of the plantlets is in agreement with the mentioned reports and may be related to accumulative effects of MWCNT and putrescine which have been assembled in the individual particle.
Effects of MWCNT-Put on the biochemical characteristics
Indisputably, photosynthesis is the most important process which influenced by nanoparticles. Some studies has been depicted that the levels of photosynthetic pigments could increase in response to application of nanotubes (Sharma et al. 2019; Gohari et al. 2020b). Also, the substances could enhance photosynthetic activity, carbon assimilation and biomass (Khodakovskaya et al. 2012; Ghafariyan et al. 2013; Banerjee and Kole 2016). Likewise, polyamines including putrescine, are now being deliberated as a specific PGR which could modulate photosynthesis (Farooq et al. 2009; Yuan et al. 2015; Abd Elbar et al. 2019). Enrichment of photosynthetic pigments in the present study may correlates with the nature of the MWCNT-Put. The outstanding role of putrescine in plants would be its implication on chloroplast bioenergetics by means of motivating ATP synthesis. Also, putrescine can increase light energy utilization through stimulation of photophosphorylation (Ioannidis et al. 2006; Ioannidis and Kotzabasis 2007). So, putrescine could enhance the growth of plants by providing more ATP as the common source of energy. In this study, intensive growth which plantlets were experienced after treatment by MWCNT-Put would be explained from bioenergetics point of view. In other respects, increased level of the GPX in our study, as the enzymatic system which plays array of physiological roles in protection of photosynthetic apparatus against oxidative damages (Herbette et al. 2002; Dayer et al. 2008), could explicates the ascending trends of Chl a and b in the case of treated plantlets with MWCNT-Put.
Form and distribution of Stomata
Definitely, stomata are important tissues due to their controlling role in simultaneous exchange of carbon dioxide and water with the atmosphere (Webb and Baker 2002). Consequently, formation and spacing pattern of the stomata are considered as determining factors in relation to carbon assimilation, transpiration and plant performance. In overall, stomata are distributed on a leaf based on the one-cell-spacing rule. According to this hypothesis, separating two adjacent stomata from each other by at least one intervening pavement cell, is critical for achieving an optimal balance between carbon assimilation and water exhaust (Larkin et al. 1997; Bergmann and Sack 2007). Moreover, obligatory dependency of guard cell functioning, gas exchange, and ion transport with stomatal spacing have been proven (Torii 2007; Papanatsiou et al. 2016).
Therefore, changes in stomatal spacing pattern have significant impact in terms of physiological approaches (Casson and Gray 2008). In the present study, treatment of in vitro apple plantlets with the MWCNT-Put significantly increased the density of the stomata in comparison to the control (Fig. 10G). Our results are in contract with some other studies indicating positive effects of CNTs on the stomatal density (Joshi et al. 2018a, b; Khorrami et al. 2020). Studying the effects of CNTs on tobacco callus culture revealed that expressions of genes responsible for encoding cell division (CycB) and tobacco aquaporin (NtPIP1) are enhanced in treated cells compared to the controls (Khodakovskaya et al. 2012). From the morphogenetic point of view, stomatal distribution and frequency are the main factors which could directly affect carbon assimilation and subsequently plant productivity (Shpak et al. 2005; Casson and Gray 2008; Torii 2015). Generally, formation of stomata is known as a complicated process at which numerous physiological and molecular factors involve (Shpak et al. 2005; Martin and Glover 2007; Bergmann and Sack 2007; Casson and Gray 2008; Serna 2009; Kim et al. 2012; Rudall et al. 2013; Franks and Casson 2014; Torii 2015; Castorina et al. 2016; Muir 2018). However, stomata is formed through a series of asymmetrical and symmetrical cell divisions being known as cell cycle dependent process (Croxdale 2000; Nadeau and Sack 2002; Torii 2007). It has been elucidated that the E2Fa–DPa transcription factor together with the D cyclins and Cyclin Dependant Kinases (CDKs) are crucial components regulating cell division prior to formation of stomata (De Veylder et al. 2002; Bergmann 2004). However, E2Fa is the main known factor for asymmetrical cell division in terms of stomatal spacing (Bergmann and Sack 2007; Kajala et al. 2014). Moreover, membrane-bound receptor-like kinases (RLKs) in pairing with two members of the epidermal patterning factor like (EPFL) protein family, namely, EPF1 and EPF2, have been recently stated to be essential for regulation of stomatal patterning. Noticeably, the EPF1-ERL pair appears to enforce the one-celled spacing rule responsible for stomatal density (Lau and Bergmann 2012). On the other hand, the main molecular mechanism involving in the formation of stomata, MUTE protein, seems to activate another member of D Cyclins (CycD5;1) proteins which causes the guard mother cells (GMC) to divide. Taking all into account, polyamines have been determined as the effective factor for enhancing cell division which is critical for stomata formation (Kono et al. 2007; Yamashita et al. 2013).
The present study indicated that application of MWCNT-Put increases the frequency of stomata, meaning low spacing between stomata and high ratio of guard per pavement cells. Accordingly, it seems that MWCNT-Put is effective on asymmetrical division of mesophylls with spacing division pattern. Research on the tomato plants treated with saline water, indicated some significant morphological and physiological changes (Romero-Aranda et al. 2001). They reported that reduction in stomatal density leads to attenuating stomatal conductance and decreasing net CO2 assimilation which indirectly confirms our results. Salt stress could decrease CDK activity and could profoundly suppress the expression levels of the cyclins genes including A and B Cyclin which leads to extensive inhibition in both S and G2/M phase (Qi and Zhang 2020). As it was mentioned above, salt stress can reduce stomatal density with suppressing cell cycle process. Another experiment conducted on the barley under salt stress, revealed that the stomata index is reduced by application of PAs (Çavuşoğlu et al. 2007). Our data, however, contradicts their results indicating negative effect of PAs in regard to stomata formation. In their work stomatal density was determined in terms of the stomata index, i.e., the ratio of stomata per total mesophyll cells. Also, in treated plants, total number of mesophyll cells was increased while their size diminished. Therefore, higher total mesophyll cells may lead to lower stomata index. The discrepancy between our data and the data of Çavuşoğlu et al. (2007) may be due to different methodologies which were exploited for calculating stomatal density. In our study, stomatal density was calculated as the observed stomata per distinct field of view, while they described the ratio of the number of stomata per surrounding cells.
The subsidiary effect of MWCNT-Put which was detected in our study is the formation of stomata over the main leaf vein in the case of nanoparticle application (Fig. 11). In general, stomata rarely develops over major veins with an exception, i.e., Selaginellaceae family (Brown and Lemmon 1985; Martin and Glover 2007). It seems the lack of stomata over leaf major veins mainly helps to prevent excess transpiration. As the control in-vitro plants conformed to the mentioned rule and stomata only appeared over the vein in regard to treated plants, it seems the MWCNT-Put is responsible for such change in the development of the studied leaves. We reported this phenomenon based on several microscopic observations and no statistical analysis was accomplished for this purpose. So, further studies are required for clarification of this morphological event.
The change in shape and form is largely a matter of importance to biologists (Cooke and Terhune 2015; Klingenberg 2016). Mostly, differentiation in the cellular levels could be demonstrated by anisotropy in the form of cell (Coen et al. 2004). The polyamines, especially putrescine, as well as nanotubes are well known for their implication in the developmental process. Geometric morphometric analysis in this study elucidated substantial anisotropic transformation of the stomatal form in the case of MWCNT-Put treatments in particular at the vertices area. So, it seems the synthetized nanotube might be involved in differentiation of the stomata.