Even when the comparisons of cladodes’ conditions and positions for 4-HBA and CGA were statistically not significant, there were some tendencies in the differences between cladode state and position that could be observed. For example, the results suggest that lighted apical cladodes produced 4-HBA and sent it to basal cladodes when they could not produce it by themselves (i.e. when shaded). That is because the incidence of this secondary metabolite was similar in lighted apical cladodes and in shaded basal cladodes, and similar in shaded apical and in lighted basal, but lower than in the former situation.
The relationship between the concentrations of secondary metabolites and cladodes’ states that were not obvious in the analysis of separate effect of a given secondary metabolites, were analyzed by us in a regression analysis. The comparison of the relationship between the concentrations of SA and 4-HBA showed that light had an effect on the concentrations of both secondary metabolites and changed the slope of this relationship between shaded and lighted states. Similarly, light changed the relationship for the concentrations of CGA and 4-HBA in apical cladodes. For example, the SMA model predicts that for high concentrations of 4-HBA, the concentrations of both SA and CGA will be higher in shaded than in lighted cladodes. For lower concentrations of 4-HBA these relationships were inverse. Analogously, for high concentrations of SA and for most but the lowest concentrations of CGA, the concentrations of 4-HBA were higher in lighted (vs. SA) or lighted apical (vs. CGA) cladodes. For the relationship CGA vs. 4-HBA most of the concentrations of the latter secondary metabolite were small, but some less common higher concentrations allowed extending the prediction function for such concentrations. Low concentrations of 4-HBA for the relation SA vs. 4-HBA and moderately low concentrations of this secondary metabolite for the relation CGA vs. 4-HBA were associated with higher concentrations of either SA or CGA in lighted than in shaded cladodes, and vice versa for higher concentrations of 4-HBA. Low or moderately low concentrations of 4-HBA were referred to the interval of all the data for this secondary metabolite.
Higher slope of the relationship CGA vs. 4-HBA for lighted than for shaded apical cladodes means that for most concentrations of 4-HBA, lighted cladodes produced higher concentrations of CGA than shaded cladodes. This result, together with a negative non-significant relationship between these two metabolites in basal cladodes and with the observed decrease of CGA concentration in basal cladodes is parallel with the tendency obtained in the comparison of the concentrations of CGA for different cladodes states that suggested an autonomous production of this secondary metabolite by apical cladodes, without a flow from neither lighted nor shaded basal cladodes. The lack of significant differences between cladode states in the comparison of CGA concentration, for example between lighted apical vs. shaded apical cladodes, was probably an effect of the fact that for lower or higher 4-HBA concentration, the relationship between the concentrations of both metabolites was inverse and thus, during the comparisons of the CGA concentrations between different cladodes states, these concentrations were averaged for high and low 4-HGA concentrations.
An analogous phenomenon existed for the relationship between SA and 4-HBA, confirming the observed tendency for higher concentration of SA in lighted apical than in lighted basal, and in lighted apical than in shaded apical cladodes were mainly the outcome of the autonomous production of this secondary metabolite by apical cladodes.
Considering that 1) cladodes are growing more slowly at least during the event of shading and thus, their tissue is more difficult to recover from damage (they are more valuable from both ODH and RAH point of view, and thus, should be better defended); 2) lighted cladodes are more valuable in terms of higher photosynthetic activity (they are more valuable from the ODH point of view and should be better defended) but at the same time are growing faster and thus, their tissue is easier to recover from damage (they are less valuable, from the RAH point of view and should be less defended); and provided that 3) the relationships between pairs of secondary metabolites have different slopes for different cladodes’ states, this is difficult to find a congruence between the concepts 1) and 2) and the predictions of either RAH or ODH.
The results of the experiment concerning QUE were concordant with the general tendency of the results of the field study, since basal cladodes from the experiment and cladodes from the field study bearing more daughter cladodes above, that is, the older ones contained this metabolite with higher probability. Both results show that rather the basal position of a cladode determined a higher probability of presence of QUE in its vegetative tissue than the access to light. However, higher and statistically significant probability of detection of QUE in shaded basal cladodes when apical cladodes were lighted, compared to lighted basal cladodes when apical cladodes were shaded and to lighted basal cladodes when apical cladodes were also lighted, suggesting that shaded cladodes received QUE or its precursor from apical cladodes. Since the lighted condition of basal cladodes did not increase the proportion of either shaded or lighted apical cladodes bearing QUE, it probably meant that basal cladodes did not transfer QUE or its precursors to apical cladodes. As the proportion of basal cladodes with QUE was lower under light than in the shade, it meant that 1) QUE was transferred from basal (penultimate order) to the cladodes parental to them, or, 2) QUE was unstable in the light and was decomposed. Similar pH in shaded and lighted cladodes suggests that differences in the probability of detection of QUE were not an effect of higher concentration of the malic acid, precursor of most secondary compounds (Bieto and Talón 2008). This result reinforces the conclusion about the production of QUE in apical cladodes and its transfer to the basal cladodes. Higher incidence of this metabolite in older cladodes obtained in field study, and the existence of significant differences between some combinations of treatment and position were a result of QUE transfer from higher to lower order cladodes that decreased the variability between cladodes and thus, smaller slope of the relationship QUE incidence vs. cladode age. This metabolite was probably not produced to promote cell growth, since higher growth intensity occurs in apical, and thus, younger cladodes: in such case the flow of the secondary metabolite should occur in the opposite direction. Neither was it produced to protect the plant from UV radiation or to rest the effect of free radicals: in both cases, QUE should be moved to both apical and lighted cladodes. These conclusions are parallel with the conclusions of our previous field study in which we did not find in hermaphrodite individuals significant relationships between the incidence of QUE and of meteorological variables that affect both cell growth (air temperature and humidity) and the response against abiotic factors like UV (Janczur and González Camarena 2018). Independently of the proximate causes, the results of both field study and field experiment do not support the predictions of the ODH, since we could not show the existence of significantly higher QUE incidence in younger cladodes even when a mechanism of transport of this secondary metabolite between cladodes existed. They rather support RAH, since slower-growing plant parts in our study had high levels of QUE. This was an analogous result to that predicted by RAH for plants growing in less productive environments (that is, slower-growing). The results showed that the mechanism of the plant is to maintain either similar incidence of this metabolite in cladodes from all levels or to maintain its higher incidence in older cladodes, rather than to transfer them to the youngest cladodes. This is why we proposed in our previous study the use of a relative value of plants’ organs (in this case cladodes) in terms of fitness rather than their age (Janczur et al. 2020b). The hierarchical and frequently unilinear architecture of the individuals of O. robusta in the population studied here makes the plants sensitive to damage. A loss of lower-order cladodes (closer to soil) brings about a loss of all higher-order cladodes, where reproductive biomass is produced. So, the defense of older cladodes would be justified from an evolutionary standpoint. However, we are aware that in plants with more complex architecture, it is difficult to decide which vegetative organ contributes more in terms of fitness: probably, plant parts that bear reproductive organs. In Opuntia robusta cladodes of any age can potentially produce flowers, however, reproduction is much more probable on apical cladodes.
No study exists concerning the movement of flavonoids between cladodes of any species of Opuntia genus. There is a controversy concerning the possibility of transfer of phenolic compounds from the site of their manufacture to other tissues (Buer et al. 2007; Waller and Nowacki 1978), however it is known that the synthesis of the flavonoid biosynthetic enzyme is light dependent (Jenkins et al. 2001; Kubasek et al. 1998; Pelletier and Shirley 1996). Some researchers state that they are synthesized in the cells in which they accumulate and serve local functions (Peer et al. 2001); however, the results from the study of Buer et al. (2007) showed in Arabidopsis thaliana that flavonoids can move long distances from the light-grown shoot tissues to the shade-grown roots. The authors also suggested the active movement of flavonoids (among them QUE) because of their tissue-specific location, their cell-to-cell movement, and their unidirectional movement when applied midroot. If the movement is diffusion mediated, the flavonoid distribution is less localized and their movement is bidirectional. Our results showed that the movement of QUE was rather unidirectional and light dependent. The stability of QUE is low and its concentration is inversely correlated to temperature and to pH: its half-life decomposition may last less than one hour at high temperature and high pH, and as long as few hours at low temperature and lower pH (Liu and Zhao 2019; Wang and Zhao 2016). Therefore, it had to either disappear or decrease its concentration (incidence) in the shade, although this did not occur in our study. This reinforces our prediction that in our study, QUE was transferred from lighted apical cladodes to shaded basal cladodes.
In field study, higher order cladodes or the ones that hosted less orders of daughter cladodes contained CGA with higher probability than cladodes basal to them. On the other hand, in the experiment, light increased the difference in the concentration of this metabolite with respect to basal cladodes (both, shaded and lighted), but this effect was statistically not significant. This result showed that apical cladodes produced CGA autonomously, and did not receive it from the basal cladodes. Significant negative relationship between the cladode age (number of daughter cladodes above) and the incidence of CGA obtained in the field study is an effect of the lack of relocation of this secondary metabolite between cladode orders, autonomous production of this compound by higher-order cladodes showed in the experiment, and of low stability of this compound showed in other studies (Gil and Wianowska 2017). Higher CGA concentration in younger (higher-order) cladodes in field study was not an effect of a relocation of this metabolite from lower order to higher order cladodes; that is, plants did not have a “policy” to protect more photosynthetically valuable plant parts, but rather these parts had a ”policy” of not sending this metabolite to lower level cladodes. The results of the relationships between the concentrations of CGA and other secondary metabolites confirmed the conclusion from the comparisons between cladodes’ states. For example, at states that promote a lower photosynthesis rate (basal, shaded, or shaded basal cladodes), the concentration of CGA either decreased with the increase of, or was non-significantly related with, the other secondary metabolites.
The outcome of this result is concordant with the predictions of ODH although the rhetoric is different: plant cladodes did not defend more valuable cladodes, but rather, these cladodes defended themselves. It was also possible that the plant used CGA to arrest the negative effect of either UV or free radicals, because of a tendency of higher CGA concentration in lighted cladodes, but in such case also basal cladodes should be protected, since a negative effect of this stress factor on basal cladodes would affect apical cladodes, and thus, future reproductive biomass. Also, in field study, higher concentration of CGA in younger cladodes could be an effect of the participation of this metabolite in cell growth (Floh and Handro 2001; Kumar and Pandey 2013; Naoumkina and Dixon 2008; Saslowsky et al. 2005) and/or lignin synthesis (Boerjan et al. 2003; Gamborg 1967). As in our previous study (Janczur et al. 2020b) we did not find a relationship between the incidence of CGA or meteorological factors that might affect growth (temperature, humidity) and increase the negative effect of UV (radiation intensity), we suggest that in this population of O. robusta, this metabolite plays a defensive role. In any case, the response to non-biotic stress factors should be similar to the response to biotic factors, that is, “respond when you are attacked”. Our study showed that, contrary to the predictions of ODH, when CGA coexisted with SA, older tissues contained higher concentrations of this metabolite. We are not sure why it occurred, however this result was contradictory to both the results of field study and the non-significant tendency in the experiment, although as the coexistence of SA and CGA was non-frequent, these data did note affect the general pattern. Independently of a low dataset for the relationship between the concentrations of SA and CGA, we can conclude that SA was not an elicitor of the CGA. Larger dataset would show if the coexistence of these two secondary metabolites is associated to a lack of relationship between them or may otherwise suggest that they are competing for the same precursor. High percentage of model explanation for basal cladodes suggests that a small increase of the sample will bring about a significant relationship between the concentrations of these two substances.
The positive cladode-position-independent relationship between the concentrations of 4-HBA and SA in field experiment was probably an effect of the conversion of the former into the latter. If it had been a case of a promoting action of the SA, this metabolite would have been more common and 4-HBA an either less or equally common metabolite. We are not sure why we did not find SA in the field study and found it during experiment. If it had been an elicitor for the production of other substances in response to the stress factor (fabric bag covering cladodes) as mentioned in several studies (Beckers and Spoel 2006; Mendoza et al. 2018; Sudha and Ravishankar 2003), it would have had a positive relationship with other secondary metabolites. However, it had negative non-significant relationship with some secondary metabolites as determined in this study. The model of positive relationship between the concentrations of SA and 4-HBA explained a considerable proportion of variance although the number of data points was low. Additionally, three out of five regression lines were significant. The non-significant relationships also explained a considerable proportion of variance (36%, 39%, and 75%) suggesting that additional data had brought about a statistical significance.
It is not clear why 4-HBA was converted into SA in only few samples (if it was a case), since the pathway involving 4-HBA as a precursor of the latter compound is very common (Lee et al. 1995). Another pathway is also possible, i.e. phenylalanine ◊ trans-cinnamic acid ◊ benzoic acid ◊ SA, together with a parallel pathway phenylalanine ◊ trans-cinnamic acid ◊ 4-coumaric acid ◊ 4-HBA. Following this pathway, the synthesis of 4-HBA may compete for precursor (trans-cinnamic acid) with SA. As SA is known mainly for its signaling properties in the induction of pathogenesis-related proteins (Bennett and Wallsgrove 1994; Pierpoint 1994; Pieterse et al. 2012) and cases of signaling function of 4-HBA are scarcely known (Schnitzler et al. 1992; Tan et al. 2004; Ulmasov et al. 1994), it is possible to put forward the following hypotheses: 1) 4-HBA was produced in O. robusta for defensive purposes; SA was produced only in few cladodes either because the response against herbivores was not SA-signaling-dependent or there were no other stress factors requiring SA signaling; 2) The production of 4-HBA was higher in priority than that of SA synthesis, thus, sharing the same precursor, the plant lacked sufficient resources to produce both of them. If the conversion of 4-HBA to SA in the cladodes were a phenomenon, it would mean that the latter secondary metabolite was not produced in response to UV, since in either the lighted or the apical cladodes, the concentration of SA increased slower than in either the shaded or the basal cladodes. This effect should be confirmed in larger sample: because of a low incidence of this secondary metabolite, only a few cladodes had high concentration of 4-HBA.
In the present study 4-HBA and QUE showed dynamics in hermaphrodite cladodes during eight-months study period. Both metabolites showed a nearly monotonic dynamics: the incidence of the 4-HBA decreased from March through September with the exception of October, and the incidence of QUE increased from March through October, with the exceptions of April and September. This result is parallel with the result of the field experiment that showed a negative non-significant relationship between these two metabolites. This dynamic occurred probably because QUE was either relocated among cladodes from different ages or cladodes stopped producing them. This reallocation may have produced the following effect: if both mutually dependent cladodes contained a given metabolite, a complete relocation from an apical to a basal cladode would bring about a lower proportion of apical cladodes bearing that given secondary metabolite. It would produce the same effect as an incomplete relocation from apical to basal cladode, together with the lack of production of new secondary metabolite and/or, decomposition of the remainder metabolite. The transfer of QUE from apical to basal cladodes shown in field experiment was concordant with this result. Higher QUE incidence in older cladodes shown in field study points out that during the season the incidence of this metabolite increased because the proportion of older cladodes bearing it also increased. As the tendency of 4-HBA to move in the same direction was slight in the field experiment, this dynamics was probably produced by the higher mobility of QUE and the dynamics of the incidence of 4-HBA followed inversely this tendency because of the reasons already described (i.e. competition for the same precursor). Independently of the proximate causes, this result is not concordant with ODH because the proportion of cladodes bearing QUE was lower when tissues were younger and it increased throughout season when tissue was older. At the same time, it was concordant with RAH, because the prevalence of QUE was higher when tissues’ growth rate was lower because they were older. The contradiction was inverse for 4-HBA with respect to ODH and RAH. The probability of detection of CGA in cladodes of O. robusta did not follow the predictions of any of the hypotheses mentioned, because it did not follow monotonic seasonal dynamics. Its dynamics were mainly a result of an atypically high prevalence in August and low prevalence in September: its incidence in other months did not follow a clear tendency. This result suggests either 1) a lower mobility and/or, 2) a higher stability of this metabolite in the tissues of O. robusta. As the study of Gil and Wianowska (2017) evidenced a low stability of this metabolite, and our experiment showed that CGA is not transferred from apical to basal cladodes, the atypical CGA incidences occurred because of a higher insulation in August and lower insulation or decomposition of this compound in September.
Even when several studies showed the involvement of SA in resistance and tolerance to many abiotic factors (i.e. ozone, UV radiation, heat, cold (Dempsey and Klessig 2017; Horváth et al. 2007; Rivas-San Vicente and Plasencia 2011; Yuan and Lin 2008)) in our study the incidence of SA was similar at light than in the shade. It meant that the production of this secondary metabolite was not a response against short-term action of UV. However, this metabolite could probably take part in tissue growth, since the proportion of apical cladodes was statistically non-significantly higher in apical cladodes. It is difficult to conclude whether the lack of significant differences in SA incidence between cladode positions and experimental treatments was a result of the lack or of only a low-intensity transport of this secondary metabolite between cladodes from different levels, since this process has not been studied in Cactaceae. There is no unique transport mechanism in different plant species (Maruri-López et al. 2019). The process of transport of this metabolite throughout vegetative tissues is known only scarcely (Maruri-López et al. 2019). However, several studies showed that SA has a weak solubility in acids and in water, and crosses through cell plasma membranes by pH-dependent diffusion and carrier-mediated mechanisms (Bonnemain et al. 2013; Chen et al. 2001; Maruri-López et al. 2019; Takanaga et al. 1994). In our study, there was no significant difference in pH between either the apical and basal or covered and uncovered cladodes. This lack of differences in pH was probably responsible for the lack of significant differences in the incidence of this SA in apical/basal and shaded/lighted cladodes. In other words, there existed only a reduced transfer of SA between cladodes, because of the lack of difference in pH between cladodes. As SA is known for its elicitor action for other secondary metabolites, the absence of biotic stress was probably responsible for the small proportion of cladodes bearing it. As experimental treatments were a kind of stress, the presence of tulle bags exerted a stressful effect on cladodes, similar to that exerted by non-biotic factors. No one out of the three other secondary metabolites studied here responded to the presence of SA in a manner that could be perceived as elicitation. Even more, the concentrations of CGA and SA were associated inversely, probably due to the competition for the same precursor, i.e. phenylalanine (Chen et al. 2009; Dempsey et al. 2011; Tuan et al. 2014).
The negative (non-significant) relationship between the concentrations of QUE and 4-HBA may be explained in two ways: 1) They shared of a common precursor for the synthesis of both secondary metabolites (Bennett and Wallsgrove 1994) that is, enhanced synthesis of 4-HBA brought about a decrease of the concentration of QUE (or, vice versa), or 2) The negative relationship is a result of the fact that high concentrations of QUE corresponded to basal (mainly, shaded basal) cladodes, and that high concentrations of 4-HBA corresponded to lighted apical and to shaded basal cladodes. Analogously, the negative (non-significant) relationships between the concentrations of QUE and CGA for shaded basal cladodes may be explained by 1), or by the fact that for shaded basal cladodes, high concentrations of QUE co-occurred with low concentrations of CGA. On the other hand, in lighted basal cladodes low and high concentrations of both QUE and CGA co-occurred. As these relationships were non-significant, it is difficult to discuss the representativeness of this result at population level. However, it showed that both secondary metabolites responded to the presence of light in independent manner and were an effect of very short exposure to experimental treatments. To the best of our knowledge, CGA is not an elicitor of the secondary metabolites studied here, since only one case of an elicitor activity of QUE is actually known (Mahady and Beecher 1994), so a possible positive relationship between the concentrations of QUE and CGA in lighted cladodes was rather an effect of an independent production of secondary metabolites as a response against different stress factors.
QUE and SA did not co-occur. This means that SA did not promote the production of QUE. Neither did it promote the production of CGA, since the relationship between theses two metabolites was negative and statistically not significant. It rather means that these two secondary metabolites competed for the same precursors.
Regardless of the proximate factors that explain the relationships between the concentrations of different secondary metabolites studied here (i.e. elicitor effect, or competition for the same precursor) either the incidence or the concentration of different secondary metabolites maintain a rather complex, frequently negative, relationship with the analogue variables of other secondary metabolites, and thus, this is difficult to expect a congruent pattern of their presence in younger or faster-growing plants’ vegetative tissues. Additionally, there does not exist a general pattern of the between-cladode dynamics for different secondary metabolites. Rather, each of them is produced for a particular purpose and its incidence or dynamics depend on the goal it was synthetized for, and of either the competition for the same precursor or a possible elicitor function. Probably, the whole response (all chemical and physical defenses) of a plant against the action of herbivores should be studied to propose a new hypothesis concerning plant defense. However, this is difficult to achieve, since a matrix of metabolites and of the herbivores against which these metabolites were produced should be studied in such case. A reductionist approach may be used, when a specific secondary metabolite is used for a specific herbivore. Neither does there exist a unified hypothesis concerning plant’s response to the joint action of herbivores and abiotic stress such as UV. If a secondary metabolite protects a plant against both factors, its concentration should increase in the organs where the stress factor is exerting the main damage. However, if different substances sharing the same precursor are used for two different kinds of defenses (i.e. herbivory and UV), the investment to herbivory may take away resources from the defense against the negative effect of UV or vice versa. In such case, the resources will be allocated to the process that affects more severely the reproductive success.
Since in the experiment the cotton coarse fabric did not change significantly cladodes’ temperature, and the pH among the covered and uncovered cladodes did not differ, it meant that covered cladodes did not open their stomata, because the temperature of cladodes’ surface was similarly high during daytime as exposed to the environmental temperature. So, the concentrations of the malic acid were similar in both experimental treatments and in both cladodes’ positions. Therefore, we consider that any difference or the lack of differences in secondary metabolite concentration between treatments was an effect of either metabolite transfer between cladodes and not of a different concentration of the precursor (malic acid).