Core features of the hormonal status in in vitro grown potato plants

ABSTRACT

Some time ago, potato transformants expressing Agrobacterium-derived auxin synthesis gene tms1 were generated. These tms1-transgenic plants, showing enhanced productivity, were studied for their hormonal status, turnover and responses in comparison with control plants. For this purpose, contents of phytohormones belonging to six different classes (auxins, cytokinins, gibberellins, abscisic, jasmonic and salicylic acids) were determined by a sensitive UPLC-MS/MS method in tubers and shoots of in vitro grown plants. To date, this study represents the most comprehensive analysis of the potato hormonal system. On the basis of obtained results, several new generalizations concerning potato hormonal status were drawn. Overall, these data can serve as a framework for forthcoming integrative studies of the hormonal system in potato plants.

Potato, like other land plants, possesses the entire spectrum of known phytohormones.¹,² These phytohormones regulate potato growth and development as well as tuber formation, therefore studies of endogenous hormone content and turnover are of special interest. To date, several classes of phytohormones are known including auxins (IAA), cytokinins (CKs), gibberellins (GAs), abscisic acid (ABA), ethylene, brassinosteroids, jasmonic acid (JA), salicylic acid (SA) and strigolactones. Each physiological process in plants is usually regulated by a number of defined hormones, some of them often exerting opposite effects. In potato, for example, distinct hormones (IAA, CKs, ABA and JA) stimulate some tuberization stages whereas GAs suppress them.^1,2 In addition, a crosstalk between phytohormones belonging to different classes becomes more and more obvious over time. ³ All this points to the importance of a comprehensive study of the potato hormonal system, with a parallel analysis of different classes of hormones.⁴

In the precedent years, several works were undertaken to study the hormonal status in potato. The greatest attention was paid to GAs as potent inhibitors of tuber formation.^5-8 Very rare studies have analyzed the content of phytohormones belonging to three or more different classes. Schmülling et al.⁹ measured IAA, CKs (iPR, ZR, DZR), GA₁ and ABA in potato shoots. However, immunoassay methods used in this study were evidently not enough sensitive to detect active forms of CKs (CK-bases) and of other active GAs. In the next studies four classes of phytohormones were analyzed in shoots of the in vitro grown potato cv. Miranda¹⁰ and organs of Solanum tuberosum ssp. andigena.¹¹ The authors were able to quantify IAA, cytokinins (Z, ZR, iP, iPR), as well as ABA and ethylene. More recently, Lulai et al.¹² analyzed IAA-, CK- and GA contents in seed mini-tubers by a modern method of ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI-MS.MS). However, in this case too, no active GAs were undoubtedly found. Among CKs, only ribosides which were proven to be per se hormonally inactive¹³ were detected. Thus, a comprehensive study of the global hormonal status in potato organs performed by a parallel quantification of the wide set of active phytohormones is still missing.

Our recent versatile research was aimed to compare hormonal status, response and turnover of tms1-transgenic and control potato plants.¹⁴ This analysis allowed us to fill the gap, at least partially, in our knowledge concerning global status and crosstalk of potato hormones. To measure phytohormone content, we have used an Acquity UPLC® System (Waters) coupled to a triple quadrupole mass spectrometer XevoTM TQ S with an electrospray interface (ESI). In our hands, this instrumental system was sensitive enough to detect minor peaks of active CKs and GAs. Altogether, we have quantified in the same plant pool six classes of phytohormones: auxins, CKs, GAs, ABA, JA and SA. This set included main hormonal players directly or indirectly involved in the regulation of tuber formation. Importantly, all phytohormones were determined in two different parts of potato: tubers and shoots. Thus, we were presented with a possibility to compare hormonal status in tubers and shoots of plants from the same experiment.

Main results of hormone content determinations are presented in Table 1. To achieve adequate comparison of the data, all values were brought to the same dimension of pmoles per g dry tissue weight. On the basis of tabulated results some general observations can be noted. Enormous differences in amounts of various hormones stand out: for example, amount of SA exceeded that of JA by several hundred times! These data are consistent with earlier studies where the high basal SA content in potato was reported.¹⁶ Furthermore, the SA level can be greatly elevated in plants under stress conditions.¹⁷ In potato cuttings growing in sterile tubes under artificial conditions stress reactions are indeed not excluded.

Table 1.

Phytohormone content in tubers and shoots of WT potato cv. Désirée*.

		Hormone content (pmol/g DW ± SE) in plant organs:
Phytohormones		Tubers	Shoots	Comments
Auxins	IAA	110 ± 17	332 ± 88	Active
Cytokinins	tZ	30.3 ± 14.2	58.5 ± 7.4	Active
	iP	48.7 ± 11.8	206 ± 30	Active
	cZ	164 ± 69	104 ± 12	Low active
	tZR	299 ± 192	404 ± 57	Inactive
	iPR	773 ± 411	3329 ± 739	Inactive
	cZR	2280 ± 1027	1535 ± 63	Inactive
	DZR	65.8 ± 29.5	56.1 ± 8.7	Inactive
	∑ cytokinins	3661	5693
Gibberellins	GA1	9.1 ± 2.1	36.6 ± 4.0	Active
	GA3	24.9 ± 7.4	37.4 ± 0.9	Active
	GA8	21.2 ± 16.4	296 ± 21	Inactive
	GA20	20.1 ± 15.4	39.4 ± 18.1	Inactive
	∑ gibberellins	75.3	409
Abscisic acid	ABA	1759 ± 416	2089 ± 283	Active
Jasmonic acid	JA	36 ± 5	724 ± 191	Active
Salicylic acid	SA	30520 ± 3090	550340 ± 198250	Active

*Potato plants were grown in vitro as described^14,15 on tuber-promoting Murasige-Skoog medium with 5% sucrose at 22–24°C, 16 h light (long day).Phytohormones were extracted from organs of 4–5-week-old potato plants.^14,25 Analytical assays were performed in triplicate, biological assays were performed at least in duplicate. Statistical analysis was accomplished using the Student’s t-test, P-value <0.05 was considered as statistically significant. Abbreviations are: IAA, indole-3-acetic acid; tZ, trans-zeatin; iP, isopentenyladenine; cZ, cis-zeatin; tZR, trans-zeatin riboside; iPR, isopentenyladenosine; cZR, cis-zeatin riboside; DZR, dihydrozeatin riboside.

When considering only active forms of phytohormones, the rankings according to their amounts are as follows: SA>ABA>IAA>CKs>JA≈GAs (in tubers) and SA>ABA>JA>IAA>CKs>GAs (in shoots). Both rankings are very similar, the only difference concerns JA which was much more abundant in shoots than in tubers. This could be expected since JA is biosynthesized in shoots, and it is not so movable due to its low solubility in water.

Not only JA but most of other tested hormones were at higher concentrations in shoots than in tubers (Figure 1). The tuber-to-shoot averaged concentration ratios of active hormones were: IAA, 0.47; CKs (iP+tZ), 0.33–0.73; GAs (GA₁+GA₃), 0.35–0.94; ABA, 1.18; JA, 0.07; SA, 0.08. Thus, JA and SA were distinguished by the lowest tuber-to-shoot ratio, ABA by the highest one, while typical hormones-activators (IAA, CKs and GAs) were moderately prevalent in shoots. Such hormonal pattern seems to be reasonable as tubers represent a storage organ that does not need an extensive metabolism to maintain a dormancy state. Notably, ABA, which is considered as a dormancy hormone, was present in tubers at a rather high concentration relative to that in shoots. The only exception from the general trend were cisZ and its riboside which showed an opposite trend by prevailing in tubers vs shoots (Figure 1). This argues for a distinct mode of cisZ-type cytokinin formation and/or trafficking, unrelated to those of tZ-type- and iP-type cytokinins. CisZ has low cytokinin activity, its affinity for potato CK-receptors is approx. 40–50-fold lower than the affinity of tZ or iP¹⁸. CisZ-type CKs often occur at a relatively high concentration in plant organs with limited growth (storage organs)^19,20 and can attenuate the cytokinin signaling wherein by competing with active cytokinins. As distinct from tubers where cisZ-type CKs prevailed over other cytokinins, in shoots iP-type CKs clearly dominated (Table 1). This feature agrees with the notion that cytokinin synthesis occurs in aboveground parts of plants by producing cytokinins of iP-type.²⁰ Interesting, the tuber-to-shoot ratios of contents of active CKs and cognate ribosides are very similar (Table 1). CK transport forms (ribosides) clearly dominated over CK-bases, the domination degree being similar in tubers and shoots, namely 6.9–9.9, 15.9–16.2 and 13.9–14.8 for tZ-, iP- and cisZ-type CKs, respectively. CKs of DZ-type were represented in potato only by DZR, at much lower content compared to other CK-ribosides (Table 1). Obviously, DZ-type CKs do not play any significant role in overall CK signaling in potato.

Figure 1.

Tuber-to-shoot concentration ratio (%) of identified hormonal substances in WT potato plants cv. Désirée. Averaged hormonal concentrations were determined on the basis of data in Table 1 and water content in tubers (~81.6%) and shoots (~86.2%) of potato cv. Désirée grown in vitro. Dark-brown and light-green colors correspond to tubers and shoots, respectively.

GAs play an important restrictive role in potato tuberization,^1,2,22,23 therefore data on the GA content are of particular value. In tubers and shoots, we were able to identify two active GAs, GA₁ and GA₃, together with their precursor GA₂₀. All these gibberellins were at rather low contents, especially in tubers (Table 1). Considering the relatively low affinity of active gibberellins to their receptors (K_D = 0.5–5 μM),^24,25 we may suggest that the revealed minor GA concentrations are hardly effective. The only GA substance found in shoots at a significant content – GA₈ – represents an inactive catabolite derived from GA₁.²⁶ The low amount of GAs in shoots may be a consequence of growth cessation of in vitro cultivated plants used for hormone analysis. A high content of GA₈ is indicative of an active catabolism of GA₁ in shoots. Interestingly, the prevalence degree of GA₁ in shoots vs tubers (2.9) markedly exceeded that of GA₂₀ (1.4) (Figure 1). This result is in accordance with the hypothesis²³ suggesting that GA₂₀ is readily transported throughout the plant whereas GA₁ preferentially remains in the vicinity of the cells where it is produced. Shoot-to-tuber ratios of two other GAs led us to the suggestion that GA₃ (ratio = 1.07) is a readily transportable gibberellin whereas GA₈ (ratio = 9.9) seems to be hardly movable.

Some other generalizations can be drawn from our¹⁴ and similar^10,11 studies. First, in the same tissue contents of IAA and sums of active CKs are usually rather similar. This auxin–CK balance is very important as it plays a crucial role in proliferative growth regulation.^10,11,27 Second, the content of ABA and especially of SA, as a rule, markedly exceeds that of IAA or sum of active CKs. Third, tubers are normally characterized by a lower content of active phytohormones compared to vegetative tissues. Of course, one should always take into account that the hormonal status of potato, as of other plants, may depend on studied organ, variety, growth stage and conditions. Nevertheless, our data can serve as a basic framework for forthcoming integrative studies of the intricate hormonal system in potato plants.

Funding Statement

This study was supported by the Russian Science Foundation, grant no 17-74-20181.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.