Nitrogen remobilisation facilitates adventitious root formation on reversible dark-induced carbohydrate depletion in Petunia hybrida
Adventitious root (AR) formation with high economic significance in horticulture, agriculture and forestry is a complex physiological process. The ornamental plant propagation relies on globalised chains for young plant production via rooting of cuttings ensuring an effective utilization of beneficial external and internal factors. The whole process includes three phases, axillary bud and shoot growth on donor plants (providing recurrent excision of mature shoot tips – i.e. cuttings), subsequent logistics (i.e. transport, storage) of cuttings and insertion of the cuttings into rooting media. During this process strong transcriptomic and metabolic changes occur with high importance of nitrogen availability, dark exposure and various irradiance levels. Thus, reciprocal regulations force adaptations in nitrogen and carbohydrate metabolism during phases of axillary bud and shoot growth, dark induced senescence of cuttings, stress recovery under diurnal light and AR formation in cuttings. It has already been shown that the level of nitrogen assimilation by donor plants changes nitrogen fluxes and rebalances the pools of carbohydrates and amino acids [1, 2]. Moreover, degradation and re-synthesis of proteins enable survival of rootless cuttings and are required for the regeneration of the missing root organs. Since AR formation relies on selective proteolysis and re-synthesis of proteins, the total nitrogen stock in the cuttings constitutes a key limiting factor [3, 4]. Interestingly, there are similarities and differences between AR formation and lateral roots [5, 6] especially for nitrogen deficiency and ethylene signalling and synthesis in planta. N deficiency stimulates lateral roots of sessile plants having already their intact root system. Then lateral root formation starts with highly cell-specific responses to external nitrogen signals that are directed towards nutrient-rich soil patches to ensure nutrient acquisition [7]. In contrast, excised axillary shoot tips (i.e. cuttings) such as petunia cuttings experience wounding and isolation and thus solely rely on shoot-born signals with specific transcriptome and metabolome responses [8–10]. When the vascular continuum collapses, auxin accumulates and induces AR formation in stem base tissue [11]. Primary auxin control of AR formation depends on secondary signals like nitric oxide, polyamines and ethylene [6, 12, 13]. Recently, an aminotransferase protein was reported to coordinate the biosynthesis of the hormones ethylene and auxin [14]. Further, auxin triggers the activation of a plant target of rapamicin complex that is expressed in primary meristems and integrates auxin and nutrient signalling by regulated protein translations [15]. Thus, nitrogen resources are pivotal for protein synthesis in the stem base of cuttings, wherein the predominant amino acids comprise glutamine (gln), glutamate (glu), asparagine (asn) and aspartate (asp) [8, 16]. Carbohydrate reserves and nitric oxide (NO) enhance resilience of plant tissues and survival of dark senescence [17–19]. As AR formation depends on protein re-synthesis [3, 4] from mobile or recycled nitrogen reserves such as asn [20, 21] these could be limiting in case of N deficiency and result in an accelerated leaf senescence [22, 23] differing from lateral roots formation, in this respect [24, 25]. So far nitrogen and carbohydrate limitations of AR formation have been shown in Pelargonium, Chrysanthemum, Poinsettia and Rosa [17, 26–28]. Enhanced AR formation at high nitrogen contents may be related to an increased basipetal transport of carbohydrates [26] and nitrogenous compounds [20] with limited knowledge of the causal mechanisms including transcriptome, hormone and metabolic adaptations. Using Petunia hybrida as a model plant three metabolic phases for AR formation were established [9] during which nitrogen supply was maintained at adequate levels. A dynamic depletion and replenishment of carbohydrates has been reported in course of dark exposure of the cuttings and their subsequent rooting under light with stimulating effect on root formation [29]. In addition, at adequate nitrogen levels a strong contribution of the polar auxin transport (PAT) to AR formation was shown by an early increase of indole-3-acetic acid (IAA) in Petunia [16]. Moreover, multiple transcriptome changes in auxin transport systems, auxin conjugation and auxin signal perception uncovered auxin as a key regulator of AR formation during sink establishment phase [9, 16, 30, 31]. At the sink side amino acids and nitrogen pools provide important N resources to meet the new demand for protein re-synthesis. In addition, variation in N resources may have an influence on auxin levels. It is supposed that prior to excision of cuttings various signalling hormones including cytokinin (CK) communicate the nitrogen availability from donor plant roots to axillary shoots [32] and that their activity can be related partially to glutamine metabolism [33]. CK’s are considered as auxin antagonists and important negative regulators of AR formation [34] that would counteract auxin distribution via down-regulation of PIN activity [35]. In contrast, CK’s are also considered as important signals for dedifferentiation processes during early induction of ARs [4] and are required for fine tuning of the auxin transport and biosynthesis during the formation of the quiescent centre in the adventitious root apex [36]. In this regard, shoot levels of both CK’s and gibberellins decline with an interrupted nitrogen supply to roots [37]. This complexity of functions of nitrogen metabolism interacting with plant hormone signalling might explain the lack of information on the influence of nitrogen nutrition of donor plants and dark exposure of cuttings on their nitrogen metabolism and AR formation. Therefore, the present study tested the hypothesis that enhanced Nt contents and dark exposure of cuttings influence their internal N-pools including free amino acids and affect early events of AR formation and further root development in Petunia hybrida.