Senescence and programmed cell death in plants: polyamine action mediated by transglutaminase

Abstract

Research on polyamines (PAs) in plants laps a long way of about 50 years and many roles have been discovered for these aliphatic cations. PAs regulate cell division, differentiation, organogenesis, reproduction, dormancy-break and senescence, homeostatic adjustments in response to external stimuli and stresses. Nevertheless, the molecular mechanisms of their multiple activities are still matter of research. PAs are present in free and bound forms and interact with several important cell molecules; some of these interactions may occur by covalent linkages catalyzed by transglutaminase (TGase), giving rise to "cationization" or cross-links among specific proteins. Senescence and programmed cell death (PCD) can be delayed by PAs; in order to re-interpret some of these effects and to obtain new insights into their molecular mechanisms, their conjugation has been revised here. The TGase-mediated interactions between proteins and PAs are the main target of this review. After an introduction on the characteristics of this enzyme, on its catalysis and role in PCD in animals, the plant senescence and PCD models in which TGase has been studied, are presented: the corolla of naturally senescing or excised flowers, the leaves senescing, either excised or not, the pollen during self-incompatible pollination, the hypersensitive response and the tuber storage parenchyma during dormancy release. In all the models examined, TGase appears to be involved by a similar molecular mechanism as described during apoptosis in animal cells, even though several substrates are different. Its effect is probably related to the type of PCD, but mostly to the substrate to be modified in order to achieve the specific PCD program. As a cross-linker of PAs and proteins, TGase is an important factor involved in multiple, sometimes controversial, roles of PAs during senescence and PCD.

Keywords: flower corolla; hypersensitive response; leaf; pollen; polyamines; programmed cell death; senescence; transglutaminase.

FIGURE 1

Polyamines (PAs) in plants exist in two main forms: free and bound to many other molecules by different types of bindings: hydrogen type, more or less polarized, ionic type, or covalent one. The covalent linkage catalyzed by transglutaminase to specific proteins is Ca²⁺ dependent. Peculiar of some families of plants is the PA linkage with hydroxycinnamic acids. PAs are also actively metabolized: from PU to SD and SM but also vice versa. In addition, they can give rise to cytotoxic derivatives. PAs can act as free radicals. SD can be metabolized, by removing the aminopropyl group, and linked to a lysyl residue of the EIF5A precursor thus forming a peculiar derivative, the hypusine.

FIGURE 2

Transglutaminase could catalyze the Ca²⁺-dependent incorporation of substrates having a primary amino group, as polyamines, to the γ-carboxamide group of a specific protein-bound glutaminyl residue, giving rise to mono- and bis-(γ-glutamyl)-PAs. Similarly, the enzyme catalyzes the acyl-transfer reaction between the γ-carboxamide group of a specific protein-bound glutaminyl residue and the ∊-amino group of a distinct protein-bound lysyl residue, giving rise to protein crosslinking by the formation of isodipeptide N^∊-(γ -glutamyl)-lysine.

FIGURE 3

(A) Different protein molecules can form a net by PA linkage catalyzed by transglutaminase. (B) PAs can also form other linkages (i.e., ionic ones) with different molecules such as proteins, polysaccharides, phenols, etc., thus forming heterogeneous complexes.

FIGURE 4

Transglutaminase activity in different senescing or PCD models. (A) Corolla petals of flowers of Nicotiana tabacum senescing on the plant, starting from stage 2 (growing) to 7 (senescent), bottom panel. Top panel: glutamyl-PAs were separated by ion-exchange chromatography of extracts of the whole corolla at stages 2, 5, and 7 incubated with [³H] SM as described in (B) [modified from Della Mea et al. (2007b)]. (B) Comparison of the TGase pattern in two models of PCD. Top panel: petals of Nicotiana tabacum during natural corolla life span (stages 1–8). The activity was detected by incubating corolla extracts in the presence of 0.2 mM [¹⁴C] SM as tracer and the labelled conjugates produced were either measured by TCA precipitation or, after separation by SDS-PAGE, detected by autoradiography; a detail of the 250 kDa immunorecognized TGase polymers is shown in the insert [modified from Della Mea et al. (2007b)]. Bottom panel: TGase activity in compatible and self-incompatible pollinated styles of Citrus grandis at different days of pollination. The activity was detected by incubating style extracts in the presence of [³H]-PU as the tracer (modified from Gentile et al., 2012). At day 6, the incompatible pollen tube growth was blocked. (C) Senescing leaves of Lactuca sativa compared with the young ones. Glutamyl-PAs were separated by ion-exchange chromatography from extracts of young and senescing leaves, sprayed with SM or water when still on the plant and extracted 15 days after the spray. Extracts were incubated for 60 min with [³H] SM as tracer in the assay mixture containing 5 mM Ca²⁺. Bottom panel: picture of the Lactuca leaves and related plastids [modified from Serafini-Fracassini et al. (2010)].

FIGURE 5

(A) Model of the potential role of transglutaminase (and PAs) during the process of self-incompatibility in pear. S-RNase would be incorporated by endocytosis. This process would trigger a series of subsequent events, including the alteration of mitochondria morphology and consequently the production of ROS. In turn, abnormal levels of ROS might modify the intracellular concentration of Ca²⁺ thus leading to substantial modifications in the structure of actin filaments and/or to interference in the activation/regulation of TGase. As a result, further changes at level of actin (formation of actin foci) and microtubules may also occur. Along with the degradation of mRNA caused by S-RNase released in the cytoplasm, these effects contribute to the death of pollen tubes. (B) Distribution of TGase in pollen tubes and relationship with cell wall components. In pollen tubes, TGase (in red) is found in the cytoplasm but also in the cell wall where the enzyme could increase the stiffness of cell wall, contributing to counteract the internal turgor pressure. A further role of TGase during the SI response could be related to the interaction between TGase and specific cell wall components (such as arabinogalactans, in green) as part of the SI response. This interaction may thus prevent the growth of incompatible pollen tubes. Bar: 10 μm. The method for immunolabeling and similar figures can be found in Del Duca et al. (2013a).

FIGURE 6

Programmed cell death (PCD) and transglutaminase activity in the storage parenchyma of the tuber of Helianthus tuberosus during dormancy release. Scattered cells either parenchymatic (A) or tracheary elements undergoing PCD (C, arrow) were positive to Tunel staining. Other cells were negative (B). Fresh tuber slices were also incubated with dansyl-cadaverine and after extensive washing, immuno cytochemically recognized by an anti-dansyl antibody. Cells positive to conjugated cadaverine were scattered among others (D and F). TGase products were found either mainly in the cell walls (E, arrows) or in the whole cells (G). Bars: 50 μm.