Molecular and physiological aspects regarding the Arabidopsis polyamine oxidase gene family

Abstract

In plants, the polyamines (PAs) putrescine (Put), spermidine (Spd), spermine (Spm) and thermospermine (Therm-Spm) are involved in several physiological processes. In particular, Spd is important for survival, while Put and Spm have been implicated in plant responses to drought, high salt stress, wounding and pathogens. Furthermore, Therm-Spm is involved in the control of xylem differentiation having an auxin antagonizing effect. PA oxidases (PAOs) are FAD-dependent enzymes involved in PA catabolism. In Arabidopsis thaliana, five PAO genes (AtPAO1–AtPAO5) have been identified. AtPAO1 and AtPAO5 are cytosolic enzymes catalyzing the back-conversion of Spm and Therm-Spm to Spd. AtPAO5 is also able to oxidize N1-acetyl-Spm. Conversely, the other three members of the Arabidopsis gene family (AtPAO2, AtPAO3 and AtPAO4) have a peroxisomal localization and are able to oxidize both Spd and Spm, but not Therm-Spm. To investigate the physiological role(s) of AtPAO5 during plant growth and development, two 35S::AtPAO5-6His Arabidopsis transgenic lines that ectopically express AtPAO5, one with 70-fold (AtPAO5-1) and the other 4-fold (AtPAO5-2) higher expression levels than the endogenous gene, were characterized. Parallel studies were also performed with two loss-of-function mutants lacking AtPAO5 expression (atpao5-1 and atpao5-2 mutants). Analysis of PA levels showed decreased levels of Spm, Therm-Spm, and N1-acetyl-Spm in AtPAO5-1 seedlings and increased levels in atpao5-1 and atpao5-2 whole seedlings, as well as in specific organs (stem and leaves), as compared to the wild-type plants. Instead, the AtPAO5-2 transgenic line does not present differences in PA levels from the wild-type plants. These data are in agreement with the AtPAO5 substrate specificity in vitro and indicate that Spm, Therm-Spm and N1-acetyl-Spm are the substrates of AtPAO5 also in vivo, and that AtPAO5 contributes in a dose-dependent way to PA homeostasis along the entire plant. Additionally, analysis of the expression levels of Therm-Spm biosynthetic genes thermospermine synthase (ACAULIS5; ACL5) and S-adenosylmethionine decarboxylase 4 (SAMDC4) showed that they are up-regulated in AtPAO5-1 plants and down-regulated in atpao5 mutants, but not affected at all in AtPAO5-2 plants. Instead, no change in spermine synthase (SPMS) expression levels was observed in any of the AtPAO5 and atpao5 plants as compared to the wild-type plants. These data suggest that AtPAO5 participates in a feedback mechanism controlling Therm-Spm homeostasis. Phenotypical analyses of AtPAO5 and atpao5 plants evidenced some developmental differences in different plant organs (stems, roots, leaves and hypocotyls). In particular, the two atpao5 mutants produce longer and thicker flowering stems, while conversely the AtPAO5-1 transgenic plants produce thinner and shorter ones compared to the wild-type plants. Similarly, AtPAO5-1 transgenic plants present shorter roots with a higher number of lateral roots than the wild-type plants, while atpao5 mutant plants longer ones. Transverse sections showed that the AtPAO5-1 transgenic plants undergo excessive xylem differentiation, while the atpao5 mutants reduced. Furthermore, AtPAO5-1 and atpao5 plants present altered vasculature thickness in hypocotyls and leaves as compared with the wild-type plants. Taken together, these phenotypical differences indicate that AtPAO5 contributes to plant development controlling xylem differentiation, which is consistent with the high expression levels of AtPAO5 in the vascular system, as shown by histochemical analysis of AtPAO5::GFP-GUS transgenic plants. In the present study it has been also shown that AtPAO5 expression is up-regulated by cytokinins and Therm-spm, specifically in the roots. Furthermore, cytokinin and Therm-Spm treatment differently affected xylem differentiation in the roots of the AtPAO5-1, atpao5 and wild-type plants, suggesting that AtPAO5 is involved in the cytokinin and Therm-Spm-mediated control of root xylem differentiation. To understand the mechanism(s) through which AtPAO5 is involved in vascular system differentiation and considering that both auxin and cytokinin have an important role in these processes, the expression of some cytokinin-, auxin- and xylem-related genes were analyzed in AtPAO5-1, atpao5 and wild-type plants by qRT-PCR. The results showed that several auxin- and xylem-related genes are up-regulated in AtPAO5-1 plants and down-regulated in atpao5 mutants compared to the wild type plants. Moreover, some cytokinin-related genes were differently regulated in AtPAO5-1, atpao5 and wild-type plants following cytokinin treatment. These data altogether suggest altered auxin and cytokinin signaling, together with altered xylem differentiation, in the atpao5 mutants and the AtPAO5-1 transgenic plants comparing with the wild-type plants. To further investigate on the auxin and cytokinin signaling in the AtPAO5-1 and atpao5 plants, sexual crossings of these plants with DR5::GUS transgenic plants, DR5 being an artificial auxin–regulated promoter, are in progress. Since AtPAO1 presents some similarities to AtPAO5, the atpao1 single mutant and the atpao1/atpao5 double mutant (DM15) were additionally analyzed. Data evidenced that AtPAO1 participates, together with AtPAO5, to Spm and Therm-Spm homeostasis, as well as to the control of stem and root development, with AtPAO5 playing however a major role in these processes. Several studies evidenced an important role of PAs in plant defense responses to biotic and abiotic stresses. To determine whether AtPAO1 and AtPAO5 contribute to these processes, the AtPAO5-1 transgenic plants, as well as the atpao5 and DM15 mutants were observed under drought and salt stress. Preliminary results indicate that the atpao5 and DM15 mutants are more tolerant to both salt and drought stress as compared to the wild-type plants, the AtPAO5-1 plants appearing more sensitive to these stresses. Experiments are still in progress to further understand the role of the AtPAO1 and AtPAO5, as well as the other members of the AtPAO gene family, under conditions of environmental stress. In conclusion, our studies further support a tightly controlled interplay between Therm-Spm, auxin and cytokinins necessary for proper xylem differentiation and plant growth. AtPAO5 and AtPAO1 redundantly contribute to this regulatory network participating in the feedback mechanisms which control Therm-Spm levels. On the other hand, AtPAO5, together with AtPAO1, participate in the control of plant responses to salt and drought stress.