Absence of Arabidopsis Polyamine Oxidase 5 Influences the Cytokinin-Induced Shoot Meristem Formation from Lateral Root Primordia

Lateral root primordia (LRPs) of Arabidopsis can be directly converted to shoot meristems (SMs) by the application of exogenous cytokinin. Here, we report that Arabidopsis POLYAMINE OXIDASE 5 (AtPAO5) contributes to this process, since the rate of SM formation from LRPs was significantly lower in the pao5-2 knockout mutant. Furthermore, the presented experiments showed that AtPAO5 influences SM formation via controlling the thermospermine (T-Spm) level. Gene expression analyses supported the view that the pao5-2 mutation as well as exogenous T-Spm downregulate the expression of the class 3 haemoglobin coding genes AtGLB1 and AtGLB2. AtGLB1 and 2 have been reported to augment cytokinin sensitivity, indirectly inhibiting the expression of type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs). In agreement, the same ARR-coding genes were found to be upregulated in the pao5-2 mutant. Although GLB proteins might also control cytokinin-induced nitric oxide (NO) accumulation, we could not find experimental evidence for it. Rather, the negative effect of NO-donor treatment on AtPAO5 gene expression and SM formation was seen. Nevertheless, a hypothetical pathway is set up explaining how AtPAO5 may affect direct shoot meristem formation, controlling cytokinin sensitivity through T-Spm and GLBs. © 2023 by the authors

Timely removal of exogenous cytokinin and the prevention of auxin transport from the shoot to the root affect the regeneration potential of Arabidopsis roots

In vitro regeneration of Arabidopsis from roots is generally achieved via indirect organogenesis. First, transdifferentiation of lateral root primordia to calli is achieved by a balanced auxin-to-cytokinin ratio that is followed by the induction of shoot meristem formation using a high cytokinin level. Here we demonstrate that if the root explants were transferred onto a hormone-free medium after a transient (4-days) cytokinin treatment, embryogenic marker genes (LEC1, LEC2, FUS3) started to be expressed. App. 50% of the regeneration foci developed into plantlets with trichome-less cotyledon-like leaves. Moreover, the somatic embryogenesis defective lec1 mutant could regenerate only shoots with trichome-bearing leaves under this condition. Based on these observations, the mixed accomplishment of shoot organogenesis and somatic embryogenesis is hypothesized in the Arabidopsis root explants cultured under hormone-free conditions following cytokinin induction. Using whole seedlings instead of root explants in the same experimental set up, no regenerates were formed on the roots. Applying the auxin transport inhibitor TIBA to the root-to-shoot junction of the seedlings, the regeneration ability of the root could be restored. The observations indicate that shoot-derived endogenous auxin blocks the cytokinin-induced regeneration process in the roots of whole seedlings. The expression of the wound-induced transcription factor WIND1 could be detected in the roots of unwounded seedlings if the shoot-to-root auxin transport was inhibited. Manipulating the exogenous cytokinin level together with the endogenous shoot-to-root auxin transport therefore could mimic the effect of wounding (removal of shoot) on plant regeneration from roots. Key message Transferring root explants from high cytokinin to hormone-free conditions resulted in the expression of embryogenic markers. Inhibiting the shoot-to-root auxin transport had similar effect on regeneration as wounding

Timely removal of exogenous cytokinin and the prevention of auxin transport from the shoot to the root affect the regeneration potential of Arabidopsis roots

In vitro regeneration of Arabidopsis from roots is generally achieved via indirect organogenesis. First, transdifferentiation of lateral root primordia to calli is achieved by a balanced auxin-to-cytokinin ratio that is followed by the induction of shoot meristem formation using a high cytokinin level. Here we demonstrate that if the root explants were transferred onto a hormone-free medium after a transient (4-days) cytokinin treatment, embryogenic marker genes (LEC1, LEC2, FUS3) started to be expressed. App. 50% of the regeneration foci developed into plantlets with trichome-less cotyledon-like leaves. Moreover, the somatic embryogenesis defective lec1 mutant could regenerate only shoots with trichome-bearing leaves under this condition. Based on these observations, the mixed accomplishment of shoot organogenesis and somatic embryogenesis is hypothesized in the Arabidopsis root explants cultured under hormone-free conditions following cytokinin induction. Using whole seedlings instead of root explants in the same experimental set up, no regenerates were formed on the roots. Applying the auxin transport inhibitor TIBA to the root-to-shoot junction of the seedlings, the regeneration ability of the root could be restored. The observations indicate that shoot-derived endogenous auxin blocks the cytokinin-induced regeneration process in the roots of whole seedlings. The expression of the wound-induced transcription factor WIND1 could be detected in the roots of unwounded seedlings if the shoot-to-root auxin transport was inhibited. Manipulating the exogenous cytokinin level together with the endogenous shoot-to-root auxin transport therefore could mimic the effect of wounding (removal of shoot) on plant regeneration from roots. Key message Transferring root explants from high cytokinin to hormone-free conditions resulted in the expression of embryogenic markers. Inhibiting the shoot-to-root auxin transport had similar effect on regeneration as wounding

Subfunctionalization of Parental Polyamine Oxidase (PAO) Genes in the Allopolyploid Tobacco Nicotiana tabacum (L.)

Polyamines play an important role in developmental and environmental stress responses in plants. Polyamine oxidases (PAOs) are flavin-adenine-dinucleotide-dependent enzymes associated with polyamine catabolism. In this study, 14 genes were identified in the tobacco genome that code for PAO proteins being named based on their sequence homology with Arabidopsis PAOs (AtPAO1-5): NtPAO1A-B; NtPAO2A-C, NtPAO4A-D, and NtPAO5A-E. Sequence analysis confirmed that the PAO gene family of the allopolyploid hybrid Nicotiana tabacum is not an exact combination of the PAO genes of the maternal Nicotiana sylvestris and paternal Nicotiana tomentosiformis ones. The loss of the N. sylvestris homeolog of NtPAO5E and the gain of an extra NtPAO2 copy, likely of Nicotiana othophora origin, was revealed. The latter adds to the few pieces of evidence suggesting that the paternal parent of N. tabacum was an introgressed hybrid of N. tomentosiformis and N. othophora. Gene expression analysis indicated that all 14 PAO genes kept their expression following the formation of the hybrid species. The homeologous gene pairs showed similar or opposite regulation depending on the investigated organ, applied stress, or hormone treatment. The data indicate that the expression pattern of the homeologous genes is diversifying in a process of subfunctionalization

Absence of Arabidopsis Polyamine Oxidase 5 Influences the Cytokinin-Induced Shoot Meristem Formation from Lateral Root Primordia

Lateral root primordia (LRPs) of Arabidopsis can be directly converted to shoot meristems (SMs) by the application of exogenous cytokinin. Here, we report that Arabidopsis POLYAMINE OXIDASE 5 (AtPAO5) contributes to this process, since the rate of SM formation from LRPs was significantly lower in the pao5-2 knockout mutant. Furthermore, the presented experiments showed that AtPAO5 influences SM formation via controlling the thermospermine (T-Spm) level. Gene expression analyses supported the view that the pao5-2 mutation as well as exogenous T-Spm downregulate the expression of the class 3 haemoglobin coding genes AtGLB1 and AtGLB2. AtGLB1 and 2 have been reported to augment cytokinin sensitivity, indirectly inhibiting the expression of type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs). In agreement, the same ARR-coding genes were found to be upregulated in the pao5-2 mutant. Although GLB proteins might also control cytokinin-induced nitric oxide (NO) accumulation, we could not find experimental evidence for it. Rather, the negative effect of NO-donor treatment on AtPAO5 gene expression and SM formation was seen. Nevertheless, a hypothetical pathway is set up explaining how AtPAO5 may affect direct shoot meristem formation, controlling cytokinin sensitivity through T-Spm and GLBs