The Sequential Action of MIDA9/PP2C.D1, PP2C.D2, and PP2C.D5 Is Necessary to Form and Maintain the Hook After Germination in the Dark

Abstract

During seedling etiolation after germination in the dark, seedlings have closed cotyledons and form an apical hook to protect the meristem as they break through the soil to reach the surface. Once in contact with light, the hook opens and cotyledons are oriented upward and separate. Hook development in the dark after seedling emergence from the seed follows three distinctly timed and sequential phases: formation, maintenance, and eventual opening. We previously identified MISREGULATED IN DARK9 (MIDA9) as a phytochrome interacting factor (PIF)-repressed gene in the dark necessary for hook development during etiolated growth. MIDA9 encodes the type 2C phosphatase PP2C.D1, and pp2c-d1/mida9 mutants exhibit open hooks in the dark. Recent evidence has described that PP2C.D1 and other PP2C.D members negatively regulate SMALL AUXIN UP RNA (SAUR)-mediated cell elongation. However, the fundamental question of the timing of PP2C.D1 action (and possibly other members of the PP2C.D family) during hook development remains to be addressed. Here, we show that PP2C.D1 is required immediately after germination to form the hook. pp2c.d1/mida9 shows reduced cell expansion in the outer layer of the hook and, therefore, does not establish the differential cell growth necessary for hook formation, indicating that PP2C.D1 is necessary to promote cell elongation during this early stage. Additionally, genetic analyses of single and high order mutants in PP2C.D1, PP2C.D2, and PP2C.D5 demonstrate that the three PP2C.Ds act collectively and sequentially during etiolation: whereas PP2C.D1 dominates hook formation, PP2C.D2 is necessary during the maintenance phase, and PP2C.D5 acts to prevent opening during the third phase together with PP2C.D1 and PP2C.D2. Finally, we uncover a possible connection of PP2C.D1 levels with ethylene physiology, which could help optimize hook formation during post-germinative growth in the dark.

Genome-wide identification and expression analysis of the PP2C gene family in Apocynum venetum and Apocynum hendersonii

“…According to phylogenetic and evolutionary relationship analysis, Apocynum hendersonii and Apocynum venetum are closely related to each other, and a total of 136 PP2C genes in Apocynum hendersonii and Apocynum venetum were divided into 13 subgroups (A-L), which is consistent with the grouping of Arabidopsis thaliana. Previous studies found that genes in the A subgroup of PP2C genes in Arabidopsis mainly inhibit ABA receptor activity and negatively regulate ABA signaling [32]; the B subgroup is involved in the mitogen-activated protein kinase signaling pathway and participates in the process of MAPK phosphorylation, and is capable of dephosphorylating and inactivating MAPK [33]; Subgroup C gene family members are involved in stem cell maintenance and differentiation [34]; Members of subgroup D negatively regulate small auxin up RNA (SAUR)-mediated cell elongation, and PP2C.D1, PP2C.D2, and PP2C.D5 in Arabidopsis act during leaf greening, and PP2C.D1 regulates hook formation by affecting ethylene accumulation [15,35,36]. Moreover, members of this group respond to saline and alkaline stress, which may be related to stomatal movement and function, by inhibiting plasma membrane H-ATPase activity.…”

“…Group B is responsible for mitogen-activated protein kinase (MAPK) signaling, while Group C is mainly involved in ower development [14]. Members of Group D may have negative effects on cell elongation, but can also have positive effects on the ABA signaling pathway in response to saline stress [15][16][17]. Group E is responsible for regulating the opening and closing of plant stomata, while Group F induces stress responses in bacteria.…”