Timing a switch in tissue integrity: In plants, sperm cells travel through the pollen tube as it grows toward the ovule. Successful fertilization depends on the pollen tube rupturing to release the sperm cells (see the Perspective by Stegmann and Zipfel). Ge et al. and Mecchia et al. elucidated the intercellular cross-talk that maintains pollen tube integrity during growth but destroys it at just the right moment. The signaling peptides RALF4 and RALF19, derived from the pollen tube, maintain its integrity as it grows. Once in reach of the ovule, a related signaling peptide, RALF34, which derives from female tissues, takes over and causes rupture of the pollen tube. Science , this issue p. 1596 , p. 1600 ; see also p. 1544

Rapid alkalinization factor (RALF) is a peptide signal that plays a basic role in cell biology and most likely regulates cell expansion. In this study, transgenic Arabidopsis thaliana lines with high and low levels of AtRALF1 transcripts were used to investigate this peptide’s mechanism of action. Overexpression of the root-specific isoform AtRALF1 resulted in reduced cell size. Conversely, AtRALF1 silencing increased root length by increasing the size of root cells. AtRALF1-silenced plants also showed an increase in the number of lateral roots, whereas AtRALF1 overexpression produced the opposite effect. In addition, four AtRALF1-inducible genes were identified: two genes encoding proline-rich proteins (AtPRP1 and AtPRP3), one encoding a hydroxyproline-rich glycoprotein (AtHRPG2), and one encoding a xyloglucan endotransglucosylase (TCH4). These genes were expressed in roots and involved in cell-wall rearrangement, and their induction was concentration dependent. Furthermore, AtRALF1-overexpressing plants were less sensitive to exogenous brassinolide (BL); upon BL treatment, the plants showed no increase in root length and a compromised increase in hypocotyl elongation. In addition, the treatment had no effect on the number of emerged lateral roots. AtRALF1 also induces two brassinosteroid (BR)-downregulated genes involved in the BR biosynthetic pathway: the cytochrome P450 monooxygenases CONSTITUTIVE PHOTOMORPHISM AND DWARFISM (CPD) and DWARF4 (DWF4). Simultaneous treatment with both AtRALF1 and BL caused a reduction in AtRALF1-inducible gene expression levels, suggesting that these signals may compete for components shared by both pathways. Taken together, these results indicate an opposing effect of AtRALF1 and BL, and suggest that RALF’s mechanism of action could be to interfere with the BR signalling pathway.

The rapid alkalinization factor (RALF) peptide negatively regulates cell expansion, and an antagonistic relationship has been demonstrated between AtRALF1, a root-specific RALF isoform in Arabidopsis, and brassinosteroids (BRs). An evaluation of the response of BR signaling mutants to AtRALF1 revealed that BRI1-associated receptor kinase1 (bak1) mutants are insensitive to AtRALF1 root growth inhibition activity. BAK1 was essential for the induction of AtRALF1-responsive genes but showed no effect on the mobilization of Ca²⁺ and alkalinization responses. Homozygous plants accumulating AtRALF1 and lacking the BAK1 gene did not exhibit the characteristic semi-dwarf phenotype of AtRALF1-overexpressors. Biochemical evidence indicates that AtRALF1 and BAK1 physically interact with a K_d of 4.6 μM and acridinium-labeled AtRALF1 was used to demonstrate that part of the specific binding of AtRALF1 to intact seedlings and to a microsomal fraction derived from the roots of Arabidopsis plants is BAK1-dependent. Moreover, AtRALF1 induces an increase in BAK1 phosphorylation, suggesting that the binding of AtRALF1 to BAK1 is functional. These findings show that BAK1 contains an additional AtRALF1 binding site, indicating that this protein may be part of a AtRALF1-containing complex as a co-receptor, and it is required for the negative regulation of cell expansion. The rapid alkalinization factor (RALF) is a small peptide hormone that binds to FERONIA receptor and inhibits cell expansion. The AtRALF1, a root-specific RALF isoform in Arabidopsis, opposes brassinosteroid (BR) effects on root cell elongation. To address the role of AtRALF1 and BRs in regulating cell expansion, we evaluated the response of BR signaling mutants to AtRALF1. We found that the BRI1-associated receptor kinase1 (bak1) mutants are insensitive to AtRALF1 root growth inhibition activity. Although BAK1 showed no effect on the mobilization of Ca²⁺ and alkalinization responses, it was required for the induction of AtRALF1-responsive genes and for the semi-dwarf phenotype of AtRALF1-overexpressing plants. Different experiments demonstrated that AtRALF1 and BAK1 interact physically in a specific manner. We also suggested that the binding of AtRALF1 to BAK1 is functional because AtRALF1 induces an increase in BAK1 phosphorylation. Our findings indicate that BAK1 plays an important role in AtRALF1 signaling.

Arabidopsis thaliana rapid alkalinization factor 1 (AtRALF1) is a small secreted peptide hormone that inhibits root growth by repressing cell expansion. Although it is known that AtRALF1 binds the plasma membrane receptor FERONIA and conveys its signals via phosphorylation, the AtRALF1 signaling pathway is largely unknown. Here, using a yeast two hybrid system to search for AtRALF1-interacting proteins in Arabidopsis, we identified calmodulin-like protein 38 (CML38) as an AtRALF1-interacting partner. We also found that CML38 and AtRALF1 are both secreted proteins that physically interact in a Ca²⁺- and pH-dependent manner. CML38- knockout mutants generated via T-DNA insertion were insensitive to AtRALF1, and simultaneous treatment with both AtRALF1 and CML38 proteins restored sensitivity in these mutants. Hybrid plants lacking CML38 and having high accumulation of the AtRALF1 peptide did not exhibit the characteristic short-root phenotype caused by AtRALF1 overexpression. Although CML38 was essential for AtRALF1-mediated root inhibition, it appeared not to have an effect on the AtRALF1-induced alkalinization response. Moreover, acridinium-labeling of AtRALF1 indicated that the binding of AtRALF1 to intact roots is CML38 dependent. In summary, we describe a new component of the AtRALF1 response pathway. The new component is a calmodulin-like protein that binds AtRALF1, is essential for root growth inhibition, and has no role in AtRALF1 alkalinization.

Rapid alkalinization factor (RALF) is a peptide signal that plays a role in plant cell expansion. We have recently proposed that AtRALF1 negatively regulates root cell elongation and lateral root formation by opposing the effects of brassinosteroid (BR). We reported six AtRALF1-inducible cell wall-related genes and two P450 monooxygenase -encoding genes involved in the BR biosynthetic pathway. The AtRALF1-inducible genes implicated in cell wall remodeling were not downregulated by brassinolide (BL) treatment alone; their induction was only compromised following simultaneous treatment with AtRALF1 and BL. We further examined the cell wall-remodeling gene EXPANSIN A5 (AtEXPA5), which is upregulated by BL and has been shown to positively affect root cell elongation. Herein, we report that AtEXPA5 expression is downregulated by AtRALF1 in a dose-dependent manner in the roots and hypocotyls of Arabidopsis plants. AtEXPA5 is also downregulated in plants that overexpress AtRALF1, and it is upregulated in...

Objetivou-se avaliar o enraizamento adventício em miniestacas de Eucalyptus benthamii em relação ao genótipo, concentrações de Zn e B, coleta de brotações e aplicação de AIB. As brotações foram oriundas de minicepas fertirrigadas com soluções nutritivas variando nas concentrações de Zn e B (S1 - isento de Zn e B, S2 - 0,5 mg L-1 de Zn, S3 - 0,5 mg L-1 de B, S4 - 0,5 mg L-1 de Zn e B, S5 - 1,0 mg L-1 de Zn e B, S6 - 2,0 mg L-1 de Zn e B). A região basal da miniestaca foi imersa durante 10 s em solução contendo 2.000 mg L-1 de AIB, sendo uma solução isenta de AIB utilizada como controle. Avaliaram-se a sobrevivência das miniestacas na saída da casa de vegetação, na saída da casa de sombra e em área de pleno sol. Em área de pleno sol foram avaliadas apenas as miniestacas enraizadas, e destas, o comprimento total do sistema radicial. O enraizamento adventício em miniestacas de Eucalyptus benthamii dependeu do material genético, da coleta de brotações, da aplicação de AIB e das concentrações de Zn e B. A porcentagem de enraizamento foi baixa, sendo que os materiais genéticos foram considerados de difícil propagação pela miniestaquia. Em termos gerais, as miniestacas obtidas de minicepas fertirrigadas com as soluções nutritivas S5 (1,0 mg L-1 de Zn e B) e S6 (2,0 mg L-1 de Zn e B) associadas a presença de AIB na concentração de 2.000 mg L-1 apresentaram os maiores índices de enraizamento.