Hormonal and gene dynamics in de novo shoot meristem formation during adventitious caulogenesis in cotyledons of Pinus pinea

Abstract

Several members of WOX and KNOX gene families and several plant growth regulators, basically cytokinins and auxins, play a key role during adventitious caulogenesis in the conifer Pinus pinea. Similar to Arabidopsis thaliana, Pinus pinea shoot organogenesis is a multistep process. However, there are key differences between both species, which may alter the underlying physiological and genetic programs. It is unknown if the genic expression models during angiosperm development may be applicable to conifers. In this work, an analysis of the endogenous content of different plant growth regulators and the expression of genes putatively involved in adventitious caulogenesis in P. pinea cotyledons was conducted. A multivariate analysis of both datasets was also realized through partial least squares regression and principal component analysis to obtain an integral vision of the mechanisms involved in caulogenesis in P. pinea. Analyses show that cotyledons cultured in the presence of benzyladenine during long times (2-6 days) cluster separately from the rest of the samples, suggesting that the benzyladenine increase observed during the first hours of culture is sufficient to trigger the caulogenic response through the activation of specific developmental programs. In particular, the most relevant factors involved in this process are the cytokinins trans-zeatin, dihydrozeatin, trans-zeatin riboside and isopentenyl adenosine; the auxin indoleacetic acid; and the genes PpWUS, PpWOX5, PpKN2, PpKN3 and PipiRR1. WUS is functional in pines and has an important role in caulogenesis. Interestingly, WOX5 also seems to participate in the process, although its specific role has not been determined.

Keywords: Conifers; De novo shoot organogenesis; Gene expression; Multivariate analyses; Plant growth regulators.

Fig. 1

Content profile of the main plant growth regulators (PGRs), extracted from Pinus pinea cotyledons cultured in the presence of benzyladenine (BA-treated cotyledons, BA) and absence of exogenous hormone (control cotyledons, C): BA benzyladenine, DHZ dihydrozeatin, DHZR dihydrozeatin riboside, tZ trans-zeatin, tZR trans-zeatin riboside, iP isopentenyl adenine, iPA isopentenyl adenosine, IAA indoleacetic acid, GA₄BK castasterone, gibberellin A4, abscisic acid, ABA; jasmonic acid, JA; salicylic acid, SA. Results are expressed as mean values ± standard error. Quantitative data were analyzed by Kruskal–Wallis test. Asterisks indicate significant differences between treatments or times of culture (*P ≤ 0.05; **P ≤ 0.01;***P ≤ 0.001

Fig. 2

Proportion of different plant growth regulators (PGRs) during de novo shoot formation in Pinus pinea expressed in percentage of each hormone with respect to the total of tested compounds and excluding benzyladenine (BA) (color figure online)

Fig. 3

Immunodetection of benzyl adenosine (BAR), dihydrozeatin riboside (DHZR) and isopentenyl adenosine (iPA) in longitudinal sections of Pinus pinea cotyledons cultured in the presence and absence of benzyladenine (BA) during 0 day, 12 h, 2 days and 6 days. Alexa 488 secondary antibody (green signal) was used. Fluorescence quantification was performed following the procedure described by Burgess et al. (2010) through ImageJ software (https://fiji.sc/). BA-treated cotyledons (BA): black bars; non-treated cotyledons (C): red bars. Results are expressed as the mean of the fluorescence values ± standard error from 3–5 cotyledons per treatment. Bar, 100 µm

Fig. 4

Quantitative real-time (RT-qPCR) analysis of the mRNA abundance of several members from WOX (PpWOX5, PpWUS) and KNOX (PpKN1, PpKN2, PpKN3, PpKN4) gene families, RESPONSE REGULATOR1 (PipiRR1) and CLAVATA1-LIKE (PipiCLV1L) in Pinus pinea cotyledons cultured in the presence and absence of benzyladenine (BA) during the induction phase of adventitious caulogenesis. Results are expressed as mean values of the relative expression ± standard error. Quantitative data were analyzed by Kruskal–Wallis test. Asterisks indicate significant differences between treatments or times of culture (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Fig. 5

Analysis of PGRs content and gene expression data by principal component analysis (PCA). BA_ST: short times (0–1 days), BA-treated cotyledons (green); BA_LT: long times (2–6 days), BA-treated cotyledons (red); C_ST: short times (0–1 days), control cotyledons (purple); C_LT: long times (2–6 days), control cotyledons (blue) (color figure online)

Fig. 6

Correlation loading plot obtained by partial least squares (PLS) regression using plant growth regulator content as the X matrix and gene expression data as the Y matrix