The GRAS gene family in pine: transcript expression patterns associated with the maturation-related decline of competence to form adventitious roots

Abstract

Background: Adventitious rooting is an organogenic process by which roots are induced from differentiated cells other than those specified to develop roots. In forest tree species, age and maturation are barriers to adventitious root formation by stem cuttings. The mechanisms behind the respecification of fully differentiated progenitor cells, which underlies adventitious root formation, are unknown.

Results: Here, the GRAS gene family in pine is characterized and the expression of a subset of these genes during adventitious rooting is reported. Comparative analyses of protein structures showed that pine GRAS members are conserved compared with their relatives in angiosperms. Relatively high GRAS mRNA levels were measured in non-differentiated proliferating embryogenic cultures and during embryo development. The mRNA levels of putative GRAS family transcription factors, including Pinus radiata's SCARECROW (SCR), PrSCR, and SCARECROW-LIKE (SCL) 6, PrSCL6, were significantly reduced or non-existent in adult tissues that no longer had the capacity to form adventitious roots, but were maintained or induced after the reprogramming of adult cells in rooting-competent tissues. A subset of genes, SHORT-ROOT (PrSHR), PrSCL1, PrSCL2, PrSCL10 and PrSCL12, was also expressed in an auxin-, age- or developmental-dependent manner during adventitious root formation.

Conclusions: The GRAS family of pine has been characterized by analyzing protein structures, phylogenetic relationships, conserved motifs and gene expression patterns. Individual genes within each group have acquired different and specialized functions, some of which could be related to the competence and reprogramming of adult cells to form adventitious roots.

Figure 1

Experimental system used for analysis. A, B) Embryogenic masses of Pinus radiata after 7 (P7) and 14 (P14) days of proliferation. Embryogenic tissue (in red) was stained with 1% acetocarmine. Bar: 2 mm. C) Early-maturation embryo at polarization stage (M1). Bar: 0.5 mm. D) Late-maturation embryo at tissue differentiation stage (M3). Bar: 0.8 mm. E) Hypocotyls from 21-day-old seedlings treated with 10 μM indole-3-butyric acid (IBA) after 28 days of culture. F, G) Hypocotyls (F) and epicotyls (G) from 90-day-old seedlings treated with 10 μM IBA.

Figure 2

Phylogenetic tree of GRAS proteins SCARECROW-LIKE (SCL), SCARECROW (SCR), and SHORT-ROOT (SHR) from conifer species. Accession no. or gene references in parentheses. Picea abies SCR (MA_1793p0010), P. abies SCL1 (MA_45656p0030), P. abies SCL2 (MA_10435790p0010), P. abies SCL3 (MA_140003p0010), P. abies SCL4 (MA_18234p0010), P. abies SCL5 (MA_73870p0010), P. abies SCL6 (MA_94287p0010), P. abies SCL8 (MA_52903p0010), P.abies SCL9 (MA_10426489p0020), P.abies SCL10 (MA_10432093p0010), P. abies SCL11 (MA_19310p0010), P. abies SCL13 (MA_96029p0010), P. abies SCL17 (MA_10255p0010), P. abies SCL18 (MA_10430319p0010), P. abies SCL23 (MA_73173p0010); Pinus pinaster SCL7 (sp_v2.0_unigene8594), P. pinaster SCL8 (sp_v2.0_unigene8378), P. pinaster SCL9 (sp_v2.0_unigene4531), P. pinaster SCL13 (sp_v2.0_unigene1634), P. pinaster SCL14 (sp_v2.0_unigene1578), P. pinaster SCL15 (sp_v2.0_unigene10599); Pinus radiata SCR (KM264388), P. radiata SHR (EU044786), P. radiata SCL1 (DQ683567), P. radiata SCL2 (KM264389), P. radiata SCL10 (KM264395), P. radiata SCL12 (KM264397); Pinus taeda SCR (PITA_000043499-RA), P. taeda SHR (PITA_000092405-RA), P. taeda SCL1 (PITA_000021589-RA), P. taeda SCL5 (PITA_000017225-RA), P. taeda SCL6 (PITA_000022609-RA), P. taeda SCL8 (PITA_000040137-RA), P. taeda SCL9 (PITA_000009055-RA), P. taeda SCL10 (PITA_000009053-RA), P. taeda SCL11 (PITA_000068827-RA), P. taeda SCL12 (PITA_000010887-RA), P. taeda SCL15 (PITA_000016257-RA), P.taeda SCL16 (PITA_000056676-RA), P.taeda SCL18 (PITA_000086415-RA), P. taeda SCL19 (PITA_000075302-RA), P. taeda SCL20 (PITA_000051405-RA), P. taeda SCL21 (PITA_000056428-RA), P. taeda SCL22 (PITA_000080766-RA), P. taeda SCL23 (PITA_000072928-RA), P.taeda SCL24 (PITA_000072831-RA), P. taeda SCL25 (PITA_000041536-RA), P. taeda SCL26 (PITA_000026833-RA), P. taeda SCL27 (PITA_000049193-RA), P. taeda SCL28 (PITA_000066307-RA), P. taeda SCL29 (PITA_000051712-RA) and P. taeda SCL30 (PITA_000035221-RA). PtSCL25 was used as the outgroup. Branches with bootstrap values lower than 500 were collapsed.

Figure 3

Expression of GRAS genes in vegetative Pinus radiata organs and at the embryonic-postembryonic develop mental transition. A) Organs from 35-day-old pine seedlings. qRT-PCR was performed using RNAs from roots (R), hypocotyls (H), cotyledons (C) or shoot apex nodal segments (A). B) Embryo development. qRT-PCR was performed using RNAs from embryogenic masses at 7 (P7) and 14 (P14) days of proliferation, early-maturation embryo (M1) and late-maturation embryo (M3). C) Embryonic-postembryonic development. qRT-PCR was performed using RNAs from embryogenic masses at 7 (P7) days of proliferation, rooting-competent hypocotyls (H21) and non-competent hypocotyls (H90) or epicotyls (E90) from seedlings of 21- and 90-day-old seedlings, respectively. A total of 1 μg RNA was reverse transcribed, and 12.5 ng of cDNA was amplified with 400 nM of specific primers. Pine Ri18S was used as the control. Results are expressed as mean values of the relative expression to roots (A) or P7 (B and C) ± SE from at least three biological replicates. Insets in B show details of early developmental stages. Results of PrSHR expression in C are expressed as mean values of relative expression to H21. Expression levels of PrSCL1 and PrSHR had already been measured in organs during vegetative development [16,17]. Expression of PrSCL16 was not detected in any of the RNA samples tested. SCL, SCARECROW-LIKE; SHR, SHORT-ROOT.

Figure 4

Expression of GRAS genes during adventitious root formation in Pinus radiata . A) qRT-PCR was performed using RNAs from rooting-competent hypocotyls (H21) and non-competent hypocotyls (H90) from 21- and 90-day-old seedlings, respectively. B) qRT-PCR was performed using RNAs from non-competent epicotyls (E90) from 90-day-old seedlings. RNA was extracted from the base of hypocotyl (H) or epicotyl (E) cuttings treated with 10 μM indole-3-butyric acid at the indicated times. Hypocotyl or epicotyl cuttings maintained in water were used as controls. A total of 1 μg RNA was reverse transcribed, and 12.5 ng of cDNA was amplified with 400 nM of specific primers. Pine Ri18S was used as the control. Results are expressed as mean values of relative expression to time 0 ± SE from at least three biological replicates. Expression levels of PrSCL1 and PrSHR had already been measured in competent hypocotyls from 21-day-old seedlings during adventitious rooting [16,17]. Expression of PrSCL16 was not detected in any of the RNA samples tested. SCL, SCARECROW-LIKE; SHR, SHORT-ROOT.

Figure 5

In situ localization of Pinus radiata SHORT-ROOT ( PrSHR ) mRNA. A, B) Transverse sections of hypocotyls from 90-day-old seedlings at time 0 (A), and after 24 h of culture in the presence of 10 μM indole-3-butyric acid (IBA) (B). C, D) Transverse sections of epicotyls from 90-day-old seedlings at time 0 (C), and after 24 h of culture in the presence of 10 μM IBA (D). The sections were hybridized with an RNA probe obtained by in vitro transcription of PrSHR in either the antisense (A, B, C, D) or sense (E, F) orientation. Note the absence of hybridization in the controls. Similar results were obtained using an RNA probe obtained by in vitro transcription of PrSCL1 in either the antisense or sense orientation. ab, axillary bud; c, cambial region; co, cortex; r, resin canal; x, xylem. In situ localization of PrSCL1 and PrSHR had already been described in competent hypocotyls from 21-day-old seedlings during adventitious rooting [17]. SCL, SCARECROW-LIKE.

Figure 6

Endogenous distribution of indole-3-acetic acid (IAA) in hypocotyl cuttings from 21-day-old Pinus radiata seedlings. Transverse sections from the base of hypocotyls after 24 h of culture in the presence of 10 μM indole-3-butyric acid (IBA) (A, B, C, D) or in the presence of 10 μM IBA + 10 μM 1-N-naphthylphthalamic acid (E, F, G, H). A, E) Differential interference contrast (DIC) image, B, F) Immunodetection of IAA, C, G) DAPI nuclear staining, D, H) merged immunodetection of IAA and DAPI staining. c, cambial region; co, cortex; r, resin canal; x, xylem.

Figure 7

Endogenous distribution of indole-3-acetic acid (IAA) in hypocotyl and epicotyl cuttings from 90-day-old Pinus radiata seedlings. Transverse sections of the base of hypocotyls (A, B, C, D) and epicotyls (E, F, G, H) after 24 of culture in the presence of 10 μM indole-3-butyric acid. A, E) Differential interference contrast (DIC) image, B, F) Immunodetection of IAA, C, G) DAPI nuclear staining, D, H) merged immunodetection of IAA and DAPI staining. c, cambial region; co, cortex; r, resin canal; x, xylem.