Transcriptome-Wide Identification of the GRAS Transcription Factor Family in Pinus massoniana and Its Role in Regulating Development and Stress Response

Abstract

Pinus massoniana is a species used in afforestation and has high economic, ecological, and therapeutic significance. P. massoniana experiences a variety of biotic and abiotic stresses, and thus presents a suitable model for studying how woody plants respond to such stress. Numerous families of transcription factors are involved in the research of stress resistance, with the GRAS family playing a significant role in plant development and stress response. Though GRASs have been well explored in various plant species, much research remains to be undertaken on the GRAS family in P. massoniana. In this study, 21 PmGRASs were identified in the P. massoniana transcriptome. P. massoniana and Arabidopsis thaliana phylogenetic analyses revealed that the PmGRAS family can be separated into nine subfamilies. The results of qRT-PCR and transcriptome analyses under various stress and hormone treatments reveal that PmGRASs, particularly PmGRAS9, PmGRAS10 and PmGRAS17, may be crucial for stress resistance. The majority of PmGRASs were significantly expressed in needles and may function at multiple locales and developmental stages, according to tissue-specific expression analyses. Furthermore, the DELLA subfamily members PmGRAS9 and PmGRAS17 were nuclear localization proteins, while PmGRAS9 demonstrated transcriptional activation activity in yeast. The results of this study will help explore the relevant factors regulating the development of P. massoniana, improve stress resistance and lay the foundation for further identification of the biological functions of PmGRASs.

Keywords: GRAS; Pinus massoniana; abiotic stresses; development; expression; hormone treatments.

Figure 1

Phylogenetic grouping of members of the GRAS family of P. massoniana. Different colored rings denote various subfamilies, while red letters denote P. massoniana GRAS proteins.

Figure 2

VHIID domain of 21 PmGRAS proteins based on sequence alignment. The most conserved GRAS domain of VHIID is boxed in red.

Figure 3

Phylogenetic grouping, motif distribution and structural domain analysis of GRAS proteins in P. massoniana.

Figure 4

Subcellular localization analysis of the PmGRAS9 and PmGRAS17 proteins. Transient expression of GFP (control), PmGRAS9-GFP and PmGRAS17-GFP in Nicotiana benthamiana leaves. The scale in the images of GFP and PmGRAS9-GFP is 10 µM, and the scale in the images of PmGRAS17-GFP is 20 µM.

Figure 5

GRAS family members’ transcriptomic profiles in P. massoniana at various drought levels: CK (80 ± 5)%, T1 (65 ± 5)%, T2 (50 ± 5)% and T3 (35 ± 5)%. Heat maps were created by performing a row scale on log2 (FPKM + 0.01) values, where the color scale denotes relative expression levels.

Figure 6

Expression of PmGRASs under different stresses. (a) PEG and (b) mechanical injury. The relative expression level is indicated as the mean ± standard deviation (SD), asterisks indicate significant differences in transcript abundance in the treated group compared with the control group (0 h) (student’s t-test, * p < 0.05, ** p < 0.01).

Figure 7

Expression profiles of PmGRASs under different hormone treatments. (a) SA, (b) MeJA, (c) ETH, (d) ABA, (e) IAA, and (f) GA3. The relative expression levels are indicated as the mean ± standard deviation (SD), asterisks indicate significant differences in transcript abundance in the treated group compared with the control group (0 h) (student’s t-test, * p < 0.05, ** p < 0.01).

Figure 7

Expression profiles of PmGRASs under different hormone treatments. (a) SA, (b) MeJA, (c) ETH, (d) ABA, (e) IAA, and (f) GA3. The relative expression levels are indicated as the mean ± standard deviation (SD), asterisks indicate significant differences in transcript abundance in the treated group compared with the control group (0 h) (student’s t-test, * p < 0.05, ** p < 0.01).

Figure 8

Relative expressions of PmGRASs in eight representative tissues. YS: young stem; OS: old stem; TB: terminal bud; YN: young needle; ON: old needle; R: root; X: xylem; P: phloem. The relative expression levels are indicated as the mean ± standard deviation (SD), asterisks indicate significant differences in transcript abundance in the treated group compared to the control group (0 h) (student’s t-test, * p < 0.05, ** p < 0.01).

Figure 9

Transcriptional activation assay of PmGRAS9. Empty pGBKT7 vector was used as a negative control.