Names of poplar GRFs according to their Arabidopsis orthologs
Nineteen candidate GRF genes were found in the Populus trichocarpa genome [11]. To enable the comparison of PtrGRFs (Ptr, Populus trichocarpa) with the well-studied AtGRFs, the 19 identified PtrGRFs were renamed according to their Arabidopsis orthologs (Fig. 1, Fig. S1). According to the phylogenetic tree, the PtrGRFs could be classified into six groups (Fig. 1a, Fig. S2), with Group VI as a supplementary group to the reported classification of AtGRFs [6]. In Group I, four PtrGRFs clustered with AtGRF1 and AtGRF2 and were named PtrGRF1/2a, PtrGRF1/2b, PtrGRF1/2c, and PtrGRF1/2d (Fig. 1a). In Group II, only one PtrGRF gene corresponded to AtGRF3 and AtGRF4 and was named PtrGRF3/4 (Fig. 1a). In Group III, AtGRF5 and AtGRF6 each have two poplar orthologs, which were named accordingly (Fig. 1a). In Group IV, three PtrGRFs were named according to their sequence similarity to AtGRF7 and AtGRF8 (Fig. 1a). In Group V, although three PtrGRFs clustered with AtGRF9, only one PtrGRF with two WRC domains was named PtrGRF9 (Fig. 1a and b). In addition, four PtrGRFs with no close Arabidopsis orthologs were clustered in Group VI and named PtrGRF10a, PtrGRF10b, PtrGRF11a, and PtrGRF11b (Fig. 1a). The two PtrGRFs that clustered with AtGRF9, but lacked WRC domains were renamed PtrGRF12a and PtrGRF12b (Fig. 1a and b). Table S1 shows the complete gene information for PtrGRFs and AtGRFs.
The regulation of PtrGRFs by miR396
Since GRFs are the major targets of miR396 [17], the relationship between miR396 and PtrGRFs was investigated. First, the sequences of PtrGRFs and the mature sequences of poplar miR396b were uploaded to RNAhybrid [25, 26] to analyze whether PtrGRFs are targets of miR396. This showed that all of the PtrGRFs, except PtrGRF12b, have the potential to hybridize with miR396b with a minimal free energy hybridization value less than -33 kcal/mol, suggesting that these PtrGRFs could be targets of miR396 (Fig. 2a). For PtrGRF12b and miR396, the number of mismatches exceeded the other hybridization pairs and the hybridization energy was -28 kcal/mol, which exceeded the values observed for most endogenous miRNA targets [27], suggesting that PtrGRF12b is not a target of miR396 (Fig. 2a). Then, we aligned the target sequences of PtrGRFs to the mature miR396b sequences (Fig. S3). The sequences of PtrGRF1/2a-PtGRF12a and miR396 matched perfectly, while a thymine to adenine change in the 3' terminal of PtrGRF12b led to a mismatch, indicating that PtrGRF12b is the only PtrGRF not targeted by miR396 (Fig. S3).
In addition, degradome sequencing data [28] were analyzed to identify miR396 cleavage sites in the PtrGRFs (Fig. 2b). As expected, the miR396 cleavage sites of most PtrGRFs were found in the degradome data and no such a site was found in the GRF12b transcript (Fig. 2b, Table S2), proving the in vivo regulation of the expression of PtrGRFs by miR396 was consistent with the RNAhybrid analysis.
Furthermore, transient expression was used to investigate the regulation of poplar GRFs by miR396. On fusing PagGRF1/2c, PagGRF9, PagGRF10b, PagGRF11b, and PagGRF12b, genes isolated from poplar 84K (see Methods), with YFP (Yellow Fluorescent Protein) and expressing them transiently in tobacco leaves, the fluorescence signals of all of the PagGRF-YFP fusion proteins were very weak (Fig. S4), except that of PagGRF12b (Fig. 3a). Considering the functional conservation of plant miRNAs, the weak fluorescence signal may be due to the cleavage of PagGRFs by tobacco miR396. To test this, miR396-resistant versions of the GRFs, which contained six point mutations within the miR396-complementary domain of the GRF sequence to increase the number of mismatches without altering the amino acid sequence, were constructed and transiently expressed in tobacco leaves (Fig. S5). As expected, the fluorescence signals of the mPagGRF-YFP fusion proteins were strong and merged with the DAPI signals (Fig. 3a), indicating that miR396 targeted all of the PagGRFs, except PagGRF12b. Furthermore, transient co-expression assays were performed and PtrmiR408 was used as a negative control to evaluate the regulation of PagGRF by PagmiR396b (Fig. 3b). Similar to the fluorescent signals of GRF1/2d [previously named GRF15 by Cao et al. (2016)] in our published results [24], the fluorescent signals of GRF12a-YFP were weak when co-expressed with PtrmiR408, but were faint and difficult to detect when co-expressed with PagmiR396b, indicating that PagmiR396b could downregulate the expression of PagGRF12a. By contrast, comparable strong fluorescence of mGRF12a-YFP, the mutated version, was detected when co-expressed with PagmiR396a or PtrmiR408. These results confirmed that PagmiR396b could target PagGRF directly in vivo.
Overexpression of PagGRF6b, PagGRF7a, PagGRF12a, and PagGRF12b led to diverse changes in leaf size in transgenic poplar
The result in our previous study [24] showed that GRF1/2a, GRF1/2b, GRF1/2c, GRF1/2d, GRF5a, GRF5b, GRF6b, GRF7a, GRF7b, GRF8, GRF9, GRF10a, GRF11a, GRF11b, and GRF12a were all highly expressed in young leaves, suggesting that these GRFs may have a role in leaf size control. In addition, although its expression was relative low in all tissues, the miR396 independent GRF, GRF12b, had higher relative expression in young leaves. Therefore, to investigate the function of poplar GRFs in leaf size control, PagGRF6b representing group III, PagGRF7a from group IV, and PagGRF12a and PagGRF12b from group V were chosen to generate transgenic plants for functional characterization (Figs. 1 and 4). The mutated versions of PagGRF6b, PagGRF7a, and PagGRF12a, with synonymous mutations in the miR396 target sites, were used to avoid degradation by miR396 (Fig. S6). Three overexpression (OE) lines each for mPagGRF6b, mPagGRF7a, mPagGRF12a, and PagGRF12b with moderately increased expression of the corresponding gene were chosen for further investigation (Fig. S7). The leaf size of the mPagGRF6b OE plants did not differ significantly (Fig. 4a), while mPagGRF7a OE plants had 26.8% smaller leaves than those of the control (CK) (Fig. 4b). By contrast, mPagGRF12a and PagGRF12b OE plants had 16.1% and 28.1% larger leaves, respectively, in comparison with CK (Fig. 4c and d).
The leaf epidermis cell area was measured and leaf cell numbers were calculated for mPagGRF6b, mPagGRF7a, mPagGRF12a, and PagGRF12b OE plants and compared with the CK. The leaf epidermis cell area of mPagGRF6b did not change significantly (Fig. 4a), while it decreased in mPagGRF7a, mPagGRF12a, and PagGRF12b OE plants (Fig. 4b-d). The number of leaf cells in the mPagGRF6b and mPagGRF7a OE plants did not differ significantly (Fig. 4a and b), but increased significantly in the mPagGRF12a and PagGRF12b OE plants (Fig. 4c and d).
Furthermore, expression of the cell proliferation marker genes CYCLINB1;1a and CYCLINB1;1b and cell expansion marker genes EXPA11a and EXPA11b (Zhou et al. 2019) was examined in the fifth leaves from mPagGRF6b, mPagGRF7a, mPagGRF12a, and PagGRF12b OE plants. Consistent with our observations, expression of CYCLINB1;1a and CYCLINB1;1b was unaltered in mPagGRF6b and mPagGRF7a OE plants, but upregulated in the mPagGRF12a and PagGRF12b OE plants (Fig. 5, Fig. S8). Meanwhile, the expression of EXPA11a and EXPA11b did not change much in the mPagGRF6b OE plants, but was downregulated significantly in mPagGRF7a, mPagGRF12a, and PagGRF12b OE plants (Fig. 5, Fig. S8).
These results indicate that PagGRF6b has no function in leaf size control; PagGRF7a functions as a negative regulator of leaf size, mainly by regulating cell expansion; and PagGRF12a and PagGRF12b positively regulate leaf size through both cell proliferation and cell expansion, but mainly through cell proliferation.