The MADS-domain transcriptional regulator AGAMOUS-LIKE15 promotes somatic embryo development in Arabidopsis and soybean

Abstract

The MADS-domain transcriptional regulator AGAMOUS-LIKE15 (AGL15) has been reported to enhance somatic embryo development when constitutively expressed. Here we report that loss-of-function mutants of AGL15, alone or when combined with a loss-of-function mutant of a closely related family member, AGL18, show decreased ability to produce somatic embryos. If constitutive expression of orthologs of AGL15 is able to enhance somatic embryo development in other species, thereby facilitating recovery of transgenic plants, then AGL15 may provide a valuable tool for crop improvement. To test this idea in soybean (Glycine max), a full-length cDNA encoding a putative ortholog of AGL15 was isolated from soybean somatic embryos. Subsequently, the corresponding genomic region of the gene was obtained. This gene, designated GmAGL15, encodes a protein with highest similarity to AGL15 from Arabidopsis (Arabidopsis thaliana) and Brassica napus that accumulates to its highest amount in embryos in these species. Like Arabidopsis and Brassica AGL15, GmAGL15 was preferentially expressed in developing embryos. When ectopically overexpressed the soybean protein was able to enhance somatic embryo development in soybean.

Figure 1.

Effect of AGL15/AGL18 accumulation on somatic embryo tissue production. A, Percentage of cultured zygotic embryos of the indicated genotypes that produce secondary embryonic tissue. Results shown are the means and se of the mean derived from 15 to 16 plates with 81 to 140 embryos per plate (with the exception of agl15-4 for which there were only six plates). Different letters indicate significance of P < 0.01. wt, Wild type. B, Percentage of embryonic foci of the indicated genotypes that were subcultured at approximately 3 weeks and continued to produce embryonic tissue approximately 2.5 to 3 weeks after subculture. Mean and se of the mean are shown. Different letters indicate significance at P < 0.01. C, Percentage of seedlings that showed somatic embryo development from the SAM when seeds of the indicated genotypes were allowed to compete germination in liquid media containing 2,4-D. Mean and se of the mean are shown. Different letters indicate significance at P < 0.05. Images are representative seedlings of the indicated genotypes that have SAM somatic embryo development.

Figure 2.

A, GeneDoc (www.pac.edu/biomed/genedoc) sequence alignment between GmAGL15, AtAGL15, BnAGL15-1, and BnAGL15-2. The shade levels represent conservation degrees. B, Schematic representation of the gene structures of GmAGL15 and AtAGL15. The numbers and positions of the introns/exons were conserved between these two species. Boxes, exons; lines, introns. C, A phylogenetic tree generated from GmAGL15, BnAGL15s, and all 39 MIKC-type Arabidopsis MADS-domain proteins. Neighbor-joining method with a bootstrap number of 1,000 was used (ClustalX 1.81, http://www-igbmc.u-strasbg.fr/BioInfo/ClustalX). AGL15 group is indicated.

Figure 3.

Expression pattern of GmAGL15. Semiquantitative RT-PCR was performed on RNA derived from various tissues of ‘Jack’ soybean. The coding region of EF1-α was amplified for normalization. L, Young leaves; S, stems; F, open flowers; P, seed pods containing very young embryos; YE, young embryos (average length 2 mm); ME, mature green embryos; SEC, somatic embryo culture. The color of the image is inverted for clarity.

Figure 4.

Effects of overexpression of AGL15 on SE in soybean. A, Schematic of the transformation and scoring scheme for soybean. Please see “Materials and Methods” for further details. B, Transformation of soybean with soybean genomic (gGm) or cDNA (cGm) versions of GmAGL15 driven by the 35S cauliflower mosaic virus promoter produced more potential transformants that were individually subcultured than did the empty vector control. However, expression of Arabidopsis AGL15 did not result in an increase in potential transformants. Different letters indicate significant difference at P < 0.05.

Figure 5.

Evaluation of expression of 35S:AGL15 in soybean. A, RT-PCR was performed using a GmAGL15 forward primer and a c-myc reverse primer on RNA extracted from young leaves. RT-PCR with EF1-α was performed as a normalization control. Total RNA was also analyzed on an agarose gel to evaluate integrity. B, Protein gel-blot analysis to evaluate accumulation of AGL15 in transgenic soybean. Protein extracts were prepared from flowers of the indicated genotypes and proteins were separated on a polyacrylamide gel, blotted to membrane, and probed using anti-AGL15 serum. Inclusion protein containing GmAGL15 (without a c-myc tag) was obtained by expression in E. coli and used as a positive control. All lanes within a section are from the same gel or blot.