Wheat AGAMOUS LIKE 6 transcription factors function in stamen development by regulating the expression of Ta APETALA3.

Previous studies have revealed the functions of rice and maize AGAMOUS LIKE 6 (AGL6) genes OsMADS6 and ZAG3, respectively, in floral development; however, the functions of three wheat (Triticum aestivum) AGL6 genes are still unclear. Here, we report the main functions of wheat AGL6 homoeologous genes in stamen development. In RNAi plants, stamens showed abnormality in number and morphology, and a tendency to transform into carpels. Consistently, the expression of the B-class gene TaAPETALA3 (AP3) and the auxin-responsive gene TaMGH3 was downregulated, whereas the wheat ortholog of the rice carpel identity gene DROOPING LEAF was ectopically expressed in RNAi stamens. TaAGL6 proteins bind to the promoter of TaAP3 directly. Yeast one-hybrid and transient expression assays further showed that TaAGL6 positively regulates the expression of TaAP3 in vivo. Wheat AGL6 transcription factors interact with TaAP3, TaAGAMOUS and TaMADS13. Our findings indicate that TaAGL6 transcription factors play an essential role in stamen development through transcriptional regulation of TaAP3 and other related genes. We propose a model to illustrate the function and probable mechanism of this regulation. This study extends our understanding of AGL6 genes.

Silencing SlAGL6, a tomato AGAMOUS-LIKE6 lineage gene, generates fused sepal and green petal.

KEY MESSAGE: Silencing SlAGL6 in tomato leads to fused sepal and green petal by influencing the expression of A-, B-class genes. AGAMOUS-LIKE6 (AGL6) lineage is an important clade MADS-box transcription factor and plays essential roles in various developmental programs especially in flower meristem and floral organ development. Here, we isolated a tomato AGL6 lineage gene SlAGL6 and successfully obtained several RNA interference (RNAi) lines. Silencing SlAGL6 led to abnormal fused sepals and light green petals with smaller size. The total chlorophyll content in transgenic petals increased and the morphology of epidermis cells altered. Further analysis showed that A-class gene MACROCALYX (MC) participating in sepal development and a NAC-domain gene GOBLET involving in boundary establishment were down-regulated in transgenic lines. In transgenic petals, two chlorophyll synthesis genes, Golden2-like1 (SlGLK1) and Golden2-like2 (SlGLK2), two photosystem-related genes, ribulose bisphosphate carboxylase small chain 3B (SlrbcS3B) and chlorophyll a/b-binding protein 7 (SlCab-7) were induced and three B-class genes TM6, TAP3 and SlGLO1 were repressed. These results suggest that SlAGL6 involves in tomato sepal and petal development.

Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution.

AP1/FUL, SEP, AGL6, and FLC subfamily genes play important roles in flower development. The phylogenetic relationships among them, however, have been controversial, which impedes our understanding of the origin and functional divergence of these genes. One possible reason for the controversy may be the problems caused by changes in the exon-intron structure of genes, which, according to recent studies, may generate non-homologous sites and hamper the homology-based sequence alignment. In this study, we first performed exon-by-exon alignments of these and three outgroup subfamilies (SOC1, AG, and STK). Phylogenetic trees reconstructed based on these matrices show improved resolution and better congruence with species phylogeny. In the context of these phylogenies, we traced evolutionary changes of exon-intron structures in each subfamily. We found that structural changes have occurred frequently following gene duplication and speciation events. Notably, exons 7 and 8 (if present) suffered more structural changes than others. With the knowledge of exon-intron structural changes, we generated more reasonable alignments containing all the focal subfamilies. The resulting trees showed that the SEP subfamily is sister to the monophyletic group formed by AP1/FUL and FLC subfamily genes and that the AGL6 subfamily forms a sister group to the three abovementioned subfamilies. Based on this topology, we inferred the evolutionary history of exon-intron structural changes among different subfamilies. Particularly, we found that the eighth exon originated before the divergence of AP1/FUL, FLC, SEP, and AGL6 subfamilies and degenerated in the ancestral FLC-like gene. These results provide new insights into the origin and evolution of the AP1/FUL, FLC, SEP, and AGL6 subfamilies.

Flower Development and Perianth Identity Candidate Genes in the Basal Angiosperm Aristolochia fimbriata (Piperales: Aristolochiaceae).

Aristolochia fimbriata (Aristolochiaceae: Piperales) exhibits highly synorganized flowers with a single convoluted structure forming a petaloid perianth that surrounds the gynostemium, putatively formed by the congenital fusion between stamens and the upper portion of the carpels. Here we present the flower development and morphology of A. fimbriata, together with the expression of the key regulatory genes that participate in flower development, particularly those likely controlling perianth identity. A. fimbriata is a member of the magnoliids, and thus gene expression detected for all ABCE MADS-box genes in this taxon, can also help to elucidate patterns of gene expression prior the independent duplications of these genes in eudicots and monocots. Using both floral development and anatomy in combination with the isolation of MADS-box gene homologs, gene phylogenetic analyses and expression studies (both by reverse transcription PCR and in situ hybridization), we present hypotheses on floral organ identity genes involved in the formation of this bizarre flower. We found that most MADS-box genes were expressed in vegetative and reproductive tissues with the exception of AfimSEP2, AfimAGL6, and AfimSTK transcripts that are only found in flowers and capsules but are not detected in leaves. Two genes show ubiquitous expression; AfimFUL that is found in all floral organs at all developmental stages as well as in leaves and capsules, and AfimAG that has low expression in leaves and is found in all floral organs at all stages with a considerable reduction of expression in the limb of anthetic flowers. Our results indicate that expression of AfimFUL is indicative of pleiotropic roles and not of a perianth identity specific function. On the other hand, expression of B-class genes, AfimAP3 and AfimPI, suggests their conserved role in stamen identity and corroborates that the perianth is sepal and not petal-derived. Our data also postulates an AGL6 ortholog as a candidate gene for sepal identity in the Aristolochiaceae and provides testable hypothesis for a modified ABCE model in synorganized magnoliid flowers.

Characterization of 10 MADS-box genes from Pyrus pyrifolia and their differential expression during fruit development and ripening.

We cloned 10 Japanese pear (Pyrus pyrifolia) MIKC-type II MADS-box genes, and analyzed their expression during fruit development and ripening. PpMADS2-1 was APETALA (AP)1-like; PpMADS3-1 was FRUITFULL (FUL)/SQUAMOSA (SQUA)-like; PpMADS4-1 was AGAMOUS-like (AGL)6; PpMADS5-1 and PpMADS8-1 were SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC)-like; PpMADS9-1, PpMADS12-1, PpMADS14-1 and PpMADS16-1 were SEPALLATA (SEP)-like; while PpMADS15-1 was AGL/SHATTERPROOF (SHP)-like. Phylogenetic analysis showed their grouping into five major clades (and 10 sub-clades) that was consistent with their diverse functional types. Expression analysis in flower tissue revealed their distinct putative homeotic functional classes: A-class (PpMADS2-1, PpMADS3-1, PpMADS4-1, and PpMADS14-1), C-class (PpMADS15-1), E-class (PpMADS9-1, PpMADS12-1, and PpMADS16-1) and E (F)-class (PpMADS5-1 and PpMADS8-1). Differential gene expression was observed in different fruit tissues (skin, cortex and core) as well as in the cortex during the course of fruit development and ripening. Collectively, our results suggest their involvement in the diverse aspects of plant development including flower development and the course of fruit development and ripening.