Proanthocyanidins (PAs) are the second most abundant plant phenolic natural products. PA biosynthesis is regulated by the well-documented MYB/bHLH/WD40 (MBW) complex, but how this complex itself is regulated remains ill defined. Here, in situ hybridization and β-glucuronidase staining show that APETALA2 (AP2), a well-defined regulator of flower and seed development, is strongly expressed in the seed coat endothelium, where PAs accumulate. AP2 negatively regulates PA content and expression levels of key PA pathway genes. AP2 activates MYBL2 transcription and interacts with MYBL2, a key suppressor of the PA pathway. AP2 exerts its function by directly binding to the AT-rich motifs near the promoter region of MYBL2. Molecular and biochemical analyses revealed that AP2 forms AP2-MYBL2-TT8/EGL3 complexes, disrupting the MBW complex and thereby repressing expression of ANR, TT12, TT19, and AHA10. Genetic analyses revealed that AP2 functions upstream of MYBL2, TT2, and TT8 in PA regulation. Our work reveals a new role of AP2 as a key regulator of PA biosynthesis in Arabidopsis. Overall, this study sheds new light on the comprehensive regulation network of PA biosynthesis as well as the dual regulatory roles of AP2 in seed development and accumulation of major secondary metabolites in Arabidopsis.

β-glucosidases (BGLUs) hydrolyze the β-D-glycosidic bond with retention of anomeric configuration. BGLUs were associated with many aspects of plant physiological processes, in particular biotic and abiotic stresses through the activation of phytohormones and defense compounds. However, studies on systematic analysis of the stress- or hormone-responsive BGLU genes in plant are still rare. In this study, total 51 BGLU genes of the glycoside hydrolase family 1 were identified in the genome of the model legume plant Medicago truncatula, and they were classified into five distinct clusters. Sequence alignments revealed several conserved and characteristic motifs among these MtBGLU proteins. Analyses of their putative signal peptides and N-glycosylation site suggested that the majority of MtBGLU members have dual targeting to the vacuole and/or chloroplast. Many regulatory elements possibly related with abiotic stresses and phytohormones were identified in MtBGLU genes. Moreover, Microarray and qPCR analyses showed that these MtBGLU genes exhibited distinct expression patterns in various tissues, and in response to different abiotic stress and hormonal treatments. Notably, MtBGLU21, MtBGLU22, MtBGLU28, and MtBGLU30 in cluster I were dramatically activated by NaCl, PEG, IAA, ABA, SA and GA3 treatments. Collectively, our genome-wide characterization, evolutionary analysis, and expression pattern analysis of MtBGLU genes suggested that BGLU genes play crucial roles in response to various abiotic stresses and hormonal cues in M. truncatula.

Epimedium pubescens Maxim. is a well-known traditional Chinese medicinal herb with flavonol glycosides as the major pharmaceutically active compounds. UDP-glycosyltransferases (UGTs) are a group of enzymes responsible for the glycosylation of flavonoid glycosides. In this study, a genome-wide analysis was performed to identify UGT family genes in E. pubescens. As a result, a total of 339 putative UGT genes were identified, which represents the largest UGT gene family known thus far, implying a significant expansion of the UGT gene family in E. pubescens. All EpUGTs were unevenly distributed across six chromosomes, and they were classified into 17 major groups. The expression profiles showed that UGT genes were differentially expressed in roots, leaves, flowers, shoots and fruits. In particular, several EpUGTs were highly induced by high light intensity, which was consistent with the accumulation level of bioactive flavonoids in E. pubescens. Six UGT79 genes that were preferentially expressed in roots or leaves were successfully expressed in E. coli, and only the recombinant EpGT60 protein was found to be active toward 8-prenylkaempferol and icaritin to produce the key bioactive compounds baohuoside II and baohuoside I. The optimal temperature, pH, k_m and V_max were determined for the recombinant EpGT60 protein. In addition, expression of recombinant EpGT60 in E. coli cell culture led to successful production of baohuoside II when fed 8-prenylkaempferol. Our study provides a foundation for further functional characterization of UGT genes in E. pubescens and provides key candidate genes for bioengineering bioactive flavonoids in E. pubescens.

Alfalfa (Medicago sativa L.) is an important legume forage crop with great economic value. However, as the growth of alfalfa is seriously affected by an inadequate supply of water, drought is probably the major abiotic environmental factor that most severely affects alfalfa production worldwide. In an effort to enhance alfalfa drought tolerance, we transformed the Arabidopsis Enhanced Drought Tolerance 1 (AtEDT1) gene into alfalfa via Agrobacterium-mediated transformation. Compared with wild type plants, drought stress treatment resulted in higher survival rates and biomass, but reduced water loss rates in the transgenic plants. Furthermore, transgenic alfalfa plants had increased stomatal size, but reduced stomatal density, and these stomatal changes contributed greatly to reduced water loss from leaves. Importantly, transgenic alfalfa plants exhibited larger root systems with larger root lengths, root weight, and root diameters than wild type plants. The transgenic alfalfa plants had reduced membrane permeability and malondialdehyde content, but higher soluble sugar and proline content, higher superoxide dismutase activity, higher chlorophyll content, enhanced expression of drought-responsive genes, as compared with wild type plants. Notably, transgenic alfalfa plants grew better in a 2-year field trial and showed enhanced growth performance with increased biomass yield. All of our morphological, physiological, and molecular analyses demonstrated that the ectopic expression of AtEDT1 improved growth and enhanced drought tolerance in alfalfa. Our study provides alfalfa germplasm for use in forage improvement programs, and may help to increase alfalfa production in arid lands.