Plant Cell Reports

The Brassica napus secondary embryogenesis system requires no exogenous growth regulator to stimulate embryo development. It is stable embryogenically over a long period of culture and has a distinct pre-embryogenic stage. This system was used to investigate the morphological and cellular changes occurring in the embryogenic tissue compared to non-embryogenic tissue using various microscopy techniques. A unique ultrastructural feature designated the extracellular matrix (ECM) was observed on the surface of pre-embryogenic embryoids but not on the non-embryogenic individuals. The ECM layer was found to be dominant in the pre-embryogenic stage and reduced to fragments during embryo growth and development in mature embryogenic tissue. This is a novel aspect of the phenotype previously unreported in the Brassica system. This structure might be linked to acquisition of embryogenic competence.

We identified 23 novel proteins that can interact with At TRN1. These proteins are potential candidates of At TRN1 cargo proteins, which will facilitate our comprehending of At TRN1 functions in Arabidopsis. Tranportin 1 (TRN1) carries out the nucleo-cytoplasmic transport of many proteins, thereby ensuring that each of them is delivered to the right compartment for its proper function. These cargo proteins involved in lots of important processes, such as alternative pre-mRNA splicing, transcriptional regulation, and protein translation. Current understanding of cargo proteins transported by Arabidopsis thaliana transportin 1 (AtTRN1) is limited. Here, first we employed the yeast two-hybrid (Y2H) screening to identify proteins that can interact with AtTRN1 in Arabidopsis, and 12 novel proteins were found. Searching for PY-NLS motif in these 12 proteins suggested that no typical PY-NLS motif was present. We next investigated the specific motifs that will mediate the interactions in these sequences, and found that thirteen truncated fragments interacted with AtTRN1, containing 8 acidic and 5 basic fragments, respectively. We also searched the Arabidopsis proteome for homologs of cargo proteins of yeast Kapl04p and mammalian Kapβ2, and PY-NLS motif-containing proteins. Among these proteins, 11 were identified to interact with AtTRN1. The interactions between all the 23 proteins and AtTRN1 were confirmed by both Y2H and bimolecular fluorescence complementation (BiFC) assays. Our results show that AtTRN1 recognizes a broad spectrum of proteins having diverse functions, which will potentially be the cargoes of AtTRN1. Taken together, these results demonstrate the feasibility and potential power of these methods to identify cargo proteins of AtTRN1, and represent a primary and significant step in interpretation of AtTRN1 functionalities.

The transcription factor CONSTANS (CO) plays a central role in the photoperiod pathway by integrating the circadian clock and light signals into a control for flowering time. CO induces FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) expression, and thereby promotes flowering. The ethylene-responsive element-binding factor associated amphiphilic repression (EAR) motif was used to construct a CONSTANS-EAR motif repressor gene (CO-Rep), which was overexpressed in Arabidopsis under the control of the Cauliflower mosaic virus 35S promoter in order to test its potential for flowering time regulation under inductive long day conditions. Morphological abnormalities in the root and cotyledon formation, and dwarfness were frequently seen in the transgenic plants, suggesting that the proper timing, location, and/or level of CO-Rep expression are important for its application. In morphologically normal CO-Rep plants, both bolting and flowering times under inductive long day conditions were twofold greater than in controls. As a result of the delay in flowering, rosette leaf number at bolting, and rosette and cauline leaf number at flowering increased significantly in CO-Rep plants. RT-PCR analysis demonstrated that FT expression was greatly reduced in the CO-Rep plants, while endogenous CO and SOC1 expression levels were not markedly affected. Conservation of CO among a diverse range of plant species, and its involvement in a variety of photoperiodic responses including flowering, suggests a high potential for use of CO-Rep to manipulate such responses in an agronomically desirable manner.

We confirmed the roles of auxin, CK, and strigolactones in apical dominance in peach and established a model of plant hormonal control of apical dominance in peach. Auxin, cytokinin, and strigolactone play important roles in apical dominance. In this study, we analyzed the effect of auxin and strigolactone on the expression of ATP/ADP isopentenyltransferase (IPT) genes (key cytokinin biosynthesis genes) and the regulation of apical dominance in peach. After decapitation, the expression levels of PpIPT1, PpIPT3, and PpIPT5a in nodal stems sharply increased. This observation is consistent with the changes in tZ-type and iP-type cytokinin levels in nodal stems and axillary buds observed after treatment; these changes are required to promote the outgrowth of axillary buds in peach. These results suggest that ATP/ADP PpIPT genes in nodal stems are key genes for cytokinin biosynthesis, as they promote the outgrowth of axillary buds. We also found that auxin and strigolactone inhibited the outgrowth of axillary buds. After decapitation, IAA treatment inhibited the expression of ATP/ADP PpIPTs in nodal stems to impede the increase in cytokinin levels. By contrast, after GR24 (GR24 strigolactone) treatment, the expression of ATP/ADP IPT genes and cytokinin levels still increased markedly, but the rate of increase in gene expression was markedly lower than that observed after decapitation in the absence of IAA (indole-3-acetic acid) treatment. In addition, GR24 inhibited basipetal auxin transport at the nodes (by limiting the expression of PpPIN1a in nodal stems), thereby inhibiting ATP/ADP PpIPT expression in nodal stems. Therefore, strigolactone inhibits the outgrowth of axillary buds in peach only when terminal buds are present.

The nonproteinogenic amino acid, cyclopentenylglycine, is found in certain Flacourtiaceae. This compound may be synthesized by two C1-chain elongations of α-ketoglutarate via α-ketopimelate (C5+2C1) or by condensation of C4 and C3 units (C4+C3), a pathway not involving α-ketopimelate. The following experimental design elucidated the biosynthetic pathway: Idesia polycarpa callus cultures were freshly established from leaf petioles; synthetic α-[1,2-14C]ketopimelate was added to the medium and cultures were incubated for 3 weeks. After isolation and separation of free amino acids from the tissues, the radioactivity incorporated into cyclopentenylglycine was determined. The results establish α-ketopimelate as a precursor for cyclopentenylglycine, thus providing evidence for the C5+2C1 biosynthetic path.

EgMADS3, a pivotal transcription factor, positively regulates MCFA accumulation via binding to the EgLPAAT promoter, advancing lipid content in mesocarp of oil palm. Lipids function as the structural components of cell membranes, which serve as permeable barriers to the external environment of cells. The medium-chain fatty acid in the stored lipids of plants is an important renewable energy. Most research on MCFA production in plant lipid synthesis is based on biochemical methods, and the importance of transcriptional regulation in MCFA synthesis and its incorporation into TAGs needs further research. Oil palm is the most productive oil crop in the world and has the highest productivity among the main oil crops. In this study, the MADS transcription factor (EgMADS3) in the mesocarp of oil palm was characterized. Through the VIGS-virus induced gene silencing, it was determined that the potential target gene of EgMADS3 was related to the biosynthesis of medium-chain fatty acid (MCFA). Transient transformation in protoplasts and qRT-PCR analysis showed that EgMADS3 positively regulated the expression of EgLPAAT. The results of the yeast one-hybrid assays and EMSA indicated the interaction between EgMADS3 and EgLPAAT promoter. Through genetic transformation and fatty acid analysis, it is concluded that EgMADS3 directly regulates the mid-chain fatty acid synthesis pathway of the potential target gene EgLPAAT, thus promotes the accumulation of MCFA and improves the total lipid content. This study is innovative in the functional analysis of the MADS family transcription factor in the metabolism of medium-chain fatty acids (MCFA) of oil palm, provides a certain research basis for improving the metabolic pathway of chain fatty acids in oil palm, and improves the synthesis of MCFA in plants. Our results will provide a reference direction for further research on improving the oil quality through biotechnology of oil palm.

The intergenic regions of banana bunchy top virus (BBTV) DNA-1 to -5 were fused to the green fluorescent protein (GFP) and uidA reporter genes and assessed for promoter activity in transgenic banana (Musa spp. cv. Bluggoe). Promoter activity associated with the BBTV-derived promoters was transgene dependent with greatest activity observed using the GFP reporter. The BBTV promoters (BT1 to BT5) directed expression primarily in vascular-associated cells, although levels of activity varied between individual promoters. Promoters BT4 and BT5 directed the highest levels of GFP expression, while activity from BT1, BT2 and BT3 promoters was considerably weaker. Intron-mediated enhancement, using the maize polyubiquitin 1 (ubi1) intron, generated a significant increase in GUS expression directed by the BBTV promoters in transgenic plants.

Hairy roots of Centaurium erythraea were obtained by infection with Agrobacterium rhizogenes strain LBA 9402. They spontaneously regenerated adventitious shoots in Woody Plant liquid medium without growth regulators. The shoots were grown continuously in Murashige and Skoog (MS) liquid or agar solidified media supplemented with 0.1 mg l−1 indole-3-acetic acid and 1.0 mg l−1 6-benzylaminopurine. These shoots produced roots 4 weeks after transfer into agar-solidified MS medium without phytohormones. Regenerated plants grown and flowered under greenhouse conditions. The transgenic value of the regenerated plants was confirmed by the polymerase chain reaction amplification. Transformation by Agrobacterium rhizogenes alters plant morphology and production of secoiridoid glucosides. The level of secoiridoids was also modified by development stage of transformed plants. The total content of the compounds (expressed as the sum of gentiopicroside, sweroside and swertiamarin) in 10-week old pRi-transformed regenerants was 280 mg g−1 dry weight and was 8-times the content in the sample of commercially available C. erythraea herb.