We developed a new model system to analyze physiological behavior at the single-cell level in whole plants. Wolffiella hyalina is a species of rootless duckweed, which has a thin and very small structure and can grow rapidly on the surface of culture medium. Epidermal and mesophyll cells were transfected with a reporter gene using particle bombardment and were observed at the single-cell level in the whole living plant. An EM-CCD camera system with a macro zoom microscope was used to capture time-lapse images of bioluminescence, and we successfully detected circadian rhythms in individual cells that expressed a luciferase gene under the control of a circadian promoter. We also detected individual S-phase cells in meristematic tissues of intact W. hyalina plants by using a 5-ethynyl-2′-deoxyuridine (EdU)-labeling assay. Our observations indicated that low-molecular-weight compounds could access the inside of the plant body. Thus, W. hyalina showed the experimental characteristics suitable for single-cell analyses that could be combined with whole-plant observations and/or pharmacological analyses/chemical biology.
In this study, two temperature-induced lipocalin genes SlTIL1 and SlTIL2, and a chloroplastic lipocalin gene SlCHL were isolated from ‘Micro-Tom’ tomato. The coding sequences of SlTIL1, SlTIL2 and SlCHL were 558, 558, and 1002 bp, respectively. By TargetP analysis, no characteristic transit peptides were predicted in the proteins of SlTIL1 and SlTIL2, while a chloroplastic transit peptide was predicted in the protein of SlCHL. The subcellular localization results indicated that SlTIL1 and SlTIL2 proteins were major localized in the plasma membrane, while SlCHL was localized in chloroplast. To understand the function of lipocalins, transgenic tomato over-expressed SlTIL1, SlTIL2 and SlCHL and their virus-induced gene silencing (VIGS) plants were generated. The phenotypes were significantly affected when the SlTIL1, SlTIL2 and SlCHL were over-expressed or silenced by VIGS, which suggested that the three lipocalins played important roles in regulating the growth and development of tomato. In addition, the level of ROS (O2− and H2O2) was low in SlTIL1, SlTIL2 and SlCHL over-expressed plants, while it was high in their silenced plants. The changes in the expression of SODs were consistent with the accumulations of ROS, which indicated that lipocalins might have an important role in abiotic oxidative stress tolerance in tomato plants. Especially SlTIL1 and SlTIL2 are localized around their membranes and protect them from ROS. The results will contribute to elucidating the functions of lipocalin in plants, and provide new strategies to improve the tolerance to abiotic stress in tomato plants.
MADS-box transcription factors (TFs) are involved in a variety of processes in flowering plants ranging from root growth to flower and fruit development. However, studies of the tolerance-related functions of MADS-box genes are very limited, and to date no such studies have been conducted on Camellia sinensis. To gain insight into the functions of genes of this family and to elucidate the role they may play in tissue development and Al and F response, we identified 45 MADS-box genes through transcriptomic analysis of C. sinensis. Phylogenetic analysis of these CsMADS-box genes, along with their homologues in Arabidopsis thaliana, enabled us to classify them into distinct groups, including: M-type (Mα), MIKC* and MIKCc (which contains the SOC1, AGL12, AGL32, SEP, ANR1, SVP, and FLC subgroups). Conserved motif analysis of the CsMADS-box proteins revealed diverse motif compositions indicating a complex evolutionary relationship. Finally, we examined the expression patterns of CsMADS-box genes in various tissues and under different Al and F concentration treatments. Our qPCR results showed that these CsMADS-box genes were involved in Al and F accumulation and root growth in C. sinensis. These findings lay the foundation for future research on the function of CsMADS-box genes and their role in response to Al and F accumulation in root tissues.
Pollen coat components are derived from tapetum cells, which contain elaioplasts derived from plastids and tapetosome derived from the endoplasmic reticulum. In Brassica napus, the main neutral lipids in the elaioplast and tapetosome have been reported to be sterol ester and triacylglycerol, respectively. Isopentenyl pyrophosphate, the structural component of sterol, is produced via the cytosolic mevalonate (MVA) and plastidic methylerythritol phosphate (MEP) pathways. Although these two pathways are compartmentalized, partial cross-talk between them has been reported. To investigate the contribution of these two pathways in elaioplast formation, we characterized mutant pollen of these two pathways. We observed the anthers of male sterile hmg1-1 and atipi1 atipi2 mutants ultrastructurally, which were deficient in MVA pathway enzymes. hmg1-1 and atipi1atipi2 showed a shrunken elaioplast inner granule at the bicellular pollen stage. Conversely, in the cla1-1 mutant, which showed a defective MEP pathway, elaioplast development was normal. The pollen of hmg1-1 and atipi1atipi2 was coatless, whereas cla1-1 had a pollen coat. These results indicate that the MVA pathway but not the MEP pathway is critical for elaioplast development though the organelle is derived from plastids.
Pongamia pinnata is a legume plant which has great potential to be used as a biofuel feedstock. Conventional propagation of P. pinnata was found to be inefficient for mass propagation. Employing plant tissue culture techniques for micropropagation and further plant improvement of P. pinnata will be the right path to fulfill future challenges in biofuel production. This study aimed to establish a plant regeneration system for potential micropropagation and genetic manipulation of P. pinnata in future. In vitro nodal explants were used and Woody Plant Medium (WPM) containing 30 µM 6-benzylaminopurine (BAP) and 1 mM phloroglucinol (PG) was able to induce higher frequency of multiple shoot buds compared to other media investigated in this study. For shoot regeneration study, WPM containing 15 µM of zeatin and 1 mM PG was able to induce longer shoots while rooting of the regenerated shoots was enhanced by WPM supplemented with indole-3-butyric acid (IBA) in combination with silver thiosulphate (STS). A simple and effective acclimatisation protocol was established with very high survival frequency of regenerated plantlets. Root nodulation of the successfully acclimatised plants was also observed. In short, multiple shoot buds were successfully induced, regenerated and rooted in vitro. The rooted plantlets were successfully acclimatised and grown healthily. It was concluded that a successful plant regeneration protocol of P. pinnata was achieved for potential application in micropropagation and genetic manipulation.
Transmission electron microscopy (TEM) combined with freeze substitution was employed to examine the ultrastructure of cells of gentian shoot tips cooled to the ultra-low temperature of slush nitrogen and liquid nitrogen. When shoot tips were cooled in ultra-low temperature without plant vitrification solution 2 (PVS2) treatment, massive ice formation was observed throughout the cells, indicating that severe injury occurred during cooling. In contrast, when shoot tips were treated with PVS2 and subsequently cooled to ultra- low temperatures, no ice crystals were observed in the cells. In addition, the cells of PVS2-treated shoot tips exhibited considerable plasmolysis and formation of small vesicles in cytoplasm. These results clearly demonstrate that the PVS2 treatment is essential for preventing damage caused by ice formation and for successful cryopreservation of plant shoot tips.
Grey mangrove (Avicennia marina) is a traditional medicine used for the treatment of various diseases, including rheumatism and ulcers; however, the compounds responsible for its curative effects remain largely unknown. Triterpenoids are a diverse group of plant-specialized metabolites derived from a common precursor, 2,3-oxidosqualene. Triterpenoids are potentially responsible for the beneficial effects of A. marina; however, the chemical profiles of triterpenoids in A. marina and their biosynthetic genes have not been identified. Cytochrome P450 monooxygenases (P450s) have key roles in the structural diversification of plant triterpenoids by catalyzing site-specific oxidation of triterpene scaffolds. Recent studies have revealed that the CYP716 family represents the most common clade of P450s involved in triterpenoid biosynthesis. In this study, we performed triterpenoid profiling and RNA sequencing of A. marina leaves. Mining of CYP716 family genes and enzymatic activity assays of encoded proteins revealed that CYP716A259 catalyzed oxidation at the C-28 position of the pentacyclic triterpene skeletons of β-amyrin, α-amyrin, and lupeol to produce oleanolic acid, ursolic acid, and betulinic acid, respectively. The other functionally defined P450, CYP716C53, catalyzed the C-2α hydroxylation of oleanolic acid and ursolic acid to produce maslinic acid and corosolic acid, respectively. The possible involvement of CYP716A259 and CYP716C53 in the biosynthesis of these health-benefiting compounds in A. marina leaves, and the possible contribution of the resulting compounds to the reported bioactivities of A. marina leaf extract, are discussed.