In Vitro Calli Production Resulted in Different Profiles of Plant-Derived Medicinal Compounds in Phyllanthus amarus.

The efficient production of plant-derived medicinal compounds (PDMCs) from in vitro plants requires improvements in knowledge about control of plant or organ development and factors affecting the biosynthesis pathway of specific PDMCs under in vitro conditions, leading to a realistic large-scale tool for in vitro secondary metabolite production. Thus, this study aimed to develop an in vitro technique, through the induction and proliferation of calli, for production of plant fresh weight, and to compare the PDMC profile obtained from the plants versus in vitro calli of Phyllanthus amarus. It was successfully possible to obtain and proliferate two types of calli, one with a beige color and a friable appearance, obtained in the dark using Murashige and Skoog (MS) medium plus 2,4-dichlorophenoxyacetic acid (2,4-D), and a second type with a green color, rigid consistency, and nonfriable appearance obtained under light conditions and MS medium plus 6-benzyladenine (6-BA). In vitro micropropagated plants that gave rise to calli were also acclimatized in a greenhouse and cultivated until obtaining the mass for PDMC analysis and used as a control. While the micropropagated-derived plants concentrated the lignans niranthin, nirtetralin, and phyllanthin, the Phyllanthus amarus calli proliferated in vitro concentrated a completely different biochemical profile and synthesis of compounds, such as betulone, squalene, stigmasterol, and ß-sitosterol, in addition to others not identified by GC-MS database. These results demonstrate the possibility of applying the calli in vitro from Phyllanthus amarus for production of important PDMCs unlike those obtained in cultures of differentiated tissues from field plants.

Dynamic Changes of Pectin Epitopes in Cell Walls during the Development of the Procambium-Cambium Continuum in Poplar.

The change of pectin epitopes during procambium-cambium continuum development was investigated by immunolocalization in poplar. The monoclonal antibody JIM5 labels homogalacturonan (HGA) with a low degree of esterification, and the monoclonal antibody JIM7 labels HGA with a high degree of methyl-esterification. Arabinan, rather than galactan, and HGA with low degree of esterification were located in the cell walls of procambial, while HGA with a low degree of esterification was located in the tangential walls, and galactan was located in both the tangential and radial walls of procambial, yet nearly no arabinan was located in the tangential walls of the cambial cells. The changes in pectin distribution took place when periclinal divisions appeared within a procambial trace. The distribution difference of pectin epitopes was also present in procambium-cambium derivatives. The arabinan existed in all cell walls of primary xylem, but was absent from the tangential walls of secondary xylem cells. The galactan existed only in mature primary phloem. Furthermore, 19 pectin methylesterases (PMEs) genes were identified by RNA sequencing, six genes presented highly differentially and were supposed to be involved in the cell wall esterification process. The results provide direct evidence of the dynamic changes of pectin epitopes during the development of the procambium-cambium continuum in poplar.

Transcriptomic profiling of taproot growth and sucrose accumulation in sugar beet (Beta vulgaris L.) at different developmental stages.

In sugar beet (Beta vulgaris L.), taproot weight and sucrose content are the important determinants of yield and quality. However, high yield and low sucrose content are two tightly bound agronomic traits. The advances in next-generation sequencing technology and the publication of sugar beet genome have provided a method for the study of molecular mechanism underlying the regulation of these two agronomic traits. In this work, we performed comparative transcriptomic analyses in the high taproot yield cultivar SD13829 and the high sucrose content cultivar BS02 at five developmental stages. More than 50,000,000 pair-end clean reads for each library were generated. When taproot turned into the rapid growth stage at the growth stage of 82 days after emergence (DAE), eighteen enriched gene ontology (GO) terms, including cell wall, cytoskeleton, and enzyme linked receptor protein signaling pathway, occurred in both cultivars. Differentially expressed genes (DEGs) of paired comparison in both cultivars were enriched in the cell wall GO term. For pathway enrichment analyses of DEGs that were respectively generated at 82 DAE compared to 59 DAE (the earlier developmental stage before taproot turning into the rapid growth stage), plant hormone signal transduction pathway was enriched. At 82 DAE, the rapid enlarging stage of taproot, several transcription factor family members were up-regulated in both cultivars. An antagonistic expression of brassinosteroid- and auxin-related genes was also detected. In SD13829, the growth strategy was relatively focused on cell enlargement promoted by brassinosteroid signaling, whereas in BS02, it was relatively focused on secondarily cambial cell division regulated by cytokinin, auxin and brassinosteroid signaling. Taken together, our data demonstrate that the weight and sucrose content of taproot rely on its growth strategy, which is controlled by brassinosteroid, auxin, cytokinin, and gibberellin.

Transcript profiling of a novel plant meristem, the monocot cambium.

While monocots lack the ability to produce a vascular cambium or woody growth, some monocot lineages evolved a novel lateral meristem, the monocot cambium, which supports secondary radial growth of stems. In contrast to the vascular cambium found in woody angiosperm and gymnosperm species, the monocot cambium produces secondary vascular bundles, which have an amphivasal organization of tracheids encircling a central strand of phloem. Currently there is no information concerning the molecular genetic basis of the development or evolution of the monocot cambium. Here we report high-quality transcriptomes for monocot cambium and early derivative tissues in two monocot genera, Yucca and Cordyline. Monocot cambium transcript profiles were compared to those of vascular cambia and secondary xylem tissues of two forest tree species, Populus trichocarpa and Eucalyptus grandis. Monocot cambium transcript levels showed that there are extensive overlaps between the regulation of monocot cambia and vascular cambia. Candidate regulatory genes that vary between the monocot and vascular cambia were also identified, and included members of the KANADI and CLE families involved in polarity and cell-cell signaling, respectively. We suggest that the monocot cambium may have evolved in part through reactivation of genetic mechanisms involved in vascular cambium regulation.

Seasonal development of cambial activity in relation to xylem formation in Chinese fir.

The vascular cambium is a lateral meristem which can differentiate into secondary phloem and xylem. The secondary growth of woody plants resulting from vascular cambium activity has been a focus of considerable attention, but the quantitative relationships between cambial activity and secondary xylem formation have been little studied. Our analysis of cytological changes in the cambium of Chinese fir (Cunninghamia lanceolata), revealed a significant positive correlation between vascular cambium cell numbers and cambium zone width through the seasonal cycle. Cambium cell numbers and the cambium cell radial diameter were closely related to xylem formation. Immuno-labeling showed that de-esterified homogalacturonan and (1-4)-ß-d-galactan epitopes were highly abundant in cell walls of dormant-stage cambium, whereas high methylesterified homogalacturonan was strongly labeled in the active stage. Raman spectroscopy detected significant changes in the chemical composition of cell walls during the active-dormant stage transition. More pectin and less monolignols occurred in radial cell walls than in tangential walls during the dormant stage, but no significant changes were found in other stages, indicating that pectin accumulation facilitates cell wall expansion, with cambium activity transition. Our quantitative analysis of the relationship between cambial activity and xylem formation, as well as the cell wall modification during the active stage provides useful information about cambial characteristics and xylogenesis.

Effects of Adding Vindoline and MeJA on Production of Vincristine and Vinblastine, and Transcription of their Biosynthetic Genes in the Cultured CMCs of Catharanthus roseus.

Vincristine and vinblastine were found by Liquid Chromatography-Mass Spectrometry (LC-MS) in Catharanthus roseuscambial meristem cells (CMCs) jointly treated with 0.25 mM vindoline and methyl jasmonate (MeJA), suggesting that C. roseus CMCs contain a complete set of the enzymes which are in response to convert vindoline into vincristine and vinblastine. Based on the facts that the transcript levels of vindoline-biosynthetic genes (STR, SGD and D4H) were up-regulated instead of being down-regulated by adding itself to the culture, and that the transcriptional factor ORCA3 was up-regulated simultaneously, we further confirmed that the transcription of STR, SGD, D4H was manipulated by ORCA3.

Plant natural products: history, limitations and the potential of cambial meristematic cells.

Humans have utilised plant derived natural products as medicines for millenia. Moreover, many contemporary pharmaceuticals are also natural products or derivatives thereof. However, the full potential of these compounds remains to be exploited because often they are: complex and difficult to synthesise; found in low quantities; produced by undomesticated and sometimes rare plants; and, their synthesis is routinely influenced by weather conditions. Potentially, the in vitro culture of cells from the corresponding plant species could circumvent some of these problems but the growth of plant cells on an industrial scale is also problematic. The recent isolation and culture of cambial meristematic cells (CMCs), stem cells which ordinarily generate the plant vasculature, may now provide a key platform technology to help realise the full potential of plant natural products.

Allocation of 14C assimilated in late spring to tissue and biochemical stem components of cork oak (Quercus suber L.) over the seasons.

Carbon distribution in the stem of 2-year-old cork oak plants was studied by (14)CO(2) pulse labeling in late spring in order to trace the allocation of photoassimilates to tissue and biochemical stem components of cork oak. The fate of (14)C photoassimilated carbon was followed during two periods: the first 72 h (short-term study) and the first 52 weeks (long-term study) after the (14)CO(2) photosynthetic assimilation. The results showed that (14)C allocation to stem tissues was dependent on the time passed since photoassimilation and on the season of the year. In the first 3 h all (14)C was found in the polar extractives. After 3 h, it started to be allocated to other stem fractions. In 1 day, (14)C was allocated mostly to vascular cambium and, to a lesser extent, to primary phloem; no presence of (14)C was recorded for the periderm. However, translocation of (14)C to phellem was observed from 1 week after (14)CO(2) pulse labeling. The phellogen was not completely active in its entire circumference at labeling, unlike the vascular cambium; this was the tissue that accumulated most photoassimilated (14)C at the earliest sampling. The fraction of leaf-assimilated (14)C that was used by the stem peaked at 57% 1 week after (14)CO(2) plant exposure. The time lag between C photoassimilation and suberin accumulation was ∼8 h, but the most active period for suberin accumulation was between 3 and 7 days. Suberin, which represented only 1.77% of the stem weight, acted as a highly effective sink for the carbon photoassimilated in late spring since suberin specific radioactivity was much higher than for any other stem component as early as only 1 week after (14)C plant labeling. This trend was maintained throughout the whole experiment. The examination of microautoradiographs taken over 1 year provided a new method for quantifying xylem growth. Using this approach it was found that there was more secondary xylem growth in late spring than in other times of the year, because the calculated average cell division time was much shorter.