Jasmonic acid is a downstream component in the modulation of somatic embryogenesis by Arabidopsis Class 2 phytoglobin.

Previous studies have shown that the beneficial effect of suppression of the Arabidopsis phytoglobin 2 gene, PGB2, on somatic embryogenesis occurs through the accumulation of nitric oxide (NO) within the embryogenic cells originating from the cultured explant. NO activates the expression of Allene oxide synthase (AOS) and Lipoxygenase 2 (LOX2), genes encoding two key enzymes of the jasmonic acid (JA) biosynthetic pathway, elevating JA content within the embryogenic tissue. The number of embryos in the single aos1-1 mutant and pgb2-aos1-1 double mutant declined, and was not rescued by increasing levels of NO stimulating embryogenesis in wild-type tissue. NO also influenced JA responses by up-regulating PLANT DEFENSIN 1 (PDF1) and JASMONATE-ZIM-PROTEIN (JAZ1), as well as down-regulating MYC2. The NO and JA modulation of MYC2 and JAZ1 controlled embryogenesis. Ectopic expression of JAZ1 or suppression of MYC2 promoted the formation of somatic embryos, while repression of JAZ1 and up-regulation of MYC2 reduced the embryogenic performance. Sustained expression of JAZ1 induced the transcription of several indole acetic acid (IAA) biosynthetic genes, resulting in higher IAA levels in the embryogenic cells. Collectively these data fit a model integrating JA in the PGB2 regulation of Arabidopsis embryogenesis. Suppression of PGB2 increases JA through NO. Elevated levels of JA repress MYC2 and induce JAZ1, favoring the accumulation of IAA in the explants and the subsequent production of somatic embryos.

Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis.

Programmed cell death (PCD) in multicellular organisms is a vital process in growth, development, and stress responses that contributes to the formation of tissues and organs. Although numerous studies have defined the molecular participants in apoptotic and PCD cascades, successful identification of early master regulators that target specific cells to live or die is limited. Using Zea mays somatic embryogenesis as a model system, we report that the expressions of two plant hemoglobin (Hb) genes (ZmHb1 and ZmHb2) regulate the cell survival/death decision that influences somatic embryogenesis through their cell-specific localization patterns. Suppression of either of the two ZmHbs is sufficient to induce PCD through a pathway initiated by elevated NO and Zn2+ levels and mediated by production of reactive oxygen species. The effect of the death program on the fate of the developing embryos is dependent on the localization patterns of the two ZmHbs. During somatic embryogenesis, ZmHb2 transcripts are restricted to a few cells anchoring the embryos to the subtending embryogenic tissue, whereas ZmHb1 transcripts extend to several embryonic domains. Suppression of ZmHb2 induces PCD in the anchoring cells, allowing the embryos to develop further, whereas suppression of ZmHb1 results in massive PCD, leading to abortion. We conclude that regulation of the expression of these ZmHbs has the capability to determine the developmental fate of the embryogenic tissue during somatic embryogenesis through their effect on PCD. This unique regulation might have implications for development and differentiation in other species.

Gene expression analysis in microdissected shoot meristems of Brassica napus microspore-derived embryos with altered SHOOTMERISTEMLESS levels.

Altered expression of Brassica napus (Bn) SHOOTMERISTEMLESS (STM) affects the morphology and behaviour of microspore-derived embryos (MDEs). While down-regulation of BnSTM repressed the formation of the shoot meristem (SAM) and reduced the number of Brassica MDEs able to regenerate viable plants at germination, over-expression of BnSTM enhanced the structure of the SAM and improved regeneration frequency. Within dissected SAMs, the induction of BnSTM up-regulated the expression of many transcription factors (TFs) some of which directly involved in the formation of the meristem, i.e. CUP-SHAPED COTYLEDON1 and WUSCHEL, and regulatory components of the antioxidant response, hormone signalling, and cell wall synthesis and modification. Opposite expression patterns for some of these genes were observed in the SAMs of MDEs down-regulating BnSTM. Altered expression of BnSTM affected transcription of cell wall and lignin biosynthetic genes. The expression of PHENYLALANINE AMMONIA LYASE2, CINNAMATE 4-4HYDROXYLASE, and CINNAMYL ALCOHOL DEHYDROGENASE were repressed in SAMs over-expressing BnSTM. Since lignin formation is a feature of irreversible cell differentiation, these results suggest that one way in which BnSTM promotes indeterminate cell fate may be by preventing the expression of components of biochemical pathways involved in the accumulation of lignin in the meristematic cells. Overall, these studies provide evidence for a novel function of BnSTM in enhancing the quality of in vitro produced meristems, and propose that this gene can be used as a potential target to improve regeneration of cultured embryos.

Carrot (Daucus carota L.).

Plants are susceptible to infection by a broad range of fungal pathogens. A range of proteins have been evaluated that can enhance tolerance to these pathogens by heterologous expression in transgenic carrot tissues. The protocols for carrot transformation with Arabidopsis NPR1 (Non-Expressor of Pathogenesis-Related Proteins 1) are described in this chapter, using the herbicide resistance gene bar, which encodes phosphinothricin acetyltransferase, as a selectable marker. In this protocol, petiole segments (0.5-1.0 cm long) from aseptically grown carrot seedlings are exposed to Agrobacterium tumefaciens strain LBA4404 for 10-30 min and cocultivated for 2-3 days. Herbicide selection is then imposed for 8-12 weeks on a series of different tissue culture media until embryogenic calli are produced. The transfer of the embryogenic calli to hormone-free medium results in embryo development which eventually gives rise to transgenic plantlets. Embryogenic calli can also be propagated in suspension cultures. This protocol has yielded transgenic carrot plants with defined T-DNA inserts at the rate of between 1 and 3 Southern-positive independent events out of 100.

Hemoglobin regulation of plant embryogenesis and plant pathogen interaction.

Plant hemoglobins are ubiquitous molecules involved in several aspects of plant development and stress responses. Studies on the functional aspects of plant hemoglobins at the cellular level in these processes are limited, despite their ability to scavenge nitric oxide (NO), an important signal molecule interfering with hormone synthesis and sensitivity. This mini-review summarizes current knowledge on plant hemoglobins, analyzes their participation in plant pathogen interaction and embryogenesis and proposes a possible model centering on jasmonic acid (JA) as a downstream component of hemoglobin responses.

Liuwei Dihuang (LWDH), a traditional Chinese medicinal formula, protects against ß-amyloid toxicity in transgenic Caenorhabditis elegans.

Liuwei Dihuang (LWDH), a classic Chinese medicinal formula, has been used to improve or restore declined functions related to aging and geriatric diseases, such as impaired mobility, vision, hearing, cognition and memory. Here, we report on the effect and possible mechanisms of LWDH mediated protection of ß-amyloid (Aß) induced paralysis in Caenorhabditis elegans using ethanol extract (LWDH-EE) and water extract (LWDH-WE). Chemical profiling and quantitative analysis revealed the presence of different levels of bioactive components in these extracts. LWDH-WE was rich in polar components such as monosaccharide dimers and trimers, whereas LWDH-EE was enriched in terms of phenolic compounds such as gallic acid and paeonol. In vitro studies revealed higher DPPH radical scavenging activity for LWDH-EE as compared to that found for LWDH-WE. Neither LWDH-EE nor LWDH-WE were effective in inhibiting aggregation of Aß in vitro. By contrast, LWDH-EE effectively delayed Aß induced paralysis in the transgenic C. elegans (CL4176) model which expresses human Aß1-42. Western blot revealed no treatment induced reduction in Aß accumulation in CL4176 although a significant reduction was observed at an early stage with respect to ß-amyloid deposition in C. elegans strain CL2006 which constitutively expresses human Aß1-42. In addition, LWDH-EE reduced in vivo reactive oxygen species (ROS) in C. elegans (CL4176) that correlated with increased survival of LWDH-EE treated N2 worms under juglone-induced oxidative stress. Analysis with GFP reporter strain TJ375 revealed increased expression of hsp16.2::GFP after thermal stress whereas a minute induction was observed for sod3::GFP. Quantitative gene expression analysis revealed that LWDH-EE repressed the expression of amy1 in CL4176 while up-regulating hsp16.2 induced by elevating temperature. Taken together, these results suggest that LWDH extracts, particularly LWDH-EE, alleviated ß-amyloid induced toxicity, in part, through up-regulation of heat shock protein, antioxidant activity and reduced ROS in C. elegans.