TWISTED DWARF1 regulates Arabidopsis stamen development by differential activation of ABCB-mediated auxin transport

Despite clear evidence that a local accumulation of auxin is likewise critical for floral organ initiation than for vegetative tissues, much less is known about the molecular key players that regulate auxin-controlled flower development. Here, by an analysis of physiological and morphological parameters and by a spatial and temporal dissection of auxin fluxes and expression of key players of ABCB-mediated auxin transport in the Arabidopsis flower, we demonstrate a crucial role for the FKBP42, TWISTED DWARF1 (TWD1), in the regulation of flower development. Our analyses revealed that TWD1 promotes flower shape and number, stamen elongation, pollen maturation, nectary functionality and seed development. Most of the described developmental defects in twd1 are shared with the abcb1 abcb19 mutant, which can be attributed to the fact that TWD1 as a described ABCB chaperon is a positive regulator of ABCB1 and ABCB19-mediated auxin transport. We predict an overall housekeeping function for ABCB1 during earlier stages, while ABCB19 seems to be responsible for the key event of rapid elongation at later stages of stamen development. Our data indicate that TWD1 controls flower development by differential activation of ABCB-mediated auxin transport.

The Cell Wall Proteome of Craterostigma plantagineum Cell Cultures Habituated to Dichlobenil and Isoxaben

The remarkable desiccation tolerance of the vegetative tissues in the resurrection species Craterostigma plantagineum (Hochst.) is favored by its unique cell wall folding mechanism that allows the ordered and reversible shrinking of the cells without damaging neither the cell wall nor the underlying plasma membrane. The ability to withstand extreme drought is also maintained in abscisic acid pre-treated calli, which can be cultured both on solid and in liquid culture media. Cell wall research has greatly advanced, thanks to the use of inhibitors affecting the biosynthesis of e.g., cellulose, since they allowed the identification of the compensatory mechanisms underlying habituation. Considering the innate cell wall plasticity of C. plantagineum, the goal of this investigation was to understand whether habituation to the cellulose biosynthesis inhibitors dichlobenil and isoxaben entailed or not identical mechanisms as known for non-resurrection species and to decipher the cell wall proteome of habituated cells. The results showed that exposure of C. plantagineum calli/cells triggered abnormal phenotypes, as reported in non-resurrection species. Additionally, the data demonstrated that it was possible to habituate Craterostigma cells to dichlobenil and isoxaben and that gene expression and protein abundance did not follow the same trend. Shotgun and gel-based proteomics revealed a common set of proteins induced upon habituation, but also identified candidates solely induced by habituation to one of the two inhibitors. Finally, it is hypothesized that alterations in auxin levels are responsible for the increased abundance of cell wall-related proteins upon habituation.

Disruption of Brachypodium Lichenase Alters Metabolism of Mixed-linkage Glucan and Starch

Mixed-linkage glucan (MLG), which is widely distributed in grasses, is a polysaccharide highly abundant in cell walls of grass endosperm and young vegetative tissues. Lichenases are enzymes that hydrolyze MLG first identified in MLG-rich lichens. In this study, we identify a gene encoding a lichenase we name Brachypodium distachyon LICHENASE 1 (BdLCH1), which is highly expressed in the endosperm of germinating seeds and coleoptiles and at lower amounts in mature shoots. RNA in situ hybridization showed that BdLCH1 is primarily expressed in chlorenchyma cells of mature leaves and internodes. Disruption of BdLCH1 resulted in an eight-fold increase in MLG content in senesced leaves. Consistent with the in situ hybridization data, immunolocalization results showed that MLG was not removed in chlorenchyma cells of lch1 mutants as it was in wild type and implicate the BdLCH1 enzyme in removing MLG in chlorenchyma cells in mature vegetative tissues. We also show that MLG accumulation in lch1 mutants was resistant to dark induced degradation, and eight-week-old lch1 plants showed a faster rate of starch breakdown than wild type in darkness. Our results suggest a role for BdLCH1 in modifying the cell wall to support highly metabolically active cells.

Overexpression of Douglas-Fir LEAFY COTYLEDON1 (PmLEC1) in Arabidopsis Induces Embryonic Programs and Embryo-Like Structures in the lec1-1 Mutant but Not in Wild Type Plants

Somatic embryogenesis (SE) is the most promising method for the quick propagation of desirable plant genotypes. However, application of SE to conifers remains challenging due to our limited knowledge about the genes involved in embryogenesis and the processes that lead to somatic embryo formation. Douglas-fir, an economically important lumber species, possesses a homolog of the angiosperm embryo-regulatory LEC1 gene. In the present study, we analyzed the potential of Douglas-fir PmLEC1 to induce embryonic programs in the vegetative cells of a heterologous host, Arabidopsis thaliana. PmLEC1 complemented the Arabidopsis lec1-1 null mutant and led to a variety of phenotypes ranging from normal morphology to developmental arrest at various stages in the T1 generation. PmLEC1 did not affect the morphology of wild type Arabidopsis T1 plants. More profound results occurred in T2 generations. PmLEC1 expression induced formation of recurrent somatic embryo-like structures in vegetative tissues of the rescued lec1-1 mutant but loss of apical dominance (bushy phenotype) in wild type plants. The activation of embryonic programs in the lec1-1PmLEC1 T2 plants was confirmed by the presence of the embryo-specific transcripts, OLEOSIN and CRUCIFERIN. In contrast, no embryo-like structures, and no OLEOSIN or CRUCIFERIN were observed in PmLEC1-expressing bushy wild type T2 plants.

Temperature and Different Organs Create Volatile Profile Differences of Edible Gynura [Gynura bicolor (Roxb. ex Willd.) DC]

The volatile profile of the edible vegetable Gynura bicolor [Gynura bicolor (Roxb. ex Willd.) DC] was analyzed using gas chromatography-mass spectrometry (GC-MS). Isocaryophyllene (23.2%), α-pinene (16.8%), α-humulene (9.1%), β-pinene (7.3%), and copaene (7.0%) were identified as the major compounds in the leaves. In the stems, α-pinene (27.1%), β-pinene (13.0%), isocaryophyllene (7.8%), β-myrceneb (7.8%), 1-undecene (5.7%), and copaene (5.3%) were the main components. G. bicolor grows best at 25 °C. When cultivated at different temperatures (20 to 35 °C in incements of 5 °C), the volatile profiles shifted. The proportion of isocaryophyllene was lower at 20 °C than at the other temperatures. The relative amounts of α-pinene and α-humulene were highest at 20 °C, whereas copaene was highest at 35 °C. Principal component analysis (PCA) was used to explore the correlation between volatile compounds identified from the vegetative tissues and temperature treatments. It reveals the same trend with the previous statements and the first principal component (PC1) and the second principal component (PC2) explains up to 90% of the variance. Experimental results revealed that both temperature and vegetative organ correlate with the volatile emission profile of G. bicolor.

Culturable Seed Microbiota of Populus trichocarpa

Plants harbor a diverse community of microbes, whose interactions with their host and each other can influence plant health and fitness. While microbiota in plant vegetative tissues has been extensively studied, less is known about members of the seed microbiota. We used culture-based surveys to identify bacteria and fungi found in the seeds of the model tree, Populus trichocarpa, collected from different sites. We found that individual P. trichocarpa seeds typically contained zero or one microbe, with common taxa including species of Cladosporium, Aureobasidium, Diaporthe, Alternaria, and Pseudomonas, a bacterium. Pseudomonas isolates were associated with seed mortality and were negatively associated with the occurrence of fungal isolates within Epicoccum, Alternaria, and Aureobasidium from the same seed. Next, we conducted an inoculation experiment with one of the isolated seed microbes, Pseudomonas syringae pv. syringae, and found that it reduced seed germination and increased seedling mortality for P. trichocarpa. Our findings highlight common fungi and bacteria in the seeds of P. trichocarpa, prompting further study of their functional consequences. Moreover, our study confirms that P. syringae pv. syringae is a seed pathogen of P. trichocarpa and is the first report that P. syringae pv. syringae is a lethal seedling pathogen of P. trichocarpa, allowing for future work on the pathogenicity of this bacterium in seedlings and potential antagonism with other seed microbes.

Fructan Structure and Metabolism in Overwintering Plants

In northern regions, annual and perennial overwintering plants such as wheat and temperate grasses accumulate fructan in vegetative tissues as an energy source. This is necessary for the survival of wintering tissues and degrading fructan for regeneration in spring. Other types of wintering plants, including chicory and asparagus, store fructan as a reserve carbohydrate in their roots during winter for shoot- and spear-sprouting in spring. In this review, fructan metabolism in plants during winter is discussed, with a focus on the fructan-degrading enzyme, fructan exohydrolase (FEH). Plant fructan synthase genes were isolated in the 2000s, and FEH genes have been isolated since the cloning of synthase genes. There are many types of FEH in plants with complex-structured fructan, and these FEHs control various kinds of fructan metabolism in growth and survival by different physiological responses. The results of recent studies on the fructan metabolism of plants in winter have shown that changes in fructan contents in wintering plants that are involved in freezing tolerance and snow mold resistance might be largely controlled by regulation of the expressions of genes for fructan synthesis, whereas fructan degradation by FEHs is related to constant energy consumption for survival during winter and rapid sugar supply for regeneration or sprouting of tissues in spring.

Ectopic overexpression of a type-II DGAT (CeDGAT2-2) derived from oil-rich tuber of Cyperus esculentus enhances accumulation of oil and oleic acid in tobacco leaves

Background Engineering triacylglycerol (TAG) accumulation in vegetative tissues of non-food crops has become a promising way to meet our increasing demand for plant oils, especially the renewable production of biofuels. The most important target modified in this regard is diacylglycerol acyltransferase (DGAT) enzyme responsible for the final rate-limiting step in TAG biosynthesis. Cyperus esculentus is a unique plant largely accumulating oleic acid-enriched oil in its underground tubers. We speculated that DGAT derived from such oil-rich tubers could function more efficiently than that from oleaginous seeds in enhancing oil storage in vegetative tissues of tobacco, a high-yielding biomass crops. Results Three CeDGAT genes namely CeDGAT1, CeDGAT2-1 and CeDGAT2-2 were identified in C. esculentus by mining transcriptome of developing tubers. These CeDGATs were expressed in tissues tested, with CeDGAT1 highly in roots, CeDGAT2-1 abundantly in leaves, and CeDGAT2-2 predominantly in tubers. Notably, CeDGAT2-2 expression pattern was in accordance with oil dynamic accumulation during tuber development. Overexpression of CeDGAT2-2 functionally restored TAG biosynthesis in TAG-deficient yeast mutant H1246. Oleic acid level was significantly increased in CeDGAT2-2 transgenic yeast compared to the wild-type yeast and ScDGA1-expressed control under culture with and without feeding of exogenous fatty acids. Overexpressing CeDGAT2-2 in tobacco led to dramatic enhancements of leafy oil by 7.15- and 1.7-fold more compared to the wild-type control and plants expressing Arabidopsis seed-derived AtDGAT1. A substantial change in fatty acid composition was detected in leaves, with increase of oleic acid from 5.1% in the wild type to 31.33% in CeDGAT2-2-expressed tobacco and accompanied reduction of saturated fatty acids. Moreover, the elevated accumulation of oleic acid-enriched TAG in transgenic tobacco exhibited no significantly negative impact on other agronomic traits such as photosynthesis, growth rates and seed germination except for small decline of starch content. Conclusions The present data indicate that CeDGAT2-2 has a high enzyme activity to catalyze formation of TAG and a strong specificity for oleic acid-containing substrates, providing new insights into understanding oil biosynthesis mechanism in plant vegetative tissues. Overexpression of CeDGAT2-2 alone can significantly increase oleic acid-enriched oil accumulation in tobacco leaves without negative impact on other agronomy traits, showing CeDGAT2-2 as the desirable target gene in metabolic engineering to enrich oil and value-added lipids in high-biomass plants for commercial production of biofuel oils.

Expression of a Bacterial Trehalose-6-phosphate Synthase otsA Increases Oil Accumulation in Plant Seeds and Vegetative Tissues

We previously demonstrated that exogenous trehalose 6-phosphate (T6P) treatment stabilized WRINKLED1 (WRI1), a master transcriptional regulator of fatty acid (FA) synthesis and increased total FA content in Brassica napus (B. napus) embryo suspension cell culture. Here, we explore Arabidopsis lines heterologously expressing the Escherichia coli T6P synthase (otsA) or T6P phosphatase (otsB) to refine our understanding regarding the role of T6P in regulating fatty acid synthesis both in seeds and vegetative tissues. Arabidopsis 35S:otsA transgenic seeds showed an increase of 13% in fatty acid content compared to those of wild type (WT), while seeds of 35:otsB transgenic seeds showed a reduction of 12% in fatty acid content compared to WT. Expression of otsB significantly reduced the level of WRI1 and expression of its target genes in developing seeds. Like Arabidopsis seeds constitutively expressing otsA, transient expression of otsA in Nicotiana benthamiana leaves resulted in strongly elevated levels of T6P. This was accompanied by an increase of 29% in de novo fatty acid synthesis rate, a 2.3-fold increase in triacylglycerol (TAG) and a 20% increase in total fatty acid content relative to empty vector (EV) controls. Taken together, these data support the heterologous expression of otsA as an approach to increasing TAG accumulation in plant seeds and vegetative tissues.