| 1. |
- Arevalo, Sergio, et al.
(author)
-
Genome Engineering by RNA-Guided Transposition for Anabaena sp. PCC 7120
- 2024
-
In: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 13:3, s. 901-912
-
Journal article (peer-reviewed)abstract
- In genome engineering, the integration of incoming DNA has been dependent on enzymes produced by dividing cells, which has been a bottleneck toward increasing DNA insertion frequencies and accuracy. Recently, RNA-guided transposition with CRISPR-associated transposase (CAST) was reported as highly effective and specific in Escherichia coli. Here, we developed Golden Gate vectors to test CAST in filamentous cyanobacteria and to show that it is effective in Anabaena sp. strain PCC 7120. The comparatively large plasmids containing CAST and the engineered transposon were successfully transferred into Anabaena via conjugation using either suicide or replicative plasmids. Single guide (sg) RNA encoding the leading but not the reverse complement strand of the target were effective with the protospacer-associated motif (PAM) sequence included in the sgRNA. In four out of six cases analyzed over two distinct target loci, the insertion site was exactly 63 bases after the PAM. CAST on a replicating plasmid was toxic, which could be used to cure the plasmid. In all six cases analyzed, only the transposon cargo defined by the sequence ranging from left and right elements was inserted at the target loci; therefore, RNA-guided transposition resulted from cut and paste. No endogenous transposons were remobilized by exposure to CAST enzymes. This work is foundational for genome editing by RNA-guided transposition in filamentous cyanobacteria, whether in culture or in complex communities. [GRAPHICS] .
|
|
| 2. |
- de Vries, Jan, et al.
(author)
-
Cytokinin-induced promotion of root meristem size in the fern Azolla supports a shoot-like origin of euphyllophyte roots.
- 2016
-
In: New Phytologist. - : Wiley. - 0028-646X .- 1469-8137. ; 209:2, s. 705-720
-
Journal article (peer-reviewed)abstract
- The phytohormones cytokinin and auxin orchestrate the root meristem development in angiosperms by determining embryonic bipolarity. Ferns, having the most basal euphyllophyte root, form neither bipolar embryos nor permanent embryonic primary roots but rather an adventitious root system. This raises the questions of how auxin and cytokinin govern fern root system architecture and whether this can tell us something about the origin of that root. Using Azolla filiculoides, we characterized the influence of IAA and zeatin on adventitious fern root meristems and vasculature by Nomarski microscopy. Simultaneously, RNAseq analyses, yielding 36 091 contigs, were used to uncover how the phytohormones affect root tip gene expression. We show that auxin restricts Azolla root meristem development, while cytokinin promotes it; it is the opposite effect of what is observed in Arabidopsis. Global gene expression profiling uncovered 145 genes significantly regulated by cytokinin or auxin, including cell wall modulators, cell division regulators and lateral root formation coordinators. Our data illuminate both evolution and development of fern roots. Promotion of meristem size through cytokinin supports the idea that root meristems of euphyllophytes evolved from shoot meristems. The foundation of these roots was laid in a postembryonically branching shoot system.
|
|
| 3. |
- Dymek, Katarzyna, et al.
(author)
-
Investigation of the metabolic consequences of impregnating spinach leaves with trehalose and applying a pulsed electric field.
- 2016
-
In: Bioelectrochemistry. - : Elsevier BV. - 1878-562X .- 1567-5394. ; 112, s. 153-157
-
Journal article (peer-reviewed)abstract
- The impregnation of leafy vegetables with cryoprotectants using a combination of vacuum impregnation (VI) and pulsed electric fields (PEF) has been proposed by our research group as a method of improving their freezing tolerance and consequently their general quality after thawing. In this study, we have investigated the metabolic consequences of the combination of these unit operations on spinach. The vacuum impregnated spinach leaves showed a drastic decrease in the porosity of the extracellular space. However, at maximum weight gain, randomly located air pockets remained, which may account for oxygen-consuming pathways in the cells being active after VI. The metabolic activity of the impregnated leaves showed a drastic increase that was further enhanced by the application of PEF to the impregnated tissue. Impregnating the leaves with trehalose by VI led to a significant accumulation of trehalose-6-phosphate (T6P), however, this was not further enhanced by PEF. It is suggested that the accumulation of T6P in the leaves may increase metabolic activity, and increase tissue resistance to abiotic stress.
|
|
| 4. |
- Ma, Jingkun, et al.
(author)
-
The sucrose-regulated Arabidopsis transcription factor bZIP11 reprograms metabolism and regulates trehalose metabolism
- 2011
-
In: New Phytologist. - : Wiley. - 0028-646X .- 1469-8137. ; 191:3, s. 733-745
-
Journal article (peer-reviewed)abstract
- • The Arabidopsis basic region-leucine zipper transcription factor 11 (bZIP11) is known to be repressed by sucrose through a translational inhibition mechanism that requires the conserved sucrose control peptide encoded by the mRNA leader. The function of bZIP11 has been investigated in over-expression studies, and bZIP11 has been found to inhibit plant growth. The addition of sugar does not rescue the growth inhibition phenotype. Here, the function of the bZIP11 transcription factor was investigated. • The mechanism by which bZIP11 regulates growth was studied using large-scale and dedicated metabolic analysis, biochemical assays and molecular studies. • bZIP11 induction results in a reprogramming of metabolism and activation of genes involved in the metabolism of trehalose and other minor carbohydrates such as myo-inositol and raffinose. bZIP11 induction leads to reduced contents of the prominent growth regulatory molecule trehalose 6-phosphate (T6P). • The metabolic changes detected mimic in part those observed in carbon-starved plants. It is proposed that bZIP11 is a powerful regulator of carbohydrate metabolism that functions in a growth regulatory network that includes T6P and the sucrose non-fermenting-1 related protein kinase 1 (SnRK1).
|
|
