Genome Engineering by RNA-Guided Transposition for Anabaena sp. PCC 7120.

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.

Identifying Molecular Chechkpoints for Adventitious Root Induction: Are We Ready to Fill the Gaps?

The molecular mechanisms underlying de novo root organogenesis have been under intense study for the last decades. As new tools and resources became available, a comprehensive model connecting the processes and factors involved was developed. Separate phases that allow for specific analyses of individual checkpoints were well defined. Physiological approaches provided information on the importance of metabolic processes and long-distance signaling to balance leaf and stem status and activation of stem cell niches to form new root meristems. The study of plant hormones revealed a series of sequential roles for cytokinin and auxin, dynamically interconnected and modulated by jasmonic acid and ethylene. The identification of genes specifying cell identity uncovered a network of sequentially acting transcriptional regulators that link hormonal control to cell fate respecification. Combined results from herbaceous model plants and the study of recalcitrant woody species underscored the need to understand the limiting factors that determine adventitious rooting competence. The relevance of epigenetic control was emphasized by the identification of microRNAs and chromatin remodeling agents involved in the process. As the different players are set in place and missing pieces become apparent, findings in related processes can be used to identify new candidates to complete the picture. Molecular knobs connecting the balance cell proliferation/differentiation to hormone signaling pathways, transcriptional control of cell fate or metabolic modulation of developmental programs can offer clues to unveil new elements in the dynamics of adventitious rooting regulatory networks. Mechanisms for cell non-autonomous signaling that are well characterized in other developmental processes requiring establishment and maintenance of meristems, control of cell proliferation and cell fate specification can be further explored. Here, we discuss possible candidates and approaches to address or elude the limitations that hinder propagation programs requiring adventitious rooting.

The GRAS gene family in pine: transcript expression patterns associated with the maturation-related decline of competence to form adventitious roots.

BACKGROUND: Adventitious rooting is an organogenic process by which roots are induced from differentiated cells other than those specified to develop roots. In forest tree species, age and maturation are barriers to adventitious root formation by stem cuttings. The mechanisms behind the respecification of fully differentiated progenitor cells, which underlies adventitious root formation, are unknown. RESULTS: Here, the GRAS gene family in pine is characterized and the expression of a subset of these genes during adventitious rooting is reported. Comparative analyses of protein structures showed that pine GRAS members are conserved compared with their relatives in angiosperms. Relatively high GRAS mRNA levels were measured in non-differentiated proliferating embryogenic cultures and during embryo development. The mRNA levels of putative GRAS family transcription factors, including Pinus radiata's SCARECROW (SCR), PrSCR, and SCARECROW-LIKE (SCL) 6, PrSCL6, were significantly reduced or non-existent in adult tissues that no longer had the capacity to form adventitious roots, but were maintained or induced after the reprogramming of adult cells in rooting-competent tissues. A subset of genes, SHORT-ROOT (PrSHR), PrSCL1, PrSCL2, PrSCL10 and PrSCL12, was also expressed in an auxin-, age- or developmental-dependent manner during adventitious root formation. CONCLUSIONS: The GRAS family of pine has been characterized by analyzing protein structures, phylogenetic relationships, conserved motifs and gene expression patterns. Individual genes within each group have acquired different and specialized functions, some of which could be related to the competence and reprogramming of adult cells to form adventitious roots.

Epigenetic regulation of adaptive responses of forest tree species to the environment.

Epigenetic variation is likely to contribute to the phenotypic plasticity and adaptative capacity of plant species, and may be especially important for long-lived organisms with complex life cycles, including forest trees. Diverse environmental stresses and hybridization/polyploidization events can create reversible heritable epigenetic marks that can be transmitted to subsequent generations as a form of molecular "memory". Epigenetic changes might also contribute to the ability of plants to colonize or persist in variable environments. In this review, we provide an overview of recent data on epigenetic mechanisms involved in developmental processes and responses to environmental cues in plant, with a focus on forest tree species. We consider the possible role of forest tree epigenetics as a new source of adaptive traits in plant breeding, biotechnology, and ecosystem conservation under rapid climate change.

Towards decoding the conifer giga-genome.

Several new initiatives have been launched recently to sequence conifer genomes including pines, spruces and Douglas-fir. Owing to the very large genome sizes ranging from 18 to 35 gigabases, sequencing even a single conifer genome had been considered unattainable until the recent throughput increases and cost reductions afforded by next generation sequencers. The purpose of this review is to describe the context for these new initiatives. A knowledge foundation has been acquired in several conifers of commercial and ecological interest through large-scale cDNA analyses, construction of genetic maps and gene mapping studies aiming to link phenotype and genotype. Exploratory sequencing in pines and spruces have pointed out some of the unique properties of these giga-genomes and suggested strategies that may be needed to extract value from their sequencing. The hope is that recent and pending developments in sequencing technology will contribute to rapidly filling the knowledge vacuum surrounding their structure, contents and evolution. Researchers are also making plans to use comparative analyses that will help to turn the data into a valuable resource for enhancing and protecting the world's conifer forests.

CsSCL1 is differentially regulated upon maturation in chestnut microshoots and is specifically expressed in rooting-competent cells.

The Castanea sativa SCL1 gene (CsSCL1) has previously been shown to be induced by auxin during adventitious root (AR) formation in rooting-competent microshoots. However, its expression has not previously been analyzed in rooting-incompetent shoots. This study focuses on the regulation of CsSCL1 during maturation and the role of the gene in the formation of AR. The expression of CsSCL1 in rooting-incompetent microshoots and other tissues was investigated by quantitative reverse transcriptase--polymerase chain reaction. The analysis was complemented by in situ hybridization of the basal segments of rooting-competent and --incompetent microshoots during AR induction, as well as in AR and lateral roots. It was found that CsSCL1 is upregulated by auxin in a cell-type- and phase-dependent manner during the induction of AR. In root-forming shoots, CsSCL1 mRNA was specifically located in the cambial zone and derivative cells, which are rooting-competent cells, whereas in rooting-incompetent shoots the hybridization signal was more diffuse and evenly distributed through the phloem and parenchyma. CsSCL1 expression was also detected in lateral roots and axillary buds. The different CsSCL1 expression patterns in rooting-competent and -incompetent microshoots, together with the specific location of transcripts in cell types involved in root meristem initiation and in the root primordia of AR and lateral roots, indicate an important role for the gene in determining whether certain cells will enter the root differentiation pathway and its involvement in meristem maintenance.

Reprogramming adult cells during organ regeneration in forest species.

The possibility of regenerating whole plants from somatic differentiated cells emphasizes the plasticity of plant development. Cell-type respecification during regeneration can be induced in adult tissues as a consequence of injuries, changes in external or internal stimuli or changes in positional information. However, in many plant species, switching the developmental program of adult cells prior to organ regeneration is difficult, especially in forest species. Besides its impact on forest productivity, basic information on the flexibility of cell differentiation is necessary for a comprehensive understanding of the epigenetic control of cell differentiation and plant development. Studies of reprogramming adult cells in terms of regulative expression changes of selected genes will be of great interest to unveil basic mechanisms regulating cellular plasticity.

Characterization and expression of a Pinus radiata putative ortholog to the Arabidopsis SHORT-ROOT gene.

We characterized a Pinus radiata D. Don putative ortholog to the Arabidopsis thaliana (L.) Heynh. SHORT--ROOT gene (AtSHR) and analyzed its expression in different organs during vegetative development and in response to exogenous auxin during adventitious rooting. The predicted protein sequence contained domains characteristic of the GRAS protein family and showed a strong similarity to the SHORT--ROOT (SHR) proteins. Quantitative reverse transcriptase--polymerase chain reaction (qRT-PCR) and in situ hybridization showed that the gene is predominantly expressed in roots, root primordia and in the cambial region of hypocotyl cuttings. Increased mRNA levels were observed, independently of the presence or absence of exogenous auxin, in the cambial region and rooting competent cells of hypocotyl cuttings within the first 24 h of adventitious rooting, before the activation of cell divisions and the organization of the adventitious root meristem. The expression pattern in organs and during adventitious rooting was similar to that of a Pinus radiata SCARECROW-LIKE (PrSCL1) gene, except that PrSCL1 is induced in response to exogenous auxin. Results suggest that the Pinus radiata SHORT-ROOT (PrSHR) gene has a role in root meristem formation and maintenance and in the cambial region of hypocotyl cuttings.

Two SCARECROW-LIKE genes are induced in response to exogenous auxin in rooting-competent cuttings of distantly related forest species.

We characterized SCARECROW-LIKE genes induced by auxin in rooting-competent cuttings of two distantly related forest species (Pinus radiata D. Don and Castanea sativa Mill.) before the activation of cell division that results in adventitious root formation. The predicted protein sequences contain domains characteristic of the GRAS protein family and show a strong similarity to the SCARECROW-LIKE proteins, indicating conserved functions of these proteins. Quantitative RT-PCR analysis showed that these genes are expressed at relatively high levels in roots. Induction of increased mRNA levels in rooting-competent cuttings of both species in response to exogenous auxin was observed within the first 24 h of the root induction process, a time when cell reorganization takes place, but before the resumption of cell division and the appearance of adventitious root primordia. These results suggest that SCARECROW-LIKE genes play a role during the earliest stages of adventitious root formation.

Differential leaf stress responses in young and senescent plants.

Responses to low temperature, mechanical wounding and salicylic acid (SA) treatments were studied in 3-week-old (young) and 6-week-old (senescent) Arabidopsis thaliana (L.) Heynh. plants by analyzing increases in Pal1 and Pr1 expression and superoxide dismutase (SOD; EC 1.15.1.1) and peroxidase (POX; EC 1.11.1.7) activities. Young plants showed higher Pal1 transcript accumulation after low temperature and wounding. In contrast, senescent plants presented higher accumulation of Pr1 transcripts after SA treatments. Similar results were obtained with the ethylene-insensitive etr1 mutant, suggesting that these differences are not related to increased ethylene content in senescent tissues. SOD activity and inducibility were lower, whereas POX activity and inducibility were higher in senescent plants. A possible relationship between senescence-associated changes in responses to stress and in the metabolism of active oxygen species is discussed.