Poplar PtabZIP1-like enhances lateral root formation and biomass growth under drought stress.

Developing drought-resistance varieties is a major goal for bioenergy crops, such as poplar (Populus), which will be grown on marginal lands with little or no water input. Root architecture can affect drought resistance, but few genes that affect root architecture in relation to water availability have been identified. Here, using activation tagging in the prime bioenergy crop poplar, we have identified a mutant that overcomes the block of lateral root (LR) formation under osmotic stress. Positioning of the tag, validation of the activation and recapitulation showed that the phenotype is caused by the poplar PtabZIP1-like (PtabZIP1L) gene with highest homology to bZIP1 from Arabidopsis. PtabZIP1L is predominantly expressed in roots, particularly in zones where lateral root primordia (LRP) initiate and LR differentiate and emerge. Transgenics overexpressing PtabZIP1L showed precocious LRP and LR development, while PtabZIP1L suppression significantly delayed both LRP and LR formation. Transgenic overexpression and suppression of PtabZIP1L also resulted in modulation of key metabolites like proline, asparagine, valine and several flavonoids. Consistently, expression of both of the poplar Proline Dehydrogenase orthologs and two of the Flavonol Synthases genes was also increased and decreased in overexpressed and suppressed transgenics, respectively. These findings suggest that PtabZIP1L mediates LR development and drought resistance through modulation of multiple metabolic pathways.

A systems biology approach identifies new regulators of poplar root development under low nitrogen.

In Populus, low nitrogen (LN) elicits rapid and vigorous lateral root (LR) proliferation, which is closely mirrored by corresponding transcriptomic changes. Using transcriptomic data, we built a genetic network encompassing a large proportion of the differentially regulated transcriptome. The network is organized in a hierarchical fashion, centered on 11 genes. Transgenic manipulations of only three of the 11 genes had a strong impact on root development under LN. These three genes encoded an F-box protein similar to Hawaiian Skirt (PtaHWS) and two transcription factors (PtaRAP2.11 and PtaNAC1). Up- and downregulation of the three genes caused increased and decreased root proliferation under LN conditions, respectively. The transgenic manipulations had a strong positive effect on growth under greenhouse conditions including increased shoot and root biomass. The three genes appeared to encompass a putative yet-unknown mechanism that underlies root development under LN. Specifically, the genes are predominantly expressed in roots and have a similar temporal response to LN. More importantly, transgenic manipulation for each of the three genes had a highly significant impact on the expression of the other two. The transgenic manipulations appear to also affect the expression of the regulatory miRNA (PtamiRNA164e) of one of the transcription factors (PtaNAC1), albeit in an opposite fashion. Consistent with a putative function of PtaHWS in proteasome degradation, treatment with proteasome inhibitor reversed the expression changes in the transgenic plants. The insights from this study will allow genetic modifications of root architecture for more efficient and dynamic nitrogen foraging in biofuel crops like poplar.

Nitrogen deprivation promotes Populus root growth through global transcriptome reprogramming and activation of hierarchical genetic networks.

We show a distinct and previously poorly characterized response of poplar (Populus tremula × Populus alba) roots to low nitrogen (LN), which involves activation of root growth and significant transcriptome reprogramming. Analysis of the temporal patterns of enriched ontologies among the differentially expressed genes revealed an ordered assembly of functionally cohesive biological events that aligned well with growth and morphological responses. A core set of 28 biological processes was significantly enriched across the whole studied period and 21 of these were also enriched in the roots of Arabidopsis thaliana during the LN response. More than half (15) of the 28 processes belong to gene ontology (GO) terms associated with signaling and signal transduction pathways, suggesting the presence of conserved signaling mechanisms triggered by LN. A reconstruction of genetic regulatory network analysis revealed a sub-network centered on a PtaNAC1 (P. tremula × alba NAM, ATAF, CUC 1) transcription factor. PtaNAC1 root-specific up-regulation increased root biomass and significantly changed the expression of the connected hub genes specifically under LN. Our results provide evidence that the root response to LN involves hierarchically structured genetic networks centered on key regulatory factors. Targeting these factors via genetic engineering or breeding approaches can allow dynamic adjustment of root architecture in response to variable nitrogen availabilities in the soil.

Gene network analysis of poplar root transcriptome in response to drought stress identifies a PtaJAZ3PtaRAP2.6-centered hierarchical network.

Using time-series transcriptomic data from poplar roots undergoing polyethylene glycol (PEG)-induced drought stress, we built a genetic network model of the involved putative molecular responses. We found that the network resembled a hierarchical structure. The highest hierarchical level in this structure is occupied by 9 genes, which we called superhubs because they were primarily connected to 18 hub genes, which are then connected to 2,934 terminal genes. We were only able to regenerate transgenic plants overexpressing two of the superhubs, suggesting that the majority of the superhubs might interfere with the regeneration process and did not allow recovery of transgenic plants. The two superhubs encode proteins with closest homology to JAZ3 and RAP2.6 genes of Arabidopsis and were consequently named PtaJAZ3 and PtaRAP2.6. PtaJAZ3 and PtaRAP2.6 overexpressing transgenic lines showed a significant increase in both root elongation and lateral root proliferation and these responses were specific for the drought stress conditions and were highly correlated with the levels of overexpression of the transgenes. Several lines of evidence suggest of regulatory interactions between the two superhubs. Both superhubs were significantly induced by methyl jasmonate (MeJA). Because jasmonate signaling involves ubiquitin-mediated proteasome degradation, treatment with proteasome inhibitor abolished the MeJA induction for both genes. PtaRAP2.6 was upregulated in PtaJAZ3 transgenics but PtaJAZ3 expression was not affected in the PtaRAP2.6 overexpressors. The discovery of the two genes and further future insights into the associated mechanisms can lead to improved understanding and novel approaches to regulate root architecture in relation to drought stress.

A network of genes associated with poplar root development in response to low nitrogen.

Deployment of the root system is highly sensitive to the levels and spatial distribution of nutrients like nitrogen. However, the genetic determinants of these sensing and deployment mechanisms are still poorly understood. Previously, using system approaches based on temporal changes in root transcriptome in relation to low nitrogen (LN), we have been able to identify a module that activates root production in poplar in response to LN conditions. Here, using comparative, gene ontology and expression analyses, we provide further evidence that the genes in this module are indeed involved in regulation of root development under LN. Better understanding of these modules will enable approaches for breeding for better nitrogen use efficiency through development of a more sensitive and plastic root system.

Genetic networks involved in poplar root response to low nitrogen.

Perception of environmental cues and adaptation to changing environmental conditions are crucial for survival of sessile organisms like plants. This is even more important for woody perennial species like trees that can occupy a site for thousands of years. We have previously shown that under low nitrogen (LN), poplar trees display a vigorous and long-lasting root growth associated with global transcriptomic reprogramming and an activation of hierarchical genetic networks. Here we use computational analysis to better understand the network among the genes showing distinct chronological patterns of expression during the response. Our analyses confirm the previous findings, define new potential signaling pathways and the possible downstream targets of these signaling events.