Genome-Wide Analysis of C/S1-bZIP Subfamilies in Populus tomentosa and Unraveling the Role of PtobZIP55/21 in Response to Low Energy.

C/S1 basic leucine zipper (bZIP) transcription factors are essential for plant survival under energy deficiency. However, studies on the responses of C/S1-bZIPs to low energy in woody plants have not yet been reported. In this study, members of C/S1-bZIP subfamilies in Populus tomentosa were systematically analyzed using bioinformatic approaches. Four C-bZIPs and 10 S1-bZIPs were identified, and their protein properties, phylogenetic relationships, gene structures, conserved motifs, and uORFs were systematically investigated. In yeast two-hybrid assays, direct physical interactions between C-bZIP and S1-bZIP members were observed, highlighting their potential functional synergy. Moreover, expression profile analyses revealed that low energy induced transcription levels of most C/S1-bZIP members, with bZIP55 and bZIP21 (a homolog of bZIP55) exhibiting particularly significant upregulation. When the expression of bZIP55 and bZIP21 was co-suppressed using artificial microRNA mediated gene silencing in transgenic poplars, root growth was promoted. Further analyses revealed that bZIP55/21 negatively regulated the root development of P. tomentosa in response to low energy. These findings provide insights into the molecular mechanisms by which C/S1-bZIPs regulate poplar growth and development in response to energy deprivation.

Homolog of Human placenta-specific gene 8, PcPLAC8-10, enhances cadmium uptake by Populus roots.

Cadmium (Cd) pollution of soil occurs worldwide. Phytoremediation is an effective approach for cleaning up Cd polluted soil. Fast growing Populus species with high Cd uptake capacities are desirable for phytoremediation. Thus, it is important to elucidate the molecular functions of genes involved in Cd uptake by poplars. In this study, PcPLAC8-10, a homolog of Human placenta-specific gene 8 (PLAC8) implicated in Cd transport was functionally characterized in Populus × canescens. PcPLAC8-10 was transcriptionally induced in Cd-treated roots and it encoded a plasma membrane-localized transporter. PcPLAC8-10 exhibited Cd uptake activity when expressed in yeast cells. No difference in growth was observed between wild type (WT) and PcPLAC8-10-overexpressing poplars. PcPLAC8-10-overexpressing poplars exhibited increases in net Cd2+ influxes by 192% and Cd accumulation by 57% in the roots. However, similar reductions in biomass were found in WT and transgenic poplars when exposed to Cd. The complete motif of CCXXXXCPC in PcPLAC8-10 was essential for its Cd transport activity. These results suggest that PcPLAC8-10 is a plasma membrane-localized transporter responsible for Cd uptake in the roots and the complete CCXXXXCPC motif of PcPLAC8-10 plays a key role in its Cd transport activity in poplars.

PcNRAMP1 Enhances Cadmium Uptake and Accumulation in Populus × canescens.

Poplars are proposed for the phytoremediation of heavy metal (HM) polluted soil. Characterization of genes involved in HM uptake and accumulation in poplars is crucial for improving the phytoremediation efficiency. Here, Natural Resistance-Associated Macrophage Protein 1 (NRAMP1) encoding a transporter involved in cadmium (Cd) uptake and transport was functionally characterized in Populus × canescens. Eight putative PcNRAMPs were identified in the poplar genome and most of them were primarily expressed in the roots. The expression of PcNRAMP1 was induced in Cd-exposed roots and it encoded a plasma membrane-localized protein. PcNRAMP1 showed transport activity for Cd2+ when expressed in yeast. The PcNRAMP1-overexpressed poplars enhanced net Cd2+ influxes by 39-52% in the roots and Cd accumulation by 25-29% in aerial parts compared to the wildtype (WT). However, Cd-induced biomass decreases were similar between the transgenics and WT. Further analysis displayed that the two amino acid residues of PcNRAMP1, i.e., M236 and P405, play pivotal roles in regulating its transport activity for Cd2+. These results suggest that PcNRAMP1 is a plasma membrane-localized transporter involved in Cd uptake and transporting Cd from the roots to aerial tissues, and that the conserved residues in PcNRAMP1 are essential for its Cd transport activity in poplars.

Nitrate/ammonium-responsive microRNA-mRNA regulatory networks affect root system architecture in Populus × canescens.

BACKGROUND: Nitrate (NO3-) and ammonium (NH4+) are the primary forms of inorganic nitrogen (N) taken up by plant roots, and a lack of these N sources commonly limits plant growth. To better understand how NO3- and NH4+ differentially affect root system architecture, we analyzed the expression profiles of microRNAs and their targets in poplar roots treated with three forms of nitrogen S1 (NO3-), S2 (NH4NO3, normal), and S3 (NH4+) via RNA sequencing. RESULTS: The results revealed a total of 709 miRNAs. Among them, 57 significantly differentially expressed miRNAs and 28 differentially expressed miRNA-target pairs showed correlated expression profiles in S1 vs. S2. Thirty-six significantly differentially expressed miRNAs and 12 differentially expressed miRNA-target pairs showed correlated expression profiles in S3 vs. S2. In particular, NFYA3, a target of upregulated ptc-miR169i and ptc-miR169b, was downregulated in S1 vs. S2, while NFYA1, a target of upregulated ptc-miR169b, was downregulated in S3 vs. S2 and probably played an important role in the changes in root morphology observed when the poplar plants were treated with different N forms. Furthermore, the miRNA-target pairs ptc-miR169i/b-D6PKL2, ptc-miR393a-5p-AFB2, ptc-miR6445a-NAC14, ptc-miR172d-AP2, csi-miR396a-5p_R + 1_1ss21GA-EBP1, ath-miR396b-5p_R + 1-TPR4, and ptc-miR166a/b/c-ATHB-8 probably contributed to the changes in root morphology observed when poplar plants were treated with different N forms. CONCLUSIONS: These results demonstrate that differentially expressed miRNAs and their targets play an important role in the regulation of the poplar root system architecture by different N forms.

Physiological Characteristics and Transcriptomic Dissection in Two Root Segments with Contrasting Net Fluxes of Ammonium and Nitrate of Poplar Under Low Nitrogen Availability.

To investigate physiological and transcriptomic regulation mechanisms underlying the distinct net fluxes of NH4+ and NO3- in different root segments of Populus species under low nitrogen (N) conditions, we used saplings of Populus × canescens supplied with either 500 (normal N) or 50 (low N) µM NH4NO3. The net fluxes of NH4+ and NO3-, the concentrations of NH4+, amino acids and organic acids and the enzymatic activities of nitrite reductase (NiR) and glutamine synthetase (GS) in root segment II (SII, 35-70 mm to the apex) were lower than those in root segment I (SI, 0-35 mm to the apex). The net NH4+ influxes and the concentrations of organic acids were elevated, whereas the concentrations of NH4+ and NO3- and the activities of NiR and GS were reduced in SI and SII in response to low N. A number of genes were significantly differentially expressed in SII vs SI and in both segments grown under low vs normal N conditions, and these genes were mainly involved in the transport of NH4+ and NO3-, N metabolism and adenosine triphosphate synthesis. Moreover, the hub gene coexpression networks were dissected and correlated with N physiological processes in SI and SII under normal and low N conditions. These results suggest that the hub gene coexpression networks play pivotal roles in regulating N uptake and assimilation, amino acid metabolism and the levels of organic acids from the tricarboxylic acid cycle in the two root segments of poplars in acclimation to low N availability.

Identification and characterization of circular RNAs during wood formation of poplars in acclimation to low nitrogen availability.

MAIN CONCLUSION: Circular RNA (circRNA) identification and expression profiles, and construction of circRNAs-miRNAs-mRNAs networks indicates that circRNAs are involved in wood formation of poplars in acclimation to low nitrogen availability. Circular RNAs (circRNAs) are covalently closed non-coding RNAs that play pivotal roles in various biological processes. However, circRNAs' roles in wood formation of poplars in acclimation to low nitrogen (N) availability are currently unknown. Here, we undertook a systematic identification and characterization of circRNAs in the wood of Populus × canescens exposed to either 50 (low N) or 500 (normal N) µM NH4NO3 using rRNA-depleted RNA-sequencing. A total of 2,509 unique circRNAs were identified, and 163 (ca. 6.5%) circRNAs were significantly differentially expressed (DE) under low N condition. We observed a positive correlation between the expression patterns of DE circRNAs and their hosting protein-coding genes. Moreover, circRNAs-miRNAs-mRNAs' networks were identified in the wood of poplars under low N availability. For instance, upregulated several circRNAs, such as circRNA1226, circRNA 1732, and circRNA392 induced increases in nuclear factor Y, subunit A1-A (NFYA1-A), NFYA1-B, and NFYA10 transcript levels via the mediation of miR169b members, which is in line with reduced xylem width and cell layers of the xylem in the wood of low N-supplied poplars. Upregulation of circRNA1006, circRNA1344, circRNA1941, circRNA901, and circRNA146 caused increased transcript level of MYB61 via the mediation of a miR5021 member, corresponding well to the higher lignin concentration in the wood of low N-treated poplars. Overall, these results indicated that DE circRNAs play an essential role in regulating gene expression via circRNAs-miRNAs-mRNAs' networks to modulate wood anatomical and chemical properties of poplars in acclimation to low N availability.

Competing Endogenous RNA Networks Underlying Anatomical and Physiological Characteristics of Poplar Wood in Acclimation to Low Nitrogen Availability.

Although poplar plantations are often established on nitrogen (N)-poor soil, the physiological and molecular mechanisms underlying wood properties of poplars in acclimation to low N availability remain largely unknown. To investigate wood properties of poplars in acclimation to low N, Populus � canescens saplings were exposed to either 50 (low N) or 500 (normal N) �M NH4NO3 for 2 months. Low N resulted in decreased xylem width and cell layers of the xylem (the number of cells counted along the ray parenchyma on the stem cross section), narrower lumina of vessels and fibers, greater thickness of double fiber walls (the walls between two adjacent fiber cells), more hemicellulose and lignin deposition, and reduced cellulose accumulation in poplar wood. Consistently, concentrations of gibberellins involved in cell size determination and the abundance of various metabolites including amino acids, carbohydrates and precursors for cell wall biosynthesis were decreased in low N-supplied wood. In line with these anatomical and physiological changes, a number of mRNAs, long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) were significantly differentially expressed. Competing endogenous RNA regulatory networks were identified in the wood of low N-treated poplars. Overall, these results indicate that miRNAs-lncRNAs-mRNAs networks are involved in regulating wood properties and physiological processes of poplars in acclimation to low N availability.