Growth-limiting drought stress induces time-of-day dependent transcriptome and physiological responses in hybrid poplar

Drought stress negatively impacts the health of long-lived trees. Understanding the genetic mechanisms that underpin response to drought stress is requisite for selecting or enhancing climate change resilience. We aimed to determine how hybrid poplars respond to prolonged and uniform exposure to drought; how responses to moderate and more severe growth-limiting drought stresses differed; and, how drought responses change throughout the day. We established hybrid poplar trees (Populus x 'Okanese') from unrooted stem cutting with abundant soil moisture for six weeks. We then withheld water to establish well-watered, moderate, and severe growth-limiting drought conditions. These conditions were maintained for three weeks during which growth was monitored. We then measured photosynthetic rates and transcriptomes of leaves that had developed during the drought treatments at two times of day. The moderate and severe drought treatments elicited distinct changes in growth and development, photosynthetic rates, and global transcriptome profiles. Notably, the time of day of sampling produced the strongest signal in the transcriptome data. The moderate drought treatment elicited global transcriptome changes that were intermediate to the severe and well-watered treatments in the early evening, but did not elicit a strong drought response in the morning, emphasizing the complex nature of drought regulation in long-lived trees.

CRISPR-Knockout of CSE Gene Improves Saccharification Efficiency by Reducing Lignin Content in Hybrid Poplar

Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1–sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

Growth and foliar nutrition of a hybrid poplar clone following the application of a mixture of papermill biosolids and lime mud

Fertilization of hybrid poplar (HP) plantations with papermill by-products is a promising solution to improve soil fertility and nutrient availability, increase plantation productivity, and provide added value to these materials that would otherwise be incinerated or sent to the landfill. We assessed the growth and foliar nutrition of a HP clone (Populus x. canadensis × P. maximowiczii) at six plantation sites aged three to five years in southern Quebec, Canada. Sites received a fertilization treatment consisting of a mixture of papermill biosolids (120 to 140 t ha-1, depending on site) and lime mud (10 to 15 t ha-1) before being planted, or no fertilization (control). Tree growth was significantly improved by fertilization, with fertilized trees showing a mean annual height increment of 1.3 m (all-site mean; SD = 0.2), compared with 0.5 m (SD = 0.4) for unfertilized trees. Foliar Ca and Mg increased following fertilization and levels met optimal thresholds at all sites, whereas N, P, and K concentrations also increased but nutritional deficiencies remained for these elements at several sites. Our results confirm the benefits of fertilizing hybrid poplars with papermill by-products, but indicate that adjustments in application rates or type of by-products could be made in order to fully satisfy nutritional requirements and thus optimize tree growth.

Overexpression of a SOC1-Related Gene Promotes Bud Break in Ecodormant Poplars

Perennial species in the boreal and temperate regions are subject to extreme annual variations in light and temperature. They precisely adapt to seasonal changes by synchronizing cycles of growth and dormancy with external cues. Annual dormancy–growth transitions and flowering involve factors that integrate environmental and endogenous signals. MADS-box transcription factors have been extensively described in the regulation of Arabidopsis flowering. However, their participation in annual dormancy–growth transitions in trees is minimal. In this study, we investigate the function of MADS12, a Populus tremula × alba SUPPRESSOR OF CONSTANS OVEREXPRESSION 1 (SOC1)-related gene. Our gene expression analysis reveals that MADS12 displays lower mRNA levels during the winter than during early spring and mid-spring. Moreover, MADS12 activation depends on the fulfillment of the chilling requirement. Hybrid poplars overexpressing MADS12 show no differences in growth cessation and bud set, while ecodormant plants display an early bud break, indicating that MADS12 overexpression promotes bud growth reactivation. Comparative expression analysis of available bud break-promoting genes reveals that MADS12 overexpression downregulates the GIBBERELLINS 2 OXIDASE 4 (GA2ox4), a gene involved in gibberellin catabolism. Moreover, the mid-winter to mid-spring RNAseq profiling indicates that MADS12 and GA2ox4 show antagonistic expression during bud dormancy release. Our results support MADS12 participation in the reactivation of shoot meristem growth during ecodormancy and link MADS12 activation and GA2ox4 downregulation within the temporal events that lead to poplar bud break.

Isolation and characterization of Bacillus velezensis EB14, an endophytic bacterial strain antagonistic to poplar stem canker pathogen Sphaerulina musiva and its interactions with the endophytic fungal microbiome in poplars.

Species of the genus Populus commonly known as poplars are one of the most widely used groups of forest trees in North America and Europe, and play a significant ecological role as a pioneer species in boreal forests, and as a dominant species in the riparian forests that serve as wildlife habitats and watersheds. Natural and artificial hybrids of poplars are being extensively used in commercial plantations. However, many hybrid poplar trees are susceptible to Sphaerulina musiva, the pathogenic fungus that causes leaf spots and stem cankers and limits the utility of hybrid poplars as a plantation trees. We isolated an endophytic bacterial strain Bacillus velezensis EB14 from a Populus hybrid which showed a strong antifungal activity against S. musiva. Through mass spectrometric analyses of co-cultured B. velezensis EB14 and S. musiva, we identified five cyclic lipopeptides produced by B. velezensis EB14 – Iturin A1, Iturin A2, Iturin A9, Subtulene A and Fengycin. In addition, B. velezensis EB14 produced four major unidentified compounds in co-cultivation with S. musiva. The cyclopeptide production by B. velezensis EB14 was more pronounced (20-1000 fold) in the co-cultured plates due to elicitation by S. musiva. We also discovered that the native endophytic B. velezensis EB14 strain exhibited different levels of interactions against the endophytic fungal microbiomes of Populus sp. Overall, our results indicate B. velezensis EB14 strain as a promising biocontrol agent that could be used against stem canker and leaf spot diseases caused by S. musiva in Poplar plantations.

Hydraulic redistribution by hybrid poplars (Populus nigra x Populus maximowiczii) in a greenhouse soil column experiment

Aims Hydraulic redistribution (HR) enhances water resources for neighboring crops in silvopastoral agroforestry (AF). Here, we tested whether and to what extent water stressed shallow-rooted neighboring plants benefit from water redistributed by deep-rooted poplar plants. Methods We conducted trace experiments with deuterated water (2H2O) in greenhouse soil column experiments. We measured hydraulic lift (HL) by poplars grown at two levels of soil drying and estimated the amount of hydraulically lifted water. In a parallel experiment we grew poplars and barley (Hordeum vulgare) in two columns connected via a small cross-rooting segment. Results Soil moisture measurements and stable isotope signatures of soil and xylem water proved the occurrence of HL in poplar. Additionally, stable isotopes proved the transport of water from deep roots of poplars to shallow roots of barley. Conclusions In conclusion, the experiments showed that poplars are capable to redistribute water during drought spells and that this water can facilitate plant growth of shallow-rooted crops. This result implies evidence for an enhanced soil water supply of plants in agroforest systems under drought conditions.

Genomic Characterization Provides an Insight into the Pathogenicity of the Poplar Canker Bacterium Lonsdalea populi

An emerging poplar canker caused by the gram-negative bacterium, Lonsdalea populi, has led to high mortality of hybrid poplars Populus × euramericana in China and Europe. The molecular bases of pathogenicity and bark adaptation of L. populi have become a focus of recent research. This study revealed the whole genome sequence and identified putative virulence factors of L. populi. A high-quality L. populi genome sequence was assembled de novo, with a genome size of 3,859,707 bp, containing approximately 3434 genes and 107 RNAs (75 tRNA, 22 rRNA, and 10 ncRNA). The L. populi genome contained 380 virulence-associated genes, mainly encoding for adhesion, extracellular enzymes, secretory systems, and two-component transduction systems. The genome had 110 carbohydrate-active enzyme (CAZy)-coding genes and putative secreted proteins. The antibiotic-resistance database annotation listed that L. populi was resistant to penicillin, fluoroquinolone, and kasugamycin. Analysis of comparative genomics found that L. populi exhibited the highest homology with the L. britannica genome and L. populi encompassed 1905 specific genes, 1769 dispensable genes, and 1381 conserved genes, suggesting high evolutionary diversity and genomic plasticity. Moreover, the pan genome analysis revealed that the N-5-1 genome is an open genome. These findings provide important resources for understanding the molecular basis of the pathogenicity and biology of L. populi and the poplar-bacterium interaction.

Genome Sequence Resource of Bacillus velezensis EB14, a native endophytic bacterial strain with biocontrol potential against the poplar stem canker causative pathogen, Sphaerulina musiva

Bacillus velezensis EB14, isolated from a leaf of Populus  jackii, possesses antagonistic activity against Sphaerulina musiva, a fungal pathogen of Populus sp. that causes leaf spots and stem cankers on Poplars limiting the utility of hybrid poplars as plantation trees. We sequenced the genome of B. velezensis EB14 to gain insights into the underlying basis of its antagonistic activity. Here, we report the complete genome sequence of B. velezensis EB14, a gram-positive bacterium of the family Bacillaceae. Through antiSMASH analysis, we predicted several gene clusters coding for the biosynthesis of antimicrobial compounds, and several genes involved in plant bacterial interactions. These findings support the potential of developing B. velezensis EB14 as a biocontrol agent against S. musiva in poplar plantations. The genome of B. velezensis EB14 along with genome sequences of closely related B. velezensis species are invaluable for comparative genomic analyses to gain insights into bacterial, fungal and host plant interactions.

The effect of cutting technique on the mortality and re-sprouting vigor of poplar stumps in short-rotation plantations

Mechanical felling is the cost-effective solution for harvesting short-rotation poplar plantations, but the damage inflicted by conventional shear cutting devices on tree stumps has raised concerns about stump mortality and re-sprouting vigor - both crucial to coppice regeneration. In order to determine if such concerns are justified, the experiment monitored the survival and resprouting vigor of eleven sample blocks, composed of two 10-stump row segments cut according to either of two methods: 1) lternately with a chainsaw (control) or 2) and with an excavator-mounted shear. The sample blocks were located within the same plantation, established 7 years earlier with hybrid poplars (Populus nigra x P. deltoides), belonging to the "AF8" clone. One year after cutting, no differences were found between treatments in terms of stump mortality, number of shoots per stump, shoot diameter at 30 cm from the insertion and shoot height. These results support the use of mechanical shears to fell short-rotation poplar coppice. However, further studies should be conducted on multiple fields and clones for a safe generalization of this preliminary study.