A comprehensive workflow for optimizing RNA-seq data analysis.

BACKGROUND: Current RNA-seq analysis software for RNA-seq data tends to use similar parameters across different species without considering species-specific differences. However, the suitability and accuracy of these tools may vary when analyzing data from different species, such as humans, animals, plants, fungi, and bacteria. For most laboratory researchers lacking a background in information science, determining how to construct an analysis workflow that meets their specific needs from the array of complex analytical tools available poses a significant challenge. RESULTS: By utilizing RNA-seq data from plants, animals, and fungi, it was observed that different analytical tools demonstrate some variations in performance when applied to different species. A comprehensive experiment was conducted specifically for analyzing plant pathogenic fungal data, focusing on differential gene analysis as the ultimate goal. In this study, 288 pipelines using different tools were applied to analyze five fungal RNA-seq datasets, and the performance of their results was evaluated based on simulation. This led to the establishment of a relatively universal and superior fungal RNA-seq analysis pipeline that can serve as a reference, and certain standards for selecting analysis tools were derived for reference. Additionally, we compared various tools for alternative splicing analysis. The results based on simulated data indicated that rMATS remained the optimal choice, although consideration could be given to supplementing with tools such as SpliceWiz. CONCLUSION: The experimental results demonstrate that, in comparison to the default software parameter configurations, the analysis combination results after tuning can provide more accurate biological insights. It is beneficial to carefully select suitable analysis software based on the data, rather than indiscriminately choosing tools, in order to achieve high-quality analysis results more efficiently.

PTI-ETI synergistic signal mechanisms in plant immunity.

Plants face a relentless onslaught from a diverse array of pathogens in their natural environment, to which they have evolved a myriad of strategies that unfold across various temporal scales. Cell surface pattern recognition receptors (PRRs) detect conserved elicitors from pathogens or endogenous molecules released during pathogen invasion, initiating the first line of defence in plants, known as pattern-triggered immunity (PTI), which imparts a baseline level of disease resistance. Inside host cells, pathogen effectors are sensed by the nucleotide-binding/leucine-rich repeat (NLR) receptors, which then activate the second line of defence: effector-triggered immunity (ETI), offering a more potent and enduring defence mechanism. Moreover, PTI and ETI collaborate synergistically to bolster disease resistance and collectively trigger a cascade of downstream defence responses. This article provides a comprehensive review of plant defence responses, offering an overview of the stepwise activation of plant immunity and the interactions between PTI-ETI synergistic signal transduction.

The miR6445-NAC029 module regulates drought tolerance by regulating the expression of glutathione S-transferase U23 and reactive oxygen species scavenging in Populus.

MicroRNAs are essential in plant development and stress resistance, but their specific roles in drought stress require further investigation. Here, we have uncovered that a Populus-specific microRNAs (miRNA), miR6445, targeting NAC (NAM, ATAF, and CUC) family genes, is involved in regulating drought tolerance of poplar. The expression level of miR6445 was significantly upregulated under drought stress; concomitantly, seven targeted NAC genes showed significant downregulation. Silencing the expression of miR6445 by short tandem target mimic technology significantly decreased the drought tolerance in poplar. Furthermore, 5' RACE experiments confirmed that miR6445 directly targeted NAC029. The overexpression lines of PtrNAC029 (OE-NAC029) showed increased sensitivity to drought compared with knockout lines (Crispr-NAC029), consistent with the drought-sensitive phenotype observed in miR6445-silenced strains. PtrNAC029 was further verified to directly bind to the promoters of glutathione S-transferase U23 (GSTU23) and inhibit its expression. Both Crispr-NAC029 and PtrGSTU23 overexpressing plants showed higher levels of PtrGSTU23 transcript and GST activity while accumulating less reactive oxygen species (ROS). Moreover, poplars overexpressing GSTU23 demonstrated enhanced drought tolerance. Taken together, our research reveals the crucial role of the miR6445-NAC029-GSTU23 module in enhancing poplar drought tolerance by regulating ROS homeostasis. This finding provides new molecular targets for improving the drought resistance of trees.

PeFUS3 Drives Lateral Root Growth Via Auxin and ABA Signalling Under Drought Stress in Populus.

Changes in root system architecture are vital for plant adaptation to drought stress, yet the underlying molecular mechanisms of this process remain largely elusive. Here, FUSCA3 (FUS3), a B3 domain transcription factor isolated from Populus euphratica, was found to be an important gene of regulating lateral root (LR) development under drought stress. The expression of PeFUS3 was strongly induced by ABA and dehydration treatments. Overexpressing PeFUS3 in poplar 84 K (P. alba × P. glandulosa) positively regulated LR growth and enhanced drought tolerance, while the knockout lines, generated by the CRISPR/Cas9 system, displayed repressed LR growth and weakened drought tolerance. Further investigation demonstrated that PeFUS3 activated the expression of PIN2, PIN6a and AUX1, which were key genes involved in auxin transport, suggesting PeFUS3 modulated LR development under drought stress through auxin signalling. Moreover, PeFUS3 directly upregulated PePYL3 expression, and overexpressing PePYL3 poplar lines exhibited significantly increased drought resistance. In addition, PeABF2, an ABA responsive transcription factor, interacted with PeFUS3 and activated its transcription, indicating PeFUS3 was involved in ABA signalling pathway. Taken together, PeFUS3 is a key regulator, maintaining root growth of poplar by modulating the crosstalk of auxin and ABA signalling under drought stress.

An alternative splicing variant of PtRD26 delays leaf senescence by regulating multiple NAC transcription factors in Populus.

During leaf senescence, the final stage of leaf development, nutrients are recycled from leaves to other organs, and therefore proper control of senescence is thus critical for plant fitness. Although substantial progress has been achieved in understanding leaf senescence in annual plants, the molecular factors that control leaf senescence in perennial woody plants are largely unknown. Using RNA sequencing, we obtained a high-resolution temporal profile of gene expression during autumn leaf senescence in poplar (Populus tomentosa). Identification of hub transcription factors (TFs) by co-expression network analysis of genes revealed that senescence-associated NAC family TFs (Sen-NAC TFs) regulate autumn leaf senescence. Age-dependent alternative splicing (AS) caused an intron retention (IR) event in the pre-mRNA encoding PtRD26, a NAC-TF. This produced a truncated protein PtRD26IR, which functions as a dominant-negative regulator of senescence by interacting with multiple hub Sen-NAC TFs, thereby repressing their DNA-binding activities. Functional analysis of senescence-associated splicing factors identified two U2 auxiliary factors that are involved in AS of PtRD26IR. Correspondingly, silencing of these factors decreased PtRD26IR transcript abundance and induced early senescence. We propose that an age-dependent increase of IR splice variants derived from Sen-NAC TFs is a regulatory program to fine tune the molecular mechanisms that regulate leaf senescence in trees.

Comparative Phylogenomic Study of Malaxidinae (Orchidaceae) Sheds Light on Plastome Evolution and Gene Divergence.

Malaxidinae is one of the most confusing groups in the Orchidaceae classification. Previous phylogenetic analyses have revealed that the relationships between the taxa in Malaxidinae have not yet been reliably established, using only a few plastome regions and nuclear ribosomal internal transcribed spacer (nrITS). In the present study, the complete plastomes of Oberonia integerrima and Crepidium purpureum were assembled using high-throughput sequencing. Combined with publicly available complete plastome data, this resulted in a dataset of 19 plastomes, including 17 species of Malaxidinae. The plastome features and phylogenetic relationships were compared and analyzed. The results showed the following: (1) Malaxidinae species plastomes possess the quadripartite structure of typical angiosperms, with sizes ranging from 142,996 to 158,787 bp and encoding from 125 to 133 genes. The ndh genes were lost or pseudogenized to varying degrees in six species. An unusual inversion was detected in the large single-copy region (LSC) of Oberonioides microtatantha. (2) Eight regions, including ycf1, matK, rps16, rpl32, ccsA-ndhD, clpP-psbB, trnFGAA-ndhJ, and trnSGCU-trnGUCC, were identified as mutational hotspots. (3) Based on complete plastomes, 68 protein-coding genes, and 51 intergenic regions, respectively, our phylogenetic analyses revealed the genus-level relationships in this subtribe with strong support. The Liparis was supported as non-monophyletic.

Aspartyl tRNA-synthetase (AspRS) gene family enhances drought tolerance in poplar through BABA-PtrIBIs-PtrVOZ signaling module.

BACKGROUND: Drought stress is a prevalent abiotic stress that significantly hinders the growth and development of plants. According to studies, ß-aminobutyric acid (BABA) can influence the ABA pathway through the AtIBI1 receptor gene to enhance cold resistance in Arabidopsis. However, the Aspartate tRNA-synthetase (AspRS) gene family, which acts as the receptor for BABA, has not yet been investigated in poplar. Particularly, it is uncertain how the AspRS gene family (PtrIBIs)r can resist drought stress after administering various concentrations of BABA to poplar. RESULTS: In this study, we have identified 12 AspRS family genes and noted that poplar acquired four PtrIBI pairs through whole genome duplication (WGD). We conducted cis-action element analysis and found a significant number of stress-related action elements on different PtrIBI genes promoters. The expression of most PtrIBI genes was up-regulated under beetle and mechanical damage stresses, indicating their potential role in responding to leaf damage stress. Our results suggest that a 50 mM BABA treatment can alleviate the damage caused by drought stress in plants. Additionally, via transcriptome sequencing, we observed that the partial up-regulation of BABA receptor genes, PtrIBI2/4/6/8/11, in poplars after drought treatment. We hypothesize that poplar responds to drought stress through the BABA-PtrIBIs-PtrVOZ coordinated ABA signaling pathway. Our research provides molecular evidence for understanding how plants respond to drought stress through external application of BABA. CONCLUSIONS: In summary, our study conducted genome-wide analysis of the AspRS family of P. trichocarpa and identified 12 PtrIBI genes. We utilized genomics and bioinformatics to determine various characteristics of PtrIBIs such as chromosomal localization, evolutionary tree, gene structure, gene doubling, promoter cis-elements, and expression profiles. Our study found that certain PtrIBI genes are regulated by drought, beetle, and mechanical damage implying their crucial role in enhancing poplar stress tolerance. Additionally, we observed that external application of low concentrations of BABA increased plant drought resistance under drought stress. Through the BABA-PtrIBIs-PtrVOZ signaling module, poplar plants were able to transduce ABA signaling and regulate their response to drought stress. These results suggest that the PtrIBI genes in poplar have the potential to improve drought tolerance in plants through the topical application of low concentrations of BABA.

Geographical variation in functional traits of leaves of Caryopteris mongholica and the role of climate.

BACKGROUND: Quantifying intra-specific variation in leaf functional traits along environmental gradients is important for understanding species' responses to climate change. In this study, we assessed the degree of among and within populations variation in leaf functional traits and explored leaf response to geographic and climate change using Caryopteris mongholica as material, which has a wide range of distribution environments. RESULTS: We selected 40 natural populations of C. mongholica, measured 8 leaf functional traits, analyzed the extent of trait variation among and within populations, and developed geographic and climatic models to explain trait variation between populations. Our results showed that the variation in leaf functional traits of C. mongholica was primarily lower within populations compared to among populations. Specifically, the leaf area (LA) exhibited higher variability both among and within populations, whereas leaf carbon content (LC) exhibited lower variation within populations but greater variation among populations. We observed a specific covariation pattern among traits and a strong linkage between morphological, economic, and mechanical traits. Increasing minimum temperature, precipitation of month, and seasonal precipitation differences all limited the growth and development of C. mongholica. However, it was observed that an increase in mean annual precipitation positively influenced the morphological development of its leaf. CONCLUSIONS: These results demonstrate the response of intra-specific trait variation to the environment and provide valuable insights into the adaptation of intra-specific leaf functional traits under changing climatic conditions.

Identification of WUSCHEL-related homeobox gene and truncated small peptides in transformation efficiency improvement in Eucalyptus.

BACKGROUND: The WUSCHEL-related Homeobox (WOX) genes, which encode plant-specific homeobox (HB) transcription factors, play crucial roles in regulating plant growth and development. However, the functions of WOX genes are little known in Eucalyptus, one of the fastest-growing tree resources with considerable widespread cultivation worldwide. RESULTS: A total of nine WOX genes named EgWOX1-EgWOX9 were retrieved and designated from Eucalyptus grandis. From the three divided clades marked as Modern/WUS, Intermediate and Ancient, the largest group Modern/WUS (6 EgWOXs) contains a specific domain with 8 amino acids: TLQLFPLR. The collinearity, cis-regulatory elements, protein-protein interaction network and gene expression analysis reveal that the WUS proteins in E. grandis involve in regulating meristems development and regeneration. Furthermore, by externally adding of truncated peptides isolated from WUS specific domain, the transformation efficiency in E. urophylla × E. grandis DH32-29 was significant enhanced. The transcriptomics data further reveals that the use of small peptides activates metabolism pathways such as starch and sucrose metabolism, phenylpropanoid biosynthesis and flavonoid biosynthesis. CONCLUSIONS: Peptides isolated from WUS protein can be utilized to enhance the transformation efficiency in Eucalyptus, thereby contributing to the high-efficiency breeding of Eucalyptus.

Crucial Abiotic Stress Regulatory Network of NF-Y Transcription Factor in Plants.

Nuclear Factor-Y (NF-Y), composed of three subunits NF-YA, NF-YB and NF-YC, exists in most of the eukaryotes and is relatively conservative in evolution. As compared to animals and fungi, the number of NF-Y subunits has significantly expanded in higher plants. The NF-Y complex regulates the expression of target genes by directly binding the promoter CCAAT box or by physical interaction and mediating the binding of a transcriptional activator or inhibitor. NF-Y plays an important role at various stages of plant growth and development, especially in response to stress, which attracted many researchers to explore. Herein, we have reviewed the structural characteristics and mechanism of function of NF-Y subunits, summarized the latest research on NF-Y involved in the response to abiotic stresses, including drought, salt, nutrient and temperature, and elaborated the critical role of NF-Y in these different abiotic stresses. Based on the summary above, we have prospected the potential research on NF-Y in response to plant abiotic stresses and discussed the difficulties that may be faced in order to provide a reference for the in-depth analysis of the function of NF-Y transcription factors and an in-depth study of plant responses to abiotic stress.

Genome-Wide Analysis of the FBA Subfamily of the Poplar F-Box Gene Family and Its Role under Drought Stress.

F-box proteins are important components of eukaryotic SCF E3 ubiquitin ligase complexes, which specifically determine protein substrate proteasomal degradation during plant growth and development, as well as biotic and abiotic stress. It has been found that the FBA (F-box associated) protein family is one of the largest subgroups of the widely prevalent F-box family and plays significant roles in plant development and stress response. However, the FBA gene family in poplar has not been systematically studied to date. In this study, a total of 337 F-box candidate genes were discovered based on the fourth-generation genome resequencing of P. trichocarpa. The domain analysis and classification of candidate genes revealed that 74 of these candidate genes belong to the FBA protein family. The poplar F-box genes have undergone multiple gene replication events, particularly in the FBA subfamily, and their evolution can be attributed to genome-wide duplication (WGD) and tandem duplication (TD). In addition, we investigated the P. trichocarpa FBA subfamily using the PlantGenIE database and quantitative real-time PCR (qRT-PCR); the results showed that they are expressed in the cambium, phloem and mature tissues, but rarely expressed in young leaves and flowers. Moreover, they are also widely involved in the drought stress response. At last, we selected and cloned PtrFBA60 for physiological function analysis and found that it played an important role in coping with drought stress. Taken together, the family analysis of FBA genes in P. trichocarpa provides a new opportunity for the identification of P. trichocarpa candidate FBA genes and elucidation of their functions in growth, development and stress response, thus demonstrating their utility in the improvement of P. trichocarpa.

Genetic diversity and population structure of Caryopteris mongholica revealed by reduced representation sequencing.

BACKGROUND: Caryopteris mongholica Bunge is a rare broad-leaved shrub distributed in the desert and arid regions of Mongol and North China. Due to land reclamation, natural habitat deterioration and anthropogenic activities in recent years, the wild resources have sharply reduced. To effectively protect and rationally use it, we investigated the genetic diversity and population structure from 18 populations across the range of C. mongholica in China by reduced representation sequencing technology. RESULTS: We found the overall average values of observed heterozygosity (Ho), expected heterozygosity (He), and average nucleotide diversity (π) were 0.43, 0.35 and 0.135, respectively. Furthermore, the NM17 population exhibited higher genetic diversity than other populations. The phylogenetic tree, principal component analysis (PCA) and structure analysis showed the sampled individuals clustered into two main groups. The NM03 population, with individuals clustered in both groups, may be a transitional population located between the two groups. In addition, most genetic variation existed within populations (90.97%) compared to that among the populations (9.03%). Interestingly, geographic and environmental distances were almost equally important to the observed genetic differences. Redundancy analysis (RDA) identified optical radiation (OR), minimum temperature (MIT) and mean annual precipitation (MAP) related variables as the most important environment factors influencing genetic variation, and the importance of MIT was also confirmed in the latent factor mixed models (LFMM). CONCLUSIONS: The results of this study facilitate research on the genetic diversity of C. mongholica. These genetic features provided vital information for conserving and sustainably developing the C. mongholica genetic resources.

The transcription factor GNC optimizes nitrogen use efficiency and growth by up-regulating the expression of nitrate uptake and assimilation genes in poplar.

Plants have evolved complex mechanisms to cope with the fluctuating environmental availability of nitrogen. However, potential genes modulating plant responses to nitrate are yet to be characterized. Here, a poplar GATA transcription factor gene PdGNC (GATA nitrate-inducible carbon-metabolism-involved) was found to be strongly induced by low nitrate. Overexpressing PdGNC in poplar clone 717-1B4 (P. tremula × alba) significantly improved nitrate uptake, remobilization, and assimilation with higher nitrogen use efficiency (NUE) and faster growth, particularly under low nitrate conditions. Conversely, CRISPR/Cas9-mediated poplar mutant gnc exhibited decreased nitrate uptake, relocation, and assimilation, combined with lower NUE and slower growth. Assays with yeast one-hybrid, electrophoretic mobility shift, and a dual-luciferase reporter showed that PdGNC directly activated the promoters of nitrogen pathway genes PdNRT2.4b, PdNR, PdNiR, and PdGS2, leading to a significant increase in nitrate utilization in poplar. As expected, the enhanced NUE promoted growth under low nitrate availability. Taken together, our data show that PdGNC plays an important role in the regulation of NUE and growth in poplar by improving nitrate acquisition, remobilization, and assimilation, and provide a promising strategy for molecular breeding to improve productivity under nitrogen limitation in trees.

The Regulation of Nitrate Reductases in Response to Abiotic Stress in Arabidopsis.

The two homologous genes, NIA1 and NIA2, encode nitrate reductases in Arabidopsis, which govern the reduction of nitrate to nitrite. This step is the rate-limiting step of the nitrate assimilation and utilization. Therefore, the regulation of NIA1 and NIA2 is important for plant development and growth. Although they are similar in sequence and structure, their regulations are different. Genetic analysis uncovers that NIA1, rather than NIA2, plays a predominant role in adopting to ABA stress. Although both long-term stress conditions can cause an improvement in NIA1 levels, a decrease in NIA1 levels under short-term treatments seems to be necessary for plants to switch from the growth status into the adopting status. Interestingly, the downregulation of the NR is distinct under different stress conditions. Under ABA treatment, the NR proteins are degraded via a 26S-proteasome dependent manner, while the transcriptional regulation is the main manner to rapidly reduce the NIA1 levels under nitrogen deficiency and NaCl stress conditions. These results indicate that under stress conditions, the regulation of NIA1 is complex, and it plays a key role in regulating the balance between growth and adaptation.

Regulation of cytokinin biosynthesis using PtRD26pro -IPT module improves drought tolerance through PtARR10-PtYUC4/5-mediated reactive oxygen species removal in Populus.

Drought is a critical environmental factor which constrains plant survival and growth. Genetic engineering provides a credible strategy to improve drought tolerance of plants. Here, we generated transgenic poplar lines expressing the isopentenyl transferase gene (IPT) under the driver of PtRD26 promoter (PtRD26pro -IPT). PtRD26 is a senescence and drought-inducible NAC transcription factor. PtRD26pro -IPT plants displayed multiple phenotypes, including improved growth and drought tolerance. Transcriptome analysis revealed that auxin biosynthesis pathway was activated in the PtRD26pro -IPT plants, leading to an increase in auxin contents. Biochemical analysis revealed that ARABIDOPSIS RESPONSE REGULATOR10 (PtARR10), one of the type-B ARR transcription factors in the cytokinin pathway, was induced in PtRD26pro -IPT plants and directly regulated the transcripts of YUCCA4 (PtYUC4) and YUCCA5 (PtYUC5), two enzymes in the auxin biosynthesis pathway. Overexpression of PtYUC4 enhanced drought tolerance, while simultaneous silencing of PtYUC4/5 evidently attenuated the drought tolerance of PtRD26pro -IPT plants. Intriguingly, PtYUC4/5 displayed a conserved thioredoxin reductase activity that is required for drought tolerance by deterring reactive oxygen species accumulation. Our work reveals the molecular basis of cytokinin and auxin interactions in response to environmental stresses, and shed light on the improvement of drought tolerance without a growth penalty in trees by molecular breeding.

PdGNC confers drought tolerance by mediating stomatal closure resulting from NO and H2 O2 production via the direct regulation of PdHXK1 expression in Populus.

Drought is one of the primary abiotic stresses, seriously implicating plant growth and productivity. Stomata play a crucial role in regulating drought tolerance. However, the molecular mechanism on stomatal movement-mediated drought tolerance remains unclear. Using genetic, molecular and biochemical techniques, we identified that the PdGNC directly activating the promoter of PdHXK1 by binding the GATC element, a hexokinase (HXK) synthesis key gene. Here, PdGNC, a member of the GATA transcription factor family, was greatly induced by abscisic acid and dehydration. Overexpressing PdGNC in poplar (Populus clone 717) resulted in reduced stomatal aperture with greater water-use efficiency and increased water deficit tolerance. By contrast, CRISPR/Cas9-mediated poplar mutant gnc exhibited increased stomatal aperture and water loss with reducing drought resistance. PdGNC activates PdHXK1 (a hexokinase synthesis key gene), resulting in a remarkable increase in hexokinase activity in poplars subjected to water deficit. Furthermore, hexokinase promoted nitric oxide (NO) and hydrogen peroxide (H2 O2 ) production in guard cells, which ultimately reduced stomatal aperture and increased drought resistance. Together, PdGNC confers drought stress tolerance by reducing stomatal aperture caused by NO and H2 O2 production via the direct regulation of PdHXK1 expression in poplars.

The Receptor-Like Kinase ERECTA Confers Improved Water Use Efficiency and Drought Tolerance to Poplar via Modulating Stomatal Density.

Poplar is one of the most important tree species in the north temperate zone, but poplar plantations are quite water intensive. We report here that CaMV 35S promoter-driven overexpression of the PdERECTA gene, which is a member of the LRR-RLKs family from Populus nigra × (Populus deltoides × Populus nigra), improves water use efficiency and enhances drought tolerance in triploid white poplar. PdERECTA localizes to the plasma membrane. Overexpression plants showed lower stomatal density and larger stomatal size. The abaxial stomatal density was 24-34% lower and the stomatal size was 12-14% larger in overexpression lines. Reduced stomatal density led to a sharp restriction of transpiration, which was about 18-35% lower than the control line, and instantaneous water use efficiency was around 14-63% higher in overexpression lines under different conditions. These phenotypic changes led to increased drought tolerance. PdERECTA overexpression plants not only survived longer after stopping watering but also performed better when supplied with limited water, as they had better physical and photosynthesis conditions, faster growth rate, and higher biomass accumulation. Taken together, our data suggest that PdERECTA can alter the development pattern of stomata to reduce stomatal density, which then restricts water consumption, conferring enhanced drought tolerance to poplar. This makes PdERECTA trees promising candidates for establishing more water use efficient plantations.

Genome-Wide Comprehensive Analysis of the GASA Gene Family in Populus.

Gibberellic acid-stimulated Arabidopsis (GASA) proteins, as cysteine-rich peptides (CRPs), play roles in development and reproduction and biotic and abiotic stresses. Although the GASA gene family has been identified in plants, the knowledge about GASAs in Populus euphratica, the woody model plant for studying abiotic stress, remains limited. Here, we referenced the well-sequenced Populus trichocarpa genome, and identified the GASAs in the whole genome of P. euphratica and P. trichocarpa. 21 candidate genes in P. trichocarpa and 19 candidate genes in P. euphratica were identified and categorized into three subfamilies by phylogenetic analysis. Most GASAs with signal peptides were located extracellularly. The GASA genes in Populus have experienced multiple gene duplication events, especially in the subfamily A. The evolution of the subfamily A, with the largest number of members, can be attributed to whole-genome duplication (WGD) and tandem duplication (TD). Collinearity analysis showed that WGD genes played a leading role in the evolution of GASA genes subfamily B. The expression patterns of P. trichocarpa and P. euphratica were investigated using the PlantGenIE database and the real-time quantitative PCR (qRT-PCR), respectively. GASA genes in P. trichocarpa and P. euphratica were mainly expressed in young tissues and organs, and almost rarely expressed in mature leaves. GASA genes in P. euphratica leaves were also widely involved in hormone responses and drought stress responses. GUS activity assay showed that PeuGASA15 was widely present in various organs of the plant, especially in vascular bundles, and was induced by auxin and inhibited by mannitol dramatically. In summary, this present study provides a theoretical foundation for further research on the function of GASA genes in P. euphratica.

Root-specific NF-Y family transcription factor, PdNF-YB21, positively regulates root growth and drought resistance by abscisic acid-mediated indoylacetic acid transport in Populus.

Root growth control plays an important role in plant adaptation to drought stress, but the underlying molecular mechanisms of this control remain largely elusive. Here, a root-specific nuclear factor Y (NF-Y) transcription factor PdNF-YB21 was isolated from Populus. The functional mechanism of PdNF-YB21 was characterised by various morphological, physiological, molecular, biochemical and spectroscopy techniques. Overexpression of PdNF-YB21 in poplar promoted root growth with highly lignified and enlarged xylem vessels, resulting in increased drought resistance. By contrast, CRISPR/Cas9-mediated poplar mutant nf-yb21 exhibited reduced root growth and drought resistance. PdNF-YB21 interacted with PdFUSCA3 (PdFUS3), a B3 domain transcription factor. PdFUS3 directly activated the promoter of the abscisic acid (ABA) synthesis key gene PdNCED3, resulting in a significant increase in root ABA content in poplars subjected to water deficit. Coexpression of poplar NF-YB21 and FUS3 significantly enhanced the expression of PdNCED3. Furthermore, ABA promoted indoylacetic acid transport in root tips, which ultimately increased root growth and drought resistance. Taken together, our data indicate that NF-YB21-FUS3-NCED3 functions as an important avenue in auxin-regulated poplar root growth in response to drought.

The GATA transcription factor GNC plays an important role in photosynthesis and growth in poplar.

GATA transcription factors are involved in the regulation of diverse growth processes and environmental responses in Arabidopsis and rice. In this study, we conducted a comprehensive bioinformatic survey of the GATA family in the woody perennial Populus trichocarpa. Thirty-nine Populus GATA genes were classified into four subfamilies based on gene structure and phylogenetic relationships. Predicted cis-elements suggested potential roles of poplar GATA genes in light, phytohormone, development, and stress responses. A poplar GATA gene, PdGATA19/PdGNC (GATA nitrate-inducible carbon-metabolism-involved), was identified from a fast growing poplar clone. PdGNC expression was significantly up-regulated in leaves under both high (50 mM) and low (0.2 mM) nitrate concentrations. The CRISPR/Cas9-mediated mutant crispr-GNC showed severely retarded growth and enhanced secondary xylem differentiation. PdGNC-overexpressing transformants exhibited 25-30% faster growth, 20-28% higher biomass accumulation, and ~25% increase in chlorophyll content, photosynthetic rate, and plant height, compared with the wild type. Transcriptomic analysis showed that PdGNC was involved in photosynthetic electron transfer and carbon assimilation in the leaf, cell division and carbohydrate utilization in the stem, and nitrogen uptake in the root. These data indicated that PdGNC plays a crucial role in plant growth and is potentially useful in tree molecular breeding.