An inducible CRISPR activation tool for accelerating plant regeneration.

The inducible CRISPR activation (CRISPR-a) system offers unparalleled precision and versatility for regulating endogenous genes, making it highly sought after in plant research. In this study, we developed a chemically inducible CRISPR-a tool for plants called ER-Tag by combining the LexA-VP16-ER inducible system with the SunTag CRISPR-a system. We systematically compared different induction strategies and achieved high efficiency in target gene activation. We demonstrated that guide RNAs can be multiplexed and pooled for large-scale screening of effective morphogenic genes and gene pairs involved in plant regeneration. Further experiments showed that induced activation of these morphogenic genes can accelerate regeneration and improve regeneration efficiency in both eudicot and monocot plants, including alfalfa, woodland strawberry, and sheepgrass. Our study expands the CRISPR toolset in plants and provides a powerful new strategy for studying gene function when constitutive expression is not feasible or ideal.

High Efficiency Regeneration System from Blueberry Leaves and Stems.

The main propagation approach is tissue culture in blueberries, and tissue culture is an effective and low-cost method with higher economic efficiency in blueberries. However, there is a lack of stable and efficient production systems of industrialization of tissue culture in blueberries. In this study, the high-efficiency tissue culture and rapid propagation technology system were established based on blueberry leaves and stems. The optimal medium for callus induction was WPM (woody plant medium) containing 2.0 mg/L Forchlorfenuron (CPPU), 0.2 mg/L 2-isopentenyladenine (2-ip) with a 97% callus induction rate and a callus differentiation rate of 71% by using blueberry leaves as explants. The optimal secondary culture of the leaf callus medium was WPM containing 3.0 mg/L CPPU with an increment coefficient of 24%. The optimal bud growth medium was WPM containing 1.0 mg/L CPPU, 0.4 mg/L 2-ip, with which the growth of the bud was better, stronger and faster. The optimal rooting medium was 1/2 Murashige and Skoog (1/2MS) medium containing 2.0 mg/L naphthylacetic acid (NAA), with which the rooting rate was 90% with shorter rooting time and more adventitious root. In addition, we established a regeneration system based on blueberry stems. The optimal preculture medium in blueberry stem explants was MS medium containing 2-(N-morpholino) ethanesulfonic acid (MES) containing 0.2 mg/L indole-3-acetic acid (IAA), 0.1 mg/L CPPU, 100 mg/L NaCl, with which the germination rate of the bud was 93%. The optimal medium for fast plant growth was MS medium containing MES containing 0.4 mg/L zeatin (ZT), 1 mg/L putrescine, 1 mg/L spermidine, 1 mg/L spermidine, which had a good growth state and growth rate. The optimal cultivation for plantlet growth was MS medium containing MES containing 0.5 mg/L isopentene adenine, with which the plantlet was strong. The optimal rooting medium for the stem was 1/2MS medium containing 2.0 mg/L NAA, with which the rooting rate was 93% with a short time and more adventitious root. In conclusion, we found that stem explants had higher regeneration efficiency for a stable and efficient production system of industrialization of tissue culture. This study provides theoretical guidance and technical support in precision breeding and standardization and industrialization in the blueberry industry.

PePYL4 enhances drought tolerance by modulating water-use efficiency and ROS scavenging in Populus.

Drought is one of the major limiting factors in the growth of terrestrial plants. Abscisic acid (ABA) and pyrabactin resistance 1/prabactin resistance-1 like/regulatory components of ABA receptors (PYR/PYL/RCARs) play a key role in response to drought stress. However, the underlying mechanisms of this control remain largely elusive in trees. In this study, PePYL4, a potential ortholog of the PYR/PYL/RCARs gene, was cloned from Populus euphratica. It was localized in the cytoplasm and nucleus, induced by ABA, osmotic and dehydration treatments. To study the potential biological functions of PePYL4, transgenic triploid white poplars (Populus tomentosa 'YiXianCiZhu B38') overexpressing PePYL4 were generated. PePYL4 overexpression significantly increased ABA sensitivity and reduced stomatal aperture. Compared with wild-type plants, transgenic plants had higher water-use efficiency (WUE) and lower transpiration. When exposed to drought stress, PePYL4 overexpression plants maintained higher photosynthetic activity and accumulated more biomass. Moreover, overexpression of PePYL4 improved antioxidant enzyme activity and ascorbate content to accelerate reactive oxygen species scavenging. Meanwhile, upregulation expression of the stress-related genes also contributed to improving the drought tolerance of transgenic plants. In conclusion, our data suggest that PePYL4 is a promising gene target for regulating WUE and drought tolerance in Populus.

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.

Regulation of WOX11 Expression Represents the Difference Between Direct and Indirect Shoot Regeneration.

Somatic cells of higher plants possess the remarkable ability to regenerate new individuals via reestablishing apical meristems. Reconstitution of shoot meristem is the vital process and is required for application of plant biotechnology. Under in vitro culture condition, shoot meristem can be formed directly or indirectly, depending on the absence or presence of callus as the intermediate status. However, the difference of regulatory mechanisms between the two regeneration types remains unknown. In this study, we established a bi-directional system in which shoots regenerated directly from lateral root primordia (LRP) and indirectly from hypocotyl-derived callus simultaneously. The results based on this system revealed that regulation of WOX11 expression represents the difference between the two regeneration types in two aspects. Firstly, number of founder cells expressing WOX11 is tightly associated with regeneration types. Relatively more founder cells gave rise to callus and produce larger meristem, whereas less founder cells produce LRP that regenerate smaller meristem. Secondly, non-CG DNA methylation specifically regulated WOX11 transcription in LRP and promoted direct shoot regeneration, but had no influence on indirect regeneration. The results provide new insights for understanding the regulatory mechanisms of cell fate transition during de novo organogenesis.

The yellowhorn AGL transcription factor gene XsAGL22 contributes to ABA biosynthesis and drought tolerance in poplar.

Regulation of abscisic acid (ABA) biosynthesis helps plants adapt to drought stress, but the underlying molecular mechanisms are largely unclear. Here, a drought-induced transcription factor XsAGL22 was isolated from yellowhorn (Xanthoceras sorbifolium Bunge). Yeast one-hybrid and electrophoretic mobility shift assays indicated that XsAGL22 can physically bind to the promoters of the ABA biosynthesis-related genes XsNCED6 and XsBG1, and a dual-luciferase assay showed that XsAGL22 activates the promoters of the later two genes. Transient overexpression of XsAGL22 in yellowhorn leaves also increased the expression of XsNCED6 and XsBG1 and increased cellular ABA levels. Finally, heterologous overexpression of XsAGL22 in poplar increased ABA content, reduced stomatal aperture and increased drought resistance. Our results suggest that XsAGL22 is a powerful regulator of ABA biosynthesis and plays a critical role in drought resistance in plants.

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.

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.

Comparative transcriptome analyses define genes and gene modules differing between two Populus genotypes with contrasting stem growth rates.

BACKGROUND: Wood provides an important biomass resource for biofuel production around the world. The radial growth of tree stems is central to biomass production for forestry and biofuels, but it is challenging to dissect genetically because it is a complex trait influenced by many genes. In this study, we adopted methods of physiology, transcriptomics and genetics to investigate the regulatory mechanisms of tree radial growth and wood development. RESULTS: Physiological comparison showed that two Populus genotypes presented different rates of radial growth of stems and accumulation of woody biomass. A comparative transcriptional network approach was used to define and characterize functional differences between two Populus genotypes. Analyses of transcript profiles from wood-forming tissue of the two genotypes showed that 1542, 2295 and 2110 genes were differentially expressed in the pre-growth, fast-growth and post-growth stages, respectively. The co-expression analyses identified modules of co-expressed genes that displayed distinct expression profiles. Modules were further characterized by correlating transcript levels with genotypes and physiological traits. The results showed enrichment of genes that participated in cell cycle and division, whose expression change was consistent with the variation of radial growth rates. Genes related to secondary vascular development were up-regulated in the faster-growing genotype in the pre-growth stage. We characterized a BEL1-like (BELL) transcription factor, PeuBELL15, which was up-regulated in the faster-growing genotype. Analyses of transgenic Populus overexpressing as well as CRISPR/Cas9-induced mutants for BELL15 showed that PeuBELL15 improved accumulation of glucan and lignin, and it promoted secondary vascular growth by regulating the expression of genes relevant for cellulose synthases and lignin biosynthesis. CONCLUSIONS: This study illustrated that active division and expansion of vascular cambium cells and secondary cell wall deposition of xylem cells contribute to stem radial increment and biomass accumulation, and it identified relevant genes for these complex growth traits, including a BELL transcription factor gene PeuBELL15. This provides genetic resources for improving and breeding elite genotypes with fast growth and high wood biomass.

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.

Structure-function analysis of human l-prostaglandin D synthase bound with fatty acid molecules.

Human prostaglandin D synthase (L-PGDS) is a lipocalin-type enzyme involved in the metabolism of arachidonic acid and plays a key role in the regulation of sleep, allergy, pain sensation, and the development of male reproductive organs. Here, using a combination of crystallographic, biochemical, mutagenesis, and kinetic studies, we have gained insights into the mode of ligand binding by human L-PGDS and have identified residues involved in catalysis. Interestingly, structural evidence reveals that 2 molecules of fatty acids, one molecule each of oleic and palmitoleic acid, bind inside the ß barrel. The oleic acid is buried and binds in a highly basic patch in proximity to the catalytically critical Cys65, mimicking the binding of prostaglandin H(2). The palmitoleic acid sits in a relatively neutral region with very few interactions with the protein. Mutating Met64, Leu79, Phe83, or Leu131 to alanine reduced the catalytic efficiency by almost 10-fold, while K59A and Y149A mutations enhanced the catalytic efficiency by >2-fold. Met64 seems to function as a kinetic clamp, pushing the thiol group of Cys65 close to the site of nucleophilic attack during catalysis.