Summary: Glycerophosphodiester phosphodiesterase (GDPD), which hydrolyzes glycerophosphodiesters into sn‐glycerol‐3‐phosphate (G‐3‐P) and the corresponding alcohols, plays an important role in various physiological processes in both prokaryotes and eukaryotes. However, little is known about the physiological significance of GDPD in plants. Here, we characterized the Arabidopsis GDPD family that can be classified into canonical GDPD (AtGDPD1‐6) and GDPD‐like (AtGDPDL1‐7) subfamilies. In vitro analysis of enzymatic activities showed that AtGDPD1 and AtGDPDL1 hydrolyzed glycerolphosphoglycerol, glycerophosphocholine and glycerophosphoethanolamine, but the maximum activity of AtGDPD1 was much higher than that of AtGDPDL1 under our assay conditions. Analyses of gene expression patterns revealed that all AtGDPD genes except for AtGDPD4 were transcriptionally active in flowers and siliques. In addition, the gene family displayed overlapping and yet distinguishable patterns of expression in roots, leaves and stems, indicating functional redundancy as well as specificity of GDPD genes. AtGDPDs but not AtGDPDLs are up‐regulated by inorganic phosphate (P_i) starvation. Loss‐of‐function of the plastid‐localized AtGDPD1 leads to a significant decrease in GDPD activity, G‐3‐P content, P_i content and seedling growth rate only under P_i starvation compared with the wild type (WT). However, membrane lipid compositions in the P_i‐deprived seedlings remain unaltered between the AtGDPD1 knockout mutant and WT. Thus, we suggest that the GDPD‐mediated lipid metabolic pathway may be involved in release of P_i from phospholipids during P_i starvation.

Genotype Nisqually-1 is the first model woody plant with an available well-annotated genome. Nevertheless, a simple and rapid transformation of Nisqually-1 remains to be established. Here, we developed a novel shoot regeneration method for Nisqually-1 using leaf petiole and stem segment explants. Numerous shoots formed in the incision of explants within two weeks. The optimized shoot regeneration medium (SRM) contained 0.03 mg l⁻¹ 6-benzylaminopurine, 0.02 mg l⁻¹ indole-3-butyric acid and 0.0008 mg l⁻¹ thidiazuron. Based on this, Agrobacterium-mediated genetic transformation of stem explants was examined using the vector pBI₁₂₁ that contains the β-glucuronidase (GUS) as a reporter gene. Consequently, factors affecting transformation frequency of GUS-positive shoots were optimized as follows: Agrobacteria cell suspension with an OD₆₀₀ of 0.4, 20 min infection time, 2 days of co-cultivation duration and the addition of 80 µM acetosyringone into Agrobacteria infective suspension and co-cultivation SRM. Using this optimized method, transgenic plantlets of Nisqually-1 – with an average transformation frequency of 26.7% – were obtained with 2 months. Southern blot and GUS activity staining confirmed the integration of the foreign GUS gene into Nisqually-1. This novel transformation system for Nisqually-1 was rapid, efficient, and simple to operate and will improve more genetic applications in this model tree.

Summary: Plant cellulose is synthesized by a large plasma membrane‐localized cellulose synthase (CesA) complex. However, an overall functional determination of secondary cell wall (SCW) CesAs is still lacking in trees, especially one based on gene knockouts. Here, the Cas9/gRNA‐induced knockouts of PtrCesA4, 7A, 7B, 8A and 8B genes were produced in Populus trichocarpa. Based on anatomical, immunohistochemical and wood composition evidence, we gained a comprehensive understanding of five SCW PtrCesAs at the genetic level. Complete loss of PtrCesA4, 7A/B or 8A/B led to similar morphological abnormalities, indicating similar and nonredundant genetic functions. The absence of the gelatinous (G) layer, one‐layer‐walled fibres and a 90% decrease in cellulose in these mutant woods revealed that the three classes of SCW PtrCesAs are essential for multilayered SCW structure and wood G‐fibre. In addition, the mutant primary and secondary phloem fibres lost the n(G + L)‐ and G‐layers and retained the thicker S‐layers (L, lignified; S, secondary). Together with polysaccharide immunolocalization data, these findings suggest differences in the role of SCW PtrCesAs‐synthesized cellulose in wood and phloem fibre wall structures. Overall, this functional understanding of the SCW PtrCesAs provides further insights into the impact of lacking cellulose biosynthesis on growth, SCW, wood G‐fibre and phloem fibre wall structures in the tree.

Maturation of chloroplast ribosomal RNAs (rRNAs) comprises several endoribonucleolytic and exoribonucleolytic processing steps. However, little is known about the specific enzymes involved and cleavage steps they catalyze. Here we report the functional characterization of the single Arabidopsis thaliana gene encoding a putative YbeY endoribonuclease (AtYbeY). AtYbeY null mutants are seedling-lethal, indicating that AtYbeY function is essential for plant growth. Knock-down plants display slow growth and show pale-green leaves. Physiological and ultrastructural analyses of ybeY mutants revealed impaired photosynthesis and defective chloroplast development. Fluorescent microcopy analysis showed that, when fused with the green fluorescence protein, AtYbeY is localized in chloroplasts. Immunoblot and RNA gel blot assays revealed that the levels of chloroplast-encoded subunits of photosynthetic complexes are reduced in atybeY mutants, but the corresponding transcripts accumulate normally. In addition, atybeY mutants display defective maturation of both the 5' and 3' ends of 16S, 23S, and 4.5S rRNAs as well as decreased accumulation of mature transcripts from the tRNA genes contained in the chloroplast rRNA operon. Consequently, mutant plants show a severe deficiency in ribosome biogenesis which in turn results in impaired plastid translational activity. Furthermore, biochemical assays show that recombinant AtYbeY is able to cleave chloroplast rRNAs as well as mRNAs and tRNAs in vitro. Taken together, our findings indicate that AtYbeY is a chloroplast-localized endoribonuclease that is required for chloroplast rRNA processing and thus for normal growth and development.

Aspartic protease (AP) is one of four large proteolytic enzyme families that are involved in plant growth and development. Little is known about the AP gene family in tree species, although it has been characterized in Arabidopsis, rice and grape. The AP genes that are involved in tree wood formation remain to be determined. A total of 67 AP genes were identified in Populus trichocarpa (PtAP) and classified into three categories (A, B and C). Chromosome mapping analysis revealed that two-thirds of the PtAP genes were located in genome duplication blocks, indicating the expansion of the AP family by segmental duplications in Populus. The microarray data from the Populus eFP browser demonstrated that PtAP genes had diversified tissue expression patterns. Semi-qRT-PCR analysis further determined that more than 10 PtAPs were highly or preferentially expressed in the developing xylem. When the involvement of the PtAPs in wood formation became the focus, many SCW-related cis-elements were found in the promoters of these PtAPs. Based on PtAPpromoter::GUS techniques, the activities of PtAP66 promoters were observed only in fiber cells, not in the vessels of stems as the xylem and leaf veins developed in the transgenic Populus tree, and strong GUS signals were detected in interfascicular fiber cells, roots, anthers and sepals of PtAP17promoter::GUS transgenic plants. Intensive GUS activities in various secondary tissues implied that PtAP66 and PtAP17 could function in wood formation. In addition, most of the PtAP proteins were predicted to contain N- and (or) O-glycosylation sites, and the integration of PNGase F digestion and western blotting revealed that the PtAP17 and PtAP66 proteins were N-glycosylated in Populus. Comprehensive characterization of the PtAP genes suggests their functional diversity during Populus growth and development. Our findings provide an overall understanding of the AP gene family in trees and establish a better foundation to further describe the roles of PtAPs in wood formation.

Wood formation is a biological process during which the most abundant lignocellulosic biomass on earth is produced. Although a number of transcription factors have been linked to the regulation of wood formation process, none of them has been demonstrated to be a higher hierarchical regulator that coordinately regulates secondary wall biosynthesis genes. Here, we identified a Populus gene, PsnSHN2, a counterpart of the Arabidopsis AP2/ERF type transcription factor, SHINE2. PsnSHN2 is predominantly expressed in xylem tissues and acted evidently as a high hierarchical transcriptional activator. Overexpression of PsnSHN2 in tobacco significantly altered the expression of both transcription factors and biosynthesis genes involved in secondary wall formation, leading to the thickened secondary walls and the changed cell wall composition. The most significant changes occurred in the contents of cellulose and hemicellulose that increased 37% and 28%, respectively, whereas the content of lignin that decreased 34%. Furthermore, PsnSHN2 activated or repressed the promoter activities of transcription factors involved in secondary wall biosynthesis and bound to five cis-acting elements enriched in the promoter regions of these transcription factors. Taken together, our results suggest PsnSHN2 coordinately regulate secondary wall formation through selective up/down-regulation of its downstream transcription factors that control secondary wall formation.