PeTGA1 enhances disease resistance against Colletotrichum gloeosporioides through directly regulating PeSARD1 in poplar.

Basic leucine zipper (bZIP) proteins play important roles in responding to biotic and abiotic stresses in plants. However, the molecular mechanisms of plant resistance to pathogens remain largely unclear in poplar. The present study isolated a TGACG-binding (TGA) transcription factor, PeTGA1, from Populus euphratica. PeTGA1 belongs to subgroup D of the bZIP family and was localized to the nucleus. To study the role PeTGA1 plays in response to Colletotrichum gloeosporioides, transgenic triploid white poplars overexpressing PeTGA1 were generated. Results showed that poplars with overexpressed PeTGA1 showed a higher effective defense response to C. gloeosporioides than the wild-type plants. A yeast one-hybrid assay and an electrophoretic mobility shift assay revealed that PeTGA1 could directly bind to the PeSARD1 (P. euphratica SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1) promoter, an important regulator for salicylic acid biosynthesis. The transactivation assays indicated that PeTGA1 activated the expression of PeSARD1, and PR1 (PATHOGENESIS-RELATED 1), a SA marker gene involved in SA signaling. Subsequently, we observed that the PeTGA1 overexpression lines showed elevated SA levels, thereby resulting in the increased resistance to C. gloeosporioides. Taken together, our results indicated that PeTGA1 may exert a key role in plant immunity not only by targeting PeSARD1 thus participating in the SA biosynthesis pathway but also by involving in SA signaling via activating the expression of PR1.

ABF3 enhances drought tolerance via promoting ABA-induced stomatal closure by directly regulating ADF5 in Populus euphratica.

Water availability is a main limiting factor for plant growth, development, and distribution throughout the world. Stomatal movement mediated by abscisic acid (ABA) is particularly important for drought adaptation, but the molecular mechanisms in trees are largely unclear. Here, we isolated an ABA-responsive element binding factor, PeABF3, in Populus euphratica. PeABF3 was preferentially expressed in the xylem and young leaves, and was induced by dehydration and ABA treatments. PeABF3 showed transactivation activity and was located in the nucleus. To study its functional mechanism in poplar responsive to drought stress, transgenic triploid white poplars (Populus tomentosa 'YiXianCiZhu B385') overexpressing PeABF3 were generated. PeABF3 overexpression significantly enhanced stomatal sensitivity to exogenous ABA. When subjected to drought stress, PeABF3 overexpression maintained higher photosynthetic activity and promoted cell membrane integrity, resulting in increased water-use efficiency and enhanced drought tolerance compared with wild-type controls. Moreover, a yeast one-hybrid assay and an electrophoretic mobility shift assay revealed that PeABF3 activated the expression of Actin-Depolymerizing Factor-5 (PeADF5) by directly binding to its promoter, promoting actin cytoskeleton remodeling and stomatal closure in poplar under drought stress. Taken together, our results indicate that PeABF3 enhances drought tolerance via promoting ABA-induced stomatal closure by directly regulating PeADF5 expression.

PeSTZ1 confers salt stress tolerance by scavenging the accumulation of ROS through regulating the expression of PeZAT12 and PeAPX2 in Populus.

ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) plays an important role in stress responses, but the transcriptional regulation of ZAT12 in response to abiotic stress remains unclear. In this study, we confirmed that a SALT TOLERANCE ZINC FINGER1 transcription factor from Populus euphratica (PeSTZ1) could regulate the expression of PeZAT12 by dual-luciferase reporter (DLR) assay and electrophoretic mobility shift assay. The expression of PeSTZ1 was rapidly induced by NaCl and hydrogen peroxide (H2O2) treatments. Overexpressing PeSTZ1 in poplar 84K (Populus alba × Populus glandulosa) plant was endowed with a strong tolerance to salt stress. Under salt stress, transgenic poplar exhibited higher expression levels of PeZAT12 and accumulated a larger amount of antioxidant than the wild-type plants. Meanwhile, ASCORBATE PEROXIDASE2 (PeAPX2) can be activated by PeZAT12 and PeSTZ1, promoting the accumulation of cytosolic ascorbate peroxidase (APX) to scavenge reactive oxygen species (ROS) under salt stress. This new regulatory model (PeSTZ1-PeZAT12-PeAPX2) was found in poplar, providing a new idea and insight for the interpretation of poplar resistance. Transgenic poplar reduced the accumulation of ROS, restrained the degradation of chlorophyll and guaranteed the photosynthesis and electron transport system. On the other hand, transgenic poplar slickly adjusted K+/Na+ homeostasis to alleviate salt toxicity in photosynthetic organs of plants under salt stress and then increased biomass accumulation. In summary, PeSTZ1 confers salt stress tolerance by scavenging the accumulation of ROS through regulating the expression of PeZAT12 and PeAPX2 in poplar.

PeSTZ1, a C2H2-type zinc finger transcription factor from Populus euphratica, enhances freezing tolerance through modulation of ROS scavenging by directly regulating PeAPX2.

In the present study, PeSTZ1, a cysteine-2/histidine-2-type zinc finger transcription factor, was isolated from the desert poplar, Populus euphratica, which serves as a model stress adaptation system for trees. PeSTZ1 was preferentially expressed in the young stems and was significantly up-regulated during chilling and freezing treatments. PeSTZ1 was localized to the nucleus and bound specifically to the PeAPX2 promoter. To examine the potential functions of PeSTZ1, we overexpressed it in poplar 84K hybrids (Populus alba × Populus glandulosa), which are known to be stress-sensitive. Upon exposure to freezing stress, transgenic poplars maintained higher photosynthetic activity and dissipated more excess light energy (in the form of heat) than wild-type poplars. Thus, PeSTZ1 functions as a transcription activator to enhance freezing tolerance without sacrificing growth. Under freezing stress, PeSTZ1 acts upstream of ASCORBATE PEROXIDASE2 (PeAPX2) and directly regulates its expression by binding to its promoter. Activated PeAPX2 promotes cytosolic APX that scavenges reactive oxygen species (ROS) under cold stress. PeSTZ1 may operate in parallel with C-REPEAT-BINDING FACTORS to regulate COLD-REGULATED gene expression. Moreover, PeSTZ1 up-regulation reduces malondialdehyde and ROS accumulation by activating the antioxidant system. Taken together, these results suggested that overexpressing PeSTZ1 in 84K poplar enhances freezing tolerance through the modulation of ROS scavenging via the direct regulation of PeAPX2 expression.

Poplar miR472a targeting NBS-LRRs is involved in effective defence against the necrotrophic fungus Cytospora chrysosperma.

The hemibiotroph Colletotrichum gloeosporioides and the necrotroph Cytospora chrysosperma cause poplar foliage and stem disease, respectively, resulting in substantial economic losses. In this study, Populus trichocarpa ptc-miR472a was down-regulated in leaves treated with salicylic acid, jasmonic acid (JA) or bacterial flagellin (flg22). Here, ptc-miR472a and a short tandem target mimic (STTM) of miR472a were overexpressed in P. alba × P. glandulosa, and overexpression lines of miR472a and silenced lines of STTM472a were generated. Compared with the STTM472a and wild type lines, lower reactive oxygen species accumulation was detected in miR472a overexpressing plants treated with flg22, C. gloeosporioides or C. chrysosperma. In addition, the miR472a overexpressing lines exhibited the highest susceptibility to the hemibiotroph, C. gloeosporioides, but the highest effective defence response to the necrotroph, C. chrysosperma. The JA/ethylene marker gene ERF1 was rapidly up-regulated in miR472a overexpressing plants. Furthermore, five phased, secondary, small interfering RNAs (phasiRNAs) were confirmed in the miR472a overexpressing and STTM472a lines, triggering phasiRNAs predicted to enhance NBS-LRR silencing. Taken together, our results revealed that ptc-miR472a exerts a key role in plant immunity to C. gloeosporioides and C. chrysosperma by targeting NBS-LRR transcripts. This study provides a new strategy and method in plant breeding to improve plant disease resistance.

PeCHYR1, a ubiquitin E3 ligase from Populus euphratica, enhances drought tolerance via ABA-induced stomatal closure by ROS production in Populus.

Drought, a primary abiotic stress, seriously affects plant growth and productivity. Stomata play a vital role in regulating gas exchange and drought adaptation. However, limited knowledge exists of the molecular mechanisms underlying stomatal movement in trees. Here, PeCHYR1, a ubiquitin E3 ligase, was isolated from Populus euphratica, a model of stress adaptation in forest trees. PeCHYR1 was preferentially expressed in young leaves and was significantly induced by ABA (abscisic acid) and dehydration treatments. To study the potential biological functions of PeCHYR1, transgenic poplar 84K (Populus alba × Populus glandulosa) plants overexpressing PeCHYR1 were generated. PeCHYR1 overexpression significantly enhanced H2 O2 production and reduced stomatal aperture. Transgenic lines exhibited increased sensitivity to exogenous ABA and greater drought tolerance than that of WT (wild-type) controls. Moreover, up-regulation of PeCHYR1 promoted stomatal closure and decreased transpiration, resulting in strongly elevated WUE (water use efficiency). When exposed to drought stress, transgenic poplar maintained higher photosynthetic activity and biomass accumulation. Taken together, these results suggest that PeCHYR1 plays a crucial role in enhancing drought tolerance via ABA-induced stomatal closure caused by hydrogen peroxide (H2 O2 ) production in transgenic poplar plants.

Evaluation of Appropriate Reference Genes for Reverse Transcription-Quantitative PCR Studies in Different Tissues of a Desert Poplar via Comparision of Different Algorithms.

Despite the unshakable status of reverse transcription-quantitative PCR in gene expression analysis, it has certain disadvantages, including that the results are highly dependent on the reference genes selected for data normalization. Since inappropriate endogenous control genes will lead to inaccurate target gene expression profiles, the validation of suitable internal reference genes is essential. Given the increasing interest in functional genes and genomics of Populus euphratica, a desert poplar showing extraordinary adaptation to salt stress, we evaluated the expression stability of ten candidate reference genes in P. euphratica roots, stems, and leaves under salt stress conditions. We used five algorithms, namely, ΔCt, NormFinder, geNorm, GrayNorm, and a rank aggregation method (RankAggreg) to identify suitable normalizers. To support the suitability of the identified reference genes and to compare the relative merits of these different algorithms, we analyzed and compared the relative expression levels of nine P. euphratica functional genes in different tissues. Our results indicate that a combination of multiple reference genes recommended by GrayNorm algorithm (e.g., a combination of Actin, EF1α, GAPDH, RP, UBQ in root) should be used instead of a single reference gene. These results are valuable for research of gene identification in different P. euphratica tissues.

A new activity of anti-HIV and anti-tumor protein GAP31: DNA adenosine glycosidase--structural and modeling insight into its functions.

We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

Crystal structure of beta-luffin, a ribosome-inactivating protein, at 2.0 A resolution.

The crystal structure of beta-luffin at 2.0 A resolution was solved by the molecular-replacement method using polyalanyl trichosanthin as the search model. The structure was refined with CNS1.1, giving R(work) = 0.162 and R(free) = 0.204. The r.m.s.d.s of the bond lengths and bond angles are 0.008 A and 1.3 degrees, respectively. The overall structure is similar to those of other type I RIPs. Three N-acetylglucosamine (Nag) molecules are linked to residues Asn2, Asn78 and Asn85 of the protein.