PagHAM4a-PagSCL21 and PagHAM4b-PagTCP20 modules positively regulate cambial activity and its differentiation into secondary xylem in poplar.

Stem secondary xylem produced by cambial division and differentiation is the main source of tree biomass. The secondary xylem formation involves a complex transcriptional regulatory network, however, the underlying mechanism is still being explored. Here, we report that PagHAM4a and PagHAM4b are positive regulators of cambial differentiation into secondary xylem in hybrid poplar (Populus alba × Populus glandulosa clone 84K). Overexpression of PagHAM4a and PagHAM4b enhanced cambial activity and increased the number of secondary xylem cells in the stems of poplar. By contrast, single or double mutations of PagHAM4a and PagHAM4b by CRISPR-Cas9 decreased cambial activity, leading to a significant reduction of secondary xylem. Neither overexpression nor mutation of the two genes affected the size of vessels and fibers in xylem. Both PagHAM4a- and PagHAM4b-regulated gene networks were mainly centered in the stage when cambium had just initiated secondary growth, but the molecular networks regulated by the two genes were distinct. Further analysis revealed that PagSCL21 and PagTCP20 are direct targets of PagHAM4a and PagHAM4b, respectively, and their overexpression also promoted cambial differentiation into secondary xylem. Taken together, we identified two novel key regulatory modules in poplar, PagHAM4a-PagSCL21 and PagHAM4b-PagTCP20, which provide new insights into the mechanism of secondary xylem formation in trees.

Transcriptome and miRNAs Profiles Reveal Regulatory Network and Key Regulators of Secondary Xylem Formation in "84K" Poplar.

Secondary xylem produced by stem secondary growth is the main source of tree biomass and possesses great economic and ecological value in papermaking, construction, biofuels, and the global carbon cycle. The secondary xylem formation is a complex developmental process, and the underlying regulatory networks and potential mechanisms are still under exploration. In this study, using hybrid poplar (Populus alba × Populus glandulosa clone 84K) as a model system, we first ascertained three representative stages of stem secondary growth and then investigated the regulatory network of secondary xylem formation by joint analysis of transcriptome and miRNAs. Notably, 7507 differentially expressed genes (DEGs) and 55 differentially expressed miRNAs (DEMs) were identified from stage 1 without initiating secondary growth to stage 2 with just initiating secondary growth, which was much more than those identified from stage 2 to stage 3 with obvious secondary growth. DEGs encoding transcription factors and lignin biosynthetic enzymes and those associated with plant hormones were found to participate in the secondary xylem formation. MiRNA-target analysis revealed that a total of 85 DEMs were predicted to have 2948 putative targets. Among them, PagmiR396d-PagGRFs, PagmiR395c-PagGA2ox1/PagLHW/PagSULTR2/PagPolyubiquitin 1, PagmiR482d-PagLAC4, PagmiR167e-PagbHLH62, and PagmiR167f/g/h-PagbHLH110 modules were involved in the regulating cambial activity and its differentiation into secondary xylem, cell expansion, secondary cell wall deposition, and programmed cell death. Our results give new insights into the regulatory network and mechanism of secondary xylem formation.

The Regulation of Xylem Development by Transcription Factors and Their Upstream MicroRNAs.

Xylem, as a unique organizational structure of vascular plants, bears water transport and supports functions necessary for plant survival. Notably, secondary xylem in the stem (i.e., wood) also has important economic and ecological value. In view of this, the regulation of xylem development has been widely concerned. In recent years, studies on model plants Arabidopsis and poplar have shown that transcription factors play important regulatory roles in various processes of xylem development, including the directional differentiation of procambium and cambium into xylem, xylem arrangement patterns, secondary cell wall formation and programmed cell death. This review focuses on the regulatory roles of widely and thoroughly studied HD-ZIP, MYB and NAC transcription factor gene families in xylem development, and it also pays attention to the regulation of their upstream microRNAs. In addition, the existing questions in the research and future research directions are prospected.

[Growth stability of four drought resistant plant species in different regions]. / 四种抗旱植物在不同区域的生长稳定性.

There are abundant germplasm resources of drought resistant trees in China. It is difficult for foresters to evaluate and screen excellent germplasm that is suitable for various drought adverse circumstances. In this study, four tree species from different provenances, namely Amygdalus davi-diana, Prunus sibirica, Salix gordejevii, and Caryopteris mongolica, were used as test materials. Four soil regions, namely Dalad Banner, Siziwang Banner, Kouhezi town and Liujiazi town of Kulun Banner in the Central and Eastern Inner Mongolia Autonomous Region were selected as multi-point experiment sites to analyze the growth and physiological status of different tree species and provenances. The additive main effects and multiplicative interaction model was used to evaluate the regional adaptability and stability of the germplasm. The growth and physiological indices of four tree species differed between provenances and locations. Soil conditions (potassium content, nitrogen content, and pH) and climate conditions (annual average temperature, precipitation, and potential evaportranspiration) in different locations all influenced the growth of different provenance species. Concerning tree species, S. gordejevii and C. mongolica are more adapted to the sandy loam and chestnut soil of Dalad Banner and Siziwang Banner. A. davidiana and P. sibirica L. are more adapted to the loess and aeolian sandy soil of Kouhezi town and Liujiazi town. Concerning tree provenances, A. davidiana of Tuzuo, P. sibirica of Ningcheng and Yuanzhou, S. gordejevii of Lanqi, and C. mongolica of Jingbian displayed higher regional stability and better growth adaptability, indicating their suitability for afforestation in similar areas.

The Control of Developmental Phase Transitions by microRNAs and Their Targets in Seed Plants.

Seed plants usually undergo various developmental phase transitions throughout their lifespan, mainly including juvenile-to-adult and vegetative-to-reproductive transitions, as well as developmental transitions within organ/tissue formation. MicroRNAs (miRNAs), as a class of small endogenous non-coding RNAs, are involved in the developmental phase transitions in plants by negatively regulating the expression of their target genes at the post-transcriptional level. In recent years, cumulative evidence has revealed that five miRNAs, miR156, miR159, miR166, miR172, and miR396, are key regulators of developmental phase transitions in plants. In this review, the advanced progress of the five miRNAs and their targets in regulating plant developmental transitions, especially in storage organ formation, are summarized and discussed, combining our own findings with the literature. In general, the functions of the five miRNAs and their targets are relatively conserved, but their functional divergences also emerge to some extent. In addition, potential research directions of miRNAs in regulating plant developmental phase transitions are prospected.

The newly developed genomic-SSR markers uncover the genetic characteristics and relationships of olive accessions.

BACKGROUND: Olive (Olea europaea L.) is an important oil and fruit crop worldwide, owning a rich germplasm with a large number of cultivars. Simple sequence repeats (SSRs) are excellent markers and have been used for the identification of olive cultivars. However, the limited number of SSR markers and the occurrence of confusion on the names of cultivars, as well as the possible appearance of clonal variation make it difficult to identify cultivars and interpret relationships among olive cultivars. METHOD: SSR markers were designed based on trinucleotide repeat sequences by screening the whole genome of olive, and the polymorphic SSR markers were developed that were applied to the identification of 53 olive accessions. The genetic characteristics and relationships of these olive accessions were evaluated based on the developed SSR markers. RESULTS: Twenty-one highly polymorphic genomic-SSR markers were developed, covering most chromosomes of olive. These SSR markers could well distinguish all 53 olive accessions, confirming their effectiveness. DNA fingerprints of the 53 olive accessions were constructed based on the 21 SSR markers. The dendrogram clearly divided the tested accessions into two main groups, which was also supported by the results of principal coordinate analysis. A total of 31 private alleles were detected in 15 olive accessions, which reflected the genetic diversity within 53 olive accessions to some extent. Six homonymy cases were also clarified by genetic analysis. These results suggest that the newly developed olive SSR markers are informative for the exploitation, preservation and breeding of olive.

Integrated transcriptome and miRNA analysis uncovers molecular regulators of aerial stem-to-rhizome transition in the medical herb Gynostemma pentaphyllum.

BACKGROUND: Gynostemma pentaphyllum is an important perennial medicinal herb belonging to the family Cucurbitaceae. Aerial stem-to-rhizome transition before entering the winter is an adaptive regenerative strategy in G. pentaphyllum that enables it to survive during winter. However, the molecular regulation of aerial stem-to-rhizome transition is unknown in plants. Here, integrated transcriptome and miRNA analysis was conducted to investigate the regulatory network of stem-to-rhizome transition. RESULTS: Nine transcriptome libraries prepared from stem/rhizome samples collected at three stages of developmental stem-to-rhizome transition were sequenced and a total of 5428 differentially expressed genes (DEGs) were identified. DEGs associated with gravitropism, cell wall biosynthesis, photoperiod, hormone signaling, and carbohydrate metabolism were found to regulate stem-to-rhizome transition. Nine small RNA libraries were parallelly sequenced, and seven significantly differentially expressed miRNAs (DEMs) were identified, including four known and three novel miRNAs. The seven DEMs targeted 123 mRNAs, and six pairs of miRNA-target showed significantly opposite expression trends. The GpmiR166b-GpECH2 module involved in stem-to-rhizome transition probably promotes cell expansion by IBA-to-IAA conversion, and the GpmiR166e-GpSGT-like module probably protects IAA from degradation, thereby promoting rhizome formation. GpmiR156a was found to be involved in stem-to-rhizome transition by inhibiting the expression of GpSPL13A/GpSPL6, which are believed to negatively regulate vegetative phase transition. GpmiR156a and a novel miRNA Co.47071 co-repressed the expression of growth inhibitor GpRAV-like during stem-to-rhizome transition. These miRNAs and their targets were first reported to be involved in the formation of rhizomes. In this study, the expression patterns of DEGs, DEMs and their targets were further validated by quantitative real-time PCR, supporting the reliability of sequencing data. CONCLUSIONS: Our study revealed a comprehensive molecular network regulating the transition of aerial stem to rhizome in G. pentaphyllum. These results broaden our understanding of developmental phase transitions in plants.

3,4-Dimethoxycinnamic Acid as a Novel Matrix for Enhanced In Situ Detection and Imaging of Low-Molecular-Weight Compounds in Biological Tissues by MALDI-MSI.

Low-molecular-weight (low-MW) compounds have many essential functions in biological processes, and the molecular imaging of as many low-MW compounds as possible is critical for understanding complex biological processes. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is an emerging molecular-imaging technology that enables determination of the spatial distributions and the relative abundances of diverse endogenous compounds in tissues. New matrices suitable for the imaging of low-MW compounds by MALDI-MSI are important for the technological advancement of tissue imaging. In this study, 3,4-dimethoxycinnamic acid (DMCA) was evaluated as a new matrix for enhanced low-MW compound detection by MALDI-MSI because of its strong ultraviolet absorption, low matrix-ion related interferences below m/ z 500, and high ionization efficiency for the analysis of low-MW compounds. DMCA was successfully used for improved in situ detection of low-molecular-weight metabolites ( m/ z < 500) and lipids in rat liver, rat brain, and germinating Chinese-yew seed tissue sections. The use of DMCA led to the successful in situ detection of 303, 200, and 248 low-MW compound ion signals from these three tissues, respectively. Both MALDI-MS/MS and LC-MS/MS were used to identify these ion signals, leading to the identification of 115 low-MW compounds from rat liver (including 53 lipids, 29 oligopeptides, and 33 metabolites), 130 low-MW compounds from rat brain (including 104 lipids, 5 oligopeptides, and 21 metabolites), and 111 low-MW compounds from germinating Chinese-yew seeds (including 77 lipids, 22 oligopeptides, 8 flavonoids, and 4 alkaloids). A larger number of low-MW compounds could be detected and imaged when DMCA was used as the MALDI matrix than with other commonly used MALDI matrices such as 2,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid, 2-mercaptobenzothiazole, graphene oxide, and silver nanoparticles. Our work provides a new and powerful matrix for enhanced MALDI-MS profiling of low-MW compounds in both animal and plant tissues.

AtPAP2, a Unique Member of the PAP Family, Functions in the Plasma Membrane.

Purple acid phosphatases (PAPs) play various physiological roles in plants. AtPAP2 was previously shown to localize to both chloroplasts and mitochondria and to modulate carbon metabolism in Arabidopsis. Over-expression of AtPAP2 resulted in faster growth and increased biomass in several plant species, indicating its great potential for crop improvement of phosphate use and yield. Here, we studied the localization of AtPAP2 by transient expression in tobacco leaves. The results showed AtPAP2 was localized to the plasma membrane through the secretory pathway, which is different from previous studies. We also found that AtPAP2 had a close relationship with fungal PAP2-like proteins based on phylogenetic analysis. In addition, the C-terminal transmembrane domain conserved in land plants is unique among other AtPAPs except AtPAP9, which is a close homolog of AtPAP2. Taken together, our results provide information for further study of AtPAP2 in understanding its special function in crop improvement.

Characterization of PM2.5-bound polycyclic aromatic hydrocarbons and its deposition in Populus tomentosa leaves in Beijing.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous constituents of air particulate matter and can be taken up by plants from the atmosphere. However, the purification of particulate-bound PAHs in the atmosphere by greening tree species has not been reported. In this study, we assess the concentrations, distribution, and sources of PM2.5-bound PAHs at three representative sites of Beijing in April, July, and November (non-heating period) and analyze the correlation between PAHs in Populus tomentosa leaves and in atmospheric PM2.5. The total PAH concentrations in PM2.5 were in the range of 19.85 ± 13.59-42.01 ± 37.17 ng/m3 with mean value of 31.35 ng/m3 at the three sites, and the PM2.5-bound PAHs concentrations in the two suburban sites (YF and YQ) were significantly higher than that in urban site (XZM) in November (autumn). At the three sites, the high molecular weight (HMW) PAHs in PM2.5 were dominant, accounting for 54.09-64.90% of total PAHs and the concentration of HMW PAHs was, on average, 9.1 times higher than that of low molecular weight (LWM) PAHs. Principal component analysis combined with diagnostic ratio analysis indicated that vehicle emission, wood combustion, and industrial processes were the main sources for PM2.5-bound PAHs in the non-heating period of Beijing. However, the LMW PAHs were dominant in P. tomentosa leaves. The concentrations of HMW PAHs (BbF, BkF, BaP, IcdP, and BghiP) in P. tomentosa leaves reached 26.11 ± 2.39, 41.42 ± 7.77, and 55.70 ± 12.33 ng/g at YQ, XZM, and YF in autumn, respectively, and were, on average, 2.1 times higher than those in April (spring) at the three sites. The ∑5PAHs concentration in P. tomentosa leaves accumulatively increased from spring to autumn, which was not related to the temporal variation of PM2.5-bound PAHs. Nevertheless, the ∑5PAHs mean concentrations followed the order of YF > XZM > YQ. This trend was consistent with spatial distribution of atmosphere PM2.5, indicating that HMW PAHs in leaves increased with the increase of atmosphere PM2.5 concentration. Our results indicated that P. tomentosa may be used as a useful species for removing PAHs from the air and biomonitoring PAHs in atmosphere.

15N-labeled ammonium nitrogen uptake and physiological responses of poplar exposed to PM2.5 particles.

Air pollution caused by particulate matter with aerodynamic diameters less than 2.5 µm (PM2.5) is a serious environmental problem. Plants can improve air quality by removing PM2.5 from the atmosphere. However, direct evidence of PM2.5 absorption and assimilation into plants has not yet been found. In this study, we demonstrate that 15NH4+ in PM2.5 was absorbed by poplar leaves in low and high PM2.5 treatment groups (namely, LPT and HPT). Then, 15N was subsequently transferred to other parts of the treated seedlings as shown by 15N tracing and simulated PM2.5 generation. 15N and total N contents were the highest in high pollution treatment (HPT), followed by that in low pollution treatment (LPT) and the control. Glutamate dehydrogenase (GDH) contributed more to NH4+ assimilation than glutamine synthetase and glutamate synthase in the leaves of treated seedlings. GDH aminating activity was induced upon NH4+ exposure whereas GDH deaminating activity was repressed in both LPT and HPT, suggesting that poplar seedlings can alleviate NH4+ toxicity by enhancing NH4+ assimilation. At the end of PM2.5 treatment period, the decreased amino acid content in the treated seedlings was attributed to the probably altered balance of amino acid metabolism. The decline in the net photosynthetic rate (Pn) was accompanied by the decrease in the stomatal conductance in poplar leaves with the extension of PM2.5 treatment time, indicating that stomatal limitation is a major reason for Pn reduction. This study may provide novel insights into the relationship between PM2.5 pollution and plants.

Molecular characterization, expression, and regulation of Gynostemma pentaphyllum squalene epoxidase gene 1.

Gynostemma pentaphyllum (Thunb.) Makino is a perennial medicinal herb widely distributed in China. This herb contains important medicinal components called gypenosides, which belong to dammarane-type triterpenoid saponins. Squalene epoxidase (SE, EC 1.14.99.7) catalyzes the epoxidation of squalene to form oxidosqualene and is a key regulatory enzyme in triterpenoid saponin biosynthesis. In this study, a SE gene designated as GpSE1 was isolated from G. pentaphyllum leaves. The deduced protein sequence of GpSE1 contained two conserved domains involved in the catalytic function of SE. GpSE1 was expressed as inclusion bodies in Escherichia coli cells, and the HIS-tagged recombinant protein was successfully purified and renatured in vitro. Immunofluorescence indicated that the polygonal reticular fluorescence signal of GpSE1 was significantly stronger in young leaves than in mature leaves and rhizomes. This finding is consistent with the tissue-specific expression pattern of GpSE1 and suggests that the young leaves of G. pentaphyllum mainly serve as the active site of gypenoside synthesis. Methyl jasmonate (MeJA) treatment upregulated GpSE1 expression in both the young and mature leaves of G. pentaphyllum, with greater upregulation in young leaves than in mature leaves. However, the expression of GpSE1 was not enhanced continually with the increase in MeJA concentration. Moreover, the GpSE1 expression was maximally regulated in response to 50 µM MeJA but not to 100 µM MeJA. This result indicates that MeJA exerts a concentration-dependent effect on GpSE1 expression.

Identification and expression of a stearoyl-ACP desaturase gene responsible for oleic acid accumulation in Xanthoceras sorbifolia seeds.

Xanthoceras sorbifolia Bunge is an oilseed tree that grows well on barren lands in dry climate. Its seeds contain a large amount of oil rich in oleic acid (18:1(Δ9)) and linoleic acid (18:2(Δ9, 12)). However, the molecular regulation of oil biosynthesis in X. sorbifolia seeds is poorly understood. Stearoyl-ACP desaturase (SAD, EC 1.14.99.6) is a plastid-localized soluble desaturase that catalyzes the conversion of stearic acid (18:0) to oleic acid, which plays a key role in determining the ratio of saturated to unsaturated fatty acids. In this study, a full-length cDNA of XsSAD was isolated from developing X. sorbifolia embryos. The XsSAD open reading frame had 1194-bp, encoding a polypeptide of 397 amino acids. XsSAD expression in Escherichia coli cells resulted in increased 18:1(Δ9) level, confirming the biological activity of the enzyme encoded by XsSAD. XsSAD expression in Arabidopsis ssi2 mutants partially restored the morphological phenotype and effectively increased the 18:1(Δ9) level. The levels of other unsaturated fatty acids synthesized with 18:1(Δ9) as the substrate also increased to some degree. XsSAD in X. sorbifolia had a much higher expression in embryos than in leaves and petals. XsSAD expression also correlated well with the oleic acid, unsaturated fatty acid, and total fatty acid levels in developing embryos. These data suggested that XsSAD determined the synthesis of oleic acid and contributed to the accumulation of unsaturated fatty acid and total oil in X. sorbifolia seeds. A preliminary tobacco rattle virus-based virus-induced gene silencing system established in X. sorbifolia can also be helpful for further analyzing the functions of XsSAD and other oil synthesis-related genes in woody plants.

XsFAD2 gene encodes the enzyme responsible for the high linoleic acid content in oil accumulated in Xanthoceras sorbifolia seeds.

BACKGROUND: Xanthoceras sorbifolia Bunge is a valuable oilseed tree that has linoleic acid-rich seed oil. Microsomal oleate desaturase (FAD2; EC 1.3.1.35) is responsible for the conversion of oleic acid to linoleic acid during fatty acid synthesis. In this study, XsFAD2 was cloned from developing embryos of X. sorbifolia. RESULTS: XsFAD2 contained three histidine boxes, a C-terminal endoplasmic reticulum retrieval motif, and five putative transmembrane domains representing the characteristics of membrane-bound fatty acid desaturase. XsFAD2 expression in yeast cells resulted in linoleic acid (18:2) and palmitolinoleic acid (16:2) production, confirming the biological activity of the enzyme encoded by XsFAD2. These fatty acids are not normally present in wild-type yeast. Phylogenetic analysis indicated that XsFAD2 is located in a subgroup of FAD2 enzymes specifically or highly expressed in developing seeds. The expression level of XsFAD2 in seeds was much higher than those in leaves and petals. Furthermore, XsFAD2 expression pattern correlated well with linoleic acid accumulated in seeds. CONCLUSION: Results suggested that XsFAD2 is responsible for the high linoleic acid content in X. sorbifolia seed oil. This study provides insight on the regulation mechanism of fatty acid synthesis in X. sorbifolia seeds and a valuable gene for improving the oil quality in oilseed trees.

Flower development under drought stress: morphological and transcriptomic analyses reveal acute responses and long-term acclimation in Arabidopsis.

Drought dramatically affects plant growth and crop yield, but previous studies primarily examined responses to drought during vegetative development. Here, to study responses to drought during reproductive development, we grew Arabidopsis thaliana plants with limited water, under conditions that allowed the plants to initiate and complete reproduction. Drought treatment from just after the onset of flowering to seed maturation caused an early arrest of floral development and sterility. After acclimation, plants showed reduced fertility that persisted throughout reproductive development. Floral defects included abnormal anther development, lower pollen viability, reduced filament elongation, ovule abortion, and failure of flowers to open. Drought also caused differential expression of 4153 genes, including flowering time genes flowering locus t, suppressor of overexpression of CO1, and leafy, genes regulating anther and pistil development, and stress-related transcription factors. Mutant phenotypes of hypersensitivity to drought and fewer differentially expressed genes suggest that dehydration response element B1A may have an important function in drought response in flowers. A more severe filament elongation defect under drought in myb21 plants demonstrated that appropriate stamen development requires MYB domain protein 21 under drought conditions. Our study reveals a regulatory cascade in reproductive responses and acclimation under drought.

Two novel diacylglycerol acyltransferase genes from Xanthoceras sorbifolia are responsible for its seed oil content.

Xanthoceras sorbifolia is an excellent model system for studying triacylglycerol (TAG) biosynthesis in woody oilseed plants due to the high amount of seed oil, which is important for food and industrial uses. TAG is the major form of stored lipids in seeds and diacylglycerol acyltransferase (DGAT; EC 2. 3. 1. 20) catalyzes the final and critical step of TAG synthesis. Here, two novel DGAT genes, designated XsDGAT1 and XsDGAT2, were cloned from developing X. sorbifolia embryos. Sequence analysis showed that XsDGAT1 had little sequence homology to XsDGAT2. Heterologous expression of XsDGAT1 and XsDGAT2 in TAG-deficient yeast mutants restored TAG synthesis, confirming their biological activity. Expression of the two genes in wild-type Arabidopsis led to TAG synthesis and an increase in total seed oil in transgenic plants, with XsDGAT1 appearing to contribute to TAG synthesis at a greater level. Comparison of the expression patterns revealed that both XsDGAT1 and XsDGAT2 were expressed in the examined tissues and had similar spatiotemporal expression patterns with higher expression in embryos than in leaves and petals. The expression patterns of both XsDGAT1 and XsDGAT2 correlated with oil accumulation in developing X. sorbifolia embryos. These data suggest that XsDGAT1 and XsDGAT2 are both responsible for TAG synthesis in X. sorbifolia seeds.