Profiling of the gene expression and alternative splicing landscapes of Eucalyptus grandis.

Eucalyptus is a widely planted hardwood tree species due to its fast growth, superior wood properties and adaptability. However, the post-transcriptional regulatory mechanisms controlling tissue development and stress responses in Eucalyptus remain poorly understood. In this study, we performed a comprehensive analysis of the gene expression profile and the alternative splicing (AS) landscape of E. grandis using strand-specific RNA-Seq, which encompassed 201 libraries including different organs, developmental stages, and environmental stresses. We identified 10 416 genes (33.49%) that underwent AS, and numerous differentially expressed and/or differential AS genes involved in critical biological processes, such as primary-to-secondary growth transition of stems, adventitious root formation, aging and responses to phosphorus- or boron-deficiency. Co-expression analysis of AS events and gene expression patterns highlighted the potential upstream regulatory role of AS events in multiple processes. Additionally, we highlighted the lignin biosynthetic pathway to showcase the potential regulatory functions of AS events in the KNAT3 and IRL3 genes within this pathway. Our high-quality expression atlas and AS landscape serve as valuable resources for unravelling the genetic control of woody plant development, long-term adaptation, and understanding transcriptional diversity in Eucalyptus. Researchers can conveniently access these resources through the interactive ePlant browser (https://bar.utoronto.ca/eplant_eucalyptus).

Insights into cryptochrome modulation of ABA signaling to mediate dormancy regulation in Marchantia polymorpha.

The acquisition of dormancy capabilities has enabled plants to survive in adverse terrestrial environmental conditions. Dormancy accumulation and release is coupled with light signaling, which is well studied in Arabidopsis, but it is unclear in the distant nonvascular relative. We study the characteristics and function on dormancy regulation of a blue light receptor cryptochrome in Marchantia polymorpha (MpCRY). Here, we identified MpCRY via bioinformatics and mutant complement analysis. The biochemical characteristics were assessed by multiple protein-binding assays. The function of MpCRY in gemma dormancy was clarified by overexpression and mutation of MpCRY, and its mechanism was analyzed via RNA sequencing and quantitative PCR analyses associated with hormone treatment. We found that the unique MpCRY protein in M. polymorpha undergoes both blue light-promoted interaction with itself (self-interaction) and blue light-dependent phosphorylation. MpCRY has the specific characteristics of blue light-induced nuclear localization and degradation. We further demonstrated that MpCRY transcriptionally represses abscisic acid (ABA) signaling-related gene expression to suppress gemma dormancy, which is dependent on blue light signaling. Our findings indicate that MpCRY possesses specific biochemical and molecular characteristics, and modulates ABA signaling under blue light conditions to regulate gemma dormancy in M. polymorpha.

Time-course transcriptome analysis reveals regulation of Arabidopsis seed dormancy by the transcription factors WOX11/12.

The induction of seed dormancy and its release involve a finely regulated genetic program controlled by various environmental and developmental cues that are critical for plant survival and population expansion. Light plays a key role in seed dormancy and germination, but the molecular mechanisms underlying the control of dormancy are unclear. In the present study, high-resolution temporal RNA-seq in Arabidopsis identified WOX11 as encoding a hub transcription factor during the seed dormancy induction and release stages. This gene might have evolved from gymnosperms and expanded in angiosperms with highly conserved expression patterns in seeds. WOX11 and its homolog WOX12 were highly expressed from 2 d after pollination, and mRNA abundance was greatly increased during the seed dormancy induction and release stages. Further, we found that WOX11 plays a role in the regulation of seed dormancy downstream of phytochrome B (PHYB)-mediated red-light signaling during the induction stage, indicating that WOX11/12 are newly identified components of red-light signaling transduction. Taken together, our results suggest that WOX11/12-mediated PHYB signaling regulates seed dormancy in Arabidopsis, and provide insights into the developmental regulation and evolutionary adaptation of plants to changes in the light environment.

The Euscaphis japonica genome and the evolution of malvids.

Malvids is one of the largest clades of rosids, includes 58 families and exhibits remarkable morphological and ecological diversity. Here, we report a high-quality chromosome-level genome assembly for Euscaphis japonica, an early-diverging species within malvids. Genome-based phylogenetic analysis suggests that the unstable phylogenetic position of E. japonica may result from incomplete lineage sorting and hybridization event during the diversification of the ancestral population of malvids. Euscaphis japonica experienced two polyploidization events: the ancient whole genome triplication event shared with most eudicots (commonly known as the γ event) and a more recent whole genome duplication event, unique to E. japonica. By resequencing 101 samples from 11 populations, we speculate that the temperature has led to the differentiation of the evergreen and deciduous of E. japonica and the completely different population histories of these two groups. In total, 1012 candidate positively selected genes in the evergreen were detected, some of which are involved in flower and fruit development. We found that reddening and dehiscence of the E. japonica pericarp and long fruit-hanging time promoted the reproduction of E. japonica populations, and revealed the expression patterns of genes related to fruit reddening, dehiscence and abscission. The key genes involved in pentacyclic triterpene synthesis in E. japonica were identified, and different expression patterns of these genes may contribute to pentacyclic triterpene diversification. Our work sheds light on the evolution of E. japonica and malvids, particularly on the diversification of E. japonica and the genetic basis for their fruit dehiscence and abscission.

Genome-wide identification, evolution and expression analysis of the aspartic protease gene family during rapid growth of moso bamboo (Phyllostachys edulis) shoots.

BACKGROUND: Aspartic proteases (APs) are a class of aspartic peptidases belonging to nine proteolytic enzyme families whose members are widely distributed in biological organisms. APs play essential functions during plant development and environmental adaptation. However, there are few reports about APs in fast-growing moso bamboo. RESULT: In this study, we identified a total of 129 AP proteins (PhAPs) encoded by the moso bamboo genome. Phylogenetic and gene structure analyses showed that these 129 PhAPs could be divided into three categories (categories A, B and C). The PhAP gene family in moso bamboo may have undergone gene expansion, especially the members of categories A and B, although homologs of some members in category C have been lost. The chromosomal location of PhAPs suggested that segmental and tandem duplication events were critical for PhAP gene expansion. Promoter analysis revealed that PhAPs in moso bamboo may be involved in plant development and responses to environmental stress. Furthermore, PhAPs showed tissue-specific expression patterns and may play important roles in rapid growth, including programmed cell death, cell division and elongation, by integrating environmental signals such as light and gibberellin signals. CONCLUSION: Comprehensive analysis of the AP gene family in moso bamboo suggests that PhAPs have experienced gene expansion that is distinct from that in rice and may play an important role in moso bamboo organ development and rapid growth. Our results provide a direction and lay a foundation for further analysis of plant AP genes to clarify their function during rapid growth.

Genome-Wide Identification and Co-Expression Analysis of ARF and IAA Family Genes in Euscaphis konishii: Potential Regulators of Triterpenoids and Anthocyanin Biosynthesis.

Euscaphis konishii is an evergreen plant that is widely planted as an industrial crop in Southern China. It produces red fruits with abundant secondary metabolites, giving E. konishii high medicinal and ornamental value. Auxin signaling mediated by members of the AUXIN RESPONSE FACTOR (ARF) and auxin/indole-3-acetic acid (Aux/IAA) protein families plays important roles during plant growth and development. Aux/IAA and ARF genes have been described in many plants but have not yet been described in E. konishii. In this study, we identified 34 EkIAA and 29 EkARF proteins encoded by the E. konishii genome through database searching using HMMER. We also performed a bioinformatic characterization of EkIAA and EkARF genes, including their phylogenetic relationships, gene structures, chromosomal distribution, and cis-element analysis, as well as conserved motifs in the proteins. Our results suggest that EkIAA and EkARF genes have been relatively conserved over evolutionary history. Furthermore, we conducted expression and co-expression analyses of EkIAA and EkARF genes in leaves, branches, and fruits, which identified a subset of seven EkARF genes as potential regulators of triterpenoids and anthocyanin biosynthesis. RT-qPCR, yeast one-hybrid, and transient expression analyses showed that EkARF5.1 can directly interact with auxin response elements and regulate downstream gene expression. Our results may pave the way to elucidating the function of EkIAA and EkARF gene families in E. konishii, laying a foundation for further research on high-yielding industrial products and E. konishii breeding.

Overexpression of PtrMYB121 Positively Regulates the Formation of Secondary Cell Wall in Arabidopsis thaliana.

MYB transcription factors have a wide range of functions in plant growth, hormone signaling, salt, and drought tolerances. In this study, two homologous transcription factors, PtrMYB55 and PtrMYB121, were isolated and their functions were elucidated. Tissue expression analysis revealed that PtrMYB55 and PtrMYB121 had a similar expression pattern, which had the highest expression in stems. Their expression continuously increased with the growth of poplar, and the expression of PtrMYB121 was significantly upregulated in the process. The full length of PtrMYB121 was 1395 bp, and encoded protein contained 464 amino acids including conserved R2 and R3 MYB domains. We overexpressed PtrMYB121 in Arabidopsis thaliana, and the transgenic lines had the wider xylem as compared with wild-type Arabidopsis. The contents of cellulose and lignin were obviously higher than those in wild-type materials, but there was no significant change in hemicellulose. Quantitative real-time PCR demonstrated that the key enzyme genes regulating the synthesis of lignin and cellulose were significantly upregulated in the transgenic lines. Furthermore, the effector-reporter experiment confirmed that PtrMYB121 bound directly to the promoters of genes relating to the synthesis of lignin and cellulose. These results suggest that PtrMYB121 may positively regulate the formation of secondary cell wall by promoting the synthesis of lignin and cellulose.

Insights of Molecular Mechanism of Xylem Development in Five Black Poplar Cultivars.

Black poplar (Populus deltoides, P. nigra, and their hybrids) is the main poplar cultivars in China. It offers interesting options of large-scale biomass production for bioenergy due to its rapid growth and high yield. Poplar wood properties were associated with chemical components and physical structures during wood formation. In this study, five poplar cultivars, P. euramericana 'Zhonglin46' (Pe1), P. euramericana 'Guariento' (Pe2), P. nigra 'N179' (Pn1), P. deltoides 'Danhong' (Pd1), and P. deltoides 'Nanyang' (Pd2), were used to explore the molecular mechanism of xylem development. We analyzed the structural differences of developing xylem in the five cultivars and profiled the transcriptome-wide gene expression patterns through RNA sequencing. The cross sections of the developing xylem showed that the cell wall thickness of developed fiber in Pd1 was thickest and the number of xylem vessels of Pn1 was the least. A total of 10,331 differentially expressed genes were identified among 10 pairwise comparisons of the five cultivars, most of them were related to programmed cell death and secondary cell wall thickening. K-means cluster analysis and Gene Ontology enrichment analysis showed that the genes highly expressed in Pd1 were related to nucleotide decomposition, metabolic process, transferase, and microtubule cytoskeleton; whereas the genes highly expressed in Pn1 were involved in cell wall macromolecule decomposition and polysaccharide binding processes. Based on a weighted gene co-expression network analysis, a large number of candidate regulators for xylem development were identified. And their potential regulatory roles to cell wall biosynthesis genes were validated by a transient overexpression system. This study provides a set of promising candidate regulators for genetic engineering to improve feedstock and enhance biofuel conversion in the bioenergy crop Populus.

Functional and morphological evolution in gymnosperms: A portrait of implicated gene families.

Gymnosperms diverged from their sister plant clade of flowering plants 300 Mya. Morphological and functional divergence between the two major seed plant clades involved significant changes in their reproductive biology, water-conducting systems, secondary metabolism, stress defense mechanisms, and small RNA-mediated epigenetic silencing. The relatively recent sequencing of several gymnosperm genomes and the development of new genomic resources have enabled whole-genome comparisons within gymnosperms, and between angiosperms and gymnosperms. In this paper, we aim to understand how genes and gene families have contributed to the major functional and morphological differences in gymnosperms, and how this information can be used for applied breeding and biotechnology. In addition, we have analyzed the angiosperm versus gymnosperm evolution of the pleiotropic drug resistance (PDR) gene family with a wide range of functionalities in plants' interaction with their environment including defense mechanisms. Some of the genes reviewed here are newly studied members of gene families that hold potential for biotechnological applications related to commercial and pharmacological value. Some members of conifer gene families can also be exploited for their potential in phytoremediation applications.

An essential role for Arabidopsis Trs33 in cell growth and organization in plant apical meristems.

KEY MESSAGE: Trafficking protein particle (TRAPP) complexes subunit gene AtTrs33 plays an important role in keeping apical meristematic activity and dominance in Arabidopsis. TRAPP complexes, composed of multimeric subunits, are guanine-nucleotide exchange factors for certain Rab GTPases and are believed to be involved in the regulation of membrane trafficking, but the cases in Arabidopsis are largely unknown. Trs33, recently proposed to be a component of TRAPP IV, is non-essential in yeast cells. A single copy of Trs33 gene, AtTrs33, was identified in Arabidopsis. GUS activity assay indicated that AtTrs33 was ubiquitously expressed. Based on a T-DNA insertion line, we found that loss-of-function of AtTrs33 is lethal for apical growth. Knock-down or knock-in of AtTrs33 affects apical meristematic growth and fertility, which indicates that AtTrs33 plays an important role in keeping apical meristematic activity and dominance in Arabidopsis. Analysis of auxin responses and PIN1/2 localization indicate that impaired apical meristematic activity and dominance were caused by altered auxin responses through non-polarized PIN1 localization. The present study reported that AtTrs33 plays an essential role in Arabidopsis cell growth and organization, which is different with its homologue in yeast. These findings provide new insights into the functional divergence of TRAPP subunits.

Identification and Characterization of the PEBP Family Genes in Moso Bamboo (Phyllostachys heterocycla).

Moso bamboo is one of the economically most important plants in China. Moso bamboo is a monocarpic perennial that exhibits poor and slow germination. Thus, the flowering often causes destruction of moso bamboo forestry. However, how control of flowering and seed germination are regulated in moso bamboo is largely unclear. In this study, we identified 5 members (PhFT1-5) of the phosphatidyl ethanolamine-binding proteins (PEBP) family from moso bamboo genome that regulate flowering, flower architecture and germination, and characterized the function of these PEBP family genes further in Arabidopsis. Phylogenetic analysis revealed that 3 (PhFT1, PhFT2 and PhFT3), 1 (PhFT4) and 1 (PhFT5) members belong to the TFL1-like clade, FT-like clade, and MFT-like clade, respectively. These PEBP family genes possess all structure necessary for PEBP gene function. The ectopic overexpression of PhFT4 and PhFT5 promotes flowering time in Arabidopsis, and that of PhFT1, PhFT2 and PhFT3 suppresses it. In addition, the overexpression of PhFT5 promotes seed germination rate. Interestingly, the overexpression of PhFT1 suppressed seed germination rate in Arabidopsis. The expression of PhFT1 and PhFT5 is significantly higher in seed than in tissues including leaf and shoot apical meristem, implying their function in seed germination. Taken together, our results suggested that the PEBP family genes play important roles as regulators of flowering and seed germination in moso bamboo and thereby are necessary for the sustainability of moso bamboo forest.

Evolutionary Divergence of Duplicated Hsf Genes in Populus.

Heat shock transcription factors (Hsfs), which function as the activator of heat shock proteins (Hsps), play multiple roles in response to environmental stress and the development of plants. The Hsf family had experienced gene expansion via whole-genome duplication from a single cell algae to higher plants. However, how the Hsf gene family went through evolutionary divergence after genome duplication is unknown. As a model wood species, Populus trichocarpa is widely distributed in North America with various ecological and climatic environments. In this study, we used P. trichocarpa as materials and identified the expression divergence of the PtHsf gene family in developmental processes, such as dormant bud formation and opening, catkins development, and in response to environments. Through the co-expression network, we further discovered the divergent co-expressed genes that related to the functional divergence of PtHsfs. Then, we studied the alternative splicing events, single nucleotide polymorphism distribution and tertiary structures of members of the PtHsf gene family. In addition to expression divergence, we uncovered the evolutionary divergence in the protein level which may be important to new function formations and for survival in changing environments. This study comprehensively analyzed the evolutionary divergence of a member of the PtHsf gene family after genome duplication, paving the way for further gene function analysis and genetic engineering.

PtWOX11 acts as master regulator conducting the expression of key transcription factors to induce de novo shoot organogenesis in poplar.

KEY MESSAGE: WUSCHEL-RELATED HOMEOBOX 11 establishes the acquisition of pluripotency during callus formation and accomplishes de novo shoot formation by regulating key transcription factors in poplar. De novo shoot regeneration is a prerequisite for propagation and genetic engineering of elite cultivars in forestry. However, the regulatory mechanism of de novo organogenesis is poorly understood in tree species. We previously showed that WUSCHEL (WUS)-RELATED HOMEOBOX 11 (PtWOX11) of the hybrid poplar clone 84K (Populus alba × P. glandulosa) promotes de novo root formation. In this study, we found that PtWOX11 also regulates de novo shoot regeneration in poplar. The overexpression of PtWOX11 enhanced de novo shoot formation, whereas overexpression of PtWOX11 fused with the transcriptional repressor domain (PtWOX11-SRDX) or reduced expression of PtWOX11 inhibited this process, indicating that PtWOX11 promotes de novo shoot organogenesis. Although PtWOX11 promotes callus formation, overexpression of PtWOX11 and PtWOX11-SRDX also produced increased and decreased numbers of de novo shoots per unit weight, respectively, implying that PtWOX11 promotes de novo shoot organogenesis partially by regulating the intrinsic mechanism of shoot development. RNA-seq and qPCR analysis further revealed that PtWOX11 activates the expression of PLETHORA1 (PtPLT1) and PtPLT2, whose Arabidopsis paralogs establish the acquisition of pluripotency, during incubation on callus-inducing medium. Moreover, PtWOX11 activates the expression of shoot-promoting factors and meristem regulators such as CUP-SHAPED COTYLEDON2 (PtCUC2), PtCUC3, WUS and SHOOT MERISTEMLESS to fulfill shoot regeneration during incubation on shoot-inducing medium. These results suggest that PtWOX11 acts as a central regulator of the expression of key genes to cause de novo shoot formation. Our studies further provide a possible means to genetically engineer economically important tree species for their micropropagation.

Sequencing of Euscaphis konishii Endocarp Transcriptome Points to Molecular Mechanisms of Endocarp Coloration.

Flower and fruit colors are of vital importance to the ecology and economic market value of plants. The mechanisms of flower and fruit coloration have been well studied, especially among ornamental flower plants and cultivated fruits. As people pay more attention to exocarp coloration, the endocarp coloration in some species has often been ignored. Here, we report on the molecular mechanism of endocarp coloration in three development stages of Euscaphis konishii. The results show that endocarp reddening is closely related to anthocyanin accumulation, and a total of 86,120 unigenes were assembled, with a mean length of 893 bp (N50 length of 1642 bp). We identified a large number of differentially expressed genes associated with endocarp coloration, including anthocyanin biosynthesis, carotenoid biosynthesis, and chlorophyll breakdown. The genes participating in each step of the anthocyanin biosynthesis were found in the transcriptome dataset, but a few genes were found in the carotenoid biosynthesis and chlorophyll breakdown. In addition, the candidate R2R3-MYB transcription factors and candidate glutathione S-transferase transport genes, which likely regulate the anthocyanin biosynthesis, were identified. This study offers a platform for E. konishii functional genomic research and provides a reference for revealing the regulatory mechanisms of endocarp reddening.

AtBET5 is essential for exine pattern formation and apical meristem organization in Arabidopsis.

BET5 is a component of trafficking protein particle (TRAPP) which has been studied extensively in non-plant organisms where they are involved in membrane trafficking within Golgi and between Golgi and early endosomes. Recent analysis of TRAPP in different classes of organisms indicates that TRAPP function might exhibit differences among organisms. A single copy of the BET5 gene named AtBET5 was found in the Arabidopsis genome based on sequence similarity. Developmental phenotype and the underlying mechanisms have been characterized upon transcriptional knock-down lines generated by both T-DNA insertion and RNAi. Pollen grains of the T-DNA insertional line present reduced fertility and pilate exine instead of tectate exine. Perturbation of the AtBET5 expression by RNAi leads to apical meristematic organization defects and reduced fertility as well. The reduced fertility was due to the pollination barrier caused by an altered composition and structure of pollen walls. Auxin response in root tip cells is altered and there is a severe disruption in polar localization of PIN1-GFP, but to a less extent of PIN2-GFP in the root tips, which causes the apical meristematic organization defects and might also be responsible for the secretion of sporopollenin precursor or polar targeting of sporopollenin precursor transporters.

Characterization of the Populus Rab family genes and the function of PtRabE1b in salt tolerance.

BACKGROUND: Rab proteins form the largest family of the Ras superfamily of small GTP-binding proteins and regulate intracellular trafficking pathways. However, the function of the Rab proteins in woody species is still an open question. RESULTS: Here, a total of 67 PtRabs were identified in Populus trichocarpa and categorized into eight subfamilies (RabA-RabH). Based on their chromosomal distribution and duplication blocks in the Populus genome, a total of 27 PtRab paralogous pairs were identified and all of them were generated by whole-genome duplication events. Combined the expression correlation and duplication date, the PtRab paralogous pairs that still keeping highly similar expression patterns were generated around the latest large-scale duplication (~ 13 MYA). The cis-elements and co-expression network of unique expanded PtRabs suggest their potential roles in poplar development and environmental responses. Subcellular localization of PtRabs from each subfamily indicates each subfamily shows a localization pattern similar to what is revealed in Arabidopsis but RabC shows a localization different from their counterparts. Furthermore, we characterized PtRabE1b by overexpressing its constitutively active mutant PtRabE1b(Q74L) in poplar and found that PtRabE1b(Q74L) enhanced the salt tolerance. CONCLUSIONS: These findings provide new insights into the functional divergence of PtRabs and resources for genetic engineering resistant breeding in tree species.

The WUSCHEL-related homeobox 5a (PtoWOX5a) is involved in adventitious root development in poplar.

Adventitious rooting is an essential step in vegetative propagation. Currently, the mechanism that regulates adventitious root (AR) development in woody plants is poorly understood. This work demonstrates that Populus tomentosa WUSCHEL-related homeobox 5a (PtoWOX5a) transcription factor is involved in AR development in poplar. PtoWOX5a was specifically expressed in the AR tip and lateral root tip during AR and lateral root regeneration from the stem segment. Phenotypic complementation experiments indicated that the PtoWOX5a can functionally complement AtWOX5 in quiescent center (QC) cells. Overexpression of PtoWOX5a introduces significant developmental phenotypes in roots and leaves, such as increased AR number, decreased AR length, swollen AR tip and lateral root tip, and decreased leaf number and area. The conserved mechanism of D-type cyclins (CYCD) repression mediated by WOX5 was confirmed in poplar. The co-expression network of PtWOX5a was constructed, which provided clues to reveal the molecular mechanism of PtoWOX5a in AR development in poplar. Taken together, our results suggest that the PtoWOX5a is involved in AR development though cooperating with a series of functional genes.

A CRY-BIC negative-feedback circuitry regulating blue light sensitivity of Arabidopsis.

Cryptochromes are blue light receptors that regulate various light responses in plants. Arabidopsis cryptochrome 1 (CRY1) and cryptochrome 2 (CRY2) mediate blue light inhibition of hypocotyl elongation and long-day (LD) promotion of floral initiation. It has been reported recently that two negative regulators of Arabidopsis cryptochromes, Blue light Inhibitors of Cryptochromes 1 and 2 (BIC1 and BIC2), inhibit cryptochrome function by blocking blue light-dependent cryptochrome dimerization. However, it remained unclear how cryptochromes regulate the BIC gene activity. Here we show that cryptochromes mediate light activation of transcription of the BIC genes, by suppressing the activity of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), resulting in activation of the transcription activator ELONGATED HYPOCOTYL 5 (HY5) that is associated with chromatins of the BIC promoters. These results demonstrate a CRY-BIC negative-feedback circuitry that regulates the activity of each other. Surprisingly, phytochromes also mediate light activation of BIC transcription, suggesting a novel photoreceptor co-action mechanism to sustain blue light sensitivity of plants under the broad spectra of solar radiation in nature.