Functional importance and divergence of plant apurinic/apyrimidinic endonucleases in somatic and meiotic DNA repair.

Apurinic/apyrimidinic (AP) sites are one of the most abundant DNA lesions and are mainly repaired by AP endonucleases (APEs). While most eukaryotic genomes encode two APEs, plants usually possess three APEs, namely APE1L, APE2, and ARP. To date, the biological relevance and functional divergence of plant APEs are unclear. Here, we show that the three plant APEs have ancient origins, with the APE1L clade being plant-specific. In Arabidopsis thaliana, simultaneously mutating APE1L and APE2, but not ARP alone or in combination with either APE1L or APE2, results in clear developmental defects linked to genotoxic stress. Genetic analyses indicated that the three plant APEs have different substrate preferences in vivo. ARP is mainly responsible for AP site repair, while APE1L and APE2 prefer to repair 3'-blocked single-stranded DNA breaks. We further determined that APEs play an important role in DNA repair and the maintenance of genomic integrity in meiotic cells. The ape1l ape2 double mutant exhibited a greatly enhanced frequency of sporulation 1 (SPO11-1)-dependent and SPO11-1-independent double-stranded DNA breaks. The DNA damage response (DDR) was activated in ape1l ape2 to trigger pollen abortion. Our findings suggest functional divergence of plant APEs and reveal important roles of plant APEs during vegetative and reproductive development.

The Arabidopsis ATR-SOG1 signaling module regulates pleiotropic developmental adjustments in response to 3'-blocked DNA repair intermediates.

Base excision repair and active DNA demethylation produce repair intermediates with DNA molecules blocked at the 3'-OH end by an aldehyde or phosphate group. However, both the physiological consequences of these accumulated single-strand DNAs break with 3'-blocked ends (DNA 3'-blocks) and the signaling pathways responding to unrepaired DNA 3'-blocks remain unclear in plants. Here, we investigated the effects of DNA 3'-blocks on plant development using the zinc finger DNA 3'-phosphoesterase (zdp) AP endonuclease2 (ape2) double mutant, in which 3'-blocking residues are poorly repaired. The accumulation of DNA 3'-blocked triggered diverse developmental defects that were dependent on the ATM and RAD3-related (ATR)-suppressor of gamma response 1 (SOG1) signaling module. SOG1 mutation rescued the developmental defects of zdp ape2 leaves by preventing cell endoreplication and promoting cell proliferation. However, SOG1 mutation caused intensive meristematic cell death in the radicle of zdp ape2 following germination, resulting in rapid termination of radicle growth. Notably, mutating FORMAMIDOPYRIMIDINE DNA GLYCOSYLASE (FPG) in zdp ape2 sog1 partially recovered its radicle growth, demonstrating that DNA 3'-blocks generated by FPG caused the meristematic defects. Surprisingly, despite lacking a functional radicle, zdp ape2 sog1 mutants compensated the lack of root growth by generating anchor roots having low levels of DNA damage response. Our results reveal dual roles of SOG1 in regulating root establishment when seeds germinate with excess DNA 3'-blocks.

Tonoplast Sucrose Trafficking Modulates Starch Utilization and Water Deficit Behavior in Poplar Leaves.

Leaf osmotic adjustment by the active accrual of compatible organic solutes (e.g. sucrose) contributes to drought tolerance throughout the plant kingdom. In Populus tremula x alba, PtaSUT4 encodes a tonoplast sucrose-proton symporter, whose downregulation by chronic mild drought or transgenic manipulation is known to increase leaf sucrose and turgor. While this may constitute a single drought tolerance mechanism, we now report that other adjustments which can occur during a worsening water deficit are damped when PtaSUT4 is constitutively downregulated. Specifically, we report that starch use and leaf relative water content (RWC) dynamics were compromised when plants with constitutively downregulated PtaSUT4 were subjected to a water deficit. Leaf RWC decreased more in wild-type and vector control lines than in transgenic PtaSUT4-RNAi (RNA-interference) or CRISPR (clustered regularly interspersed short palindromic repeats) knockout (KO) lines. The control line RWC decrease was accompanied by increased PtaSUT4 transcript levels and a mobilization of sucrose from the mesophyll-enriched leaf lamina into the midvein. The findings suggest that changes in SUT4 expression can increase turgor or decrease RWC as different tolerance mechanisms to reduced water availability. Evidence is presented that PtaSUT4-mediated sucrose partitioning between the vacuole and the cytosol is important not only for overall sucrose abundance and turgor, but also for reactive oxygen species (ROS) and antioxidant dynamics. Interestingly, the reduced capacity for accelerated starch breakdown under worsening water-deficit conditions was correlated with reduced ROS in the RNAi and KO lines. A role for PtaSUT4 in the orchestration of ROS, antioxidant, starch utilization and RWC dynamics during water stress and its importance in trees especially, with their high hydraulic resistances, is considered.

Nitrogen starvation induces genome-wide activation of transposable elements in Arabidopsis.

Nitrogen (N) availability is a major limiting factor for plant growth and agricultural productivity. Although the gene regulation network in response to N starvation has been extensively studied, it remains unknown whether N starvation has an impact on the activity of transposable elements (TEs). Here, we report that TEs can be transcriptionally activated in Arabidopsis under N starvation conditions. Through genetic screening of idm1-14 suppressors, we cloned GLU1, which encodes a glutamate synthase that catalyzes the synthesis of glutamate in the primary N assimilation pathway. We found that glutamate synthase 1 (GLU1) and its functional homologs GLU2 and glutamate transport 1 (GLT1) are redundantly required for TE silencing, suggesting that N metabolism can regulate TE activity. Transcriptome and methylome analyses revealed that N starvation results in genome-wide TE activation without inducing obvious alteration of DNA methylation. Genetic analysis indicated that N starvation-induced TE activation is also independent of other well-established epigenetic mechanisms, including histone methylation and heterochromatin decondensation. Our results provide new insights into the regulation of TE activity under stressful environments in planta.

Indole-3-acetic acid has long-term effects on long non-coding RNA gene methylation and growth in Populus tomentosa.

DNA methylation and long non-coding RNAs (lncRNAs) regulate plant growth and development, but their relationship and effect on responses to the auxin phytohormone indole-3-acetic acid (IAA) remain largely unknown, particularly in woody plants such as poplar (Populus tomentosa). Following treatment of 1-year-old clonal plants with 100 µM IAA, key poplar lncRNA genes showed changes in methylation, but whole-genome methylation levels showed no significant change. Moreover, 100 µM IAA inhibited growth of the 1-year-old poplar clones, possibly through the suppression of photosynthesis. This inhibition had a long-term effect, persisting at 1 month after removal of the exogenous IAA. Transcriptome analysis identified two candidate lncRNA genes that show changes in expression following IAA treatment, TCONS_00003480 and TCONS_00004832. TCONS_00003480 contains the same microRNA target sites of ptc-miR6464 as the 4-coumarate: CoA ligase 2 transcript, which encodes a lignin biosynthesis enzyme. And TCONS_00004832 shares the same target sites of ptc-miR6437a with the Photosystem II reaction center protein D and Cytochrome C Oxidase 17 transcripts, which are related to photosynthesis. The two lncRNAs as the mimics to corresponding target genes of miRNAs to prevent them from degrading. Examination of lncRNA gene expression and methylation revealed a negative relationship (r = - 0.29, P < 0.05); moreover, hypermethylation of the two candidate lncRNA genes remained 1 month after IAA treatment, suggesting that changes in methylation might be involved in the long-term effects of plant hormones. Therefore, our study reveals a long-term effect of IAA on the growth of P. tomentosa, possibly via methylation-mediated epigenetic changes in lncRNA gene expression and the interaction with corresponding miRNAs, leading to regulation of genes related to photosynthesis and growth.

Osmotic stress-responsive promoter upstream transcripts (PROMPTs) act as carriers of MYB transcription factors to induce the expression of target genes in Populus simonii.

Complex RNA transcription and processing produces a diverse range catalog of long noncoding RNAs (lncRNAs), important biological regulators that have been implicated in osmotic stress responses in plants. Promoter upstream transcript (PROMPT) lncRNAs share some regulatory elements with the promoters of their neighbouring protein-coding genes. However, their function remains unknown. Here, using strand-specific RNA sequencing, we identified 209 differentially regulated osmotic-responsive PROMPTs in poplar (Populus simonii). PROMPTs are transcribed bidirectionally and are more stable than other lncRNAs. Co-expression analysis of PROMPTs and protein-coding genes divided the regulatory network into five independent subnetworks including 27 network modules. Significantly enriched PROMPTs in the network were selected to validate their regulatory roles. We used delaminated layered double hydroxide lactate nanosheets (LDH-lactate-NS) to transport synthetic nucleic acids into live tissues to mimic overexpression and interference of a specific PROMPT. The altered expression of PROMPT_1281 induced the expression of its cis and trans targets, and this interaction was governed by its secondary structure rather than just its primary sequence. Based on this example, we proposed a model that a concentration gradient of PROMPT_1281 is established, which increases the probability of its interaction with targets near its transcription site that shares common motifs. Our results firstly demonstrated that PROMPT_1281 act as carriers of MYB transcription factors to induce the expression of target genes under osmotic stress. In sum, our study identified and validated a set of poplar PROMPTs that likely have regulatory functions in osmotic responses.

Conserved noncoding sequences conserve biological networks and influence genome evolution.

Comparative genomics approaches have identified numerous conserved cis-regulatory sequences near genes in plant genomes. Despite the identification of these conserved noncoding sequences (CNSs), our knowledge of their functional importance and selection remains limited. Here, we used a combination of DNA methylome analysis, microarray expression analyses, and functional annotation to study these sequences in the model tree Populus trichocarpa. Methylation in CG contexts and non-CG contexts was lower in CNSs, particularly CNSs in the 5'-upstream regions of genes, compared with other sites in the genome. We observed that CNSs are enriched in genes with transcription and binding functions, and this also associated with syntenic genes and those from whole-genome duplications, suggesting that cis-regulatory sequences play a key role in genome evolution. We detected a significant positive correlation between CNS number and protein interactions, suggesting that CNSs may have roles in the evolution and maintenance of biological networks. The divergence of CNSs indicates that duplication-degeneration-complementation drives the subfunctionalization of a proportion of duplicated genes from whole-genome duplication. Furthermore, population genomics confirmed that most CNSs are under strong purifying selection and only a small subset of CNSs shows evidence of adaptive evolution. These findings provide a foundation for future studies exploring these key genomic features in the maintenance of biological networks, local adaptation, and transcription.

Stable methylation of a non-coding RNA gene regulates gene expression in response to abiotic stress in Populus simonii.

DNA methylation plays important roles in responses to environmental stimuli. However, in perennial plants, the roles of DNA methylation in stress-specific adaptions to different abiotic stresses remain unclear. Here, we present a systematic, comparative analysis of the methylome and gene expression in poplar under cold, osmotic, heat, and salt stress conditions from 3h to 24h. Comparison of the stress responses revealed different patterns of cytosine methylation in response to the four abiotic stresses. We isolated and sequenced 1376 stress-specific differentially methylated regions (SDMRs); annotation revealed that these SDMRs represent 1123 genes encoding proteins, 16 miRNA genes, and 17 long non-coding RNA (lncRNA) genes. The SDMR162 region, consisting of Psi-MIR396e and PsiLNCRNA00268512, is regulated by epigenetic pathways and we speculate that PsiLNCRNA00268512 regulates miR396e levels by acting as a target mimic. The ratios of methylated cytosine declined to ~35.1% after 1 month of recovery from abiotic stress and to ~15.3% after 6 months. Among methylated miRNA genes, only expression of the methylation-regulated gene MIRNA6445a showed long-term stability. Our data provide a strong basis for future work and improve our understanding of the effect of epigenetic regulation of non-coding RNA expression, which will enable in-depth functional analysis.

Variation in genomic methylation in natural populations of Populus simonii is associated with leaf shape and photosynthetic traits.

DNA methylation, one of the best-studied types of chromatin modification, suppresses the expression of transposable elements, pseudogenes, repetitive sequences, and individual genes. However, the extent and variation of genome-wide DNA methylation in natural populations of plants remain relatively unknown. To investigate variation in DNA methylation and whether this variation associates with important plant traits, including leaf shape and photosynthesis, 20 413 DNA methylation sites were examined in a poplar association population (505 individuals) using methylation-sensitive amplification polymorphism (MSAP) technology. Calculation of epi-population structure and kinships assigned individuals into subsets (K=3), revealing that the natural population of P. simonii consists of three subpopulations. Population epigenetic distance and geographic distance showed a significant correlation (r=0.4688, P<0.001), suggesting that environmental factors may affect epigenetics. Single-marker approaches were also used to identify significant marker-trait associations, which found 1087 high-confidence DNA methylation markers associated with different phenotypic traits explaining ~5-15% of the phenotypic variance. Among these loci, 147 differentially methylated fragments were obtained by sequencing, representing 130 candidate genes. Expression analysis of six candidate genes indicated that these genes might play important roles in leaf development and regulation of photosynthesis. This study provides association analysis to study the effects of DNA methylation on plant development and these data indicate that epigenetics bridges environmental and genetic factors in affecting plant growth and development.

Population genomic analysis of gibberellin-responsive long non-coding RNAs in Populus.

Long non-coding RNAs (lncRNAs) participate in a wide range of biological processes, but lncRNAs in plants remain largely unknown; in particular, we lack a systematic identification of plant lncRNAs involved in hormone responses. Moreover, allelic variation in lncRNAs remains poorly characterized at a large scale. Here, we conducted high-throughput RNA-sequencing of leaves from control and gibberellin (GA)-treated Populus tomentosa and identified 7655 reliably expressed lncRNAs. Among the 7655 lncRNAs, the levels of 410 lncRNAs changed in response to GA. Seven GA-responsive lncRNAs were predicted to be putative targets of 18 miRNAs, and one GA-responsive lncRNA (TCONS_00264314) was predicted to be a target mimic of ptc-miR6459b. Computational analysis predicted 939 potential cis-regulated target genes and 965 potential trans-regulated target genes for GA-responsive lncRNAs. Functional annotation of these potential target genes showed that they participate in many different biological processes, including auxin signal transduction and synthesis of cellulose and pectin, indicating that GA-responsive lncRNAs may influence growth and wood properties. Finally, single nucleotide polymorphism (SNP)-based association analysis showed that 112 SNPs from 52 GA-responsive lncRNAs and 1014 SNPs from 296 potential target genes were significantly associated with growth and wood properties. Epistasis analysis also provided evidence for interactions between lncRNAs and their potential target genes. Our study provides a comprehensive view of P. tomentosa lncRNAs and offers insights into the potential functions and regulatory interactions of GA-responsive lncRNAs, thus forming the foundation for future functional analysis of GA-responsive lncRNAs in P. tomentosa.

Methylation of microRNA genes regulates gene expression in bisexual flower development in andromonoecious poplar.

Previous studies showed sex-specific DNA methylation and expression of candidate genes in bisexual flowers of andromonoecious poplar, but the regulatory relationship between methylation and microRNAs (miRNAs) remains unclear. To investigate whether the methylation of miRNA genes regulates gene expression in bisexual flower development, the methylome, microRNA, and transcriptome were examined in female and male flowers of andromonoecious poplar. 27 636 methylated coding genes and 113 methylated miRNA genes were identified. In the coding genes, 64.5% of the methylated reads mapped to the gene body region; by contrast, 60.7% of methylated reads in miRNA genes mainly mapped in the 5' and 3' flanking regions. CHH methylation showed the highest methylation levels and CHG showed the lowest methylation levels. Correlation analysis showed a significant, negative, strand-specific correlation of methylation and miRNA gene expression (r=0.79, P <0.05). The methylated miRNA genes included eight long miRNAs (lmiRNAs) of 24 nucleotides and 11 miRNAs related to flower development. miRNA172b might play an important role in the regulation of bisexual flower development-related gene expression in andromonoecious poplar, via modification of methylation. Gynomonoecious, female, and male poplars were used to validate the methylation patterns of the miRNA172b gene, implying that hyper-methylation in andromonoecious and gynomonoecious poplar might function as an important regulator in bisexual flower development. Our data provide a useful resource for the study of flower development in poplar and improve our understanding of the effect of epigenetic regulation on genes other than protein-coding genes.

Comparison of the physiological effects and transcriptome responses of Populus simonii under different abiotic stresses.

In the field, perennial plants such as poplar (Populus spp.) must adapt to simultaneous exposure to various abiotic stresses, which can affect their growth and survival. However, the mechanisms for stress-specific adaption in response to different abiotic stresses remain unclear. Thus, understanding the unique acclimation process for each abiotic treatment will require a comprehensive and systematic comparison of the responses of poplar to different abiotic stresses. To compare the responses to multiple stresses, we compared physiological effects and transcriptome changes in poplar under four abiotic stresses (salinity, osmotic, heat and cold). Photosynthesis and antioxidant enzymes changed significantly after 6 h abiotic stress treatment. Therefore, using 6 h abiotic stress treatment groups for transcriptome analysis, we identified a set of 863 differentially expressed genes (653 up-regulated and 210 down-regulated) common to osmotic, salinity, heat and cold treatment. We also identified genes specific to osmotic (1,739), salinity (1,222), cold (2,508) and heat (3,200), revealing that salinity stress has the fewest differently-expressed genes. After gene annotation, we found differences in expression of genes related to electron transport, stomatal control, antioxidant enzymes, cell wall alteration, and phytohormone biosynthesis and signaling in response to various abiotic stresses. This study provides new insights to improve our understanding of the mechanisms by which poplar adapts under different abiotic stress conditions and provides new clues for further studies.

Effects of high temperature on photosynthesis and related gene expression in poplar.

BACKGROUND: High temperature, whether transitory or constant, causes physiological, biochemical and molecular changes that adversely affect tree growth and productivity by reducing photosynthesis. To elucidate the photosynthetic adaption response and examine the recovery capacity of trees under heat stress, we measured gas exchange, chlorophyll fluorescence, electron transport, water use efficiency, and reactive oxygen-producing enzyme activities in heat-stressed plants. RESULTS: We found that photosynthesis could completely recover after less than six hours of high temperature treatment, which might be a turning point in the photosynthetic response to heat stress. Genome-wide gene expression analysis at six hours of heat stress identified 29,896 differentially expressed genes (15,670 up-regulated and 14,226 down-regulated), including multiple classes of transcription factors. These interact with each other and regulate the expression of photosynthesis-related genes in response to heat stress, controlling carbon fixation and changes in stomatal conductance. Heat stress of more than twelve hours caused reduced electron transport, damaged photosystems, activated the glycolate pathway and caused H2O2 production; as a result, photosynthetic capacity did not recover completely. CONCLUSIONS: This study provides a systematic physiological and global gene expression profile of the poplar photosynthetic response to heat stress and identifies the main limitations and threshold of photosynthesis under heat stress. It will expand our understanding of plant thermostability and provides a robust dataset for future studies.

Biochemical, physiological and gene expression analysis reveals sex-specific differences in Populus tomentosa floral development.

The productivity, distribution and population structure of poplar are affected by temperature transitions. Poplar floral buds develop in a fluctuating environment and the molecular basis of temperature-dependent flowering regulation has been extensively studied, but little is known about how sex-specific floral bud development responds to temperature transitions. Here, morphological observations indicated that floral bud growth rates were affected by maximum and minimum air temperature at the later stages of enlargement (stage 4) and later stage of dormancy (stage 8), respectively. We investigated the physiological, biochemical and gene expression changes in floral development and in response to temperature treatment (heat and chilling stress). Male floral buds showed more adverse effects than female floral buds under temperature treatment. Temperature treatment experiments revealed that temperature treatment significantly increased catalase, peroxidase, superoxide dismutase activities and transcription of related genes in female floral buds, whereas malondialdehyde (MDA) significantly increased only in males. Soluble sugars and protein increased both in female and male floral buds but were higher in males. Temperature treatment also caused significant increases in Ca(2+) content and transcription of genes related to calcium transport in female flowers. These results revealed sex-specific floral developmental responses to seasonal temperature transitions and suggest that in Populus tomentosa, female floral buds possess better mechanisms for environment adaptation than do males.

Sexual dimorphic floral development in dioecious plants revealed by transcriptome, phytohormone, and DNA methylation analysis in Populus tomentosa.

Dioecious plants have evolved sex-specific floral development mechanisms. However, the precise gene expression patterns in dioecious plant flower development remain unclear. Here, we used andromonoecious poplar, an exceptional model system, to eliminate the confounding effects of genetic background of dioecious plants. Comparative transcriptome and physiological analysis allowed us to characterize sex-specific development of female and male flowers. Transcriptome analysis identified genes significantly differentially expressed between the sexes, including genes related to floral development, phytohormone synthesis and metabolism, and DNA methylation. Correlation analysis revealed a significant correlation between phytohormone signaling and gene expression, identifying specific phytohormone-responsive genes and their cis-regulatory elements. Two genes related to DNA methylation, METHYLTRANSFERASE1 (MET1) and DECREASED DNA METHYLATION 1 (DDM1), which are located in the sex determination region of Chromosome XIX, have differential expression between female and male flowers. A time-course analysis revealed that MET1 and DDM1 expression may produce different DNA methylation levels in female and male flowers. Understanding the interactions of phytohormone signaling, DNA methylation and target gene expression should lead to a better understanding of sexual differences in floral development. Thus, this study identifies a set of candidate genes for further studies of poplar sexual dimorphism and relates sex-specific floral development to physiological and epigenetic changes.

Transcriptome profiling reveals differential transcript abundance in response to chilling stress in Populus simonii.

KEY MESSAGE: We report global gene expression patterns of poplar in response to chilling stress. A total of 1,085 significantly differentially expressed genes, involved in photosynthesis, signal transduction, and regulation of transcription, were identified. To understand the gene network underlying the response to chilling stress in the poplar, Populus simonii, we determined the genome transcript expression profile using an Affymetrix GeneChip with 56,000 genes. Our results revealed 11,626 cold-responsive genes, with 5,267 upregulated and 6,359 downregulated. In terms of biological processes, gene ontology (GO) analysis indicated that cold-induced genes were enriched in response to temperature stimulus, reactive oxygen species, and hormone stimulus. GO terms including cellular nitrogen compound metabolic processes, photosynthesis, and generation of precursor metabolites and energy were enriched in the cold-repressed genes. The functional annotation of differentially expressed genes revealed genes involved in photosynthesis, calcium/calmodulin-mediated signal transduction, abscisic acid (ABA) homeostasis and transport, and antioxidant defense systems. Gene expression analysis showed that the majority of genes involved in photosynthesis were repressed, but the THF1 gene was induced, suggesting that it may play an important role in the production of vesicles for leaf development under low-temperature conditions. Several genes involved in calcium/calmodulin-mediated signal transduction, ABA homeostasis and transport, and antioxidant defense systems were significantly induced under chilling stress, suggesting that they may act as positive regulators in the enhanced low-temperature tolerance of poplar. Several transcription factors had divergent expression patterns, suggesting they have variable functional responses to abiotic stress. This profile of global gene expression patterns during chilling stress will be valuable for future studies on the molecular mechanisms of chilling tolerance in woody plants.

The SUPERMAN gene family in Populus: nucleotide diversity and gene expression in a dioecious plant.

KEY MESSAGES: SUP gene family expression and regulation patterns reported in dioecious woody plant. Phylogenetic and nucleotide diversity analysis indicated PtoSUP1 is highly conserved and has undergone strong purifying selection. The molecular basis of SUPERMAN (SUP) regulation during floral development in monoecious plants has been extensively studied, but little is known of the SUP gene family in dioecious woody plants. In this study, we systematically examined the diversification of the SUP gene family in Populus, integrating genomic organization, expression, and phylogeny data. SUP family members showed sex-specific expression throughout flower development. Transcript profiling of rare gynomonoecious poplar flowers revealed that a significant reduction in PtoSUP1 mRNA might be important for stamen development in gynomonoecious poplar flowers. We found that the coding regions of Populus SUP genes are very highly conserved and that synonymous sites in exon regions have undergone strong purifying selection during SUP evolution in Populus. These results indicate that SUP genes play an important role in floral development of dioecious plants. Expression analysis of SUP suggested possible regulatory mechanisms for gynomonoecious poplar flower development. These findings provide an important insight into the mechanisms of the evolution of SUP function and may help enable engineered regulation of flower development for breeding improved tree varieties.

ALBA proteins confer thermotolerance through stabilizing HSF messenger RNAs in cytoplasmic granules.

High temperature is one of the major environmental stresses affecting plant growth and fitness. Heat stress transcription factors (HSFs) play critical roles in regulating the expression of heat-responsive genes. However, how HSFs are regulated remains obscure. Here, we show that ALBA4, ALBA5 and ALBA6, which phase separate into stress granules (SGs) and processing bodies (PBs) under heat stress, directly bind selected messenger RNAs, including HSF mRNAs, and recruit them into SGs and PBs to protect them from degradation under heat stress in Arabidopsis. The alba456 triple mutants, but not single and double mutants, display pleiotropic developmental defects and hypersensitivity to heat stress. Mutations in XRN4, a cytoplasmic 5' to 3' exoribonuclease, can rescue the observed developmental and heat-sensitive phenotypes of alba456 seedlings. Our study reveals a new layer of regulation for HSFs whereby HSF mRNAs are stabilized by redundant action of ALBA proteins in SGs and PBs for plant thermotolerance.

Genome Editing in Trees: From Multiple Repair Pathways to Long-Term Stability.

The CRISPR technology continues to diversify with a broadening array of applications that touch all kingdoms of life. The simplicity, versatility and species-independent nature of the CRISPR system offers researchers a previously unattainable level of precision and control over genomic modifications. Successful applications in forest, fruit and nut trees have demonstrated the efficacy of CRISPR technology at generating null mutations in the first generation. This eliminates the lengthy process of multigenerational crosses to obtain homozygous knockouts (KO). The high degree of genome heterozygosity in outcrossing trees is both a challenge and an opportunity for genome editing: a challenge because sequence polymorphisms at the target site can render CRISPR editing ineffective; yet an opportunity because the power and specificity of CRISPR can be harnessed for allele-specific editing. Examination of CRISPR/Cas9-induced mutational profiles from published tree studies reveals the potential involvement of multiple DNA repair pathways, suggesting that the influence of sequence context at or near the target sites can define mutagenesis outcomes. For commercial production of elite trees that rely on vegetative propagation, available data suggest an excellent outlook for stable CRISPR-induced mutations and associated phenotypes over multiple clonal generations.

The Role of DNA Methylation in Xylogenesis in Different Tissues of Poplar.

In trees, xylem tissues play a key role in the formation of woody tissues, which have important uses for pulp and timber production; also DNA methylation plays an important part in gene regulation during xylogenesis in trees. In our study, methylation-sensitive amplified polymorphism (MSAP) analysis was used to analyze the role cytosine methylation plays in wood formation in the commercially important tree species Populus tomentosa. This analysis compared the methylation patterns between xylem tissues (developing xylem and mature xylem) and non-xylem tissues (cambium, shoot apex, young leaf, mature leaf, phloem, root, male catkin, and female catkin) and found 10,316 polymorphic methylation sites. MSAP identified 132 candidate genes with the same methylation patterns in xylem tissues, including seven wood-related genes. The expression of these genes differed significantly between xylem and non-xylem tissue types (P < 0.01). This indicated that the difference of expression of specific genes with unique methylation patterns, rather than relative methylation levels between the two tissue types plays a critical role in wood biosynthesis. However, 46.2% of candidate genes with the same methylation pattern in vascular tissues (cambium, phloem, and developing xylem) did not have distinct expression patterns in xylem and non-xylem tissue. Also, bisulfite sequencing and transcriptome sequencing of MYB, NAC and FASCICLIN-LIKE AGP 13 revealed that the location of cytosine methylation in the gene might affect the expression of different transcripts from the corresponding gene. The expression of different transcripts that produce distinct proteins from a single gene might play an important role in the regulation of xylogenesis.