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bioRxiv2018

CMT3 and SUVH4/KYP silence the exonic retroelement Evelknievel to allow for reconstitution of CMT1 mRNA

Highlights: O_LISilencing of the intragenic Evelknievel (EK) retroelement is maintained by CMT3-KYP/SUVH4 independently of DDM1 and the RdDM pathways.\nC_LIO_LIMethylation and silencing of EK is required for transcription through the EK retroelement.\nC_LIO_LISilencing of EK allows for splicing out of the entire EK and reconstitution of an intact CMT1 mRNA.\nC_LI\n\nBackgroundCHROMOMEHYLASE1 (CMT1) has long been considered a non-essential gene because, in certain Arabidopsis ecotypes, the CMT1 gene is disrupted by the retroelement Evelknievel (EK), inserted within exon 13, or contains frame-shift mutations resulting in a truncated, non-functional protein. Here, we wanted to explore the regulatory pathway responsible for EK silencing in the Ler ecotype and its effect on CMT1 transcription.\n\nResultsMethylome databases confirmed that EK retroelement is heavily methylated but methylation is extended toward CMT1 downstream region. Strong transcriptional activation of EK accompanied by significant reduction in non-CG methylation was found in cmt3 and kyp2, but not in ddm1 or RdDM mutants. EK activation in cmt3 and kyp2 did not interfere with upstream CMT1 expression but abolish transcription through the EK. We identified, in wild type Ler, three spliced variants in which the entire EK is spliced out; one variant (25% of splicing incidents) facilitates proper reconstitution of an intact CMT1 mRNA. We could recover very low amount of the full length CMT1 mRNA from WT Ler and Col but not from cmt3 mutant..\n\nConclusionsOur findings highlight CMT3-SUVH4/KYP as the major pathway silencing the intragenic EK via inducing non-CG methylation. Furthermore, retroelement insertion within exons (e.g., CMT1) may not lead to a complete abolishment of the gene product when the element is kept silent. Rather the element can be spliced out to bring about reconstruction of a very low level of an intact, functional mRNA and possibly to retrieval of an active protein.

plant biology↗

bioRxiv2018

UDP-glucose:anthocyanidin 3-O-glucoside-2”-O-glucosyltransferase catalyzes further glycosylation of anthocyanins in purple Ipomoea batatas

Glycosylation contributes to the diversity and stability of anthocyanins in plants. The process is catalyzed by various glucosyltransferases using different anthocyanidin aglycones and glycosyl donors. An anthocyanidin 3-O-glucoside-2\"-O-glucosyltransferase (3GGT) from purple sweetpotato (cv. Ayamurasaki) served for the catalytic conversion of anthocyanidin 3-O-glucoside into anthocyanidin 3-O-sophoroside, which is functionally different from the 3GGT ortholog of Arabidopsis. The phylogenetic analysis indicates regioselectivity of 3GGT using UDP-xylose or UDP-glucose as the glycosyl is divergent between Convolvulaceae and Arabidopsis. Homology-based protein modeling and site-directed mutagenesis of Ib3GGT and At3GGT suggested that the Thr-138 of Ib3GGT is a key amino acid residue for UDP-glucose recognition and plays a major role in sugar donor selectivity. The wild type and ugt79b1 mutants of Arabidopsis plants overexpressing Ib3GGT produced the new component cyanidin 3-O-sophoroside. Moreover, Ib3GGT expression was associated with anthocyanin accumulation in different tissues during Ayamurasaki plant development and was regulated by the transcription factor IbMYB1. The localization assay of Ib3GGT showed that further glycosylation occurs in the cytosol and not endoplasmic reticulum. The present study revealed the function of Ib3GGT in further glycosylation of anthocyanins and its Thr-138 is the key amino acid residue for UDP-glucose recognition.

plant biology↗

bioRxiv2018

Characteristics and origins of non-functional Pm21 alleles in Dasypyrum villosum and wheat genetic stocks

Most Dasypyrum villosum resources are highly resistant to wheat powdery mildew that carries Pm21 alleles. However, in the previous studies, four D. villosum lines (DvSus-1 [~] DvSus-4) and two wheat-D. villosum addition lines (DA6V#1 and DA6V#3) were reported to be susceptible to powdery mildew. In the present study, the characteristics of non-functional Pm21 alleles in the above resources were analyzed after Sanger sequencing. The results showed that loss-of-functions of Pm21 alleles Pm21-NF1 [~] Pm21-NF3 isolated from DvSus-1, DvSus-2/DvSus-3 and DvSus-4 were caused by two potential point mutations, a 1-bp deletion and a 1281-bp insertion, respectively. The non-functional Pm21 alleles in DA6V#1 and DA6V#3 were same to that in DvSus-4 and DvSus-2/DvSus-3, respectively, indicating that the susceptibilities of the two wheat genetic stocks came from their D. villosum donors. The origins of non-functional Pm21 alleles were also investigated in this study. Except the target variants involved, the sequences of Pm21-NF2 and Pm21-NF3 were identical to that of Pm21-F2 and Pm21-F3 in the resistant D. villosum lines DvRes-2 and DvRes-3, derived from the accessions GRA961 and GRA1114, respectively. It was suggested that the non-functional alleles Pm21-NF2 and Pm21-NF3 originated from the wild-type alleles Pm21-F2 and Pm21-F3. In summary, this study gives an insight into the sequence characteristics of non-functional Pm21 alleles and their origins in natural population of D. villosum.

plant biology↗

bioRxiv2018

Transcriptome-wide analysis of auxin-induced carotenoid accumulation in Chlorella microalgae

Microalgae are a commercially viable route for the production of carotenoids, including {beta}-carotene and astaxanthin. In the current study, the commercially relevant microalga, Chlorella sp. BR2 was treated with four plant hormones: indole-3-acetic acid, salicylic acid, abscisic acid and methyl jasmonate, over a range of dosages and screened for enhanced carotenoid production. Indole-3-acetic acid was the only hormone with an inductive effect on carotenoid accumulation. As such, the transcriptome under the condition with the highest carotenoid increase was profiled using RNA-Seq and expressed sequences reconstructed with de novo assembly. This allowed for the profiling of transcriptome-wide changes following auxin treatment, revealing the active pathway components of auxininduced carotenogenesis. Data analysis specified the differentially expressed genes involved in auxin biosynthesis and signal transduction, which suggest a close relationship to equivalent pathways in higher plants. However unlike in plants, the ancient ABP1/SCFSKP2A/IBR5-mediated pathways for auxin response likely acted as the primary signaling route in Chlorella. As carotenoids are precursors for abscisic acid, the findings suggest a causative link between auxin signaling and abiotic stress tolerance.\n\nHighlightTranscriptomics of plant hormone-treated Chlorella revealed the active pathway components of auxin-induced carotenogenesis and included the ancient ABP1/SCFSKP2A/IBR5-mediated pathways. The manuscript presents the first documented transcriptomic data of auxin-treated microalgae.

plant biology↗

bioRxiv2018

Natural variation in expression of the HECT E3 ligase UPL3 influences seed size and crop yields in Brassica napus by altering regulatory gene expression.

Identifying genetic variation that increases crop yields is a primary objective in plant breeding. We have used association analyses of Brassica napus (oilseed rape/canola) accessions to identify variation in the expression of a HECT E3 ligase gene, BnaUPL3.C03, that influences seed size and final yield. We establish a mechanism in which UPL3 mediates the proteasomal degradation of LEC2, a master regulator of seed maturation. Reduced UPL3 expression increases LEC2 protein levels and prolongs expression of lipid biosynthetic genes and seed maturation. Natural variation in BnaUPL3.C03 expression has not yet been exploited in current Brassica napus breeding lines and can therefore be used as a new approach to maximize future yields in this important oil crop.

plant biology↗

bioRxiv2018

Endoplasmic Reticulum Retention Motif Fused to Recombinant Anti-cancer Monoclonal Antibody (mAb) CO17-1A Affects mAb Expression and Plant Stress Response

The endoplasmic reticulum (ER) is the main site of protein synthesis, folding, and secretion to other organelles. The capacity of the ER to process proteins is limited, and excessive accumulation of unfolded and misfolded proteins can induce ER stress, which is associated with plant diseases. Here, a transgenic Arabidopsis system was established to express anti-cancer monoclonal antibodies (mAbs) that recognize the tumor-associated antigen GA733-2. The ER retention Lys-Asp-Glu-Leu (KDEL) motif sequence was added to the C-terminus of the heavy chain to retain anti-colorectal cancer mAbs in the ER, consequently boosting mAb production. Agrobacterium-mediated floral dip transformation was used to generate T1 transformants, and homozygous T4 seeds obtained from transgenic Arabidopsis plants expressing anti-colorectal cancer mAbs were used to confirm the physiological effects of KDEL tagging. Germination rates were not significantly different between mAb CO and mAb COK. However, mAb COK primary root lengths were shorter than those of mAb CO plants and non-transgenic Arabidopsis plants in in vitro media. Most ER stress-related genes, with the exception of bZIP28 and IRE1a, were upregulated in mAb COK plants compared to mAb CO plants. Western blot and SDS-PAGE analyses showed that mAb COK plants exhibited up to five-times higher expression and mAb amounts than mAb CO plants. Enhanced expression in mAb COK plants was confirmed by immunohistochemical analyses. mAb COK was distributed across most of the area of leaf tissues, whereas mAb CO was mainly distributed in extracellular areas. Surface plasmon resonance analyses revealed that both mAb CO and mAb COK possessed equivalent or slightly better binding activities to antigen EpCAM compared to a commercially available parental antibody. These results suggest that the introduction of the KDEL motif is a promising strategy for obtaining enhanced amounts of recombinant therapeutic proteins, but the KDEL sequence may induce ER stress and slightly reduce plant biomass.

plant biology↗

bioRxiv2018

Complexity of ABA signaling for stomatal development and aperture regulation

Stomata, small pores on the surfaces of leaves formed by a pair of guard cells, adapt rapidly to changes in the environment by adjusting the aperture width. As a long term response, the number of stomata is regulated during stomatal development. The hormone abscisic acid (ABA) regulates both processes. In ABA mediated guard cell signaling the protein kinase OPEN STOMATA1 (OST1) has a central role, as stomatal closure in the ost1 mutant is impaired in response to ABA and to different environmental stimuli. We aimed to dissect the contribution of different ABA-related regulatory mechanisms in determining stomatal conductance, a combination of stomatal density and aperture width, and crossed the ost1 mutant with mutants that either decreased (aba3) or increased (cyp707a1/a3) the concentration of ABA in plants. The double mutant ost1 aba3 had higher stomatal conductance than either parent due to a combination of increased stomatal aperture width and higher stomatal density. In the triple mutant ost1 cyp707a1/a3 stomatal conductance was significantly lower compared to ost1-3 due to lower stomatal density. Further characterization of the single, double and triple mutants showed that responses to treatments that lead to stomatal closure were impaired in ost1 as well as ost1 aba3 and ost1 cyp707a1/a3 mutants, supporting a critical role for OST1 in stomatal aperture regulation. Based on our results, we suggest that there are two signaling pathways to regulate water flux from leaves i.e. stomatal conductance: an ABA-dependent pathway that determines stomatal density independent of OST1; and an OST1-dependent pathway that regulates rapid changes in stomatal aperture.

plant biology↗

bioRxiv2018

Vascular bundle sheath and mesophyll regulation of leaf water balance in response to chitin

Plants can detect pathogen invasion by sensing pathogen-associated molecular patterns (PAMPs). This sensing process leads to the induction of defense responses. Most PAMP mechanisms of action have been described in the guard cells. Here, we describe the effects of chitin, a PAMP found in fungal cell walls, on the cellular osmotic water permeability (Pf) of the leaf vascular bundle-sheath (BS) and mesophyll cells and its subsequent effect on leaf hydraulic conductance (Kleaf).\n\nThe BS is a parenchymatic tissue that tightly encases the vascular system. BS cells have been shown to control Kleaf through changes in their Pf, for example, in response to ABA. It was recently reported that, in Arabidopsis, the chitin receptors chitin elicitor receptor kinase 1 (CERK1) and LYSINE MOTIF RECEPTOR KINASE 5 (LYK5) are highly expressed in the BS, as well as the neighboring mesophyll. Therefore, we studied the possible impact of chitin on these cells.\n\nOur results revealed that both BS cells and mesophyll cells exhibit a sharp decrease in Pf in response to chitin treatment. In addition, xylem-fed chitin decreased Kleaf and led to stomatal closure. However, an Atlyk5 mutant showed none of these responses. Complimenting AtLYK5 specifically in the BS cells (using the SCARECROW promoter) and transient expresion in mesophyll cells each resulted in a response to chitin that was similar to that observed in the wild type. These results suggest that BS and mesophyll cells each play a role in the perception of apoplastic chitin and in initiating chitin-triggered immunity.\n\nSignificance StatementPAMP perception by plant receptors triggers various defense responses important for plant immunity. Here we provide new insights into a topic that has received a great deal of previous attention, revealing that a chitin immune response is present in additional leaf tissues other than the stomata. Chitin perception by the bundle sheath cells enwrapping the whole leaf vascular system decrease its cellular osmotic permeability and leaf hydraulic conductance. This in turn, leads to hydraulic signals being sent to the stomata and regulates whole-leaf water balance in response to chitin application and, perhaps, during fungal infection. Emphasizing the dynamic role of the BS in chitin-sensing and water balance regulation.

plant biology↗

bioRxiv2018

A variably imprinted epiallele impacts seed development

The contribution of epigenetic variation to phenotypic variation is unclear. Imprinted genes, because of their strong association with epigenetic modifications, represent an opportunity for the discovery of such phenomena. In mammals and flowering plants, a subset of genes are expressed from only one parental allele in a process called gene imprinting. Imprinting is associated with differential DNA methylation and chromatin modifications between parental alleles. In flowering plants imprinting occurs in a seed tissue - endosperm. Proper endosperm development is essential for the production of viable seeds. We previously showed that in Arabidopsis thaliana intraspecific imprinting variation is correlated with naturally occurring DNA methylation polymorphisms. Here, we investigated the mechanisms and function of allele-specific imprinting of the class IV homeodomain-Leucine zipper (HD-ZIP) transcription factor HDG3. In imprinted strains, HDG3 is expressed primarily from the methylated paternally inherited allele. We manipulated the methylation state of endogenous HDG3 in a non-imprinted strain and demonstrated that methylation of a proximal transposable element is sufficient to promote HDG3 expression and imprinting. Gain of HDG3 imprinting was associated with earlier endosperm cellularization and changes in seed weight. These results indicate that epigenetic variation alone is sufficient to explain imprinting variation and demonstrate that epialleles can underlie variation in seed development phenotypes.\n\nAuthor SummaryThe contribution of genetic variation to phenotypic variation is well-established. By contrast, it is unknown how frequently epigenetic variation causes differences in organismal phenotypes. Epigenetic information is closely associated with but not encoded in the DNA sequence. In practice, it is challenging to disentangle genetic variation from epigenetic variation, as what appears to be epigenetic variation might have an underlying genetic basis. DNA methylation is one form of epigenetic information. HDG3 encodes an endosperm specific transcription factor that exists in two states in A. thaliana natural populations: methylated and expressed and hypomethylated and repressed. We show that pure epigenetic variation is sufficient to explain expression variation of HDG3 - a naturally lowly expressed allele can be switched to a higher expressed state by adding DNA methylation. We also show that expression of HDG3 in strains where it is normally hypomethylated and relatively repressed causes a seed development phenotype. These data indicate that naturally circulating epialleles have consequences for seed phenotypic variation.

plant biology↗

bioRxiv2018

Physical and transcriptional organisation of the bread wheat intracellular immune receptor repertoire

Disease resistance genes encoding intracellular immune receptors of the nucleotide-binding and leucine-rich repeat (NLR) class of proteins detect pathogens by the presence of pathogen effectors. Plant genomes typically contain hundreds of NLR encoding genes. The availability of the hexaploid wheat cultivar Chinese Spring reference genome now allows a detailed study of its NLR complement. However, low NLR expression as well as high intra-family sequence homology hinders their accurate gene annotation. Here we developed NLR-Annotator for in silico NLR identification independent of transcript support. Although developed for wheat, we demonstrate the universal applicability of NLR-Annotator across diverse plant taxa. Applying our tool to wheat and combining it with a transcript-validated subset of genes from the reference gene annotation, we characterized the structure, phylogeny and expression profile of the NLR gene family. We detected 3,400 full-length NLR loci of which 1,540 were confirmed as complete genes. NLRs with integrated domains mostly group in specific sub-clades. Members of another subclade predominantly locate in close physical proximity to NLRs carrying integrated domains suggesting a paired helper-function. Most NLRs (88%) display low basal expression (in the lower 10 percentile of transcripts), which may be tissue-specific and/or induced by biotic stress. As a case study for applying our tool to the positional cloning of resistance genes, we estimated the number of NLR genes within the intervals of mapped rust resistance genes. Our study will support the identification of functional resistance genes in wheat to accelerate the breeding and engineering of disease resistant varieties.

plant biology↗

bioRxiv2018

Long-term acclimation to different stress types: revealing tradeoffs between mesophyll and stomatal conductance

Ziziphus spina-christi, a thermophilic tree, became more abundant in the Mediterranean, presumably due to increased winter temperatures. In order to predict the plant acclimation to future climate changes, we attempted to understand which factors underlie photosynthetic stress acclimation.\n\nStress acclimation to three major long-term abiotic stresses (drought, salinity and temperature) was investigated by measuring growth, gas exchange, chlorophyll fluorescence and leaf structure. We developed a stress index that allowed to precisely define stress level, enabling a comparison between stress types. For each stress, photosynthesis-limiting factors were compared: stomatal conductance (gs), mesophyll conductance (gm) and maximum capacity for Rubisco carboxylation (Vcmax).\n\nPhotosynthesis under all stresses was limited mostly by gs and gm (80-90%); whereas biochemistry (Vcmax) made a minor contribution (10-20%). The relative contribution of gs and gm on photosynthetic limitation was influenced by stress type. During acclimation to drought or salinity, photosynthesis was limited by a decline in gs, while intolerance to low temperatures was driven by decline in gm. Low mesophyll-imposed limitation was the best predictor of abiotic stress tolerance.\n\nThe results demonstrate how warming climate benefits thermophilic species. Moreover, current work gives methodology for stress studies, and defines the main factors underlying the plant response to climate change.\n\nHighlightWe have compared the photosynthesis limitation resulting from long-term acclimation to three major a-biotic stresses (drought, salinity and temperature) to understand which limiting-factor are dominant under each type of stress.

plant biology↗

bioRxiv2018

Identification of genes affecting saturated fat acid content in Elaeis guineensis by genome-wide association analysis

Oil palm is the highest yielding oil crop per unit area worldwide. Unfortunately, palm oil is often considered unhealthy. In particular, palmic acid (C16:0) is a major component of palm oil. In this study a total of 1 261 501 SNP markers were produced in a diversity panel of 200 oil palm individuals. Oil content in this population varied from 29.8% to 70.3%, palmic acid varied from 31.3% to 48.8%, and oleic acid varied from 31.3% to 50.1%. We identified 274 SNP markers significantly associated with fatty acid compositions; 44 candidate genes in the flanking regions of these SNPs were involved in fatty acid biosynthesis and metabolism. Among them, two acyl-ACP thioesterase B genes had differential expression patterns between the mesocarp and kernel, tissues which show different oil profiles in oil palm (high palmic acid and high lauric acid respectively). Overexpression of both genes caused a significant increase in palmic acid content, while overexpression of the EgFatB2 gene also caused an accumulation of lauric acid and myristic acid. Our research provides genome-wide SNPs, a set of markers significantly associated with fatty acid content, and validated candidate genes for future targeted breeding of lower saturated fat content in palm oil.

plant biology↗

bioRxiv2018

Tobacco TGA7 mediates gene expression dependent and independent of salicylic acid

Basic region leucine zipper (bZIP) transcription factors of the TGA family control gene expression in response to diverse stimuli. Arabidopsis clade II and clade III TGA factors mediate salicylic acid (SA)-induced expression of PATHOGENESIS-RELATED GENE1 (PR-1) via interplay with NONEXPRESSOR OF PR GENES1 (NPR1, a.k.a. NIM1). Interaction with TGA factors occurs through the central ankyrin repeat domain of NPR1. In a yeast two-hybrid screen with the NPR1 bait, we identified TGA7, a novel member of the tobacco (Nt) TGA family grouping to clade III. TGA7 is most similar to NtTGA1a, and, like NtTGA1a, TGA7 displays transcription activity in yeast. Unexpectedly, TGA7 preferentially and uniquely interacts with the SA-sensitive C-terminal region of NtNPR1, demonstrating that NtNPR1 harbors multiple distinct TGA factor binding sites. Interaction with NPR1 impairs TGA7 transcription activity in yeast. Furthermore, TGA7 binding to the NtNPR1 C-terminus is outcompeted by SA-induced type 2 NIM1-INTERACTING (NIMIN) proteins. In tobacco plants, a TGA7-Gal4 DNA-binding domain chimeric protein (TGA7GBD) mediates SA-responsive reporter gene expression in young leaf tissue and spontaneous reporter activation in older leaves displaying PR-1 gene expression. Astonishingly, TGA7GBD is also able to activate the reporter independent from PR-1 gene expression in noninduced cotyledons of tobacco seedlings. Together, our findings support a model in which TGA7 mediates both SA-dependent and SA-independent gene activation controlled by the plants developmental stage and by the C-terminal region of constitutively accumulating NtNPR1.

plant biology↗

bioRxiv2018

Arabidopsis species deploy distinct strategies to cope with drought stress

O_LIBackground and Aims Water limitation is an important determinant of the distribution, abundance and diversity of plant species. Yet, little is known about how the response to limiting water supply changes among closely related plant species with distinct ecological preferences. Comparison of the model annual species A. thaliana to its close perennial relatives A. lyrata and A. halleri, can help disentangle the molecular and physiological changes contributing to tolerance and avoidance mechanisms, because these species must maintain tolerance and avoidance mechanisms to increase long-term survival, but they are exposed to different levels of water stress and competition in their natural habitat.\nC_LIO_LIMethods We conducted a dry-down experiment that mimics a period of missing precipitation. We quantified the covariation of progressive decrease in soil water content (SWC) with various physiological and morphological plant traits across a set of representative genotypes in Arabidopsis thaliana, A. lyrata and A. halleri. To quantify the degree of plant stress, transcriptome changes were also monitored.\nC_LIO_LIKey Results The analysis of trait co-variation demonstrates that the three species differ in the strategies they deploy to respond to drought stress. A. thaliana showed drought avoidance reaction but failed to survive wilting. A. lyrata efficiently combined avoidance and tolerance mechanisms. By contrast, A. halleri showed some degree of tolerance to wilting but it did not seem to protect itself from the stress imposed by drought. Transcriptome data collected just before plant wilting and after recovery corroborated the phenotypic analysis, with A. lyrata and A. halleri showing a stronger activation of recovery- and stress-related genes, respectively.\nC_LIO_LIConclusions We conclude that these three Arabidopsis species have evolved distinct strategies to face drought stress, and discuss the extent to which these strategic differences reflect their respective ecological priorities.\nC_LI

plant biology↗

bioRxiv2018

RNA Binding Motif Protein 48 is required for U12 splicing and maize endosperm differentiation

The last eukaryotic common ancestor had two classes of introns that are still found in most eukaryotic lineages. Common U2-type and rare U12-type introns are spliced by the major and minor spliceosomes, respectively. Relatively few splicing factors have been shown to be specific to the minor spliceosome. We found that the maize RNA Binding Motif Protein48 (RBM48) is a U12 splicing factor that functions to promote cell differentiation and repress cell proliferation. RBM48 is coselected with the U12 splicing factor, ZRSR2/RGH3. Protein-protein interactions between RBM48, RGH3, and U2 Auxiliary Factor (U2AF) subunits suggest major and minor spliceosome factors may form complexes during intron recognition. Human RBM48 interacts with ARMC7. Maize RBM48 and ARMC7 have a conserved protein-protein interaction. These data predict that RBM48 is likely to function in U12 splicing throughout eukaryotes and that U12 splicing promotes endosperm cell differentiation in maize.

plant biology↗

bioRxiv2018

TOL proteins mediate vacuolar sorting of the borate transporter BOR1 in Arabidopsis thaliana.

Boron (B) is an essential micronutrient for plants, however, it shows cytotoxicity at high concentrations. A borate transporter BOR1 is required for efficient transport of boron (B) toward the root stele in Arabidopsis thaliana. BOR1 shows polar localization in the plasma membrane of various root cells toward the stele-side under B limitation. To avoid over-accumulation of B, BOR1 in the plasma membrane is rapidly internalized and transported into the vacuole for proteolysis after high-B supply in an ubiquitination-dependent manner. Although BOR1 has been predicted to be transported into multi-vesicular bodies/late endosomes (MVB/LEs) via the endosomal sorting complex required for transport (ESCRT) machinery, experimental evidence was absent so far. In this study, we investigated the intracellular localization of BOR1 by visualizing endomembrane compartments, and tested the involvement of ESCRT-0-like proteins TOM1-LIKEs (TOLs) in the vacuolar sorting of BOR1. Under low-B conditions, a large portion of cytoplasmic BOR1 was localized in the trans-Golgi networks/early endosomes (TGN/EEs) labeled with VHA-a1 subunit. Pharmacological interference of endosomal recycling using brefeldin A induced colocalization of BOR1 with RabA5D, which labels recycling vesicles associated with the TGN. These data suggest that BOR1 cycles between plasma membrane and TGN/EE via RabA5D-positive endomembrane compartments under low-B conditions. On the other hand, under high-B conditions, BOR1 was localized in the inside of TOL5-positive MVB/LEs. To examine the roles of TOL proteins in intracellular trafficking of BOR1, we analyzed BOR1-GFP localization in the TOL quintuple mutant (tolQ; tol2-1tol3-1tol5-1tol6-1tol9-1) after high-B supply. In the tolQ mutant, vacuolar sorting of BOR1 was delayed, while the polar localization of BOR1 was not disturbed. Taken together, BOR1 is constantly transported to the TGN/EE by endocytosis and recycled to the plasma membrane likely via RabA5D-positive endomembrane compartments under low-B conditions. On the other hand, BOR1 is transported to the vacuole via TOL5-positive MVB/LEs under high-B conditions. TOL proteins are required for sorting of ubiquitinated BOR1 into MVB/LE for vacuolar degradation but not for the polar trafficking of BOR1.

plant biology↗

bioRxiv2018

Synchrotron FTIR and Raman spectroscopy provide unique spectral fingerprints for Arabidopsis floral stem vascular tissues

Cell walls are highly complex structures that are modified during plant growth and development. For example, the development of phloem and xylem vascular cells, which participate in the transport of sugars and water as well as support, can be influenced by cell-specific cell wall composition. Here, we used synchrotron radiation-based infrared (SR-FTIR) and Raman spectroscopy to analyze the cell wall composition of wild-type and double mutant sweet11-1sweet12-1, which impairs sugar transport, Arabidopsis floral stem vascular tissue. The SR-FTIR spectra showed that in addition to modified xylem cell wall composition, phloem cell walls in the double mutant line were characterized by modified hemicellulose composition. Moreover, combining Raman spectroscopy with a Classification and Regression Tree (CART) method identified combinations of Raman shifts that could distinguish xylem vessels and fibers. Additionally, the disruption of SWEET11 and SWEET12 genes impacts xylem cell wall composition in a cell-specific manner, with changes in hemicelluloses and cellulose observed at the xylem vessel interface. These results suggest that the facilitated transport of sugars by transporters that exist between vascular parenchyma cells and conducting cells is important to ensuring correct phloem and xylem cell wall composition.\n\nHighlightCombining vibrational spectroscopy techniques and multivariate analysis shows that the disruption of SWEET genes impacts phloem cell wall composition and that the effect on xylem cell wall composition is cell-specific.

plant biology↗

bioRxiv2018

RNA-Seq analysis of compatible and incompatible styles of Pyrus species at the beginning of pollination

In Rosaceae, incompatible pollen can penetrate into the style during the gametophytic self-incompatibility response. It is therefore considered a stylar event rather than a stigmatic event. In this study, we explored the differences in gene expression between compatibility and incompatibility in the early stage of pollination. The self-compatible pear variety \"Jinzhuili\" is a naturally occurring bud mutant from \"Yali\", a leading Chinese native cultivar exhibiting typical gametophytic self-incompatibility. We collected the styles of Yali and Jinzhuili at 0.5 and 2 h after self-pollination and then performed high-throughput sequencing. According to the pathway enrichment analysis of the differentially expressed genes, \"plant-pathogen interaction\" was the most represented pathway. Quantitative PCR was used to validate these differential genes. The expression levels of genes related to pollen growth and disease inhibition, such as LRR (LEUCINE-RICH REPEAT EXTENSIN), resistance, and defensin, differed significantly between compatible and incompatible pollination. Interestingly, at 0.5 h, most of these genes were upregulated in the compatible pollination system compared with the incompatible pollination system. Calcium ion transport, which requires ATPase, also demonstrated upregulated expression. In summary, the self-incompatibility reaction was initiated when the pollen came into contact with the stigma.

plant biology↗