Arabidopsis Leaf Research Articles

Interplay among ZF-HD and GRF transcription factors during Arabidopsis leaf development.

The growth-regulating factor (GRF) family of transcriptional factors are involved in the control of leaf size and senescence, inflorescence and root growth, grain size, and plant regeneration. However, there is limited information about the genes regulated by these transcriptional factors, which are in turn responsible for their functions. Using a meta-analysis approach, we identified genes encoding Arabidopsis (Arabidopsis thaliana) zinc-finger homeodomain (ZF-HD) transcriptional factors, as potential targets of the GRFs. We further showed that GRF3 binds to the promoter of one of the members of the ZF-HD family, HOMEOBOX PROTEIN 33 (HB33), and activates its transcription. Increased levels of HB33 led to different modifications in leaf cell number and size that were dependent on its expression levels. Furthermore, we found that expression of HB33 for an extended period during leaf development increased leaf longevity. To cope with the functional redundancy among ZF-HD family members, we generated a dominant repressor version of HB33, HB33-SRDX. Expression of HB33-SRDX from HB33 regulatory regions was seedling-lethal, revealing the importance of the ZF-HD family in plant development. Misexpression of HB33-SRDX in early leaf development caused a reduction in both cell size and number. Interestingly, the loss-of-function of HB33 in lines carrying a GRF3 allele insensitive to miR396 reverted the delay in leaf senescence characteristic of these plants. Our results revealed functions for ZF-HDs in leaf development and linked them to the GRF pathway.

Assaying plant formate-tetrahydrofolate ligase with monoglutamylated and polyglutamylated substrates using a fluorescence-HPLC based method.

Formate-tetrahydrofolate ligase catalyzes reversible, ATP-dependent conversion of tetrahydrofolate and formate to 10-formyltetrahydrofolate, simultaneously releasing ADP and inorganic phosphate. This enzyme has traditionally been assayed in the direction of 10-CHO-tetrahydrofolate formation by lowering pH of the reaction post-incubation, thus converting the product of the reaction to 5,10-methenyltetrahydrofolate, which is then quantified spectrophotometrically. To increase sensitivity of the product detection, which is particularly useful when determining the kinetic parameters of the enzyme with polyglutamylated substrates, we have replaced the spectrophotometric detection with HPLC separation and fluorescence detection. In addition to the modified enzyme assay protocol, we are also providing protocols for producing recombinant formate-tetrahydrofolate ligase from Arabidopsis in Escherichia coli cells, producing crude Arabidopsis leaf and root extracts suitable for assaying this enzyme, and for synthesis of polyglutamylated tetrahydrofolate substrates.

SHORT-ROOT and SCARECROW homologs regulate patterning of diverse cell types within and between species.

The roles of SHORT-ROOT (SHR) and SCARECROW (SCR) in ground tissue patterning and differentiation have been well established in the root of Arabidopsis thaliana. Recently, work in additional organs and species revealed the extensive functional diversification of these genes, including regulation of cortical divisions essential for nodule organogenesis in legume roots, bundle sheath specification in the Arabidopsis leaf, patterning of inner leaf cell layers in maize, and stomatal development in rice. The co-option of distinct functions and cell types is attributed to different mechanisms, including paralog retention, spatiotemporal changes in gene expression, and novel protein functions. Elaborating our knowledge of the SHR-SCR module further unravels the developmental regulation that controls diverse forms and functions within and between species.

Single-Cell RNA Sequencing of Arabidopsis Leaf Tissues Identifies Multiple Specialized Cell Types: Idioblast Myrosin Cells and Potential Glucosinolate-Producing Cells.

The glucosinolate-myrosinase defense system (GMDS), characteristic of Brassicales, is involved in plant defense. Previous single-cell transcriptomic analyses have reported the expression profiles of multiple GMDS-related cell types (i.e. myrosinase-rich myrosin idioblasts and multiple types of potential glucosinolate synthetic cells as well as a candidate S-cell for glucosinolate accumulation). However, differences in plant stages and cell-type annotation methods have hindered comparisons among studies. Here, we used the single-cell transcriptome profiles of extended Arabidopsis leaves and verified the distribution of previously used markers to refine the expression profiles of GMDS-associated cell types. Moreover, we performed beta-glucuronidase promoter assays to confirm the histological expression patterns of newly obtained markers for GMDS-associated candidates. As a result, we found a set of new specific reporters for myrosin cells and potential glucosinolate-producing cells.

Proteomic Profiling of Plant and Pathogen Interaction on the Leaf Epidermis.

The plant epidermis is the first line of plant defense against pathogen invasion, and likely contains important regulatory proteins related to the plant–pathogen interaction. This study aims to identify the candidates of these regulatory proteins expressed in the plant epidermis. We performed comparative proteomic studies to identify rapidly and locally expressed proteins in the leaf epidermis inoculated with fungal phytopathogen. The conidia solutions were dropped onto the Arabidopsis leaf surface, and then, we collected the epidermal tissues from inoculated and mock-treated leaves at 4 and 24 hpi. The label-free quantification methods showed that expressions of Arabidopsis proteins, which are related to defense signals, such as BAK1, MKK5, receptor-like protein kinases, transcription factors, and stomatal functions, were rapidly induced in the epidermal tissues of inoculated leaves. In contrast, most of them were not differentially regulated by fugal inoculation in the whole leaves. These findings clearly indicate that epidermal proteomics can monitor locally expressed proteins in inoculated areas of plant tissues. We also identified the 61 fungal proteins, including effector-like proteins specifically expressed on the Arabidopsis epidermis. Our new findings suggested that epidermal proteomics is useful for understanding the local expressions of plant and fungal proteins related to their interactions.

Nondestructive circadian profiling of starch content in fresh intact Arabidopsis leaf with two-photon fluorescence and second-harmonic generation imaging

Plant chloroplasts conduct photosynthesis to convert solar energy into sugars for the carbon source essential for cell living and growth during the day. One fraction of photosynthetic products is stored in chloroplasts by forming starch granules to continue the provision of carbon energy during the night. Currently, profiling the starch temporal pattern requires either: (i) sacrificing the leaves, or (ii) generating transgenic plants at the risk of changing the metabolisms by incorporating a genetically modified granule-bound starch synthase (GBSS). In this paper, we demonstrated a nondestructive method using two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging to quantify starch granules within chloroplasts of fresh intact leaves across a day-night cycle. We did so using two Arabidopsis lines having normal and excess starch contents: wild-type (Columbia-0) and starch excess 1 (sex1). The starch granules were visualized by SHG imaging, while the chloroplasts in mesophyll cells were visualized by TPF imaging. Our results provided micron scale spatial resolution of starch distribution within leaves and showed starch circadian patterns consistent with those profiled by enzymatic assays in previous studies. We demonstrated that TPF-SHG imaging is a potential tool for revealing the real-time heterogeneity of starch circadian rhythm in leaf cells, without the need for destructive sample preparation.

Oxicam-type nonsteroidal anti-inflammatory drugs enhance Agrobacterium-mediated transient transformation in plants.

Agrobacterium-mediated transformation is a key innovation for plant breeding, and routinely used in basic researches and applied biology. However, the transformation efficiency is often the limiting factor of this technique. In this study, we discovered that oxicam-type nonsteroidal anti-inflammatory drugs, including tenoxicam (TNX), increase the efficiency of Agrobacterium-mediated transient transformation. TNX treatment increased the transformation efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana mature leaves by agroinfiltration. The increase of efficiency by TNX treatment was not observed in dde2/ein2/pad4/sid2 quadruple mutant, indicating that TNX inhibits the immune system mediated by jasmonic acid, ethylene, and salicylic acid against to Agrobacterium. We also found that TNX-treatment is applicable for the transient expression and subcellular localization analysis of fluorescent-tagged proteins in Arabidopsis leaf cells. In addition, we found that TNX increases the efficiency of Agrobacterium-mediated transient transformation of Jatropha. Given that treatment with oxicam compounds is a simple and cost effective method, our findings will provide a new option to overcome limitations associated with Agrobacterium-mediated transformation of various plant species.

Ethylene Acts as a Local and Systemic Signal to Mediate UV-B-Induced Nitrate Reallocation to Arabidopsis Leaves and Roots via Regulating the ERFs-NRT1.8 Signaling Module.

Nitrate is the preferred nitrogen source for plants and plays an important role in plant growth and development. Under various soil stresses, plants reallocate nitrate to roots to promote stress tolerance through the ethylene-ethylene response factors (ERFs)-nitrate transporter (NRT) signaling module. As a light signal, ultraviolet B (UV-B) also stimulates the production of ethylene. However, whether UV-B regulates nitrate reallocation in plants via ethylene remains unknown. Here, we found that UV-B-induced expression of ERF1B, ORA59, ERF104, and NRT1.8 in both Arabidopsis shoots and roots as well as nitrate reallocation from hypocotyls to leaves and roots were impaired in ethylene signaling mutants for Ethylene Insensitive2 (EIN2) and EIN3. UV-B-induced NRT1.8 expression and nitrate reallocation to leaves and roots were also inhibited in the triple mutants for ERF1B, ORA59, and ERF104. Deletion of NRT1.8 impaired UV-B-induced nitrate reallocation to both leaves and roots. Furthermore, UV-B promoted ethylene release in both shoots and roots by enhancing the gene expression and enzymatic activities of ethylene biosynthetic enzymes only in shoots. These results show that ethylene acts as a local and systemic signal to mediate UV-B-induced nitrate reallocation from Arabidopsis hypocotyls to both leaves and roots via regulating the gene expression of the ERFs-NRT1.8 signaling module.

Identification and Functional Analysis of Key Autophosphorylation Residues of Arabidopsis Senescence Associated Receptor-like Kinase.

Reversible protein phosphorylation mediated by protein kinases and phosphatases plays important roles in the regulation of leaf senescence. We previously reported that the senescence-associated leucine-rich repeat receptor-like kinase AtSARK autophosphorylates on both serine/threonine and tyrosine residues and functions as a positive regulator of Arabidopsis leaf senescence; the senescence-suppressed protein phosphatase SSPP interacts with and dephosphorylates the cytoplasmic domain of AtSARK, thereby negatively regulating leaf senescence. Here, 27 autophosphorylation residues of AtSARK were revealed by mass spectrometry analysis, and six of them, including two Ser, two Thr, and two Tyr residues, were further found to be important for the biological functions of AtSARK. All site-directed mutations of these six residues that resulted in decreased autophosphorylation level of AtSARK could significantly inhibit AtSARK-induced leaf senescence. In addition, mutations mimicking the dephosphorylation form of Ser384 (S384A) or the phosphorylation form of Tyr413 (Y413E) substantially reduced the interaction between AtSARK and SSPP. All results suggest that autophosphorylation of AtSARK is essential for its functions in promoting leaf senescence. The possible roles of S384 and Y413 residues in fine-tuning the interaction between AtSARK and SSPP are discussed herein.

High temperature restricts cell division and leaf size by coordination of PIF4 and TCP4 transcription factors.

High ambient temperature suppresses Arabidopsis (Arabidopsis thaliana) rosette leaf area and elongates the stem and petiole. While the mechanism underlying the temperature-induced elongation response has been extensively studied, the genetic basis of temperature regulation of leaf size is largely unknown. Here, we show that warm temperature inhibits cell proliferation in Arabidopsis leaves, resulting in fewer cells compared to the control condition. Cellular phenotyping and genetic and biochemical analyses established the key roles of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and TEOSINTE BRANCHED1/CYCLOIDEA/PCF4 (TCP4) transcription factors in the suppression of Arabidopsis leaf area under high temperature by a reduction in cell number. We show that temperature-mediated suppression of cell proliferation requires PIF4, which interacts with TCP4 and regulates the expression of the cell cycle inhibitor KIP-RELATED PROTEIN1 (KRP1) to control leaf size under high temperature. Warm temperature induces binding of both PIF4 and TCP4 to the KRP1 promoter. PIF4 binding to KRP1 under high temperature is TCP4 dependent as TCP4 regulates PIF4 transcript levels under high temperature. We propose a model where a warm temperature-mediated accumulation of PIF4 in leaf cells promotes its binding to the KRP1 promoter in a TCP4-dependent way to regulate cell production and leaf size. Our finding of high temperature-mediated transcriptional upregulation of KRP1 integrates a developmental signal with an environmental signal that converges on a basal cell regulatory process.

Development of a GFP biosensor reporter for the unfolded protein response-signaling pathway in plants: incorporation of the bZIP60 intron into the GFP gene

ABSTRACT The ability to measure the activation of the unfolded protein response (UPR) in plants is important when they are exposed to stressful environments. To this end, we developed a unique and versatile biosensor-reporter system to indicate the activation of UPR in living plant cells. The small cytoplasmically spliced intron from the bZIP60 locus was incorporated into the 5’ end of the GFP gene, creating the 35S::bZIP60 intron:GFP construct. When this construct is transiently expressed in Arabidopsis protoplasts, the presence of the bZIP60 intron prevents GFP mRNA from being translated under non-UPR conditions. However, when UPR is activated, the IRE1 kinase/ribonuclease splices this intron from the GFP mRNA and its translation proceeds, generating GFP fluorescence. We demonstrated the utility of the system in Arabidopsis leaf protoplasts treated with DTT, which is a chemical inducer of UPR, followed by visualization and quantification using confocal microscopy. The 35S::bZIP60 intron:GFP construct was also expressed in protoplasts from an overexpressor line containing the coding sequence for the UPR-induced, protein folding chaperone, protein disulfide isomerase-9 (PDI9). PDI9 also influences the strength of the UPR signaling pathway. Protoplasts from WT and PDI9 overexpressor plants treated with DTT exhibited significantly higher GFP fluorescence relative to untreated protoplasts, indicating that the bZIP60 intron was spliced from the GFP mRNA in response to activation of UPR. RT-PCR further confirmed the higher induction of PDI9 and bZIP60 (total and spliced) mRNA levels in DTT-treated protoplasts relative to controls. This system can be adapted for monitoring crop stress and for basic studies dissecting the UPR signaling pathway.

CALMODULIN1 and WRKY53 Function in Plant Defense by Negatively Regulating the Jasmonic Acid Biosynthesis Pathway in Arabidopsis.

Jasmonic acid (JA) is an important hormone that functions in plant defense. cam1 and wrky53 mutants were more resistant to Spodoptera littoralis than in the wild-type (WT) Arabidopsis group. In addition, JA concentration in cam1 and wrky53 mutants was higher compared with the WT group. To explore how these two proteins affect the resistance of Arabidopsis plants, we used a yeast two-hybrid assay, firefly luciferase complementation imaging assay and in vitro pull-down assay confirming that calmodulin 1 (CAM1) interacted with WRKY53. However, these two proteins separate when calcium concentration increases in Arabidopsis leaf cells. Then, electrophoretic mobility shift assay and luciferase activation assay were used to verify that WRKY53 could bind to lipoxygenases 3 (LOX3) and lipoxygenases 4 (LOX4) gene promoters and negatively regulate gene expression. This study reveals that CAM1 and WRKY53 negatively regulate plant resistance to herbivory by regulating the JA biosynthesis pathway via the dissociation of CAM1-WRKY53, then the released WRKY53 binds to the LOXs promoters to negatively regulate LOXs gene expression. This study reveals WRKY53′s mechanism in insect resistance, a new light on the function of WRKY53.

The Transcription Factor MYB37 Positively Regulates Photosynthetic Inhibition and Oxidative Damage in Arabidopsis Leaves Under Salt Stress.

MYB transcription factors (TFs) mediate plant responses and defenses to biotic and abiotic stresses. The effects of overexpression of MYB37, an R2R3 MYB subgroup 14 transcription factors in Arabidopsis thaliana, on chlorophyll content, chlorophyll fluorescence parameters, reactive oxygen species (ROS) metabolism, and the contents of osmotic regulatory substances were studied under 100 mM NaCl stress. Compared with the wild type (Col-0), MYB37 overexpression significantly alleviated the salt stress symptoms in A. thaliana plants. Chlorophyll a (Chl a) and chlorophyll b (Chl b) contents were significantly decreased in OE-1 and OE-2 than in Col-0. Particularly, the Chl a/b ratio was also higher in OE-1 and OE-2 than in Col-0 under NaCl stress. However, MYB37 overexpression alleviated the degradation of chlorophyll, especially Chl a. Salt stress inhibited the activities of PSII and PSI in Arabidopsis leaves, but did not affect the activity of PSII electron donor side oxygen-evolving complex (OEC). MYB37 overexpression increased photosynthesis in Arabidopsis by increasing PSII and PSI activities. MYB37 overexpression also promoted the transfer of electrons from QA to QB on the PSII receptor side of Arabidopsis under NaCl stress. Additionally, MYB37 overexpression increased Y(II) and Y(NPQ) of Arabidopsis under NaCl stress and decreased Y(NO). These results indicate that MYB37 overexpression increases PSII activity and regulates the proportion of energy dissipation in Arabidopsis leaves under NaCl stress, thus decreasing the proportion of inactivated reaction centers. Salt stress causes excess electrons and energy in the photosynthetic electron transport chain of Arabidopsis leaves, resulting in the release of reactive oxygen species (ROS), such as superoxide anion and hydrogen peroxide, leading to oxidative damage. Nevertheless, MYB37 overexpression reduced accumulation of malondialdehyde in Arabidopsis leaves under NaCl stress and alleviated the degree of membrane lipid peroxidation caused by ROS. Salt stress also enhanced the accumulation of soluble sugar (SS) and proline (Pro) in Arabidopsis leaves, thus reducing salt stress damage to plants. Salt stress also degraded soluble protein (SP). Furthermore, the accumulation of osmoregulation substances SS and Pro in OE-1 and OE-2 was not different from that in Col-0 since MYB37 overexpression in Arabidopsis OE-1, and OE-2 did not significantly affect plants under NaCl stress. However, SP content was significantly higher in OE-1 and OE-2 than in Col-0. These results indicate that MYB37 overexpression can alleviate the degradation of Arabidopsis proteins under NaCl stress, promote plant growth and improve salt tolerance.

ROP Interactive Partners are Involved in the Control of Cell Division Patterns in Arabidopsis Leaves.

Animal Rho GTP-binding proteins and their plant counterparts, Rho of plants (ROPs), regulate cell polarity, but they do so through different effector proteins. A class of ROP effectors, interactor of constitutive active ROPs (ICRs)/ROP interactive partners (RIPs), has been implicated in diverse biological processes; however, there are limited analyses of RIP loss-of-function mutants. Here, we report an analysis of the functions of the Arabidopsis thaliana RIPs in the leaf epidermis. Green Fluorescent Protein (GFP) fusion proteins of all the RIPs colocalized to cortical microtubules. RIP1, RIP3 and RIP4, but not RIP2 and RIP5, colocalized with the preprophase band (PPB), spindles and phragmoplasts. RIP2 and RIP5 did not colocalize with the PPB, spindles or phragmoplasts even when they were expressed under a promoter active in proliferative cells, indicating that there are differences among RIP protein properties. The overexpression of RIP1 or RIP4 resulted in the fragmentation of cortical microtubules, and the rip1 2 3 4 5 quintuple mutant showed increased growth rate of microtubules at their plus ends compared with the wild type. The rip1 2 3 4 5 mutant leaves and petals were narrow, which was explained by the decreased cell number along the transverse axis compared with that of the wild type. The rip1 2 3 4 5 mutant leaf epidermis possessed fewer PPBs oriented close to the long axis of the leaf compared with wild type, indicating the involvement of RIPs in cell division plane regulation and leaf shape determination.

Characterization of Organellar-Specific ABA Responses during Environmental Stresses in Tobacco Cells and Arabidopsis Plants.

Abscisic acid (ABA) is a critical phytohormone involved in multifaceted processes in plant metabolism and growth under both stressed and nonstressed conditions. Its accumulation in various tissues and cells has long been established as a biomarker for plant stress responses. To date, a comprehensive understanding of ABA distribution and dynamics at subcellular resolution in response to environmental cues is still lacking. Here, we modified the previously developed ABA sensor ABAleon2.1_Tao3 (Tao3) and targeted it to different organelles including the endoplasmic reticulum (ER), chloroplast/plastid, and nucleus through the addition of corresponding signal peptides. Together with the cytosolic Tao3, we show distinct ABA distribution patterns in different tobacco cells with the chloroplast showing a lower level of ABA in both cell types. In a tobacco mesophyll cell, organellar ABA displayed specific alterations depending on osmotic stimulus, with ABA levels being generally enhanced under a lower and higher concentration of salt and mannitol treatment, respectively. In Arabidopsis roots, cells from both the meristem and elongation zone accumulated ABA considerably in the cytoplasm upon mannitol treatment, while the plastid and nuclear ABA was generally reduced dependent upon specific cell types. In Arabidopsis leaf tissue, subcellular ABA seemed to be less responsive when stressed, with notable increases of ER ABA in epidermal cells and a reduction of nuclear ABA in guard cells. Together, our results present a detailed characterization of stimulus-dependent cell type-specific organellar ABA responses in tobacco and Arabidopsis plants, supporting a highly coordinated regulatory network for mediating subcellular ABA homeostasis during plant adaptation processes.

CML8 and GAD4 function in (Z)–3–hexenol–mediated defense by regulating γ–aminobutyric acid accumulation in Arabidopsis

(Z)–3–hexenol, a small gaseous molecule, is produced in plants under biotic stress and induces defense responses in neighboring plants. However, little is known about how (Z)–3–hexenol induces plant defense–related signaling. In this study, we uncovered how (Z)–3–hexenol treatment enhances plant resistance to insect attacks by increasing γ–aminobutyric acid (GABA) contents in Arabidopsis leaves. First, (Z)–3–hexenol increases the intracellular content of calcium as secondary messenger in Arabidopsis leaf mesophyll cells. Both intracellular and extracellular calcium stores regulate changes in calcium content. Then, CML8 and GAD4 transmit calcium signaling to affect (Z)–3–hexenol induced GABA content and plant resistance. Herein, CML8 interaction with GAD4 was examined via yeast two–hybrid assays, firefly luciferase complementation imaging, and GST pull–down assays. These results indicate that (Z)–3–hexenol treatment increased the GABA contents in Arabidopsis leaves based on CML8 and GAD4, thus increasing plant resistance to the insect Plutella xylostella. This study revealed the mechanism of activating plant insect defense induced by (Z)–3–hexenol, which guides the study of volatiles as biological pest control.

Shade suppresses wound-induced leaf repositioning through a mechanism involving PHYTOCHROME KINASE SUBSTRATE (PKS) genes.

Shaded plants challenged with herbivores or pathogens prioritize growth over defense. However, most experiments have focused on the effect of shading light cues on defense responses. To investigate the potential interaction between shade-avoidance and wounding-induced Jasmonate (JA)-mediated signaling on leaf growth and movement, we used repetitive mechanical wounding of leaf blades to mimic herbivore attacks. Phenotyping experiments with combined treatments on Arabidopsis thaliana rosettes revealed that shade strongly inhibits the wound effect on leaf elevation. By contrast, petiole length is reduced by wounding both in the sun and in the shade. Thus, the relationship between the shade and wounding/JA pathways varies depending on the physiological response, implying that leaf growth and movement can be uncoupled. Using RNA-sequencing, we identified genes with expression patterns matching the hyponastic response (opposite regulation by both stimuli, interaction between treatments with shade dominating the wound signal). Among them were genes from the PKS (Phytochrome Kinase Substrate) family, which was previously studied for its role in phototropism and leaf positioning. Interestingly, we observed reduced shade suppression of the wounding effect in pks2pks4 double mutants while a PKS4 overexpressing line showed constitutively elevated leaves and was less sensitive to wounding. Our results indicate a trait-specific interrelationship between shade and wounding cues on Arabidopsis leaf growth and positioning. Moreover, we identify PKS genes as integrators of external cues in the control of leaf hyponasty further emphasizing the role of these genes in aerial organ positioning.

BrABF3 promotes flowering through the direct activation of CONSTANS transcription in pak choi.

Drought stress triggers the accumulation of the phytohormone abscisic acid (ABA), which in turn activates the expression of the floral integrator gene CONSTANS (CO), accelerating flowering. However, the molecular mechanism of ABA-induced CO activation remains elusive. Here, we conducted a yeast one-hybrid assay using the CO promoter from Brassica campestris (syn. Brassica rapa) ssp. chinensis (pak choi) to screen the ABA-induced pak choi library and identified the transcription activator ABF3 (BrABF3). BrABF3, the expression of which was induced by ABA in pak choi, directly bound to the CO promoter from both pak choi and Arabidopsis. The BrABF3 promoter is specifically active in the Arabidopsis leaf vascular tissue, where CO is mainly expressed. Impaired BrABF3 expression in pak choi decreased BrCO expression levels and delayed flowering, whereas ectopic expression of BrABF3 in Arabidopsis increased CO expression and induced earlier flowering under the long-day conditions. Electrophoretic mobility shift assay analysis showed that BrABF3 was enriched at the canonical ABA-responsive element-ABRE binding factor (ABRE-ABF) binding motifs of the BrCO promoter. The direct binding of BrABF3 to the ABRE elements of CO was further confirmed by chromatin immunoprecipitation quantitative PCR. In addition, the induction of BrCO transcription by BrABF3 could be repressed by BrCDF1 in the morning. Thus, our results suggest that ABA could accelerate the floral transition by directly activating BrCO transcription through BrABF3 in pak choi.

Signatures of mRNA Alternative Polyadenylation in Arabidopsis Leaf Development.

Alternative polyadenylation (APA) of pre-mRNA is an important co-transcriptional mechanism that modulates gene expression, leading to transcriptomic and functional diversities. The role of APA in Arabidopsis leaf development, however, remains elusive. We applied a poly(A)-tag sequencing (PAT-seq) technique to characterize APA-mediated regulation events in cotyledon and in five stages of true leaf development. Over 60% APA was identified in genes expressed in leaves, consistent with the results in previous publications. However, a reduced APA level was detected in younger leaves, reaching 44% in the 18th true leaf. Importantly, we also found that >70% of the poly(A) site usages were altered in the second true leaf relative to the cotyledon. Compared with the cotyledon, more genes in the second true leaf tended to use the distal site of 3′UTR, but this was not found in pairwise comparison among other true leaves. In addition, a significant APA gene was found to be decreased in a pairwise comparison among true leaves, including differentially expressed genes. The APA genes identified herein were associated with specific biological processes, including metabolic and cellular processes and response to stimuli and hormones. These results provide a new insight into the regulation of Arabidopsis leaf development through APA.

CLE42 delays leaf senescence by antagonizing ethylene pathway in Arabidopsis.

Leaf senescence is the final stage of leaf development and is influenced by numerous internal and environmental factors. CLE family peptides are plant-specific peptide hormones that regulate various developmental processes. However, the role of CLE in regulating Arabidopsis leaf senescence remains unclear. Here, we found that CLE42 is a negative regulator of leaf senescence by using a CRISPR/Cas9-produced CLE mutant collection. The cle42 mutant displayed earlier senescence phenotypes, while overexpression of CLE42 delayed age-dependent and dark-induced leaf senescence. Moreover, application of the synthesized 12-amino-acid peptide (CLE42p) also delayed leaf senescence under natural and dark conditions. CLE42 and CLE41/44 displayed functional redundancy in leaf senescence, and the cle41 cle42 cle44 triple mutant displayed more pronounced earlier senescence phenotypes than any single mutant. Analysis of differentially expressed genes obtained by RNA-Seq methodology revealed that the ethylene pathway was suppressed by overexpressing CLE42. Moreover, CLE42 suppressed ethylene biosynthesis and thus promoted the protein accumulation of EBF, which in turn decreased the function of EIN3. Accordingly, mutation of EIN3/EIL1 or overexpression of EBF1 suppressed the earlier senescence phenotypes of the cle42 mutant. Together, our results reveal that the CLE peptide hormone regulates leaf senescence by communicating with the ethylene pathway.