Transcriptional activation by WRKY23 and derepression by removal of bHLH041 coordinately establish callus pluripotency in Arabidopsis regeneration.

Induction of the pluripotent cell mass termed callus from detached organs or tissues is an initial step in typical in vitro plant regeneration, during which auxin-induced ectopic activation of root stem cell factors is required for subsequent de novo shoot regeneration. While Arabidopsis (Arabidopsis thaliana) AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 and their downstream transcription factors LATERAL ORGAN BOUNDARIES DOMAIN (LBD) are known to play key roles in directing callus formation, the molecules responsible for activation of root stem cell factors and thus establishment of callus pluripotency are unclear. Here, we identified Arabidopsis WRKY23 and BASIC HELIX-LOOP-HELIX 041 (bHLH041) as a transcriptional activator and repressor, respectively, of root stem cell factors during establishment of auxin-induced callus pluripotency. We show that auxin-induced WRKY23 downstream of ARF7 and ARF19 directly activates the transcription of PLETHORA 3 (PLT3) and PLT7 and thus that of the downstream genes PLT1, PLT2, and WUSCHEL-RELATED HOMEOBOX 5 (WOX5), while LBD-induced removal of bHLH041 derepresses the transcription of PLT1, PLT2, and WOX5. We provide evidence that transcriptional activation by WRKY23 and loss of bHLH041-imposed repression act synergistically in conferring shoot-regenerating capability on callus cells. Our findings thus disclose a transcriptional mechanism underlying auxin-induced cellular reprogramming, which, together with previous studies, outlines the molecular framework of auxin-induced pluripotent callus formation for in vitro plant regeneration programs.

The calcium signaling module CaM-IQM destabilizes IAA-ARF interaction to regulate callus and lateral root formation.

Induction of a pluripotent cell mass, called callus, from detached organs is an initial step in in vitro plant regeneration, during which phytohormone auxin-induced ectopic activation of a root developmental program has been shown to be required for subsequent de novo regeneration of shoots and roots. However, whether other signals are involved in governing callus formation, and thus plant regeneration capability, remains largely unclear. Here, we report that the Arabidopsis calcium (Ca2+) signaling module CALMODULIN IQ-MOTIF CONTAINING PROTEIN (CaM-IQM) interacts with auxin signaling to regulate callus and lateral root formation. We show that disruption of IQMs or CaMs retards auxin-induced callus and lateral root formation by dampening auxin responsiveness, and that CaM-IQM complexes physically interact with the auxin signaling repressors INDOLE-3-ACETIC ACID INDUCIBLE (IAA) proteins in a Ca2+-dependent manner. We further provide evidence that the physical interaction of CaM6 with IAA19 destabilizes the repressive interaction of IAA19 with AUXIN RESPONSE FACTOR 7 (ARF7), and thus regulates auxin-induced callus formation. These findings not only define a critical role of CaM-IQM-mediated Ca2+ signaling in callus and lateral root formation, but also provide insight into the interplay of Ca2+ signaling and auxin actions during plant regeneration and development.

Biological active ingredients of Astragali Radix and its mechanisms in treating cardiovascular and cerebrovascular diseases.

BACKGROUND: With the rising age of the global population, the incidence rate of cardiovascular and cerebrovascular diseases (CCVDs) is increasing, which causes serious public health burden. The efforts for new therapeutic approaches are still being sought since the treatment effects of existing therapies are not quite satisfactory. Chinese traditional medicine proved to be very efficient in the treatment of CCVDs. Well described and established in Chinese medicine, Astragali Radix, has been commonly administered in the prophylaxis and cure of CCVDs for thousands of years. PURPOSE: This review summarized the action mode and mechanisms of Astragali Radix phytochemicals on CCVDs, hoping to provide valuable information for the future application, development and improvement of Astragali Radix as well as CCVDs treatment. METHODS: A plenty of literature on biological active ingredients of Astragali Radix used for CCVDs treatment were retrieved from online electronic PubMed and Web of Science databases. RESULTS: This review highlighted the effects of five main active components in Astragali Radix including astragaloside Ⅳ, cycloastragenol, astragalus polysaccharide, calycosin-7-O-ß-d-glucoside, and calycosin on CCVDs. The mechanisms mainly involved anti-oxidative damage, anti-inflammatory, and antiapoptotic through signaling pathways such as PI3K/Akt, Nrf2/HO-1, and TLR4/NF-κB pathway. In addition, the majority active constituents in AR have no obvious toxic side effects. CONCLUSION: The main active components of Astragali Radix, especially AS-IV, have been extensively summarized. It has been proved that Astragali Radix has obvious therapeutic effects on various CCVDs, including myocardial and cerebral ischemia, hypertension, atherosclerosis, cardiac hypertrophy, chronic heart failure. CAG possesses anti-ischemia activity without toxicity, indicating a worthy of further development. However, high-quality clinical and pharmacokinetic studies are required to validate the current studies.

Catabolism of strigolactones by a carboxylesterase.

Strigolactones (SLs) are carotenoid-derived plant hormones that control shoot branching and communications between host plants and symbiotic fungi or root parasitic plants. Extensive studies have identified the key components participating in SL biosynthesis and signalling, whereas the catabolism or deactivation of endogenous SLs in planta remains largely unknown. Here, we report that the Arabidopsis carboxylesterase 15 (AtCXE15) and its orthologues function as efficient hydrolases of SLs. We show that overexpression of AtCXE15 promotes shoot branching by dampening SL-inhibited axillary bud outgrowth. We further demonstrate that AtCXE15 could bind and efficiently hydrolyse SLs both in vitro and in planta. We also provide evidence that AtCXE15 is capable of catalysing hydrolysis of diverse SL analogues and that such CXE15-dependent catabolism of SLs is evolutionarily conserved in seed plants. These results disclose a catalytic mechanism underlying homoeostatic regulation of SLs in plants, which also provides a rational approach to spatial-temporally manipulate the endogenous SLs and thus architecture of crops and ornamental plants.

The molecular regulation of cell pluripotency in plants.

Plants have a remarkably regenerative capability to replace the damaged organs or form the new organs and individuals both in vivo and in vitro, which is fundamental for their developmental plasticity and the agricultural practices. The regenerative capacities of plants are highly dependent on the totipotency or pluripotency of somatic cells, whose fates are directed by phytohormones, wounding, and other stimuli. Recent studies have revealed that the two types of cellular reprogramming are involved in the acquisition of cell pluripotency during plant in vitro and in vivo regeneration programs. This review focuses on the recent advances of the cellular origin, molecular characteristic, and genetic and epigenetic regulations of cell pluripotency acquisition in plants, highlighting the molecular frameworks of cellular reprogramming activated by diverse stimuli and their possible potentials in regeneration-based plant biotechnologies.

Control of auxin-induced callus formation by bZIP59-LBD complex in Arabidopsis regeneration.

Induction of pluripotent cells termed callus by auxin represents a typical cell fate change required for plant in vitro regeneration; however, the molecular control of auxin-induced callus formation is largely elusive. We previously identified four Arabidopsis auxin-inducible Lateral Organ Boundaries Domain (LBD) transcription factors that govern callus formation. Here, we report that Arabidopsis basic region/leucine zipper motif 59 (AtbZIP59) transcription factor forms complexes with LBDs to direct auxin-induced callus formation. We show that auxin stabilizes AtbZIP59 and enhances its interaction with LBD, and that disruption of AtbZIP59 dampens auxin-induced callus formation whereas overexpression of AtbZIP59 triggers autonomous callus formation. AtbZIP59-LBD16 directly targets a FAD-binding Berberine (FAD-BD) gene and promotes its transcription, which contributes to callus formation. These findings define the AtbZIP59-LBD complex as a critical regulator of auxin-induced cell fate change during callus formation, which provides a new insight into the molecular regulation of plant regeneration and possible developmental programs.

Genome-Wide Identification of Arabidopsis LBD29 Target Genes Reveals the Molecular Events behind Auxin-Induced Cell Reprogramming during Callus Formation.

Auxin-induced callus formation represents an important cell reprogramming process during in vitro regeneration of plants, in which the pericycle or pericycle-like cells within plant organs are reprogrammed into the pluripotent cell mass termed callus that is generally required for subsequent regeneration of root or shoot. However, the molecular events behind cell reprogramming during auxin-induced callus formation are largely elusive. We previously identified that auxin-induced LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factors act as the master regulators to trigger auxin-induced callus formation. Here, by ChIP-seq (chromatin immunoprecipitation-based sequencing) and RNA sequencing approaches, we identified the potential LBD29 target genes at the genome-wide level and outlined the molecular events of LBD-triggered cell reprogramming during callus formation. We showed that LBD29 preferentially bound to the G-box (CACGTG) and TGGGC[C/T] motifs and potentially targeted >350 genes, among which the genes related to methylation, reactive oxygen species (ROS) metabolism, cell wall hydrolysis and lipid metabolism were rapidly activated, while most of the light-responsive genes were suppressed by LBD29. Further examination of a few representative genes validated that they were targeted by LBD29 and participated in the regulation of cell reprogramming during callus formation. Our data not only outline a framework of the early molecular events behind auxin-induced cell reprogramming of callus formation, but also provide a valuable resource for identification of genes that regulate cell fate switch during in vitro regeneration of plants.

Very-long-chain fatty acids restrict regeneration capacity by confining pericycle competence for callus formation in Arabidopsis.

The already differentiated organs in plants have a remarkable capacity to regenerate new individuals under culture conditions. Plant in vitro regeneration practically starts with the induction of a pluripotent cell mass, the callus, from detached organs on auxin-rich callus-inducing medium (CIM), which is generally required for subsequent regeneration of new bodies. Recent studies show that CIM-induced callus formation occurs from the pericycle or pericycle-like cells through a root developmental pathway, whereas the signals involved in governing callus-forming capacity of pericycle cells remain unknown. Here we report that very-long-chain fatty acids (VLCFAs) play a critical role in confining the pericycle competence for callus formation and thus the regeneration capacity of Arabidopsis By genetic screening, we identified the callus formation-related 1 (cfr1) mutant, which bypasses the inhibition of callus-forming capacity in roots by solitary-root (slr/iaa14). We show that CFR1 encodes 3-ketoacyl-CoA synthase 1 (KCS1), which catalyzes a rate-limiting step of VLCFA biosynthesis. Our biochemical and genetic analyses demonstrate that VLCFAs restrict the pericycle competence for callus formation, at least in part, by regulating the transcription of Aberrant Lateral Root Formation 4 (ALF4). Moreover, we provide evidence that VLCFAs act as cell layer signals to mediate the pericycle competence for callus formation. Taken together, our results identify VLCFAs or their derivatives as the confining signals for mediating the pericycle competence for callus formation and thus the regeneration capacity of plant organs.

A genome-wide transcriptome profiling reveals the early molecular events during callus initiation in Arabidopsis multiple organs.

Induction of a pluripotent cell mass termed callus is the first step in an in vitro plant regeneration system, which is required for subsequent regeneration of new organs or whole plants. However, the early molecular mechanism underlying callus initiation is largely elusive. Here, we analyzed the dynamic transcriptome profiling of callus initiation in Arabidopsis aerial and root explants and identified 1342 differentially expressed genes in both explants after incubation on callus-inducing medium. Detailed categorization revealed that the differentially expressed genes were mainly related to hormone homeostasis and signaling, transcriptional and post transcriptional regulations, protein phosphorelay cascades and DNA- or chromatin-modification. Further characterization showed that overexpression of two transcription factors, HB52 or CRF3, resulted in the callus formation in transgenic plants without exogenous auxin. Therefore, our comprehensive analyses provide some insight into the early molecular regulations during callus initiation and are useful for further identification of the regulators governing callus formation.

LATERAL ORGAN BOUNDARIES DOMAIN transcription factors direct callus formation in Arabidopsis regeneration.

The remarkable regeneration capability of plant tissues or organs under culture conditions has underlain an extensive practice for decades. The initial step in plant in vitro regeneration often involves the induction of a pluripotent cell mass termed callus, which is driven by the phytohormone auxin and occurs via a root development pathway. However, the key molecules governing callus formation remain unknown. Here we demonstrate that Arabidopsis LATERAL ORGAN BOUNDARIES DOMAIN (LBD)/ASYMMETRIC LEAVES2-LIKE (ASL) transcription factors are involved in the control of callus formation program. The four LBD genes downstream of AUXIN RESPONSE FACTORs (ARFs), LBD16, LBD17, LBD18 and LBD29, are rapidly and dramatically induced by callus-inducing medium (CIM) in multiple organs. Ectopic expression of each of the four LBD genes in Arabidopsis is sufficient to trigger spontaneous callus formation without exogenous phytohormones, whereas suppression of LBD function inhibits the callus formation induced by CIM. Moreover, the callus triggered by LBD resembles that induced by CIM by characteristics of ectopically activated root meristem genes and efficient regeneration capacity. These findings define LBD transcription factors as key regulators in the callus induction process, thereby establishing a molecular link between auxin signaling and the plant regeneration program.

The Arabidopsis SMO2, a homologue of yeast TRM112, modulates progression of cell division during organ growth.

Cell proliferation is integrated into developmental progression in multicellular organisms, including plants, and the regulation of cell division is of pivotal importance for plant growth and development. Here, we report the identification of an Arabidopsis SMALL ORGAN 2 (SMO2) gene that functions in regulation of the progression of cell division during organ growth. The smo2 knockout mutant displays reduced size of aerial organs and shortened roots, due to the decreased number of cells in these organs. Further analyses reveal that disruption of SMO2 does not alter the developmental timing but reduces the rate of cell production during leaf and root growth. Moreover, smo2 plants exhibit a constitutive activation of cell cycle-related genes and over-accumulation of cells expressing CYCB1;1:beta-glucuronidase (CYCB1;1:GUS) during organogenesis, suggesting that smo2 has a defect in G(2)-M phase progression in the cell cycle. SMO2 encodes a functional homologue of yeast TRM112, a plurifunctional component involved in a few cellular events, including tRNA and protein methylation. In addition, the mutation of SMO2 does not appear to affect endoreduplication in Arabidopsis leaf cells. Taken together we postulate that Arabidopsis SMO2 is a conserved yeast TRM112 homologue and SMO2-mediated cellular events are required for proper progression of cell division in plant growth and development.

Molecular cloning and characterization of wheat calreticulin (CRT) gene involved in drought-stressed responses.

Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca(2+)-binding protein in multicellular eukaryotes. CRT plays a crucial role in many cellular processes including Ca(2+) storage and release, protein synthesis, and molecular chaperone activity. To elucidate the function of CRTs in plant responses against drought, a main abiotic stress limiting cereal crop production worldwide, a full-length cDNA encoding calreticulin protein namely TaCRT was isolated from wheat (Triticum aestivum L.). The deduced amino acid sequence of TaCRT shares high homology with other plant CRTs. Phylogenetic analysis indicates that TaCRT cDNA clone encodes a wheat CRT3 isoform. Southern analysis suggests that the wheat genome contains three copies of TaCRT. Subcellular locations of TaCRT were the cytoplasm and nucleus, evidenced by transient expression of GFP fused with TaCRT in onion epidermal cells. Enhanced accumulation of TaCRT transcript was observed in wheat seedlings in response to PEG-induced drought stress. To investigate further whether TaCRT is involved in the drought-stress response, transgenic plants were constructed. Compared to the wild-type and GFP-expressing plants, TaCRT-overexpressing tobacco (Nicotiana benthamiana) plants grew better and exhibited less wilt under the drought stress. Moreover, TaCRT-overexpressing plants exhibited enhanced drought resistance to water deficit, as shown by their capacity to maintain higher WUE (water use efficiency), WRA (water retention ability), RWC (relative water content), and lower MDR (membrane damaging ratio) (P < or = 0.01) under water-stress conditions. In conclusion, a cDNA clone encoding wheat CRT was successfully isolated and the results suggest that TaCRT is involved in the plant response to drought stress, indicating a potential in the transgenic improvements of plant water-stress.

A wheat (Triticum aestivum) protein phosphatase 2A catalytic subunit gene provides enhanced drought tolerance in tobacco.

BACKGROUND AND AIMS: Multiple copies of genes encoding the catalytic subunit (c) of protein phosphatase 2A (PP2A) are commonly found in plants. For some of these genes, expression is up-regulated under water stress. The aim of this study was to investigate expression and characterization of TaPP2Ac-1 from Triticum aestivum, and to evaluate the effects of TaPP2Ac-1 on Nicotiana benthamiana in response to water stress. METHODS: TaPP2Ac-1 cDNA was isolated from wheat by in silico identification and RT-PCR amplification. Transcript levels of TaPP2Ac-1 were examined in wheat responding to water deficit. Copy numbers of TaPP2Ac-1 in wheat genomes and subcellular localization in onion epidermal cells were studied. Enzyme properties of the recombinant TaPP2Ac-1 protein were determined. In addition, studies were carried out in tobacco plants with pCAPE2-TaPP2Ac-1 under water-deficit conditions. KEY RESULTS: TaPP2Ac-1 cDNA was cloned from wheat. Transcript levels of TaPP2Ac-1 in wheat seedlings were up-regulated under drought condition. One copy for this TaPP2Ac-1 was present in each of the three wheat genomes. TaPP2Ac-1 fused with GFP was located in the nucleus and cytoplasm of onion epidermis cells. The recombinant TaPP2Ac-1 gene was over-expressed in Escherichia coli and encoded a functional serine/threonine phosphatase. Transgenic tobacco plants over-expressing TaPP2Ac-1 exhibited stronger drought tolerance than non-transgenic tobacco plants. CONCLUSIONS: Tobacco plants with pCAPE2-TaPP2Ac-1 appeared to be resistant to water deficit, as shown by their higher capacity to maintain leaf relative water content, leaf cell-membrane stability index, water-retention ability and water use efficiency under water stress. The results suggest that the physiological role of TaPP2Ac-1 is related to drought stress response, possibly through its involvement in drought-responding signal transduction pathways.