Arabidopsis thaliana MYC2 and MYC3 Are Involved in Ethylene-Regulated Hypocotyl Growth as Negative Regulators.

The ethylene-regulated hypocotyl elongation of Arabidopsis thaliana involves many transcription factors. The specific role of MYC transcription factors in ethylene signal transduction is not completely understood. The results here revealed that two MYCs, MYC2 and MYC3, act as negative regulators in ethylene-suppressed hypocotyl elongation. Etiolated seedlings of the loss-of-function mutant of MYC2 or MYC3 were significantly longer than wild-type seedlings. Single- or double-null mutants of MYC2 and MYC3 displayed remarkably enhanced response to ACC(1-aminocyclopropane-1-carboxylate), the ethylene precursor, compared to wild-type seedlings. MYC2 and MYC3 directly bind to the promoter zone of ERF1, strongly suppressing its expression. Additionally, EIN3, a key component in ethylene signaling, interacts with MYC2 or MYC3 and significantly suppresses their binding to ERF1's promoter. MYC2 and MYC3 play crucial roles in the ethylene-regulated expression of functional genes. The results revealed the novel role and functional mechanism of these transcription factors in ethylene signal transduction. The findings provide valuable information for deepening our understanding of their role in regulating plant growth and responding to stress.

Spatial specificity of metabolism regulation of abscisic acid-imposed seed germination inhibition in Korean pine (Pinus koraiensis sieb et zucc).

Introduction: Abscisic acid (ABA) can negatively regulate seed germination, but the mechanisms of ABA-mediated metabolism modulation are not well understood. Moreover, it remains unclear whether metabolic pathways vary with the different tissue parts of the embryo, such as the radicle, hypocotyl and cotyledon. Methods: In this report, we performed the first comprehensive metabolome analysis of the radicle and hypocotyl + cotyledon in Pinus koraiensis seeds in response to ABA treatment during germination. Results and discussion: Metabolome profiling showed that following ABA treatment, 67 significantly differentially accumulated metabolites in the embryo were closely associated with pyrimidine metabolism, phenylalanine metabolism, cysteine and methionine metabolism, galactose metabolism, terpenoid backbone biosynthesis, and glutathione metabolism. Meanwhile, 62 metabolites in the hypocotyl + cotyledon were primarily involved in glycerophospholipid metabolism and glycolysis/gluconeogenesis. We can conclude that ABA may inhibit Korean pine seed germination primarily by disrupting the biosynthesis of certain plant hormones mediated by cysteine and methionine metabolism and terpenoid backbone biosynthesis, as well as reducing the reactive oxygen species scavenging ability regulated by glutathione metabolism and shikimate pathway in radicle. ABA may strongly disrupt the structure and function of cellular membranes due to alterations in glycerophospholipid metabolism, and weaken glycolysis/gluconeogenesis in the hypocotyl + cotyledon, both of which are major contributors to ABA-mediated inhibition of seed germination. These results highlight that the spatial modulation of metabolic pathways in Pinus koraiensis seeds underlies the germination response to ABA.

CCaP1/CCaP2/CCaP3 interact with plasma membrane H+-ATPases and promote thermo-responsive growth by regulating cell wall modification in Arabidopsis.

Arabidopsis plants adapt to warm temperatures by promoting hypocotyl growth primarily through the basic helix-loop-helix transcription factor PIF4 and its downstream genes involved in auxin responses, which enhance cell division. In the current study, we discovered that cell wall-related calcium-binding protein 2 (CCaP2) and its paralogs CCaP1 and CCaP3 function as positive regulators of thermo-responsive hypocotyl growth by promoting cell elongation in Arabidopsis. Interestingly, mutations in CCaP1/CCaP2/CCaP3 do not affect the expression of PIF4-regulated classic downstream genes. However, they do noticeably reduce the expression of xyloglucan endotransglucosylase/hydrolase genes, which are involved in cell wall modification. We also found that CCaP1/CCaP2/CCaP3 are predominantly localized to the plasma membrane, where they interact with the plasma membrane H+-ATPases AHA1/AHA2. Furthermore, we observed that vanadate-sensitive H+-ATPase activity and cell wall pectin and hemicellulose contents are significantly increased in wild-type plants grown at warm temperatures compared with those grown at normal growth temperatures, but these changes are not evident in the ccap1-1 ccap2-1 ccap3-1 triple mutant. Overall, our findings demonstrate that CCaP1/CCaP2/CCaP3 play an important role in controlling thermo-responsive hypocotyl growth and provide new insights into the alternative pathway regulating hypocotyl growth at warm temperatures through cell wall modification mediated by CCaP1/CCaP2/CCaP3.

Comprehensive analysis of the UDP-glucuronate decarboxylase (UXS) gene family in tobacco and functional characterization of NtUXS16 in Golgi apparatus in Arabidopsis.

BACKGROUND: UDP-glucuronate decarboxylase (also named UXS) converts UDP-glucuronic acid (UDP-GlcA) to UDP-xylose (UDP-Xyl) by decarboxylation of the C6-carboxylic acid of glucuronic acid. UDP-Xyl is an important sugar donor that is required for the synthesis of plant cell wall polysaccharides. RESULTS: In this study, we first carried out the genome-wide identification of NtUXS genes in tobacco. A total of 17 NtUXS genes were identified, which could be divided into two groups (Group I and II), and the Group II UXSs can be further divided into two subgroups (Group IIa and IIb). Furthermore, the protein structures, intrachromosomal distributions and gene structures were thoroughly analyzed. To experimentally verify the subcellular localization of NtUXS16 protein, we transformed tobacco BY-2 cells with NtUXS16 fused to the monomeric red fluorescence protein (mRFP) at the C terminus under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The fluorescent signals of NtUXS16-mRFP were localized to the medial-Golgi apparatus. Contrary to previous predictions, protease digestion analysis revealed that NtUXS16 is not a type II membrane protein. Overexpression of NtUXS16 in Arabidopsis seedling in darkness led to a significant increase in hypocotyl length and a reduction in root length compared with the wild type. In summary, these results suggest Golgi apparatus localized-NtUXS16 plays an important role in hypocotyl and root growth in the dark. CONCLUSION: Our findings facilitate our understanding of the novel functions of NtUXS16 and provide insights for further exploration of the biological roles of NtUXS genes in tobacco.

Promotive effect of phytosulfokine - peptide growth factor - on protoplast cultures development in Fagopyrum tataricum (L.) Gaertn.

BACKGROUND: Fagopyrum tataricum (Tartary buckwheat) is a valuable crop of great nutritional importance due to its high level of bioactive compounds. Excellent opportunities to obtain plants with the high level or the desired profile of valuable metabolites may be provided by in vitro cultures. Among known in vitro techniques, protoplast technology is an exciting tool for genetic manipulation to improve crop traits. In that context, protoplast fusion may be applied to generate hybrid cells between different species of Fagopyrum. To apply protoplast cultures to the aforementioned approaches in this research, we established the protoplast-to-plant system in Tartary buckwheat. RESULTS: In this work, cellulase and pectinase activity enabled protoplast isolation from non-morphogenic and morphogenic callus (MC), reaching, on average, 2.3 × 106 protoplasts per g of fresh weight. However, to release protoplasts from hypocotyls, the key step was the application of driselase in the enzyme mixture. We showed that colony formation could be induced after protoplast embedding in agarose compared to the alginate matrix. Protoplasts cultured in a medium based on Kao and Michayluk supplemented with phytosulfokine (PSK) rebuilt cell walls, underwent repeated mitotic division, formed aggregates, which consequently led to callus formation. Plating efficiency, expressing the number of cell aggregate formed, in 10-day-old protoplast cultures varied from 14% for morphogenic callus to 30% for hypocotyls used as a protoplast source. However plant regeneration via somatic embryogenesis and organogenesis occurred only during the cultivation of MC-derived protoplasts. CONCLUSIONS: This study demonstrated that the applied protoplast isolation approach facilitated the recovery of viable protoplasts. Moreover, the embedding of protoplasts in an agarose matrix and supplementation of a culture medium with PSK effectively stimulated cell division and further development of Tartary buckwheat protoplast cultures along with the plant regeneration. Together, these results provide the first evidence of developing a protoplast-to-plant system from the MC of Fagopyrum tataricum used as source material. These findings suggest that Tartary buckwheat's protoplast cultures have potential implications for the species' somatic hybridization and genetic improvement.

A Small Compound, HYGIC, Promotes Hypocotyl Growth Through Ectopic Ethylene Response.

Plant seedlings adjust the growth of the hypocotyl in response to surrounding environmental changes. Genetic studies have revealed key players and pathways in hypocotyl growth, such as phytohormones and light signaling. However, because of genetic redundancy in the genome, it is expected that not-yet-revealed mechanisms can be elucidated through approaches different from genetic ones. Here, we identified a small compound, HYGIC (HG), that simultaneously induces hypocotyl elongation and thickening, accompanied by increased nuclear size and enlargement of cortex cells. HG-induced hypocotyl growth required the ethylene signaling pathway activated by endogenous ethylene, involving CONSTITUTIVE PHOTOMORPHOGENIC 1, ETHYLENE INSENSITIVE 2 (EIN2) and redundant transcription factors for ethylene responses, ETHYLENE INSENSITIVE 3 (EIN3) and EIN3 LIKE 1. By using EBS:GUS, a transcriptional reporter of ethylene responses based on an EIN3-binding-cis-element, we found that HG treatment ectopically activates ethylene responses at the epidermis and cortex of the hypocotyl. RNA-seq and subsequent gene ontology analysis revealed that a significant number of HG-induced genes are related to responses to hypoxia. Indeed, submergence, a representative environment where the hypoxia response is induced in nature, promoted ethylene-signaling-dependent hypocotyl elongation and thickening accompanied by ethylene responses at the epidermis and cortex, which resembled the HG treatment. Collectively, the identification and analysis of HG revealed that ectopic responsiveness to ethylene promotes hypocotyl growth, and this mechanism is activated under submergence.

Nitric oxide induces S-nitrosylation of CESA1 and CESA9 and increases cellulose content in Arabidopsis hypocotyls.

Nitric oxide (NO), a small signaling gas molecule, participates in several growth and developmental processes in plants. However, how NO regulates cell wall biosynthesis remains unclear. Here, we demonstrate a positive effect of NO on cellulose content that may be related to S-nitrosylation of cellulose synthase 1 (CESA1) and CESA9. Two S-nitrosylated cysteine (Cys) residues, Cys562 and Cys641, which are exposed on the surface of CESA1 and CESA9 and located in the cellulose synthase catalytic domain, were identified to be S-nitrosylated. Meanwhile, Cys641 was located on the binding surface of CESA1 and CESA9, and Cys562 was very close to the binding surface. Cellulose synthase complexes (CSCs) dynamics are closely associated with cellulose content. S-nitrosylation of CESA1 and CESA9 improved particles mobility and thus increased the accumulation of cellulose in Arabidopsis hypocotyl cells. An increase in hemicellulose content as well as an alteration in pectin content facilitated cell wall extension and contributed to cell growth, finally promoting elongation of Arabidopsis hypocotyls. Overall, our work provides a path to investigate the way NO affects the cellulose content of plants.

Convergence between Development and Stress: Ectopic Xylem Formation in Arabidopsis Hypocotyl in Response to 24-Epibrassinolide and Cadmium.

Ectopic xylary element (EXE) formation in planta is a poorly investigated process, and it is unknown if it occurs as a response to the soil pollutant Cadmium (Cd). The pericycle cells of Arabidopsis thaliana hypocotyl give rise to EXEs under specific hormonal inputs. Cadmium triggers pericycle responses, but its role in EXE formation is unknown. Brassinosteroids (BRs) affect numerous developmental events, including xylogenesis in vitro, and their exogenous application by 24-epibrassinolide (eBL) helps to alleviate Cd-stress by increasing lateral/adventitious rooting. Epibrassinolide's effects on EXEs in planta are unknown, as well as its relationship with Cd in the control of the process. The research aims to establish an eBL role in pericycle EXE formation, a Cd role in the same process, and the possible interaction between the two. Results show that 1 nM eBL causes an identity reversal between the metaxylem and protoxylem within the stele, and its combination with Cd reduces the event. All eBL concentrations increase EXEs, also affecting xylary identity by changing from protoxylem to metaxylem in a concentration-dependent manner. Cadmium does not affect EXE identity but increases EXEs when combined with eBL. The results suggest that eBL produces EXEs to form a mechanical barrier against the pollutant.

The mungbean VrP locus encoding MYB90, an R2R3-type MYB protein, regulates anthocyanin biosynthesis.

Anthocyanins are water-soluble pigments present in several tissues/parts of plants. The pigments provide color and are wildly known for health benefits for human, insect attraction for plant pollination, and stress resistance in plants. Anthocyanin content variations in mungbean [Vigna radiata (L.) Wilczek] were first noticed a long time ago, but the genetic mechanism controlling the anthocyanins in mungbean remains unknown. An F2 population derived from the cross between purple-hypocotyl (V2709) and green-hypocotyl (Sulv1) mungbeans was used to map the VrP locus controlling purple hypocotyl. The VrP locus was mapped to a 78.9-kb region on chromosome 4. Sequence comparison and gene expression analysis identified an R2R3-MYB gene VrMYB90 as the candidate gene for the VrP locus. Haplotype analysis using 124 mungbean accessions suggested that 10 single nucleotide polymorphisms (SNPs) in exon 3 may lead to an abolished expression of VrMYB90 and an absence of anthocyanin accumulation in the hypocotyl of Sulv1 and KPS2. The overexpression of VrMYB90 in mungbean hairy root, tobacco leaf, and Arabidopsis resulted in anthocyanin accumulation (purple color). Gene expression analysis demonstrated that VrMYB90 regulated anthocyanin accumulation in the hypocotyl, stem, petiole, and flowers, and the expression was sensitive to light. VrMYB90 protein may upregulate VrDFR encoding dihydroflavonol 4-reductase at the late biosynthesis step of anthocyanins in mungbeans. These results suggest that VrMYB90 is the dominator in the spatiotemporal regulation of anthocyanin biosynthesis. Our results provide insight into the biosynthesis mechanism of anthocyanin and a theoretical basis for breeding mungbeans.

SCaBP3/CBL7 negatively regulates the plasma membrane H+-ATPase and modulates hypocotyl elongation in Arabidopsis.

The regulation of hypocotyl elongation is an important process in plant growth and development and depends on the activity of the plasma membrane (PM) H+-ATPase. In this study, we found that the Arabidopsis protein SOS3-LIKE CALCIUM BINDING PROTEIN3 (SCaBP3) negatively regulates PM H+-ATPase activity in yeast and hypocotyl elongation in dark-grown seedlings. Yeast two-hybrid assays showed that SCaBP3 interacts with representative members of the Arabidopsis PM H+-ATPase family. Experiments in RS-72 yeast showed that SCaBP3 negatively regulates PM H+-ATPase activity-dependent yeast cell growth. Hypocotyl elongation was promoted in the loss-of-function mutant scabp3 and inhibited in SCaBP3 overexpression lines of Arabidopsis. We propose that SCaBP3 modulates hypocotyl elongation by negatively regulating PM H+-ATPase activity.

Ethylene Insensitive 3-Like 2 is a Brassicaceae-specific transcriptional regulator involved in fine-tuning ethylene responses in Arabidopsis thaliana.

Ethylene signaling directs a pleiotropy of developmental processes in plants. In Arabidopsis, ethylene signaling converges at the master transcription factor Ethylene Insensitive 3 (EIN3), which has five homologs, EIN3-like 1-5 (EIL1-EIL5). EIL1 is most fully characterized and operates similarly to EIN3, while EIL3-5 are not involved in ethylene signaling. EIL2 remains less investigated. Our phylogenetic analysis revealed that EIL2 homologs have only been retrieved in the Brassicaceae family, suggesting that EIL2 diverged to have specific functions in the mustard family. By characterizing eil2 mutants, we found that EIL2 is involved in regulating ethylene-specific developmental processes in Arabidopsis thaliana, albeit in a more subtle way compared with EIN3/EIL1. EIL2 steers ethylene-triggered hypocotyl elongation in light-grown seedlings and is involved in lateral root formation. Furthermore, EIL2 takes part in regulating flowering time as eil2 mutants flower on average 1 d earlier and have fewer leaves. A pEIL2:EIL2:GFP translational reporter line revealed that EIL2 protein abundance is restricted to the stele of young developing roots. EIL2 expression, and not EIL2 protein stability, is regulated by ethylene in an EIN3/EIL1-dependent way. Despite EIL2 taking part in several developmental processes, the precise upstream and downstream regulation of this ethylene- and Brassicaceae-specific transcription factor remains to be elucidated.

Highly efficient Agrobacterium-mediated transformation and plant regeneration system for genome engineering in tomato.

Tomato (Solanum lycopersicum L.) is an important vegetable and nutritious crop plant worldwide. They are rich sources of several indispensable compounds such as lycopene, minerals, vitamins, carotenoids, essential amino acids, and bioactive polyphenols. Plant regeneration and Agrobacterium-mediated genetic transformation system from different explants in various genotypes of tomato are necessary for genetic improvement. Among diverse plant growth regulator (PGR) combinations and concentrations tested, Zeatin (ZEA) at 2.0 mg l-1 in combination with 0.1 mg l-1 indole-3-acetic acid (IAA) generated the most shoots/explant from the cotyledon of Arka Vikas (36.48 shoots/explant) and PED (24.68 shoots/explant), respectively. The hypocotyl explant produced 28.76 shoots/explant in Arka Vikas and 19.44 shoots/explant in PED. In contrast, leaf explant induced 23.54 shoots/explant in Arka Vikas and 17.64 shoots/explant in PED. The obtained multiple shoot buds from three explant types were elongated on a medium fortified with Gibberellic acid (GA3) (1.0 mg l-1), IAA (0.5 mg l-1), and ZEA (0.5 mg l-1) in both the cultivars. The rooting was observed on a medium amended with 0.5 mg l-1 indole 3-butyric acid (IBA). The transformation efficiency was significantly improved by optimizing the pre-culture of explants, co-cultivation duration, bacterial density and infection time, and acetosyringone concentration. The presence of transgenes in the plant genome was validated using different methods like histochemical GUS assay, Polymerase Chain Reaction (PCR), and Southern blotting. The transformation efficiency was 42.8% in PED and 64.6% in Arka Vikas. A highly repeatable plant regeneration protocol was established by manipulating various plant growth regulators (PGRs) in two tomato cultivars (Arka Vikas and PED). The Agrobacterium-mediated transformation method was optimized using different explants like cotyledon, hypocotyl, and leaf of two tomato genotypes. The present study could be favourable to transferring desirable traits and precise genome editing techniques to develop superior tomato genotypes.

CINCINNATA-Like TCP Transcription Factors in Cell Growth - An Expanding Portfolio.

Post-mitotic cell growth is a key process in plant growth and development. Cell expansion drives major growth during morphogenesis and is influenced by both endogenous factors and environmental stimuli. Though both isotropic and anisotropic cell growth can contribute to organ size and shape at different degrees, anisotropic cell growth is more likely to contribute to shape change. While much is known about the mechanisms that increase cellular turgor and cell-wall biomass during expansion, the genetic factors that regulate these processes are less studied. In the past quarter of a century, the role of the CINCINNATA-like TCP (CIN-TCP) transcription factors has been well documented in regulating diverse aspects of plant growth and development including flower asymmetry, plant architecture, leaf morphogenesis, and plant maturation. The molecular activity of the CIN-TCP proteins common to these biological processes has been identified as their ability to suppress cell proliferation. However, reports on their role regulating post-mitotic cell growth have been scanty, partly because of functional redundancy among them. In addition, it is difficult to tease out the effect of gene activity on cell division and expansion since these two processes are linked by compensation, a phenomenon where perturbation in proliferation is compensated by an opposite effect on cell growth to keep the final organ size relatively unaltered. Despite these technical limitations, recent genetic and growth kinematic studies have shown a distinct role of CIN-TCPs in promoting cellular growth in cotyledons and hypocotyls, the embryonic organs that grow solely by cell expansion. In this review, we highlight these recent advances in our understanding of how CIN-TCPs promote cell growth.

Tripartite hormonal regulation of plasma membrane H+-ATPase activity.

The enzyme activity of the plasma membrane (PM) proton pump, well known as arabidopsis PM H+-ATPase (AHA) in the model plant arabidopsis (Arabidopsis thaliana), is controlled by phosphorylation. Three different classes of phytohormones, brassinosteroids (BRs), abscisic acid (ABA), and auxin regulate plant growth and responses to environmental stimuli, at least in part by modulating the activity of the pump through phosphorylation of the penultimate Thr residue in its carboxyl terminus. Here, we review the current knowledge regarding this tripartite hormonal AHA regulation and highlight mechanisms of activation and deactivation, as well as the significance of hormonal crosstalk. Understanding the complexity of PM H+-ATPase regulation in plants might provide new strategies for sustainable agriculture.

The CBL-Interacting Protein Kinase NtCIPK23 Positively Regulates Seed Germination and Early Seedling Development in Tobacco (Nicotiana tabacum L.).

CBL-interacting protein kinase (CIPK) family is a unique group of serine/threonine protein kinase family identified in plants. Among this family, AtCIPK23 and its homologs in some plants are taken as a notable group for their importance in ions transport and stress responses. However, there are limited reports on their roles in seedling growth and development, especially in Solanaceae plants. In this study, NtCIPK23, a homolog of AtCIPK23 was cloned from Nicotiana tabacum. Expression analysis showed that NtCIPK23 is mainly expressed in the radicle, hypocotyl, and cotyledons of young tobacco seedlings. The transcriptional level of NtCIPK23 changes rapidly and spatiotemporally during seed germination and early seedling growth. To study the biological function of NtCIPK23 at these stages, the overexpressing and CRISPR/Cas9-mediated knock-out (ntcipk23) tobacco lines were generated. Phenotype analysis indicated that knock-out of NtCIPK23 significantly delays seed germination and the appearance of green cotyledon of young tobacco seedling. Overexpression of NtCIPK23 promotes cotyledon expansion and hypocotyl elongation of young tobacco seedlings. The expression of NtCIPK23 in hypocotyl is strongly upregulated by darkness and inhibited under light, suggesting that a regulatory mechanism of light might underlie. Consistently, a more obvious difference in hypocotyl length among different tobacco materials was observed in the dark, compared to that under the light, indicating that the upregulation of NtCIPK23 contributes greatly to the hypocotyl elongation. Taken together, NtCIPK23 not only enhances tobacco seed germination, but also accelerate early seedling growth by promoting cotyledon greening rate, cotyledon expansion and hypocotyl elongation of young tobacco seedlings.

Insights Into Plant Surgery: An Overview of the Multiple Grafting Techniques for Arabidopsis thaliana.

Plant grafting, the ancient practice of cutting and joining different plants, is gaining popularity as an elegant way to generate chimeras that combine desirable traits. Grafting was originally developed in woody species, but the technique has evolved over the past century to now encompass a large number of herbaceous species. The use of plant grafting in science is accelerating in part due to the innovative techniques developed for the model plant Arabidopsis thaliana. Here, we review these developments and discuss the advantages and limitations associated with grafting various Arabidopsis tissues at diverse developmental stages.

Development of a Direct in vitro Plant Regeneration Protocol From Cannabis sativa L. Seedling Explants: Developmental Morphology of Shoot Regeneration and Ploidy Level of Regenerated Plants.

In vitro shoot regeneration can efficiently contribute to the improvement of recalcitrant Cannabis sativa L. We aimed at developing a highly efficient protocol for in vitro direct regeneration of C. sativa plants from different explants (cotyledon, hypocotyl, and true leaf) from seedlings of monoecious C. sativa short-day varieties Ferimon, Felina32, Fedora17, and USO31, together with dioecious neutral-day variety Finola. Ten regeneration media, including already published protocols, and self-designed combinations of plant growth regulators were tested. The developmental morphology since germination of seeds to the development of rooted plantlets was followed. Additionally, the ploidy level of explants and in vitro regenerants was analyzed. We concluded that hypocotyl is the best explant for in vitro direct regeneration of C. sativa plants with 49.45% of responding explants, while cotyledon and true leaf had a poor response with, respectively, 4.70 and 0.42% of explants developing plantlets. In terms of shoot regeneration, we found significant differences among the culture media evaluated and the varieties studied. Overall, the best regeneration media were ZEARIB 2.0 (mg/L) and ZEARIB 1.0 (mg/L) + NAA 0.02 (mg/L) with 66.67% of responding hypocotyls. Amazingly, hypocotyls cultured in medium without plant growth regulators showed an excellent response (61.54% of responding hypocotyls) and spontaneous rooting of regenerants (17.94%). In vitro regenerated plants were acclimatized just 6 weeks after culture initiation. The developmental morphology study suggests that regenerated shoots originate from pericycle cells adjacent to xylem poles. Polysomaty was detected in hypocotyls and cotyledons of all varieties studied, and diploid (>80%) and mixoploid (with diploid and tetraploid cells) plants were regenerated. Our protocol allows a high shoot organogenesis efficiency in different C. sativa varieties. The fact that a significant percentage of plants are mixoploid may provide an alternative way to develop polyploids in C. sativa. Our results show that direct in vitro regeneration may make a significant contribution to the development of improved C. sativa materials for medical applications.

Target of Rapamycin (TOR) Negatively Regulates Ethylene Signals in Arabidopsis.

Target of rapamycin (TOR) acts as a master regulator in coordination of cell growth with energy and nutrient availability. Despite the increased appreciation of the essential role of the TOR complex in interaction with phytohormone signaling, little is known about its function on ethylene signaling. Here, through expression analysis, genetic and biochemical approaches, we reveal that TOR functions in the regulation of ethylene signals. Transcriptional analysis indicates that TOR inhibition by AZD8055 upregulated senescence- and ethylene-related genes expression. Furthermore, ethylene insensitive mutants like etr1-1, ein2-5 and ein3 eil1, showed more hyposensitivity to AZD8055 than that of WT in hypocotyl growth inhibition. Similarly, blocking ethylene signals by ethylene action inhibitor Ag+ or biosynthesis inhibitor aminoethoxyvinylglycine (AVG) largely rescued hypocotyl growth even in presence of AZD8055. In addition, we also demonstrated that Type 2A phosphatase-associated protein of 46 kDa (TAP46), a downstream component of TOR signaling, physically interacts with 1-aminocy-clopropane-1-carboxylate (ACC) synthase ACS2 and ACS6. Arabidopsis overexpressing ACS2 or ACS6 showed more hypersensitivity to AZD8055 than WT in hypocotyl growth inhibition. Moreover, ACS2/ACS6 protein was accumulated under TOR suppression, implying TOR modulates ACC synthase protein levels. Taken together, our results indicate that TOR participates in negatively modulating ethylene signals and the molecular mechanism is likely involved in the regulation of ethylene biosynthesis by affecting ACSs in transcription and protein levels.

PIN-LIKES Coordinate Brassinosteroid Signaling with Nuclear Auxin Input in Arabidopsis thaliana.

Auxin and brassinosteroids (BR) are crucial growth regulators and display overlapping functions during plant development. Here, we reveal an alternative phytohormone crosstalk mechanism, revealing that BR signaling controls PIN-LIKES (PILS)-dependent nuclear abundance of auxin. We performed a forward genetic screen for imperial pils (imp) mutants that enhance the overexpression phenotypes of PILS5 putative intracellular auxin transport facilitator. Here, we report that the imp1 mutant is defective in the BR-receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1). Our set of data reveals that BR signaling transcriptionally and post-translationally represses the accumulation of PILS proteins at the endoplasmic reticulum, thereby increasing nuclear abundance and signaling of auxin. We demonstrate that this alternative phytohormonal crosstalk mechanism integrates BR signaling into auxin-dependent organ growth rates and likely has widespread importance for plant development.

Submergence stress-induced hypocotyl elongation through ethylene signaling-mediated regulation of cortical microtubules in Arabidopsis.

Plant growth is significantly altered in response to submergence stress. However, the molecular mechanisms used by seedlings in response to this stress, especially for hypocotyl growth, are largely unknown in terrestrial plants such as Arabidopsis thaliana. The microtubule cytoskeleton participates in plant cell growth, but it remains unclear whether submergence-mediated plant growth involves the microtubule cytoskeleton. We demonstrated that in Arabidopsis submergence induced underwater hypocotyl elongation through the activation of ethylene signaling, which modulated cortical microtubule reorganization. Submergence enhanced ethylene signaling, which then activated and stabilized its downstream transcription factor, phytochrome-interacting factor 3 (PIF3), to promote hypocotyl elongation. In particular, the regulation of microtubule organization was important for this physiological process. Microtubule-destabilizing protein 60 (MDP60), which was previously identified as a downstream effector of PIF3, played a positive role in submergence-induced hypocotyl elongation. Submergence induced MDP60 expression through ethylene signaling. The effects of submergence on hypocotyl elongation and cortical microtubule reorganization were suppressed in mdp60 mutants. These data suggest a potential mechanism by which submergence activates ethylene signaling to promote underwater hypocotyl elongation via alteration of the microtubule cytoskeleton in Arabidopsis.