Cadmium negatively affects the growth and physiological status and the alleviation effects by exogenous selenium in silage maize (Zea mays L.).

Increasing soil cadmium (Cd) contamination is a serious threat to human food health and safety. In order to reduce Cd uptake and Cd toxicity in silage maize, hydroponic tests were conducted to investigate the effect of exogenous Cd on the toxicity of silage maize in this study. In the study, a combination of Cd (5, 20, 50, 80, and 10 µM) treatments was applied in a hydroponic system. With increasing Cd concentration, Cd significantly inhibited the total root length (RL), root surface area (SA), root volume (RV), root tip number (RT), and branching number (RF) of maize seedlings, which were reduced by 28.1 to 71.3%, 20.2 to 64.9%, 11.2 to 56.5%, 43.7 to 63.4%, and 38.2 to 72.6%, respectively. The excessive Cd accumulation inhibited biomass accumulation and reduced silage maize growth, photosynthesis, and chlorophyll content and activated the antioxidant systems, including increasing lipid peroxidation and stimulating catalase (CAT) and peroxidase (POD), but reduced the activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in the root. Besides, selenium (Se) significantly decreased the Cd concentration of the shoot and root by 27.1% and 35.1% under Cd50, respectively. Our results reveal that exogenously applied Cd reduced silage maize growth and impaired photosynthesis. Whereas silage maize can tolerate Cd by increasing the concentration of ascorbate and glutathione and activating the antioxidant defense system, the application of exogenous selenium significantly reduced the content of Cd in silage maize.

Physiological and iTRAQ-based quantitative proteomics analyses reveal the similarities and differences in stress responses between short-term boron deficiency and toxicity in wheat roots.

BACKGROUND: Boron (B) is a trace element that is essential for normal wheat development, such as root growth. In wheat, roots are important organs that absorb nutrients and water. However, at present, there is insufficient research on the molecular mechanism underlying how short-term B stress affects wheat root growth. METHODS AND RESULTS: Here, the optimal concentration of B for wheat root growth was determined, and the proteomic profiles of roots under short-term B deficiency and toxicity were analyzed and compared by the isobaric tag for relative and absolute quantitation (iTRAQ) technique. A total of 270 differentially abundant proteins (DAPs) that accumulated in response to B deficiency and 263 DAPs that accumulated in response to B toxicity were identified. Global expression analysis revealed that ethylene, auxin, abscisic acid (ABA), and Ca2+ signals were involved in the responses to these two stresses. Under B deficiency, DAPs related to auxin synthesis or signaling and DAPs involved in calcium signaling increased in abundance. In striking contrast, auxin and calcium signals were repressed under B toxicity. Twenty-one DAPs were detected under both conditions, including RAN1 that played a core role in the auxin and calcium signals. Overexpression of RAN1 was shown to confer plant resistance to B toxicity by activating auxin response genes, including TIR and those identified by iTRAQ in this research. Moreover, growth of the primary roots of tir mutant was significantly inhibited under B toxicity. CONCLUSION: Taken together, these results indicate that some connections were present between RAN1 and the auxin signaling pathway under B toxicity. Therefore, this research provides data for improving the understanding of the molecular mechanism underlying the response to B stress.

Control of lateral root initiation by DA3 in Arabidopsis.

Lateral root (LR) initiation is controlled by the pericycle and the neighboring endodermis in Arabidopsis. Here, we demonstrate that UBIQUITIN-SPECIFIC PROTEASE14/DA3 regulates LR initiation by modulating auxin signaling in the pericycle and endodermis. DA3 negatively affects the mRNA and protein levels of AUXIN RESPONSE FACTOR7 (ARF7) and ARF19 in the pericycle and endodermis but positively regulates the protein stability of SHORT HYPOCOTYL 2 (SHY2/IAA3), an auxin signaling repressor, in the endodermis. We show that DA3 interacts with ARF7 and ARF19, inhibiting their binding to the locus of LATERAL ORGAN BOUNDARY DOMAIN16 (LBD16) to repress its expression in the pericycle. SHY2 also interacts with ARF7 and ARF19 in the endodermis and enhances the DA3 repressive effect on ARF7 and ARF19, thus modulating LBD16 expression in the pericycle. Overall, our findings show that DA3 acts with SHY2, ARF7, and ARF19 to coordinate auxin signaling in the pericycle and endodermis to control LR initiation in Arabidopsis.

Exogenous ascorbic acid application alleviates cadmium toxicity in seedlings of two wheat (Triticum aestivum L.) varieties by reducing cadmium uptake and enhancing antioxidative capacity.

The aggravation of soil cadmium (Cd) pollution is a serious threat to human food health and safety. To reduce Cd uptake and alleviate Cd toxicity in staple food of wheat, a completely random experiment was performed to investigate the effect of exogenous ascorbic acid (AsA) on Cd toxicity in two wheat varieties (L979 and H27). In this study, the treatments with combinations of Cd (0, 5, and 10 µmol L-1) and AsA (0, 50, and 200 µmol L-1) were applied in a hydroponic system. Toxicity induced by Cd inhibited biomass accumulation; decreased wheat growth, photosynthesis, and chlorophyll content; increased lipid peroxidation; and reduced activity of superoxide dismutase (SOD), but stimulated catalase (CAT) and peroxidase (POD). The addition of AsA significantly improved the growth status by increasing the wheat biomass, chlorophyll content, photosynthetic rate, protein concentrations, and antioxidant enzyme activity. Besides, AsA significantly decreased Cd concentration of shoot and root by 14.1-53.9% and 20.8-59.5% in L979 and 23.7-58.8% and 22.1-58.1% in H27 under Cd5, and 23.7-53.6% and 16.6-57.1% in L979 and 21.5-51.6% and 15.3-54.0% in H27 under Cd10, respectively. Malondialdehyde (MDA) accumulation was decreased remarkably with the addition of AsA by 31.2-32.9% in L979 and 27.1-45.2% in H27 under Cd10, respectively. Overall, exogenous application of AsA alleviated the Cd toxicity in wheat plants by improving the wheat growth, soluble protein content, photosynthesis, and antioxidant defense systems, and decreasing MDA accumulation.

Proteomic Analysis of Generative and Vegetative Nuclei Reveals Molecular Characteristics of Pollen Cell Differentiation in Lily.

In plants, the cell fates of a vegetative cell (VC) and generative cell (GC) are determined after the asymmetric division of the haploid microspore. The VC exits the cell cycle and grows a pollen tube, while the GC undergoes further mitosis to produce two sperm cells for double fertilization. However, our understanding of the mechanisms underlying their fate differentiation remains limited. One major advantage of the nuclear proteome analysis is that it is the only method currently able to uncover the systemic differences between VC and GC due to GC being engulfed within the cytoplasm of VC, limiting the use of transcriptome. Here, we obtained pure preparations of the vegetative cell nuclei (VNs) and generative cell nuclei (GNs) from germinating lily pollens. Utilizing these high-purity VNs and GNs, we compared the differential nucleoproteins between them using state-of-the-art quantitative proteomic techniques. We identified 720 different amount proteins (DAPs) and grouped the results in 11 fate differentiation categories. Among them, we identified 29 transcription factors (TFs) and 10 cell fate determinants. Significant differences were found in the molecular activities of vegetative and reproductive nuclei. The TFs in VN mainly participate in pollen tube development. In comparison, the TFs in GN are mainly involved in cell differentiation and male gametogenesis. The identified novel TFs may play an important role in cell fate differentiation. Our data also indicate differences in nuclear pore complexes and epigenetic modifications: more nucleoporins synthesized in VN; more histone variants and chaperones; and structural maintenance of chromosome (SMC) proteins, chromatin remodelers, and DNA methylation-related proteins expressed in GN. The VC has active macromolecular metabolism and mRNA processing, while GC has active nucleic acid metabolism and translation. Moreover, the members of unfolded protein response (UPR) and programmed cell death accumulate in VN, and DNA damage repair is active in GN. Differences in the stress response of DAPs in VN vs. GN were also found. This study provides a further understanding of pollen cell differentiation mechanisms and also a sound basis for future studies of the molecular mechanisms behind cell fate differentiation.

Identification of a Novel NtLRR-RLK and Biological Pathways That Contribute to Tolerance of TMV in Nicotiana tabacum.

Tobacco mosaic virus (TMV) infection can causes serious damage to tobacco crops. To explore the approach of preventing TMV infection of plants, two tobacco cultivars with different resistances to TMV were used to analyze transcription profiling before and after TMV infection. The involvement of biological pathways differed between the tolerant variety (Yuyan8) and the susceptible variety (NC89). In particular, the plant-virus interaction pathway was rapidly activated in Yuyan8, and specific resistance genes were enriched. Liquid chromatography tandem mass spectrometry analysis detected large quantities of antiviral substances in the tolerant Yuyan8. A novel Nicotiana tabacum leucine-rich repeat receptor kinase (NtLRR-RLK) gene was identified as being methylated and this was verified using bisulfite sequencing. Transient expression of TMV-green fluorescent protein in pRNAi-NtLRR-RLK transgenic plants confirmed that NtLRR-RLK was important for susceptibility to TMV. The specific protein interaction map generated from our study revealed that levels of BIP1, E3 ubiquitin ligase, and LRR-RLK were significantly elevated, and all were represented at node positions in the protein interaction map. The same expression tendency of these proteins was also found in pRNAi-NtLRR-RLK transgenic plants at 24 h after TMV inoculation. These data suggested that specific genes in the infection process can activate the immune signal cascade through different resistance genes, and the integration of signal pathways could produce resistance to the virus. These results contribute to the overall understanding of the molecular basis of plant resistance to TMV and in the long term could identify new strategies for prevention and control virus infection.

ZmCCA1a on Chromosome 10 of Maize Delays Flowering of Arabidopsis thaliana.

Maize (Zea mays) is a major cereal crop that originated at low latitudes, and thus photoperiod sensitivity is an important barrier to the use of tropical/subtropical germplasm in temperate regions. However, studies of the mechanisms underlying circadian regulation in maize are at an early stage. In this study we cloned ZmCCA1a on chromosome 10 of maize by map-based cloning. The gene is homologous to the Myb transcription factor genes AtCCA1/AtLHY in Arabidopsis thaliana; the deduced Myb domain of ZmCCA1a showed high similarity with that of AtCCA1/AtLHY and ZmCCA1b. Transiently or constitutively expressed ZmCCA1a-YFPs were localized to nuclei of Arabidopsis mesophyll protoplasts, agroinfiltrated tobacco leaves, and leaf and root cells of transgenic seedlings of Arabidopsis thaliana. Unlike AtCCA1/AtLHY, ZmCCA1a did not form homodimers nor interact with ZmCCA1b. Transcripts of ZmCCA1a showed circadian rhythm with peak expression around sunrise in maize inbred lines CML288 (photoperiod sensitive) and Huangzao 4 (HZ4; photoperiod insensitive). Under short days, transcription of ZmCCA1a in CML288 and HZ4 was repressed compared with that under long days, whereas the effect of photoperiod on ZmCCA1a expression was moderate in HZ4. In ZmCCA1a-overexpressing A. thaliana (ZmCCA1a-ox) lines, the circadian rhythm was disrupted under constant light and flowering was delayed under long days, but the hypocotyl length was not affected. In addition, expression of endogenous AtCCA1/AtLHY and the downstream genes AtGI, AtCO, and AtFt was repressed in ZmCCA1a-ox seedlings. The present results suggest that the function of ZmCCA1a is similar, at least in part, to that of AtCCA1/AtLHY and ZmCCA1b, implying that ZmCCA1a is likely to be an important component of the circadian clock pathway in maize.

Physiological and transcriptomic analyses reveal the molecular networks of responses induced by exogenous trehalose in plant.

It is well known that exogenous trehalose can improve resistances of plants to some abiotic and biotic stresses. Nonetheless, information respecting the molecular responses of tobacco leaves to Tre treatment is limited. Here we show that exogenous Tre can rapidly reduce stomatal aperture, up-regulate NADPH oxidase genes and increase O2•-andH2O2 on tobacco leaves at 2 h after treatment. We further demonstrated that imidazole and DPI, inhibitors of NADPH oxidase, can promote recovery of stomatal aperture of tobacco leaves upon trehalose treatment. Exogenous trehalose increased tobacco leaf resistance to tobacco mosaic disease significantly in a concentration-dependent way. To elucidate the molecular mechanisms in response to exogenous trehalose, the transcriptomic responses of tobacco leaves with 10 (low concentration) or 50 (high concentration) mM of trehalose treatment at 2 or 24h were investigated through RNA-seq approach. In total, 1288 differentially expressed genes (DEGs) were found with different conditions of trehalose treatments relative to control. Among them, 1075 (83.5%) were triggered by low concentration of trehalose (10mM), indicating that low concentration of Tre is a better elicitor. Functional annotations with KEGG pathway analysis revealed that the DEGs are involved in metabolic pathway, biosynthesis of secondary metabolites, plant hormone signal transduction, plant-pathogen interaction, protein processing in ER, flavonoid synthesis and circadian rhythm and so on. The protein-protein interaction networks generated from the core DEGs regulated by all conditions strikingly revealed that eight proteins, including ClpB1, HSP70, DnaJB1-like protein, universal stress protein (USP) A-like protein, two FTSH6 proteins, GolS1-like protein and chloroplastics HSP, play a core role in responses to exogenous trehalose in tobacco leaves. Our data suggest that trehalose triggers a signal transduction pathway which involves calcium and ROS-mediated signalings. These core components could lead to partial resistance or tolerance to abiotic and biotic stresses. Moreover, 19 DEGs were chosen for analysis of quantitative real-time polymerase chain reaction (qRT-PCR). The qRT-PCR for the 19 candidate genes coincided with the DEGs identified via the RNA-seq analysis, sustaining the reliability of our RNA-seq data.

NtWRKY-R1, a Novel Transcription Factor, Integrates IAA and JA Signal Pathway under Topping Damage Stress in Nicotiana tabacum.

Topping damage can induce the nicotine synthesis in tobacco roots, which involves the activation of JA and auxin signal transduction. It remains unclear how these hormone signals are integrated to regulate nicotine synthesis. Here we isolated a transcription factor NtWRKY-R1 from the group IIe of WRKY family and it had strong negative correlation with the expression of putrescine N-methyltransferase, the key enzyme of nicotine synthesis pathway. NtWRKY-R1 was specifically and highly expressed in tobacco roots, and it contains two transcriptional activity domains in the N- and C-terminal. The promoter region of NtWRKY-R1 contains two cis-elements which are responding to JA and auxin signals, respectively. Deletion of NtWRKY-R1 promoter showed that JA and auxin signals were subdued by NtWRKY-R1, and the expression of NtWRKY-R1 was more sensitive to auxin than JA. Furthermore, Yeast two-hybrid experiment demonstrated that NtWRKY-R1 can interact with the actin-binding protein. Our data showed that the intensity of JA and auxin signals can be translated into the expression of NtWRKY-R1, which regulates the balance of actin polymerization and depolymerization through binding actin-binding protein, and then regulates the expression of genes related to nicotine synthesis. The results will help us better understand the function of the WRKY-IIe family in the signaling crosstalk of JA and auxin under damage stress.

UBIQUITIN-SPECIFIC PROTEASE14 Interacts with ULTRAVIOLET-B INSENSITIVE4 to Regulate Endoreduplication and Cell and Organ Growth in Arabidopsis.

Organ growth is determined by a coordinated combination of cell proliferation and cell growth and differentiation. Endoreduplication is often coupled with cell growth and differentiation, but the genetic and molecular mechanisms that link endoreduplication with cell and organ growth are largely unknown. Here, we describe UBIQUITIN-SPECIFIC PROTEASE14 (UBP14), encoded by the DA3 gene, which functions as a negative regulator of endoreduplication. The Arabidopsis thaliana da3-1 mutant shows large cotyledons, leaves, and flowers with higher ploidy levels. UBP14 acts along with UV-B-INSENSITIVE4 (UVI4), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase, to repress endoreduplication. Also, UBP14 functions antagonistically with CELL CYCLE SWITCH52 A1 (CCS52A1), an activator of APC/C, to regulate endoreduplication. UBP14 physically associates with UVI4 both in vitro and in vivo but does not directly interact with CCS52A1. Further results reveal that UBP14 influences the stability of cyclin A2;3 (CYCA2;3) and cyclin-dependent kinase B1;1 (CDKB1;1), two downstream components of the APC/C Thus, our findings show how endoreduplication is linked with cell and organ growth by revealing important genetic and molecular functions for the ubiquitin-specific protease UBP14 and for the key cell cycle regulators UVI4, CCS52A1, CYCA2;3, and CDKB1;1.

Control of root meristem size by DA1-RELATED PROTEIN2 in Arabidopsis.

The control of organ growth by coordinating cell proliferation and differentiation is a fundamental developmental process. In plants, postembryonic root growth is sustained by the root meristem. For maintenance of root meristem size, the rate of cell differentiation must equal the rate of cell division. Cytokinin and auxin interact to affect the cell proliferation and differentiation balance and thus control root meristem size. However, the genetic and molecular mechanisms that determine root meristem size still remain largely unknown. Here, we report that da1-related protein2 (dar2) mutants produce small root meristems due to decreased cell division and early cell differentiation in the root meristem of Arabidopsis (Arabidopsis thaliana). dar2 mutants also exhibit reduced stem cell niche activity in the root meristem. DAR2 encodes a Lin-11, Isl-1, and Mec-3 domain-containing protein and shows an expression peak in the border between the transition zone and the elongation zone. Genetic analyses show that DAR2 functions downstream of cytokinin and SHORT HYPOCOTYL2 to maintain normal auxin distribution by influencing auxin transport. Further results indicate that DAR2 acts through the PLETHORA pathway to influence root stem cell niche activity and therefore control root meristem size. Collectively, our findings identify the role of DAR2 in root meristem size control and provide a novel link between several key regulators influencing root meristem size.

[Control of organ shape and size by YUAN1 in Arabidopsis thaliana].

Control of organ shape and size by cell proliferationandcell expansion is a fundamental process in plant development. However, little is known about the molecular mechanisms that set the shape and size of determinate organs in plants. We have previously demonstrated that the Arabidopsis gene DA1 controls the final size of organs by restricting cell proliferation. Through an activation tagging screen for modifiers of da1-1, we have identified a semi-dominant mutant (yuan1-1D) with altered leaf shape and size. The yuan1-1D mutation results in reduced plant height, short and round leaves and short petioles due to defects in cell elongation. YUAN1 encodes a PHD zinc finger domain-containing protein. The GFP-YUAN1 fusion protein is localized to the nucleus. Overexpression of YUAN1 leads to round leaves and short petioles. Genetic analyses show that YUAN1 acts independently of DA1, ROTUNDIFOLIA 3 (ROT3) and ROTUNDIFOLIA4 (ROT4) to influence leaf shape and size. Collectively, our findings show that Arabidopsis YUAN1, a PHD zinc finger domain-containing protein, controls organ shape and size by restricting cell elongation, and give insight into how plants control their organ shape and size.