A Novel Senescence-Specific Gene (ZmSAG39) Negatively Regulates Darkness and Drought Responses in Maize

The papain-like cysteine proteases (PLCPs) is a subfamily of cysteine proteases that plays an important role in leaf senescence, and some of its members are involved in the regulation of plant growth and development under stress. In this study, we cloned a new gene, ZmSAG39, from maize. Expression profile analysis showed that ZmSAG39 was induced by darkness and drought treatments. In addition, the ZmSAG39 overexpression in maize accelerated the senescence of maize leaves under darkness and drought treatments. However, the knockout of ZmSAG39 in maize enhanced the resistance of maize to darkness and drought stresses and reduced the degree of senescence of maize leaves. Under drought stress, compared with WT plants, the knockout lines had a higher seed germination rate, seedling survival rate and chlorophyll content, and lower reactive oxygen species (ROS) level and malondialdehyde (MDA) content. In addition, quantitative real-time PCR (qRT-PCR) analysis showed that ZmSAG39 negatively regulated some stress-related genes but positively regulated senescence-related genes under darkness and drought stress conditions. To summarize, these results indicate that ZmSAG39 is a senescence-related gene and plays a negative role in response to darkness and drought stresses. This study laid a theoretical foundation for the innovation of maize germplasm resources with high quality, high yield and strong stress resistance.     

Genome-Wide Identification of Maize Protein Arginine Methyltransferase Genes and Functional Analysis of ZmPRMT1 Reveal Essential Roles in Arabidopsis Flowering Regulation and Abiotic Stress Tolerance

Histone methylation, as one of the important epigenetic regulatory mechanisms, plays a significant role in growth and developmental processes and stress responses of plants, via altering the methylation status or ratio of arginine and lysine residues of histone tails, which can affect the regulation of gene expression. Protein arginine methyltransferases (PRMTs) have been revealed to be responsible for histone methylation of specific arginine residues in plants, which is important for maintaining pleiotropic development and adaptation to abiotic stresses in plants. Here, for the first time, a total of eight PRMT genes in maize have been identified and characterized in this study, named as ZmPRMT1-8. According to comparative analyses of phylogenetic relationship and structural characteristics among PRMT gene family members from several representative species, all maize 8 PRMT proteins were categorized into three distinct subfamilies. Further, schematic structure and chromosome location analyses displayed evolutionarily conserved structure features and an unevenly distribution on maize chromosomes of ZmPRMT genes, respectively. The expression patterns of ZmPRMT genes in different tissues and under various abiotic stresses (heat, drought, and salt) were determined. The expression patterns of ZmPRMT genes indicated that they play a role in regulating growth and development and responses to abiotic stress. Eventually, to verify the biological roles of ZmPRMT genes, the transgenic Arabidopsis plants overexpressing ZmPRMT1 gene was constructed as a typical representative. The results demonstrated that overexpression of ZmPRMT1 can promote earlier flowering time and confer enhanced heat tolerance in transgenic Arabidopsis. Taken together, our results are the first to report the roles of ZmPRMT1 gene in regulating flowering time and resisting heat stress response in plants and will provide a vital theoretical basis for further unraveling the functional roles and epigenetic regulatory mechanism of ZmPRMT genes in maize growth, development and responses to abiotic stresses.     

TaKLU Plays as a Time Regulator of Leaf Growth via Auxin Signaling

The growth of leaves is subject to strict time regulation. Several genes influencing leaf growth have been identified, but little is known about how genes regulate the orderly initiation and growth of leaves. Here, we demonstrate that TaKLU/TaCYP78A5 contributes to a time regulation mechanism in leaves from initiation to expansion. TaKLU encodes the cytochrome P450 CYP78A5, and its homolog AtKLU has been described whose deletion is detrimental to organ growth. Our results show that TaKLU overexpression increases leaf size and biomass by altering the time of leaf initiation and expansion. TaKLU-overexpressing plants have larger leaves with more cells. Further dynamic observations indicate that enlarged wheat leaves have experienced a longer expansion time. Different from AtKLU inactivation increases leaf number and initiation rates, TaKLU overexpression only smooths the fluctuations of leaf initiation rates by adjusting the initiation time of local leaves, without affecting the overall leaf number and initiation rates. In addition, complementary analyses suggest TaKLU is functionally conserved with AtKLU in controlling the leaf initiation and size and may involve auxin accumulation. Our results provide a new insight into the time regulation mechanisms of leaf growth in wheat.     

Maize Transcription Factor ZmARF4 Confers Phosphorus Tolerance by Promoting Root Morphological Development

Plant growth and development are closely related to phosphate (Pi) and auxin. However, data regarding auxin response factors (ARFs) and their response to phosphate in maize are limited. Here, we isolated ZmARF4 in maize and dissected its biological function response to Pi stress. Overexpression of ZmARF4 in Arabidopsis confers tolerance of Pi deficiency with better root morphology than wild-type. Overexpressed ZmARF4 can partially restore the absence of lateral roots in mutant arf7 arf19. The ZmARF4 overexpression promoted Pi remobilization and up-regulated AtRNS1, under Pi limitation while it down-regulated the expression of the anthocyanin biosynthesis genes AtDFR and AtANS. A continuous detection revealed higher activity of promoter in the Pi-tolerant maize P178 line than in the sensitive 9782 line under low-Pi conditions. Meanwhile, GUS activity was specifically detected in new leaves and the stele of roots in transgenic offspring. ZmARF4 was localized to the nucleus and cytoplasm of the mesophyll protoplast and interacted with ZmILL4 and ZmChc5, which mediate lateral root initiation and defense response, respectively. ZmARF4 overexpression also conferred salinity and osmotic stress tolerance in Arabidopsis. Overall, our findings suggest that ZmARF4, a pleiotropic gene, modulates multiple stress signaling pathways, and thus, could be a candidate gene for engineering plants with multiple stress adaptation.     

The Role of Pyoluteorin from Pseudomonas protegens Pf-5 in Suppressing the Growth and Pathogenicity of Pantoea ananatis on Maize

The rhizospheric bacterium Pseudomonas protegens Pf-5 can colonize the seed and root surfaces of plants, and can protect them from pathogen infection. Secondary metabolites, including lipopeptides and polyketides produced by Pf-5, are involved in its biocontrol activity. We isolated a crude extract from Pf-5. It exhibited significant surface activity and strong antibacterial activity against Pantoea ananatis DZ-12, which causes maize brown rot on leaves. HPLC analysis combined with activity tests showed that the polyketide pyoluteorin in the crude extract participated in the suppression of DZ-12 growth, and that the lipopeptide orfamide A was the major biosurfactant in the crude extract. Further studies indicated that the pyoluteorin in the crude extract significantly suppressed the biofilm formation of DZ-12, and it induced the accumulation of reactive oxygen species in DZ-12 cells. Scanning electron microscopy and transmission electron microscopy observation revealed that the crude extract severely damaged the pathogen cells and caused cytoplasmic extravasations and hollowing of the cells. The pathogenicity of DZ-12 on maize leaves was significantly reduced by the crude extract from Pf-5 in a dose-dependent manner. The polyketide pyoluteorin had strong antibacterial activity against DZ-12, and it has the potential for development as an antimicrobial agent.     

Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance

Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.     

Characterization of Stem Nodes Associated with Carbon Partitioning in Maize in Response to Nitrogen Availability

Stem node has been found to be a hub for controlling mineral nutrient distribution in gramineous plants. However, the characteristics of stem nodes associated with whole-plant carbon partitioning in maize (Zea mays L.) and their responses to nitrogen (N) availability remains elusive. Maize plants were grown in greenhouse under low to high N supply. Plant growth, sugar accumulation, and sugar transporters in nodes and leaves, as well as the anatomical structure of nodes, were investigated at vegetative phase. When compared to N-sufficient plants, low-N availability stunted growth and resulted in 49–64% less sugars in leaves, which was attributed to low photosynthesis or the accelerated carbon export, as evidenced by more ¹³C detected further below leaf tips. Invariably higher sugar concentrations were found in the stem nodes, rather than in the leaves across N treatments, indicating a crucial role of nodes in facilitating whole-plant carbon partitioning. More and smaller vascular bundles and phloem were observed in stem nodes of N-deficient plants, while higher sugar levels were found in the bottom nodes than in the upper ones. Low-N availability upregulated the gene expressions of sugar transporters, which putatively function in nodes such as ZmSWEETs and ZmSUTs at the bottom stem, but suppressed them in the upper ones, showing a developmental impact on node function. Further, greater activity of sugar transporters in the bottom nodes was associated with less sugars in leaves. Overall, these results highlighted that stem nodes may play an important role in facilitating long-distance sugar transport in maize.     

Roles of a Cysteine Desulfhydrase LCD1 in Regulating Leaf Senescence in Tomato

Hydrogen sulfide (H₂S), a novel gasotransmitter in both mammals and plants, plays important roles in plant development and stress responses. Leaf senescence represents the final stage of leaf development. The role of H₂S-producing enzyme L-cysteine desulfhydrase in regulating tomato leaf senescence is still unknown. In the present study, the effect of an L-cysteine desulfhydrase LCD1 on leaf senescence in tomato was explored by physiological analysis. LCD1 mutation caused earlier leaf senescence, whereas LCD1 overexpression significantly delayed leaf senescence compared with the wild type in 10-week tomato seedlings. Moreover, LCD1 overexpression was found to delay dark-induced senescence in detached tomato leaves, and the lcd1 mutant showed accelerated senescence. An increasing trend of H₂S production was observed in leaves during storage in darkness, while LCD1 deletion reduced H₂S production and LCD1 overexpression produced more H₂S compared with the wild-type control. Further investigations showed that LCD1 overexpression delayed dark-triggered chlorophyll degradation and reactive oxygen species (ROS) accumulation in detached tomato leaves, and the increase in the expression of chlorophyll degradation genes NYC1, PAO, PPH, SGR1, and senescence-associated genes (SAGs) during senescence was attenuated by LCD1 overexpression, whereas lcd1 mutants showed enhanced senescence-related parameters. Moreover, a correlation analysis indicated that chlorophyll content was negatively correlated with H₂O₂ and malondialdehyde (MDA) content, and also negatively correlated with the expression of chlorophyll degradation-related genes and SAGs. Therefore, these findings increase our understanding of the physiological functions of the H₂S-generating enzyme LCD1 in regulating leaf senescence in tomato.     

Silencing GmBIR1 in Soybean Results in Activated Defense Responses

Receptor-like kinases (RLKs) are a large group of pattern recognition receptors (PRRs) and play a critical role in recognizing pathogens, transducing defense signals, and mediating the activation of immune defense responses. Although extensively studied in the model plant Arabidopsis, studies of RLKs in crops, including soybean, are limited. When a BAK1-interacting receptor-like kinase (BIR1) homolog (referred to as GmBIR1 hereafter) was silenced by the BPMV (Bean pod mottle virus)-induced gene silencing (BPMV-VIGS), it resulted in phenotypes that were reminiscent of constitutively activated defense responses, including a significantly stunted stature with observable cell death on the leaves of the silenced plants. In addition, both SA and H₂O₂ were over-accumulated in the leaves of the GmBIR1-silenced plants. Consistent with this autoimmune phenotype, GmBIR1-silenced plants exhibited significantly enhanced resistance to both Pseudomonas syringae pv. glycinea (Psg) and Soybean mosaic virus (SMV), two different types of pathogens, compared to the vector control plants. Together, our results indicated that GmBIR1 is a negative regulator of immunity in soybean and the function of BIR1 homologs is conserved in different plant species.     

The Anthocyanin Accumulation Related ZmBZ1, Facilitates Seedling Salinity Stress Tolerance via ROS Scavenging

Anthocyanins are a class of antioxidants that scavenge free radicals in cells and play an important role in promoting human health and preventing many diseases. Here, we characterized a maize Bronze gene (BZ1) from the purple colored W22 introgression line, which encodes an anthocyanin 3-O-glucosyltransferase, a key enzyme in the anthocyanin synthesis pathway. Mutation of ZmBZ1 showed bronze-colored seeds and reduced anthocyanins in seeds aleurone layer, seedlings coleoptile, and stem of mature plants by comparison with purple colored W22 (WT). Furthermore, we proved that maize BZ1 is an aleurone layer-specific expressed protein and sub-located in cell nucleus. Real-time tracing of the anthocyanins in developing seeds demonstrated that the pigment was visible from 16 DAP (day after pollination) in field condition, and first deposited in the crown part then spread all over the seed. Additionally, it was transferred along with the embryo cell activity during seed germination, from aleurone layer to cotyledon and coleoptile, as confirmed by microscopy and real-time qRT-PCR. Finally, we demonstrated that the ZmBZ1 contributes to stress tolerance, especially salinity. Further study proved that ZmBZ1 participates in reactive oxygen scavenging (ROS) by accumulating anthocyanins, thereby enhancing the tolerance to abiotic stress.     

SDG102, a H3K36-Methyltransferase-Encoding Gene,Plays Pleiotropic Roles in Growth and Development of Maize (Zea mays L.)

Although histone lysine methylation has been studied in thale cress (Arabidopsis thaliana (L.) Heynh.) and rice (Oryza sativa L.) in recent years, its function in maize (Zea mays L.) remains poorly characterized. To better understand the function of histone lysine methylation in maize, SDG102, a H3 lysine 36 (H3K36) methylase, was chosen for functional characterization using overexpressed and knockout transgenic plants. SDG102-deficiency in maize caused multiple phenotypes including yellow leaves in seedlings, late-flowering, and increased adult plant height, while the overexpression of SDG102 led to reduced adult plant height. The key flowering genes, ZCN8/ZCN7 and MADS4/MADA67, were downregulated in SDG102-deficient plants. Chromatin immunoprecipitation (ChIP) experiments showed that H3 lysine 36 trimethylation (H3K36me3) levels were reduced at these loci. Perturbation of SDG102 expression caused the misexpression of multiple genes. Interestingly, the overexpression or knockout of SDG102 also led to genome-wide decreases and increases in the H3K36me3 levels, respectively. Together, our results suggest that SDG102 is a methyltransferase that catalyzes the trimethylation of H3K36 of many genes across the maize genome, which are involved in multiple biological processes including those controlling flowering time.     

ZmGLP1, a Germin-like Protein from Maize,Plays an Important Role in the Regulation of Pathogen Resistance

A gene encoding a protein similar to germin-like proteins (GLPs) was obtained from maize (Zea mays) and designated as ZmGLP1. Based on the ZmGLP1 conserved domain and phylogenetic status, ZmGLP1 was grouped into GLP subfamily b and has high similarity to OsGLP8-14 from Oryza sativa. ZmGLP1 is expressed in different maize tissues during different growth stages and is mainly expressed in the stems and leaves. The induced expression patterns confirmed that ZmGLP1 is differentially expressed under abiotic and hormone stress; it had an early response to jasmonic acid (JA) and ethephon (ET) but a late response to salicylic acid (SA) and was significantly upregulated under Bipolaris maydis infection. The overexpression of ZmGLP1 in Arabidopsis improved the resistance to biotrophic Pseudomonas syringae pv. tomato DC3000 (PstDC3000) and necrotrophic Sclerotinia sclerotiorum by inducing the expression of JA signaling-related genes. Moreover, the hydrogen peroxide (H₂O₂) content increased due to the overexpression of ZmGLP1 in Arabidopsis after pathogen infection. Compared to the wild-type control, the H₂O₂ content of ZmGLP1-overexpressing Arabidopsis infected by PstDC3000 increased significantly but was lower in transgenic plants infected with S. sclerotiorum. Furthermore, high-performance liquid chromatography–tandem mass (HPLC-MS/MS) spectrometry showed that the JA contents of ZmGLP1-overexpressing Arabidopsis markedly increased after pathogen infection. However, the improved resistance of ZmGLP1-overexpressing Arabidopsis pretreated with the JA biosynthetic inhibitor, sodium diethyldithiocarbamate trihydrate (DIECA), was suppressed. Based on these findings, we speculate that ZmGLP1 plays an important role in the regulation of Arabidopsis resistance to biotrophic PstDC3000 and necrotrophic S. sclerotiorum; the regulatory effects are achieved by inducing plant oxidative burst activity and activation of the JA signaling pathway.