The R2R3-MYB Transcriptional Repressor TgMYB4 Negatively Regulates Anthocyanin Biosynthesis in Tulips (Tulipa gesneriana L.).

Anthocyanins play a paramount role in color variation and significantly contribute to the economic value of ornamental plants. The conserved activation complex MYB-bHLH-WD40 (MBW; MYB: v-myb avian myeloblastosis viral oncogene homolog; bHLH: basic helix-loop-helix protein; WD40:WD-repeat protein) involved in anthocyanin biosynthesis has been thoroughly researched, but there have been limited investigations into the function of repressor factors. In this study, we characterized TgMYB4, an R2R3-MYB transcriptional repressor which is highly expressed during petal coloration in red petal cultivars. TgMYB4-overexpressing tobaccos exhibited white or light pink petals with less anthocyanin accumulation compared to control plants. TgMYB4 was found to inhibit the transcription of ANTHOCYANIDIN SYNTHASE (TfANS1) and DIHYDRO-FLAVONOL-4-REDUCTASE (AtDFR), although it did not bind to their promoters. Moreover, the TgMYB4 protein was able to compete with the MYB activator to bind to the :bHLHprotein, thereby suppressing the function of the activator MBW complex. These findings demonstrate that TgMYB4 plays a suppressive role in the regulation of anthocyanin synthesis during flower pigmentation.

Functional conservation and divergence of SEPALLATA-like genes in the development of two-type florets in marigold.

Marigold (Tagetes erecta), as one member of Asteraceae family, bears a typical capitulum with two morphologically distinct florets. The SEPALLATA genes are involved in regulating the floral meristem determinacy, organ identity, fruit maturation, seed formation, and plant architecture. Here, five SEP-like genes were cloned and identified from marigold. Sequence alignment and phylogenetic analysis demonstrated that TeSEP3-1, TeSEP3-2, and TeSEP3-3 proteins were grouped into SEP3 clade, and TeSEP1 and TeSEP4 proteins were clustered into SEP1/2/4 clade. Quantitative real-time PCR analysis revealed that TeSEP1 and TeSEP3-3 were broadly expressed in floral organs, and that TeSEP3-2 and TeSEP4 were mainly expressed in pappus and corollas, while TeSEP3-1 was mainly expressed in two inner whorls. Ectopic expression of TeSEP1, TeSEP3-2, TeSEP3-3, and TeSEP4 in arabidopsis and tobacco resulted in early flowering. However, overexpression of TeSEP3-1 in arabidopsis and tobacco caused no visible phenotypic changes. Notably, overexpression of TeSEP4 in tobacco decreased the number of petals and stamens. Overexpression of TeSEP1 in tobacco led to longer sepals and simpler inflorescence architecture. The comprehensive pairwise interaction analysis suggested that TeSEP proteins had a broad interaction with class A, C, D, E proteins to form dimers. The yeast three-hybrid analysis suggested that in ternary complexes, class B proteins interacted with TeSEP3 by forming heterodimer TePI-TeAP3-2. The regulatory network analysis of MADS-box genes in marigold further indicated that TeSEP proteins played a "glue" role in regulating floral organ development, implying functional conservation and divergence of MADS box genes in regulating two-type floret developments. This study provides an insight into the formation mechanism of floral organs of two-type florets, thus broadening our knowledge of the genetic basis of flower evolution.

Integrating physiological and metabolites analysis to identify ethylene involvement in petal senescence in Tulipa gesneriana.

Flower senescence is classified into ethylene-dependent and ethylene-independent manners and determines the flower longevity which is valuable for ornamental plants. However, the manner of petal senescence in tulip is still less defined. In this study, we characterized the physiological indexes in the process of petal senescence, as well as metabolic and ethylene responses in tulip cultivar 'American Dream', and further identified the role of ethylene biosynthesis genes TgACS by transgenic and transient assays. Primary metabolites profiling revealed that sugars, amino acids and organic acids preferentially accumulated in senescent petals. Additionally, senescence-associated genes were identified and significantly up-regulated, coupled with increased ROS contents, rapid water loss and accelerated cell membrane breakdown. Moreover, ethylene production was stimulated as evidenced by increasing in ACS activity and ethylene biosynthesis-related genes expression. Exogenous treatment of cutting flowers with 1-MCP or ethephon resulted in delayed or enhanced petal senescence, respectively. Transient down-regulation of TgACS by VIGS assay in tulip petals delayed senescence, while over-expressed TgACS1 in tobacco promoted leaf senescence. Taken together, this study provides evidences to certify ethylene roles and TgACS functions during flower senescence in tulip.

Endogenous Cytokinin Overproduction Modulates ROS Homeostasis and Decreases Salt Stress Resistance in Arabidopsis Thaliana.

Cytokinins in plants are crucial for numerous biological processes, including seed germination, cell division and differentiation, floral initiation and adaptation to abiotic stresses. The salt stress can promote reactive oxygen species (ROS) production in plants which are highly toxic and ultimately results in oxidative stress. However, the correlation between endogenous cytokinin production and ROS homeostasis in responding to salt stress is poorly understood. In this study, we analyzed the correlation of overexpressing the cytokinin biosynthetic gene AtIPT8 (adenosine phosphate-isopentenyl transferase 8) and the response of salt stress in Arabidopsis. Overproduction of cytokinins, which was resulted by the inducible overexpression of AtIPT8, significantly inhibited the primary root growth and true leaf emergence, especially under the conditions of exogenous salt, glucose and mannitol treatments. Upon cytokinin overproduction, the salt stress resistance was declined, and resulted in less survival rates and chlorophyll content. Interestingly, ROS production was obviously increased with the salt treatment, accompanied by endogenously overproduced cytokinins. The activities of catalase (CAT) and superoxide dismutase (SOD), which are responsible for scavenging ROS, were also affected. Transcription profiling revealed that the differential expressions of ROS-producing and scavenging related genes, the photosynthesis-related genes and stress responsive genes were existed in transgenic plants of overproducing cytokinins. Our results suggested that broken in the homeostasis of cytokinins in plant cells could modulate the salt stress responses through a ROS-mediated regulation in Arabidopsis.

INDOLE-3-ACETIC ACID INDUCIBLE 17 positively modulates natural leaf senescence through melatonin-mediated pathway in Arabidopsis.

Melatonin (N-acetyl-5-methoxytryptamine) functions as a ubiquitous modulator in multiple plant developmental processes and various stress responses. However, the involvement of melatonin in natural leaf senescence and the underlying molecular mechanism in Arabidopsis remain unclear. In this study, we found that the endogenous melatonin level was significantly induced in a developmental stage-dependent manner, and exogenous melatonin treatment delayed natural leaf senescence in Arabidopsis. The expression level of AUXIN RESISTANT 3 (AXR3)/INDOLE-3-ACETIC ACID INDUCIBLE 17 (IAA17) was significantly downregulated by exogenous melatonin treatment and decreased with developmental age in Arabidopsis. Further investigation indicated that AtIAA17-overexpressing plants showed early leaf senescence with lower chlorophyll content in rosette leaves compared with wild-type plants, while AtIAA17 knockout mutants displayed delayed leaf senescence with higher chlorophyll content. Notably, exogenous melatonin-delayed leaf senescence was largely alleviated in AtIAA17-overexpressing plants, and AtIAA17-activated senescence-related SENESCENCE 4 (SEN4) and SENESCENCE-ASSOCIATED GENE 12 (SAG12) transcripts might have contributed to the process of natural leaf senescence. Taken together, the results indicate that AtIAA17 is a positive modulator of natural leaf senescence and provides direct link between melatonin and AtIAA17 in the process of natural leaf senescence in Arabidopsis.

Hydrogen sulfide regulates abiotic stress tolerance and biotic stress resistance in Arabidopsis.

Hydrogen sulfide (H2S) is an important gaseous molecule in various plant developmental processes and plant stress responses. In this study, the transgenic Arabidopsis thaliana plants with modulated expressions of two cysteine desulfhydrases, and exogenous H2S donor (sodium hydrosulfide, NaHS) and H2S scavenger (hypotaurine, HT) pre-treated plants were used to dissect the involvement of H2S in plant stress responses. The cysteine desulfhydrases overexpressing plants and NaHS pre-treated plants exhibited higher endogenous H2S level and improved abiotic stress tolerance and biotic stress resistance, while cysteine desulfhydrases knockdown plants and HT pre-treated plants displayed lower endogenous H2S level and decreased stress resistance. Moreover, H2S upregulated the transcripts of multiple abiotic and biotic stress-related genes, and inhibited reactive oxygen species (ROS) accumulation. Interestingly, MIR393-mediated auxin signaling including MIR393a/b and their target genes (TIR1, AFB1, AFB2, and AFB3) was transcriptionally regulated by H2S, and was related with H2S-induced antibacterial resistance. Moreover, H2S regulated 50 carbon metabolites including amino acids, organic acids, sugars, sugar alcohols, and aromatic amines. Taken together, these results indicated that cysteine desulfhydrase and H2S conferred abiotic stress tolerance and biotic stress resistance, via affecting the stress-related gene expressions, ROS metabolism, metabolic homeostasis, and MIR393-targeted auxin receptors.

The cysteine2/histidine2-type transcription factor ZINC FINGER OF ARABIDOPSIS THALIANA 6-activated C-REPEAT-BINDING FACTOR pathway is essential for melatonin-mediated freezing stress resistance in Arabidopsis.

Melatonin (N-acetyl-5-methoxytryptamine) is not only a widely known animal hormone, but also an important regulator in plant development and multiple abiotic stress responses. Recently, it has been revealed that melatonin alleviated cold stress through mediating several cold-related genes, including C-REPEAT-BINDING FACTORs (CBFs)/Drought Response Element Binding factors (DREBs), COR15a, and three transcription factors (CAMTA1, ZINC FINGER OF ARABIDOPSIS THALIANA 10 (ZAT10), and ZAT12). In this study, we quantified the endogenous melatonin level in Arabidopsis plant leaves and found the endogenous melatonin levels were significantly induced by cold stress (4 °C) treatment. In addition, we found one cysteine2/histidine2-type zinc finger transcription factor, ZAT6, was involved in melatonin-mediated freezing stress response in Arabidopsis. Interestingly, exogenous melatonin enhanced freezing stress resistance was largely alleviated in AtZAT6 knockdown plants, but was enhanced in AtZAT6 overexpressing plants. Moreover, the expression levels of AtZAT6 and AtCBFs were commonly upregulated by cold stress (4 °C) and exogenous melatonin treatments, and modulation of AtZAT6 expression significantly affected the induction AtCBFs transcripts by cold stress (4 °C) and exogenous melatonin treatments. Taken together, AtZAT6-activated CBF pathway might be essential for melatonin-mediated freezing stress response in Arabidopsis.

The Cysteine2/Histidine2-Type Transcription Factor ZINC FINGER OF ARABIDOPSIS THALIANA6 Modulates Biotic and Abiotic Stress Responses by Activating Salicylic Acid-Related Genes and C-REPEAT-BINDING FACTOR Genes in Arabidopsis.

The cysteine2/histidine2-type zinc finger proteins are a large family of transcription regulators, and some of them play essential roles in plant responses to biotic and abiotic stress. In this study, we found that expression of C2H2-type ZINC FINGER OF ARABIDOPSIS THALIANA6 (AtZAT6) was transcriptionally induced by salt, dehydration, cold stress treatments, and pathogen infection, and AtZAT6 was predominantly located in the nucleus. AtZAT6-overexpressing plants exhibited improved resistance to pathogen infection, salt, drought, and freezing stresses, while AtZAT6 knockdown plants showed decreased stress resistance. AtZAT6 positively modulates expression levels of stress-related genes by directly binding to the TACAAT motifs in the promoter region of pathogen-related genes (ENHANCED DISEASE SUSCEPTIBILITY1, PHYTOALEXIN DEFICIENT4, PATHOGENESIS-RELATED GENE1 [PR1], PR2, and PR5) and abiotic stress-responsive genes (C-REPEAT-BINDING FACTOR1 [CBF1], CBF2, and CBF3). Moreover, overexpression of AtZAT6 exhibited pleiotrophic phenotypes with curly leaves and small-sized plant at vegetative stage and reduced size of floral organs and siliques at the reproductive stage. Modulation of AtZAT6 also positively regulates the accumulation of salicylic acid and reactive oxygen species (hydrogen peroxide and superoxide radical). Taken together, our findings indicated that AtZAT6 plays important roles in plant development and positively modulates biotic and abiotic stress resistance by activating the expression levels of salicylic acid-related genes and CBF genes.

AtHAP5A modulates freezing stress resistance in Arabidopsis through binding to CCAAT motif of AtXTH21.

Several eukaryotic Heme-associated proteins (HAPs) have been reported to bind specifically to DNA fragments containing CCAAT-box; however, the physiological functions and direct targets of these HAP proteins in plants remain unclear. In this study, we showed that AtHAP5A as a transcription factor interacted with CCAAT motif in vivo, and AtXTH21, one direct target of AtHAP5A, was involved in freezing stress resistance. The AtHAP5A overexpressing plants were more tolerant, whereas the loss-of-function mutant of AtHAP5A was more sensitive to freezing stress than wild-type plants. Chromatin immunoprecipitation (ChIP) assay demonstrated that AtHAP5A could bind to five fragments that contained CCAAT motifs in the AtXTH21 promoter. Similarly, the AtXTH21 overexpressing plants exhibited improved freezing resistance, while xth21 knockdown mutants displayed decreased freezing resistance. Notably, the modulated freezing resistance of AtHAP5A overexpressing plants and knockout mutant could be reversed by the xth21 mutant and AtXTH21 overexpressing plants, respectively, indicating that AtHAP5A might act upstream of AtXTH21 in freezing stress. Additionally, modulation of AtHAP5A and AtXTH21 expression had the same effects on abscisic acid (ABA) sensitivity and reactive oxygen species (ROS) metabolism. Taken together, these results demonstrated that AtHAP5A modulates freezing stress resistance in Arabidopsis through binding to the CCAAT motif of AtXTH21.

Nitric oxide-activated hydrogen sulfide is essential for cadmium stress response in bermudagrass (Cynodon dactylon (L). Pers.).

Nitric oxide (NO) and hydrogen sulfide (H2S) are important gaseous molecules, serving as important secondary messengers in plant response to various biotic and abiotic stresses. However, the interaction between NO and H2S in plant stress response was largely unclear. In this study, endogenous NO and H2S were evidently induced by cadmium stress treatment in bermudagrass, and exogenous applications of NO donor (sodium nitroprusside, SNP) or H2S donor (sodium hydrosulfide, NaHS) conferred improved cadmium stress tolerance. Additionally, SNP and NaHS treatments alleviated cadmium stress-triggered plant growth inhibition, cell damage and reactive oxygen species (ROS) burst, partly via modulating enzymatic and non-enzymatic antioxidants. Moreover, SNP and NaHS treatments also induced the productions of both NO and H2S in the presence of Cd. Interestingly, combined treatments with inhibitors and scavengers of NO and H2S under cadmium stress condition showed that NO signal could be blocked by both NO and H2S inhibitors and scavengers, while H2S signal was specifically blocked by H2S inhibitors and scavengers, indicating that NO-activated H2S was essential for cadmium stress response. Taken together, we assigned the protective roles of endogenous and exogenous NO and H2S in bermudagrass response to cadmium stress, and speculated that NO-activated H2S might be essential for cadmium stress response in bermudagrass.

Exogenous application of hydrogen sulfide donor sodium hydrosulfide enhanced multiple abiotic stress tolerance in bermudagrass (Cynodon dactylon (L). Pers.).

As a gaseous molecule, hydrogen sulfide (H2S) has been recently found to be involved in plant responses to multiple abiotic stress. In this study, salt (150 and 300 mM NaCl), osmotic (15% and 30% PEG6000) and cold (4 °C) stress treatments induced accumulation of endogenous H2S level, indicating that H2S might play a role in bermudagrass responses to salt, osmotic and cold stresses. Exogenous application of H2S donor (sodium hydrosulfide, NaHS) conferred improved salt, osmotic and freezing stress tolerances in bermudagrass, which were evidenced by decreased electrolyte leakage and increased survival rate under stress conditions. Additionally, NaHS treatment alleviated the reactive oxygen species (ROS) burst and cell damage induced by abiotic stress, via modulating metabolisms of several antioxidant enzymes [catalase (CAT), peroxidase (POD) and GR (glutathione reductase)] and non-enzymatic glutathione antioxidant pool and redox state. Moreover, exogenous NaHS treatment led to accumulation of osmolytes (proline, sucrose and soluble total sugars) in stressed bermudagrass plants. Taken together, all these data indicated the protective roles of H2S in bermudagrass responses to salt, osmotic and freezing stresses, via activation of the antioxidant response and osmolyte accumulation. These findings might be applicable to grass and crop engineering to improve abiotic stress tolerance.